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

发布时间：2018-06-04 22:44

本文选题：RhoA + 卵巢肿瘤　；参考：《广州医科大学》2014年硕士论文

【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。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（P0.05）；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（P0.001），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（P0.001）； 4. TUNEL结果提示，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AI明显升高（P0.001）。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10d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RhoAmRNA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空白对照组，差异均有统计学意义(P=0.000)。治疗结束后10d，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RhoAmRNA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。
[Abstract]:Research background
Invasion and migration are an important feature of malignant tumor metastasis. About 90% of patients die of tumor recurrence and metastasis. In gynecologic malignancies, ovarian cancer mortality ranks the first. 75% of the patients are advanced at the time of diagnosis, although more than 80% of the patients benefit from one line treatment, but for 5 years of III/IV ovarian cancer patients. The survival rate is hovering in the 25%-30%., so it is urgent to understand the molecular mechanism of ovarian cancer development and seek new treatment methods. Currently, gene therapy provides new hope for the treatment of ovarian cancer. In recent years, the emerging RNA interference technology has a strong post transcriptional gene silencing effect, due to its high efficiency and specificity in the base. There is a strong advantage in basic research, but how to bring its advantages into clinical targeted therapy is a difficult problem.
RhoA gene is one of the members of the Rho family. As a "switch" gene for malignant tumor invasion and metastasis, the activation of.RhoA gene in the occurrence and development of a variety of malignant tumors can lead to cell carcinogenesis, and the RhoA signaling pathway is involved in the regulation of tumor growth and proliferation, invasion and metastasis, cell apoptosis and neovascularization of tumor. Therefore, gene therapy targeting RhoA gene is expected to become a new direction for targeted therapy of ovarian cancer.
In the previous study, the RNA interfering technology has been used to successfully construct a RhoA gene for the lentivirus interference vector and to establish a stable and silent RhoA gene cell line for ovarian cancer. In vitro experiments have proved that the lentivirus mediated down regulation of RhoA gene expression by RNAi can inhibit the proliferation, invasion and migration of ovarian cancer cells. It is not completely true to evaluate the feasibility and effectiveness of the ovarian cancer intervention, and it is necessary to establish a suitable model of nude mice transplantation tumor to simulate the pathological state of ovarian cancer and to evaluate the treatment measures. Therefore, on the basis of this study, the model of xenograft in nude mice of ovarian cancer is established, and Rho is first discussed. The effect of A gene silencing on the invasion and metastasis of ovarian cancer in nude mice and other malignant biological behaviors, followed by the use of lentivirus mediated RhoA shRNA to study the anticancer treatment of subcutaneous transplanted tumor in nude mice of ovarian cancer, in order to explore its feasibility, effectiveness and anti-tumor mechanism.
Part 1 RhoA gene silencing inhibits malignant biological behavior of ovarian cancer xenografts in nude mice
Objective to construct an intraperitoneal tumor model in nude mice of ovarian cancer, and to study the effect of lentivirus mediated RhoA gene silencing on the growth, invasion and metastasis of human ovarian cancer in nude mice.
Method
1. SPF female BALB/C nude mice were divided into three groups by using random digital table method, using the established HO8910-RhoA-shRNA cells with stable silent RhoA, negative control group HO8910-RhoA-NC cells, and HO8910 cells in blank control group, respectively, to establish the abdominal xenograft tumor model of ovarian cancer in nude mice respectively.
2. the abdominal circumference was measured every 2 days and the nude mice were dissected after 4 weeks. Gross observation: the amount of ascites, the number of organs scattered and the number of nodules; weighing the tumor body weight, and calculating the tumor suppressor rate.
3. microscopic observation: HE staining was used to analyze the pathomorphological characteristics of transplanted tumor.
4. real-time fluorescence quantitative PCR and Western blot were used to detect RhoA mRNA and protein expression in transplanted tumors.
The apoptotic index (AI) was detected by 5. deoxynucleotidyl transferase mediated nick end labeling (TUNEL) technique.
Result
The tumorigenesis rate of nude mice in group 1. HO8910-RhoA-NC and HO8910 group was 100%, but the tumor rate of nude mice with stable silent RhoA gene was only 71.4%, but the difference was not statistically significant (P=0.231).
2. compared with the negative control HO8910-RhoA-NC group and the blank control HO8910 group, the abdominal circumference growth of the HO8910-RhoA-shRNA group was significantly lagged (P0.05), the amount of ascites decreased significantly (P=0.01), the number of tumor disseminated organs, the number of nodules and the weight of the tumor decreased significantly (P0.001), and the tumor suppressor rate was up to 70.62%.
3. the results of real-time fluorescence quantitative PCR and Western Blot showed that the level of mRNA and protein expression of the RhoA gene in the transplanted tumor of HO8910-RhoA-shRNA group decreased significantly compared with the negative control group and the blank control group HO8910 group (P0.001).
4. TUNEL results showed that compared with the negative control group HO8910-RhoA-NC and the blank control HO8910 group, the AI in the HO8910-RhoA-shRNA group increased significantly (P0.001).
conclusion
The growth, invasion and metastasis of HO8910 in ovarian cancer cells are affected by RhoA. When the lentivirus mediates the stable silence of the RhoA gene, the weight, quantity and dissemination of the transplanted tumor in nude mice decrease correspondingly. To some extent, the malignant biological line of ovarian cancer is delayed.
The second part is the study of shRNA targeting RhoA gene in the treatment of ovarian cancer xenografts in nude mice and its mechanism.
Objective to investigate the effect of shRNA targeting RhoA gene on ovarian cancer xenografts in nude mice and its possible anti-tumor mechanism.
Method
1. the human ovarian cancer cell HO8910 was inoculated into the armpit of the right forelimb of 18 female BALB/C nude mice to establish a nude mouse axillary transplantation tumor model. After the nude mice model was built, the randomized digital table was used to divide the model into 3 groups for the treatment experiment, that is, the experimental group (given the target RhoA based shRNA lentivirus solution) and the negative control group. With the shRNA lentivirus solution for random unrelated sequences, the blank control group (given phosphate buffer solution) was injected into the 3 groups of nude mice at the first 1,5,9,13 day to compare the growth of nude mice, including the growth rate of the tumor, the volume of the tumor, and the growth curve of the transplanted tumor. After the treatment of 10d, the nude mice were killed and swollen. The tumor was called tumor mass and the tumor inhibition rate was calculated.
2. HE staining was used to observe the histopathological characteristics of the 3 groups of transplanted tumors, and to determine whether there was metastasis in the abdominal viscera and the toxic reaction of lentiviral fluid in nude mice.
3. the expression of RhoAmRNA and protein in transplanted tumor tissues was detected by real-time quantitative PCR, immunohistochemistry and SP blotting.
4. TUNEL assay was used to detect the apoptosis of 3 groups of transplanted tumor cells in nude mice [apoptosis index (AI)].
Result
1. the growth rate of the transplanted tumor in the experimental group lags behind the negative control group and the blank control group on the ninth day of the beginning of the treatment. The difference is statistically significant (P=0.000). After the treatment, the volume of the transplanted tumor in the experimental group is (338 + 114) mm3, less than that of the negative control group and the blank control group [1190 + 332) and (1101 + 396) mm3] respectively. The difference was statistically significant (P 0.000,0.001), and the average mass of tumor in the experimental group was (0.23 + 0.11) g, which was lower than that in the negative control group and the blank control group (0.79 + 0.19) and (0.74 + 0.17) g] respectively. The difference was statistically significant (P=0.000). The growth rate, the volume of transplanted tumor and the average tumor mass in nude mice were negative. The difference was not statistically significant compared with the blank control group (P > 0.05).
2. HE staining optical microscope observed that the tumor cells in the experimental group showed large area of necrosis and the nuclear pyknosis was more common, but there was no obvious change in the tumor cells in the negative control group and the blank control group. In addition, the results of the three groups of nude mice abdominal liver, spleen, lung and kidney tissue were found to be normal, and no tumor cell infiltration was found.
3. in nude mice, the expression level of RhoA mRNA was (0.30 + 0.05), which was lower than that of negative control group (0.95 + 0.06) and blank control group (1 + 0.11), and the difference was statistically significant (P=0.000); the expression level of RhoA protein was (0.14 + 0.06), lower than that of negative control group (0.78 + 0.14) and blank control group (0.75 + 0.13), poor The difference was statistically significant (P=0.000), but the expression level of RhoAmRNA and protein in the transplanted tumor tissues of the negative control group was no significant difference compared with the blank control group (P > 0.05).
The AI of nude mice in 4. experimental group was (20.9 + 3.4)%, higher than that of negative control group (5.2 + 2)% and (6 + 2.1)% in blank control group. The difference was statistically significant (P=0.000), but there was no significant difference between AI and blank control group in nude mice (P > 0.05).
conclusion
1. the gene therapy of the target silencing RhoA mediated by lentivirus can effectively reduce the expression of RhoA gene in the subcutaneous transplanted tumor tissues of the ovarian cancer nude mice and inhibit the growth of the subcutaneous xenografts in nude mice. The experiment in nude mice proved that the RhoA gene silencing therapy was effective.
2. lentivirus mediated silencing of RhoA gene may inhibit the growth and metastasis of ovarian cancer.
3. in vivo experiment in animals found that the treatment of lentivirus has no obvious toxicity to experimental animals. It is a safe and reliable treatment method, which is helpful to the design of the antitumor treatment strategy of ovarian cancer and provides the experimental basis for the development of new type of gene targeting drug for ovarian cancer.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

【参考文献】