CAJ | 학술논문

目的构建人Cfos启动子调控的单纯疱疹病毒(HSV)-胸苷激酶(TK)自杀基因重组腺病毒载体,观察其介导的HSV-TK/更昔洛韦(GCV)系统对胶质瘤细胞的杀伤作用. 方法构建重组腺病毒载体Ad-Cfos-TK-IRES-hr绿色荧光蛋白(GFP)、Ad-CMV-TK-IRES-hrGFP,转染至HEK293细胞获得重组腺病毒Ad-Cfos-TK-IRES-hrGFP、Ad-CMV-TK-IRES-hrGFP,荧光计数法检测病毒滴度;将病毒感染U251、U87细胞,荧光显微镜下观察GFP的表达.将病毒感染U251、U87细胞,24 h后加入1000、100、10、1、0.1、0.01、0μmol/L GCV作用48 h,CCK-8法检测各组细胞抑制率. 结果成功构建重组腺病毒载体pDC315-CMV-TK-IRES-hrGFP、pDC315-CFOS-TK-IRES-hrGFP.荧光计数法检测显示2种腺病毒滴度均达1×1010 PFU/mL.在病毒感染复数(MOI)为100、GCV浓度为1、10、100、1000 μmol/L时,转染Ad-Cfos-TK-IRES-hrGFP组U251细胞抑制率分别为(24.18±6.01)％、(30.39±9.67)％、(57.07±9.29)％、(94.50±3.48)％,转染Ad-Cfos-TK-IRES-hrGFP组U87细胞细胞抑制率分别为(9.78±2.24)％、(86.33±5.06)％、(98.48±0.79)％、(98.76±0.93)％. 结论腺病毒介导的HSV-TK/GCV系统在Cfos启动子调控下在体外可以有效杀伤胶质瘤细胞.
목적 구건인Cfos계동자조공적단순포진병독(HSV)-흉감격매(TK)자살기인중조선병독재체,관찰기개도적HSV-TK/경석락위(GCV)계통대효질류세포적살상작용. 방법 구건중조선병독재체Ad-Cfos-TK-IRES-hr록색형광단백(GFP)、Ad-CMV-TK-IRES-hrGFP,전염지HEK293세포획득중조선병독Ad-Cfos-TK-IRES-hrGFP、Ad-CMV-TK-IRES-hrGFP,형광계수법검측병독적도;장병독감염U251、U87세포,형광현미경하관찰GFP적표체.장병독감염U251、U87세포,24 h후가입1000、100、10、1、0.1、0.01、0μmol/L GCV작용48 h,CCK-8법검측각조세포억제솔. 결과 성공구건중조선병독재체pDC315-CMV-TK-IRES-hrGFP、pDC315-CFOS-TK-IRES-hrGFP.형광계수법검측현시2충선병독적도균체1×1010 PFU/mL.재병독감염복수(MOI)위100、GCV농도위1、10、100、1000 μmol/L시,전염Ad-Cfos-TK-IRES-hrGFP조U251세포억제솔분별위(24.18±6.01)％、(30.39±9.67)％、(57.07±9.29)％、(94.50±3.48)％,전염Ad-Cfos-TK-IRES-hrGFP조U87세포세포억제솔분별위(9.78±2.24)％、(86.33±5.06)％、(98.48±0.79)％、(98.76±0.93)％. 결론 선병독개도적HSV-TK/GCV계통재Cfos계동자조공하재체외가이유효살상효질류세포.
Objective To construct recombinant adenovirus vectors containing herpes simple virus thymidine kinase (HSV-TK) controlled by human Cfos promoter and cytomegalovirus (CMV) promoter and observe the expression of adenovirus in human glioma U251 cells and their specific cytotoxic effect on U251 cells in combination with ganciclovir (GCV) in vitro.Methods Two recombinant adenovirus vectors containing herpes simplex virus thymidine kinase gene controlled by human Cfos promoter and CMV promoter were constructed (Ad-Cfos-TK-IRES-hr green fluorescent protein [GFP] and Ad-CMV-TK-IRES-hrGFP),respectively.For packaging virus,two adenovirus vectors were transfected into HEK293 cells.Ad-CMV-TK-IRES-hrGFP and Ad-Cfos-TK-IRES-hrGFP were collected,purified and transfected into U251 cells.The expression of EGFP was observed under a fluorescence microscope.Recombinant adenovirus infected U251 cells were cultured with 1,10,100 and 1000 μmol/L GCV to observe specific cytotoxic effect on glioma.Results The recombinant plasmid vectors Ad-CMV-TK-IRES-hrGFP and Ad-cfos-TK-IRES-hrGFP were verified by restriction endonuclease digestion digestion and sequencing.Ad-CMV-TK-IRES-hrGFP and Ad-Cfos-TK-IRES-hrGFP adenovirus were collected and purified,successfully.The titers of adenovirus reached 1 ×1010 IU/ml.In company with the rising of GCV concentration gradient (1,10,100 and 1000 μmol/L),the inhibition rate of U251 cells gradually increased (24.18％±6.01％,30.39％±9.67％,57.07％±9.29％ and 94.50％±3.48％) at mutiplicity of infection (MOI) 100.In company with the rising of GCV concentration gradient (1,10,100 and 1000 μmol/L),the inhibition rate of U87 cells gradually increased (9.78％± 2.24％,86.33％ ±5.06％,98.48％ ±0.79％ and 98.76％ ±0.93) at MOI 100.Conclusion Adenoviral-mediated delivery of herpes simplex virus thymidine kinase gene controlled by Cfos promoter can confer cytotoxic effect on human glioma cells in vitro.