HepaCAM通过阻碍PKCε从胞浆转位到胞膜抑制肾癌786-O细胞增殖及迁移

发布时间：2018-05-19 10:22

  本文选题：HepaCAM + PKCε　； 参考：《重庆医科大学》2014年硕士论文

【摘要】：第一部分HepaCAM和PKCε蛋白在肾透明细胞癌组织中的表达 目的：检测HepaCAM和PKCε蛋白在肾透明细胞癌组织中的表达。 方法：免疫组化检测36例人肾透明细胞癌组织HepaCAM和PKCε蛋白在癌组织和癌旁组织的表达水平，于倒置显微镜下成像，IPP6.0软件分析平均光密度。 结果：肾透明细胞癌组织HepaCAM表达显著缺失，而PKCε呈高表达状态；癌旁组织HepaCAM表达增高，而PKCε低表达。PKCε和HepaCAM表达成反相关。其中PKCε表达与肿瘤分级分期相关。差异有统计学意义（p0.05）。 结论：在组织水平验证了PKCε和HepaCAM蛋白的表达状况和分析了二者的关系。 第二部分HepaCAM过表达影响PKCε胞浆和胞膜定位 目的：探究感染Ad-GFP-HepaCAM腺病毒后对肾癌786-O细胞株PKCε蛋白表达和定位的影响。 方法：感染Ad-GFP-HepaCAM腺病毒、Ad-GFP空载腺病毒后， western blot验证HepaCAM的表达，检测PKCε细胞膜、细胞浆蛋白和总蛋白的表达情况。细胞免疫荧光进一步验证HepaCAM表达对PKCε定位的影响。 结果：实验组HepaCAM成功表达后，PKCε细胞膜蛋白降低、浆蛋白升高，总蛋白无显著差异。细胞免疫荧光实验定位实验组胞浆PKCε较空白和空载组增多（p0.05）。 结论：HepaCAM表达能影响PKCε在细胞膜和细胞浆中的定位变化，但不影响总蛋白表达。 第三部分PKCε特异性转位抑制剂εV1-2对786-O细胞增殖和迁移的影响 目的：验证εV1-2对786-O细胞株增殖和迁移力的影响；比较HepaCAM与εV1-2抑制克隆形成和细胞迁移的能力。 方法：应用CCK-8实验摸索PKCε特异性转位抑制剂εV1-2对786-O细胞的最佳作用浓度和时间点。Western blot实验验证不同处理后MMP-9，cyclinD1和p-AKT蛋白表达。平板克隆和划痕实验验证HepaCAM与εV1-2对增殖和迁移力的影响。 结果：10μmol/L和24h为εV1-2对786-O细胞的最佳作用浓度和时间点。Western blot证实HepaCAM表达和εV1-2处理后MMP-9，，cyclinD1和p-AKT蛋白表达降低。平板克隆和划痕实验显示HepaCAM表达比εV1-2组更能有效抑制克隆形成和细胞迁移，且二者合用抑制效应更强（p0.05）。 结论：外源性HepaCAM表达和εV1-2处理都降低了增殖和迁移相关分子的表达，HepaCAM比εV1-2处理更有效抑制克隆细胞数和细胞迁移力。HepaCAM可能作为PKCε上游分子调节肾癌786-O细胞的增殖和迁移。
[Abstract]:The expression of HepaCAM and PKC 蔚 protein in renal clear cell carcinoma Objective: to detect the expression of HepaCAM and PKC 蔚 protein in renal clear cell carcinoma. Methods: the expression levels of HepaCAM and PKC 蔚 proteins in 36 cases of clear cell carcinoma of kidney were detected by immunohistochemistry. The mean optical density was analyzed with the software of IPP6.0 under inverted microscope. Results: the expression of HepaCAM was significantly absent in renal clear cell carcinoma, but the expression of PKC 蔚 was high, the expression of HepaCAM in adjacent tissues was higher, but the expression of PKC 蔚 was lower. PKC 蔚 and HepaCAM expression were inversely correlated. The expression of PKC 蔚 was correlated with tumor grade and stage. The difference was statistically significant (P 0.05). Conclusion: the expression of PKC 蔚 and HepaCAM protein was verified and the relationship between them was analyzed at tissue level. Part two: overexpression of HepaCAM affects the localization of PKC 蔚 cytoplasm and membrane Aim: to investigate the effect of Ad-GFP-HepaCAM adenovirus infection on the expression and localization of PKC 蔚 protein in 786-O cell line. Methods: Ad-GFP-HepaCAM adenovirus was infected with Ad-GFP. Western blot was used to verify the expression of HepaCAM and to detect the expression of PKC 蔚 cell membrane, cytoplasmic protein and total protein. The effect of HepaCAM expression on the localization of PKC 蔚 was further verified by cellular immunofluorescence. Results: after the successful expression of HepaCAM in the experimental group, the membrane protein of PKC 蔚 decreased, the plasma protein increased, but there was no significant difference in total protein. The number of PKC 蔚 in cytoplasm of the experimental group was higher than that of the blank group and the no-load group by immunofluorescence assay (P 0.05). Conclusion the expression of PKC 蔚 can affect the localization of PKC 蔚 in cell membrane and cytoplasm, but it does not affect the expression of total protein. The effect of PKC 蔚 specific translocation inhibitor 蔚 V1-2 on proliferation and migration of 786-O cells Aim: to investigate the effect of 蔚 V1-2 on proliferation and migration of 786-O cell line and to compare the ability of HepaCAM and 蔚 V1-2 to inhibit clone formation and cell migration. Methods: the optimal concentration and time point of PKC 蔚 specific translocation inhibitor 蔚 V1-2 on 786-O cells were explored by CCK-8 assay. Western blot assay was used to verify the expression of MMP-9 cyclin D1 and p-AKT protein after different treatments. The effects of HepaCAM and 蔚 V1-2 on proliferation and mobility were verified by plate cloning and scratch test. Results the optimal concentration and time point of 10 渭 mol/L and 24 h for 蔚 V1-2 on 786-O cells. Western blot confirmed that the expression of HepaCAM and the expression of MMP-9 cyclin D1 and p-AKT protein decreased after treatment with 蔚 V1-2. Plate cloning and scratch assay showed that HepaCAM expression was more effective than 蔚 V1-2 in inhibiting clone formation and cell migration, and the inhibition effect was stronger than that in 蔚 V1-2 group. Conclusion: both exogenous HepaCAM expression and 蔚 V1-2 treatment can reduce the expression of proliferative and migration-related molecules. HepaCAM is more effective than 蔚 V1-2 in inhibiting the number of cloned cells and cell migration. HepaCAM may be used as upstream of PKC 蔚 to regulate the proliferation and migration of 786-O cells in renal cell carcinoma.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.11

【参考文献】

相关期刊论文 前3条

1 张巧琳;罗春丽;颜令;吴小候;;肝细胞黏附分子诱导肾癌786-0细胞系凋亡[J];基础医学与临床;2011年01期

2 张巧琳;罗春丽;颜令;;HepaCAM对肾癌786-0细胞Caspase-3活性和表达的影响[J];生物技术通报;2010年11期

3 荀春华;罗春丽;吴小候;谢建红;杨淑哲;潘翠翠;;HepaCAM基因转染对肾癌细胞侵袭活性的影响[J];肿瘤;2008年12期

本文编号：1909730