ADAM17-shRNA对裸小鼠MCF-7乳腺癌移植瘤的影响

发布时间：2018-02-12 11:57

本文关键词： 解聚素-金属蛋白酶17 乳腺癌 RNA干扰基因治疗　出处：《华北理工大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的利用人MCF-7乳腺癌移植型裸小鼠模型,探讨解聚素-金属蛋白酶17-sh RNA(ADAM17-sh RNA)的体内抑瘤效果。方法常规培养人MCF-7乳腺癌细胞,用ADAM17-sh RNA转染MCF-7细胞,利用激光共聚焦检测慢病毒Lentivirus-ADAM17-sh RNA-RFP介导的RFP在MCF-7中的表达情况。30只裸小鼠随机分为对照组(接种正常MCF-7细胞)、无意义序列组(接种转染空载体的MCF-7乳腺癌细胞)和转染组(接种转染ADAM17-sh RNA的人MCF-7乳腺癌细胞)。制备实验所需三组细胞悬液,0.2 ml/只(5×107/ml)种植在裸小鼠右侧鼷部皮下。种植后约第10天待接种部位出现明显的肿瘤结节,结节质地较硬后,每4天一次测量肿瘤瘤体大小,并观察裸小鼠的一般情况,种植后第26天测量后处死所有动物。HE染色观察三组细胞的形态变化;免疫组化法检测ADAM17、Ki-67两种蛋白在三组肿瘤中的表达情况;Western blotting法检测ADAM17蛋白在三组中的表达。结果激光共聚焦检测显示慢病毒介导的ADAM17-shRNA可成功转入到MCF-7细胞内,转染率达80%。30只裸小鼠全部成瘤,成瘤率达到100%(30/30);种植10天后可见裸小鼠瘤体表面皮肤呈粉红色,肿瘤局限隆起于皮肤表面,形状不规则,呈分叶状,边界清楚,质韧,活动度差;观察发现无意义序列组和对照组的裸小鼠肿瘤明显大于转染组,无意义序列组和对照组裸小鼠进食明显减少、消瘦严重,转染组裸小鼠觅食、饮水、活动等状况良好。实验结束时转染组、对照组和无意义序列组的移植瘤体积分别为241.96±17.14 mm3、609.50±15.13mm3和611.40±15.97 mm3,三组之间差异有统计学意义(F=1765.96、P0.001),与对照组和无意义序列组比较,转染组的最大抑瘤率分别为63.11%、63.26%。HE染色镜下可见,三组移植瘤呈典型的人乳腺浸润性导管癌的特征,乳腺癌细胞呈条索状分布,浸润间质,间质伴出血症状;转染组相比于其他两组,移植瘤组织出现局部大片坏死区,坏死区细胞崩解,细胞结构消失等情况明显有所改善。免疫组织化学结果显示:ADAM17蛋白阳性表达于胞浆,转染组移植瘤染色指数评分为2.65±0.49,其与对照组(7.17±0.27)和无意义序列组(7.11±0.21)比较,差异具有统计学意义(F=174.12,P0.001);Ki-67蛋白阳性表达于胞核,转染组瘤染色指数评分为3.76±0.23,其与对照组(9.05±0.34)和无意义序列组(8.94±0.42)比较,差异具有统计学意义(F=490.93,P0.001)。Western-blotting结果显示转染组ADAM17蛋白OD值为0.367±0.034,对照组为0.611±0.037,无意义序列组为0.618±0.028,转染组的ADAM17蛋白的水平明显低于无意义序列组和对照组,差异有统计学意义(F=56.23,P0.001)。结论转染ADAM17-shRNA的人MCF-7乳腺癌细胞在裸小鼠体内的生长受到了明显地抑制。
[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 卤17.14 mm ~ 3, 609.50 卤15.13 mm ~ 3 and 611.40 卤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 卤0.27) and the meaningless sequence group (7.11 卤0.21), the staining index of transplanted tumor in the transfection group was 2.65 卤0.49, and the difference was statistically significant. The tumor staining index score of the transfected group was 3.76 卤0.23, which was compared with that of the control group (9.05 卤0.34) and the meaningless sequence group (8.94 卤0.42). The OD value of ADAM17 protein was 0.367 卤0.034 in transfection group, 0.611 卤0.037 in control group and 0.618 卤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.
【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.9

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