中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2011年
2期
97-102
,共6页
高峰%林雨佳%李国良%吴德全
高峰%林雨佳%李國良%吳德全
고봉%림우가%리국량%오덕전
间充质干细胞%一氧化氮%Th1/Th2%免疫调节
間充質榦細胞%一氧化氮%Th1/Th2%免疫調節
간충질간세포%일양화담%Th1/Th2%면역조절
Mesenchymal stem cells%Nitric oxide%Th1/Th2%Immune regulation
目的 研究一氧化氮(nitric oxide,NO)在骨髓间充质干细胞(mesenchymal stem cells,MSCs)的免疫调节作用.方法 贴壁筛选法分离培养Lewis大鼠骨髓间充质干细胞.以刀豆蛋白A(concanavalin A,ConA)作为刺激因素,SD大鼠外周血分离的T淋巴细胞作为反应细胞,采用CCK-8法检测T淋巴细胞与MSCs共培养后其增殖能力的变化.然后采用RT-PCR、Western blot检测MSCs诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)mRNA与蛋白的表达;Griess法检测上清液亚硝酸盐含量;ELISA检测上清液Th1细胞因子IFN-γ和Th2细胞因子IL-4的水平.结果 MSCs明显抑制ConA诱导的T淋巴细胞增殖,抑制作用因细胞比例的不同而有所区别.当实验组MSC:T为1:10时最明显,增殖率为(33.83±2.10)%,而1:80组[(78.62±3.80)%]与不含MSCs的阳性对照组[(79.03±1.70)%]差异无统计学意义(P＞0.05).实验组MSCs的iNOS mRNA与蛋白的表达以及上清液中亚硝酸盐含量均较阳性对照组明显上调,1:10～1:40的范围内呈明显递增趋势,但1:80组与1:40组差异无统计学意义(P＞0.05).在加入iNOS特异性抑制剂2-甲基-2-巯基硫酸脲(S-methylisothiourea sulfate,SMT)后,各组T淋巴细胞增殖率基本恢复.实验组IFN-γ含量随MSCs所占比例的降低而增加,各组IL-4含最差异无统计学意义(P＞0.05).结论 MSCs对同种异体外周血T淋巴细胞的增殖有免疫调节作用,机制可能与其分泌的可溶性因子NO影响了 Th1/Th2平衡有关.
目的 研究一氧化氮(nitric oxide,NO)在骨髓間充質榦細胞(mesenchymal stem cells,MSCs)的免疫調節作用.方法 貼壁篩選法分離培養Lewis大鼠骨髓間充質榦細胞.以刀豆蛋白A(concanavalin A,ConA)作為刺激因素,SD大鼠外週血分離的T淋巴細胞作為反應細胞,採用CCK-8法檢測T淋巴細胞與MSCs共培養後其增殖能力的變化.然後採用RT-PCR、Western blot檢測MSCs誘導型一氧化氮閤酶(inducible nitric oxide synthase,iNOS)mRNA與蛋白的錶達;Griess法檢測上清液亞硝痠鹽含量;ELISA檢測上清液Th1細胞因子IFN-γ和Th2細胞因子IL-4的水平.結果 MSCs明顯抑製ConA誘導的T淋巴細胞增殖,抑製作用因細胞比例的不同而有所區彆.噹實驗組MSC:T為1:10時最明顯,增殖率為(33.83±2.10)%,而1:80組[(78.62±3.80)%]與不含MSCs的暘性對照組[(79.03±1.70)%]差異無統計學意義(P＞0.05).實驗組MSCs的iNOS mRNA與蛋白的錶達以及上清液中亞硝痠鹽含量均較暘性對照組明顯上調,1:10～1:40的範圍內呈明顯遞增趨勢,但1:80組與1:40組差異無統計學意義(P＞0.05).在加入iNOS特異性抑製劑2-甲基-2-巰基硫痠脲(S-methylisothiourea sulfate,SMT)後,各組T淋巴細胞增殖率基本恢複.實驗組IFN-γ含量隨MSCs所佔比例的降低而增加,各組IL-4含最差異無統計學意義(P＞0.05).結論 MSCs對同種異體外週血T淋巴細胞的增殖有免疫調節作用,機製可能與其分泌的可溶性因子NO影響瞭 Th1/Th2平衡有關.
목적 연구일양화담(nitric oxide,NO)재골수간충질간세포(mesenchymal stem cells,MSCs)적면역조절작용.방법 첩벽사선법분리배양Lewis대서골수간충질간세포.이도두단백A(concanavalin A,ConA)작위자격인소,SD대서외주혈분리적T림파세포작위반응세포,채용CCK-8법검측T림파세포여MSCs공배양후기증식능력적변화.연후채용RT-PCR、Western blot검측MSCs유도형일양화담합매(inducible nitric oxide synthase,iNOS)mRNA여단백적표체;Griess법검측상청액아초산염함량;ELISA검측상청액Th1세포인자IFN-γ화Th2세포인자IL-4적수평.결과 MSCs명현억제ConA유도적T림파세포증식,억제작용인세포비례적불동이유소구별.당실험조MSC:T위1:10시최명현,증식솔위(33.83±2.10)%,이1:80조[(78.62±3.80)%]여불함MSCs적양성대조조[(79.03±1.70)%]차이무통계학의의(P＞0.05).실험조MSCs적iNOS mRNA여단백적표체이급상청액중아초산염함량균교양성대조조명현상조,1:10～1:40적범위내정명현체증추세,단1:80조여1:40조차이무통계학의의(P＞0.05).재가입iNOS특이성억제제2-갑기-2-구기류산뇨(S-methylisothiourea sulfate,SMT)후,각조T림파세포증식솔기본회복.실험조IFN-γ함량수MSCs소점비례적강저이증가,각조IL-4함최차이무통계학의의(P＞0.05).결론 MSCs대동충이체외주혈T림파세포적증식유면역조절작용,궤제가능여기분비적가용성인자NO영향료 Th1/Th2평형유관.
Objective To observe the role of nitric oxide(NO) in the immune regulatory effect of bone marrow mesenchymal stem cells(MSCs). Methods Bone marrow MSCs were isolated from Lewis rats by adherence screening. Concanavalin A (ConA) was adopted as the stimulator and T-lymphocyte isolated from peripheral blood of SD rats as the reactive cells. The changes of the ability of T-lymphocyte proliferation, when co-cultured with MSCs, were measured by CCK-8 assay. The inducible nitric oxide synthase (iNOS) mRNA and protein expression on MSCs and were detected by RT-PCR, Western blot. The contents of nitrites and the levels of Th1 type cytokine IFN-γand Th2 type cytokine IL-4 were measured by Griess test and ELISA respectively, in the co-cultured supernatant. Results T-lymphocyte proliferation was inhibited by co-cultured MSCs, which was concentration-dependent. In this study, the inhibition was most obviously group[ (79.03 ± 1.70)% ] (P ＞ 0.05 ). The I NOS mRNA expression, protein and nitrite levels were signifigroups, the proliferation rate of T-lymphocyte recovered. The content of IFN-γwas increased with the ratio decline of MSCs in the experimental group and IL-4 in each group has no significant difference( P ＞ 0.05 ).Conclusion MSCs inhibited T-lymphocyte proliferation by influencing Th1/Th2 balance, and the secretion of soluble factor NO, which secreted by MSCs, may plays an important role in the immune regulation.
