本文關(guān)鍵詞： 解聚素-金屬蛋白酶17 乳腺癌 RNA干擾 基因治療　出處：《華北理工大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的利用人MCF-7乳腺癌移植型裸小鼠模型,探討解聚素-金屬蛋白酶17-sh RNA(ADAM17-sh RNA)的體內(nèi)抑瘤效果。方法常規(guī)培養(yǎng)人MCF-7乳腺癌細(xì)胞,用ADAM17-sh RNA轉(zhuǎn)染MCF-7細(xì)胞,利用激光共聚焦檢測慢病毒Lentivirus-ADAM17-sh RNA-RFP介導(dǎo)的RFP在MCF-7中的表達(dá)情況。30只裸小鼠隨機(jī)分為對(duì)照組(接種正常MCF-7細(xì)胞)、無意義序列組(接種轉(zhuǎn)染空載體的MCF-7乳腺癌細(xì)胞)和轉(zhuǎn)染組(接種轉(zhuǎn)染ADAM17-sh RNA的人MCF-7乳腺癌細(xì)胞)。制備實(shí)驗(yàn)所需三組細(xì)胞懸液,0.2 ml/只(5×107/ml)種植在裸小鼠右側(cè)鼷部皮下。種植后約第10天待接種部位出現(xiàn)明顯的腫瘤結(jié)節(jié),結(jié)節(jié)質(zhì)地較硬后,每4天一次測量腫瘤瘤體大小,并觀察裸小鼠的一般情況,種植后第26天測量后處死所有動(dòng)物。HE染色觀察三組細(xì)胞的形態(tài)變化;免疫組化法檢測ADAM17、Ki-67兩種蛋白在三組腫瘤中的表達(dá)情況;Western blotting法檢測ADAM17蛋白在三組中的表達(dá)。結(jié)果激光共聚焦檢測顯示慢病毒介導(dǎo)的ADAM17-shRNA可成功轉(zhuǎn)入到MCF-7細(xì)胞內(nèi),轉(zhuǎn)染率達(dá)80%。30只裸小鼠全部成瘤,成瘤率達(dá)到100%(30/30);種植10天后可見裸小鼠瘤體表面皮膚呈粉紅色,腫瘤局限隆起于皮膚表面,形狀不規(guī)則,呈分葉狀,邊界清楚,質(zhì)韌,活動(dòng)度差;觀察發(fā)現(xiàn)無意義序列組和對(duì)照組的裸小鼠腫瘤明顯大于轉(zhuǎn)染組,無意義序列組和對(duì)照組裸小鼠進(jìn)食明顯減少、消瘦嚴(yán)重,轉(zhuǎn)染組裸小鼠覓食、飲水、活動(dòng)等狀況良好。實(shí)驗(yàn)結(jié)束時(shí)轉(zhuǎn)染組、對(duì)照組和無意義序列組的移植瘤體積分別為241.96±17.14 mm3、609.50±15.13mm3和611.40±15.97 mm3,三組之間差異有統(tǒng)計(jì)學(xué)意義(F=1765.96、P0.001),與對(duì)照組和無意義序列組比較,轉(zhuǎn)染組的最大抑瘤率分別為63.11%、63.26%。HE染色鏡下可見,三組移植瘤呈典型的人乳腺浸潤性導(dǎo)管癌的特征,乳腺癌細(xì)胞呈條索狀分布,浸潤間質(zhì),間質(zhì)伴出血癥狀;轉(zhuǎn)染組相比于其他兩組,移植瘤組織出現(xiàn)局部大片壞死區(qū),壞死區(qū)細(xì)胞崩解,細(xì)胞結(jié)構(gòu)消失等情況明顯有所改善。免疫組織化學(xué)結(jié)果顯示:ADAM17蛋白陽性表達(dá)于胞漿,轉(zhuǎn)染組移植瘤染色指數(shù)評(píng)分為2.65±0.49,其與對(duì)照組(7.17±0.27)和無意義序列組(7.11±0.21)比較,差異具有統(tǒng)計(jì)學(xué)意義(F=174.12,P0.001);Ki-67蛋白陽性表達(dá)于胞核,轉(zhuǎn)染組瘤染色指數(shù)評(píng)分為3.76±0.23,其與對(duì)照組(9.05±0.34)和無意義序列組(8.94±0.42)比較,差異具有統(tǒng)計(jì)學(xué)意義(F=490.93,P0.001)。Western-blotting結(jié)果顯示轉(zhuǎn)染組ADAM17蛋白OD值為0.367±0.034,對(duì)照組為0.611±0.037,無意義序列組為0.618±0.028,轉(zhuǎn)染組的ADAM17蛋白的水平明顯低于無意義序列組和對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(F=56.23,P0.001)。結(jié)論轉(zhuǎn)染ADAM17-shRNA的人MCF-7乳腺癌細(xì)胞在裸小鼠體內(nèi)的生長受到了明顯地抑制。
[Abstract]:Objective to investigate the inhibitory effect of depolymerin-metalloproteinase-17-sh RNA(ADAM17-sh on human MCF-7 breast cancer xenografted nude mice. Methods Human MCF-7 breast cancer cells were cultured routinely and transfected with ADAM17-sh RNA into MCF-7 cells. The expression of RFP mediated by lentivirus Lentivirus-ADAM17-sh RNA-RFP in MCF-7 was detected by laser confocal focusing. Thirty nude mice were randomly divided into control group (inoculated with normal MCF-7 cells), meaningless sequence group (inoculated with empty vector MCF-7 breast cancer cells), and. The transfected group (inoculated with human MCF-7 breast cancer cells transfected with ADAM17-sh RNA. 3 groups of cell suspension 0.2 ml / 5 脳 10 7 / ml) was implanted in the subcutaneous part of the right side of nude mice. The tumor nodules appeared at the inoculation site about 10 days after implantation. After the nodules were hard, the tumor size was measured once every 4 days, and the general condition of nude mice was observed. After 26 days of implantation, all animals were killed by HE staining to observe the morphological changes of the three groups. The expression of ADAM17 Ki-67 protein in the three groups was detected by immunohistochemistry and the expression of ADAM17 protein in the three groups was detected by Western blotting. Results Laser confocal detection showed that lentivirus-mediated ADAM17-shRNA could be successfully transferred into MCF-7 cells. The transfection rate reached 80.30% in all nude mice, and the tumorigenesis rate reached 100% 30%. 10 days after implantation, the skin of nude mice could be seen to be pink, the tumor was localized on the skin surface, irregular shape, lobular shape, clear boundary, tough quality and poor mobility. The results showed that the tumor of nude mice in the meaningless sequence group and control group was significantly larger than that in the transfection group, while the diet of nude mice in the meaningless sequence group and control group was significantly reduced, and the weight loss was serious. The nude mice in the transfection group were feeding and drinking water. At the end of the experiment, the volume of transplanted tumor in transfection group, control group and meaningless sequence group were 241.96 鹵17.14 mm ~ 3, 609.50 鹵15.13 mm ~ 3 and 611.40 鹵15.97 mm ~ 3, respectively. The maximum tumor inhibition rate in the transfection group was 63.11 and 63.26 respectively. He staining showed that the transplanted tumors in the three groups showed typical characteristics of human breast infiltrating ductal carcinoma, and the breast cancer cells distributed in stripe shape, infiltrating stroma and stroma with bleeding symptoms. Compared with the other two groups, the tumor tissue in the transfection group showed a large area of necrosis, cell disintegration in the necrotic area, and cell structure disappeared. The immunohistochemical results showed that the expression of 1% ADAM17 protein was positive in the cytoplasm. Compared with the control group (7.17 鹵0.27) and the meaningless sequence group (7.11 鹵0.21), the staining index of transplanted tumor in the transfection group was 2.65 鹵0.49, and the difference was statistically significant. The tumor staining index score of the transfected group was 3.76 鹵0.23, which was compared with that of the control group (9.05 鹵0.34) and the meaningless sequence group (8.94 鹵0.42). The OD value of ADAM17 protein was 0.367 鹵0.034 in transfection group, 0.611 鹵0.037 in control group and 0.618 鹵0.028 in meaningless sequence group. The level of ADAM17 protein in transfection group was significantly lower than that in meaningless sequence group and control group. Conclusion the growth of human MCF-7 breast cancer cells transfected with ADAM17-shRNA was significantly inhibited in nude mice.
【學(xué)位授予單位】：華北理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R737.9

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本文編號(hào)：1505577