Search

Objective: To investigate the effect of cell-to-cell communication amongst single-cell clones from healthy periodontium with different osteogenic differentiation potentials on change of osteogenic differentiation capabilities and the role histone acetyltransferase partaken in this process. Methods: In order to research the change of osteogenic differentiation ability via cell-to-cell communication, indirect co-culture method was used by placing two single-cell clones with different osteogenesis potentials in each of the 6-well plates. Blank control, weak and strong osteogenic groups were set up, corresponding to Transwell chambers with blank, cells of weak osteogenesis ability and cells of strong osteogenesis ability, respectively. Each group was made in triplicate. After co-culture for four days, Transwell chamber was removed. Quantitative real-time PCR (qPCR) and alizarin red staining were employed to detect the change of osteogenic differentiation ability. The acetylation level of H3 was measured by using Western blotting. Histone acetyltransferases were detected by qPCR. Results: Single-cell clones were ensured from mesenchymal stem cells by flow cytometer, the positive expression of CD29, CD90, CD105, CD146 was (99.80±0.02)%, (99.36±0.18)%, (99.41±0.05)% and (95.10±2.11)%, respectively. And CD31 and CD34 expression were (0.29±0.11)% and (0.22±0.13)%, respectively. Alizarin red and oil red O staining confirmed that single-cell clones had the abilities of adipogenesis and osteogenesis. Alkaline phosphatase (ALP) and alizarin red staining indicated that different single-cell clones were heterogeneity in osteogenesis differentiation. Indirect co-culture indicated that the mRNA expression of osteocalcin (OCN) were 14.24±5.60 and 4.78±2.90, respectively and Runt-related transcription factor 2 (RUNX2) were 2.75±1.44 and 1.61±0.44, respectively, in strong and weak osteogenic groups. They were significantly higher compared to the blank group (the mRNA expression of OCN and RUNX2 were 1.00±0.47 and 1.00±0.39, respectively). The expression of OCN and RUNX2 were also higher in strong osteogenic group than that in weak osteogenic group ( P< 0.05). The mean gray level of the acetylation of H3 in strong osteogenic group (0.76±0.09) and weak osteogenic group (0.54±0.12) were also higher than that in the blank group (0.30±0.04)( P< 0.05). qPCR results showed that KAT6A in strong osteogenic group exhibiting higher expression ( P< 0.05) compared to weak osteogenic group and the blank group, which were corresponding to the changes of acetylation levels. Conclusions: Single-cell clones from healthy periodontium showed heterogeneity in osteogenic differentiation abilities. Single-cell clones with strong osteogenesis abilities had an advantage over others by promoting others' osteogenesis differentiation and this change mediated by cell-to-cell communication might be caused by modulating KAT6A to affect the acetylation level of histone.

Objective: To probe the expression level of insulin-like growth factor-I receptor (IGF-IR) in malignant clone cells of myelodysplastic syndromes (MDS)., Method: The combination of fluorescence in situ hybridization (FISH) and immunochemistry (alkaline phosphatase anti-alkaline phosphatase, APAAP) was used to detect the expression of IGF-IR in the bone marrow cells of 40 MDS patients with abnormal karyotypes., Results: The average IGF-IR expression level on the clone cells \[(84.5 ± 14.2)%\] from the MDS cases was markedly elevated compared to the corresponding level on normal cells \[(11.4 ± 12.1)%\]. And the percentages of malignant clone cells in all 40 MDS cases were significantly correlated with the relevant percentages of IGF-IR positive nucleated cells (r = 0.929, P < 0.01)., Conclusions: IGF-IR might be taken as a marker of clone cells in MDS for its trait to malignant proliferation.

Objective: To establish a colorectal cancer cell line with stable PRL-3 and CDH22 gene knock-down for investigating the role of PRL-3 and CDH22 genes in the carcinogenesis and metastasis of colorectal cancer., Methods: A recombinant lentiviral vector targeting CDH22 gene was obtained using the pENTRTM/U6 construct and pLenti6/BLOCK-iT (TM)-DEST vector. The recombinant lentivirus was harvested from 293FT cells cotransfected with the optimized ViraPower(TM) Packaging Mix and the pLenti6/BLOCK-iT(TM)-DEST expression construct. SW480/PRL-3- cells were infected with the recombinant lentivirus targeting CDH22, and SW480 cells with stable PRL-3 and CDH22 knock-down were screened by blasticidin selection. PRL-3 expression in the cells was determined by real-time RT-PCR. RESULTS The titer of the lentivirus for the second infection was 8 x 10(5) U/ml. Seventeen positive clones were selected, among which the Clone 1 exhibited substantially down-regulated CDH22 and PRL-3 mRNA expressions., Conclusions: A human colorectal cancer cell line with stable PRL-3 and CDH22 gene knock-down has been established.

Objective: To investigate whether there are cancer stem cells in the MCF-7 human breast cancer cell line., Methods: Flow cytometry was applied to separate different subpopulation cells from MCF-7 cells, and their ability of clone in vitro and reconstruction tumor in vivo were determined., Results: The ability of clone in vitro and reconstruction tumor in vivo were observed in some MCF-7 cells. Contrast with CD44+ CD24+ cells, the proportion of tumorigenic cancer cells in CD44+ CD24- cells is higher., Conclusion: Breast cancer stem cell exists in MCF-7 and it mainly locates the subpopulation of CD44+ CD24- cells, CD44+ CD24+ cell possibly is breast cancer progenitor cell.

This study was aimed to investigate the distribution of abnormal clone in marrow cell lineages and apoptosis cells in myelodysplastic syndrome (MDS) with deletion of chromosome 20q. Monoclonal antibodies recognizing myeloid precursors (CD15), erythroid precursors (GPA), T cells (CD3(+)CD56(-)CD16(-)), B cells (CD19), NK cells (CD3(-)CD56(+)CD16(+)) were used to sort bone marrow cells in a MDS patient with del (20q) by fluorescence activated cell sorting (FACS). Annexin V-FITC and PI were used to sort bone marrow Annexin V(+)PI(-) and Annexin V(-)PI(-) cells by FACS. The sorted positive cells were detected by interphase dual-color fluorescence in situ hybridization (D-FISH) using a LSI D20S108 probe (Spectrum Orange) and a Telvysion TM 20p probe (Spectrum Green). FACS and FISH analysis were also performed on the samples from 4 cases with normal karyotype. The results showed that the proportions of MDS clone in the myeloid and erythroid precursors were 70.50% and 93.33% respectively, in the RAEB-1 patient with del (20q) and were obviously higher than that in control group (5.39% and 6.17%). The proportions of abnormal clone in T, B and NK cells were 3.23%, 4.32% and 5.77% respectively and were less than that in control group (5.76%, 4.85%, 6.36%). The percentage of apoptotic cells in the bone marrow nucleated cells was 16.09%. The proportions of MDS clone in Annexin V(+)PI(-) and Annexin V(-)PI(-) cells were 32.48% and 70.11%, respectively. It is concluded that most myeloid and erythroid precursors are originated from the abnormal clone in MDS with del (20q). A little part of apoptotic cells are derived from the abnormal clone.

Objective: To study the clonality status of peripheral papilloma (peri-MP), ductal carcinoma in situ (DCIS), and normal tissue of the breast using an assay based on inactivation mosaicism of the length-polymorphic X-chromosomes at the androgen receptor (AR) locus and to explore a reliable way to distinguish the benign and malignant (or pre-malignant) cases judged morphologically., Methods: Specimens of breast tissues were obtained from 26 cases of peri-PM, 25 cases of peri-PM with atypical ductal hyperplasia (ADH), and 27 cases pf DCIS, 16 cases of developed canceration, and 20 normal women. DNA was extracted and amplified via nested-PCR with or without previous digestion by the methylation-sensitive restriction endonuclease Hha I. The products were resolved on denaturing polyacrylamide gels and visualized through silver staining. The clonality of these samples was analyzed by showing the lanes., Results: Loss of polymorphism at the AR locus was found in all the cases with DCIS and in 10 cases (10/25, 40.0%) of peri-PM with ADH, indicating the monoclonality of the tumor. Twenty-four cases (92.3%) of the 26 cases with peri-PM and the 20 specimens of normal tissue were shown to be polyclonal. In the 16 cases of developed canceration identical X chromosome inactivation (monoclonal alterations) was observed in the cancer focus, parts of peri-PM with ADH, and the part of DCIS., Conclusion: Normal breast tissue and peri-PM show polyclonality and the peri-PM with ADH shows monoclonality. Clonality analysis may be a useful modality to screen high-risk cases from precancerous lesions or to distinguish between the benign hyperplasia and early carcinoma.

Aim: To screen and identify differentially expressed genes in subcloned lines from a same human bladder transitional cell carcinoma (TCC)., Methods: Two bladder TCC cell lines (BLX and BLS-211) with different phenotypes but same origin were used to screen for differentially expressed genes by suppression subtractive hybridization (SSH)., Results: 9 over-expressed genes in BLX and 15 in BLS-211 cells were obtained, respectively. Some Bacillus Galmette-Guerin(BCG)- associated genes, such as BCG induced integral membrane protein(BIGM103), fibronectin(FN), complement factor B(BF), were over-expressed in BLX cells. And 8 new ESTs(Expressed Sequence Tag) in BLS-211 cells were collected by GenBank dbEST database with the accession number of DY505708-13, DY230447-8., Conclusion: SSH is a powerful method for the identification of differentially expressed genes in different cell lines or clones. Some BCG-associated genes, which are differentially expressed in different cells may contribute to the different response to clinical BCG therapy. The identified new ESTs can be cloned for full length to further study their functions.

Objective: To study the clonality of palmar fibromatosis by molecular genetic analysis of X chromosome inactivation pattern at a polymorphic site of human androgen receptor gene (HUMARA)., Methods: Twelve female cases of palmar fibromatosis were enrolled into this study. Hematoxylin and eosin-stained sections of paraffin-embedded, formalin-fixed tissues were microdissected by laser capture microdissection technology in order to obtain the proliferative spindle cells. Tumor cells isolated from rectal adenocarcinoma in a female patient were used as positive control. The genomic DNAs were extracted with phenol and chloroform, digested with methylation-sensitive restriction endonuclease HpaII, and amplified by polymerase chain reaction (PCR) using primers targeted to a highly polymorphic short tandem repeat of HUMARA. The amplimers were separated on vertical 8% non-denaturing polyacrylamide gels and the patterns were visualized with ethidium bromide stain., Results: The methodology for clonality analysis was validated in the positive control using rectal adenocarcinoma cells. Among the 12 cases studied, PCR amplification failed in 3 samples and 1 sample showed homozygosity which was not suitable for further analysis. Eight samples were successfully amplified and showed a random X chromosome inactivation pattern, suggesting polyclonality in these lesions., Conclusions: Palmar fibromatosis is a polyclonal condition and should be considered as a form of non-neoplasmic fibroblastic proliferation.

Objective: To screen a sub-clone of human breast cancer cell of the MCF-7 line with high metastasis potential., Methods: Human breast cancer cells of the MCF-7 line were injected subcutaneously into 10 severe combined immunodeficiency (SCID) mice. Sixty-eight days after the mice were killed and their lungs were taken out. Primary cell culture was conducted. When the cells were passed on to the third generation a sub-clone was screened from the lung tissue and termed LM-MCF-7. Microscopy was performed on the lung tissues. The growth curve was drawn. Flow cytometry was used to examine the cell cycle. Chromosome analysis was done. Immunohistochemistry was used to detect the expression of breast cancer specific antigen CAI5-3. Western blotting was used to detect the protein expression of the protein associated with tumor metastasis: nm23 (a metastasis-suppressing gene), myosin light chain kinase (MLCK, a kinase related to cell movement), survivin, bcl-2 and p27 (a gene related to cell cycle). LM-MCF-7 cells were injected into other SCID mice and these mice were killed 30 days later to observe the metastasis of cancer so as to detect the tumorigenic ability of the LM-MCF-7 cells., Results: When the cells from the mouse lung tissues were passed on to the third generation a sub-clone with high metastasis potential was screened and termed LM-MCF-7. The morphology of the new cell line was typically epithelioid. Flow cytometry showed that the DNA relatively contents were 53.40% of the LM-MCF-7 cells were in the G(0)/G(1) phase, a lower percentage than that of the MCF-7 cells, and 17.10% in the S phase and 23.20% in the G(2+)M phase, both percentages higher than those of the MCF-7 cells. The proliferating time of the LM-MCF-7 cell population was about 20 +/- 14 hours, much shorter than that of the parent strain cells. The chromosomes of the LM-MCF-7 cells, numbering 16-123, showed the morphology characteristic c of human chromosomes. The marker of human breast cancer CA15-3 was detected in both MCF-7 and LM-MCF-7 cells. The protein expression of nm23 and p27 was down-regulated, but the protein expression of MLCK, bcl-2 and survivin was up-regulated in LM-MCF-7 cells in comparison with those in MCF-7 cells. The tumorigenesis rate of LM-MCF-7 cells was 100% (5/5), with a latent period of 5.0 +/- 0.0 d, and the tumor metastasized to lung, kidney, spleen, bone marrow, lymph node and heart., Conclusion: A human breast cancer line, LM-MCF-7 cell line, with high metastasis potential has been derived from the human breast cancer cells of MCF-7 line.

Objective: To study the random amplified polymorphic DNA (RAPD) characteristics of five S180, clonal cell strains with 23 primers and the biological characteristics of passage cells from S180-S2D9., Methods: The DNA of 5 clone strains of S180 including S180-S2D9, S180-S2D7, S180-S1F11, S180-S1H10 and S180-S1B11, was amplified with RAPD-PCR using 23 single primers. The PCR products were resolved with electrophoresis on agarose gel. To analyze genomic DNA with RAPD, the cultured cells in vitro were treated with colchicine for 6 hours. Then, the chromosome numbers of 100 cells were examined under the microscope. The KM mice were injected intraperitoneally with 0.2 ml solution of S180-S2D9 cell, and the growth of ascitic fluid and the life span of the mice were observed. The cultured cells were fixed with 750 ml/L ethanol, and DNA analysis was made by Flow Cytometry., Results: Three of the 23 single primers resulted in diversity between amplified products from different clonal strains. RAPD character of S180-S2D9 was analyzed by the 3 single primers; RAPD bands of the first generation, 25th generation, 50th generation and 75th generation showed no difference; ANOVA showed that the average numbers of chromosomes of four generations were of no significant difference (P>0.05). The ascites of the KM mice subjected to the intraperitoneal injection of first generation and 50th generation S180-S2D9 cells was bloody, and the survival days of mice were 13-23 d and 13-20 d respectively; ANOVA showed there was no significant difference between the first generation and 50th generation (P>0.05). DNA contents assayed by flow cytometry revealed that DNA corresponding content of the cells is individually 0.3890, 0.3542, 0.3575 and 0. 3984. These results imply that the S180-S2D9 passage cells have not been found to vary a lot., Conclusion: It is adaptable to give quality control to the clonal cell strains with RAPD.

T cells recognize antigens through TCRs (T cell receptor). T cell clones can be sorted into 24 gene subfamilies based on the usage of the segments of TCR BV in gene rearrangement. Application of the various segments of TCR BV may establish TCR BV spectrotyping that can be used to analyze and recognize the different functional T cell clones, and understand the function and proliferation of various T cell clones in malignancy and autoimmune disease. In vitro expansion of a great deal of the specific antitumor T cells and transfusing them to patients will be able to develop a new method for tumor immunotherapy. Through analyze the character of the TCR BV gene, McAb against TCR or DNA vaccine to inhibit the growth of T cell clones associated-autoimmune disease and tumors might be developed. The McAb and vaccine may be used to cure these diseases. The commo T cells can also be modifed to specific antitumor T cells by method of TCR gene transfer. In this review, the characteristics of TCR, analysis method for gene spectrotyping of TCR BV, segments of TCR BV and autoimmue distase, T cell clones in hematologic maligrancies, recognition of T cell oligoclone expansion of T cells, and application of TCR BV gene spectrotyping in bone marrow transplantation were discussed and summarised.

Objective: To investigate clonal evolution of abnormal Philadelphia chromosome-negative cells (Ph- CE) after imatinib mesylate therapy in patients with Philadelphia chromosome-positive chronic myelogenous leukemia (Ph+ CML)., Methods: Bone marrow cells G-banding karyotype was evaluated every 3 months in 100 patients with Ph+ CML after achieving hematologic responses on the course of imatinib therapy. There were 54 patients in chronic phase (CP), 37 in accelerated phase (AP) and 9 in blast phase (BP)., Results: After a median follow-up of 32 months (ranged 25-34 months), 11 patients, including 5 cases in CP, 5 in AP and 1 in BP, developed transient, interrupted or continuous Ph- CE after 3 - 29 months on imatinib therapy. Ph- CE emerged at the beginning of Ph+ cells decreasing or after Ph+ cells disappearing. The proportion of Ph- CE, was negatively correlated with the proportion of Ph+ cells (P < 0.05). Ph- CE commonly included +8 (45.5%) and +Y (27.3%). Five patients had additional cytogenetic abnormalities besides Ph+ in Ph- CE. Seven of the patients with Ph- CE achieved a major cytogenetic response while 9 of them achieved a complete hematologic response. One patient with Ph- CE in AP progressed to BP 20 months after the initiation of the therapy while the rests remained in hematologic or cytogenetic responses., Conclusion: Ph- CE occurred in about 11% of the patients with Ph+ CML who achieved major or minor cytogenetic responses on imatinib therapy. After a median follow-up of more than 2 years, most of the patients with Ph- CE were in a stable status with no disease progression.

Objective: To investigate the clonal origin of the apoptotic hematopoietic cells in myelodysplastic syndromes (MDS)., Methods: Nineteen patients with MDS whose karyotypes had been analysed with G- or R-binding were studied. ISEL (DNA in situ end labelling) and FISH (fluorescence in situ hybridization) (simplified as ISEL/FISH) were performed simultaneously in a single nucleated cells on cytospin slides in 10/19 of the patients. The ratio of FISH positive cells to all nucleated cells and to apoptotic cells were counted. Eight slides from 4 normal donors induced apoptosis by Fas monoclonal antibody (McAb) were used as methodology control. For the rest 9 patients, different fractions of cells (PI-, Annexin V+PI-, Annexin V-PI-) were sorted by flow cytometry and then cytospined for FISH analysis (simplified as FCM/FISH)., Results: The mean percentage of abnormal clonal cells in nuclear cells in the 10 cases detected by ISEL/FISH was 37.1%, whereas it was 24.0% in apoptotic cells. Induction by Fas McAb didn't lead to change of karyotype. Moreover, 8/9 patients analysed by FCM/FISH showed higher cell proportion with normal karyotypes in apoptosis-undergoing cells than that in non-apoptotic cells., Conclusion: Over apoptotic hematopoietic cells in MDS are mainly residual normal hematopoietic cells.

Objective: To construct-the eukaryotic expression clone for human amelogenin., Methods: Total RNA was isolated from human fetal tooth buds. RT-PCR was used to amplify the amelogenin encoding region, and the amplified fragment for human amelogenin was inserted into eukaryotic expression vector PcDNA 3.1. The positive clones were selected and analyzed by restriction endonuclease mapping and DNA sequencing., Results: 570 bp fragment was produced by RT-PCR; it was of the same size as expected based on human ameloginin mRNA encoding area length. The sequence of the inserted fragment from the recombinant clone PcDNA 3.1-AMG was consistent with that of AMELX from GenBank with one mismatch on 485 from G to C, without affecting the amino acid sequence., Conclusion: The eukaryotic expression clone PcDNA 3.1-AMG was successfully constructed with the properly inserted DNA sequence encoding mature human amelogenin.

Objective: The purpose of this study was to construct a eukaryotic expression vector for human amelogenin (AMG)., Methods: PCR was performed to amplify the AMG encoding region. Amplified fragments for human AMG were recovered and inserted into eukaryotic expression vectors PsecTaq2A. The recombinant plasmid PsecTaq2A-AMG was constructed and their positive clones were identified., Results: 1. Amplified products were checked by electrophoresis and the results were satisfactory. 2. The recombinant plasmid PsecTaq2A-AMG was analyzed by restriction endonuclease mapping and DNA sequencing. The results of sequencing were consistent with those from GenBank., Conclusion: The recombinant plasmid PsecTaq2A-AMG was successfully constructed with properly inserted DNA sequence encoding mature amelogenin.

Objective: To study the origin of aberrant clones with chromosome abnormalities in MDS., Methods: Antibodies recognizing myeloid cells (CD33), B cells (CD19) and T cells (CD2) were used to sort bone marrow cells of ten MDS patients with chromosome abnormalities (trisomy 8 five cases, trisomy 8 accompanying trisomy 9 one case, trisomy 8 accompanying 7p- one case, 20q- two cases, t (1; 7) one case) by Immuno-Magnetic Bead technique, then the positive cells for CD33, CD19 or CD2 were detected respectively by interphase FISH using the centromeric probes for chromosomes 1, 7and 8, and the locus specific probe for 20q12. 200 to 300 cells were scored for each detection., Results: FISH showed that trisomy 8, t (1; 7) and 20q- all affected CD33 positive cells, but not CD2 positive cells in these ten MDS patients. Only in one MDS patient, 20q- affected the CD19 positive cells., Conclusion: Our data suggest that the chromosomal aberrations in MDS are mostly restricted to myeloid cell lineages, but B lymphocytes are involved in minor MDS implying that abnormal clone originated from a multipotent hematopoietic progenitor.

To analyze the capability of IL-1 and IL-6 in the induction of chemokine (CF) production by mouse thymus epithelial cell (MTEC1) clones, MTEC1 cells were cloned through one cell culture and individual cell clones were established in long term culture referred to as MTEC1-DW clones. The constitutive production of IL-1, IL-6 and CF by MTEC1-DW clones was evaluated and the patterns of the cytokine production determined. Addition of exogenous IL-1 or IL-6 or both to the cultures of those MTEC1-DW clones that are unable to produce CF, and incubated for 2 days, then, to assess the chemotactic activity in the cell culture supernatants (SNs). In the opposite, addition of anti-IL-1 mAb(s) to the cultures of those MTEC1-DW clones that can produce IL-1 and CF to neutralize secreted IL-1 then, to test chemotactic activity in the SNs after 2-day incubation. The results showed that in the MTEC1-DW clones which were unable to constitutively produce IL-1 or CF, addition of IL-1 could induce these cloned cells to produce CF with high chemotactic activity. By constrast, addition of anti-IL-1 mAb(s) to those MTEC1-DW clones that constitutively produce IL-1 and CF could significantly inhibit them to produce CF. IL-6 only exhibited weak activity in the induction of CF production by those cloned cells. Therefore, in the cytokine network regulation, CF production is mainly induced by endogenously produced IL-1 in MTEC1 cells.