中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
CHINESE JOURNAL OF HEPATOLOGY
2012年
11期
853-858
,共6页
冯吉%熊吉%熊全%陈潇迪%王斌%陈东风
馮吉%熊吉%熊全%陳瀟迪%王斌%陳東風
풍길%웅길%웅전%진소적%왕빈%진동풍
间质干细胞%骨髓细胞%胰岛素样生长因子1%细胞增殖%L02细胞
間質榦細胞%骨髓細胞%胰島素樣生長因子1%細胞增殖%L02細胞
간질간세포%골수세포%이도소양생장인자1%세포증식%L02세포
Mesenchymal stem cells%Bone marrow cells%Insulin-like growth factor 1%Cell proliferation%L02 cell
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(IGF-1)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-GalN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,Western blot 检测损伤L02细胞的IGF-1受体(IGF-IR)表达水平及外源性和BM-MSCs源性IGF-1对D-GalN损伤L02细胞增殖的影响.两组间数据比较采用t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-GalN损伤组L02细胞增殖,其在24、48、72 h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±0.06(t值分别为2.493、3.116、2.285,P值均＜0.05).经D-GalN损伤组L02细胞分泌上清液处理48 h的BM-MSCs的IGF-1分泌量为(156±24) ng/ml,与D-GalN损伤组L02细胞共培养48 h的BM-MSC IGF-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160 ng/ml时达到最大效应,处理D-GalN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0 ng/ml组的0.79±0.09(t=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、72 h时的吸光度值分别为1.80±0.11比1.30±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.
目的 探討骨髓間充質榦細胞(BM-MSCs)徬分泌胰島素樣生長因子1(IGF-1)促受損肝L02細胞增殖作用的分子機製. 方法 利用Transwell小室建立MSCs與D-氨基半乳糖(D-GalN)損傷L02肝細胞的非接觸式共培養模型,四甲基偶氮唑鹽法檢測BM-MSCs對D-GalN損傷組L02細胞增殖的影響,酶聯免疫吸附法檢測共培養體繫中IGF-1的錶達水平及其主要來源細胞,Western blot 檢測損傷L02細胞的IGF-1受體(IGF-IR)錶達水平及外源性和BM-MSCs源性IGF-1對D-GalN損傷L02細胞增殖的影響.兩組間數據比較採用t檢驗,兩組以上數據的比較採用方差分析. 結果 共培養條件下的BM-MSCs能非接觸依賴性地促進D-GalN損傷組L02細胞增殖,其在24、48、72 h的吸光度值分彆為0.60±0.09、0.82±0.05、0.90±0.06,明顯高于D-GalN損傷組在相應時間點的0.36±0.08、0.52±0.06、0.68±0.06(t值分彆為2.493、3.116、2.285,P值均＜0.05).經D-GalN損傷組L02細胞分泌上清液處理48 h的BM-MSCs的IGF-1分泌量為(156±24) ng/ml,與D-GalN損傷組L02細胞共培養48 h的BM-MSC IGF-1分泌量為(185±36) ng/ml,均較正常BM-MSCs分泌量(19±9)ng/ml明顯上升(t值分彆為5.345和4.473,P值均＜0.01).外源性rh-IGF-1能明顯促進L02細胞增殖,且在160 ng/ml時達到最大效應,處理D-GalN損傷L02細胞96h的吸光度值為1.70±0.09,明顯高于rh-IGF-1濃度0 ng/ml組的0.79±0.09(t=9.460,P＜0.05).BM-MSCs與D-GalN損傷L02細胞共培養組在加入IGF-1R單剋隆抗體處理前後,24、48、72 h時的吸光度值分彆為1.80±0.11比1.30±0.16、1.70±0.12比1.30±0.10、1.69±0.11比1.25±0.12,IGF-1R單剋隆抗體處理後的L02細胞增殖明顯被抑製(t值分彆為2.909、2.328、2.560,P值均＜0.05).結論 BM-MSCs源性IGF-1通過結閤損傷L02細胞的IGF-1R,在BM-MSCs的徬分泌促受損肝L02細胞增殖中髮揮主要作用.
목적 탐토골수간충질간세포(BM-MSCs)방분비이도소양생장인자1(IGF-1)촉수손간L02세포증식작용적분자궤제. 방법 이용Transwell소실건립MSCs여D-안기반유당(D-GalN)손상L02간세포적비접촉식공배양모형,사갑기우담서염법검측BM-MSCs대D-GalN손상조L02세포증식적영향,매련면역흡부법검측공배양체계중IGF-1적표체수평급기주요래원세포,Western blot 검측손상L02세포적IGF-1수체(IGF-IR)표체수평급외원성화BM-MSCs원성IGF-1대D-GalN손상L02세포증식적영향.량조간수거비교채용t검험,량조이상수거적비교채용방차분석. 결과 공배양조건하적BM-MSCs능비접촉의뢰성지촉진D-GalN손상조L02세포증식,기재24、48、72 h적흡광도치분별위0.60±0.09、0.82±0.05、0.90±0.06,명현고우D-GalN손상조재상응시간점적0.36±0.08、0.52±0.06、0.68±0.06(t치분별위2.493、3.116、2.285,P치균＜0.05).경D-GalN손상조L02세포분비상청액처리48 h적BM-MSCs적IGF-1분비량위(156±24) ng/ml,여D-GalN손상조L02세포공배양48 h적BM-MSC IGF-1분비량위(185±36) ng/ml,균교정상BM-MSCs분비량(19±9)ng/ml명현상승(t치분별위5.345화4.473,P치균＜0.01).외원성rh-IGF-1능명현촉진L02세포증식,차재160 ng/ml시체도최대효응,처리D-GalN손상L02세포96h적흡광도치위1.70±0.09,명현고우rh-IGF-1농도0 ng/ml조적0.79±0.09(t=9.460,P＜0.05).BM-MSCs여D-GalN손상L02세포공배양조재가입IGF-1R단극륭항체처리전후,24、48、72 h시적흡광도치분별위1.80±0.11비1.30±0.16、1.70±0.12비1.30±0.10、1.69±0.11비1.25±0.12,IGF-1R단극륭항체처리후적L02세포증식명현피억제(t치분별위2.909、2.328、2.560,P치균＜0.05).결론 BM-MSCs원성IGF-1통과결합손상L02세포적IGF-1R,재BM-MSCs적방분비촉수손간L02세포증식중발휘주요작용.
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 cells using a transwell assay platform.Secreted levels of insulin-like growth factor-1 (IGF-1) were measured by enzyme-linked immunosorbent assay of the co-culture supematant.Expression of the IGF-1 receptor (IGF-1R) was assessed by Western blot.The effect of exogenous IGF-1 on proliferation of D-GalN-injured L02 cells was examined by MTT assay.Results Upon co-culture,BM-MSCs promoted proliferation of D-GalN-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; all P ＜ 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-GalN-injured L02 cells.Constitutive expression of IGF-1R was found in the D-GalN-injured L02 cells and blocking of IGF-1 R 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 paracrine pro-proliferative effect on D-GalN-injured L02 hepatocytes by engaging with the constitutively expressed IGF-1R on L02 cells.
