普伐他汀对人骨髓间充质干细胞增殖分化及分泌功能的影响

Effect of pravastatin on the proliferation, differentiation and secretion of human bone marrow mesenchymal stem cells

背景：针对骨髓干细胞移植治疗心肌梗死及心力衰竭的效果报道不一，如何提高移植细胞在心肌的存活率，并促进其生长，从而增强移植的疗效，是一个值得深入研究且具有重要临床价值的问题。目的：观察普伐他汀在体外对人骨髓间充质干细胞增殖分化及分泌功能的影响。设计、时间及地点：细胞学体外观察，于2006—05／2007—05在上海市疾病预防控制中心毒理实验室完成。材料：人骨髓间充质干细胞由上海第九人民医院组织工程实验室提供。他汀类药物普伐他汀为旅贵宝制药公司产品，国药准字H10950315。方法：将冻存细胞解冻，加入含10^5U／L青霉素、100mg／L链霉素、10％胎牛血清的低糖DMEM，体外扩增培养骨髓间充质于细胞。传至第6代细胞随机分为2组：空白对照组换用低糖DMEM培养基，普伐他汀组分别加入含0．2，1，10，100μmol／L普伐他汀的低糖DMEM培养基，作用96h。MTT比色法检测细胞增殖情况，流式细胞仪检测细胞表面抗原表达，ELISA法测定细胞上清液中血管内皮生长因子含量。主要观察指标：普伐他汀对人骨髓问充质于细胞形态、增殖、表面抗原、血管内皮生长因子分泌的影响。结果：传代后细胞贴壁生长，呈梭形或条形，形态较均一，经100μmol／L普伐他汀作用24h后细胞形态无明显变化。细胞传代后潜伏期约为48h，对数增殖期为接种后第4～6天，7d后进入平台期。与空白对照组比较，0．2，1，10μmol／L普伐他汀组均可明显刺激骨髓问充质干细胞增殖（t=3．154～5．837，P〈0．05或0．01）。空白对照组CD34呈阴性表达，CD105及CD106阳性细胞百分率分别为32．55％，39．30％；普伐他汀作用2周后细胞表明抗原的表达无明显变化。与空白对照组比较，0．2μmol／L普伐他汀组血管内皮生长因子浓度无明显变化（t=-1．449，P〉0．05）；100μmol／L普伐他汀组血管内皮生长因子浓度明显升高（t=-2．325，P〈0．05）。结论：普伐他汀不影响人骨髓间充质干细胞的形态与分化，浓度为0．2～10μmol／L时可促进细胞增殖，浓度过高则对细胞产生一定毒性而抑制其增殖。此外普伐他汀对骨髓间充质干细胞分泌血管内皮生长因子具有促进作用。

BACKGROUND： There are different studies on the bone marrow stem cell transplantation for treatment of myocardial infarction and heart failure. How to enhance the survival rate and growth of transplanted cells in the myocardium to accelerate the curative effect of transplantation is a problem deserving further study and having a key clinical value. OBJECTIVE： To investigate the effect of pravastatin on proliferation, differentiation and secretion of human bone marrow mesenchymal stem cells （BMSCs） in vitro. DESIGN, TIME AND SETTING： The cytology in vitro experiment was performed at the Laboratory of Toxicology of Shanghai Municipal Center for Disease Control and Prevention from May 2006 to May 2007. MATERIALS： Human BMSCs were obtained from Laboratory of Tissue Engineering of Shanghai Ninth People＇s Hospital. Pravastatin was purchased from Shiguibao China, No. H 10950315. METHODS： Freezing cells were defrosted, and in vitro incubated in low glucose DMEM containing 10% fetal bovine serum, 105 U/L penicillin and 100 mg/L streptomycin. At the 6th passage, cells were randomly divided into 2 groups. Cells in the blank control group were incubated in the low glucose DMEM. Cells in the pravastatin group were incubated in the low glucose DMEM containing 0.2, 1, 10, 100 it mol/L pravastatin for 96 hours. Cell proliferation was assessed by MTT colorimetric assay. Cell surface antigen expression was detected by flow cytometry. Levels of vascular endothelial growth factor （VEGF） from the cells were detected by enzyme-linked immunoadsordent assay （ELISA）. MAIN OUTCOME MEASURES： Effects of pravastatin on the morphous, proliferation, surface antigen and VEGF levels of human BMSCs. RESULTS： After passage, cells adhered, showing uniformly spindle or stripe, with no obvious morphology alternation observed after treated with pravastatin 100 μmol/L for 24 hours. After passage, incubation period lasted for about 48 hours, logarithmic multiplication period appeared at 4 to 6 days, and plateau period showed after 7 days. Compared with the blank control group, 0.2, 1, 10 μmol/L pravastatin could significantly stimulate the proliferation of BMSCs （t=3.154-5.837, P 〈 0.05 or 0.01）. In the blank control group, cells were negative for CD34, positive for CD105 and CD106 （percentage rate of 32.55% and 39.30% respectively）. Pravastatin treated for 2 weeks had no obvious effect on cell surface antigens. Compared with the blank control group, no significant difference in VEGF levels was detected in the 0.2 μmol/L pravastatin group （t=-1.449, P 〉 0.05）. VEGF levels significantly increased in the 100 μmol/L pravastatin group （t=-2.325, P 〈 0.05）. CONCLUSION： Pravastatin does not affect the morphous and differentiation of human BMSCs. At concentration of 0.2- 10 μ mol/L, pravastatin can improve BMSCs proliferation. Higher dose of pravastatin can have toxicity and inhibit cell proliferation. In addition, pravastatin promotes VEGF secretion of BMSCs.