生肌玉红膏提取液促进脂肪干细胞增殖的实验研究  

Experiment Researches on Proliferation of Adipose Stem Cells with Extracting Solution of Shenji Yuhong Gao

作　　者：冯泽宇 许岩磊[2] 张媞[1] 顾铭[1] 陈佳静[1] 叶小舟[1] 姚昶[2] 

机构地区：[1]南京中医药大学附属医院,江苏南京210029 [2]南京中医药大学,江苏南京210023

出　　处：《中华中医药学刊》2017年第2期355-358,I0005,共5页Chinese Archives of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(81373646);康缘基金项目(HZ1009KY);江苏省高校优势学科建设II期项目(012062003010);江苏省中医院高峰学术人才培养项目(y2014cr06)

摘　　要：目的:探查生肌玉红膏提取液对脂肪干细胞的增殖作用与机制,为构建中医药组织工程血管化支架提供前期基础。方法:实验分为空白组:仅加入细胞培养液与CCK-8溶液,对照组:空白组基础上加入细胞密度为4×104/m L P3代的脂肪干细胞悬液0.2 m L,实验组:对照组基础上加入生肌玉红膏梯度浓度为0.012 5、0.025、0.05、0.1 mg/m L的生肌玉红膏提取液。表面抗原及脂滴油红O染色与细胞茜素红S染色鉴定脂肪干细胞;CCK法测定脂肪干细胞增殖状况;流式细胞仪测定脂肪干细胞增殖周期及蛋白质印迹法(Western blot)测定脂肪干细胞凋亡相关蛋白Bcl-2,Bax及促血管生长因子VEGF的表达。结果:脂肪干细胞P3代细胞表面抗原CD34阴性,CD90阳性,油红O染色可见细胞中的脂滴且细胞茜素红S染色阳性;生肌玉红膏提取液浓度由低到高刺激脂肪干细胞增殖率逐渐增加,0.05 mg/m L时增殖达到高峰,明显高于对照组及浓度为0.012 5 mg/m L与0.025 mg/m L组(P<0.01)及对照组;浓度为0.05 mg/m L生肌玉红膏提取液在培养后24 h与48 h脂肪干细胞细胞周期更多位于DNA合成期与合成后期,比例分别达24%与28%,高于对照组8%左右(P<0.01)并显著促进抑制凋亡蛋白Bcl-2与凋亡蛋白Bax的比例与刺激VEGF表达。结论:生肌玉红膏具有促进脂肪干细胞增殖疗效且0.05 mg/m L生肌玉红膏提取液疗效较佳,其作用机制与抗脂肪干细胞凋亡相关。Objective ： To explore the effects and mechanisms of extracting solution of Shenji Yuhong Gao on enhancing the proliferation of adipose stem cells for establishment of vascularized biomaterials of TCM. Methods ： Such groups as required ware set up on the following assays which were divided as blank group with cell culture solution and CCK -8 solution, control group with furthermore the amounts of 0.2 mL of 4 × 10^4/mL P3 adipose stem ceils, experimental groups with additional 0.2 mL of 0. 012 5,0. 025,0.05 and 0.1 mg/mL of extracting solution of Shenji Yuhong Gao on the basis of control group. The biological characters of ADSCs were determined with flow cytometry detection. The porosities and adhesive rates of ADSCs were detected. The degrees of proliferation of ADSCs were determined by CCK assay. Cell proliferation cycles and apoptosis - related proteins of Bcl - 2, Bax and VEGF were detected with flow cytometry and western blot respectively. Results： The surface signs of P3 generation of ADSCs expressed CD34 negatively and CD90 positively as well as lipid droplet secretion and alizarin red which were proved by biological properties of ADSCs. The effects of proliferation rates increased with the improvement of concentrations of extracting solution of Shenji Yuhong Gao and up to the concentration of 0. 05 mg/mL which reached to the peak plate with significant differences less than 0. 01. The cell proliferation cycles in the experimental group distributed on the cycle of S and G2 with ratio were 24% and 28% respectively. The proportion of Bcl - 2/Bax as well as the expression of VEGF was improved. Conclusions ： The rate of proliferation of adipose stem cell can be enhanced with the extraction solution of Shengji Yuhong Gao with best effects produced by the concentration of 0.05 mg/mL. The mechanisms should be related with the regulations of apoptosis - related proteins.

关 键 词：生肌玉红膏 脂肪干细胞 增殖 细胞周期 凋亡 

分 类 号：R285.5[医药卫生—中药学]