siRNA沉默YKL-40基因表达对子宫内膜癌HEC-1A细胞体外生物学特性的影响

发布时间：2018-01-05 03:30

本文关键词：siRNA沉默YKL-40基因表达对子宫内膜癌HEC-1A细胞体外生物学特性的影响　出处：《广西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:检测4株子宫内膜癌细胞株HEC-1A、HEC-1B、ishikawa、RL-952细胞中YKL-40基因的表达水平,筛选相对高表达YKL-40基因的子宫内膜癌细胞株。同时通过RNA干扰(RNAi)技术靶向抑制子宫内膜癌HEC-1A细胞中的YKL-40基因的表达,构建稳定沉默YKL-40基因的子宫内膜癌细胞株。方法:1、用荧光定量PCR(q PCR)的实验方法检测子宫内膜癌细胞株HEC-1A、HEC-1B、ishikawa、RL-952细胞中YKL-40基因的表达水平。2、通过RNAi技术构建YKL-40 siRNA慢病毒载体。3、细胞转染:用特异性YKL-40 siRNA基因序列制备的慢病毒载体,转染人子宫内膜癌HEC-1A细胞。4、实验分组:将人子宫内膜癌细胞HEC-1A细胞分为3组。实验组(E.G):转染含有YKL-40 siRNA的慢病毒,即siRNA实验组;空载体组(mock):转染只含有绿色荧光蛋白(GFP)的慢病毒;空白对照组(blank):未进行转染的人子宫内膜癌细胞。将人子宫内膜癌HEC-1A细胞置于5%CO2、37°C培养箱中常规培养。取生长状态良好的癌细胞进行转染。5、构建稳定转染的子宫内膜癌细胞株:用嘌呤霉素筛选稳定沉默YKL-40基因表达的子宫内膜癌细胞株。耐嘌呤霉素的细胞则用于后续实验。6、用q PCR实验检测转染前后子宫内膜癌细胞株HEC-1A细胞中YKL-40基因的表达水平,即YKL-40基因的沉默水平。7、用Western Blotting实验检测转染前后子宫内膜癌细胞株HEC-1A细胞中YKL-40蛋白的表达水平。结果:1、4株子宫内膜癌细胞株细胞中YKL-40基因的表达水平比较:HEC-1A、HEC-1B、ishikawa、RL-952细胞中YKL-40 m RNA的相对表达水平分别为:1.0052±0.13,0.0024±0.00,0.0017±0.00,0.0032±0.00。4株细胞中均有YKL-40基因表达,HEC-1A细胞中YKL-40基因的表达相对最高,用于后续基因沉默细胞模型的建立。2、成功构建YKL-40 siRNA慢病毒载体,病毒滴度为1×108PFU/ml。3、细胞转染:转染成功的细胞则带有绿色荧光蛋白(GFP),未转染成功的细胞则不带有绿色荧光蛋白。转染效率(带绿色荧光的癌细胞)达80%以上。4、构建稳定转染的子宫内膜癌细胞株:用嘌呤霉素筛选的转染后的细胞,未成功转染未表达绿色荧光蛋白(GFP)的细胞则被嘌呤霉素杀死,表达GFP蛋白的细胞则被成功筛选出来。5、转染后HEC-1A细胞中YKL-40 m RNA表达水平的比较:实时荧光定量RT-PCR(q PCR)技术检测显示,siRNA组、空载组、空白对照组细胞中YKL-40 m RNA相对表达水平分别为0.31±0.27、1.33±0.62、1.01±0.12,siRNA组明显低于空载体组和空白对照组(P0.05);而空载体组与空白对照组比较,差异则无统计学意义(P0.05)。6、转染后HEC-1A细胞中的YKL-40蛋白的表达水平比较:经过Western Blotting实验检测转染前后子宫内膜癌细胞株HEC-1A细胞中YKL-40蛋白的表达水平未见明显差异(P=0.061),可能有基因翻译出来的蛋白质有关,或者检测的时候YKL-40蛋白降解。结论:子宫内膜癌HEC-1A细胞有相对表达量高的YKL-40基因水平,可用于沉默细胞中YKL-40基因的表达水平,作为后续实验的基础。构建的慢病毒载体可成功抑制干扰人子宫内膜癌HEC-1A细胞中YKL-40基因的表达水平,为后续细胞模型的建立奠定基础。目的:探讨siRNA沉默YKL-40基因表达对人子宫内膜癌HEC-1A细胞体外生物学性状(增殖、迁移、侵袭以及凋亡)的影响以及对顺铂化疗敏感性的影响。方法:1、实验分组同前。MTT实验检测3组细胞的增殖能力。2、Transwell小室迁移和侵袭实验检测3组细胞的迁移和侵袭能力。3、MTT实验检测基因转染前后人子宫内膜癌HEC-1A细胞对顺铂化疗的敏感性。4、顺铂处理子宫内膜癌HEC-1A细胞对细胞中YKL-40基因的影响。5、流式细胞仪检测技术(Annexin V-PE/7AAD双染法)检测转染前后3组细胞对顺铂化疗的总凋亡率。结果:1、转染后3组HEC-1A细胞增殖能力的比较:MTT比色法检测显示,与空载体组和空白对照组比较,siRNA组细胞的增殖能力明显受到抑制(P0.05);而空载组与空白对照组比较,差异则无统计学意义(P0.05)。2、转染后3组HEC-1A细胞迁移能力的比较:体外transwell小室迁移实验显示,siRNA组穿膜细胞数为(133±14)个,明显少于空载体组和空白对照组[分别为(178±11)和(179±19)个,P0.05];而空载体组与空白对照组比较,差异则无统计学意义(P=0.872)。3、转染后3组HEC-1A细胞侵袭能力的比较:体外transwell小室侵袭实验显示,siRNA组穿膜细胞数为(143±13)个,明显少于空载体组和空白对照组[分别为(238±26)和(227±18)个,P0.05];而空载体组与空白对照组比较,差异则无统计学意义(P=0.411)。4、顺铂对子宫内膜癌HEC-1A细胞YKL-40基因降低前后细胞生长的影响:加入不同浓度梯度的顺铂培养细胞48h后,细胞的生长明显抑制,实验组细胞(转染siRNA后)比空白对照组和空载组细胞生长抑制更显著(P0.05),但空白对照组组和空载组无明显差异(P0.05)5、顺铂处理前后对子宫内膜癌细胞中YKL-40表达的影响比较:经过相同浓度的顺铂处理子宫内膜癌后,子宫内膜癌中YKL-40基因的表达量上调(P=0.000)。6、经相同浓度顺铂处理各组细胞48小时3组HEC-1A细胞总凋亡率的比较:经过相同浓度的顺铂处理子宫内膜癌48小时后siRNA实验组的增殖能力低于空白对照组和空载组(P0.05)。siRNA组、空载体组、空白对照组细胞总体凋亡率分别为(38.07±4.88)、(13.3±1.01)、(12.5±0.17),siRNA实验组的总体凋亡率高于空白对照组和空载体组,差异均有统计学意义(P0.05),而空白对照组和空载组无明显差异(P=0.776)。结论:siRNA沉默HEC-1A细胞中YKL-40基因表达可以降低HEC-1A细胞的增殖、迁移和侵袭能力,并且可以提高HEC-1A细胞对顺铂化疗的敏感性,经过顺铂处理细胞48小时后其凋亡率明显降低。因此,YKL-40具有促进子宫内膜癌细胞增殖、促进血管形成以及抗凋亡能力。YKL-40可能是未来子宫内膜癌分子治疗的潜在靶点。
[Abstract]:Objective: to detect 4 strains of endometrial carcinoma cell lines HEC-1A, HEC-1B, Ishikawa, YKL-40 gene expression level in RL-952 cells, screening of relatively high expression in endometrial carcinoma cell lines YKL-40 gene. At the same time by RNA interference (RNAi) targeting YKL-40 gene expression of HEC-1A cells of endometrial carcinoma, endometrial carcinoma construction of cell lines stably silencing YKL-40 gene. Methods: 1, using fluorescence quantitative PCR (Q PCR) of the experimental method for the detection of endometrial carcinoma cell lines HEC-1A, HEC-1B, Ishikawa, YKL-40 gene in RL-952 cells, the expression level of.2, through building YKL-40 siRNA lentiviral vector.3 RNAi cells transfected with lentiviral vector with specific YKL-40 siRNA gene sequences were prepared by transfection of human endometrial carcinoma HEC-1A cells.4 group: human endometrial carcinoma cell line HEC-1A cells were divided into 3 groups. The experimental group (E.G): chronic disease with YKL-40 siRNA transfection Poison, namely siRNA experimental group; vector group (mock): transfection only containing green fluorescent protein (GFP) lentivirus; blank control group (blank): no human endometrial carcinoma cells. The transfection of human endometrial carcinoma HEC-1A cells in 5%CO2,37 ~ C cultured routinely in the box. Get by.5 good growth state of cancer cells, endometrial cancer cell lines stably transfected construct: endometrial carcinoma cell line with puromycin and stable expression of YKL-40 gene silencing. Cell resistance to puromycin was used for subsequent experiments with.6, the expression level of Q before and after PCR transfection assay in endometrial carcinoma cell lines YKL-40 HEC-1A cells the gene, YKL-40 gene silencing.7 expression level, YKL-40 cells HEC-1A protein in endometrial carcinoma cell lines before and after Western Blotting transfection assay. Results: 1,4 strain in endometrial carcinoma cell line YKL-40 cells Comparison of gene expression level: HEC-1A, HEC-1B, Ishikawa, the relative expression level of RL-952 cells in YKL-40 m RNA were: the expression of YKL-40 gene were 1.0052 + 0.13,0.0024 + 0.00,0.0017 + 0.00,0.0032 + 0.00.4 cells, the expression of YKL-40 gene in HEC-1A cells is the highest, for subsequent gene silencing cell model.2, successfully constructed YKL-40 siRNA lentiviral vector, the virus titer was 1 * 108PFU/ml.3, transfected cells: cells transfected with green fluorescent protein (GFP), non transfected cells were not with green fluorescent protein. The transfection efficiency (green fluorescent cells) was more than 80%.4 in endometrial carcinoma cell lines to construct stable transfection: transfection with puromycin to the cells after transfection was not successful expression of green fluorescent protein (GFP) cells by puromycin kill, expression of GFP protein in the cells were formed Work out.5 screening, after transfection in HEC-1A cells YKL-40 m expression level of RNA: real time fluorescence quantitative RT-PCR (Q PCR) detected, siRNA group, empty vector group, blank control group cells in YKL-40 m RNA relative expression levels were 0.31 + 0.27,1.33 + 0.62,1.01 + 0.12, siRNA group was significantly lower than that of empty vector group and the blank control group (P0.05); and the empty vector group and blank control group, no significant difference (P0.05.6), compare the expression level of HEC-1A cells in YKL-40 protein after transfection: after Western Blotting assay in HEC-1A cells transfected with YKL-40 protein in endometrial carcinoma cell line expression level showed no significant difference (P=0.061), the protein may have a gene translation, or detection of YKL-40 protein degradation. Conclusion: the relative expression of YKL-40 gene high level HEC-1A endometrial cancer cells, can be used for The expression level of YKL-40 gene silencing in the cell, as a basis for subsequent experiments. The constructed lentiviral vector can successfully suppress the interference level of YKL-40 expression in human endometrial carcinoma cells HEC-1A gene, for the subsequent cell model foundation. Objective: To investigate the siRNA silencing of YKL-40 gene expression traits on human endometrial carcinoma HEC-1A cells in vitro (proliferation, migration, invasion and apoptosis) and the influence of sensitivity to cisplatin. Methods: 1 experimental groups, the proliferation ability of.2 with.MTT assay, 3 groups of cells, migration and invasion of.3 Transwell cell migration and invasion assay in 3 groups of cells, HEC-1A human endometrial carcinoma cell line.4 to cisplatin sensitivity chemotherapy detected before and after MTT gene transfection experiments, cisplatin treatment effect on HEC-1A cells in endometrial carcinoma cells YKL-40 gene.5, flow cytometry (Anne Xin V-PE/7AAD double staining) rate of apoptosis 3 group cells to cisplatin detection after transfection. Results: 1. Comparison of 3 groups of HEC-1A after transfection, the cell proliferation: MTT assay showed that, compared with the control group and the blank vector group, siRNA cell proliferation was significantly suppressed (P0.05); and no load group compared with the control group, no significant difference (P0.05.2), compared 3 groups of migration ability of HEC-1A cells after transfection in vitro Transwell cell migration assay showed that siRNA cells were (133 + 14), significantly less than the empty vector group and blank control group. For (178 + 11) and (179 + 19), P0.05]; and the empty vector group and blank control group, no significant difference (P=0.872.3), compared 3 groups of invasion ability of HEC-1A cells after transfection in vitro by Transwell invasion assay showed that siRNA cells were (143 + 13) a, Less than the empty vector group and blank control group respectively [(238 + 26) and (227 + 18), P0.05]; and the empty vector group and blank control group, no significant difference (P=0.411).4, cisplatin on reducing the endometrial carcinoma HEC-1A cells YKL-40 gene before and after cell growth: different concentrations of cisplatin in cultured cells after 48h cells could inhibit the growth of cells in the experimental group (siRNA after transfection) than the blank control group and empty vector group of cell growth inhibition was more significant (P0.05), but the control group and control group had no significant difference (P0.05 5), cisplatin treatment on the expression of YKL-40 of uterus endometrial cancer cells in comparison: after treatment with cisplatin in endometrial carcinoma at the same concentration, expression of YKL-40 gene in endometrial carcinoma (P=0.000) of.6, compared with the same concentration of cisplatin treated cells were 48 hours of total HEC-1A cell apoptosis rate was 3: After 48 hours the same concentration of cisplatin treatment in endometrial carcinoma after siRNA in experimental group the proliferation ability is lower than the blank control group and empty vector group (P0.05).SiRNA group, vector group, blank control group overall cell apoptotic rates were (38.07 + 4.88), (13.3 + 1.01), (12.5 + 0.17), total apoptosis the siRNA group was higher than those of control group and empty vector group, the differences were statistically significant (P0.05), but no significant difference between the control group and empty vector group (P=0.776). Conclusion: HEC-1A can reduce the cell proliferation of YKL-40 gene silence of siRNA expression in HEC-1A cells, migration and invasion, and can improve the sensitivity of HEC-1A cells to cisplatin, cisplatin treated cells after 48 hours after the apoptosis rate was significantly reduced. Therefore, YKL-40 can promote the proliferation of endometrial cancer cells, promote angiogenesis and anti apoptosis ability of.YKL-40 may be the future of endometrial cancer Potential targets for molecular therapy.

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33

【相似文献】