慢病毒介导RNAi沉默RhoA基因对人卵巢癌裸鼠移植瘤生长的影响和基因治疗研究

目录中文摘要 (1)中文摘要 (6)英文缩略语一览表 (11)前言 (12)第一部分 RhoA基因沉默抑制卵巢癌裸鼠腹腔移植瘤恶性生物学行为的研究14一、材料和方法 (14)二、结果 (27)三、讨论 (33)四、结论 (35)第二部分靶向RhoA基因的shRNA对卵巢癌裸鼠皮下移植瘤治疗的研究及其作用机制探讨 (36)一、材料及方法 (36)二、结果 (39)三、讨论 (45)四、结论 (48)展望 (49)参考文献 (50)攻读硕士学位期间发表论文及参与科研活动 (64)致谢 (65)学位论文原创性声明 (66)学位论文知识产权权属声明 (66)关于学位论文使用授权的说明 (66)中文摘要慢病毒介导RNAi沉默RhoA基因对人卵巢癌裸鼠移植瘤生长的影响及基因治疗研究中文摘要研究背景侵袭和迁移是恶性肿瘤转移的重要特征，大约90%的患者死于肿瘤的复发和转移。

在妇科恶性肿瘤中，卵巢癌死亡率位居首位。

因其临床症状隐匿，诊断时75％的患者已属晚期，虽然超过80%的患者经一线治疗受益,但III/IV期卵巢癌患者的5年生存率徘徊在25%-30%。

因此，深入了解卵巢癌发生发展的分子机制，并寻求新的治疗方法刻不容缓。

目前基因治疗为卵巢癌的治疗提供了新的希望。

近年来兴起的RNA干扰技术因具有很强的转录后基因沉默作用，由于其高效性和特异性在基础研究方面展现出强大的优势，然而如何将其优势发挥到临床靶向治疗却是一个难题。

RhoA基因是Rho家族成员之一，作为恶性肿瘤侵袭及转移的“开关”基因，参与多种恶性肿瘤的发生发展。

RhoA基因激活可以导致细胞癌变，且RhoA信号通路在参与调控肿瘤的生长和增殖、侵袭和转移、细胞凋亡及肿瘤新生血管形成等方面发挥重要作用。

因此，以RhoA基因为靶点的基因治疗将有望成为卵巢癌靶向治疗的一个新方向。

本研究组在前期研究中，已利用RNA干扰技术成功构建了针对RhoA基因的慢病毒干扰载体并建立了卵巢癌稳定沉默RhoA基因细胞株，且体外实验证实慢病毒介导RNAi下调RhoA基因表达可抑制卵巢癌细胞增殖、侵袭及迁移能力。

由于体外实验并不能完全真实的评价对于卵巢癌干预措施的可行性和有效性，而建立合适的裸鼠移植瘤模型对于模拟卵巢癌的病理状态并评价治疗措施是必须的，且有着非常重要的作用。

故本研究在此基础上，建立卵巢癌裸鼠移植瘤模型，首先探讨RhoA基因沉默对卵巢癌裸鼠腹腔侵袭转移等恶性生物学行为的影响；其次采用慢病毒介导RhoA shRNA对其卵巢癌裸鼠皮下移植瘤进行抗癌治疗的研究，以探讨其可行性、有效性及抗肿瘤机制。

广州医科大学硕士学位论文第一部分RhoA基因沉默抑制卵巢癌裸鼠腹腔移植瘤恶性生物学行为的研究目的构建卵巢癌裸鼠腹腔移植瘤模型，研究慢病毒介导RhoA基因沉默对人卵巢癌裸鼠腹腔移植瘤生长、侵袭及转移等恶性生物学行为的影响。

方法1.SPF级雌性BALB/C裸鼠21只应用随机数字表法分为三组,利用已建立的稳定沉默RhoA的HO8910-RhoA-shRNA细胞，阴性对照组HO8910-RhoA-NC 细胞，和空白对照组HO8910细胞这3组细胞分别建立裸鼠卵巢癌腹腔移植瘤模型。

2.每2天测量腹围，4周后解剖裸鼠。

大体观察：测定腹水量；统计肿瘤播散器官数及瘤结节数；称量瘤体重量，并计算抑瘤率。

3.镜下观察：应用HE染色分析移植瘤病理形态学特点。

4.实时荧光定量PCR和Western blot检测移植瘤RhoA mRNA和蛋白表达情况。

5.脱氧核苷酸末端转移酶介导的核苷酸缺口末端标记（TUNEL）技术检测移植瘤凋亡指数（apoptotic index，AI）。

结果1.HO8910-RhoA-NC组和HO8910组的裸鼠成瘤率均为100%，而稳定沉默RhoA基因的HO8910-RhoA-shRNA组裸鼠成瘤率仅为71.4%,但差异无统计学意义(P=0.231)。

2.与阴性对照HO8910-RhoA-NC组和空白对照HO8910组比较，RhoA基因稳定沉默HO8910-RhoA-shRNA组裸鼠腹围增长明显滞后（P<0.05）；腹水量明显减少（P=0.01），肿瘤播散器官数、瘤结节数及瘤体重量均明显减少（P<0.001），抑瘤率达70.62%；3.实时荧光定量PCR及Western Blot结果显示，与阴性对照HO8910-RhoA-NC组和空白对照HO8910组比较，HO8910-RhoA-shRNA组移植瘤RhoA基因mRNA 和蛋白表达水平均显著下降（P<0.001）；4.TUNEL结果提示，与阴性对照HO8910-RhoA-NC组和空白对照HO8910组2中文摘要比较，HO8910-RhoA-shRNA组移植瘤AI明显升高（P<0.001）。

结论卵巢癌细胞HO8910在腹腔内的生长、侵袭及转移均受到RhoA的影响，慢病毒介导RhoA基因稳定沉默时，裸鼠腹腔移植瘤的重量、数量及播散转移均相应减少，一定程度上延缓了卵巢癌的恶性生物学行为。

第二部分靶向RhoA基因的shRNA对卵巢癌裸鼠皮下移植瘤治疗的研究及其作用机制探讨目的探讨靶向RhoA基因的shRNA对卵巢癌裸鼠移植瘤的影响及可能的抗肿瘤作用机制。

方法1.采用细胞接种法将人卵巢癌细胞HO8910接种于18只雌性BALB/C裸鼠右前肢腋下建立裸鼠腋下移植瘤模型。

荷瘤裸鼠模型建成后，应用随机数字表法分为3组进行治疗实验，即实验组（予携带靶向RhoA基因的shRNA慢病毒液治疗）、阴性对照组(予携带针对随机无关序列的shRNA慢病毒液治疗)、空白对照组（予磷酸盐缓冲液），分别于治疗开始第1，5，9,13天进行注射，比较3组裸鼠的移植瘤生长情况，包括移植瘤生长速度、肿瘤体积，并绘制移植瘤生长曲线。

治疗结束10 d后处死裸鼠，剥离肿瘤，称肿瘤质量并计算抑瘤率，同时留取各组裸鼠腹腔主要脏器。

2.应用HE染色，镜下观察3组移植瘤组织病理形态学特征，并判断裸鼠腹腔脏器有无转移及慢病毒液的毒性反应。

3.采用实时荧光定量PCR技术、免疫组化SP法和蛋白印迹法检测移植瘤组织中RhoA mRNA和蛋白的表达；4.TUNEL法检测3组裸鼠移植瘤细胞的凋亡情况[以凋亡指数（AI）表示]。

结果1.自治疗开始的第9天起，实验组裸鼠移植瘤的生长速度滞后于阴性对照组和广州医科大学硕士学位论文空白对照组，差异均有统计学意义(P=0.000)。

治疗结束后10 d，实验组裸鼠的移植瘤体积为(338±114) mm3，小于阴性对照组和空白对照组[分别为（1190±332）和（1101±396）mm3]，差异均有统计学意义(P分别为0.000、0.001)；实验组裸鼠平均肿瘤质量为（0.23±0.11）g，低于阴性对照组和空白对照组[分别为（0.79±0.19）、（0.74±0.17）g]，差异均有统计学意义(P=0.000)。

而阴性对照组裸鼠移植瘤的生长速度、移植瘤体积、平均肿瘤质量分别与空白对照组比较，差异均无统计学意义（P＞0.05）。

2.HE染色光学显微镜下观察发现，实验组肿瘤细胞呈大片坏死区域且核固缩多见，而阴性对照组和空白对照组肿瘤细胞无明显变化。

此外，观察发现三组裸鼠腹腔肝、脾、肺及肾组织HE染色结果显示形态正常，且均未见肿瘤细胞浸润。

3.实验组裸鼠移植瘤组织中，RhoA mRNA的表达水平为(0.30±0.05)，低于阴性对照组的(0.95±0.06)和空白对照组的(1.00±0.11)，差异均有统计学意义(P=0.000)；RhoA蛋白的表达水平为(0.14±0.06)，低于阴性对照组的(0.78±0.14)和空白对照组的(0.75±0.13)，差异均有统计学意义(P=0.000)；而阴性对照组裸鼠移植瘤组织中RhoA mRNA和蛋白的表达水平分别与空白对照组比较，差异均无统计学意义（P＞0.05）。

4.实验组裸鼠移植瘤组织AI为（20.9±3.4）%，高于阴性对照组的（5.2±2.0）%和空白对照组的（6.0±2.1）%，差异均有统计学意义(P=0.000)。

而阴性对照组裸鼠移植瘤组织中AI与空白对照组比较，差异均无统计学意义（P＞0.05）。

结论1.通过慢病毒介导靶向沉默RhoA的基因治疗有效下调卵巢癌裸鼠皮下移植瘤组织中RhoA基因的表达，抑制了卵巢癌裸鼠皮下移植瘤的生长，裸鼠动物体内实验证明RhoA基因沉默治疗有效。

2.慢病毒介导靶向沉默RhoA基因抑制卵巢癌生长转移的机制可能与促进细胞凋亡有关。

3.动物体内试验发现慢病毒治疗对实验动物未见明显的毒性，是一种比较安全可靠的治疗方法，有助于卵巢癌抗癌治疗策略的设计，为卵巢癌新型的基因靶向药物研发提供了体内实验依据。

4中文摘要关键词RhoA；卵巢肿瘤；慢病毒载体；异种移植模型抗肿瘤试验；肿瘤侵润；基因治疗；凋亡广州医科大学硕士学位论文Effect and mechanism of lentivirusmediated silence of RhoA gene on the growth of ovarian cancer xenograft in vivoABSTRACTBACKGROUNDInvasion and migration are the important characteristics of the metastasis of malignant tumor, account for about 90% of all ovarian carcinomas patients died from the tumor recurrence and metastasis. Ovarian cancer is one of the most severe gynecological malignancies known and has now become the most lethal of all gynecological malignancies. Because of hidden clinical symptoms, 75% of patients has been in later period when diagnosis. Although more than 80% of the patients has received benefit after first-line treatment, whereas the 5-year survival rate of patients with stage III/IV ovarian cancer is between25% and 30%.Therefore, to understand deeply the molecular mechanism of the the occurrence and development of ovarian cancer and seek new treatment methods are urgent.Current gene therapy has provided a new hope for the treatment of ovarian cancer. In recent years the rise of RNA interference technology which has strong gene silencing effect after transcriptional. Due to its high efficiency and specificity show a strong advantage in basic research. However,how to exert the most advantages of clinical targeted therapy will still be a problem.RhoA gene is one of the members of the Rho protein family, which acted as a "switch" gene of invasion and metastasis of malignant tumor is involved in metastasis of many malignances. The activation of RhoA gene may cause cells transform to become cancer. Recent study indicate that RhoA is associated with the movement and shape of the cell,cell growth and proliferation,invasion and migration, cell apoptosis and Tumor angiogenesis formation. Therefore, gene therapy that target RhoA gene is expected to become a new direction in the therapy targeting ovarian cancer.6英文摘要In our previous study, we has successfully cloned a novel RhoA gene stable down-regulation cell line by using lentivirus mediated gene silencing technology called RNA interference. Reduce expression of RhoA gene by lentivirus vector mediated RNAi may inhibit the proliferation, invasion and migration ability of ovarian cancer cell. Because of evaluating the feasibility and effectiveness of interventions for ovarian cancer in vitro experiment is not entirely true，to establish a nude mice transplantation tumor model for simulation of the ovarian cancer pathology and evaluating treatment is necessary and vital. Therefore, on the basis of precious study, ovarian cancer nude mice transplantation tumor model was built to expore the inhibitory effects of RhoA gene silence on malignant biological behaviors of human ovarian cancer in nude mice intraperitoneal xenograft, and expore the effect and mechanism of gene therapy of lentivirus mediated RhoA shRNA on ovarian cancer in nude mice subcutaneous xenograft in vivo.Chapter I Inhibitory effects of RhoA gene Silence on malignant biological behaviors of human ovarian cancer in nude mice intraperitoneal xenograftOBJECTIVE:To establish intraperitoneal xenograft model of human ovarian cancer and investigate effects of RhoA gene silence on the malignant biological behaviors of ovarian xenograft in nude mice in vivo.METHODS:1.21 female nude mice were randomly assigned to three groups:HO8910-RhoA-shRNA group, HO8910-RhoA-NC group and HO8910 group. The stable knockdown of RhoA cell line, negative control cell line and ovarian cancer cell line HO8910 were inoculated respectively into abdominal cavity of each group to establish intraperitoneal xenograft model of human ovarian cancer.2.Abdominal circumference were recorded every two days, the nude mice weresacrificed four weeks post-inoculation. The ascitic volume, dissemination position广州医科大学硕士学位论文number, disseminated tumor number, tumor weight and tumor growth inhibition rate were recorded.3.Histopathological analysis for xenograft tissues were observed by HE staining.4.The expression of RhoA mRNA and protein of xenograft tissues were detectedrespectively by real-time qPCR and Western blot.5.Cell apoptosis in tumor tissues were observed by TUNEL method and apoptoticindex (AI) were counted.RESULTS:1.Abdominal circumference in HO8910-RhoA-shRNA group growth delay werestatistically significant (P<0.05).2.The HO8910-RhoA-shRNA group had decreased in ascitic volume (P=0.01),dissemination position number (P<0.001), disseminated tumor number (P<0.001) and tumor weight (P<0.001), with tumor growth inhibition rate of 70.62%.3.The relative expression of RhoA mRNA and protein were both significantlydecreased in HO8910-RhoA-shRNA group (P<0.001).4.AI were significantly increased in HO8910-RhoA-shRNA group (P<0.001). CONCLUSION: RhoA gene silenced by Lentivirus-mediated RNAi can significantly suppress the malignant biological behaviors of human ovarian cancer in nude mice xenograft.Chapter II Effect and mechanism of gene therapy of lentivirus mediated RhoA shRNA on ovarian cancer xenograft in vivoOBJECTIVE: To investigate treatment effect of lentivirus mediated RhoA shRNA on xenograft tumor of ovarian cancer in nude mice in vivo and underlying mechanism.METHODS:1.Human ovarian cancer cell line HO8910 were inoculated to establishsubcutaneous xenograft model of human ovarian cancer. Tumor-bearing nudemice were abandonly assigned to three groups: Lenti-RhoA-sh group,8英文摘要Lenti-NC( Negative control）group and PBS（Phosphate Buffered Saline）group;lentivirus mediated RhoA shRNA, negative control lentivirus and PBS wererespectively injected in the three groups. Effects of treatment were observed by tumor growth curve, tumor volume ，tumor weight, and tumor inhibition rate. 2.Xenograft tissues and liver, spleen, lung, and renal tissues were histopathologicalanalysis by HE staining.3.The changes of RhoA gene expression in xenograft tissues after lentivirusmediated RhoA shRNA treated were detected by real-time qPCR,immunohischemistry and Western blot assay.4.Cell apoptosis in xenograft tissues were examined by TUNEL method andapoptotic index (AI) were counted.RESULTS:pared with Lenti-NC group and PBS group, the growth speed of xenograft inLenti-RhoA-sh group delayed significantly from post-injection 9 days（P=0.000）.Tumor volume (338±114) mm3 statistically significant decreased in theLenti-RhoA-sh group when compared with Lenti-NC group（1190±332）mm3 and PBS group（1101±396）mm3 (P=0.000,P=0.001).Tumor weight (0.23±0.11）g statistically significant decreased in the Lenti-RhoA-sh group when comparedwith Lenti-NC group（0.79±0.19）g and PBS group（0.74±0.17）g （P=0.000）.2.Real-time qPCRresult displayed that the expression of RhoA mRNA（0.30±0.05）statistically significant decreased in the Lenti-RhoA-sh group compared withLenti-NC group (0.95±0.06) and PBS group(1.00±0.11)(P=0.000) .Western blot result displayed that the expression of RhoA protein (0.14±0.06 ) statisticallysignificant decreased in the Lenti-RhoA-sh group compared with Lenti-NCgroup(0.78±0.14) and PBS group (0.75±0.13)3.TUNEL staining displayed that AI significantly increased in theLenti-RhoA-sh(（20.9±3.4）% compared with Lenti-NC group（5.2±2.0）% and PBS group（6.0±2.1）%（P=0.000）.CONCLUSIONS:1.Lentivirus mediated RhoA shRNA may effectively down-regulate of theexpression of RhoA in xenograft, inhibit the growth of subcutaneous xenograft tumor of ovarian cancer in nude mice in vivo广州医科大学硕士学位论文2.The underlying mechanism of treatment of Lentivirus mediated RhoA shRNA inhuman ovarian cancer xenograge in vivo is by the means of increasing the cell apoptosis.3.No obvious toxicity in lentivirus mediated gene therapy was found in animalexpresiments in vivo, it indicates lentivirus mediated gene therapy may be arelatively safe treatment.KEY WORDS：RhoA; Ovarian neoplasms；Lentivirus vector；Xenograft model antitumor assays；Tumor invasion; Gene therapy; Apoptosis英文缩略语一览表英文缩略语一览表英文缩写英文全文中文RhoA Ras homologue member A Ras超家族成员A RNAi RNA interference RNA干扰PBS Phosphate buffered saline 磷酸盐缓冲液shRNA Short hairpin RNA 短发夹RNART-qPCR Real-time quantitative Polymerase ChainReaction 实时荧光定量聚合酶链式反应HRP Horseradish peroxidase 辣根过氧化物酶MOI Multiple of infection 感染复数SDS Sodium dodecyl sulfate 十二烷基硫酸钠PAGE Polyacrylamide gel electrophoresis 聚丙烯酰胺凝胶电泳DEPC Diethlpyrocarbonate 焦炭酸二乙酯CBB-R250 Coomassie Brilliant Biue R250 考马斯亮蓝-R250 DMSO Dimethyl sulfoxide 二甲基亚砜LV Lentivirus based vector 慢病毒载体TUNEL Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nickend labeling 脱氧核苷酸末端转移酶介导的核苷酸缺口末端标记EOC Epithelial Ovarian Cancer 上皮性卵巢癌DAB Diaminobenzidine二氨基联苯胺ECL Enhanced chemiluminescence , 增强化学发光SPF Specefic pathogen Free 无特定病原体TMEMD Tetramethylenediamine 四甲基乙二胺PVDF Polyvinylidene difluoride 聚偏二氟乙烯Tris Tris(hydroxymethyl) aminomethane 三(羟甲基)氨基甲烷EDTA Ethylene diamine tetracetic acid 乙二胺四乙酸AI Apoptotic index 凋亡指数广州医科大学硕士学位论文前言侵袭和迁移是恶性肿瘤转移的重要特征，大约90%的患者死于肿瘤的复发和转移[1]。