【作者】 刘敏；

【导师】 王陆飞；

【作者基本信息】 吉林大学 ， 临床医学硕士（眼科学）（专业学位）， 2021， 硕士

【摘要】 目的:1.应用转化生长因子-β2(transforming growth factor,TGF-β2)诱导人来源的晶状体上皮细胞(human lens epithelial cells,HLECs)发生间质化转变,构建间质化细胞模型,检测晶状体上皮细胞中长链非编码RNA结肠癌相关转录本1(Long non-coding RNA colon cancer-associated transcript-1,lncRNA CCAT1)的表达量。2.敲低CCAT1的表达,检测各分子水平间质化标志物的表达量的变化。3.探讨C CAT1在TGF-β2诱导的HLECs的间质化过程中的作用。方法:本实验以TGF-β2诱导HLECs永生系SRA01/04细胞发生间质化,建立间质化细胞模型。含有10ng/ml TGF-β2的低糖DMEM培养液培养SRA01/04细胞48小时作为实验组,以普通低糖DMEM培养基培养SRA01/04细胞作空白对照组。利用倒置显微镜观察细胞的形态学变化,通过实时荧光定量PCR以及Western blot法检测lncRNA CCAT1以及上皮间质转化标志物的相对表达量的变化。采用小干扰RNA(si RNA)沉默CCAT1的表达,采用实时荧光定量PCR以及Western blot法检测lncRNA CCAT1以及上皮间质转化标志物的相对表达量的变化。探讨LncRNA CCAT1在TGF-β2诱导的HLECs间质化过程中的作用及其对PCO形成的影响。结果:1.TGF-β2诱导人晶状体SRA01/04细胞,成功构建间质化细胞模型。倒置显微镜下,实验组细胞形态由多边形变为长梭形。实时荧光定量PCR以及western blot法检测发现,与空白对照组相比较,实验组上皮细胞标志蛋白E-钙黏蛋白(E-cadh erin)表达明显降低(p<0.05),间质细胞标志物波形蛋白(vimentin)(p<0.05),α-平滑肌肌动蛋白(α-SMA)(p<0.05)表达量明显增加,差异有统计学意义。荧光显微镜下发现,与空白对照组相比较,实验组晶状体上皮细胞内α-SMA蛋白以及波形蛋白表达量增加,荧光强度增加,同时细胞形态呈现间质化转变。实时荧光定量PCR检测不同浓度的TGF-β2因子对lncRNA CCAT1表达的影响,发现CCA T1的表达具有浓度依赖性,随着TGF-β2浓度的增加,CCAT1的表达量增加,10ng/ml时CCAT1的表达提高最为明显,所以选用10ng/ml作为处理浓度进行后续实验。2.成功筛选出高效率沉默CCAT1表达的si CCAT1,实时荧光定量PCR结果显示,si CC AT1-1,si CCAT1-2,si CCAT1-3均可沉默CCAT1的表达,但si CCAT1-2的干扰效果最明显,最终我们选择si CCAT1-2进行后续实验。3.运用实时荧光定量PCR以及w estern blot法检测不同CCAT1表达水平下上皮细胞标志蛋白E-钙黏蛋白,以及间质细胞标志蛋白波形蛋白,α-平滑肌肌动蛋白的表达变化。结果发现:与si NC组(标记为NC组)相比,经TGF-β2处理的si NC(标记为Tnc),波形蛋白,α-平滑肌肌动蛋白的表达量增加(P<0.05),E-钙黏蛋白的表达量降低(P<0.05),差异有统计学意义。与Tnc组相比,TGF-β2处理的si CCAT1敲低实验组(标记为Tl nc组),波形蛋白,α-平滑肌肌动蛋白的表达量降低(P<0.05),E-钙黏蛋白的表达量增加(P<0.05),差异有统计学意义。结论:LncRNA CCAT1参与TGF-β2诱导的HLECs间质化过程;沉默LncRNA CCAT1的表达,可逆转TGF-β2引起的HLECs间质化进程。更多还原

【Abstract】 Objective: 1.Transforming growth factor-β2(TGF-β2)was applied to induce the interstitial transformation of human lens epithelial cells(HLECs)to construct the interstitial cell model.Then,the expression level of Long non-coding RNA colon cancerassociated transcript-1(lncRNA CCAT1)in lens epithelial cells was detected.2.The expression of CCAT1 was knocked down to detect the changes in the expression of interstitial markers at the molecular level.3.To investigate the role of CCAT1 in the interstitial process of HLECs induced by TGF-β2.Methods: In this study,TGF-β2 was used to induce the mesenchymal development of HLECs immortal cell line SRA01/04,and the mesenchymal cell model was established.SRA01/04 cells were cultured in low-glucose DMEM medium containing 10ng/ml TGF-β2 for 48 h as the experimental group,and SRA01/04 cells were cultured in normal low-glucose DMEM medium as the blank control group.The morphological changes of cells were observed by inverted microscope,and the changes in the relative expression levels of lncRNA CCAT1 and epithelial mesenchymal transformation markers were detected by real-time fluorescence quantitative PCR and Western blot.The expression of CCAT1 was silenced by small interfering RNA(si RNA),and the relative expression levels of lncRNA CCAT1 and epithelial mesenchymal transformation markers were detected by real-time fluorescence quantitative PCR and Western blot.To investigate the role of lncRNA CCAT1 in the interstitial process of HLECs induced by TGF-β2 and its effect on the formation of PCO.Results: 1.TGF-β2 induced human lens SRA01/04 cells,and successfully constructed mesenchymal cell model.Under inverted microscope,the morphology of cells in the experimental group changed from polygon to long spindle.Real-time quantitative PCR and Western blot analysis showed that the expression of the epithelial marker protein E-cadherin was significantly decreased in the experimental group compared with the blank control group(P< 0.05),the interstitial cell marker vimentin(p< 0.05),α-smooth muscle actin(α-SMA)(P< 0.05),and the difference was statistically significant.Fluorescence microscopy showed that compared with the blank control group,the expression of α-SMA protein and vimentin in the lens epithelial cells of the experimental group increased,the fluorescence intensity increased,and the cell morphology showed interstitial transformation.The effect of TGF-β2 at different concentrations on lncRNA CCAT1 expression was detected by real-time fluorescence quantitative PCR,and the expression of CCAT1 was concentration dependent.With the increase of TGF-β2 concentration,the expression of CCAT1 increased,and the increase was most obvious at 10ng/ml.Therefore,10ng/ml was selected as the treatment concentration for subsequent experiments.2.The si CCAT1 with high efficiency silencing expression of CCAT1 was successfully screened out.Realtime quantitative PCR results showed that siccat1-1,siccat1-2 and siccat1-3 could all silence the expression of CCAT1,but the interference effect of siccat1-2 was the most obvious.Finally,we chose siccat1-2 for subsequent experiments.3.The expression changes of epithelial marker protein E-cadcadin,interstitial marker protein vimentin and α-smooth muscle actin under different CCAT1 expression levels were detected by real-time fluorescence quantitative PCR and Western blot.The results showed that TGF-β2-treated SINC(labeled TNC),vimentin,and α-smooth muscle actin expression increased compared with SINC(labeled NC)(P < 0.05),the expression of E-cadherin decreased(P<0.05),the difference was statistically significant.Compared with the TNC group,the expression of vimentin and α-smooth muscle actin decreased in the TGF-β2-treated si CCAT1 knockdown group(labeled TLNC group)(P< 0.05),the expression of E-cadherin increased(P<0.05),the difference was statistically significant.Conclusion: LncRNA CCAT1 is involved in the interstitial differentiation of h LECs induced by TGF-β2.Silencing the expression of lncRNA CCAT1 can reverse the interstitial process of HLECs induced by TGF-β2.更多还原