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shRNA靶向血管生成素2基因沉默联合奥沙利铂对子宫内膜癌生长抑制作用的研究
作者:周凌1  孙慧婷2  杨思慧3  荆秀娟4  周怀君4 
单位:1. 南京医科大学 鼓楼临床医学院, 江苏 南京 210008;
2. 常州市第二人民医院 生殖中心, 江苏 常州 213003;
3. 南京中医药大学 中西医结合鼓楼临床医学院, 江苏 南京 210008;
4. 南京医科大学鼓楼临床医学院 妇产科, 江苏 南京 210008
关键词:子宫内膜癌 血管生成素2基因沉默 奥沙利铂 抗血管生成 
分类号:R737.33;R73-362
出版年·卷·期(页码):2021·40·第二期(148-157)
摘要:

目的:探讨shRNA靶向血管生成素2(Ang2)基因沉默联合奥沙利铂对子宫内膜癌(endometrial cancer,EC)生长的抑制作用。方法:设计Ang2特异性shRNA,构建携带Ang2-shRNA的pRNAT-CMV3.2-Neo质粒和阴性对照pRNAT-CMV3.2-Neo-neg质粒,并构建阳性和阴性质粒的稳转Ishikawa细胞系。使用未进行任何处理的Ishikawa细胞系、携带阳性和阴性质粒的稳转Ishikawa细胞系分5组进行细胞学实验,分别为生理盐水组、奥沙利铂组、空载质粒组、Ang2敲低组、Ang2敲低联合奥沙利铂组。采用qRT-PCR、蛋白质印迹法检测各组细胞中Ang2 mRNA和蛋白的表达,采用细胞凋亡、侵袭实验检测各组细胞生物学行为的变化。利用上述细胞系建立裸鼠移植瘤模型,分5组(同细胞分组)给予不同的试剂干预,观察肿瘤生长情况,测量移植瘤体积,计算肿瘤生长抑制率;使用免疫组织化学方法检测各组瘤体内vWF蛋白,测算微血管密度;采用qRT-PCR、蛋白质印迹法检测瘤体组织Ang2 mRNA和蛋白的表达。结果:(1)5组Ishikawa细胞Ang2表达检测:奥沙利铂组与生理盐水组比较Ang2 mRNA和蛋白表达分别降低了21.8%和22.9%(均P<0.05);而Ang2敲低组、Ang2敲低联合奥沙利铂组与空载质粒组相比,Ang2 mRNA含量分别降低了76.54%、80.24%(均P<0.001),Ang2蛋白表达分别降低60.79%、65.09%(均P<0.001),差异均具有统计学意义。(2)Ishikawa细胞行为学实验:奥沙利铂组、Ang2敲低组、Ang2敲低联合奥沙利铂组与生理盐水组和空载质粒组相比细胞凋亡数量都有明显增加(均P<0.001),侵袭细胞数量明显减少(均P<0.001)。(3)动物实验:Ang2敲低联合奥沙利铂组与奥沙利铂组及Ang2敲低组相比肿瘤体积明显减小(均P<0.001),抑瘤率高达92.46%,微血管密度显著降低(P<0.05或P<0.001)。结论:敲低Ang2联合奥沙利铂能更有效地抑制Ishikawa细胞中Ang2的表达,促进Ishikawa细胞凋亡、抑制其侵袭能力,抑制移植瘤的生长,减少肿瘤血管的生成。

Objective: To investigate the inhibitory effect of shRNA targeting angiopoigenin 2(Ang2) gene silencing combined with oxaliplatin on tumor growth of endometrial carcinoma(EC). Methods: Ang2-shRNA was designed, and pRNAT-CMV3.2-neo plasmid carrying Ang2-shRNA and the negative control pRNAT-CMV3.2-neo plasmid were constructed, and the stable transgenic Ishikawa cell lines with positive and negative plasmid were constructed. The Ishikawa cell lines without any treatment and the stable Ishikawa cell lines carrying positive and negative plasmids were divided into 5 groups for cytological experiments. The groups were normal saline group, oxaliplatin group, no-load plasmid group, Ang2 knockdown group, and Ang2 knockdown combined with oxaliplatin group. The mRNA and protein expressions of Ang2 in each group were detected by qRT-PCR and Western blotting assay. Cell apoptosis and invasion assay were used to detect the changes of cell biological behavior in each group. The cell lines above were used to establish the xenograft tumor model in nude mice. Tumor growth was observed in 5 groups of nude mice after intervention with different reagents, the volume of the xenograft tumor was measured, and the tumor growth inhibition rate was calculated. vWF protein in each group was detected by immunohistochemical method to measure the microvessel density(MVD). The expression of Ang2 mRNA and protein in the tumor tissues were detected by qRT-PCR and Western blotting. Results: (1) Compared with the normal saline group, the expression of Ang2 mRNA and protein in oxaliplatin group was decreased by 21.8% and 22.9%, respectively(all P<0.05), while Ang2 knockdown group and Ang2 knockdown combined with oxaliplatin group reduced the content of Ang2 mRNA by 76.54% and 80.24% compared with no-load plasmid group, respectively(all P<0.001), the expression of Ang2 protein was decreased by 60.79% and 65.09%(all P<0.001).(2) Compared with the normal saline group and the no-load plasmid group, the number of cell apoptosis were significantly increased in the oxaliplatin group, the Ang2 knockdown group and the Ang2 knockdown combined with oxaliplatin group(all P<0.001), and the number of invaded cells were significantly decreased(all P<0.001).(3) Animal experiments showed, compared with the oxaliplatin group and the Ang2 knockdown group, the tumor volume of the Ang2 knockdown combined with oxaliplatin group was significantly reduced(all P<0.001), the tumor inhibition rate was as high as 92.46%, and the microvessel density was significantly reduced(P<0.05 or P<0.001). Conclusion: Ang2 knockdown combined with oxaliplatin can more effectively inhibit the expression of Ang2 in Ishikawa cells,promote the apoptosis of Ishikawa cells and inhibit its invasion ability. It significantly inhibits the growth of transplanted tumor and reduces the angiogenesis of tumor.

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