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Acetylcholinesterase (AChE) inhibitors are mainly used in the treatment of Alzheimer's disease (AD). The inhibitory effect of icariin on the activity of AChE was investigated by inhibition kinetics. The binding interaction and binding sites between icariin and AChE were also studied by using fluorimetry and molecular docking, respectively. The results showed that icariin could potently inhibit the activity of AChE, the IC50 value was determined to be 3.50 x 10(-8) mol x L(-1), and the determined IC50 value to tacrine was 0.75 x 10(-8) mol x L(-1). Kinetic analyses showed that icariin is a reversible and mixed type AChE inhibitor. The inhibition constants K1 and K(IS) were determined to be 2.67 x 10(-8) and 4.43 x 10(-8) mol x L(-1), respectively. Icariin binds selectively to the AChE peripheral anionic site via hydrogen bonds and Van der Waals forces.

To construct the recombinant adenoviral vectors expressing human endostatin, K5 and endostatin-K5 gene respectively, and study their bioactivity in vitro.Human endostatin, K5 and endostatin-K5 gene were amplified by PCR, which were then subcloned into shuttle vector pAd5-CMV-H1H2-MCS-6His by enzyme and ligation respectively. The positive recombinant plasmids linearized by Pac I were cotransfected into HEK 293 cells with the Pac I linearized adenoviral backbone plasmid using calcium phosphate precipitation method. The recombinant viruses were purified by CsCl density gradient centrifugation. The protein expression at different time points (24 h, 48 h and 72 h) was determined by Western blot. The inhibitory effect of the protein on ECV-304 growth was detected by MTT.The recombinant adenoviral vectors expressing human endostatin, K5 and endostatin-K5 gene were successfully constructed in HEK293 cells. Protein expression was detected by Western blot. The level of protein expression was increased with the prolonged incubation of the infected HeLa cells. Three kinds of the protein expressed by the recombinant adenoviral vectors showed obvious inhibitory effect on ECV-304 cell growth.The protein expressed by adenoviral vectors carrying endostatin, K5 and endostatin-K5 gene has an obvious inhibitory effect on ECV-304 cell growth.

To analyze and optimize the gene rearrangement primers of different frame regions (FR) of immunoglobulin heavy chain (IgH) genes by bioinformatic methods and explore the application of these primers in the detection of paraffin-embedded lymphoma tissues.Three pairs of primers from IgH FR1, FR2 and FR3 regions (P1c, P2A and P31, respectively) were selected as the B cell gene rearrangement primers after comparison of the gene fragments in 44 IgH variable and 6 joining regions. Using one pair of T cell receptor (TCR) gamma primer as the T cell gene rearrangement primer, 101 histopathologically confirmed lymphoproliferative samples including 80 B cell lymphomas, 14 T cell lymphomas, and 7 reactive proliferative lymph nodes were examined by PCR for gene arrangement. The DNAs from DG75 and Jurkat cell lines were used as the positive controls for B and T cell lymphoma, respectively, with those from reactive proliferative lymph nodes as the negative control.The positivity rates of IgH primers (P1c, P2A and P31) in the 80 B cell lymphomas were 37.5% (30/80), 52.5% (42/80) and 70.0% (56/80), respectively, and only one of the 14 T cell lymphoma cases was positive for the primers, suggesting significant differences in the detection rates of B cell lymphomas by the 3 primers. The detection rate was increased to 83.9% by combining the results by P31 and P2A primers. No positivity was found in the proliferative reaction tissues.Primers from IgH FR3 region genes are more sensitive than that from the FR1 and FR2 regions in the detection of gene rearrangement in paraffin-embedded lymphoma tissues. The detection rates can be increased by combining the results with the primers for IgH FR3 with that of FR2.

To construct the prokaryotic plasmid of FUS1 gene for efficient FUS1 expression in E.coli strain Rosetta(DE3)2plys.The full-length FUS1 gene was amplified by PCR from the total RNA of umbilical mesenchymal stem cells and cloned into pET-32a(+) vector followed by identification with PCR and sequencing. The recombinant plasmid pET-32a(+)-FUS1 was transformed into the E.coli strain Rosetta(DE3)2plys and the target protein expression was induced by IPTG.The plasmid pET-32a(+)-FUS1 was obtained successfully as verified by PCR and sequence analysis. High expression of the fused FUS1 protein was achieved after induction by low-concentration IPTG (25 micromol/L) for 3 h, and the recombinant FUS1 protein accounted for 40% of the total bacterial protein of Rosetta(DE3)2plys.The recombinant FUS1 plasmid has been successfully cloned, which allows highly efficient FUS1 expression in Rosetta (DE3)2 plys.

Clonality detection through amplifying immunoglobulin heavy chain (IgH) by polymerase chain reaction (PCR) is a useful tool in diagnosis of lymphoma, but the false negative rate is high, especially in paraffin-embedded tissues. This study was to explore the value of tumor tissue microdissection and combinational detection of IgH and immunoglobulin light chain (Ig kappa or Ig lambda) in diagnosis of non-Hodgkin's lymphoma (NHL).Two pairs of conventional primers for IgH and T-cell receptor gamma (TCRgamma), 2 novel designed pairs of primers for Ig kappa and Ig lambda were used to detect 58 paraffin-embedded blocks, which had been diagnosed by pathology and histochemistry. Of the 58 cases of lymph node tissues, 39 were B-cell lymphoma, 16 were T-cell lymphoma, and 3 were reactive proliferative lymph node tissue. Lymphoma cell lines DG75 and Jurkat were used as control.The positive rates of IgH primers (P1) and IgL primers (P kappa/P lambda) were 79.5% and 71.8% in the 39 cases of B-cell lymphoma (P0.05), 6.3% and 12.5% in the 16 cases of T-cell lymphoma, respectively. The positive rate was greatly increased to 92.3% in combinationally detecting the primers for IgH and P kappa/P lambda. There was no positive detection among the reactive proliferative lymph node tissues.B-cell lymphoma detection rate can be significantly improved by the combination of IgH and IgL gene rearrangement primers, which provides efficient assistant method for the diagnosis and differential diagnosis of lymphoma.

To explore the value of detecting clonal T cell receptor gamma (TCR-gamma) gene rearrangement with touch-down PCR and single-strand conformational polymorphism analysis (SSCP) in the diagnosis of lymphoid leukemia.The DNA of peripheral blood leucocytes from lymphoid leukemia patients were extracted for amplification of the TCR-gamma gene rearrangement with the consensus primers and touch-down PCR. The PCR products were analyzed by agarose gel electrophoresis, direct DNA sequencing and SSCP analysis. The positive control cell line DNA was mixed in different proportions with the DNA extracted from reactive lymphoid tissue to test the sensitivity of the touch-down PCR.Fifteen of 18 T lymphoid leukemia and 2 of the 4 B lymphoid leukemia patients were identified to be positive by agarose electrophoresis. The positive PCR products were further analyzed by SSCP analysis, which showed discrete bands. Direct DNA sequencing confirmed the clones to be TCR-gamma gene rearrangement, and the sensitivity of touch-down PCR was 1%.Consensus primers for studying TCR-gamma gene rearrangement in combination with touch-down PCR can effectively amplify the clonal TCR-gamma gene rearrangement in T lymphoid leukemia.

To study the expressions of latent membrane protein 1 (LMP1), p53 and bcl-2 proteins and investigate their significance in the pathogenesis of Hodgkin's lymphoma.Immunohistochemical staining was used to examine the expressions of LMP1, bcl-2, and p53 proteins in 31 paraffin-embedded tissue samples from Hodgkin's lymphoma.The positivity rates of LMP1, p53 and bcl-2 proteins were 58.1% (18/31), 61.3% (19/31) and 35.5% (11/31), respectively, showing a statistically significant difference between the expressions of the former 2 proteins.p53 may be involved as an important agent in the pathogenesis of Hodgkin's lymphoma induced by LMP1, whereas bcl-2 appears unrelated to the development of this disease.

To investigate the relationship between Epstein-Barr virus (EBV) and the pathogenesis of human colorectal cancer.The small-molecule RNA fragments of EBV was examined by in situ hybridization in colorectal cancer specimens obtained from 130 patients.EBV was found in the 6 of 130 colorectal cancer cases. EBV positivity was more prevalent in male patients (4/6), and the tumor tissues of the 4 EBV-positive cases were featured by obvious stromal infiltration by the lymphocytes (4/6).EBV infection might be related to the pathogenesis of some colorectal adenocarcinomas in China, and intensive lymphocyte infiltration in the stroma of the tumor tissues can be an important pathological indication of EBV infection in colorectal tumors.