Objective To construct an eukaryotic expression vector of pre-miR-15a,and to investigate the inhibitory effect of pre-miR-15a to Raji cells. Methods The pGCSIL-GFP vector encoding pre-miR-15a nucleotides was transfected into the bacterial competent cells,and then confirmed by PCR and sequencing analysis. The identified vector was transfected into Raji cells with oligofectamine 2000. The cells were divided into 3 groups,blank,negative control and pre-miR-15a group. Semi-quantitative RT-PCR was used to detect the expression of Bcl-2 mRNA,and immunofluorescence indirect for Bcl-2 protein expression. The growth of Raji cells was assayed by trypan blue dye exclusion method. Results PCR and sequences analysis indicated that the recombinant clones was identical with target sequences. Many green fluorescent cells were observed under fluorescent microscopy. The levels of Bcl-2 mRNA at every group had no obviously difference. Bcl-2 protein expression was obviously decreased at pre-miR-15a group compared with the other groups. Trypan blue dye exclusion method showed the cell growth was inhibited at 48 h and 72 h post-transfection. Conclusion We successfully construct the eukaryotic expression vector of pre-miR-15a,and it can inhibit the growth of Raji cells.

Objective To investgate the expression of Survivin and Caspase-3 in primary ovarian carcinoma, and to explore their relationship. Methods The expression of Survivin and Caspase-3 was detected by flow cytometry (FCM) . Results FCM showed that the expression of Survivin in ovarian serous carcinoma was higher than that in serous cystadenoma and normal ovarian tissues (F=19.80;P=0.00) and correlated with the clinical stage( t=2.882,P=0.009).The expression of Caspase-3 in ovarian serous carcinoma was higher than that in serous cystadenoma and normal ovarian tissues (F=16.32,P=0.00)and has no correlativity with clinical stage,histological grade,and lymph metastasis(P＞0.05).Survivin expression was negatively correlated with Caspase-3 expression in ovarian serous tumors (r=0.43,P=0.029).Conclusion Survivin was negatively correlated with Caspase-3 in ovarian serous tumors.Combination of detecting the two protein expressions might be valuable in diagnosis and determining the malignant degree in ovarian serous carcinoma.

Objective:To determine whether the bcl-2 antisense oligonucleotide (ASODN) can increase the sensitivity of NCI-H460 cell lines to docetaxel or radiation. Methods: Docetaxel in combination with bcl-2 ASODN or non-sense oligonucleotide (NSODN) was used to treat NCI-H460 cells, and then IC_~50 of docetaxel against NCI-H460 cells was determined with MTT method. In radiation group, NCI-H460 cells were divided into pure radiation, radiation plus bcl-2 ASODN, and radiation plus NSODN groups. The inhibition rates of cell growth were assayed by MTT, and the expression levels of Bcl-2 protein were assayed by immunofluorescence. Results: IC_~50 in bcl-2 ASODN plus docetaxel, pure docetaxel and NSODN plus docetaxel groups was (0.101?0.009) ?mol/L,(0.183?0.018)?mol/L, and (0.179?0.016)?mol/L, respectively. The differences between the first group and the latter 2 groups were significant (P

AIM:To explore the effects of the combined use of human telomerase reverse transcriptase (hTERT) gene antisense oligodeoxynucleotide (ASODN) and cisplatin on apoptosis of HL-60 cells. METHODS:PCR-ELISA was used to determine telomerase activity in HL-60 cells untreated or treated with ASODN, the expression levels of hTERT protein were assayed by immunofluorescence using fluoresce isothiocyanate (FITC) label. Apoptosis was detected by DNA electrophoresis and flow cytomertric cell cycle analysis. RESULTS:The expression levels of hTERT protein and telomerase activity in HL-60 cells treated with ASODN were shown to decrease with time, the DNA fragments were obviously found and the percentage of apoptosis cells was significantly enhanced in HL-60 cells with the combined use of hTERT ASODN and cisplatin compared with the combined use of hTERT sense oligodeoxynucleotide and cisplatin or cisplatin alone, respectively. CONCLUSION:Inhibition of telomerase activity by hTERT ASODN increases the cisplatin-induced apoptosis of HL-60 cells.

AIM: To explore the effects of the combined use of human telomerase reverse transcriptase (hTERT) gene antisense oligodeoxynucleotide (ASODN) and cisplatin on apoptosis of HL-60 cells. METHODS: PCR-ELISA was used to determine telomerase activity in HL-60 cells untreated or treated with ASODN, the expression levels of hTERT protein were assayed by immunofluorescence using fluoresce isothiocyanate (FITC) label. Apoptosis was detected by DNA electrophoresis and flow cytomertric cell cycle analysis. RESULTS: The expression levels of hTERT protein and telomerase activity in HL-60 cells treated with ASODN were shown to decrease with time, the DNA fragments were obviously found and the percentage of apoptosis cells was significantly enhanced in HL-60 cells with the combined use of hTERT ASODN and cisplatin compared with the combined use of hTERT sense oligodeoxynucleotide and cisplatin or cisplatin alone, respectively. CONCLUSION: Inhibition of telomerase activity by hTERT ASODN increases the cisplatin-induced apoptosis of HL-60 cells.

Objective It was designed to investigate the relationship between the productions of p16 and Rb genes expression and gastric carcinogenesis and evaluate the correlation between the expression of p16 and Rb genes in gastric carcinoma.Methods The expression of P16 and Rb proteins was examined in gastric carcinoma and precancerous lesion and normal gastric mucosa by streptavidin-peroxidase immunohistochemical method (S-P).Results The positive rates of P16 and Rb proteins expression showed as below: There were 96 25%(77/80) and 98 75%(79/80) in normal gastric mucosa, 92 00%(46/50) and 80 00%(40/50) in atypical hyperplasia gastric mucosa and 47 54%(58/122) and 59 84%(73/122) in gastric carcinoma respectively. The positive rates of P16 and Rb proteins expression in gastric carcinoma were significantly lower than that in normal gastric mucosa and atypical hyperplasia gastric mucosa (P

Objective To optimized the culture conditions of the human umbilical cord blood mesenchymal stem cells.Methods The umbilical cord blood was taken in Guangzhou Hua Qiao Hospital between Oct.2004 to Feb.2005.The isolation method,planting density,the time of the first medium changing,the influence of culture medium on the growth of MSC were analyzed. Mesenchymal stem cells were identified using the surface marker by flow cytometry.Osteoblast was identified by Von Kossa staining and alkaline phosphatase staining,adipocyte was identified by Oil Red O staining. Results When the other conditions were the same,the hespan isolation was better than Ficoll isolation;5?10~6/cm~2 was the best planting density;the best first medium changing was on the seventh day at primary culture. Under optimized conditions,the MSC expressed adhesion molecules CD_ 13 ,CD_ 29 and CD_ 44 ,but not antigens of hematopoietic CD_ 34 ,CD_ 45 ,CD_ 14 and not antigens of endothelia CD_ 106 . Exposure of these cells to osteogenic inductive agents resulted in an increase in expression of alkaline phosphatase and the appearance of hydroxyapatite nodules. Incubation with adipogenic inductive agents resulted in morphological change and staining with Oil Red O. Conclusion Mesenchymal stem cells exist in Cord blood,but slower to establish in culture.Cord blood may prove to be a new source of cells for cellular therapeutics.

Objective:To investigate the effects of Bcl-2 shRNA on the growth of liver carcinoma cell line BEL-7402 and colorectal carcinoma cell line Caco2.Methods: Bcl-2 shRNA was cloned into Pgenesil-1 plasmid labeled with fluorescent protein and the product was transfected into BEL-7402 and Caco2 cells with Lipofectamine 2000.The study also included shRNA as negative control,Pgenesil-1,Lipofectamine and blank control groups.Transfected cells were visualized by inverted fluorescent microscope and then assayed by flow cytometry.The expression levels of Bcl-2 protein were assayed by Western-blot and cell proliferation was measured by MTT method.Results: There was no difference in transfection rate among cells in Bcl-2 shRNA,shRNA and Pgenesil-1 vector groups.Western-blot assay showed that the expression levels of Bcl-2 protein in BEL-7402 and Caco2 cells were significantly decreased in Bcl-2 shRNA group compared with those in other 4 groups(P

Objective T To investigate the effect of sirolimus (SRL) on the differentiation,development,and maturation of mice bone marrow-derived dendritic cells (DC),and the synergic effects of them in prolonging survival time of skin allograft.Methods (1) DC of C57BL/6 mice were derived from bone marrow cells upon culture with SRL. The expression of CD11c,CD86 and major histocompatibility complex (MHCⅡ) molecules was assessed with or without lipopolysaccharide (LPS) stimulation by flow cytometry; (2) The capacity of DC administrated by SRL to stimulate allogeneic T lymphocyte proliferation was examined by mixed lymphocyte culture (MLR); (3) A skin transplantation model was established with the recipients BALB/c mice and the donor C57BL/6 mice. Recipients were divided into control group (there was no administration before skin transplantation),immature DC group (injection of donor C57BL/6 mice immature dendritic cells 2?10 6 via tail vein before skin transplantation),SRL group (receiving oral SRL 3 mg/kg every day for 7 days before skin transplantation),combined group (receiving an injection of donor C57BL/6 mice immature DC via tail vein and of oral SRL before skin transplantation),and isogeneic group (in which the donors and recipients were both BALB/c mice and there was no administration before skin transplantation). Survival time and histological changes of skin allograft were observed in different groups.Results (1) CD11c expression on the DC in the presence of SRL was slightly decreased,but CD86 and MHCⅡ molecules significantly decreased,and SRL treatment could resist the stimulation of LPS; (2) MLR revealed that DC administrated by SRL could inhibit allogeneic T lymphocyte proliferation; (3) SRL treatment in combination with donor immature DC before transplantation could alleviate inflammation and prolong survival time of skin allograft in mice.Conclusions SRL does not alter differentiation but inhibit the maturation of DC. Sirolimus can cooperate with immature DC to prolong survival time of skin allograft in mice.

Objective:To study the mechanism of tretinoin-induced neural differentiation in fetal liver CD34+ cells in vitro. Methods :CD34+ cells from fetal liver were isolated with a magnetic cell sorting kit and were cultured. Cells pretreated with or without protein kinase C(PKC) inhibitor chelerythrine chloride(3 ?mol/L) were induced by 5?10-7mol/L tretinoin for 24 h, and then incubated in serum-free medium. Expressions of genes in treated cells were assayed by Western blotting and RT-PCR. Results: Tretinoin significantly promoted expression of neural specific genes such as Nestin, neuron-specific nuclear protein (NeuN) , neuron-specific neuronfilament-M (NF-M) , and MAP-2 in fetal liver CD34+ cells, with Nestin increased by 4. 09 folds, NeuN by 5. 12 folds, and NF-M by 7. 27 folds (all P