中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2011年
12期
1071-1076
,共6页
牛静宜%刘婧%李晓霞%陈建苏%徐锦堂%钟敬祥
牛靜宜%劉婧%李曉霞%陳建囌%徐錦堂%鐘敬祥
우정의%류청%리효하%진건소%서금당%종경상
转化生长因子β诱导基因%角膜上皮细胞%基质金属蛋白酶%真核表达载体
轉化生長因子β誘導基因%角膜上皮細胞%基質金屬蛋白酶%真覈錶達載體
전화생장인자β유도기인%각막상피세포%기질금속단백매%진핵표체재체
Transforming growth factor beta-induced gene%Corneal epithelial cell%Matrix metalloproteinase%Eukaryotic plasmid vector
背景 人转化生长因子-β诱导(TGFBI)基因是第一个被确定的角膜营养不良的致病基因,而TGFBI引起角膜营养不良的机制目前尚不清楚,研究TGFBI的功能对于揭示角膜营养不良的发病机制及了解角膜生理及病理功能都具有重要意义.目的 构建人TGFBI基因的真核表达载体并将其转染于人角膜上皮细胞,探讨其对角膜上皮细胞增生及相关基因表达的影响.方法 从角膜移植后剩余的供体角膜中提取人正常角膜组织总RNA,经逆转录聚合酶链反应(RT-PCR)合成TGFBI cDNA,将TGFBI胶回收产物与载体pCMV-N-HA分别进行双酶切,取连接产物10 μl转化100μl大肠杆菌感受态DH5α克隆入真核表达载体pCMV-N-HA,以菌落PCR和EcoRV、XhoI双酶切法测序鉴定.设置重组质粒pCMV-N-HA-TGFBI转染组、空质粒pCMV-N-HA转染组、阴性对照组及pGFP-C2转染对照组,以测定重组质粒转染率.重组质粒pCMV-N-HA-TGFBI转染人角膜上皮细胞,激光共焦显微镜下观察转染pGFP-C2后增强型绿色荧光蛋白(EGFP)的表达,用细胞计数试剂盒8(CCK-8法)检测转染细胞的增生情况,SYBR荧光实时定量PCR和Western blot法检测TGFBI、基质金属蛋白酶(MMPs)、组织金属蛋白酶抑制剂(TIMP)蛋白及其mRNA在转染后角膜上皮细胞的表达.结果 pCMV-N-HA-TGFBI阳性克隆质粒进行经EcoRV和XhoI双酶切鉴定测序结果显示,扩增的TGFBI cDNA以正确序列和方式插入载体,pGFP-C2载体转染人角膜上皮细胞后48 h共焦显微镜下可见EGFP的表达,转染效率为70％.重组质粒pCMV-N-HA-TGFBI转染组TGFBI mRNA的表达明显高于空质粒pCMV-N-HA转染组和阴性对照组,TGFBI蛋白仅在pCMV-N-HA-TGFBI转染组表达.CCK-8法显示重组质粒pCMV-N-HA-TGFBI转染组、空质粒pCMV-N-HA转染组和阴性对照组间角膜上皮的吸光度(A450)值的差异无统计学意义(F=3.34,P＞0.05).荧光实时定量PCR结果显示,重组质粒pCMV-N-HA-TGFBI转染组中MMP1mRNA、MMP3mRNA转录水平比空质粒pCMV-N-HA转染组和阴性对照组均升高(P＜0.05),而TIMP1mRNA则明显下降(P＜0.05)；Western blot结果证实MMP1、MMP3、TIMP1蛋白表达规律相同(P＜0.05).结论 人TGFBI基因真核表达载体可被成功构建并在人角膜上皮细胞中呈过表达.TGFBI可能是通过MMP1、MMP3及TIMP1的表达变化来增加细胞外基质的降解,从而参与角膜的某些生理病理过程.
揹景 人轉化生長因子-β誘導(TGFBI)基因是第一箇被確定的角膜營養不良的緻病基因,而TGFBI引起角膜營養不良的機製目前尚不清楚,研究TGFBI的功能對于揭示角膜營養不良的髮病機製及瞭解角膜生理及病理功能都具有重要意義.目的 構建人TGFBI基因的真覈錶達載體併將其轉染于人角膜上皮細胞,探討其對角膜上皮細胞增生及相關基因錶達的影響.方法 從角膜移植後剩餘的供體角膜中提取人正常角膜組織總RNA,經逆轉錄聚閤酶鏈反應(RT-PCR)閤成TGFBI cDNA,將TGFBI膠迴收產物與載體pCMV-N-HA分彆進行雙酶切,取連接產物10 μl轉化100μl大腸桿菌感受態DH5α剋隆入真覈錶達載體pCMV-N-HA,以菌落PCR和EcoRV、XhoI雙酶切法測序鑒定.設置重組質粒pCMV-N-HA-TGFBI轉染組、空質粒pCMV-N-HA轉染組、陰性對照組及pGFP-C2轉染對照組,以測定重組質粒轉染率.重組質粒pCMV-N-HA-TGFBI轉染人角膜上皮細胞,激光共焦顯微鏡下觀察轉染pGFP-C2後增彊型綠色熒光蛋白(EGFP)的錶達,用細胞計數試劑盒8(CCK-8法)檢測轉染細胞的增生情況,SYBR熒光實時定量PCR和Western blot法檢測TGFBI、基質金屬蛋白酶(MMPs)、組織金屬蛋白酶抑製劑(TIMP)蛋白及其mRNA在轉染後角膜上皮細胞的錶達.結果 pCMV-N-HA-TGFBI暘性剋隆質粒進行經EcoRV和XhoI雙酶切鑒定測序結果顯示,擴增的TGFBI cDNA以正確序列和方式插入載體,pGFP-C2載體轉染人角膜上皮細胞後48 h共焦顯微鏡下可見EGFP的錶達,轉染效率為70％.重組質粒pCMV-N-HA-TGFBI轉染組TGFBI mRNA的錶達明顯高于空質粒pCMV-N-HA轉染組和陰性對照組,TGFBI蛋白僅在pCMV-N-HA-TGFBI轉染組錶達.CCK-8法顯示重組質粒pCMV-N-HA-TGFBI轉染組、空質粒pCMV-N-HA轉染組和陰性對照組間角膜上皮的吸光度(A450)值的差異無統計學意義(F=3.34,P＞0.05).熒光實時定量PCR結果顯示,重組質粒pCMV-N-HA-TGFBI轉染組中MMP1mRNA、MMP3mRNA轉錄水平比空質粒pCMV-N-HA轉染組和陰性對照組均升高(P＜0.05),而TIMP1mRNA則明顯下降(P＜0.05)；Western blot結果證實MMP1、MMP3、TIMP1蛋白錶達規律相同(P＜0.05).結論 人TGFBI基因真覈錶達載體可被成功構建併在人角膜上皮細胞中呈過錶達.TGFBI可能是通過MMP1、MMP3及TIMP1的錶達變化來增加細胞外基質的降解,從而參與角膜的某些生理病理過程.
배경 인전화생장인자-β유도(TGFBI)기인시제일개피학정적각막영양불량적치병기인,이TGFBI인기각막영양불량적궤제목전상불청초,연구TGFBI적공능대우게시각막영양불량적발병궤제급료해각막생리급병리공능도구유중요의의.목적 구건인TGFBI기인적진핵표체재체병장기전염우인각막상피세포,탐토기대각막상피세포증생급상관기인표체적영향.방법 종각막이식후잉여적공체각막중제취인정상각막조직총RNA,경역전록취합매련반응(RT-PCR)합성TGFBI cDNA,장TGFBI효회수산물여재체pCMV-N-HA분별진행쌍매절,취련접산물10 μl전화100μl대장간균감수태DH5α극륭입진핵표체재체pCMV-N-HA,이균락PCR화EcoRV、XhoI쌍매절법측서감정.설치중조질립pCMV-N-HA-TGFBI전염조、공질립pCMV-N-HA전염조、음성대조조급pGFP-C2전염대조조,이측정중조질립전염솔.중조질립pCMV-N-HA-TGFBI전염인각막상피세포,격광공초현미경하관찰전염pGFP-C2후증강형록색형광단백(EGFP)적표체,용세포계수시제합8(CCK-8법)검측전염세포적증생정황,SYBR형광실시정량PCR화Western blot법검측TGFBI、기질금속단백매(MMPs)、조직금속단백매억제제(TIMP)단백급기mRNA재전염후각막상피세포적표체.결과 pCMV-N-HA-TGFBI양성극륭질립진행경EcoRV화XhoI쌍매절감정측서결과현시,확증적TGFBI cDNA이정학서렬화방식삽입재체,pGFP-C2재체전염인각막상피세포후48 h공초현미경하가견EGFP적표체,전염효솔위70％.중조질립pCMV-N-HA-TGFBI전염조TGFBI mRNA적표체명현고우공질립pCMV-N-HA전염조화음성대조조,TGFBI단백부재pCMV-N-HA-TGFBI전염조표체.CCK-8법현시중조질립pCMV-N-HA-TGFBI전염조、공질립pCMV-N-HA전염조화음성대조조간각막상피적흡광도(A450)치적차이무통계학의의(F=3.34,P＞0.05).형광실시정량PCR결과현시,중조질립pCMV-N-HA-TGFBI전염조중MMP1mRNA、MMP3mRNA전록수평비공질립pCMV-N-HA전염조화음성대조조균승고(P＜0.05),이TIMP1mRNA칙명현하강(P＜0.05)；Western blot결과증실MMP1、MMP3、TIMP1단백표체규률상동(P＜0.05).결론 인TGFBI기인진핵표체재체가피성공구건병재인각막상피세포중정과표체.TGFBI가능시통과MMP1、MMP3급TIMP1적표체변화래증가세포외기질적강해,종이삼여각막적모사생리병리과정.
Background The human transforming growth factor beta-induced gene (TGFBI) is the first determined pathogenic gene to corneal dystrophy.But the molecular genetic mechanism is completely unknown.The study of concerning role of TGFBI is very important for us understand the physiological function of cornea,and the pathogenesis of corneal dystrophy.Objective The vector of human transforming growth factor beta-induced gene (TGFBI) in eukaryotic expression was constructed and transfected into the human corneal epithelial cells in order to explore its influence on the growth of human corneal epithelial cells.Methods Total RNA was extracted from normal donor cornea tissue and cDNA was obtained by reverse transcription.TGFBI cDNA was synthesized by reverse transcription-PCR and cloned into pCMV-N-HA vector and identified by sequencing with PCR and EcoRV,XhoI double restriction endonuclease.The cells were grouped into recombinant pCMV-N-HA-TGFBI plasmid group,pCMVN-HA plasmid group,non-transfected group and pGFP-C2 transfected group.The recombinant pCMV-N-HA-TGFBI plasmid was transfected to human corneal epithelial cells and identified by observing the expression of enhanced green fluorescence protein(EGFP) in the cells.The TGFBI mRNA and proteins were harvested from the cells for real-time PCR analysis and Western blot assay respectively in 58 hours after transfection.The growth of the transfected cells was assessed by Cell Counting Kit-8.The expressions of matrix metalloproteinase(MMP) and tissue inhibitors of matrix metalloproteinase (TIMP) proteins and their mRNA in transfected cells were detected using SYBR fluorescence realtime PCR analysis and Western blot assay.Results The sequencing result of pCMV-N-HA-TGFBI positive clone plasmid showed that amplified TGFBI eDNA inserted into the vector at the correct sequence.EGFP was expressed in transfected cells in 48 hours after transfer of pGFP-C2 with the transfer efficacy 70％.The expression intensity of TGFBI mRNA was significantly higher in recombinant pCMV-N-HA-TGFBI plasmid group compared with pCMV-N-HA plasmid group and non-transfected group,and TGFBI protein was expressed in recombinant pCMV-N-HA-TGFBI plasmid group.No significant difference was found in the A450value among recombinant pCMV-N-HA-TGFBI plasmid group,pCMV-N-HA plasmid group and non-transfected group ( F=3.34,P＞0.05 ).The mRNA level of MMP1,MMP3in the transfected cells was significant elevated but that of TIMP1 was declined in the recombinant pCMV-N-HA-TGFBI plasmid group compared with pCMV-N-HA plasmid group and non-transfected group (all P ＜ 0.05 ).Meanwhile,the expressions of MMP1,MMP3 and TIMP1 proteins appeared the same tendency( all P＜0.05).Conclusions Eukaryotic expression vector harboring human TGFBI eDNA can be successfully constructed and efficiently overexpressed in human corneal epithelial cells.TGFBI gene is involved in the physical and pathological conditions of human corneal epithelial cells by regulating the activity of MMP1,MMP3 and TIMP1.The results offer a new approach for the study of the role of TGFBI in pathogenesis of corneal transparency.
