| Bladder cancer was the most common cancer in urology. The incidence of bladder cancer in the male was the eighth in all tumor in the world, and standards incidence of male was 9.9/10 million; while twenty fifth in female, 2.3/10 million. Bladder cancer was still the most common cancer in urology in our country. BTCC (bladder transitional cell carcinoma) was the most common malignant tumor of the bladder, account for 92.8%. The produce,invasion,metastasis and recurrence of BTCC were a multi-stage,complex process, having relationship with many oncogene activation. More than 50% of the oncogene,oncogene product had tyrosine kinase activity. HER-2/neu played a central role in the oncogenes type of RTK including HER-1 (EGFR),HER-2/neu,HER-3,HER-4. Signals activated by HER-2/neu could transduct to downstream molecules, mediate variety of biochemical reactions, produce a wide range of biological effects, and play an important role in cell proliferation,differentiation,anti-apoptosis. Over-expression of HER-2/neu had relationship with development,chemotherapy resistance,radiation resistance of a variety tumors. What kind of role HER-2/neu playing was not yet very clear in development of BTCC. Experimental study on siRNA of HER-2/neu transfecting BTCC cell lines was not yet reported at home and abroad. The first time in the world we used siRNA to inhibit BTCC cell line T24 to reduce the HER-2/neu gene expression, to study function of HER-2/neu in BTCC generation and development, then to explore a new way of gene therapy in BTCC.Chapter I The expression of HER-2/neu mRNA and protein in bladder transitional cell carcinomaObjective: To study the expression of HER-2/neu mRNA,HER-2/neu protein in BTCC and the normal bladder mucosa, evaluate the possible role in BTCC development by the clinical,pathological features.Methods: HER-2/neu mRNA and HER-2/neu protein were examined by RT-PCR and immunohistochemistry respectively in BTCC and the normal bladder mucosa. The correlations between the expression of HER-2/neu and tumor grade,stage were closely studied.Results: The expression of HER-2/neu mRNA in BTCC was 57.1%, higher than the normal bladder mucosa. Along with clinical stage,pathological stage increasing, the expression of HER-2/neu mRNA was more and more. The expression of HER-2/neu protein in BTCC was 43.1 %, higher than the normal bladder mucosa. The expression of HER-2/neu protein in II,III BTCC was higher than the I BTCC. The expression of HER-2/neu protein in T2-T4 BTCC was higher than Tis-T1 BTCC. Along with pathological stage increasing, the expression of HER-2/neu protein was more and more. There was the positive correlation between HER-2/neu protein,PCNA protein in BTCC, and the correlation coefficient was 0.336. HER-2/neu mRNA,HER-2/neu protein had no relationship with gender,age,recurrence,multiple and reperfusion therapy.Conclusion: The abnormal expression of HER-2/neu was common in BTCC, which playing an important role in the development of BTCC. HER-2/neu would be a useful predictor of diagnosis,prognosis,treatments follow-up testing. HER-2/neu gene would be a new therapeutic target.Chapter II Construction and identification vector expressing siRNA on HER-2/neuObjective: To study the effect of siRNA on HER-2/neu gene expression and biological behavior of BTCC cell line, we established and identified the recombinant plasmid pGenesil-1.Method: Complete sequence of HER-2/neu gene as a template, designed two siRNA sequences for the CDS online, BLAST confirming its specificity. Negative control shHK and positive control shGAPDH were used at the same time. Annealing,phosphorylating oligonucleotide chain, dsDNA connected linearized plasmid pGenesil-1. Plasmid transformated state feelings E. coli. The constructed vectors expressing siRNA were digested with enzymes, and positive clones having been inserted sequences were confirmed by DNA sequencing.Result: DNA sequencing results of positive clones were the same with our designation.Conclusion: Recombinant plasmid pGenesil-1 including siRNA on HER-2/neu gene was constructed successfully.ChapterIII Surpression of HER-2/neu gene expression by using siRNA in T24 cell lineObjective: To explore the effect of siRNA on HER-2/neu on the BTCC cell in vitro, T24 that stable expressing siRNA was constructed. HER-2/neu mRNA and protein expression of T24 were detected by RT-PCR,Western Blot. Method: Five plasmids: P0(Empty plasmid),P1(shHER-2/neu-1),P2(shHER-2/neu-2),P3(shHK),P4(shGAPDH) were transferred respectively into T24 in vitro by using lipfectamine. Transfection efficiency was detected by fluorescence microscopy. Cells were selected by G418 and the positive cell clones were chosen,expanded culture. The expression of HER-2/neu gene on the levels of mRNA and protein were determined respectively by RT-PCR,western blot.Result: The clones of T24-P1,T24-P2 were smaller than T24, and grew slowly. HER-2/neu mRNA of T24,T24-P0,T24-P3 were same by RT-PCR, but HER-2/neu mRNA of T24-P1,T24-P2 were less, T24-P2 specially. HER-2/neu mRNA of T24-P2 was inhibited about 54.45%. HER-2/neu protein of T24-P2 was less than T24,T24-P0, but HER-2/neu protein of the latter two were same.Conclusion: We successfully established BTCC cells that could stably express the siRNA on HER-2/neu gene and empty plasmid pGenesil-1 respectively. The expression of HER-2/neu mRNA and protein were inhibited by P1,P2. It provided a foundation for post experimentation. Objective: To investigate whether the RNA interference on HER-2/neu could inhibited the growth,apotosis,motility,invasion of BTCC cell lines in vitro and to explore the possible mechanism.Methods: BTCC cell lines T24-P2,T24-P0 and T24 were taken as our object. Cell growth curve was obtained by cell counter and cell doubling time was computed. Cell survival rate was measured by MTT assay. Flow cytometry was used to analyze the cell cycle and cell apoptosis. Cell proliferation was measured by colony-forming test. The motility and invasion ability in vitro were obtained by cell adhere assay,scratch assay and matrigel invasion assay.Results: T24 and T24-P0 had no obvious different in all indicators, morphology shrinkaged,edge incomplete,apoptotic bodies appearing in T24. Compared with the other two groups, T24-P2 grew more slowly, having lower survival rate,higher proportion of cells in G0/G1 phase and higher in the apoptosis,lower in colony formation rate,motility and invasion ability.Conclusions: Transferring P2 could inhibit the growth,motility,invasion of BTCC cell T24 in vitro. This effect was possibly atributed to suppressing HER-2/neu mRNA and protein.
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