【作者】 王志华；

【导师】 叶章群； 胡志全；

【作者基本信息】 华中科技大学 ， 外科学， 2007， 博士

【摘要】 第一部分Survivin基因在TCCB患者组织和尿脱落细胞中的表达及其临床意义目的:检测Survivin基因在膀胱移行细胞癌(TCCB)患者癌组织与尿脱落细胞中的表达情况,并探讨二者的相关性及其临床意义。方法:48例标本来自武汉同济医院2004年9月～2006年9月期间所有病理证实为TCCB,其中男35例,女13例,年龄56.4+2.6岁,I级7例、II级30例、III级11例,Tis~T1期40例和T2~T4期8例;初发患者30例,复发18例。分别采用巢式逆转录聚合酶链反应(Nested RT-PCR)法检测48例TCCB和16例非TCCB对照组(其中8例腺性膀胱炎、5例BPH、3例膀胱结核患者)组织及尿脱落细胞中Survivin mRNA表达情况。采用免疫组织化学SP法复检所有TCCB患者癌组织Survivin蛋白的表达情况并确定其临床病理分期和分级。统计学方法分析了Survivin表达与TCCB的分期分级及预后等病理学参数之间的关系。结果:48例TCCB患者癌组织中Survivin基因的阳性表达率为(36/48)75.0%,mRNA表达结果与蛋白免疫组织化学结果一致。在尿液中的mRNA阳性表达率为(34/48)70.8%;16例非TCCB组织中Survivin基因的阳性表达率仅为(1/16)6.3%而尿液中未见Survivin基因表达。以上表明Survivin基因在TCCB组织中的表达显著高于非肿瘤组织中的表达(P<0.01);而组织标本与尿液标本对Survivin基因的检出率无明显差异(P>0.05)。Survivin基因在TCCB患者中呈不同程度的表达,其表达随TCCB的病理分级、临床分期的增高而相应增高,复发TCCB表达明显增高。I、II、III级阳性表达率分别为55.6%,76.7%,88.9%;浅表性(Tis~T1)、浸润性(T2~T4)中阳性表达率分别为72.5%和87.5%;初发及复发TCCB阳性表达率为65.5%和89.5% (P<0.01)。Survivin的阳性表达与膀胱癌的病理分级分期或是否复发关系密切(P<0.05),而与患者年龄、性别、肿瘤大小或生长方式无明显相关性(P>0.05)。结论:细胞凋亡抑制因子Survivin在TCCB组织中表达上调,且表达水平与TCCB分级分期及复发正相关,提示Survivin基因可能通过抑制细胞凋亡在TCCB的发生发展和肿瘤转移及预后等方面起重要作用,而检测尿脱落细胞中Survivin表达可作为TCCB的筛查、早期诊断、术后监测的新方法,膀胱肿瘤细胞中Survivin基因也可能为生物学治疗提供新的靶点。第二部分Survivin反义寡核苷酸诱导膀胱癌细胞凋亡及增强其化疗敏感性目的:探讨Survivin反义寡核苷酸(Survivin-ASODN)对化疗药丝裂霉素C(MMC)诱导膀胱癌T24细胞凋亡的影响。方法:将已构建成功的Survivin-ASODN真核表达载体pcDNA3-SVVas通过脂质体介导转染膀胱癌细胞系T24,并筛选转染成功的阳性克隆;采用RT-PCR法检测其Survivin mRNA的表达;台盼蓝拒染法观察Survivin-ASODN与MMC联合应用对癌细胞生存的影响;细胞计数和MTT试验测定MMC对转染细胞增殖抑制情况;琼脂糖凝胶电泳分析凋亡细胞DNA断裂情况;核染色检测凋亡细胞的细胞核变化;流式细胞术检测细胞凋亡率。结果:转染Survivin-ASODN真核表达载体pcDNA3-SVVas的T24-SVVas细胞Survivin mRNA表达水平下降、细胞增殖明显受抑,与转染pcDNA3空载体T24-neo细胞及未转染载体的T24细胞进行比较,具有显著性差异(P<0.05);经琼脂糖凝胶电泳,T24-SVVas细胞组均可见到DNA梯形条带,而其他对照组未发现;与对照组细胞相比,转染阳性克隆细胞的细胞核呈致密浓染;加入MMC的T24-SVVas细胞凋亡率大幅度增加(P<0.05)。结论:Survivin-ASODN可促进MMC诱导膀胱癌细胞凋亡及增加其对化疗药物的敏感性,为膀胱癌的生物学治疗研究奠定了实验基础。第三部分siRNA靶向Survivin对膀胱癌细胞增殖与凋亡的影响目的:采用RNA干扰技术(siRNA )阻断Survivin基因的表达,观察其抑制膀胱癌细胞T24增殖及诱导细胞凋亡的作用。方法:本研究使用的pGenesil-1-SVVsi质粒含有U6启动子、EGFP标记蛋白和三条针对Survivin基因的siRNA表达载体,采用LipofectamineTM2000转染至人膀胱癌细胞系T24,并筛选转染成功的阳性克隆(T24-SVVsi);同时设置空载体(T24-neo)和未转染T24作为对照组,采用RT-PCR、Westernblot技术检测各组肿瘤细胞Survivin的mRNA和蛋白表达变化;采用MTT法和聚落形成试验检测对T24细胞增殖抑制作用;透射电镜观察凋亡细胞形态变化;采用Annexin-V/PI双色标记的流式细胞术(FCM)检测Survivin-siRNA诱导的细胞凋亡情况;并采用MTT测定转染的T24细胞对化疗药物MMC的敏感性。结果:透射电镜下可观察到转染组凋亡的征象,而各对照组未见凋亡证据;肿瘤细胞的转染组Survivin的mRNA和蛋白质表达降低至少50%;T24细胞接种48 h后其增殖抑制率达47%;FCM分析显示转染组细胞均阻滞在G2/M期,细胞复制时间延长1.4倍,凋亡率(AR)明显增加,由3.38%增加到33.86%。具有显著意义(P<0.05)。MTT试验显示,与对照组比较,细胞转染组MMC对肿瘤半数抑制浓度(IC50)明显降低(P<0.05)。结论:靶向Survivin基因的重组siRNA载体pGenesil1-SVVsi有效抑制了Survivin基因表达,可显著抑制T24细胞增殖并在一定程度上诱导其凋亡,并能更有效增强化疗药物的敏感性。这表明据抗Survivin的RNA干扰技术在膀胱癌的基因治疗中具有重要价值。第四部分Survivin-siRNA靶向复合物系统治疗TCCB的实验研究目的:制备含有Survivin-siRNA双表达载体和靶向分子TfRscFv-GAL4的复合物系统,并研究该靶向复合物系统(TfRScFv-GAL4-GAL4rec-SurvivinsiRNA -pGes)对膀胱癌细胞系T24的生物学效应。方法:①以摩尔比为1:2.5的比例将GAL4rec-SurvivinsiRNA- pGes和TfRScFv-GAL4在体外混合,并以4.5%非变性的聚丙烯凝胶电泳检测制备的靶向复合物系统;②流式细胞仪检测在靶细胞内绿色荧光蛋白表达率;③倒置荧光显微镜观察TfRScFv-GAL4介导的靶向复合物内吞作用及目的基因的表达;④采用萤光定量RT-PCR和Western-blot分别检测阳性表达细胞Survivin mRNA和蛋白的变化;⑤MTT法检测靶向复合物系统对不同组细胞增殖抑制情况;⑥靶向复合物系统与膀胱癌细胞株T24共孵育48h后,Hochest染细胞核观察细胞凋亡现象;⑦Annexin-V/PI双色标记的流式细胞术分析靶向复合物系统对不同组细胞周期的影响结果:①凝胶电泳中可见TfRScFv蛋白加入不同浓度GAL4rec-SurvivinsiRNA-pGes载体,蛋白染色只有一条条带;而GAL4蛋白和TfRScFv-GAL4蛋白随加入的GAL4rec-SurvivinsiRNA-pGes载体量的增加,向上移位的蛋白增多,表明TfRscFv-GAL4融合蛋白可通过其中GAL4片断与GAL4rec-SurvivinsiRNA-pGes载体特异性连接,并在最适摩尔比例为1:2.5条件下成功制备TfRScFv-GAL4-GAL4rec-SurvivinsiRNA-pGes靶向复合物系统;②FCM检测表明靶向复合物系统在靶细胞内绿色荧光蛋白表达率为44.30%。③靶向复合物系统与肿瘤细胞共孵育后,在荧光显微镜下可见细胞内有绿色荧光蛋白表达,而其他各实验对照组均未见细胞内有绿色荧光蛋白表达。在光镜下可见靶向复合物系统组有部分贴壁细胞胞质回缩,胞体趋于变圆,似凋亡细胞的形态,而其他各实验组细胞贴壁,生长良好;④萤光定量RT-PCR和West-blotting检测显示,靶向复合物系统组Survivin mRNA和蛋白表达量降低至少53%;⑤MTT检测表明靶向复合物系统组肿瘤细胞生长明显抑制(P<0.05);⑥Hochest染色可见,靶向复合物系统组可见大量的凋亡细胞,而其他组凋亡细胞较少,叠加照片显示光镜中所见胞质回缩,胞体趋于变圆的细胞,均为凋亡的细胞;⑦双色标记的流式细胞术检测结果显示,靶向复合物系统组肿瘤细胞周期阻滞在G2/M期,凋亡率明显增加(P<0.05)。结论:①靶向复合物系统TfRscFv-GAL4-GAL4rec-SurvivinsiRNA-pGes与肿瘤细胞孵育后能被内吞进入细胞内表达绿色荧光蛋白,并诱导肿瘤细胞凋亡。表明TfRscFv-GAL4具有较好的肿瘤靶向性,并能介导双基因表达载体在肿瘤细胞中的表达。②靶向复合物系统表达的Survivin-siRNA能有效抑制膀胱癌细胞增殖并诱导细胞凋亡,对正常细胞无影响。更多还原

【Abstract】 Part I Clinical significance of Survivin expression in tumor tissues and urine exfoliate cells of patinets with transitional cell carcinoma of bladderBackground: Transitional cell carcinoma (TCC) of bladder is the most common malignant tumor in uropoiesis system. Up to date, there is still lack of an ideal marker for the diagnosis of TCC except CT and MRI imaging and cystoscopy. Cystoscopy is an invasive examination, which increases the possibility of urinary tract infection. Urine cytology has low sensitivity (21%~40%) in diagnosis of bladder cancer, especially for those with medium or high differentiation. Urologists are in the ongoing search for novel markers for the improvement of bladder cancer diagnosis and prognosis. Survivin is a 16.5kDa protein overexpressed in almost all malignancies but rarely detected in normal differentiated adult tissues. Functionally, Survivin has been shown to inhibit apoptosis, promote cell proliferation and enhance angiogenesis, even consistent with its role in these processes. Because of the large difference in expression between normal and malignant tissue and its causal role in cancer progression, Survivin is currently undergoing intensive investigation as a potential tumor marker. Emerging data suggests that measurement of Survivin can aid the early diagnosis of bladder cancer, determine prognosis in multiple cancer types and predict response to diverse anti-cancer therapies. These preliminary findings on the diagnostic, prognostic and predictive potential of Survivin should now be confirmed in large prospective trials. However, only a few studies have dealt with Survivin expression in TCCB with a small number of patients, and the results were controversial, moreover, the correlations between Survivin gene expression and pathological factors for bladder cancer remain unclear. Furthermore, assays for the measurement of Survivin should be simplified, standardized and evaluated in external quality assurance schemes. In the present study, both immunohistochemistry and RT-PCR were to examine Survivin expression in urine exfoliate cells and tumor tissues of patients for the purpose of comparing the differential expression of Survivin in non-TCCB, TCCB of different clinical or pathological parameters, and of determining whether expression of Survivin is associated with TCCB clinical outcomes. Objective:To investigate correlation of detections by two methods that were used to measure Survivin expression in urine exfoliate cells and tumor tissues of patients with TCCB, and further develop their clinical significances.Methods: All 48 specimens of pathologically confirmed TCCB in experimental group were collected in Wuhan Tongji Hospital during the surgical operation from 2004 Sep. to 2006 Sep.. Among those patients of 35 male and 13 female, their mean age was 56.4±2.6 years. Of 48 cases, 30 cases were first discovered and 18 cases relapsed. The pathological grade showed Grade I was 9 cases, Grade II was 30 and Grade III was 9 cases. The clinical stage was that Tis~T1 40 cases, T2~T4 8 cases. 16 specimens in control group were obtained from the patients (8 cases with glandular cystitis, 5 cases with BPH, 3 cases with cystophthisis) during bladder operations and were pathologically confirmed to be normal bladder tissue. Nested RT-PCR were respectively used to determine Survivin mRNA in urine exfoliate cells and tumor tissues of all cases in both experimental and control groups, meanwhile, immunohistochemistry was used to detect Survivin protein and identify the pathological grade and clinical stage in tumor tissues, as well as analysis of the relationship of Survivin expression and pathological parameters by statistics methods.Results:The positive expression rate of Survivin was 75.0%, and the mRNA expression detected by RT-PCR parallelly correlated with protein expression by immunohistochemistry in bladder tumor tissues from 48 TCCBs. The positive result of urine detection by RT-PCR was (34/48)70.8%, there was no significant difference of the expression rate between tissue specimen and urine specimen in TCCB patients(P>0.05). While no positive expression was found in urine and 1case of positive expression in non-neoplastic bladder tissues from 16 control cases. This indicated that the positive expression rate of Survivin in tumor tissues of TCCBs was significantly higher than that in bladder tissues of control group(P<0.05). The expression level of Survivin elevated as the grade and stage got higher in TCCBs. The positive rates in grade I, II, III were 55.6%, 76.7%, and 88.9%; in stage of Tis~T1, T2~4 72.5%, 87.5%; and primary, recurrent cases 65.5%, 89.5%, respectively. Multivariate Cox proportional hazards model analysis identified Survivin expression as an independent prognostic factor of disease-free survival (P=0.009, r=3.12). The positive expression of Survivin was strongly associated with pathological grade, clinical stage or relapse (P<0.05), but not with individual age, sex, tumor number, size, or growth mode.Conclusions:The up-regulated expression of Survivin in TCCB suggests that Survivin may play an important role in tumorigenesis and progression through inhibiting cell apoptosis. Besides, the higher the grade and stage are, the higher the expression level of Survivin is. Survivin is correlated with recurrence of TCCB. And detecting the Survivin expression in urine may be a new method for screening and diagnosis in early stage or monitoring recurrence after operation. High and specific expression of Survivin in tumor tissue would contribute it to be a new targeted gene in biotherapy of TCCB.Part II Antisense Oligodeoxynucleotide Targeting Survivin Expression Induces and Sensitizes Bladder Cancer Cells to ChemotherapyBackground: Diminished apoptosis plays a critical role in tumor initiation, progression, and drug resistance. Several IAPs that inhibit apoptosis have been identified. Survivin, a novel gene encoding a structurally unique apoptosis inhibitor IAP, is abundantly expressed during fetal development, and almost undetectable in terminally differentiated adult tissues. However, Survivin is prominently expressed in transformed cell lines and in all the most common human cancers in vivo, including transitional cell carcinoma of bladder (TCCB). It is also a bifunctional protein that acts as a cell division regulator and an apoptosis suppressor. To evaluate the abilities of inhibiting Survivin expression and enhancing sensibility to chemotherapy, A Survivin-ASODN eukaryotic vector pcDNA3-SVVas was prepared in previous study.Objective: To evaluate the effects of antisense oligodeoxynucleotide(ASODN) of Survivin gene on MMC-induced apoptosis in bladder cancer cell line T24.Methods: A Survivin-ASODN eukaryotic vector pcDNA3-SVVas prepared in previous study was delivered into T24 mediated by liposomal reagent. The mRNA expression of Survivin measured by RT-PCR. Cell survival fraction was determined using the trypan blue dye exclusion assay. Cell counting method and MTT colorimetry. were tested to investigate the sensibility of transfected cells to MMC. Induced Apoptosis was detected by DNA gel electrophoresis and nuclear staining,as well as apoptosis rate by FCM.Results: We obtained two positive cell clone T24-SVVas and T24-neo by liPofectin. Compared to T24 and T24-neo cells, the mRNA expression of Survivin in T24-SVVas cells decreased, as well as significantly inhibited in T24-SVVas cell growth (p<0.05). The sensibility of T24-SVVas cells to MMC increased significantly. Agarose gel electrophoresis of genomic DNA from T24 SVVas displayed typical DNA ladder, but DNA from control groups did not. Nuclear staining found cellular nuclei become condense in T24-SVVas cells. FCM showed that cell apoptosis rate of T24-SVVas with MMC is significantly higher than that of other groups.Conclusion: As the human Survivin-ASODN could decrease the expression of Surviving to enhance MMC-induced apoptosis in bladder cancer cell line T24 in vitro and sensitize T24 cells to chemotherapy, this may lay an experimental foundation for further research of biotherapy in bladder cancer, it also deserves further investigation as a novel approach to selective tumor therapy. Part III Survivin-targeted siRNA induces apoptosis in human bladder cancer T24 cell linesBackground: Bladder cancer has been known as the most common type of urinary system tumours in China. It is frequently associated with genetic mutations that deregulate the cell cycle and render these tumours resistant to apoptosis. Survivin is recognized as a general target in cancer therapy because of its selective overexpression in the majority of tumors. In bladder cancer (BCa), its expression correlates with tumor grade, recurrence risk and survival. By inducing cell proliferation and inhibiting apoptosis is frequently activated in bladder cancer. RNA interference (RNAi) has recently emerged as a powerful reverse genetic tool to silence gene expression, which would cause strong and stable degradation of the specific mRNA and decreased protein expression. For these reasons, we studied the influence of small interfering RNA (siRNA) targeting Survivin on the growth and apoptosis of bladder cancer cells.Objective: This study was aimed to investigate the effect of T24 cell growth, apoptosis, and sensitivity to chemotheraputic drug MMC by small interferencing RNA silencing Survivin gene in human bladder transitional cell carcinoma (BTCC).Methods: The eukaryotic vector pGenesil-1 was designed to contain 3 Survivin-targeted siRNA, promoter U6, and marked protein EGFP. Liposomal transfection by LipofectamineTM2000 was respectively performed for pGenesil-1-SVVsi and a blank vector pGenesil-1 delivered into T24, positively transfected cells (T24-SVVsi and T24-neo) were harvested to continue next tests after transfection for 48h, as well as no transfection T24 as control. Survivin protein detected by using Westernbloting and mRNA expression was quantified by real time RT-PCR; The viability of 3 groups of treated cell was examined by using MTT and Colony formation assays were performed to evaluate the long-term survival of treated cells; Morphological change was observed with transmission electron microscope, Cell cycle analysis and apoptosis dectection of treated cells was carried out by FACS after double staining cells with propidium iodide (PI) and FITC-labeled Annexin V; MTT assay and cell counting method was performed to evaluate the sensibility of T24 cells to chemotherapeutics MMC.Results: Abnormal cell morphological change was found in T24-SVVsi, no apoptotic evidences were observed in 2 control groups; In T24-SVVsi group the mRNA and protein expression quantified by real time RT-PCR and Westernbloting was reduced by at least 50%; T24-SVVsi showed a significant reduction (up to 47%) of viability; Cell cycle analysis and quantitation of apoptosis revealed both a specific G2/M arrest and an induction of apoptosis, as well as the occurrence of multinucleated cells. only T24-SVVsi group exhibited a prolonged duplication time up to 1.4-fold at 48 h after treatment. Furthermore, compared to control groups, the half inhibition concentration (IC50) of MMC to bladder cancer cells in T24-SVVsi group was significantly reduced by MTT assay, the difference was significant by statistical analysis.Conclusion: Survivin-siRNA may silence the Survivin expression to induce T24 cells apoptosis, inhibit cell proliferation and sensitize T24 cells to chemotheraputic drug (MMC). This suggested that the anti-Survivin siRNA treatment might represent a suitable therapeutic approach to selectively inhibit growth of BCa cells in addition to commonly applied therapy schemesPart IV Effect of Survivin-targeted complex system on transitional cell carcinoma of the bladderBackground: Bladder cancer is the most common cancer of urinary deseases in China. Survivin, a member of inhibitor of apoptosis protein (IAP) gene family, is abundantly expressed in a wide range of malignancies, including carcinoma of the bladder urothelium. Our previous study demonstrated Survivin as a novel intervention target to suppress the growth and induce apoptosis in cancer cells by antisense oligodeoxynucleotide (ASODN) or small interfering RNA(siRNA). But the efficiency of their inducing gene knockdown is hampered by low transfection efficiency; also several gene-transected approaches limited in vivo application due to their toxicity or damage. Furthermore, the mechanism by which Survivin plays a role in the regulation of cell death is still unknown. In the present study, basing the previous foundation that TfR is overexpressed on the surface of TCC and TfR-ScFv is a ideal tumor-targeted molecule by carrying expression vector to intra-cellular internalization,we established the Survivin-targeted complex system to investigate its biological effect on bladder cancer cell.Objective: By mixing double expression vector and target molecule TfRscFv-GAL4 at a adopted ratio in vitro experiment, we first established the Survivin-targeted complex system(TfRScFv-GAL4-GAL4rec-SurvivinsiRNA–pGes) to incubate with T24 cell line and investigate its biological effectMethods:①4.5% native polyacrylamide gel electrophoresis was used to identify the conjugated target system(TfRScFv-GAL4-GAL4rec-SurvivinsiRNA–pGes) of renatured fusion protein TfRScFv-GAL4 and recombinant vector GAL4rec-SurvivinSiRNA-pGes at molar ratio of 1:2.5;②The expression rate of green fluorescent protein(GFP) in T24 was determined by FCM;③The targeted complex intake mediated by the fusion protein TfRScFv-GAL4 and effector expression were observed through inversted fluophot.④Survivin protein measured by Western-blot and mRNA expression was quantified by real time RT-PCR;⑤Growth inhibition of T24 cells was determined by use of the colorimetric MTT⑥It was observed using Hochest staining kit that tumor cell apoptosis induced by the targeted complex system TfRScFv-GAL4- GAL4rec-SurvivinSiRNA-pGes.⑦Cell cycle analysis and apoptosis dectection of treated cells was carried out by FACS with propidium iodide (PI) and FITC-labeled Annexin V Results:①The only one protein band could be seen in the TfRScFv group with different concentration of GAL4rec-SurvivinSiRNA-pGes in 4.5 % native polyacrylamide gel electrophoresis, but the moved protein band was dose-dependent with addition of vector GAL4rec- SurvivinSiRNA-pGes in GAL4 and TfRScFv-GAL4 group, these results showed fusion protein TfRScFv-GAL4 could specially bind to GAL4rec-SurvivinSiRNA-pGes by GAL4 fragment, and the molar rate is 1:2.5.②The expression rate of GFP is 44.30%.③Of all groups, two groups of the targeted complex system and TfRScFv-GAL4-GAL4rec-pGes were co-cultured with TCCB T24 cell lines for 48h, it was observed the green fluorescin in the tumor cell endochylema but not in other control groups through fluorescent microscope. Also microscope could find that cytoplasm condensed in some adherent cells and cell bodies appeared rotund like the apoptosis cell morphous in the targeted complex system group, whereas the adherent cells grow well in the control groups.④In targeted complex system group the mRNA and protein expression was reduced by at least 53%;⑤It showed a significant reduction of viability and strong inhibition in cells of targeted complex system group (P<0.05);⑥in the cell nucleus staining test, it was found that numerous apoptosis cell as well as the cells with condensed cytoplasm and rotund bodies by rebuilding their pictures in the targeted complex system group.⑦Cell cycle analysis and quantitation of apoptosis revealed both a specific G2/M arrest and an induction of apoptosis by 48%. Conclusion: It firstly demonstrated the complex system TfRscFv-GAL4-GAL4rec-SurvivinsiRNA-pGes could be intaken into endochylema to express the GFP, and then siRNA induce malignant cells apoptosis. This suggested that TfRScFv-GAL4 has the tumor-targeted characteristic and could induce the expression of eukaryotic bifuction vector in the tumor cells. The investigation also suggested the targeted complex system against Survivin could remarkably inhibit proliferation of T24 and induce apoptosis. But not affect the normal cells.更多还原