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Objective: To confirm the direct regulatory effect of WTAP -mediated RNA m 6 A modification on the KDM4B gene in t (8;21) acute myeloid leukemia (AML) cells through MeRIP combined with reverse transcription real-time quantitative PCR (RT-qPCR) technology., Methods: The lentivirus-mediated shRNA target WTAP or KDM4B gene was used to transfect the t (8;21) AML cell lines: Kasumi-1 and SKNO-1, and cells transfected with randomly shuffled shRNA as the control. Using the Ultrapure RNA Extraction Kit (DNase I) to extract RNA. The Magna MeRIP TM m 6 A Kit was used to enrich methylated modified fragments, and detect the m 6 A methylated RNA regions by RT-qPCR, and the protein and mRNA expression levels of WTAP and KDM4B in cells were detected by Western blot and reverse transcription real-time quantitative PCR (RT-qPCR). Colony formation assays were used to detect the colony ability of cells in vitro ., Results: Silencing the expression of WTAP in Kasumi-1 cells, the enrichment of m 6 A methylation modification was significantly decreased in the 3'UTR of KDM4B mRNA( P < 0.01), and the protein( P < 0.001) and mRNA (Kasumi-1: P < 0.001; SKNO-1: P < 0.01) expression levels of KDM4B were also significantly inhibited in Kasumi-1 and SKNO-1 cells upon WTAP knockdown (all P < 0.01), accompanied by a significant decrease in the colony-forming ability of both cell lines (both P < 0.01)., Conclusion: In t(8;21) AML cell lines, WTAP could regulate the expression of KDM4B by regulating the m 6 A modification of the 3'UTR of KDM4B mRNA, and silencing the expression of KDM4B could inhibit the cellular proliferation in vitro .

Objective: To study the distribution characteristics of thalassemia genotype in Han Population in Sanya of Hainan Province., Methods: Gap PCR and reverse dot hybridization were used to detect and analyze the thalassemia gene in 572 suspected thalassemia carriers of Han Population in Sanya., Results: Among the 572 Han Population in Sanya, 271 cases of thalassemia gene abnormality were detected, among which 161 cases were founded to be carriers of α-thalassemia gene. A total of 9 genotypes were detected, in the following order of the detection rate was －－ SEA /αα，－α 3.7 /αα，－α 4.2 /αα，－－ SEA /－α 3.7 ，－－ SEA /－α 4.2 ，－α 4.2 /－α 4.2 ，－α 3.7 /－α 4.2 ，－α 3.7 /－α 3.7 ，－－ SEA /－－ SEA . Among them, the deletion type (－－ SEA /αα) in southeast Asia was the most common, accounting for 66 cases. 99 cases of β-thalassemia were detected, there were 7 genotypes, all of which were heterozygous. The order of the detection rate was CD41-42/βN, IVS-II-654/βN, CD17/βN, CD71-72/βN, -28/βN, －29/βN, CD27-28/βN. Among them, CD41-42/βN was the most common, accounting for 51 cases. In addition, 11 cases of combined α and β thalassemia were detected. Five kinds of genotypes were checked out, the order of detection rate was －α 3.7 /αα composite CD41-42/βN, －－ SEA /αα composite IVS-II-654/βN, －α 4.2 /－α 4.2 composite CD41-42/βN, －α 4.2 /αα composite －29/βN , －－ SEA / －α 4.2 composite CD41-42/βN., Conclusion: Han Population in Sanya of Hainan Province is a high-risk population of thalassemia, the genotype characteristics are different from other areas with high incidence of thalassemia in China. The main type of α-thalassemia is the deficiency mutation of southeast Asia, while CD41-42 heterozygous mutation is the main type of β-thalassemia.

Objective: To analyze the clinical characteristics and risk factors of invasive fungal infection (IFI) occurenced in patients with acute leukemia (AL) during treatment in tropical regions., Methods: The clinical data of 68 AL patients admitted to the Hainan Hospital of PLA General Hospital from April 2012 to April 2019 was retrospectively analyzed. Logistic regression analysis was used to analyze the factors affecting the occurrence of IFI in AL patients., Results: Among the 68 patients, 44 were acute myeloid leukemia, 24 were acute lymphoblastic leukemia, 39 were male, 29 were female and the median age was 41(13-75) years old. The 68 patients received 242 times of chemotherapy or hematopoietic stem cell transplantation(HSCT), including 73 times of initial chemotherapy or inducting chemotherapy after recurrence, 14 times of HSCT, 155 times of consolidating chemotherapy. Patients received 152 times of anti-fungal prophylaxis, including 77 times of primary anti-fungal prophylaxis and 75 times of secondary anti-fungal prophylaxis. Finally, the incidence of IFI was 31 times, including 24 times of probable diagnosis, 7 times of proven diagnosis, and the total incidence of IFI was 12.8%(31/242), the incidence of IFI in inducting chemotherapy was 24.66%(18/73), the incidence of IFI in HSCT patients was 28.57% (4/14), the incidence of IFI in consolidating chemotherapy was 5.80% (9/155). Multivariate analysis showed that inducting chemotherapy or HSCT, the time of agranulocytosis ≥7 days, risk stratification of high risk were the independent risk factors for IFI in AL patients during treatment in tropical regions., Conclusion: The incidence of IFI in patients with AL in the tropics regions is significantly higher than that in other regions at homeland and abroad. Anti-fungal prophylaxis should be given to the patients with AL who have the high risk factors of inducting chemotherapy or HSCT, time of agranulocytosis ≥7 days and risk stratification of high risk.

Objective: To investigate the relationship between hypoxia inducible factor 1 (HIF1α) and Wilms' tumor 1associating protein (WTAP) expression level in t(8;21) acute myeloid leukemia cells., Methods: The t(8;21) acute myeloid leukemia cell lines, including SKNO-1 and Kasumi-1 were treated by Echinomycin for 24 h, RT-qPCR and Western blot were used to detect the expression levels of WTAP mRNA and the protein. The CoCl 2 was used to induce the hypoxia of the cells for 24 h, the expression levels of HIF1α, WTAP protein were detected by Western blot., Results: The expression level of WTAP mRNA and the protein in the echinomycin treated group was significantly lower than those in the control group (P<0.01). The expression level of WTAP protein in the CoCl 2 treated group was significantly higher than those in the control group (P<0.05)., Conclusion: The inhibition of HIF1-α could down-regulates the expression of WTAP, while the up-regulation of HIF1α could up-regulates the expression of WTAP, which shows that there is a positive correlation of HIF1α and WTAP expression. This result suggesting that HIF1α may be involves in the expression regulation of WTAP gene.

Objective: To analyze the characteristics, prognosis and risk factors of bloodstream infection in patients with hematological malignancies in the tropics, so as to provide evidence for the prevention and treatment of bloodstream infection., Methods: The clinical features, blood culture results and prognosis of patients with bloodstream infection in patients with hematological malignancies admitted to Hainan Hospital of PLA General Hospital were retrospectively studied., Results: The most common primary infection site of the 81 patients with hematological malignancies was lung (46.91%), followed by PICC (11.11%). The detection rate of Gram-positive bacteria and Gram-negative bacteria in the blood culture was 60.98% and 30.02%, respectively. Coagulase-negative staphylococci was the most common Gram-positive bacteria resulting in bloodstream infection in our study. Of the Gram-negatives, Klebsiella pneumoniae (34.38%) was predominant, followed by Escherichia coli (18.75%) and Pseudomonas aeruginosa (18.75%). Gram-positive bacteria was highly sensitive (100%) to vancomycin, linezolid and tigecycline. Study showed that Gram-negative bacteria had low sensitive to quinolones, in particular, the resistance rate of Escherichia coli to quinolones was as high as 83.33%. In terms of overall survival (OS), the 30-days OS of patients with Gram-negative and Gram-positive septicemia was 77.42% and 92.00%, respectively. There was no statistically significant difference between the two groups. Multivariate analysis revealed that septic shock (P=0.001, RR=269.27) was an independent risk factor for 30-day mortality, and remission status (P=0.027, RR=0.114) was an independent predictor of a favourable outcome of bloodstream infection in patients with hematological malignancies., Conclusion: Gram-positive bacteria are the main pathogens causing bloodstream infections in patients with hematological malignancies in the tropics. Improving the care of PICC is an important measure to reduce the incidence of bloodstream infection in patients with hematological malignancies in the tropics. A correct treatment relieving disease and effective prevention and treatment of septic shock can reduce mortality of patients with bloodstream infection in patients with hematological malignancies in the tropics.

Objective: To investigate the clinical significance of post-transplantation serum immunoglobulin level in the outcome of patients with hemalologic malignancies treated by haploidentical peripheral hematopoietic stem cell transplanta-tion(Haplo-HSCT)., Methods: The clinical data of 157 patients treated by haplo-HSCT were analyzed retrospectively. The overall survival rate (OS), graft versus host disease (GVHD) incidence, infection incidence, serum immunoglobulin level, the relationship of immunoglobulin levels with OS and transplant complications were analyzed., Results: The 2-year OS rate was 59.2%(95%CI:51.6%-66.9%), 2-year relapse mortality was 11.5%(95%CI: 6.4%-16.6%), and non-relapse mortality was 29.3%(95%CI:21.7%-36.9%). The cumulative incidence of III-IV aGVHD was 16.6%(95%CI:10.8%-22.9%); the cumulative incidence of extensive cGVHD was 21.7%(95%CI:15.3%-28.6%); the cumulative incidence of severe bacterial infection within 1 year was 59.2%(95%CI:51.6%-66.2%); the cumulative incidence of invasive fungal infection was 47.1%(95%CI:38.9%-54.8%). The occurrence of extensive cGVHD after haplo-HSCT related with the gender match of donor-recipient and bacterial infection. The levels of IgG in patients with 0-II aGVHD and patients with III-IV aGVHD for 1 month after haplo-HSCT were (6.96±2.47) and (4.27±2.42) g/L （P=0.003）, IgG levels at 3 months afte haplo-HSCT were (8.71±4.47) and (6.65±2.95) g/L (P=0.038); IgG levels at 1 month after haplo-HSCT showed predictive value for III-IV aGVHD susceptibility(P=0.003); for patients with IgG<4 g/L at any time after haplo-HSCT, the incidence of extensive cGVHD was significantly increased (35.5% vs 18.3%) (P=0.037), the incidence of fungal infection within 1 year after haplo-HSCT was significantly increased(71.0% vs 41.3%) (P=0.003), and the 2-year survival rate was reduced significantly (P=0.035)., Conclusion: Haplo-HSCT is effective for the treatment of hematologic malignancies. Patients with lower IgG at 1 month after haplo-HSCT are more likely to develop III-IV aGVHD, and IgG levels at 1 month after haplo-HSCT can predict its susceptibility to a certain extent. Patients with severe hypoimmunoglobulinemia (IgG<4 g/L) after haplo-HSCT are more likely to develop extensive cGVHD, fungal infection and show worse survival prognosis.

Objective: To investigate the clinical characteristics of relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL) and the factors affecting overall survival (OS) time., Methods: The clinical data of 14 R/R DLBCL patients admitted to the Hainan Hospital of Chinese PLA General Hospital from April 2012 to March 2019 were analyzed retrospectively and the overall response rate (ORR) after the end of different treatments was estimated. Kaplan-Meier method was used to describe the survival curve, and Log-rank test was used to compare whether different survival curves showed statistically different., Results: There were 8 males and 6 females with a median age of 51 (26-75) years old and the median course of treatment before R/R was 7 (4-13). Finally, 11 patients achieved remission, 6 patients of which showed complete remission, and 5 patients showed partial remission, with the median ORR duration at 2.5 (0-51) months. All patients in the group of ibrutinib combined with second-line chemotherapy achieved remission (4/4), it was equivalent to the high-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (HDC-AHSCT) group (4/4), which was significantly higher than that of the other second-line group (3/6). The median OS time of patients was 17 (6-76) months. The survival of patients receiving ibrutinib combined with second-line chemotherapy and HDC-AHSCT was significantly better than that of patients not receiving ibrutinib combined with second-line chemotherapy and HDC-AHSCT. Normal lactate dehydrogenase, IPI score＜3 at diagnosis, and CR/PR after treatment could improve the survival time of patients., Conclusion: The duration of remission for R/R DLBCL patients is short and the prognosis is very poor. The survival time of patients with high level of lactate dehydrogenase, IPI score≥3 at diagnosis and SD/PD after treatment is significantly shortened. Ibrutinib combined second-line chemotherapy and HDC-AHSCT can improve the efficacy and survival of R/R DLBCL patients.

Objective: To analyze the efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for treatment of acute leukemia in the tropical area., Methods: Twelve acute leukemia patients who were underwent allo-HSCT from April 2013 to November 2018 in Hainan Hospital of Chinese PLA General Hospital were selected, including 5 cases of acute lymphoblastic leukemia (ALL) and 7 case of acute myeloid leukemia (AML). Three cases received HLA matched sibling hematopoietic stem cell transplantation, 8 cases received haploidentical hematopoietic stem cell transplantation, 1 cases received partially mismatched unrelated hematopoietic stem cell transplantation. Pretreatment regimen: 9 cases received modified BU/CY+ATG pretreatment regimen, 3 cases received BU/CY pretreatment regimen. Graft-versus-host disease (GVHD) prevention regimen: all patients received cyclosporine A, mycophenolate mofetil combined with short-term methotrexate regimen. The clinical efficacy of allo-HSCT in treatment of acute leukemia in the tropical area was analyzed by detecting hematopoietic reconstitution, GVHD, infection, relapse and survival after transplantation., Results: All the 12 patients achieved granulocyte reconstruction and megakaryocyte reconstruction. The median time of granulocyte reconstruction was 11.5 (6-14) days, and the median time of megakaryocytic reconstruction was 12.5 (10-22) days. Within 100 days after transplantation, the acute GVHD occurved in 8 cases, including 6 cases of Ⅱ-Ⅳ degree acute GVHD and 2 cases of Ⅲ-Ⅳ degree acute GVHD, 11 cases survived more than 100 days after transplantation, and the chronic GVHD occurred in 1 case, which was mildly limited. Pulmonary infection occurred in 7 cases, cytomegaloviremia occurred in 6 cases, EB viremia occurred in 6 cases, and hemorrhagic cystitis occurred in 5 cases. 2 cases relapsed and eventually died, and the remaining 10 patients survived without disease until the date of follow-up. The median follow-up time was 4 (1-68) months, 83.3% (10/12) survived without disease, and 16.7% (2/12) relapsed., Conclusion: Allo-HSCT is an effective method for the treatment of acute leukemia in adults. Leukemia patients should be transplanted as soon as possible after remission. The incidence of pulmonary fungal infection in transplanted patients in tropics is high, therefore the prevention and treatment of fungal infection should be strengthened.

Objective: To study the clinical characteristics of patients with post-transplantation lymphoproliferative disease (PTLD) after allogeneic peripheral blood hematopoietic stem cell transplantation, and to improve the understanding and diagnosis of PTLD., Methods: The clinical data of 244 patients underwent allogeneic hematopoietic stem cell transplantation in the General Hospital of PLA from May 2014 to April 2017 were analyzed retrospectively. The follow-up time was up to November 30, 2017. The incidence, risk factors, treatment and survival of patients with PTLD were statistically analyzed., Results: Among the 244 cases the PTLD occurred in 22 cases, the incidence rate was 9.02%, 5 of them were diagnosed by pathology, and 17 were diagnosed clinically. All of them had EB virus infection. They were all ATG user, either underwent related haploidentical hematopoietic stem cell transplantation or unrelated hematopoietic stem cell transplantation, 20 cases were treated with rituximab or rituximab combined with γ-globulin, glucocorticoid, ERV+CTL, chemotherapy and 17 showed the effective response, with a total effective rate of 85%. The median follow-up time was 122 days, the median survival time was 5 months (1-22 months) and the total survival rate was 50%., Conclusion: The incidence of PTLD after allogeneic peripheral blood hematopoietic stem cell transplantation closely relates with EB virus infection. The application of ATG in the preconditioning scheme is a high risk factor for the onset of PTLD. In the case of no pathological diagnosis, clinical and laboratory examinations should be actively combined so as to define clinical diagnosis. The riturimab should be used more and more for patients with PTLD.

Objective: To summarize the clinical characteristics of peripheral blood, immune phenotypes, fusion genes and cytogenetics of patients with t(8;21) acute myeloid leukemia(AML) through the retrospective analysis of 586 patients with t(8;21) AML from 15 blood disease research centers in Northern area of China., Methods: The factors affecting prognosis of patients with t(8;21) AML were investigated by using univariate and multivariate COX regression., Results: The immune type of t(8;21) AML patients was mainly with HLA-DR + , CD117 + , CD34 + , MPO + , CD38 + , CD13 + and CD33 + (>95%), part of them with CD19 + and CD56 + ; the most common accompanied mutation of t(8;21) AML patients was C-KIT mutation (37.8%); in addition to t(8;21) ectopic, the most common chromosomal abnormality was sex chromosome deletions (38.9%). The univariate analysis revealed a significant survival superiority of OS and PFS in t(8;21) AML patients of WBC≤3.5×10 9 /L without C-KIT mutation, the newly diagnosed ones achieved HSCT(P<0.05), only survival superiority on OS in t(8;21) AML patients with extramedullary infiltration and CD19 positive; the results of multivariate analysis showed a significant survival superiority on OS and PFS in t(8;21) AML patients with WBC≤3.5×10 9 /L(P<0.05)., Conclusion: The clinical features of t(8;21) AML patients in China are similar to those in other countries, WBC≤3.5×10 9 /L is a good prognostic factor while the C-KIT mutation is a poor one in t(8;21) AML patients.

Muscle-derived stem cells (MDSC) are a population of multipotent stem cells in the muscular tissue. It provide an excellent prospect of hemopathy treatment due to their superiorities, such as rich sources, convenient material resource and a high survival rate after transplantation and so on. However, there are great differences in sampling, separation, purification, and proliferation when MDSC were cultured in vitro. In addition, the proliferation conditions of the MDSC in vitro are yet unclear. The related regulatory mechanisms, which MDSC transformed into haematopoietic cells, need to be investigated. In this article, the experimental researches on the differentiation of MDSC into haematopoietic lineages are reviewed, the concrete problems discussed in this review are culture of MDSC in vitro, identification of MDSC, proleferation of MDSC, differention of MDSC in to hematopoietic lineages and so on.

Objective: To investigate the inductive therapeutic effects of imatinib combined with VP low dose regiment on adult patients with Ph-positive acute lymphoblastic leukemia (Ph(+) ALL)., Methods: Fourteen newly diagnosed adult patients with Ph(+) ALL were treated with VP regimen, and imatinib (400 mg/d) was added at the 8(th) day. VP regimen would be stopped when neutropenia lasted for 1 week or complicated with infection, fever, etc. Therapeutic effects were assessed by bone marrow morphology and quantitative analysis of BCR/ABL:ABL at the 28(th) - 33(rd) day. Patients could be treated with imatinib combined with chemotherapy for consolidation and maintenance therapy or were treated with allogeneic hematopoietic stem cell transplantation after complete remission., Results: Fourteen cases obtained CR1 after first course of treatment, the median decline of BCR/ABA:ABL was 55.89 (10.25 -180.97) %; during the induction chemotherapy, pulmonary infection occurred in 3 patients, diarrhea in 1 patients, facial edema in 3 patients, however, all these patients were cured after symptomatic treatment, only 1 patient died of relapse after transplantation., Conclusion: In the period of tyrosine kinase inhibitor (TKI), inductive chemotherapy combined with imatinib and low dose VP can obtaine satisfactory CR rate and decrease the toxicity of the traditional drugs. It is suggested that TKI combined with VP regimen chemotherapy can achieve CR1 and make possible for allo-HSCT, from which patients can achieve the long-term survival.

Muscle-derived stem cells (MDSC) are defined as myogenic stem cells endowed with their ability to self-renew and differentiate into multiple cell types of their derivative tissue, and are proved to be over 10 times more efficient in hematopoiesis than hematopoietic stem cells (HSC). Although the mechanism which MDSC differentiate into blood cells is still unclear, MDSC were considered to replace HSC to treat the patients suffering from bone marrow diseases such as aplastic anemia and tumor. MDSC are different from HSC in a variety aspects like biological characteristics, protein expression and cell proliferation. On the other hand, MDSC contain multiple distinct stem cell populations. Among these, there is only a small part with the ability to repopulate hematopoietic cells, and it is still uncertain whether their origin is same as HSC. This review summarizes the difference between MDSC and HSC, the ability of MDSC to repopulate hematopoietic cells, and the prospect of MDSCs' transplantation.

The aim of this study was to investigate the effect of microRNA-193b (miR-193b) on C-KIT protein expression and biological behaviors in K562 cells. The FAM-labeled miR-193b mimic and negative control were respectively transfected into K562 cells using HiPerFect transfection reagent. The percentage of FAM-positive cells was monitored by flow cytometry. The levels of C-KIT and phosphorylated C-KIT protein were detected by Western blot. The cell growth was measured by CCK-8 reagent. The apoptosis of cells were analyzed by flow cytometry with Annexin V staining. The differentiation of cells were analyzed by flow cytometry with anti-CD11b or anti-CD15 staining. The results demonstrated that the percentage of FAM-positive cells was about 80% in miR-193b or negative control-transfected K562 cells. Compared with the negative control group, overexpression of miR-193b in K562 cells significantly inhibited the levels of C-KIT and phosphorylated C-KIT protein. Meanwhile, the cell growth was inhibited and the percentages of apoptotic cells, CD11b- or CD15-positive cells increased. It is concluded that miR-193b can reduce C-KIT expression and inhibit cell growth in K562 cells. The growth-inhibitory activity of miR-193b is associated with apoptosis and granulocytic differentiation. This study contributed to further investigate the role of miR-193b in leukemogenesis.

The aim of the research was to find the up-regulated microRNA (miRNA) in K562 cells treated by 5-aza, to detect the miRNA expression in healthy people, CML patients and leukemia cell lines and to investigate the influence of miRNA on K562 cell proliferation. Up-regulated miRNA in K562 cells after 5-aza treatment was screened by microarray. The up-regulated miR-638, miR-663 and miR-92b with CpG islands in upstream region were screened by microarray in combination with bioinformatics. The miRNA mentioned above was further ascertained by SYBR-green real-time PCR. Up-regulated miR-663 was confirmed by real-time PCR. Expression level of miR-663 was detected in K562, U937 and Kasumi cell lines, and white blood cells from bone marrow of normal donor and from peripheral blood of newly diagnosed CML patient. Methylation-specific PCR (MSP) was applied to analyze the methylated status of miR-663 CpG island in K562 cells. Proliferation of K562 cells was observed after miR-663 was over expressed by transient transfection. The results showed that the expression level of miR-663 in K562 cells was up-regulated after 5-aza treatment, and the expressions of miR-663 were lower in K562, U937, Kasumi cell lines and newly diagnosed patients, compared with healthy people. The CpG island of miR-663 was methylated in K562 cell line according to detection result of MSP. The proliferation of K562 cell could be suppressed by over-expression of miR-663 in vitro. It is concluded that miR-663 CpG island is methylated in K562 cell line. The miR-663 is down-regulated in K562, U937, Kasumi cell lines and CML patients, compared with healthy people. miRNA-663 in K562 cells is up-regulated after 5-aza treatment. Over-expression of miR-663 can suppress the proliferation of K562 cells, which suggests that miR-663 may possesses suppressive effect for leukaemia.

This study was purposed to investigate the difference of zo-1 gene promoter methylation between healthy individuals and acute leukemia patients. BS-PCR method was used to detect the status of zo-1 gene methylation in healthy individuals, acute leukemia patients and leukemic cell line NB4 cells. The results showed that zo-1 gene was hypomethylated in bone marrow samples from healthy individuals (1.9%). In newly diagnosed AL and relapsed patients, the rate of zo-1 gene methylation was 93.2% and 66.9% respectively, while it was 16.4% in AL patients in complete remission, which was much higher than that in healthy individuals. There was significant difference between them. It is concluded that as compared with healthy individuals, zo-1 gene in acute leukemia patients is hypermethylated and with different degrees in various phases of leukemia. Analysis of zo-1 gene methylation status may be useful to monitor the development of acute leukemia.

To analyze the possible microRNA (miRNA) target sites in the 3' untranslated region (3'-UTR) of FMS-like tyrosine kinase 3 (FLT3) gene, a FLT3 3'-UTR-luciferase reporter vector was constructed and the effect of miRNA on its activity was evaluated in 293T cell line. The 3'-UTR fragment of FLT3 gene was amplified by PCR from genomic DNA of HepG2 cells. PCR products were cloned into PstI/EcoRI-modified pGL3-control reporter vector (pGL3-control-m). The miRNA targeting FLT3 3'-UTR was predicted by Target Scan 5.1 software. The luciferase reporter vector and miRNA eukaryotic expression vector were transferred into 293T cells using FuGENE HD transfection reagent. The Dual-Luciferase Reporter Assay System was used to quantitate the reporter activity. The results showed that a 804-bp 3'-UTR fragment of FLT3 gene was successfully cloned into the pGL3-control-m reporter vector, which authenticated by PstI/EcoRI digestion and DNA sequencing. The predicted miRNA targeting FLT3 3'-UTR included miRNA-15a, miRNA-15b, miRNA-16, miRNA-195, miRNA-424 and miRNA-497. The luciferase activity of reporter construct treated with miRNA-15a, miRNA-15b or miRNA-195 was decreased respectively about 20% compared with the control group. It is concluded that the FLT3 3'-UTR-luciferase reporter vector has been successfully constructed. The luciferase activity of the reporter can be suppressed by miRNA-15a, miRNA-15b or miRNA-195.

In order to investigate the effect of demethylating treatment on the expression of inhibitory KIR and the cytolytic activity of NK-92MI cells, and to study the possible relationship between the demethylation of inhibitory kir gene and the function of NK cells. NK-92MI cells were treated with 5-azacytidine to induce DNA demethylation. The expression of KIR3DL1, KIR2DL2/KIR2DL3, KIR2DL1 and the viability of NK-92MI cells were detected by flow cytometry. The KIR3DL1 positive and the KIR3DL1 negative NK-92MI cells were also sorted by flow cytometry. The cytotoxicity of NK-92MI against K562 cells was detected by the LDH release assay. The results demonstrated that the expressions of KIR3DL1, KIR2DL2/KIR2DL3 and KIR2DL1 in NK-92MI cells all increased after treating with 1.0, 2.5 and 5 micromol/L of 5-azacytidine for 72 hours. And the cytotoxicity of NK-92MI against K562 cells decreased. In these dose range, 5-azacytidine did not influence the viability of NK-92MI cells. Additionally, the cytotoxicity of KIR3DL1 positive NK-92MI cells was lower than that of the KIR3DL1 negative cells. It is concluded that the demethylating treatment suppresses the cytotoxicity of NK-92MI cells through increasing the expression of inhibitory KIR.

The aim of this study was to analyze the promoter methylation patterns of inhibitory killer cell immunoglobulin-like receptor (KIR) which gene expression and the effect of demethylation treatment were studied, and to explore the possible regulation mechanism of inhibitory kir gene expression. The promoter methylation levels of kir2DL1 and kir2DL2/kir2DL3 in NK-92MI cell line were detected by bisulfite sequencing technique. Then NK-92MI cells were treated with 5-azacytidine to induce the demethylation of CpG islands. The levels of gene expression of kir were determined by RT-PCR. The results demonstrated that the methylation frequencies of CpG dinucleotides surrounding the promoter regions of kir2DL1 and kir2DL2/kir2DL3 genes were 25% to 88% and 5% to 80% respectively. DNA-demethylating treatment with 5-azacytidine resulted in re-expression of kir2DL1 gene and increased expressions of kir2DL1, kir2DL2 and kir2DL3 genes in NK-92MI cells. In conclusion, the promoter DNA methylation participates in the regulation of kir gene expression in NK-92MI cells.

Objective: To study the role of transcription factor E2F1 in the transcription of KIR3DL1 promoter, and to identify its molecular mechanism of regulation of KIR3DL1 gene expression., Methods: The mutant promoter fragment of KIR3DL1 gene was amplified from genomic DNA of K562 cells by PCR. The PCR product was cloned into pGL3-basic reporter vector to construct KIR3DL1 promoter-luciferase reporter plasmid (PLRP). The binding of E2F1 to KIR3DL1 promoter was detected by chromatin immunoprecipitation (CHIP) assays. The KIR3DL1 PLRP construction was transfected into K562 cells using cationic liposome SuperFect. The binding of E2F1 to the construction was detected by CHIP assays and reporter activity was quantitated by the dual-luciferase reporter assay system. The mammalian expression vector containing E2F1 cDNA was co-transfected into K562 cells with wild-type KIR3DL1 PLR construction and reporter activity was quantitated., Results: The mutant KIR3DL1 PLR recombinant was constructed successfully and a naturally point mutation (TTTGGCGC-->TTCGGCGC) within a putative E2F1 binding site in the KIR3DL1 promoter region was authenticated by DNA sequencing. E2F1 absolutely could not bind to the mutant KIR3DL1 promoter in K562 cells, but could bind to the wild-type one in NK-92MI cells. The binding of E2F1 to the mutant KIR3DL1 PLR construction was partially reserved, however, its relative luciferase activity was decreased by 50% than that of wild-type. On the other hand, when co-transfected with E2F1 mammalian expression vector, the relative luciferase activity of wild-type construction was increased, over 2-fold higher than that of control group., Conclusion: E2F1 participates in the regulation of the transcriptional activation of KIR3DL1 gene. The number of CpG dinucleotide and methylation pattern within the E2F1 binding site probably influence the binding of E2F1 to target sequence.

Aim: To investigate the methylation pattern of KIR3DL1 promoter region in NK cell line NK-92MI and the effect of demethylation and histone acetylation on gene expression, and to study the possible regulation mechanism of KIR3DL1 expression., Methods: The methylation status of KIR3DL1 promoter in NK-92MI cells was detected by bisulfite sequencing technique. Then NK-92MI cells were treated with the DNA-demethylating compound 5-azacytidine and (or) the inhibitor of histone deacetylase trichostatin A, and the expression of KIR3DL1 gene was observed., Results: The CpG dinucleotides surrounding promoter region of KIR3DL1 gene in NK-92MI cells were consistently methylated with a frequency of 70%-100%. After 72 h treatment with 2.5 micromol/L and 5 micromol/L of 5-azacytidine, the mRNA expression of KIR3DL1 gene in NK-92MI cells increased 66.6% and 114.6%, respectively. However, after 72 h treatment with 50 nmol/L of trichostatin A, the mRNA expression of KIR3DL1 gene in NK-92MI cells did not increase. Additionally, combined treatment with 5-azacytidine and trichostatin A did not lead to a synergistic effect compared with 5-azacytidine alone., Conclusion: KIR3DL1 expression in NK-92MI cells is regulated by promoter methylation.

To analyze the methylation pattern of kir3dl1 promoter and its relationship with gene expression, and to study the possible regulation mechanism of kir3dl1 gene expression, the methylation status of kir3dl1 promoter in K562 cell line was detected by bisulfite sequencing technique. Then K562 cells were treated with 5-azacytidine so as to induce de-methylation of CpG island, and the relationship of CpG island methylation with kir3dl1 gene expression was investigated. The results demonstrated that CpG dinucleotides surrounding core promoter region of kir3dl1 gene was methylated at a frequency of 20% to 100% in K562 cell line. Comparison of promoter sequence with GenBank database revealed a base substitution within a putative binding site for the transcription factor E2F in K562 cell line. This mutation forms a new methylation site in kir3dl1 promoter. DNA-demethylating treatment resulted in de novo expression of kir3dl1 gene in formerly non-expressed K562 cell line. It is concluded that kir3dl1 expression in K562 cells is regulated by DNA methylation. Deeply to study E2F function contributes to profound understanding of its role in kir3dl1 gene expression regulation.

Objective: To explore the distribution of leptin expression and the effect of sepsis on leptin protein and mRNA levels., Methods: Vital organ samples including hypothalamus, lung, liver, spleen, stomach, duodenum, kidney, epididymal fat pad and testis of normal rats were collected. The mRNA expressions of leptin in those samples were determined by RT-PCR. The sepsis rat model induce by cecal ligation and perforation (CLP) was established, setting groups of sham-operation, CLP model, CLP + intralipid injection, CLP + estradiol injection and CLP + insulin injection, as the latter three groups were set to intervene energy metabolism and neuroendocrine function. Radioimmunoassay was applied to measure serum leptin concentrations in each group at 12 h after injury, while RT-PCR was also used to detect Leptin mRNA expressions in hypothalamus, fat and lung after injury., Results: Leptin mRNA expressions were confirmed in all the above nine vital organs, with the highest in kidney but the lowest in testis. The serum leptin level showed no significant difference between sham operation group and other four groups. Compared with sham operation group, the Leptin mRNA level in CLP group decreased significantly in hypothalamus, fat and lung, while that in the other three groups showed different changes. The effect of intralipid on Leptin mRNA expression was found to be a dual-direction pattern, with central stimulation but peripheral inhibition., Conclusion: Leptin is widely expressed in multiple vital organs, and it may be a protective factor to promote recovery of sepsis-induced internal disorders.

To analyze the function of kir3dl1 core promoter and study the possible regulation mechanism of kir3dl1 gene expression, a kir3dl1 promoter-luciferase reporter vector was constructed and the promoter activity was evaluated in the K562 cell line. A core promoter fragment of the kir3dl1 5'-untranslated region was amplified by PCR. PCR products were cloned into BglII/NcoI-digested pGL3-basic reporter vector; the polycationic compound SuperFect-reporter vector complexes were transferred into K562 cells. The Dual-Luciferase Reporter Assay System was used to quantitate the reporter vector luciferase activity. MTT method was used to measure the influence of SuperFect-DNA complexes on the survival rate of K562 cells. The results indicated that a 254-bp promoter fragment of kir3dl1 gene was successfully constructed and cloned into the pGL3-basic reporter vector, which was authenticated by BglII/NcoI digestion and DNA sequencing. The luciferase activity of the minimal promoter construct was significantly higher than that of the pGL3-Basic promoter in K562 cells. Transiently transfected cells presented continuously optimal luciferase activity and relative luciferase activity up to 3 days. The cell activity was between 76% and 92%. It is concluded that a kir3dl1 promoter-luciferase reporter vector is successfully constructed, the transfection system used in this study can effectively transfer gene in K562 cells. The kir3dl1 core promoter possesses higher activity in K562 cells, and can promote significantly expression of luciferase reporter gene in K562 cells.

Objective: To investigate the clinical features, treatment modalities and the prognosis of advanced neuroblastoma in children., Methods: The medical records of 63 children with stage III or IV neuroblastoma from January 1996 to December 2005 were retrospectively reviewed. Sixty patients were treated by tumor resection and (or) chemotherapy and (or) radiation. Fourteen out of the 60 patients received another autologous peripheral blood stem cell transplantation., Results: Of the 63 patients with advanced neuroblastoma, the male/female ratio was 2.7:1 and the median age at diagnosis was 4 years old. Most of the initial symptoms included pyrexia, abdominal pain, abdominal mass, and leg or articular pain. Primary tumor sites were adrenal (38%), retroperitoneal (35%), mediastinal (17%), pelvic (6%) and cervical (2%). The sites of metastasis at diagnosis included local (41%) and (or) distant (37%) lymph nodes, bone marrow (60%), bone (46%) and liver (16%). The median survival time of the 63 patients was 32.7 months. The 2-year survival rate was 44.3%. Statistical analysis demonstrated that unfavorable survival prognostic factors were the following: age > 1 year at diagnosis (P < 0.05); serum neuro-specific enolase > 100 mg/L (P < 0.05); serum lactic dehydrogenase > 1500 U/L (P < 0.01); serum ferritin >150 mg/L (P < 0.05). The overall survival period of the patients was prolonged through total resection of the primary tumor (P < 0.05). Intensive chemotherapy in combination with autologous peripheral blood stem cell transplantation could also result in a prolonged overall survival period (P < 0.01)., Conclusions: Neuroblastoma with advanced stages often presents with various clinical manifestations and has a poor prognosis. It is beneficial to improve the prognosis of neuroblastoma through an early diagnosis and a comprehensive therapy including total resection of the primary tumor, autologous peripheral blood stem cell transplantation and intensive chemotherapy.

The purpose of this study was to evaluate the safety of cryopreserved and thawed peripheral blood stem cell (PBSC) fractionated return infusions in children. 35 children patients with malignant tumors (13 acute leukaemias, 15 neuroblastomas and 7 malignant lymphomas) received fractionated return infusions of cryopreserved stem cells after undergoing high-dose chemotherapy without or with total body irradiation. The toxicities of 70 return infusions were evaluated. All patients were mobilized by chemotherapy plus recombination human granulocyte colony-stimulating factor (rhG-CSF), and then PBSCs were collected by a separator CS-3000 plus or COBE spectra-4. The grafts were cryopreserved in 10% dimethyl sulfoxide (DMSD) and stored in liquid nitrogen. There were totally 70 PBSC transfusions. The total volume of PBSCs transfused: 190 - 420 ml (265 +/- 73 ml or 13.7 +/- 4.2 ml/kg) with a mean of (4.43 +/- 1.91) x 10(8)/kg of PBSCs, and 0.94 +/- 0.18 g/kg of DMSO. The single dose: 90 - 300 ml (132 +/- 37 ml or 6.6 +/- 5.2 ml/kg) with a mean of 0.68 +/- 0.12 g/kg of DMSO. Symptoms occurring during the infusions were recorded. All patients were monitored for 24 hours after infusion. Pulse, blood pressure, body temperature, and respiratory rate were recorded every 15 minutes. At four hours before and 8 hours after infusion, urinalysis was performed. Serum potassium, sodium, creatinine, total bilirubin, aspartate amino transferase (AST), and alanine amino transferase (ALT) levels were examined within 24 hours before and after the first infusion. The results showed that the toxicities observed included hemoglobinuria in 54 return infusions (77.1%), headache in 28 (40.0%), nausea in 24 (34.3%), vomiting in 17 (24.3%), and abdominal pain in 8 (11.4%). Patients who received a graft > 200 ml tended to have a higher frequency of hemoglobinuria, headache, nausea, vomiting, or abdominal pain (P<0.01), and they disappeared quickly, too. Total bilirubin increased after the first return infusion (P<0.01), and there was a significant correlation between the volume of infusion and the degree of total bilirubin increase (r=0.8977, P<0.01). No renal failure or shock occurred. It is concluded that transient hemoglobinuria, headache, nausea, vomiting, and abdominal pain are common toxicities associated with PBSC autograft, and these toxicities are related with a single volume of PBSCs transfused. Total bilirubin increase is correlated with the volume of infusion. In a word, the toxicity is less frequent and lower severe in children with fractionated infusions of cryopreserved peripheral blood stem cell.

Objective: To examine the feasibility and practicability of quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) with SYBR GREEN I fluorescence for detecting the MYCN mRNA expression in neuroblastoma cell line LA-N-5., Methods: MYCN mRNA expression in LA-N-5 cells was measured using real time RT-PCR with SYBR GREEN I. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as internal control. The level of the MYCN mRNA was calculated as MYCN copies/GAPDH copies., Results: Standard curves were linear and showed high correlations (R2>0.99). The ratio of MYCN mRNA copies to GAPDH mRNA copies was calculated based on specific PCR products. The MYCN mRNA level in LA-N-5 cells was obtained (17.4 +/- 1.2)., Conclusions: Quantitative RT-PCR with SYBR GREEN I fluorescence may be a sensitive and reliable method for detecting the MYCN mRNA expression. It may also be potential applicable for detecting the MYCN mRNA expression in the small amount neuroblastoma tissues.

To explore the clinical and pathological characteristics of hepatosplenic gammadelta T-cell lymphoma and its relationship with Epstein-Barr virus infection, the clinical features of a 9-year-old girl with hepatosplenic gammadelta T-cell lymphoma were investigated, the smears of bone marrow was stained with Wright' s stain, biopsies of bone marrow and liver specimen were embedded in plastic and sliced about 4 microm in thickness and routinely stained with HE staining, the immunohistochemical staining was used to mark the tumor cells, and EBER probes were used to detect Epstein-Barr virus RNA. The results showed that the girl presented with prolonged fever, anemia, thrombocytopenia, hepatosplenomegaly, chronic active Epstein-Barr virus infection, and elevated levels of serum ferritin and lactate dehydrogenase. Bone marrow aspirate revealed the infiltration of atypical lymphocytes in the bone marrow stroma. The liver biopsy specimen revealed the infiltration of lymphocytes in the sinusoids, which was positive for the T-cell associated marker CD3 and activated cytotoxicity-associated marker granzyme B. In-situ hybridization analysis with EBER probes revealed that the above-mentioned characteristics were negative in neoplastic cells. It is concluded that hepatosplenic gammadelta T-cell lymphoma is a disease with distinctive clinical, histopathologic, and phenotypic characteristics. Hepatic and/or splenic and/or bone marrow biopsy with combined phenotype is beneficial to diagnosis. Epstein-Barr virus infection is late event involving an already transformed gammadelta T-cell clone.

Objective: To explore the effect of intestinal ischemia/reperfusion (I/R) injury on leptin and orexin-A levels in peripheral blood and central secretory tissues, and investigate the roles of leptin and orexin-A in acute inflammatory responses., Methods: An intestinal I/R injury rat model was established, and the rats were grouped according to duration of the reperfusion time following a 60-min ischemia. Radioimmunoassay was used to examine the protein levels of leptin in the serum and adipose tissue, and the protein levels of orexin-A in the plasma and hypothalamus. Reverse transcriptase-polymerase chain reaction was also performed to detect the mRNA expressions of adipose leptin and hypothalamus orexin-A., Results: Compared with that before injury, serum leptin level of 60-min ischemia with 30-min reperfusion (I60'R30') group decreased significantly and that of I60'R360' increased significantly. Compared with the sham-operation group (sham) after injury, serum leptin level of I60'R360' group increased significantly, and adipose leptin protein levels of I60'R30' and I60'R90' groups decreased significantly, whereas that of I60'R360' group increased obviously. Compared with sham group after injury, adipose leptin mRNA expressions of I60'R30', I60'R240' and I60'R360' groups all increased significantly, while that of I60'R150' showed significant decrease. No significant changes were noted in the protein levels of orexin-A either in the plasma or hypothalamus after I/R injury. In comparison with sham group after injury, hypothalamus orexin-A mRNA expressions of I60'R30' and I60'R90' groups showed gradual but significant decrease, and till 150 min of reperfusion, the expression reached its lowest, followed then by slow recovery at 240 and 360 min, though still remaining significantly lower than that of sham group., Conclusion: Leptin and orexin-A have a time-dependent response to intestinal I/R injury, but the former appears to exhibit a faster response, and they may play a certain role in the metabolic disorders of acute inflammation.

A series of receptors expressed in the surface of tumor cells, which are able to mediate internalizing effect by specially connecting with corresponding ligands. These receptors are potential targets for drugs combined with conjugates. So the drug-conjugate compounds can be targeted delivery to tumor cells. The folate receptor is a promising target because of its marrow tissue specificity, its overexpression in malignant tissues, especially in myeloid leukemic cells, and its ability to bind and internalize folic acid conjugates. It is a promising potential method to apply folate receptor in the receptor-mediated targeting therapy of leukemic cells. In this review, the biological features of folate receptor, its chromosome location and its interaction with ligands, the distribution characteristics of folate receptor in normal and tumor tissues, especially in myeloid leukemic cells, and progress of research on folate receptor mediated targeting tumor cells, especially leukemic cells were summarized.