中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2014年
1期
92-94
,共3页
张继勤%刘作金%李钺%杨慷%陈玉培
張繼勤%劉作金%李鉞%楊慷%陳玉培
장계근%류작금%리월%양강%진옥배
骨髓间充质干细胞%枯否细胞%共培养%重塑
骨髓間充質榦細胞%枯否細胞%共培養%重塑
골수간충질간세포%고부세포%공배양%중소
Bone marrow stromal cells%Kupffer cells%Co-culture%Reprogram
目的 建立骨髓间充质干细胞(BMSCs)与枯否细胞(KCs)的体外共培养体系,并观察BMSCs对KCs表型的影响,为BMSCs在肝脏移植中的应用提供依据.方法 建立KCs与BMSCs(1:1)直接接触共培养体系作为实验组,单独培养KCs为对照组.两组细胞均加入内毒素(LPS,1 mg/L),培养24h后,应用实时定量聚合酶链反应(Real-time PCR)法及Western blot法检测白细胞介素(IL)-10基因及蛋白的表达、酶联免疫吸附试验(ELISA)法检测细胞IL-10、前列腺素E2(PGE2)、IL-6分泌量、流式细胞仪检测人类主要组织相容性复合体(MHC)Ⅱ、CD40、CD80、CD86表达.结果 Real-time PCR及Western blot检测结果显示,与对照组比较,共培养体系能明显增加IL-10基因和蛋白质的表达(P＜0.05).LPS刺激后,共培养组IL-10分泌量(2 307.44±308.97) ng/L与PGE2分泌量(424.48 ±47.86) ng/L均比对照组高[IL-10:(1 000.12±49.06) ng/L;PGE2:(244.48 ±28.15) ng/L,P ＜0.01]；IL-6分泌量低于对照组[BMSCs+ KCs:(78.14±10.58) ng/L;KCs:(283.18 ±20.50)ng/L,P＜0.01].流式细胞细胞仪检测显示,LPS刺激24h后,与对照组高表达MHCⅡ(99.80％)、CD40(99.26％)、CD80(99.93％)及CD86(99.92％)比较,共培养组MHCⅡ (95.18％)、CD40(89.92％)、CD80(99.89％)、CD86(94.75％)表达下调.结论 BMSCs能干扰KCs功能表达,重塑KCs表型,诱导KCs向M2型巨噬细胞转变,产生抑制免疫应答效应.
目的 建立骨髓間充質榦細胞(BMSCs)與枯否細胞(KCs)的體外共培養體繫,併觀察BMSCs對KCs錶型的影響,為BMSCs在肝髒移植中的應用提供依據.方法 建立KCs與BMSCs(1:1)直接接觸共培養體繫作為實驗組,單獨培養KCs為對照組.兩組細胞均加入內毒素(LPS,1 mg/L),培養24h後,應用實時定量聚閤酶鏈反應(Real-time PCR)法及Western blot法檢測白細胞介素(IL)-10基因及蛋白的錶達、酶聯免疫吸附試驗(ELISA)法檢測細胞IL-10、前列腺素E2(PGE2)、IL-6分泌量、流式細胞儀檢測人類主要組織相容性複閤體(MHC)Ⅱ、CD40、CD80、CD86錶達.結果 Real-time PCR及Western blot檢測結果顯示,與對照組比較,共培養體繫能明顯增加IL-10基因和蛋白質的錶達(P＜0.05).LPS刺激後,共培養組IL-10分泌量(2 307.44±308.97) ng/L與PGE2分泌量(424.48 ±47.86) ng/L均比對照組高[IL-10:(1 000.12±49.06) ng/L;PGE2:(244.48 ±28.15) ng/L,P ＜0.01]；IL-6分泌量低于對照組[BMSCs+ KCs:(78.14±10.58) ng/L;KCs:(283.18 ±20.50)ng/L,P＜0.01].流式細胞細胞儀檢測顯示,LPS刺激24h後,與對照組高錶達MHCⅡ(99.80％)、CD40(99.26％)、CD80(99.93％)及CD86(99.92％)比較,共培養組MHCⅡ (95.18％)、CD40(89.92％)、CD80(99.89％)、CD86(94.75％)錶達下調.結論 BMSCs能榦擾KCs功能錶達,重塑KCs錶型,誘導KCs嚮M2型巨噬細胞轉變,產生抑製免疫應答效應.
목적 건립골수간충질간세포(BMSCs)여고부세포(KCs)적체외공배양체계,병관찰BMSCs대KCs표형적영향,위BMSCs재간장이식중적응용제공의거.방법 건립KCs여BMSCs(1:1)직접접촉공배양체계작위실험조,단독배양KCs위대조조.량조세포균가입내독소(LPS,1 mg/L),배양24h후,응용실시정량취합매련반응(Real-time PCR)법급Western blot법검측백세포개소(IL)-10기인급단백적표체、매련면역흡부시험(ELISA)법검측세포IL-10、전렬선소E2(PGE2)、IL-6분비량、류식세포의검측인류주요조직상용성복합체(MHC)Ⅱ、CD40、CD80、CD86표체.결과 Real-time PCR급Western blot검측결과현시,여대조조비교,공배양체계능명현증가IL-10기인화단백질적표체(P＜0.05).LPS자격후,공배양조IL-10분비량(2 307.44±308.97) ng/L여PGE2분비량(424.48 ±47.86) ng/L균비대조조고[IL-10:(1 000.12±49.06) ng/L;PGE2:(244.48 ±28.15) ng/L,P ＜0.01]；IL-6분비량저우대조조[BMSCs+ KCs:(78.14±10.58) ng/L;KCs:(283.18 ±20.50)ng/L,P＜0.01].류식세포세포의검측현시,LPS자격24h후,여대조조고표체MHCⅡ(99.80％)、CD40(99.26％)、CD80(99.93％)급CD86(99.92％)비교,공배양조MHCⅡ (95.18％)、CD40(89.92％)、CD80(99.89％)、CD86(94.75％)표체하조.결론 BMSCs능간우KCs공능표체,중소KCs표형,유도KCs향M2형거서세포전변,산생억제면역응답효응.
Objective To investigate the phenomenon of bone marrow stromal cells (BMSCs) regulating the function of Kupffer cells (KCs) under direct contact co-culture in vitro.Methods In co-culture experiments,pure rat BMSCs were directly added to the KCs plates with the ratio of 1∶ 1.KCs were separately cultured in a well as control group.After stimulation with lipopolysaccharide (LPS,1 mg/L) for 24 h,the gene and protein expression of interleukin (IL)-10 was detected by using real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting,respectively.The abundance of IL-10,prostaglandin E2 (PGE2) and IL-6 was detected by using enzyme linked immunosorbent assay (ELISA) kits.Flow cytometry was used to detect the expression of major histocompatibility complex (MHC) Ⅱ,CD40,CD80 and CD86.Results As compared with control group,the gene and protein expression of IL-10 in co-culture system was significantly increased (P ＜ 0.05).After co-culture with BMSCs,the levels of IL-10 [KCs:(1000.12±49.06) ng/L; KCs + BMSCs:(2307.44 ±308.97) ng/L] and PGE2[KCs:(244.48 ±28.15) ng/L; KCs + BMSCs:(424.48 ± 47.86) ng/L] were significantly increased (P ＜ 0.01),while the abundance of IL-6 [KCs:(283.18 ± 20.50) ng/L; KCs + BMSCs:(78.14 ± 10.58) ng/L] was significantly down-regulated (P ＜ 0.01).In co-culture system,the expression of MHC Ⅱ,CD40,CD80 and CD86 was lower than in control group.Conclusion BMSCs may reprogram the expression patterns of KCs,regulate the function of KCs,and then induce immune tolerance.
