Objective:To explore the apoptosis inducing effect of matrine on C6 glioma cells and the related mechanisms.Methods:MTT assay was used to examine the inhibition of C6 glioma cell line by matrine at various concentrations and the IC50 was calculated.Inverted microscope and TEM(transmission electron microscope) were employed to observe the morphological alterations of C6 glioma cells after exposure to matrine;FCM(Flow cytometry) was used to detect the apoptosis rate of C6 glioma cells;and real-time PCR was used to examine the differential expression of related genes.ICC and Western blotting was used to detect the expression of caspase-3.Results:MTT showed that the cell inhibition effect of matrine increased with its concentration(0.1-1.0 mg/ml)(P

Objective:To investigate the inhibitory effect of inhibitor of differentiation 3(Id3)on growth of human lung adenocarcinoma cell line A549.Methods:Recombinant eukaryotic expression vector pEGFP/Id3 was constructed and transfected into A549 cells by liposome-mediated method.Expression of pEGFP/Id3 in A549 cells was analyzed by flow cytometry(FCM),fluorescence microscopy,semi-quantitative RT-PCR and immunocytochemistry.The growth inhibitory rate of A549 cells was examined by MTT assay;cell cycle change was evaluated by PI(propidium iodide)staining method.Cell apoptotic rate and nuclear morphology were detected by Annexin V/7-AAD and Hoechst33258 staining. Results:The recombinant eukaryotic expression vector pEGFP/Id3 was successfully constructed.The expression of EGFP reached the peak 48-72 h after transfection;the expresion of pEGFP-transfected group was higher than that of the pEGFP/ Id3 group.RT-PCR and immunocytochemistry staining showed that Id3 mRNA and protein were effectively expressed in pEGFP/Id3-transfected A549 cells.The growth of cells in pEGFP/Id3 transfeeted cells was significantly inhibited 48-72 h after transfection(P

Objective:To investigate the inhibitory effect of breast cancer-associated antigen 1(BRCAA1)gene silencing on gastric cancer MGC-803 cells and the related mechanism.Methods:Plasmid shRNA-BRCAA1 and shRNA-N were constructed and transfected with FuGene HD into gastric cancer cell line MGC-803.The transfection efficiency was examined using fluorescent microscope 24 h later.The total RNAs was extracted 48 h 'after transfection and the expression of BRCAA1 and GAPDH gene were analyzed by real-time PCR.The cell proliferation was assessed by MTT assay 24 h,48 h,and 72 h after transfection.The cell apoptosis was determined by Annexin V-PE/TAAD.The expression of Rb,Bax, Bcl-2 and BRCAA1 proteins was analyzed by Western blotting 48 h after transfection.Results:We found that the transfection efficiency of shRNA-BRCAA1 was(81.2?2.6)%24 h after transfection.Forty-eight hours after transfection with shRNA-BRCAA1 the expression of BRCAAI mRNA decreased by 61.4%;the inhibition rate of MGC-803 cells growth was 45.0%.The cell apoptosis rate of shRNA-BRCAA1 transfection group was significantly higher than those of untransfected group and mock plasmid transfected group([14.4?1.6]%vs[5.4?2.0]%,[4.4?2.5]%,P

Objective:To observe the inhibitory effects of survivin-specific siRNA on human lung cancer cell lines with different P53 statuses,and to analyse the relationship between P53 and survivin in vitro.Methods:A549(wild-type P53)and H1299(P53 absent)cells were transfected with chemosynthetic survivin siRNA.The effect of siRNA on cell proliferation was analysed by MTT assay.The changes of cell cycle and apoptosis were examined by flow cytometry.The expression of survivin mRNA was detected by RT-PCR.The protein expression of survivin,P53,P21 and PARP was examined by Western blotting.Results:Endogenous survivin level in A549 cells was lower than that in H1299 cells.The mRNA and protein expression of survivin was significantly lower in siRNA group than in blank control group and non-silencing siRNA group(P

Objective: To provide an efficeut protocol for constructing recombinant adenovirus, an in vitro ligation was used instead of homologous recombination. Methods: Gene BMP-2 was ligated into pShuttle 2 vector ( pShuttle 2-BMP-2 ) and then fragment containing BMP-2 gene and promoter pcmvie excised by PI-SCe Ⅰ and Ⅰ-Ceu Ⅰ endonuclease. The fragment was further combined with adenovirus vector (Adeno-X-BMP-2) , which was finally linearized with Pac Ⅰ and trans-fered to HEK293 to package adenovirus particles. Results: Both PCR assay and restiction analysis showed that the recombined rectors pShuttle2-BMP-2 and Adeno-X-BMP-2 contains the target BMP-2 gene. THe packaged adenovirus was also i-dentified by PCR assay with specific primers for BMP-2. Conclusions: The BMP-2 incorporated recombinant adenovirus was obtained and this laid a foundation for further study on BMP-2 mediated gene therapy. The in vitro ligation method de-scinbed here for constructing recombined adenovirus was more efficient than traditional homologous recombination.

Objective: To verify that COX-2 promoter can drive its downstream genes specifically in COX-2-positive o-varian cancer cells; Moreover, comparing with CMV promoter, analyze the transcript efficiency of COX-2 promoter. Methods: Contacting the recombinant plasmids named COX-2-BAX and CMV-Luc. After transient transfection liposome-mediated with the plasmids COX-2-Luc and CMV-Luc, respectively, the expression of Luciferase reporter gene was measured in COX-2-positive ovarian cancer cell line-SKOV3 and COX-2-negative colon cancer cell line-SW480. SKOV-3 and SW480 were transfected with COX-2-BAX and CMV-BAX in the same way, respectively. The apoptosis rates were measured through flow cytometry. Results: The recombinant plasmids named COX-2-BAX and CMV-Luc were constructed successfully. The expression efficiency of reporter gene was 1554 ? 86. 5 in SKOV3 and 53. 7 ? 10.9 in SW480 after 24 hours transfected with phPES2, 9851. 7 ? 129. 5 in SKOV3 and 8831. 0 ? 167. 3 in SW480 after 24 hours transfected with CMV-Luc in the same way. The apoptosis rate was 10.4% in SKOV3 and 3.7% in SW480 after transfected with COX-2-BAX, 21.7%in SKOV3 and 25. 6% in SW480 after 36 hours transfected with pcDNA3-BAX in the same way. Conclusions: COX-2 promoter can drive its downstream genes specifically in COX-2-positive ovarian cancer cell lines, but its expression efficiency wasmarkedly lower than CMV promoters. With proper modification, COX-2 promoter is expected to be useful in gene therapy of ovarian cancers.

Objective: To investigate the inhibitory effects and mechanism of a novel anti-human DR5 ( death receptor 5 of TRAIL) monoclonal antibody to glioma cell lines U343. Methods: DR5 protein was tested quantitative through FCM: DR5 mRNA was observed through RT-PCR and distribution was tested by immunocytochemistry. Inhibitory effects and inducing apoptosis of anti-human DR5 monoclonal antibody to U343 were analysed by MTT, DNA Ladder, FCM. Results: The expression of death receptor 5 ( DR5 ) was certificated in U343 , DR5 appeared to be located in intracellular perinucle-ar compartment. Inhibitory effects of anti-human DR5 monoclonal antibody on U343 were achieved by 3 ?g/ml at 4 hours and the mechanism was associated with apoptosis. Conclusion: Apoptosis of glioma cell lines U343 can be induced by anti-human DR5 monoclonal antibody, and targeted on DR will provide new way to treating cancer.

Objective: To study the effect on cytotoxicity and drug resistance by blocking ERCCl gene expression in o-varian cancer cell lines. Methods: ERGC1 antisense expression vector was constructed and transfected into human ovarian cancer cell lines. Northern blotting and Western blotting were used to detect the RNA and protein expression level in the cells. Luciferase assay system was used to reveal the host cell reactivation function. MTT assay was used to study the effect on cytotoxicity and drug resistance in the cell lines. Results: After transfection of the antisense ERCCl, Northern and Western blotting indicated that the RNA and protein expression level of ERCCl was obviously decreased. Luciferease assay showed reduced DNA-damage repair capacity as assessed by host cell reactivation. MTT assay also showed decreased drug resistance to cisplatin in the lines. Conclusion: Transfection of antisense ERCCl may enhance the cisplatin cytotoxicity by disturbing the NER pathway in cisplatin-resistant cell lines.

Objective: To investigate the effect of helper T cells (Th1 and Th2) and its important significance in judging prognosis of patients with advanced lung cancer. Methods: Interferon-? (INF-?) and interleukin-4 (IL-4) in plasma and pleural effusion were detected by enzyme-linked immunoassay in patients with advanced lung cancer, IFN-? and IL-4 reflected activity of Thl and Th2 respectively. Results: The activity of Th1 in patients with advanced lung cancer was lower than that in normal persons in peripheral blood. For non-outstanding curative effect or progressive state of illness, the activity of Thl in patients of above 1 year survival time decreased in post-treatment than in pretreatment, the activity of Th2 increased in post-treatment. Conclusion: Activity of Helper T cells ( Th1, Th2 ) could be an important marker to diagnose lung cancer and judge prognosis in patients with advanced lung cancer.

Objective: To investigate the inhibitory effect of survivin antisense oligonucleotide (ASODN) on the expression of survivin gene and proliferation of human hepatocellular carcinoma cell line SMMC-7721. Methods: The 20 mer antisense oligonucleotide (ASODN) targeted to the promoter region of survivin mRNA was designed and synthesized. The expression of survivin gene in hepatocellular carcinoma cell line SMMC-7721 was blocked by means of ASODN transfection mediated by DOTAP liposomal reagent. The changes of survivin protein and mRNA expression after transfection were as-sessd by Western blot and in situ hybridization, respectively. The apoptotic rate was detected by flow cytometer. The changes of cell adherent rate, cell growth activity, and the inhibitory rate of cell growth were also studied. Results: The expression of survivin protein and mRNA were decreased markedly after survivin ASODN transfection. Meanwhile, the cell adherent rate also decreased markedly while the apoptotic rate increased markedly. Conclusions: Transfection of ASODN targeted to the promotor region of survivin mRNA by DOTAP liposomal transfection reagent could down-regulated the expression of survivin protein and mRNA significantly in 7721 cell line and inhibit the proliferation of cancer cells. Survivin could be an important target in the therapy of hepatocellular carcinoma.