本文選題：RhoA + 卵巢腫瘤�。� 參考：《廣州醫(yī)科大學(xué)》2014年碩士論文

【摘要】：研究背景 侵襲和遷移是惡性腫瘤轉(zhuǎn)移的重要特征，大約90%的患者死于腫瘤的復(fù)發(fā)和轉(zhuǎn)移。在婦科惡性腫瘤中，卵巢癌死亡率位居首位。因其臨床癥狀隱匿，診斷時75％的患者已屬晚期，雖然超過80%的患者經(jīng)一線治療受益,但I(xiàn)II/IV期卵巢癌患者的5年生存率徘徊在25%-30%。因此，深入了解卵巢癌發(fā)生發(fā)展的分子機(jī)制，并尋求新的治療方法刻不容緩。目前基因治療為卵巢癌的治療提供了新的希望。近年來興起的RNA干擾技術(shù)因具有很強(qiáng)的轉(zhuǎn)錄后基因沉默作用，由于其高效性和特異性在基礎(chǔ)研究方面展現(xiàn)出強(qiáng)大的優(yōu)勢，然而如何將其優(yōu)勢發(fā)揮到臨床靶向治療卻是一個難題。 RhoA基因是Rho家族成員之一，作為惡性腫瘤侵襲及轉(zhuǎn)移的“開關(guān)”基因，參與多種惡性腫瘤的發(fā)生發(fā)展。RhoA基因激活可以導(dǎo)致細(xì)胞癌變，且RhoA信號通路在參與調(diào)控腫瘤的生長和增殖、侵襲和轉(zhuǎn)移、細(xì)胞凋亡及腫瘤新生血管形成等方面發(fā)揮重要作用。因此，以RhoA基因?yàn)榘悬c(diǎn)的基因治療將有望成為卵巢癌靶向治療的一個新方向。 本研究組在前期研究中，已利用RNA干擾技術(shù)成功構(gòu)建了針對RhoA基因的慢病毒干擾載體并建立了卵巢癌穩(wěn)定沉默RhoA基因細(xì)胞株，且體外實(shí)驗(yàn)證實(shí)慢病毒介導(dǎo)RNAi下調(diào)RhoA基因表達(dá)可抑制卵巢癌細(xì)胞增殖、侵襲及遷移能力。由于體外實(shí)驗(yàn)并不能完全真實(shí)的評價對于卵巢癌干預(yù)措施的可行性和有效性，而建立合適的裸鼠移植瘤模型對于模擬卵巢癌的病理狀態(tài)并評價治療措施是必須的，且有著非常重要的作用。故本研究在此基礎(chǔ)上，建立卵巢癌裸鼠移植瘤模型，首先探討RhoA基因沉默對卵巢癌裸鼠腹腔侵襲轉(zhuǎn)移等惡性生物學(xué)行為的影響；其次采用慢病毒介導(dǎo)RhoA shRNA對其卵巢癌裸鼠皮下移植瘤進(jìn)行抗癌治療的研究，以探討其可行性、有效性及抗腫瘤機(jī)制。 第一部分RhoA基因沉默抑制卵巢癌裸鼠腹腔移植瘤惡性生物學(xué)行為的研究 目的構(gòu)建卵巢癌裸鼠腹腔移植瘤模型，研究慢病毒介導(dǎo)RhoA基因沉默對人卵巢癌裸鼠腹腔移植瘤生長、侵襲及轉(zhuǎn)移等惡性生物學(xué)行為的影響。 方法 1. SPF級雌性BALB/C裸鼠21只應(yīng)用隨機(jī)數(shù)字表法分為三組,利用已建立的穩(wěn)定沉默RhoA的HO8910-RhoA-shRNA細(xì)胞，陰性對照組HO8910-RhoA-NC細(xì)胞，和空白對照組HO8910細(xì)胞這3組細(xì)胞分別建立裸鼠卵巢癌腹腔移植瘤模型。 2.每2天測量腹圍，4周后解剖裸鼠。大體觀察：測定腹水量；統(tǒng)計(jì)腫瘤播散器官數(shù)及瘤結(jié)節(jié)數(shù)；稱量瘤體重量，并計(jì)算抑瘤率。 3.鏡下觀察：應(yīng)用HE染色分析移植瘤病理形態(tài)學(xué)特點(diǎn)。 4.實(shí)時熒光定量PCR和Western blot檢測移植瘤RhoA mRNA和蛋白表達(dá)情況。 5.脫氧核苷酸末端轉(zhuǎn)移酶介導(dǎo)的核苷酸缺口末端標(biāo)記（TUNEL）技術(shù)檢測移植瘤凋亡指數(shù)（apoptotic index，AI）。 結(jié)果 1. HO8910-RhoA-NC組和HO8910組的裸鼠成瘤率均為100%，而穩(wěn)定沉默RhoA基因的HO8910-RhoA-shRNA組裸鼠成瘤率僅為71.4%,但差異無統(tǒng)計(jì)學(xué)意義(P=0.231)。 2.與陰性對照HO8910-RhoA-NC組和空白對照HO8910組比較，RhoA基因穩(wěn)定沉默HO8910-RhoA-shRNA組裸鼠腹圍增長明顯滯后（P0.05）；腹水量明顯減少（P=0.01），腫瘤播散器官數(shù)、瘤結(jié)節(jié)數(shù)及瘤體重量均明顯減少（P0.001），抑瘤率達(dá)70.62%； 3.實(shí)時熒光定量PCR及Western Blot結(jié)果顯示，與陰性對照HO8910-RhoA-NC組和空白對照HO8910組比較， HO8910-RhoA-shRNA組移植瘤RhoA基因mRNA和蛋白表達(dá)水平均顯著下降（P0.001）； 4. TUNEL結(jié)果提示，與陰性對照HO8910-RhoA-NC組和空白對照HO8910組比較， HO8910-RhoA-shRNA組移植瘤AI明顯升高（P0.001）。 結(jié)論 卵巢癌細(xì)胞HO8910在腹腔內(nèi)的生長、侵襲及轉(zhuǎn)移均受到RhoA的影響，慢病毒介導(dǎo)RhoA基因穩(wěn)定沉默時，裸鼠腹腔移植瘤的重量、數(shù)量及播散轉(zhuǎn)移均相應(yīng)減少，一定程度上延緩了卵巢癌的惡性生物學(xué)行為。 第二部分靶向RhoA基因的shRNA對卵巢癌裸鼠皮下移植瘤治療的研究及其作用機(jī)制探討 目的探討靶向RhoA基因的shRNA對卵巢癌裸鼠移植瘤的影響及可能的抗腫瘤作用機(jī)制。 方法 1.采用細(xì)胞接種法將人卵巢癌細(xì)胞HO8910接種于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，應(yīng)用隨機(jī)數(shù)字表法分為3組進(jìn)行治療實(shí)驗(yàn)，即實(shí)驗(yàn)組（予攜帶靶向RhoA基因的shRNA慢病毒液治療）、陰性對照組(予攜帶針對隨機(jī)無關(guān)序列的shRNA慢病毒液治療)、空白對照組（予磷酸鹽緩沖液），分別于治療開始第1，5，9,13天進(jìn)行注射，比較3組裸鼠的移植瘤生長情況，包括移植瘤生長速度、腫瘤體積，并繪制移植瘤生長曲線。治療結(jié)束10d后處死裸鼠，剝離腫瘤，稱腫瘤質(zhì)量并計(jì)算抑瘤率，同時留取各組裸鼠腹腔主要臟器。 2.應(yīng)用HE染色，鏡下觀察3組移植瘤組織病理形態(tài)學(xué)特征，并判斷裸鼠腹腔臟器有無轉(zhuǎn)移及慢病毒液的毒性反應(yīng)。 3.采用實(shí)時熒光定量PCR技術(shù)、免疫組化SP法和蛋白印跡法檢測移植瘤組織中RhoAmRNA和蛋白的表達(dá)； 4. TUNEL法檢測3組裸鼠移植瘤細(xì)胞的凋亡情況[以凋亡指數(shù)（AI）表示]。 結(jié)果 1.自治療開始的第9天起，實(shí)驗(yàn)組裸鼠移植瘤的生長速度滯后于陰性對照組和空白對照組，差異均有統(tǒng)計(jì)學(xué)意義(P=0.000)。治療結(jié)束后10d，實(shí)驗(yàn)組裸鼠的移植瘤體積為(338±114) mm3，小于陰性對照組和空白對照組[分別為（1190±332）和（1101±396）mm3]，差異均有統(tǒng)計(jì)學(xué)意義(P分別為0.000、0.001)；實(shí)驗(yàn)組裸鼠平均腫瘤質(zhì)量為（0.23±0.11）g，低于陰性對照組和空白對照組[分別為（0.79±0.19）、（0.74±0.17）g]，差異均有統(tǒng)計(jì)學(xué)意義(P=0.000)。而陰性對照組裸鼠移植瘤的生長速度、移植瘤體積、平均腫瘤質(zhì)量分別與空白對照組比較，差異均無統(tǒng)計(jì)學(xué)意義（P＞0.05）。 2. HE染色光學(xué)顯微鏡下觀察發(fā)現(xiàn)，實(shí)驗(yàn)組腫瘤細(xì)胞呈大片壞死區(qū)域且核固縮多見，而陰性對照組和空白對照組腫瘤細(xì)胞無明顯變化。此外，觀察發(fā)現(xiàn)三組裸鼠腹腔肝、脾、肺及腎組織HE染色結(jié)果顯示形態(tài)正常，且均未見腫瘤細(xì)胞浸潤。 3.實(shí)驗(yàn)組裸鼠移植瘤組織中，RhoA mRNA的表達(dá)水平為(0.30±0.05)，低于陰性對照組的(0.95±0.06)和空白對照組的(1.00±0.11)，差異均有統(tǒng)計(jì)學(xué)意義(P=0.000)；RhoA蛋白的表達(dá)水平為(0.14±0.06)，低于陰性對照組的(0.78±0.14)和空白對照組的(0.75±0.13)，差異均有統(tǒng)計(jì)學(xué)意義(P=0.000)；而陰性對照組裸鼠移植瘤組織中RhoAmRNA和蛋白的表達(dá)水平分別與空白對照組比較，差異均無統(tǒng)計(jì)學(xué)意義（P＞0.05）。 4.實(shí)驗(yàn)組裸鼠移植瘤組織AI為（20.9±3.4）%，高于陰性對照組的（5.2±2.0）%和空白對照組的（6.0±2.1）%，差異均有統(tǒng)計(jì)學(xué)意義(P=0.000)。而陰性對照組裸鼠移植瘤組織中AI與空白對照組比較，差異均無統(tǒng)計(jì)學(xué)意義（P＞0.05）。 結(jié)論 1.通過慢病毒介導(dǎo)靶向沉默RhoA的基因治療有效下調(diào)卵巢癌裸鼠皮下移植瘤組織中RhoA基因的表達(dá)，抑制了卵巢癌裸鼠皮下移植瘤的生長，裸鼠動物體內(nèi)實(shí)驗(yàn)證明RhoA基因沉默治療有效。 2.慢病毒介導(dǎo)靶向沉默RhoA基因抑制卵巢癌生長轉(zhuǎn)移的機(jī)制可能與促進(jìn)細(xì)胞凋亡有關(guān)。 3.動物體內(nèi)試驗(yàn)發(fā)現(xiàn)慢病毒治療對實(shí)驗(yàn)動物未見明顯的毒性，是一種比較安全可靠的治療方法，有助于卵巢癌抗癌治療策略的設(shè)計(jì)，，為卵巢癌新型的基因靶向藥物研發(fā)提供了體內(nèi)實(shí)驗(yàn)依據(jù)。
[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.
【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.31

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相關(guān)期刊論文 前4條

1 薛妍,畢鋒,劉娜,潘陽林,時永全,張學(xué)庸;Rho GTPases對腫瘤血管生成相關(guān)分子的作用[J];中國生物化學(xué)與分子生物學(xué)報(bào);2004年05期

2 李娟;唐良萏;湯為學(xué);王輝;Shu-Wing Ng;;RhoA在上皮性卵巢癌細(xì)胞中的表達(dá)與侵襲性的相關(guān)性[J];細(xì)胞生物學(xué)雜志;2008年04期

3 林英姿;陳仁;彭江龍;潘婉;陳錦龍;盧偉英;;RhoA在卵巢癌組織中的表達(dá)及意義[J];醫(yī)學(xué)綜述;2008年11期

4 楊文娟;康佳麗;王小霞;劉啟才;王冬昱;賈菡;;慢病毒介導(dǎo)RhoA基因沉默對卵巢癌細(xì)胞侵襲和遷移的影響[J];中國腫瘤生物治療雜志;2012年06期

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