中华眼科杂志
中華眼科雜誌
중화안과잡지
Chinese Journal of Ophthalmology
2012年
9期
829-835
,共7页
蒋永祥%卢奕%刘天津%杨晋%吴新华
蔣永祥%盧奕%劉天津%楊晉%吳新華
장영상%로혁%류천진%양진%오신화
慢病毒属%整合酶类%基因表达调控%基因,转基因,自杀%DNA结合蛋白质类%晶体%上皮细胞
慢病毒屬%整閤酶類%基因錶達調控%基因,轉基因,自殺%DNA結閤蛋白質類%晶體%上皮細胞
만병독속%정합매류%기인표체조공%기인,전기인,자살%DNA결합단백질류%정체%상피세포
Lentivirus%Integrases%Gene expression regulation%Genes,transgenic,suicide%DNA-binding proteins%Lens,crystalline%Epithelial cells
目的 探讨人晶状体上皮细胞(HLEC)特异性启动子LEP503调控的Cre/LoxP增强型自杀基因(HSV-tk/GCV)系统在眼内主要细胞中的特异性表达及杀伤作用.方法 实验研究.利用分子克隆技术,构建HLEC特异性启动子LEP503调控的HSV-tk/GCV系统并进行慢病毒包装.分别在HLEC、视网膜色素上皮细胞(RPEC)、小鼠成纤维细胞(NIH3T3)、293T细胞中转人此系统,3d后观察4组细胞增强型绿色荧光蛋白(EGFP)表达情况,并收集HLEC和RPEC,通过RT-PCR检测HSV-tk的表达差异.20 mg/L丙氧鸟苷(GCV)作用后,CCK-8试剂盒和流式细胞仪检测HSV-tk/GCV系统对HLEC和RPEC的杀伤作用.采用单因素方差分析Scheffe两两比较法对各组RPEC活性进行比较.结果 HSV-tk/GCV系统在RPEC、NIH3T3、293T细胞中EGFP表达效率分别为62.3％,68.3％,75.8％；在HLEC中EGFP表达效率为17.5％.RT-PCR检测显示HLEC内有较高的HSV-tk表达,相对灰度值为4.01,而RPEC中HSV-tk表达微弱,相对灰度值为0.29.20 mg/L GCV作用72 h后流式细胞仪检测:HLEC组的细胞凋亡率为76.51％,而RPEC组仅为2.44％.CCK-8试剂盒检测细胞增殖抑制情况显示:20 mg/L GCV作用72 h后LEP503调控的HSV-tk/GCV转染的RPEC活性0.9198 ±0.06与正常RPE组0.9672±0.02和阴性对照RPE组0.8927±0.07相比,差异无统计学意义[组间均数差值(MD)分别为MD1=-0.047,P=0.671；MD2 =0.027,P=0.912].结论 HLEC特异性启动子LEP503调控的Cre/LoxP增强型自杀基因系统在HLEC中能特异性表达HSV-tk,GCV作用后可特异性抑制HLEC增殖,但对RPEC无明显杀伤作用.
目的 探討人晶狀體上皮細胞(HLEC)特異性啟動子LEP503調控的Cre/LoxP增彊型自殺基因(HSV-tk/GCV)繫統在眼內主要細胞中的特異性錶達及殺傷作用.方法 實驗研究.利用分子剋隆技術,構建HLEC特異性啟動子LEP503調控的HSV-tk/GCV繫統併進行慢病毒包裝.分彆在HLEC、視網膜色素上皮細胞(RPEC)、小鼠成纖維細胞(NIH3T3)、293T細胞中轉人此繫統,3d後觀察4組細胞增彊型綠色熒光蛋白(EGFP)錶達情況,併收集HLEC和RPEC,通過RT-PCR檢測HSV-tk的錶達差異.20 mg/L丙氧鳥苷(GCV)作用後,CCK-8試劑盒和流式細胞儀檢測HSV-tk/GCV繫統對HLEC和RPEC的殺傷作用.採用單因素方差分析Scheffe兩兩比較法對各組RPEC活性進行比較.結果 HSV-tk/GCV繫統在RPEC、NIH3T3、293T細胞中EGFP錶達效率分彆為62.3％,68.3％,75.8％；在HLEC中EGFP錶達效率為17.5％.RT-PCR檢測顯示HLEC內有較高的HSV-tk錶達,相對灰度值為4.01,而RPEC中HSV-tk錶達微弱,相對灰度值為0.29.20 mg/L GCV作用72 h後流式細胞儀檢測:HLEC組的細胞凋亡率為76.51％,而RPEC組僅為2.44％.CCK-8試劑盒檢測細胞增殖抑製情況顯示:20 mg/L GCV作用72 h後LEP503調控的HSV-tk/GCV轉染的RPEC活性0.9198 ±0.06與正常RPE組0.9672±0.02和陰性對照RPE組0.8927±0.07相比,差異無統計學意義[組間均數差值(MD)分彆為MD1=-0.047,P=0.671；MD2 =0.027,P=0.912].結論 HLEC特異性啟動子LEP503調控的Cre/LoxP增彊型自殺基因繫統在HLEC中能特異性錶達HSV-tk,GCV作用後可特異性抑製HLEC增殖,但對RPEC無明顯殺傷作用.
목적 탐토인정상체상피세포(HLEC)특이성계동자LEP503조공적Cre/LoxP증강형자살기인(HSV-tk/GCV)계통재안내주요세포중적특이성표체급살상작용.방법 실험연구.이용분자극륭기술,구건HLEC특이성계동자LEP503조공적HSV-tk/GCV계통병진행만병독포장.분별재HLEC、시망막색소상피세포(RPEC)、소서성섬유세포(NIH3T3)、293T세포중전인차계통,3d후관찰4조세포증강형록색형광단백(EGFP)표체정황,병수집HLEC화RPEC,통과RT-PCR검측HSV-tk적표체차이.20 mg/L병양조감(GCV)작용후,CCK-8시제합화류식세포의검측HSV-tk/GCV계통대HLEC화RPEC적살상작용.채용단인소방차분석Scheffe량량비교법대각조RPEC활성진행비교.결과 HSV-tk/GCV계통재RPEC、NIH3T3、293T세포중EGFP표체효솔분별위62.3％,68.3％,75.8％；재HLEC중EGFP표체효솔위17.5％.RT-PCR검측현시HLEC내유교고적HSV-tk표체,상대회도치위4.01,이RPEC중HSV-tk표체미약,상대회도치위0.29.20 mg/L GCV작용72 h후류식세포의검측:HLEC조적세포조망솔위76.51％,이RPEC조부위2.44％.CCK-8시제합검측세포증식억제정황현시:20 mg/L GCV작용72 h후LEP503조공적HSV-tk/GCV전염적RPEC활성0.9198 ±0.06여정상RPE조0.9672±0.02화음성대조RPE조0.8927±0.07상비,차이무통계학의의[조간균수차치(MD)분별위MD1=-0.047,P=0.671；MD2 =0.027,P=0.912].결론 HLEC특이성계동자LEP503조공적Cre/LoxP증강형자살기인계통재HLEC중능특이성표체HSV-tk,GCV작용후가특이성억제HLEC증식,단대RPEC무명현살상작용.
Objective To explore the specific expression of HSV-tk gene and killing effects on ocular leading cells of the enhanced specific HSV-tk/GCV gene therapy system regulated by lens-specific promoter LEP503.Methods Experimental research.The enhanced specific HSV-tk/GCV gene system of two vectors were constructed (Lenti-LEP503-HSVtk-Cre and Lenti-HPGK-Loxp-EGFP-pA-Loxp-HSVtk).The lentiviral vectors were produced by transient transduction of transfering vectors,packaging vectors and enveloping vector into 293T cells.Virus was collected with ultracentrifugation and resuspended with 1 ml phosphate buffered saline and stored at -80 ℃.The HLEC and RPEC,NIH3T3,293T cells were transduced with the enhanced specific HSV-tk gene system.The specific expressions of EGFP and HSV-tk were detected by fluorescence microscopy,flow cytometry and RT-PCR. The killing effects of HLEC and RPEC at the concentration of 20 mg/L GCV were assayed and compared by flow cytometry and CCK-8 kit.Difference of RPE cell viability among groups was evaluated by analysis of variance (ANOVA). Results Expression efficiency of EGFP in RPEC group was 62.3％,68.3％ in NIH3T3 group,75.8％ in 293T group,whereas 17.5％ in HLEC group.There was higher expression of HSV-tk at mRNA level in HLEC group than that in RPEC group.The relative intensity of HSV-tk mRNA in HLEC group transduced with the enhanced specific HSV-tk gene system was 4.01,whereas 0.29 in RPEC group.At the concentration of 20 mg/L GCV after 72 hours,the percentage of apoptosis detected by the flow cytometry in HLEC group transduced by the enhanced specific HSV-tk gene system was 76.51％,and 2.44％ in RPEC group.There was no significant difference in the RPE cell viability among the enhanced specific HSV-tk gene combination-RPE group,normal-RPE group and negative-RPE control group at the concentration of 20 mg/L GCV after 72 hours (MD1 =-0.047,P=0.671;MD2 =0.027,P=0.912).Conclusions The enhanced specific HSV-tk gene system express HSV-tk selectively in HLEC.At the concentration of 20 mg/L GCV,it is effective against the proliferation of HLEC in vitro,but has less kill effect on RPEC.
