骨髓间充质干细胞旁分泌胰岛素样生长因子1促受损肝L02细胞增殖的效应及其分子机制

Insulin-like growth factor-l-mediated paracrine pro-proliferative effect of bone marrow mesenchymal stem cells on injured L02 hepatoeytes

目的探讨骨髓间充质干细胞（BM-MSCs）旁分泌胰岛素样生长因子1（IGFq）促受损肝L02细胞增殖作用的分子机制。方法利用Transwell小室建立MSCs与D-氨基半乳糖（D-GalN）损伤L02肝细胞的非接触式共培养模型，四甲基偶氮唑盐法检测BM-MSCs对D—GaIN损伤组L02细胞增殖的影响，酶联免疫吸附法检测共培养体系中IGF-1的表达水平及其主要来源细胞，Westemblot检测损伤L02细胞的IGF-1受体（IGF-1R）表达水平及外源性和BM-MSCs源性IGF-1对D-GaIN损伤L02细胞增殖的影响。两组间数据比较采用f检验，两组以上数据的比较采用方差分析。结果共培养条件下的BM—MSCs能非接触依赖性地促进D-GaIN损伤组L02细胞增殖，其在24、48、72h的吸光度值分别为0．60±0．09、0．82±0．05、0．90±0．06，明显高于D-GalN损伤组在相应时间点的0．36±0．08、0．52±0．06、0．68土0．06（t值分别为2．493、3．116、2．285，尸值均〈0．05）。经D—GalN损伤组L02细胞分泌上清液处理48h的BM-MSCs的IGF一1分泌量为（156±24）ng／ml，与D-GalN损伤组L02细胞共培养48h的BM—MSCIGF-1分泌量为（185±36）ng／ml，均较正常BM—MSCs分泌量（19±9）ng／ml明显上升（t值分别为5．345和4．473，P值均〈0．01）。外源性rh-IGF-1能明显促进L02细胞增殖，且在160ng／ml时达到最大效应，处理D—GaIN损伤L02细胞96h的吸光度值为1．70±0．09，明显高于rh_IGF-1浓度0ng／ml组的0．79±0．09（f=9．460，P〈0．05）。BM-MSCs与D-GalN损伤L02细胞共培养组在加入IGF-1R单克隆抗体处理前后，24、48、72h时的吸光度值分别为1．80±0．11比1．30士0．16、1．70±0．12比1．30±0．10、1．69±0．11比1．25±0．12，IGF-1R单克隆抗体处理后的L02细胞增殖明显被抑制（t值分别为2．909、2．328、2．560，P值均〈0．05）。结论BM-MSCs源性IGF-1通过结合损伤L02细胞的IGF-1R，在BM-MSCs的旁分泌促受损肝L02细胞增殖中发挥主要作用。

Objective To explore the therapeutic effect of bone marrow mesenchymal stem cells （BM- MSCs） on injured hepatocytes mediated by paracrine mechanisms and to investigate the potential molecular mechanism of this action. Methods A contact-independent model of aberrant hepatic microenvironment was established by co-culturing BM-MSCs with D-galactosamine （D-GalN）-injured human L02 hepatic ceils using a transwell assay platform. Secreted levels of insulin-like growth factor-I （IGF-1） were measured by enzyme-linked immunosorbent assay of the co-culture supernatant. Expression of the IGF-1 receptor （IGF-1R） was assessed by Western blot. The effect of exogenous IGF-1 on proliferation of D-GaiN-injured L02 ceils was examined by MTT assay. Results Upon co-culture, BM-MSCs promoted proliferation of D-GaiN-injured L02 cells in a contact-independent manner （absorbance values of at 24 h： 0.36±0.08, 48 h： 0.52 ± 0.06, and 96 h： 0.68 ± 0.06; vs. uninjured cells t = 2.493, 3.116, and 2.285, respectively; allP 〈 0.05）. Robust expression of IGF-1 was identified in the supematants of co-cultures and was demonstrated to have been secreted mainly from BM-MSCs under the influence of D-GaiN-injured L02 cells. Constitutive expression of IGF-1R was found in the D-GaiN-injured L02 cells and blocking of IGF-1R by a neutralizing antibody significantly inhibited the paracrine pro-proliferative effect of co-cultured BM-MSCs at 24 h, 48 h, and 72 h （t = 2.909, 2.328, and 2.560, respectively; allP 〈 0.05）. Conclusion BM-MSC-derived IGF-1 plays an important role in the paraerine pro-proliferative effect on D-GaiN-injured L02 hepatoeytes by engaging with the constitutively expressed IGF- 1R on L02 cells.