CAJ | 학술논문

背景:在肝纤维化发生发展过程中,肝星状细胞起着关键作用.研究证实骨髓间充质干细胞移植可作为治疗肝纤维化的方法,但其逆转肝纤维化的机制不明.目的:探讨体外共培养条件下,大鼠骨髓间充质干细胞对肝星状细胞增殖的调控机制.方法:实验组在半透膜上接种大鼠骨髓间充质干细胞,在6孔塑料培养板上接种肝星状细胞,建立上下双层细胞共培养体系;对照组将大鼠骨髓间充质干细胞更换为成纤维细胞;空白组仅行肝星状细胞单独培养.WST-8法检测肝星状细胞生长抑制率,流式细胞仪分析细胞周期,RT-PCR检测肝星状细胞CyclinD1和P27 mRNA的表达,Western blot检测肝星状细胞CyclinD1和P27蛋白的表达.结果与结论:与空白组、对照组比较,实验组共培养24,48,72 h肝星状细胞牛长抑制率均显著升高(P＜0.01):共培养72 h G_0/G_1期细胞显著增加(P＜0.01),S期细胞显著减少(P＜0.01),可阻滞肝星状细胞由G_0/G_1期向S期转换.共培养24 h,实验组CyclinD1 mRNA及蛋白表达开始下调,至72 h时表达量显著低于对照组、空白组(P＜0.01);共培养全过程中各组p27mRNA的表达无明显差异(P＞0.05),共培养24 h时实验组p27蛋白表达较对照组、空白组均显著上调(P＜0.01).结果证实大鼠骨髓间充质干细胞能够抑制肝星状细胞的增殖,其机制可能是通过下调CyclinD1表达,上调P27蛋白表达,使细胞周期停滞于G_0/G_1期实现的.
배경:재간섬유화발생발전과정중,간성상세포기착관건작용.연구증실골수간충질간세포이식가작위치료간섬유화적방법,단기역전간섬유화적궤제불명.목적:탐토체외공배양조건하,대서골수간충질간세포대간성상세포증식적조공궤제.방법:실험조재반투막상접충대서골수간충질간세포,재6공소료배양판상접충간성상세포,건립상하쌍층세포공배양체계;대조조장대서골수간충질간세포경환위성섬유세포;공백조부행간성상세포단독배양.WST-8법검측간성상세포생장억제솔,류식세포의분석세포주기,RT-PCR검측간성상세포CyclinD1화P27 mRNA적표체,Western blot검측간성상세포CyclinD1화P27단백적표체.결과여결론:여공백조、대조조비교,실험조공배양24,48,72 h간성상세포우장억제솔균현저승고(P＜0.01):공배양72 h G_0/G_1기세포현저증가(P＜0.01),S기세포현저감소(P＜0.01),가조체간성상세포유G_0/G_1기향S기전환.공배양24 h,실험조CyclinD1 mRNA급단백표체개시하조,지72 h시표체량현저저우대조조、공백조(P＜0.01);공배양전과정중각조p27mRNA적표체무명현차이(P＞0.05),공배양24 h시실험조p27단백표체교대조조、공백조균현저상조(P＜0.01).결과증실대서골수간충질간세포능구억제간성상세포적증식,기궤제가능시통과하조CyclinD1표체,상조P27단백표체,사세포주기정체우G_0/G_1기실현적.
BACKGROUND:The hepatic stellate cells(HSCs)plays a key role in the development of liver fibrosis.Studies have shown that bone marrow-derived mesenchymal stem cell(BMSCs)transplantation can be used to treat liver fibrosis,but the mechanism for reversal of liver fibrosis remains unknown.OBJECTIVE:To explore the mechanism of bone marrow mesenchymal stem cells to regulate the proliferation of HSCs under co-culture in vitro.METHODS:Rat BMSCs and HSCs in the experimental group were cultured in the plastic culture plate(6 holes)to establish the upper and lower double-cell co-culture system.Rat normal fibroblast cell lines were seeded as control group;HSCs were cultured alone as blank group.Cell proliferation was determined by WST8 and cell cycle was determined by flow cytometry.The Cyclin D1 and P27 mRNA expression in HSC was determined by reverse transcription-polymerase chain reaction(RT-PCR)and the level of Cyclin D1 and P27 protein by Western blot.RESULTS AND CONCLUSION:HSCs co-cultured with BMSCs significantly inhibited HSC proliferation compared with the blank and control groups at 24,48,and 72 hours(P ＜ 0.01);Flow cytometry showed that the percentage of G_0/G_1 phase cells of co-culture group was increased but the S phase cells reduced(P ＜ 0.01)compared with the other groups at 72 hours,and BMSCs blocked HSC to convert from G_0/G_1 period to S phase.After HSCs co-cultured with BMSCs for 24 hours,the expression of Cyclin D1 mRNA and protein was reduced,and significantly less than the blank and control groups at 72 hours(P ＜ 0.01);no differences were detected in P27 mRNA expression in each group during the co-culture(P ＞ 0.05).After co-culture of 24 hours,the p27 protein expression was significantly increased compared with the blank and control groups(P ＜ 0.01).BMSCs inhibited the proliferation of HSCs,possibly through inhibiting CydinDI expression,increasing the p27 protein expression to cause cell cyde arresting in G_0/G_1 phase.