本文關(guān)鍵詞：siRNA沉默YKL-40基因表達(dá)對(duì)子宮內(nèi)膜癌HEC-1A細(xì)胞體外生物學(xué)特性的影響　出處：《廣西醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)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(xué)性狀 順鉑 化療

【摘要】：目的:檢測(cè)4株子宮內(nèi)膜癌細(xì)胞株HEC-1A、HEC-1B、ishikawa、RL-952細(xì)胞中YKL-40基因的表達(dá)水平,篩選相對(duì)高表達(dá)YKL-40基因的子宮內(nèi)膜癌細(xì)胞株。同時(shí)通過(guò)RNA干擾(RNAi)技術(shù)靶向抑制子宮內(nèi)膜癌HEC-1A細(xì)胞中的YKL-40基因的表達(dá),構(gòu)建穩(wěn)定沉默YKL-40基因的子宮內(nèi)膜癌細(xì)胞株。方法:1、用熒光定量PCR(q PCR)的實(shí)驗(yàn)方法檢測(cè)子宮內(nèi)膜癌細(xì)胞株HEC-1A、HEC-1B、ishikawa、RL-952細(xì)胞中YKL-40基因的表達(dá)水平。2、通過(guò)RNAi技術(shù)構(gòu)建YKL-40 siRNA慢病毒載體。3、細(xì)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(xì)胞。4、實(shí)驗(yàn)分組:將人子宮內(nèi)膜癌細(xì)胞HEC-1A細(xì)胞分為3組。實(shí)驗(yàn)組(E.G):轉(zhuǎn)染含有YKL-40 siRNA的慢病毒,即siRNA實(shí)驗(yàn)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(duì)照組(blank):未進(jìn)行轉(zhuǎn)染的人子宮內(nèi)膜癌細(xì)胞。將人子宮內(nèi)膜癌HEC-1A細(xì)胞置于5%CO2、37°C培養(yǎng)箱中常規(guī)培養(yǎng)。取生長(zhǎng)狀態(tài)良好的癌細(xì)胞進(jìn)行轉(zhuǎn)染。5、構(gòu)建穩(wěn)定轉(zhuǎn)染的子宮內(nèi)膜癌細(xì)胞株:用嘌呤霉素篩選穩(wěn)定沉默YKL-40基因表達(dá)的子宮內(nèi)膜癌細(xì)胞株。耐嘌呤霉素的細(xì)胞則用于后續(xù)實(shí)驗(yàn)。6、用q PCR實(shí)驗(yàn)檢測(cè)轉(zhuǎn)染前后子宮內(nèi)膜癌細(xì)胞株HEC-1A細(xì)胞中YKL-40基因的表達(dá)水平,即YKL-40基因的沉默水平。7、用Western Blotting實(shí)驗(yàn)檢測(cè)轉(zhuǎn)染前后子宮內(nèi)膜癌細(xì)胞株HEC-1A細(xì)胞中YKL-40蛋白的表達(dá)水平。結(jié)果:1、4株子宮內(nèi)膜癌細(xì)胞株細(xì)胞中YKL-40基因的表達(dá)水平比較:HEC-1A、HEC-1B、ishikawa、RL-952細(xì)胞中YKL-40 m RNA的相對(duì)表達(dá)水平分別為:1.0052±0.13,0.0024±0.00,0.0017±0.00,0.0032±0.00。4株細(xì)胞中均有YKL-40基因表達(dá),HEC-1A細(xì)胞中YKL-40基因的表達(dá)相對(duì)最高,用于后續(xù)基因沉默細(xì)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(xì)胞轉(zhuǎn)染:轉(zhuǎn)染成功的細(xì)胞則帶有綠色熒光蛋白(GFP),未轉(zhuǎn)染成功的細(xì)胞則不帶有綠色熒光蛋白。轉(zhuǎn)染效率(帶綠色熒光的癌細(xì)胞)達(dá)80%以上。4、構(gòu)建穩(wěn)定轉(zhuǎn)染的子宮內(nèi)膜癌細(xì)胞株:用嘌呤霉素篩選的轉(zhuǎn)染后的細(xì)胞,未成功轉(zhuǎn)染未表達(dá)綠色熒光蛋白(GFP)的細(xì)胞則被嘌呤霉素殺死,表達(dá)GFP蛋白的細(xì)胞則被成功篩選出來(lái)。5、轉(zhuǎn)染后HEC-1A細(xì)胞中YKL-40 m RNA表達(dá)水平的比較:實(shí)時(shí)熒光定量RT-PCR(q PCR)技術(shù)檢測(cè)顯示,siRNA組、空載組、空白對(duì)照組細(xì)胞中YKL-40 m RNA相對(duì)表達(dá)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(duì)照組(P0.05);而空載體組與空白對(duì)照組比較,差異則無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。6、轉(zhuǎn)染后HEC-1A細(xì)胞中的YKL-40蛋白的表達(dá)水平比較:經(jīng)過(guò)Western Blotting實(shí)驗(yàn)檢測(cè)轉(zhuǎn)染前后子宮內(nèi)膜癌細(xì)胞株HEC-1A細(xì)胞中YKL-40蛋白的表達(dá)水平未見(jiàn)明顯差異(P=0.061),可能有基因翻譯出來(lái)的蛋白質(zhì)有關(guān),或者檢測(cè)的時(shí)候YKL-40蛋白降解。結(jié)論:子宮內(nèi)膜癌HEC-1A細(xì)胞有相對(duì)表達(dá)量高的YKL-40基因水平,可用于沉默細(xì)胞中YKL-40基因的表達(dá)水平,作為后續(xù)實(shí)驗(yàn)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(xì)胞中YKL-40基因的表達(dá)水平,為后續(xù)細(xì)胞模型的建立奠定基礎(chǔ)。目的:探討siRNA沉默YKL-40基因表達(dá)對(duì)人子宮內(nèi)膜癌HEC-1A細(xì)胞體外生物學(xué)性狀(增殖、遷移、侵襲以及凋亡)的影響以及對(duì)順鉑化療敏感性的影響。方法:1、實(shí)驗(yàn)分組同前。MTT實(shí)驗(yàn)檢測(cè)3組細(xì)胞的增殖能力。2、Transwell小室遷移和侵襲實(shí)驗(yàn)檢測(cè)3組細(xì)胞的遷移和侵襲能力。3、MTT實(shí)驗(yàn)檢測(cè)基因轉(zhuǎn)染前后人子宮內(nèi)膜癌HEC-1A細(xì)胞對(duì)順鉑化療的敏感性。4、順鉑處理子宮內(nèi)膜癌HEC-1A細(xì)胞對(duì)細(xì)胞中YKL-40基因的影響。5、流式細(xì)胞儀檢測(cè)技術(shù)(Annexin V-PE/7AAD雙染法)檢測(cè)轉(zhuǎn)染前后3組細(xì)胞對(duì)順鉑化療的總凋亡率。結(jié)果:1、轉(zhuǎn)染后3組HEC-1A細(xì)胞增殖能力的比較:MTT比色法檢測(cè)顯示,與空載體組和空白對(duì)照組比較,siRNA組細(xì)胞的增殖能力明顯受到抑制(P0.05);而空載組與空白對(duì)照組比較,差異則無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。2、轉(zhuǎn)染后3組HEC-1A細(xì)胞遷移能力的比較:體外transwell小室遷移實(shí)驗(yàn)顯示,siRNA組穿膜細(xì)胞數(shù)為(133±14)個(gè),明顯少于空載體組和空白對(duì)照組[分別為(178±11)和(179±19)個(gè),P0.05];而空載體組與空白對(duì)照組比較,差異則無(wú)統(tǒng)計(jì)學(xué)意義(P=0.872)。3、轉(zhuǎn)染后3組HEC-1A細(xì)胞侵襲能力的比較:體外transwell小室侵襲實(shí)驗(yàn)顯示,siRNA組穿膜細(xì)胞數(shù)為(143±13)個(gè),明顯少于空載體組和空白對(duì)照組[分別為(238±26)和(227±18)個(gè),P0.05];而空載體組與空白對(duì)照組比較,差異則無(wú)統(tǒng)計(jì)學(xué)意義(P=0.411)。4、順鉑對(duì)子宮內(nèi)膜癌HEC-1A細(xì)胞YKL-40基因降低前后細(xì)胞生長(zhǎng)的影響:加入不同濃度梯度的順鉑培養(yǎng)細(xì)胞48h后,細(xì)胞的生長(zhǎng)明顯抑制,實(shí)驗(yàn)組細(xì)胞(轉(zhuǎn)染siRNA后)比空白對(duì)照組和空載組細(xì)胞生長(zhǎng)抑制更顯著(P0.05),但空白對(duì)照組組和空載組無(wú)明顯差異(P0.05)5、順鉑處理前后對(duì)子宮內(nèi)膜癌細(xì)胞中YKL-40表達(dá)的影響比較:經(jīng)過(guò)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(dá)量上調(diào)(P=0.000)。6、經(jīng)相同濃度順鉑處理各組細(xì)胞48小時(shí)3組HEC-1A細(xì)胞總凋亡率的比較:經(jīng)過(guò)相同濃度的順鉑處理子宮內(nèi)膜癌48小時(shí)后siRNA實(shí)驗(yàn)組的增殖能力低于空白對(duì)照組和空載組(P0.05)。siRNA組、空載體組、空白對(duì)照組細(xì)胞總體凋亡率分別為(38.07±4.88)、(13.3±1.01)、(12.5±0.17),siRNA實(shí)驗(yàn)組的總體凋亡率高于空白對(duì)照組和空載體組,差異均有統(tǒng)計(jì)學(xué)意義(P0.05),而空白對(duì)照組和空載組無(wú)明顯差異(P=0.776)。結(jié)論:siRNA沉默HEC-1A細(xì)胞中YKL-40基因表達(dá)可以降低HEC-1A細(xì)胞的增殖、遷移和侵襲能力,并且可以提高HEC-1A細(xì)胞對(duì)順鉑化療的敏感性,經(jīng)過(guò)順鉑處理細(xì)胞48小時(shí)后其凋亡率明顯降低。因此,YKL-40具有促進(jìn)子宮內(nèi)膜癌細(xì)胞增殖、促進(jìn)血管形成以及抗凋亡能力。YKL-40可能是未來(lái)子宮內(nèi)膜癌分子治療的潛在靶點(diǎn)。
[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(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.33

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