中华器官移植杂志
中華器官移植雜誌
중화기관이식잡지
CHINESE JOURNAL OF ORGAN TRANSPLANTATION
2009年
1期
14-16
,共3页
汪慧访%秦磊%胡明政%钱海鑫
汪慧訪%秦磊%鬍明政%錢海鑫
왕혜방%진뢰%호명정%전해흠
血红素氧化酶(脱环)%腺病毒科%细胞低氧%再灌注损伤%肝细胞
血紅素氧化酶(脫環)%腺病毒科%細胞低氧%再灌註損傷%肝細胞
혈홍소양화매(탈배)%선병독과%세포저양%재관주손상%간세포
Heine oxygenase%Adenoviridae%Cell hypoxia%Reperfusion injury%Hepatocytes
目的 探讨血红素氧合酶-1重组腺病毒载体(Ad-HO-1)体外转染人肝细胞的转染效果及其对肝细胞缺氧-复氧损伤的影响.方法 取人肝细胞系L-02细胞,滴加低温保存的Ad-HO-1,分别培养24 h、48 h和72 h(24 h组、48 h组和72 h组),并以加入空载体腺病毒共培养的L-02细胞为空白对照.采用逆转录聚合酶链反应法检测各组肝细胞HO-1 mRNA的表达水平;以间接免疫荧光标记法检测各组肝细胞的HO-1表达率;在倒置荧光显微镜下观察转染24 h和72 h的肝细胞中绿色荧光蛋白(EGFP)的表达.取空白对照组肝细胞(培养48 h)和48 h组肝细胞,缺氧培养4 h后再有氧培养8 h,采用四甲基偶氮唑盐法测定两组肝细胞存活率.结果 24 h组、48 h组和72 h组HO-1 mRNA表达水平明显高于空白对照组,且随着转染时间的延长,HO-1 mRNA的表达水平逐渐升高.空白对照组HO-1的表达率为2.0%,24 h组为29%,48 h组为85.6%,72 h组为84.6%.基因转染后24 h和72 h,可以观察到L-02细胞中EGFP的表达.经历缺氧-复氧实验后,空白对照组肝细胞的存活率为(37.7±3.5)%,48 h组肝细胞的存活率为(89.4±5.2)%,二者相比较,差异有统计学意义(P<0.01).结论 Ad-HO-1在体外能有效的转染人肝细胞;与未转染者相比,转染肝细胞的缺氧-复氧损伤程度较轻.
目的 探討血紅素氧閤酶-1重組腺病毒載體(Ad-HO-1)體外轉染人肝細胞的轉染效果及其對肝細胞缺氧-複氧損傷的影響.方法 取人肝細胞繫L-02細胞,滴加低溫保存的Ad-HO-1,分彆培養24 h、48 h和72 h(24 h組、48 h組和72 h組),併以加入空載體腺病毒共培養的L-02細胞為空白對照.採用逆轉錄聚閤酶鏈反應法檢測各組肝細胞HO-1 mRNA的錶達水平;以間接免疫熒光標記法檢測各組肝細胞的HO-1錶達率;在倒置熒光顯微鏡下觀察轉染24 h和72 h的肝細胞中綠色熒光蛋白(EGFP)的錶達.取空白對照組肝細胞(培養48 h)和48 h組肝細胞,缺氧培養4 h後再有氧培養8 h,採用四甲基偶氮唑鹽法測定兩組肝細胞存活率.結果 24 h組、48 h組和72 h組HO-1 mRNA錶達水平明顯高于空白對照組,且隨著轉染時間的延長,HO-1 mRNA的錶達水平逐漸升高.空白對照組HO-1的錶達率為2.0%,24 h組為29%,48 h組為85.6%,72 h組為84.6%.基因轉染後24 h和72 h,可以觀察到L-02細胞中EGFP的錶達.經歷缺氧-複氧實驗後,空白對照組肝細胞的存活率為(37.7±3.5)%,48 h組肝細胞的存活率為(89.4±5.2)%,二者相比較,差異有統計學意義(P<0.01).結論 Ad-HO-1在體外能有效的轉染人肝細胞;與未轉染者相比,轉染肝細胞的缺氧-複氧損傷程度較輕.
목적 탐토혈홍소양합매-1중조선병독재체(Ad-HO-1)체외전염인간세포적전염효과급기대간세포결양-복양손상적영향.방법 취인간세포계L-02세포,적가저온보존적Ad-HO-1,분별배양24 h、48 h화72 h(24 h조、48 h조화72 h조),병이가입공재체선병독공배양적L-02세포위공백대조.채용역전록취합매련반응법검측각조간세포HO-1 mRNA적표체수평;이간접면역형광표기법검측각조간세포적HO-1표체솔;재도치형광현미경하관찰전염24 h화72 h적간세포중록색형광단백(EGFP)적표체.취공백대조조간세포(배양48 h)화48 h조간세포,결양배양4 h후재유양배양8 h,채용사갑기우담서염법측정량조간세포존활솔.결과 24 h조、48 h조화72 h조HO-1 mRNA표체수평명현고우공백대조조,차수착전염시간적연장,HO-1 mRNA적표체수평축점승고.공백대조조HO-1적표체솔위2.0%,24 h조위29%,48 h조위85.6%,72 h조위84.6%.기인전염후24 h화72 h,가이관찰도L-02세포중EGFP적표체.경력결양-복양실험후,공백대조조간세포적존활솔위(37.7±3.5)%,48 h조간세포적존활솔위(89.4±5.2)%,이자상비교,차이유통계학의의(P<0.01).결론 Ad-HO-1재체외능유효적전염인간세포;여미전염자상비,전염간세포적결양-복양손상정도교경.
Objective To investigate the effects of transfection of heme oxygenase-1 adenovirus vector (Ad-HO-1) in vitro on hypoxia-reoxygenation injury of human liver ceils. Methods Human liver cell line L-02 cells were cultured for 24, 48 and 72 h (24-h, 48-h and 72-h groups), after adding cryopreserved Ad-HO-1. And L-02 cells were co-cultured with the adenovirus vector vehicle as controls. The HO-1 mRNA expression level of liver cells in each group was detected by reverse transcription PCR. HO-1 expression was detected by indirect immunofluorescence labeling. An inverted fluorescence microscope was applied to observe enhanced green fluorescent protein (EGFP) expression in liver cells 24 and 72 h after transfection. The liver cells in blank control group (culture for 48 h) and 48-h group were cultured for 4 h under hypoxia, followed by culture for 8 h under oxygen condition. The survival rate of cells in two groups was tested by MTT assay. Results HO-1 rnRNA expression in 24-h, 48-h and 72-h groups was significantly higher than in control group, and with the prolongation of the transfection time, HO-1 mRNA expression level was gradually increased. In blank control, 24-h, 48-h, and 72-h groups, HO-1 expression rate was 2.0%, 29%, 85.6%, and 84.6%, respectively. After hypoxia-reoxygenation culture, survival rate in blank control and 48-h groupswas (37.7±3.5)% and (89.4±5.2)% respectively (P<0.01).Conclusion Ad-HO-1 in vitro can be effectively transfected into human liver cells. The hypoxia-reoxygenation injury to liver cells with transfection was milder than that to control cells.
