中华眼科杂志
中華眼科雜誌
중화안과잡지
Chinese Journal of Ophthalmology
2010年
1期
56-61
,共6页
葛红岩%宋甄%施展%刘平
葛紅巖%宋甄%施展%劉平
갈홍암%송견%시전%류평
转化生长因子β%细胞外基质蛋白质类%诱变%定点%上皮%角膜%上皮细胞%转染%细胞凋亡
轉化生長因子β%細胞外基質蛋白質類%誘變%定點%上皮%角膜%上皮細胞%轉染%細胞凋亡
전화생장인자β%세포외기질단백질류%유변%정점%상피%각막%상피세포%전염%세포조망
Transforming growth factor beta%Extracellular matrix proteins%Mutagenesis%site-directed%Epithelium%corneal%Epithelial cells%Transfection%Apoptosis
目的 构建野生型BIGH3和突变型R555W-BIGH3基因的真核表达载体,探讨其过表达对人角膜上皮细胞的影响.方法 以pMD18-T/BIGH3载体为模板扩增野生型BIGH3基因全长编码序列,克隆至真核表达载体pIRES2-EGFP上,采用快速体外定点诱变技术获得R555W突变体.瞬时转染体外培养的人角膜上皮细胞,免疫印迹检测野生与突变型BIGH3基因的表达,流式细胞仪和透射电镜定量和定性检查细胞凋亡,分析caspase3活性.多组间数据采用单因素方差分析进行统计学处理.结果 PCR成功扩增了野生型和R555W突变型的BIGH3基因.荧光显微镜下可见成功转染BIGH3/R555W-BIGH3-pIRES2-EGFP的CRL-11135细胞发绿色荧光,转染率为30.1%.瞬时转染48 h后的HCE细胞的上清液及其细胞裂解液免疫印迹分析显示,BIGH3/R555W-BIGH3-pIRES2-EGFP两组免疫印迹条带的密度比分别为1.26±0.06和1.27±0.08.正常HCE细胞组、转染pIRES2-EGFP空质粒及BIGH3-pIRES2-EGFP细胞凋亡率分别为(1.49 ±0.02)%、(1.53 ±0.02)%、(1.50 ±0.06)%,转染R555W-BIGH3-pIRES2-EGFP组细胞凋亡率达到19.46%±0.08%.差异有统计学意义(F=175 261.23,p<0.01).透射电镜下可见转染组细胞核内染色质凝集,固缩,分布于核膜下,核仁消失,有的胞核碎裂,细胞呈典型的凋亡改变.转染R555W-BIGH3-pIRES2-EGFP组caspase-3活性为561.03 ±1.05,与其他组相比,差异有统计学意义(F=629 347.38,P<0.01).结论 应用快速体外定点诱变技术,成功获得突变型R555W-BIGH3基因,转染人角膜上皮细胞后,通过caspase3途径引起细胞凋亡.
目的 構建野生型BIGH3和突變型R555W-BIGH3基因的真覈錶達載體,探討其過錶達對人角膜上皮細胞的影響.方法 以pMD18-T/BIGH3載體為模闆擴增野生型BIGH3基因全長編碼序列,剋隆至真覈錶達載體pIRES2-EGFP上,採用快速體外定點誘變技術穫得R555W突變體.瞬時轉染體外培養的人角膜上皮細胞,免疫印跡檢測野生與突變型BIGH3基因的錶達,流式細胞儀和透射電鏡定量和定性檢查細胞凋亡,分析caspase3活性.多組間數據採用單因素方差分析進行統計學處理.結果 PCR成功擴增瞭野生型和R555W突變型的BIGH3基因.熒光顯微鏡下可見成功轉染BIGH3/R555W-BIGH3-pIRES2-EGFP的CRL-11135細胞髮綠色熒光,轉染率為30.1%.瞬時轉染48 h後的HCE細胞的上清液及其細胞裂解液免疫印跡分析顯示,BIGH3/R555W-BIGH3-pIRES2-EGFP兩組免疫印跡條帶的密度比分彆為1.26±0.06和1.27±0.08.正常HCE細胞組、轉染pIRES2-EGFP空質粒及BIGH3-pIRES2-EGFP細胞凋亡率分彆為(1.49 ±0.02)%、(1.53 ±0.02)%、(1.50 ±0.06)%,轉染R555W-BIGH3-pIRES2-EGFP組細胞凋亡率達到19.46%±0.08%.差異有統計學意義(F=175 261.23,p<0.01).透射電鏡下可見轉染組細胞覈內染色質凝集,固縮,分佈于覈膜下,覈仁消失,有的胞覈碎裂,細胞呈典型的凋亡改變.轉染R555W-BIGH3-pIRES2-EGFP組caspase-3活性為561.03 ±1.05,與其他組相比,差異有統計學意義(F=629 347.38,P<0.01).結論 應用快速體外定點誘變技術,成功穫得突變型R555W-BIGH3基因,轉染人角膜上皮細胞後,通過caspase3途徑引起細胞凋亡.
목적 구건야생형BIGH3화돌변형R555W-BIGH3기인적진핵표체재체,탐토기과표체대인각막상피세포적영향.방법 이pMD18-T/BIGH3재체위모판확증야생형BIGH3기인전장편마서렬,극륭지진핵표체재체pIRES2-EGFP상,채용쾌속체외정점유변기술획득R555W돌변체.순시전염체외배양적인각막상피세포,면역인적검측야생여돌변형BIGH3기인적표체,류식세포의화투사전경정량화정성검사세포조망,분석caspase3활성.다조간수거채용단인소방차분석진행통계학처리.결과 PCR성공확증료야생형화R555W돌변형적BIGH3기인.형광현미경하가견성공전염BIGH3/R555W-BIGH3-pIRES2-EGFP적CRL-11135세포발록색형광,전염솔위30.1%.순시전염48 h후적HCE세포적상청액급기세포렬해액면역인적분석현시,BIGH3/R555W-BIGH3-pIRES2-EGFP량조면역인적조대적밀도비분별위1.26±0.06화1.27±0.08.정상HCE세포조、전염pIRES2-EGFP공질립급BIGH3-pIRES2-EGFP세포조망솔분별위(1.49 ±0.02)%、(1.53 ±0.02)%、(1.50 ±0.06)%,전염R555W-BIGH3-pIRES2-EGFP조세포조망솔체도19.46%±0.08%.차이유통계학의의(F=175 261.23,p<0.01).투사전경하가견전염조세포핵내염색질응집,고축,분포우핵막하,핵인소실,유적포핵쇄렬,세포정전형적조망개변.전염R555W-BIGH3-pIRES2-EGFP조caspase-3활성위561.03 ±1.05,여기타조상비,차이유통계학의의(F=629 347.38,P<0.01).결론 응용쾌속체외정점유변기술,성공획득돌변형R555W-BIGH3기인,전염인각막상피세포후,통과caspase3도경인기세포조망.
Objective To construct the eukaryotic express vector of wild type and R555W mutated transforming growth factor β induced ( BIGH3 ) gene, and to determine the effects of overexpression of R555W mutated BIGH3 in human corneal epithelial (HCE) cells.Methods The full coding domain sequence of BIGH3 gene was cloned from human corneal tissue from operative spaceman with RT-PCR.Mutagenesis of R555W-BIGH3 was performed in rapid site-directed mutagenesis technique in the expression vector pIRES2-EGFP.The two types of BIGH3 gene were transient transfected to HCE cells.Immunoblotting was used to detect the expression of BIGH3.The cell apoptosis rates were observed by flow cytometry.The morphological changes of the cells were examined by electron microscopy.The viability of caspase-3 was examined.Results Wild type and mutated BIGH3 gene were successfully amplified by PCR.After HCE cells transfected with two types eukaryotic expression plasmind, the BIGH3 were detected in the HCE cells.The cell apoptosis were observed by flow cytometry and electron microscopy.The viability of caspase-3 was increased in the RSSSW mutated type.Conclusions The R555W mutated BIGH3 is successfully obtained by rapid site-directed mutagenesis technique.Overexpression of R555W mutated BIGH3 induces apoptosis in HCE cells through activation of caspase-3.
