中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2011年
10期
1674-1676
,共3页
邱明链%吴燕斌%陈丽红%方航荣%曾金华%刘景丰
邱明鏈%吳燕斌%陳麗紅%方航榮%曾金華%劉景豐
구명련%오연빈%진려홍%방항영%증금화%류경봉
树突状细胞%FasL%转化生长因子-β1%基因修饰%肝移植
樹突狀細胞%FasL%轉化生長因子-β1%基因脩飾%肝移植
수돌상세포%FasL%전화생장인자-β1%기인수식%간이식
Dendritic cells%FasL%TGF-β1%Gene modification%Liver transplantation
目的 观察人凋亡相关因子配体(hFasL)及人转化生长因子-β1( hTGF-β1)基因共转染的大鼠未成熟树突状细胞(imDC)对其生物学活性影响.方法 实验分6组:mDC组、imDC组、空载体组、hFasL基因转染组、hTGF-β1基因转染组及hFasL-hTGF-β1基因共同转染组(即共转染组).用hFasL或(和)hTGF-β1真核表达载体(pIRES2-EGFP-hFasL、pIRES2-EGFP-hTGF-β1)转染培养6d后大鼠骨髓来源的imDC,转染24 ～48 h后应用荧光显微镜、酶联免疫吸附试验(ELISA)、Western blot、流式细胞术及混合淋巴细胞反应(MLR)检测不同基因修饰的imDC的生物学活性.结果 荧光显微镜观察显示pIRES2-EGFP-hFasL及pIRES2-EGFP-hTGF-β1质粒转染imDC后可见绿色荧光；ELISA检测共转染组和转化生长因子(TGF)组TGF-β1基因表达均高于mDC组、imDC组、空载体组及FasL组(P值均＜0.01),imDC组、空载体组及FasL组的TGF-β1基因表达均高于mDC组(P＜0.05).hFasL基因表达各组间的差异均无统计学意义(P值均＞0.05)；经Westen blot检测hFasL及hTGF-β1基因转染组有均有相应蛋白表达(相对分子质量分别为31×103和43×103)；流式细胞术显示表面分子CD86、CD80在mDC表达高于imDC及各基因转染组和空载体组；转染后混合淋巴细胞反应(MLR)显示,共染组T细胞的增殖反应均明显弱于TGF组和FasL组(P＜0.05).结论 联合FasL和TGF-β1基因转染的imDC在体外诱导同种异体免疫耐受的能力强于FasL或TGF-β1单基因转染.
目的 觀察人凋亡相關因子配體(hFasL)及人轉化生長因子-β1( hTGF-β1)基因共轉染的大鼠未成熟樹突狀細胞(imDC)對其生物學活性影響.方法 實驗分6組:mDC組、imDC組、空載體組、hFasL基因轉染組、hTGF-β1基因轉染組及hFasL-hTGF-β1基因共同轉染組(即共轉染組).用hFasL或(和)hTGF-β1真覈錶達載體(pIRES2-EGFP-hFasL、pIRES2-EGFP-hTGF-β1)轉染培養6d後大鼠骨髓來源的imDC,轉染24 ～48 h後應用熒光顯微鏡、酶聯免疫吸附試驗(ELISA)、Western blot、流式細胞術及混閤淋巴細胞反應(MLR)檢測不同基因脩飾的imDC的生物學活性.結果 熒光顯微鏡觀察顯示pIRES2-EGFP-hFasL及pIRES2-EGFP-hTGF-β1質粒轉染imDC後可見綠色熒光；ELISA檢測共轉染組和轉化生長因子(TGF)組TGF-β1基因錶達均高于mDC組、imDC組、空載體組及FasL組(P值均＜0.01),imDC組、空載體組及FasL組的TGF-β1基因錶達均高于mDC組(P＜0.05).hFasL基因錶達各組間的差異均無統計學意義(P值均＞0.05)；經Westen blot檢測hFasL及hTGF-β1基因轉染組有均有相應蛋白錶達(相對分子質量分彆為31×103和43×103)；流式細胞術顯示錶麵分子CD86、CD80在mDC錶達高于imDC及各基因轉染組和空載體組；轉染後混閤淋巴細胞反應(MLR)顯示,共染組T細胞的增殖反應均明顯弱于TGF組和FasL組(P＜0.05).結論 聯閤FasL和TGF-β1基因轉染的imDC在體外誘導同種異體免疫耐受的能力彊于FasL或TGF-β1單基因轉染.
목적 관찰인조망상관인자배체(hFasL)급인전화생장인자-β1( hTGF-β1)기인공전염적대서미성숙수돌상세포(imDC)대기생물학활성영향.방법 실험분6조:mDC조、imDC조、공재체조、hFasL기인전염조、hTGF-β1기인전염조급hFasL-hTGF-β1기인공동전염조(즉공전염조).용hFasL혹(화)hTGF-β1진핵표체재체(pIRES2-EGFP-hFasL、pIRES2-EGFP-hTGF-β1)전염배양6d후대서골수래원적imDC,전염24 ～48 h후응용형광현미경、매련면역흡부시험(ELISA)、Western blot、류식세포술급혼합림파세포반응(MLR)검측불동기인수식적imDC적생물학활성.결과 형광현미경관찰현시pIRES2-EGFP-hFasL급pIRES2-EGFP-hTGF-β1질립전염imDC후가견록색형광；ELISA검측공전염조화전화생장인자(TGF)조TGF-β1기인표체균고우mDC조、imDC조、공재체조급FasL조(P치균＜0.01),imDC조、공재체조급FasL조적TGF-β1기인표체균고우mDC조(P＜0.05).hFasL기인표체각조간적차이균무통계학의의(P치균＞0.05)；경Westen blot검측hFasL급hTGF-β1기인전염조유균유상응단백표체(상대분자질량분별위31×103화43×103)；류식세포술현시표면분자CD86、CD80재mDC표체고우imDC급각기인전염조화공재체조；전염후혼합림파세포반응(MLR)현시,공염조T세포적증식반응균명현약우TGF조화FasL조(P＜0.05).결론 연합FasL화TGF-β1기인전염적imDC재체외유도동충이체면역내수적능력강우FasL혹TGF-β1단기인전염.
Objective To investigate the biological activities of immature dendritic cells (imDCs)modified by hFasL and hTGF-β1 gene,from bone marrow stem cells of rats.Methods The experiment was divided into six groups:mDC group,imDC group,empty vector group,hFasL gene modification group (FasL group),hTGF-β1 gene modification group (TGF group) and hFasL-hTGF-β1 co-transfection group (namely F-T group).The plasmids of pIRES2-EGFP-hFasL or(and) pIRES2-EGFP-hTGF-β1 were transfected into imDCs which were cultivated for six days.The expression of hFasL and hTGF-β1 gene was detected by using fluorescence microscopy,ELISA,Western blotting,flow cytometry and MLR after imDCs were transfected for 24-48 h.Results After transfection,green fluorescence was observed in the transfected imDCs under the fluorescence microscopy.ELISA revealed that the expression of hTGF-β1 in F-T group and TGF group was significantly higher than that in other groups (P ＜0.01 ) ; the expression of hTGF-β1 in imDC group,empty vector group and FasL group was significantly higher than that in mDC group (P ＜0.05) ; and the hFasL gene expression among groups had no significant difference ( P ＞ 0.05 ).Western blotting showed the positive expression of hFasL and hTGF-β1 protein in hFasL and hTGF-β1 gene modification groups.After the plasmids of pIRES2-EGFP-hFasL or(and) pIRES2-EGFP-hTGF-β1 were transfected into imDCs,CD86 and CD80 expression on the imDCs surface were lower than in mDC group by flow cytometry.The result of MLR demonstrated that the imDCs induced lower T-cells in F-T group than in TGF group (P ＜ 0.05 ) and FasL group ( P ＜ 0.05 ).Conclusion The ability of imDCs with hFasL and hTGF-β1 co-transfection was stronger than that transfected by single hFasL or hTGF-β1 in inducing allogeneic immunology tolerance.
