【作者】 万光霞；

【导师】 蒋先镇；

【作者基本信息】 中南大学 ， 外科学， 2008， 博士

【摘要】 膀胱肿瘤是泌尿外科最常见的肿瘤,在全世界范围内膀胱肿瘤的发病率在男性占全身肿瘤的第8位,标准发病率在男性为9.9/10万,在女性为第25位,标准发病率2.3/10万。在我国膀胱肿瘤也是泌尿外科最常见的肿瘤,膀胱移行细胞癌(Bladder Transitional CellCarcinoma,BTCC)是最常见的膀胱恶性肿瘤,占其中的92.8%。膀胱肿瘤的产生、浸润、转移和复发是一个多阶段复杂的过程,与许多癌基因的激活有关。超过50%的癌基因、原癌基因产物都具有酪氨酸激酶(receptor tyrosine kinase,RTK)活性,RTK类癌基因,包括HER-1(EGFR)、HER-2/neu(human epidermal growth factorreceptor-2)、HER-3、HER-4等,HER-2/neu起核心作用。HER-2/neu的活化信号可以转导下游分子,介导多种生化反应,产生广泛的生物学效应,在细胞的增殖、分化、抗凋亡等过程中起重要作用。HER-2/neu过表达与多种肿瘤的发生发展、化疗耐受、放疗抵抗等相关。HER-2/neu基因在膀胱肿瘤的发生、发展中扮演何种角色仍不十分清楚。国内外现在仍未见HER-2/neu的小干扰RNA(small interfering RNA,siRNA)转染BTCC细胞系的实验研究报道。我们的实验首次使用siRNA抑制膀胱癌细胞株T24中HER-2/neu基因的表达,研究HER-2/neu在膀胱癌发生、发展中的作用,探讨膀胱癌基因治疗的新途径。第一章人膀胱移行细胞癌组织中HER-2/neu表达的研究目的:从mRNA和蛋白水平研究癌基因HER-2/neu在BTCC及正常膀胱粘膜组织中的表达,结合临床病理特征研究HER-2/neu在BTCC发生发展中的作用。方法:采用RT-PCR和免疫组化从mRNA和蛋白水平检测BTCC及正常膀胱粘膜组织中HER-2/neu的表达,分析HER-2/neu表达与肿瘤发生、肿瘤临床分期、病理分级的关系。结果:BTCC中HER-2/neu mRNA表达率为57.1%,较正常膀胱粘膜组织(表达率5.0%)明显升高。随着临床分期、病理分期的升高,HER-2/neu mRNA在BTCC表达量增多。BTCC中HER-2/neu蛋白阳性率为43.1%,较正常膀胱粘膜组织(阳性率5.0%)明显升高。HER-2/neu蛋白在中低分化(Ⅱ、Ⅲ)BTCC中的阳性率明显高于高分化(Ⅰ)BTCC。HER-2/neu蛋白在浸润性(T2-T4)BTCC中的阳性率明显高于表浅性(Tis-T1)BTCC。随着病理分期的升高,HER-2/neu蛋白在BTCC阳性率增多。HER-2/neu蛋白、PCNA蛋白在BTCC中表达为正相关性,相关系数为0.336。以上差异均有显著性(P＜0.05)。BTCC中HER-2/neu mRNA、HER-2/neu蛋白表达与性别、年龄、初复发、单多发、复发前是否灌注治疗等因素无关。结论:HER-2/neu mRNA、HER-2/neu蛋白均在BTCC中表达异常增强,在BTCC发生、发展过程中起重要作用。HER-2/neu可作为诊断、判断预后、指导治疗、随访检测的指标。HER-2/neu基因可能成为BTCC新的治疗靶点。第二章针对HER-2/neu基因的siRNA表达载体的构建及鉴定目的:为研究针对HER-2/neu基因的siRNA对BTCC细胞系体外生物学行为的影响,构建插入siRNA的重组质粒pGenesil-1。方法:HER-2/neu mRNA完整序列为模板,网上在线设计两条针对CDS区的siRNA序列,BLAST同源分析证实其特异性,同时设阴性对照shHK和阳性对照shGAPDH。寡核苷酸链合成,退火磷酸化后生成双链DNA,与线性化的质粒pGenesil-1连接。转化感受态的大肠杆菌,鉴定阳性克隆,质粒酶切、测序鉴定。结果:质粒酶切鉴定可见约400bp的DNA小带,与预期相符。测序也证明重组质粒序列与设计序列完全一致。证明针对HER-2/neu基因的siRNA表达载体已构建成功。结论:成功构建了包含针对HER-2/neu基因的siRNA的重组质粒pGenesil-1,这为后期的稳定转染、抗性筛选挑取单克隆细胞株奠定了试验基础。第三章RNA干扰抑制膀胱癌细胞株T24 HER-2/neu的表达目的:为研究针对HER-2/neu基因的siRNA对BTCC细胞系体外生物学特性的影响,建立了稳定表达siRNA的BTCC细胞株T24,并探讨siRNA对HER-2/neu mRNA、HER-2/neu蛋白表达的影响。方法:将质粒P0(空质粒),质粒P1(包含shHER-2/neu-1)、质粒P2(包含shHER-2/neu-2)、质粒P3(包含shHK)、质粒P4(包含shGAPDH)瞬时转染T24细胞,分别命名为T24-P0、T24-P1、T24-P2、T24-P3、T24-P4细胞。荧光显微镜观察转染效率,RT-PCR筛选出RNA干扰效果最好的重组质粒。G418筛选阳性克隆,建立稳转细胞系,RT-PCR、Western Blot检测HER-2/neu基因沉默效果。结果:T24-P1、T24-P2克隆较T24-P0克隆小,克隆生长较为缓慢,细胞的形态无明显差别。RT-PCR发现T24、空载体转染的T24-P0、阴性对照T24-P3三者目的条带亮度相当。而T24-P1、T24-P2的目的条带较前三者弱,T24-P2尤为明显。与T24相比,T24-P2细胞HER-2/neu mRNA的表达量被抑制了54.45%。T24-P2 HER-2/neu蛋白的表达比T24、T24-P0明显下降。T24 HER-2/neu蛋白表达量约为T24-P2的2.12倍,而T24、T24-P0 HER-2/neu蛋白表达量无明显差异。结论:成功构建了稳定表达针对HER-2/neu基因的siRNA的BTCC细胞株,质粒P1、P2转染对细胞HER-2/neu mRNA和蛋白的表达量均有抑制,为下一步HER-2/neu的功能研究提供了实验基础。第四章RNA干扰HER-2/neu表达对膀胱癌细胞生物学行为的影响目的:研究HER-2/neu siRNA对BTCC细胞系T24体外增殖、凋亡、迁移侵袭能力的影响及其可能机理。方法:以成功构建的稳定表达siRNA的T24-P2、T24-P0为研究对象,以未转染的T24为对照,绘制三组细胞的生长曲线比较生长差异,计算细胞倍增时间;MTT法比较三组细胞存活率的差异;流式细胞仪检测三组细胞周期和凋亡的改变;集落形成试验检验三组细胞增殖能力的差异;细胞粘附实验、划痕实验、Transwell小室检测三组细胞体外侵袭力的变化。结果:T24、T24-P0的各项指标无明显差异。与前两组相比,T24-P2生长明显缓慢,从第三天开始生长速度明显低于前两组,至对数生长期更明显,生长曲线较平缓;T24-P2细胞倍增时间30.06小时左右,延长约5小时。T24-P2存活率明显下降,最低时细胞生长被抑制了将近一半。T24-P2处于细胞前期(G0/G1期)的比例明显增加(80.13%),细胞中期(S期)的比例明显减少,凋亡细胞比例明显增多(31.08%),在G1期前出现明显的凋亡峰。T24-P2克隆速度明显减慢,细胞形态皱缩、边缘不整,出现凋亡小体,集落形成率明显低于前两组(31.70%),而集落抑制率明显高于前两组(56.93%)。T24-P2细胞粘附力明显下降。T24-P2迁移到划痕区的细胞数明显低于前两组,说明侵袭力下降。T24-P2穿过Matrigel滤膜的细胞数明显低于前两组,说明侵袭力受抑制。以上差异均具有显著性(P＜0.05)。结论:HER-2/neu干扰质粒P2稳定转染可抑制BTCC细胞株T24的体外生长,使细胞倍增时间延长,细胞周期停滞在G1/G2期,促进癌细胞凋亡,抑制癌细胞的体外粘附、迁移侵袭能力。这种抑制作用可能与siRNA下调HER-2/neu表达有关。更多还原

【Abstract】 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.更多还原