中华器官移植杂志
中華器官移植雜誌
중화기관이식잡지
CHINESE JOURNAL OF ORGAN TRANSPLANTATION
2014年
6期
370-373
,共4页
赵越%王璐%阮永乐%王筱啸%贾钰%向莹%陈刚
趙越%王璐%阮永樂%王篠嘯%賈鈺%嚮瑩%陳剛
조월%왕로%원영악%왕소소%가옥%향형%진강
脂肪干细胞%体液免疫%异种抗原
脂肪榦細胞%體液免疫%異種抗原
지방간세포%체액면역%이충항원
Adipose-derived stem cell%Humoral immunity%Xenoantigen
目的 研究大鼠脂肪来源干细胞(rASC)抵抗人血清介导的异种体液性杀伤的作用及其主要机制.方法 分离培养SD大鼠ASC,将2～8代的rASC作为实验材料,以大鼠淋巴细胞(rLC)作为对照.采用流式细胞技术,检测两种细胞异种抗原α-Gal的表达情况,体积分数20％正常人血清对两种细胞的杀伤作用、两种细胞分别与正常人血清中天然抗体IgM和IgG的结合情况,以及与正常人血清作用后补体C3c、C4c、C5b-9在两种细胞上的沉积情况.结果 成功分离和培养rASC,rASC表面标志检测呈CD44阳性、CD45阴性、CD90阳性及主要组织相容性抗原Ⅱ阴性,并成功诱导rASC向成脂细胞和成骨细胞分化.相对于rLC,rASC对人血清介导的异种体液性杀伤具有明显的抵抗作用,其几何平均荧光强度(Gmean)值为(20.42±2.80)％,低于rLC的(51.84±6.70)％(P＜0.01);rASC上α-Gal的表达量为13.97±0.33,显著低于rLC的24.47±3.03 (P＜0.05);rASC与人血清中IgG和IgM的结合量分别为9.4±2.0和5.73±1.0,显著低于rLC的107.2±4.8和27.49±3.9(P＜0.01);与正常人血清作用后,rLC可见显著的C3c、C4c和C5b-9沉积(Gmean值分别为1 924±509.4、177.3±37.17和37.05±7.4),但rASC仅有较少C3c和C4c沉积(Gmean值分别为294.6±38.02和35.23±3.1),C5b-9沉积几乎为阴性(Gmean值为5.63±1.74),两种细胞间比较,差异均有统计学意义(P＜0.05或P＜0.01).结论 rASC能明显抵抗人血清介导的异种体液免疫杀伤作用,其机制可能与rASC低表达异种抗原α-Gal及抑制了补体攻膜复合物形成有关.
目的 研究大鼠脂肪來源榦細胞(rASC)牴抗人血清介導的異種體液性殺傷的作用及其主要機製.方法 分離培養SD大鼠ASC,將2～8代的rASC作為實驗材料,以大鼠淋巴細胞(rLC)作為對照.採用流式細胞技術,檢測兩種細胞異種抗原α-Gal的錶達情況,體積分數20％正常人血清對兩種細胞的殺傷作用、兩種細胞分彆與正常人血清中天然抗體IgM和IgG的結閤情況,以及與正常人血清作用後補體C3c、C4c、C5b-9在兩種細胞上的沉積情況.結果 成功分離和培養rASC,rASC錶麵標誌檢測呈CD44暘性、CD45陰性、CD90暘性及主要組織相容性抗原Ⅱ陰性,併成功誘導rASC嚮成脂細胞和成骨細胞分化.相對于rLC,rASC對人血清介導的異種體液性殺傷具有明顯的牴抗作用,其幾何平均熒光彊度(Gmean)值為(20.42±2.80)％,低于rLC的(51.84±6.70)％(P＜0.01);rASC上α-Gal的錶達量為13.97±0.33,顯著低于rLC的24.47±3.03 (P＜0.05);rASC與人血清中IgG和IgM的結閤量分彆為9.4±2.0和5.73±1.0,顯著低于rLC的107.2±4.8和27.49±3.9(P＜0.01);與正常人血清作用後,rLC可見顯著的C3c、C4c和C5b-9沉積(Gmean值分彆為1 924±509.4、177.3±37.17和37.05±7.4),但rASC僅有較少C3c和C4c沉積(Gmean值分彆為294.6±38.02和35.23±3.1),C5b-9沉積幾乎為陰性(Gmean值為5.63±1.74),兩種細胞間比較,差異均有統計學意義(P＜0.05或P＜0.01).結論 rASC能明顯牴抗人血清介導的異種體液免疫殺傷作用,其機製可能與rASC低錶達異種抗原α-Gal及抑製瞭補體攻膜複閤物形成有關.
목적 연구대서지방래원간세포(rASC)저항인혈청개도적이충체액성살상적작용급기주요궤제.방법 분리배양SD대서ASC,장2～8대적rASC작위실험재료,이대서림파세포(rLC)작위대조.채용류식세포기술,검측량충세포이충항원α-Gal적표체정황,체적분수20％정상인혈청대량충세포적살상작용、량충세포분별여정상인혈청중천연항체IgM화IgG적결합정황,이급여정상인혈청작용후보체C3c、C4c、C5b-9재량충세포상적침적정황.결과 성공분리화배양rASC,rASC표면표지검측정CD44양성、CD45음성、CD90양성급주요조직상용성항원Ⅱ음성,병성공유도rASC향성지세포화성골세포분화.상대우rLC,rASC대인혈청개도적이충체액성살상구유명현적저항작용,기궤하평균형광강도(Gmean)치위(20.42±2.80)％,저우rLC적(51.84±6.70)％(P＜0.01);rASC상α-Gal적표체량위13.97±0.33,현저저우rLC적24.47±3.03 (P＜0.05);rASC여인혈청중IgG화IgM적결합량분별위9.4±2.0화5.73±1.0,현저저우rLC적107.2±4.8화27.49±3.9(P＜0.01);여정상인혈청작용후,rLC가견현저적C3c、C4c화C5b-9침적(Gmean치분별위1 924±509.4、177.3±37.17화37.05±7.4),단rASC부유교소C3c화C4c침적(Gmean치분별위294.6±38.02화35.23±3.1),C5b-9침적궤호위음성(Gmean치위5.63±1.74),량충세포간비교,차이균유통계학의의(P＜0.05혹P＜0.01).결론 rASC능명현저항인혈청개도적이충체액면역살상작용,기궤제가능여rASC저표체이충항원α-Gal급억제료보체공막복합물형성유관.
Objective To investigate whether rat adipose-derived stem cells (rASCs) could resist human xenoantibody-dependent complement-mediated lysis and to explore its possible mechanisms.Method SD rat ASCs were isolated,rASCs at passage 2 to 8 were used for the following studies and rat lymphocytes were harvested as control cells.α-Gal expression was detected by flow cytometry.After incubation of rASCs with 20％ normal human serum (NHS) or heat inactivated normal human serum (HINHS),flow cytometry was used to detect cytotoxicity,IgG or IgM binding,and C3c,C4c and C5b-9 deposition.Result We successfully established the method to isolate and culture rASCs.The morphology of rASCs remained unchanged after passages.rASCs were positive for tell surface markers of CD44 and CD90,while negative for CD45 and MHC-Ⅱ.As compared with rLCs,rASCs significantly resisted human natural antibody and complement-mediated lysis when incubated with 20％ NHS in vitro (20.42％ ± 2.80％ vs 51.84％ ± 6.70％,P ＜ 0.01).Mechanistically,rASCs expressed lower level of α-Gal (13.97 ± 0.33 vs.24.47 ± 3.03,P＜0.05),which was correlated with decreased binding of human xenoreactive IgG and IgM (IgM:9.4 ± 2.0 vs.107.2± 4.8,P＜0.01; IgG:5.73 ± 1.0 vs.27.49 ± 3.9,P＜0.01) and reduced deposition of complements C3c,C4c and C5b-9 (C3c:294.6 ± 38.02 vs.1924 ± 509.4,P＜0.05; C4c:35.23 ± 3.1vs.177.3 ± 37.17,P＜0.05; C5b-9:5.63 ± 1.74 vs.37.05 ± 7.4,P＜0.01).Conclusion These data demonstrated that the resistance of rASCs to human xenoantibody and complement-mediated lysis is associated with low expression of xenoantigen a-Gal and inhibition of MAC (membrane attack complex) formation.
