【作者】 张立力；

【导师】 罗荣城；

【作者基本信息】 南方医科大学 ， 肿瘤学， 2008， 博士

【摘要】 肝细胞性肝癌（Hepatocullular carcinoma,HCC）作为人类最常见、恶性程度最高的恶性肿瘤之一,有着较高的发病率和死亡率。我国是世界上肝癌发病集中的国家之一,全球每年新发生的肝癌患者中有45%在我国大陆地区,严重地威胁着人民的身体健康。手术切除和肝脏移植是目前治疗肝癌较为有效的方法,但由于肝癌具有恶性程度高、发展迅速、容易复发和转移等特点,肝癌患者中仅有一部分人适合外科手术治疗。绝大部分肝癌患者只能依赖于放疗、化疗及其它的一些姑息疗法。发展新的肝癌治疗手段和治疗途径已成为广大医务工作者迫切需要研究和解决的问题。近年来,随着肿瘤分子生物学和免疫学理论及技术的发展,人们对癌基因的认识不断深入,随着转基因技术的日趋完善,肝癌的基因治疗已显示出较好的应用前景。Gankyrin基因作为近年来发现的第一个在肝细胞癌中普遍激活的癌基因,相关的研究证实几乎所有肝细胞癌患者的癌组织中均存在Gankyrin的高表达,且Gankyrin可通过依赖泛素的蛋白酶解系统介导多种细胞周期调控蛋白、转录因子以及p53、p16等抑癌蛋白分子降解参与肝细胞癌的发生发展。提示Gankyrin可作为肝细胞癌诊断和治疗的靶点,具有重要的临床应用价值。有鉴于此,本课题采用目前国际上较先进的RNA干扰技术一慢病毒介导的靶向沉默Gankyrin基因表达系统,通过体内、体外实验观察该表达系统沉默Gankyrin基因对肝癌细胞株生物学行为的影响,并探讨其相关机制。希望为该系统最终应用于肝细胞性肝癌的临床治疗提供科学的理论和实验依据。实验目的1.制备兔抗Gankyrin多克隆抗体,为后续实验中Gankyrin蛋白表达的定性、定量检测提供实验工具。2.构建并制备携带靶向沉默Gankyrin基因shRNA的慢病毒载体,为后续验证采用该表达系统进行肝癌基因治疗有效性的体内和体外实验提供基本的实验工具。3.探讨慢病毒介导靶向沉默Gankyrin基因表达系统对肝癌细胞生物学特性的影响,为该系统最终应用于肝癌的临床治疗提供科学的理论和实验依据。实验方法1.兔抗Gankyrin多克隆抗体的制备、纯化及鉴定1.1采用基因克隆技术构建pET30a（+）-Gankyrin表达载体质粒并进行酶切鉴定;1.2通过IPTG诱导Gankyrin蛋白原核表达获得His6-Gankyrin融合蛋白;1.3以纯化的His6-Gankyrin融合蛋白作为抗原进行免疫反应制备兔抗Gankyrin多克隆抗体,并进行抗体特异性和反应性检测。2.靶向沉默Gankyrin基因shRNA慢病毒载体的构建和制备2.1根据Gankyrin基因（NM₀02814）CDS区编码序列设计、选择三段19nt的寡核苷酸序列作为靶向Gankyrin基因的shRNA片段序列,并以此合成三对长引物单链DNA片段,经退火、双酶切后与同样双酶切的质粒pDC316-EGFP-U6连接、转化,获得三个质粒;2.2将获得的质粒分别转染肝癌细胞株SMMC-7721,以RT-PCR和Westernblot方法筛选对Gankyrin基因表达沉默作用最为明显的质粒;再以该质粒为基础通过基因克隆技术构建携带有靶向沉默Gankyrin基因shRNA片段的慢病毒包装质粒plenti6/V5-shRNA（Gankyrin）-EGFP;2.3采用Invitrogen公司的四质粒共转染系统,制备Lenti-shRNA（Gankyrin）-EGFP慢病毒表达载体系统,并进行相关质量检测。3.重组慢病毒介导靶向沉默Gankyrin基因对肝癌细胞生物学特性的影响3.1采用构建的Lenti-shRNA（Gankyrin）-EGFP慢病毒表达载体系统感染肝癌细胞株SMMC-7721,分别检测该载体系统在体外对肝癌细胞生长抑制率、凋亡率、Gankyrin mRNA和蛋白表达的影响;3.2建立肝癌细胞SMMC-7721移植瘤模型,成瘤后使用Lenti-shRNA（Gankyrin）-EGFP慢病毒表达载体进行瘤体注射,观察其对荷瘤裸鼠移植瘤生长、移植瘤Gankyrin mRNA的影响,以及移植瘤形态学的改变。实验结果1.兔抗Gankyrin多克隆抗体的制备、纯化及鉴定1.1酶切鉴定结果显示构建的pET30a（+）-Gankyrin表达载体质粒正确,转化入宿主大肠杆菌后通过IPTG可诱导Gankyrin蛋白;1.2 ELISA和Western Blot检测结果显示,以纯化的His6-Gankyrin融合蛋白作为抗原制备的兔抗Gankyrin多克隆抗体有较好的反应性和特异性,可以满足后续实验的要求。2.靶向沉默Gankyrin基因shRNA慢病毒载体的构建和制备2.1鉴定结果显示3个携带靶向沉默Gankyrin基因shRNA片段的质粒构建正确,均携带有启动EGFP转录的CMV启动子和启动shRNA序列转录的U6启动子。RT-PCR和Western blot方法筛选结果显示其中的pDC316-EGFP-U6-shRNA（Gankyrin）1质粒剔降SMMC-7721细胞Gankyrin表达效果最好。以该质粒为基础构建的慢病毒包装质粒pLenti6/V5-shRNA（Gankyrin）-EGFP经鉴定显示质粒构建准确。2.2通过四质粒共转染策略制备的高滴度慢病毒表达载体系统Lenti-shRNA（Gankyrin）-EGFP,经PCR鉴定和EGFP的活性观察等检测方法,确认该慢病毒构建正确、滴度高、感染活性高。3.重组慢病毒介导靶向沉默Gankyrin基因对肝癌细胞生物学特性的影响3.1 MTT法检测体外培养SMMC-7721细胞生长结果显示空白对照组与慢病毒对照组细胞之间相比,细胞生长率差异无显著性意义（P＞0.05）;以不同MOI值加入Lenti-shRNA（Gankyrin）-EGFP感染的SMMC-7721细胞分别与空白对照组和慢病毒对照组相比,细胞生长率差异有统计学意义（P＜0.05）,Lenti-shRNA（Gankyrin）-EGFP可抑制SMMC-7721细胞生长（P＜0.05）,且随着MOI值的增加,抑制作用逐渐增加,但MOI值增加到一定程度,其细胞生长抑制率变化不明显。3.2流式细胞仪对体外培养SMMC-7721细胞凋亡率的检测结果显示,Lenti-shRNA（Gankyrin）-EGFP感染细胞48h后,肝癌SMMC-7721细胞凋亡率明显高于空白对照组、慢病毒对照组,差异有统计学意义（P＜0.05）。3.3 Lenti-shRNA（Gankyrin）-EGFP载体表达系统对体外培养SMMC-7721细胞Gankyrin mRNA水平和Gankyrin蛋白表达影响的检测结果显示,空白对照组、慢病毒对照组之间Gankyrin mRNA和Gankyrin蛋白表达水平差异无统计学意义（P＞0.05）;Lenti-shRNA（Gankyrin）-EGFP感染细胞后,其Gankyrin mRNA水平较前述对照组显著降低,差异有统计学意义（P＜0.05）;Gankyrin蛋白表达水平较对照组亦同样显著降低,差异有统计学意义（P＜0.05）。3.4全部裸鼠接种肝癌细胞系SMMC-7721单细胞悬液后,均存活并有皮下移植瘤形成。3.5荷瘤裸鼠使用Lenti-shRNA（Gankyrin）-EGFP慢病毒表达载体进行瘤体内注射,3w后处死,结果显示Lenti-shRNA（Gankyrin）-EGFP慢病毒表达载体瘤体内注射可显著抑制移植瘤的生长,其移植瘤体积和重量均明显低于对照组移植瘤的体积和重量,差异有显著性意义（P＜0.05）。3.6荷瘤裸鼠移植瘤Gankyrin mRNA水平检测结果显示,Lenti-shRNA（Gankyrin）-EGFP瘤体内注射3w后检测裸鼠移植瘤组织Gankyrin mRNA水平低于对照组,差异有显著性意义（P＜0.05）,Lenti-shRNA（Gankyrin）-EGFP慢病毒载体系统可显著抑制肝细胞癌组织中Gankyrin表达。3.7荷瘤裸鼠移植瘤HE及免疫组化染色结果显示,Lenti-shRNA（Gankyrin）-EGFP瘤体内注射裸鼠移植瘤可抑制肝细胞癌组织中Gankyrin蛋白表达。结论1.本课题成功构建了pET30a（+）-Gankyrin原核表达载体,将该质粒转化大肠杆菌后通过IPTG诱导原核表达的方法获得了His6-tagged-Gankyrin融合重组蛋白。2.以纯化的His6-tagged-Gankyrin融合蛋白为抗原制备的兔抗Gankyrin抗血清,经Western blot检测表明其对His6-tagged-Gankyrin融合重组蛋白和SMMC-7721肝癌细胞中的Gankyrin蛋白具有较好的反应性和特异性。该抗血清可作为免疫反应中的抗体用于检测Gankyrin蛋白的表达。3.利用成功构建的慢病毒穿梭质粒载体plenti6/V5-shRNA（Gankyrin）-EGFP,通过四质粒共转染的策略制备了靶向沉默Gankyrin基因表达的高滴度重组慢病毒载体Lenti-shRNA（Gankyrin）-EGFP,经PCR鉴定和EGFP的活性观察等检测方法,确认该慢病毒载体系统构建正确、滴度高、感染活性高,为下一步开展靶向Gankyrin基因的肝癌基因治疗临床前研究提供了必须的实验工具。4.慢病毒载体系统对于肝癌细胞具有较高的转染效率,是以肝细胞为靶细胞的合适的基因转移载体。利用慢病毒载体系统实现基因转移的治疗技术可成为进行肝癌基因治疗的有效手段。5.本课题中构建的靶向沉默Gankyrin基因慢病毒载体通过RNAi在体外可有效抑制肝癌细胞系SMMC-7721增殖,剔降肝癌SMMC-7721细胞系GankyrinmRNA和Gankyrin蛋白的表达。6.体内注射靶向沉默Gankyrin基因慢病毒载体Lenti-shRNA（Gankyrin）-EGFP可显著抑制裸鼠肝癌SMMC-7721细胞皮下移植瘤的生长速度和移植瘤组织Gankyrin mRNA及蛋白表达水平,该表达系统可能是肝癌治疗的一种有效工具。本课题的创新性在于:1.从基因治疗的角度探讨了慢病毒介导靶向沉默Gankyrin基因表达系统对肝细胞癌的影响及治疗意义,为以Gankyrin基因为靶点进行肝细胞癌的基因治疗研究和相关基因治疗产品最终进入肝细胞癌的临床治疗提供了不可或缺的实验数据;2.为在肝脏通过慢病毒载体介导RNAi的技术手段进行包括肿瘤在内的肝脏相关疾病的基因治疗研究提供了通用的基因治疗策略。3.为利用慢病毒介导基因转移的病毒载体表达系统制造肝脏疾病动物模型的研究奠定了实验基础。更多还原

【Abstract】 BackgroundHepatocullular carcinoma（HCC）,one of the common carcinomas,contributes to the higher morbility and mortality among caner patients because of its highest malignancy.HCC endangers the lives and health of Chinese people because each year the newly developed hepatic cancer patients in the inland of China account for 45 percent of the global total.In current perspective,surgical therapy and liver transplantation are effective,but very few patients are indicative of surgical operations due to high malignancy,fast growth,susceptibility for relapse and metabasis and a great portion of them are tentatively treated with chemotherapy, radiotherapy as well as other alleviafive treatments.Therefore,innovations for hepatic cancer treatments await our medical researchers and doctors in this field.Recently,the developments in oncomolecularbiology and immunology have brought our sight into deep understanding of oncogene.What is more,transgenic technology has been on its track to perfection,which opens a window for gene therapy of hepatic cancer.So far,the recently detected Gankyrin has been found to an oncogene comprehensively activated in HCC.Other findings indicate Gankyrin is over-expressed in the cancer tissues of nearly all HCC patients and participates in the carcinogenesis and development of HCC by way of systematically mediating multiple cell life cycle modulins and transcription factors,and degrading tumor suppression proteins like p53 and p16.As a result,Gankyrin has its clinical prospect when it is used as a target for diagnosis and treatment of HCC.Therefore,we aim at studying the effect of silencing Gankyrin in vivo and in vitro via lentiviral mediation on the biological behaviors of hepatoma carcinoma cell lines and exploring the relevant mechanism so as to build a scientific and experimental basis for its final application in the clinical treatment of hepatic cancer.Aims1.To prepare rabbit-anti-Gankyrin polyclonal antibody used in the quantitative and qualitative detection of Gankyrin expression.2.To construct and prepare lentiviral vectors of target-silenced Gankyrin shRNA for the following in vivo and in vitro experiments on the effectiveness of HCC gene therapy based on Gankyrin expression.3.To explore the effect of lentivirus-mediated target-silenced Gankyrin expression on the bionomics of hepatoma carcinoma cells and construct a theoretical and experimental basis for its use in the clinical treatment of HCC.Methods1.Preparation,purification and identification of Rabbit-anti-Gankyrin polycional antibody.1.1 pET30a（+）-Gankyrin expression vector plasmid was constructed using gene cloning and identification of enzyme digestion.1.2 His6-Gankyrin fusion protein was harvested using IPTG to induce Gankyrin prokaryotic expression.1.3 Rabit-anti-Gankyrin polyclonal antibody was prepared using purified His6-Gankyrin fusion protein as antigen to conduct immune reaction and antibody specificity and reactance were detected.2.Construction and preparation of lentiviral vectors of target-silenced Gankyrin shRNA2.1 Three segments of 19nt oligonucleotide sequence were selected as shRNA segment sequence of targeted Gankyrin based on Gankyrin（NM₀02814） CDS exon,which was used to synthesize three pairs of DNA segments of long primers,to gain three plasmids by way of renaturation,double enzyme digestion as well as connection and inversion with plasmid pDC316-U6 from the same double enzyme digestion.2.2 The gained plasmids were transfected into SMMC-7721.The plasmid most effectively silencing the Gankyrin expression was screened by PT-PCR and Western blot and used to construct lentiviral packaging plasmid of plenti6/V5-shRNA （Gankyrin）-EGFP using gene cloning.2.3 Lenti-shRNA（Gankyrin）-EGFP expression vectors were prepared using quadri-plasmid contransfection and the related quality test was performed.3.The effect of reconstructed lentivirus-mediated target-silenced Gankyrin on the bionomics of hepatoma carcinoma cells3.1 After the Lenti-shRNA（Gankyrin）-EGFP expression vectors were used to infect SMMC-7721,their reactions on the growth inhibition and apoptosis of hepatoma carcinoma cell,Gankyrin mRNA and protein expressions were detected.3.2 SMMC-7721 transplantation tumor model was constructed.After tumor generation,Lenti-shRNA（Gankyrin）-EGFP expression vectors were injected into the tumor.Their reactions on the growth of the tumors transplanted into the cancer-bearing mice as well as on Gankyrin mRNA of transplanted tumor were detected and the morphologic changes of the transplanted tumors were observed.Results1.Preparation,purification and identification of Rabbit-anti-Gankyrin polyclonal antibody.1.1 Enzyme digestion identification showed the constructed pET30a（+）-Gankyrin expression vector plasmids could induce Gankyrin protein via IPTG after inversion into the host of Bacillus coli.1.2 ELISA and Western blot verified better reactivity and specificity of the rabbit-anti-Gankyrin polyclonal antibody prepared from the purified His6-Gankyrin infusion protein as antigen,which met the demands of following experiments.2.Construction and preparation of lentiviral vectors of target-silenced Gankyrin shRNA2.1 The three target-silenced Gankyrin shRNA plasmids were satisfactorily constructed,all carrying CMV promoters for EGFP transcription and U6 promoters for shRNA sequence transcription.By RT-PCR and Western blot,pDC316-EGFP-U6-shRNA （Gankyrin） 1 plasmid knock clown SMMC-7721cells Gankyrin,based on which the pLenti6/V5-shRNA（Gankyrin）-EGFP,the lentiviral packaging plasmid, was accurately constructed.2.2 Detection by PCR and EGFP activity observation demonstrated Lenti-shRNA （Gankyrin）-EGFP with high titer and high infectious activity,was satisfactorily constructed by using quadri-plasmid contransfection system. 3.The effect of reconstructed lentivirus-mediated target silenced Gankyrin on the bionomics of hepatoma carcinoma cells3.1 By MTT,the growth rates of the in vitro cultured SMMC-7721 growth cells were insignificantly different between the blank control group and the lentivirus control group（P＞0.05）,while the differences were significant when MOI at various values was added into Lenti-shRNA（Gankyrin）-EGFP-infected SMMC-7721 cells （P＞0.05）.Lenti-shRNA（Gankyrin）-EGFP inhibited the growth of SMMC-7721 cells（P＜0.05） and the inhibitive escalated with the increase of MOI value until MOI went up to a certain value.3.2 Detection under flow cytometry showed infected by Lenti-shRNA（Gankyrin）-EGFP for 48 hours,the in vitro cultured SMMC-7721 growth cells had higher apoptosis rate than those in the blank control group and in the lentivirus control group,the differences significant statistically（P＜0.05）.3.3 Expressions of Lenti-shRNA（Gankrin）-EGFP on Gankyrin mRNA and Gankyrin protein were of no difference between the blank control and lentivirus control group statistically（P＞0.05）.After SMMC-7721 growth cells were infected,the expressions on Gankyrin mRNA and Gankyrin proteins were both significantly repressed compared to the above-mentioned two control groups（P＜0.05）.3.4 Transplanted with SMMC-7721 cells suspension solution into the athymic mice, the SMMC-7721 cells grew and subcutaneous transplantation tumors formed.3.5 The transplantation tumor-carrying athymic mice received Lenti-shRNA （Gankyrin）-EGFP injection into the subcutaneous transplantation tumors and sacrificed at the third week.The subcutaneous transplantation tumor growth was notably inhibited by Lenti-shRNA（Gankyrin）-EGFP,their sizes and weights significantly smaller and lower than those of the control groups（P＜0.05）.3.6 Three weeks following Lenti-shRNA（Gankyrin）-EGFP injection into the subcutaneous transplantation tumors,Gankyrin mRNA levels were detected to be significantly lower than those at the control groups（P＜0.05）,indicating that Lenti-shRNA（Gankyrin）-EGFP repressed the Gankyrin expressions on liver cell carcinoma cells.3.7 HE and immunohistochemical staining on the transplantation carcinomas from the tumor-carrying mice showed Lenti-shRNA（Gankyrin）-EGFP injected into the transplantation carcinomas repressed the Gankyrin protein expressions on liver cell carcinoma cells.Conclusions1.In this study,pET30a（+）-Gankyrin prokaryotic expression vectors were successfully constructed.The reconstructed His6-tagged-Gankyrin infusion protein was gained by inverting plasmids into Bacillus coli and inducing by IPTG.2.The rabbit anti-Gankyrin antiserum prepared with purified His6-tagged-Gankyrin infusion protein as antigen was detected by Western blot to be reactive and specific against the reconstructed His6-tagged-Gankyrin infusion protein and Gankyrin proteins from SMMC-7721.The antiserum can be used as an antibody for immune response to detect the expressions of Gankyrin protein.3.Lenti-shRNA（Gankyrin）-EGFP with high titer,which was prepared and reconstructed from plenti6/V5-shRNA（Gankyrin）-EGFP contransfected with quadri-plasmid,was detected to be of satisfaction and with high titer and high activity for infection,which provides a necessary experimental basis for further clinical research on targeted Gankyrin gene therapy of hepatic cancer.4.Lenti-shRNA（Gankyrin）-EGFE as a satisfactory gene transfer vector,is effective in transfection of hepatic cancer cells.Gene transfer therapy based on Lenti-shRNA（Gankyrin）-EGFP may be a possibly effective choice for gene therapy of hepatic cancer.5.Lenti-shRNA（Gankyrin）-EGFP may repress the proliferation of SMMC-7721 and knock down inhibit Gankyrin mRNA and Gankyrin expressions in vitro by way of RNA interference（RNAi）.6.Lenti-shRNA（Gankyrin）-EGFP injected into the transplantation tumors can suppress the growth of SMMC-7721-induced subcutaneous transplantation tumors and inhibit the expression Gankyrin mRNA and protein,indicating the lentiviral vector of target-silenced Gankyrin may be a prospective therapy for hepatic cancer.The innovations in this study include:1.From the perspective of gene therapy,we have explored the effect of Lenti-shRNA（Gankyrin）-EGFP from lentivirus-mediated target-silenced Gankyrin on hepatic cellular cancer,as well as the curative significance,which provides necessary experimental data for the Gankyrin-targeted gene therapy of HCC and the products for gene therapy into clinical treatment of HCC.2.This study has suggested a general strategy for gene therapy for liver-involved diseases by lentivirus-mediated RNAi.3.Finally,our study has provided an experimental basis for constructing animal models of hepatic diseases using lentivirus-mediated transgenic viral vectors.更多还原