大鼠脂肪来源干细胞抵抗人血清介导的异种体液免疫杀伤作用及其机制

Resistance of rat adipose-derived stem cells to human xenoantibody-dependant complement-mediated lysis and its mechanism

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”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、CAc,CSb-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）Vo（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、CAc和C5b-9沉积（Gmean值分别为1924±509．4、177．3±37．17和37．05±7．4），但rASC仅有较少C3c和CAc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。

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, CAc 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 CIN4 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）. Mechanistieally, 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, CAc and C5b-9 （C3c： 294. 6 ± 38. 02 vs. 1924 ± 509. 4, P〈 0. 05; CAc.. 35.23 ± 3. 1 vs. 177.3±37.17,P〈0.05; C5b-9 ： 5. 63 ±1. 74 vs. 37. 05± 7. 4 , P〈0. 01）. Conclusion Thesedata demonstrated that the resistance of rASCs to human xenoantibody and complemenvmediated lysis is associated with low expression of xenoantigen α-Gal and inhibition of MAC （membrane attack complex） formation.