本文關(guān)鍵詞：siRNA沉默YKL-40基因表達對子宮內(nèi)膜癌HEC-1A細胞體外生物學特性的影響　出處：《廣西醫(yī)科大學》2017年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 子宮內(nèi)膜癌 siRNA YKL-40 RNA干擾 慢病毒載體 PCR YKL-40 siRNA 子宮內(nèi)膜癌 生物學性狀 順鉑 化療

【摘要】：目的:檢測4株子宮內(nèi)膜癌細胞株HEC-1A、HEC-1B、ishikawa、RL-952細胞中YKL-40基因的表達水平,篩選相對高表達YKL-40基因的子宮內(nèi)膜癌細胞株。同時通過RNA干擾(RNAi)技術(shù)靶向抑制子宮內(nèi)膜癌HEC-1A細胞中的YKL-40基因的表達,構(gòu)建穩(wěn)定沉默YKL-40基因的子宮內(nèi)膜癌細胞株。方法:1、用熒光定量PCR(q PCR)的實驗方法檢測子宮內(nèi)膜癌細胞株HEC-1A、HEC-1B、ishikawa、RL-952細胞中YKL-40基因的表達水平。2、通過RNAi技術(shù)構(gòu)建YKL-40 siRNA慢病毒載體。3、細胞轉(zhuǎn)染:用特異性YKL-40 siRNA基因序列制備的慢病毒載體,轉(zhuǎn)染人子宮內(nèi)膜癌HEC-1A細胞。4、實驗分組:將人子宮內(nèi)膜癌細胞HEC-1A細胞分為3組。實驗組(E.G):轉(zhuǎn)染含有YKL-40 siRNA的慢病毒,即siRNA實驗組;空載體組(mock):轉(zhuǎn)染只含有綠色熒光蛋白(GFP)的慢病毒;空白對照組(blank):未進行轉(zhuǎn)染的人子宮內(nèi)膜癌細胞。將人子宮內(nèi)膜癌HEC-1A細胞置于5%CO2、37°C培養(yǎng)箱中常規(guī)培養(yǎng)。取生長狀態(tài)良好的癌細胞進行轉(zhuǎn)染。5、構(gòu)建穩(wěn)定轉(zhuǎn)染的子宮內(nèi)膜癌細胞株:用嘌呤霉素篩選穩(wěn)定沉默YKL-40基因表達的子宮內(nèi)膜癌細胞株。耐嘌呤霉素的細胞則用于后續(xù)實驗。6、用q PCR實驗檢測轉(zhuǎn)染前后子宮內(nèi)膜癌細胞株HEC-1A細胞中YKL-40基因的表達水平,即YKL-40基因的沉默水平。7、用Western Blotting實驗檢測轉(zhuǎn)染前后子宮內(nèi)膜癌細胞株HEC-1A細胞中YKL-40蛋白的表達水平。結(jié)果:1、4株子宮內(nèi)膜癌細胞株細胞中YKL-40基因的表達水平比較:HEC-1A、HEC-1B、ishikawa、RL-952細胞中YKL-40 m RNA的相對表達水平分別為:1.0052±0.13,0.0024±0.00,0.0017±0.00,0.0032±0.00。4株細胞中均有YKL-40基因表達,HEC-1A細胞中YKL-40基因的表達相對最高,用于后續(xù)基因沉默細胞模型的建立。2、成功構(gòu)建YKL-40 siRNA慢病毒載體,病毒滴度為1×108PFU/ml。3、細胞轉(zhuǎn)染:轉(zhuǎn)染成功的細胞則帶有綠色熒光蛋白(GFP),未轉(zhuǎn)染成功的細胞則不帶有綠色熒光蛋白。轉(zhuǎn)染效率(帶綠色熒光的癌細胞)達80%以上。4、構(gòu)建穩(wěn)定轉(zhuǎn)染的子宮內(nèi)膜癌細胞株:用嘌呤霉素篩選的轉(zhuǎn)染后的細胞,未成功轉(zhuǎn)染未表達綠色熒光蛋白(GFP)的細胞則被嘌呤霉素殺死,表達GFP蛋白的細胞則被成功篩選出來。5、轉(zhuǎn)染后HEC-1A細胞中YKL-40 m RNA表達水平的比較:實時熒光定量RT-PCR(q PCR)技術(shù)檢測顯示,siRNA組、空載組、空白對照組細胞中YKL-40 m RNA相對表達水平分別為0.31±0.27、1.33±0.62、1.01±0.12,siRNA組明顯低于空載體組和空白對照組(P0.05);而空載體組與空白對照組比較,差異則無統(tǒng)計學意義(P0.05)。6、轉(zhuǎn)染后HEC-1A細胞中的YKL-40蛋白的表達水平比較:經(jīng)過Western Blotting實驗檢測轉(zhuǎn)染前后子宮內(nèi)膜癌細胞株HEC-1A細胞中YKL-40蛋白的表達水平未見明顯差異(P=0.061),可能有基因翻譯出來的蛋白質(zhì)有關(guān),或者檢測的時候YKL-40蛋白降解。結(jié)論:子宮內(nèi)膜癌HEC-1A細胞有相對表達量高的YKL-40基因水平,可用于沉默細胞中YKL-40基因的表達水平,作為后續(xù)實驗的基礎(chǔ)。構(gòu)建的慢病毒載體可成功抑制干擾人子宮內(nèi)膜癌HEC-1A細胞中YKL-40基因的表達水平,為后續(xù)細胞模型的建立奠定基礎(chǔ)。目的:探討siRNA沉默YKL-40基因表達對人子宮內(nèi)膜癌HEC-1A細胞體外生物學性狀(增殖、遷移、侵襲以及凋亡)的影響以及對順鉑化療敏感性的影響。方法:1、實驗分組同前。MTT實驗檢測3組細胞的增殖能力。2、Transwell小室遷移和侵襲實驗檢測3組細胞的遷移和侵襲能力。3、MTT實驗檢測基因轉(zhuǎn)染前后人子宮內(nèi)膜癌HEC-1A細胞對順鉑化療的敏感性。4、順鉑處理子宮內(nèi)膜癌HEC-1A細胞對細胞中YKL-40基因的影響。5、流式細胞儀檢測技術(shù)(Annexin V-PE/7AAD雙染法)檢測轉(zhuǎn)染前后3組細胞對順鉑化療的總凋亡率。結(jié)果:1、轉(zhuǎn)染后3組HEC-1A細胞增殖能力的比較:MTT比色法檢測顯示,與空載體組和空白對照組比較,siRNA組細胞的增殖能力明顯受到抑制(P0.05);而空載組與空白對照組比較,差異則無統(tǒng)計學意義(P0.05)。2、轉(zhuǎn)染后3組HEC-1A細胞遷移能力的比較:體外transwell小室遷移實驗顯示,siRNA組穿膜細胞數(shù)為(133±14)個,明顯少于空載體組和空白對照組[分別為(178±11)和(179±19)個,P0.05];而空載體組與空白對照組比較,差異則無統(tǒng)計學意義(P=0.872)。3、轉(zhuǎn)染后3組HEC-1A細胞侵襲能力的比較:體外transwell小室侵襲實驗顯示,siRNA組穿膜細胞數(shù)為(143±13)個,明顯少于空載體組和空白對照組[分別為(238±26)和(227±18)個,P0.05];而空載體組與空白對照組比較,差異則無統(tǒng)計學意義(P=0.411)。4、順鉑對子宮內(nèi)膜癌HEC-1A細胞YKL-40基因降低前后細胞生長的影響:加入不同濃度梯度的順鉑培養(yǎng)細胞48h后,細胞的生長明顯抑制,實驗組細胞(轉(zhuǎn)染siRNA后)比空白對照組和空載組細胞生長抑制更顯著(P0.05),但空白對照組組和空載組無明顯差異(P0.05)5、順鉑處理前后對子宮內(nèi)膜癌細胞中YKL-40表達的影響比較:經(jīng)過相同濃度的順鉑處理子宮內(nèi)膜癌后,子宮內(nèi)膜癌中YKL-40基因的表達量上調(diào)(P=0.000)。6、經(jīng)相同濃度順鉑處理各組細胞48小時3組HEC-1A細胞總凋亡率的比較:經(jīng)過相同濃度的順鉑處理子宮內(nèi)膜癌48小時后siRNA實驗組的增殖能力低于空白對照組和空載組(P0.05)。siRNA組、空載體組、空白對照組細胞總體凋亡率分別為(38.07±4.88)、(13.3±1.01)、(12.5±0.17),siRNA實驗組的總體凋亡率高于空白對照組和空載體組,差異均有統(tǒng)計學意義(P0.05),而空白對照組和空載組無明顯差異(P=0.776)。結(jié)論:siRNA沉默HEC-1A細胞中YKL-40基因表達可以降低HEC-1A細胞的增殖、遷移和侵襲能力,并且可以提高HEC-1A細胞對順鉑化療的敏感性,經(jīng)過順鉑處理細胞48小時后其凋亡率明顯降低。因此,YKL-40具有促進子宮內(nèi)膜癌細胞增殖、促進血管形成以及抗凋亡能力。YKL-40可能是未來子宮內(nèi)膜癌分子治療的潛在靶點。
[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.

【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.33

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