【作者】 杜虎；

【导师】 吴小候；

【作者基本信息】 重庆医科大学 ， 外科学， 2008， 博士

【摘要】 目的筛选和鉴定重组表达质粒pshRNA-survivin。方法重新克隆pshRNA-survivin重组质粒后,Amp筛选阳性克隆提取质粒DNA,用Sal I、EcoR I和Hind III分别酶切阳性克隆,并用相同的条件酶切载体pTZU6+1作为对照,1%琼脂糖凝胶电泳鉴定;将筛选的阳性克隆质粒进行序列分析。结果经EcoR I和Hind III双酶切和序列分析证实阳性克隆中含有和设计片段完全一致的序列,插入DNA片段为:TCGAGCTGAGAACGAGCCAGACTTGTTAGTACTCAAGTCTGGCTCGTTCTCAGTTTTT(SUR-siRNA suqunce),所得测序结果与GeneBank文献报道的一致,未见缺失和突变,表明成功筛选和鉴定了含目的DNA片段的重组质粒pshRNA-survivin。结论重组质粒pshRNA-survivin筛选和鉴定成功,可用于后续实验。目的观察重组质粒pshRNA-survivin对TCCB细胞株T24细胞中survivin基因和蛋白表达的抑制作用。方法将pshRNA-survivin质粒转染TCCB细胞株T24细胞,采用RT-PCR和Western blot检测T24细胞内survivin基因和蛋白的表达。结果pshRNA-survivin转染T24细胞后,能明显抑制survivin基因和蛋白的表达,应用Quantity One软件分析结果,survivin mRNA表达抑制率达61.73%,survivin蛋白表达抑制率达53.37%。结论pshRNA-survivin能够抑制T24细胞survivin基因和蛋白的表达。目的观察pshRNA-survivin对T24细胞的生物学行为的影响。方法pshRNA-survivin质粒转染TCCB细胞株T24细胞,MTT法观察pshRNA-survivin对细胞的增殖抑制作用;细胞运动实验、细胞侵袭实验检测psh-survivin对T24细胞的生长、运动、侵袭的抑制作用;流式细胞仪和AO/EB荧光染色法检测T24细胞的凋亡;透射电镜观察T24细胞超微结构变化。结果MTT结果显示,转染组在24h、48h、72h时肿瘤抑制率分别为15.69%、27.64%和59.13%,细胞增殖受到抑制;流式细胞结果表明pshRNA-survivin组的细胞凋亡率为(16.76±1.31)%,与未转染组凋亡率(2.82±1.44)%、空载体组凋亡率(3.59±1.23)%比较有显著差异(P<0.01);AO/EB荧光染色法测定转染24h后,未转染组凋亡率为(2.37±1.67)%,空载体组的凋亡率为(5.15±2.28)%,pshRNA-survivin组的细胞凋亡率为(14.26±2.97)%,转染组与未转染组、空载体组相比较有显著性差异(P<0.01),转染48h后,转染组细胞死亡率(40.03±6.32)%和其他两组比较差异有显著性(P<0.01);细胞运动实验、细胞侵袭实验表明pshRNA-survivin组的细胞侵袭力与运动能力均有显著的降低(穿膜细胞数分别为10.34±1.85、41.32±3.47),与未转染组(27.62±2.06、62.84±4.97)和空载体组(26.07±1.73、64.15±6.71)比较有显著差异(P<0.01);透射电镜可观察到pshRNA-survivin组发生大量T24细胞的凋亡。结论当沉默survivin基因后,T24细胞增殖受到抑制、细胞侵袭力与运动能力均有明显的下降,细胞凋亡增加,提示survivin可能是TCCB生物学治疗的一个潜在靶点。目的建立TCCB裸鼠异位肿瘤模型,观察pshRNA-survivin对TCCB生长抑制作用。方法筛选稳定表达survivin-siRNA的膀胱移行细胞癌细胞株(T24/survivin-siRNA);实验共分3组:pshRNA-survivin转染组、空载体组、未转染组(每组5只);建立裸鼠皮下种植瘤模型,观察肿瘤形成时间,计算抑瘤率;HE染色行组织学检查;免疫组化检测survivin蛋白表达。结果pshRNA-survivin组种植瘤形成的时间显著地延长,与其它两组比较有显著性差异(P<0.01);在14d、21d,pshRNA-survivin组种植瘤体积(分别为54.19±3.81、149.54±11.49),与未转染组(138.40±12.44、311.02±37.01)和空载体组(167.15±13.15、338.24±19.62)比较有显著性差异(P<0.01);pshRNA-survivin组21d时的抑瘤率为68.93%,与未转染组、空载体组有明显差异(P<0.01);光镜下可见未转染组和空载体组发生肌肉侵润、肌纤维的溶解、断裂;免疫组化显示pshRNA-survivin组survivin蛋白的表达与未转染组比较有显著的降低(IOD分别为8705.4、3814932.1)。结论在TCCB裸鼠异位肿瘤模型中,pshRNA-survivin能够显著抑制移植瘤的生长、浸润和survivin蛋白的表达。更多还原

【Abstract】 Objective To identified the plasmid containing short hairpin RNA(shRNA)of survivin .Methods PshRNA-survivin expression plasmid get from Dr Huang Ai-long;pshRNA-survivin was emplified in system and identitified by enzyme digestion and sequencing method.Results The recombinant plasmid pshRNA-survivin was identified successfully by enzyme digestion and sequencing.Conclusion The results show that the short hairpin RNA of survivin can be efficiently identified. Objective To observe the plasmid containing short hairpin RNA(shRNA)of survivin suppressed the expression of exogenous survivin gene in T24 cells.Methods The plasmid pshRNA-survivin was stably transfected into TCCB cells T24 to detect effect of survivin expression and analyze the inhibition of survivin gene with semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot. Results The expression of surviving in transfected T24 cells was markedly depressed at both mRNA and protein levels (61.73%, 53.37%, respectively) as compared with control.Conclusion The results showed that down-regulate the expression of survivin with RNA interference technology can significantly suppress expression of survivin to a certain degree. Objective To assess the effects of pshRNA-survivin on the biological behaviors of T24 cells by gene siliencing.Methods The biological effects were observed, including anchorage-independent growth by MTT method, and the effects of apoptosis inducing on T24 cells were detected by flow cytometry assay and comfired by electron microscope, the ability of mobility and invasion in vitro by cell migration assay and tranwell chamber assay.Results The growth of tumor cells was retared by anchorage-independent growth assay. The inhibitory percentages of T24 cells of the experimental groups correlated with the negative control groups were 15.69%、27.64%、59.13% in 24hour、48hour and 72hour, respectively. Meanwhile, 24h after transfection, it was observed that silencing survivin by RNAi could significantly induce the spontaneous apoptosis of T24 cells at the rate 16.76±1.31%, detected by flow cytometry assay and comfirmed by electron microscope. In addition, fewer penetrating cells of pshRNA-survivin group were observed along with a marked inhibition of invasion by tranwell chamber assay (27.62±2.06,26.07±1.73,10.34±1.85*, respectively).Conclusion The results showed that blocking the expression of survivin with RNA interference technology can significantly suppress expression of proliferation,mobility and invasion of T24 cells, and induce apoptosis to a certain degree. RNAi targeted to survivin has a potential value in gene therapy of bladder cancer. Objective To observe the effects of pshRNA-Survivin on human bladder cancer T24 cell line and to suppress the expression of survivin gene in the animal model of BALB/c xenograft tumor.Methods Establishment of TCCB T24 lines with expressing siRNA-survivin stably. Then T24 cells were inoculated into the right-back legs of BALB/c nude mice to establish bladder cancer model. The inhibitory effects were observed on the growth of tumor and analyzed the inhibition of survivin gene with SABC of immunohistochemistry; Microscope were used to observe the morphological changes.Results Nude mice were injected subcutaneously with siRNA-survivin cells and xenograft tumor formed 17.0 days later,no subcutaneous metastasis.While the other two groups formed tumor at 12.4days,11.8days,respectively,six/all with subcutaneous metastasis.Tumor size were measured with caliper every seven days.21days after formed xenograft tumor,mice were sacrificed and their tumors were weighted for evaluative antitumor ratio.Result showed that the volume of T24/ siRNA-survivin xenografts in mice was effectively inhibited by pshRNA-survivin.The Ratio of antitumor was significantly higher in pshRNA-survivin group than those in control groups. The expression of survivin in pshRNA-survivin group in vivo was markedly depressed as compared.Conclusion pshRNA-survivin exerts its antitumor effect on human bladder cancer T24 cell lines in vivo. The mechanisms may be changing tumor cell cycle and inducing tumor cell apoptosis by down-regulating the expression of survivin.更多还原