摘要
目的:茶黄素是红茶中的主要成分,具有调节血脂、抗氧化、抗肿瘤、抗心脑血管疾病等多种药理活性。以往茶黄素对人体内脂肪代谢的影响多数属于宏观分析,实验在细胞水平上探讨茶黄素对兔骨髓基质干细胞向脂肪细胞分化的影响。方法:实验于2007-05/09在安徽医科大学附属省立医院中心实验室完成。①动物:清洁级4～8周龄家兔10只,由安徽医科大学动物试验中心提供,实验过程中对动物的处置符合动物伦理学标准。②实验方法:兔全身肝素化后麻醉状态下处死,取双侧股骨胫骨和肱骨,去除软组织,切除包括骺板在内的两侧骺端,采用全骨髓法分离培养骨髓基质干细胞,按2×10~8 L~(-1)密度接种,当细胞生长至80%～90%融合时消化传代。取传至3代的细胞按1×10~5/cm~2密度接种,加入含重组人胰岛素10mg/L、地塞米松10~(-6)mol/L、0.5mmol/L IBMX的DMEM成脂诱导液培养2周。设立3组:脂肪对照组单纯放入成脂诱导液1ml;茶黄素组向多少1ml成脂诱导液中加入500μg/L茶黄素;空白对照组仅加入等量普通DMEM培养液。③实验评估:取第2代培养细胞绘制生长曲线;油红O染色对诱导的脂肪细胞进行鉴定,计算脂肪细胞转化率。
结果:①细胞生长曲线:骨髓基质干细胞具有旺盛的增殖能力,培养1d为细胞适应期,3d后为对数增长期,8d时进入平台期,之后细胞增殖迅速减慢,细胞数下降。②脂肪细胞油红O染色鉴定结果:脂肪细胞中的脂滴被染成橙红色,胞核为蓝色。脂肪对照组多数骨髓基质干细胞诱导为脂肪细胞,转化率为(64.8±4.8)%,茶黄素组仅为(32.0±3.4)%,空白对照组未见明显脂肪细胞形成。结论:茶黄素可明显抑制兔骨髓基质干细胞向脂肪细胞方向的分化。
Abjective: Theaflavins are the major constituent of black tea,and have a lot of pharmacological activity,for example,regulating blood fat, antioxygen , anti-tumor, resisting cerebrovascular disease ,and so on.To go forward,theaflavins’effect on mankind fat metabolism in vivo trenmendously belongs to macro analysis,but our experiments try to find out the effect of theaflavins on the transformation of rabbit mesenchymal stem cells (MSCs) into adipocytes in vitro.Method:Our experiment completed in center laboratory of Anhui provincial hospital affiliated to Anhui medical university.①Animals: 10 rabbits of cleaning grade and 4-8 weeks old are provided by animal experiment center of Anhui medical university.The disposal of animals in process of experiment refer to ethical standard of animals.②experical method:The rabbits are heparinized and put to death in drugged state,we obtain its sideway femur,tibia and humerus,remove its soft tissue,cut epiphysis of two sides including epiphyseal plate, isolate and culture the MSCs with whole bone marrow culture method technology,inoculate it according to the density of 2×10~(-8) L~(-1) ,and when the cells grew to the fusion of 80%-90%,digesting and going down to the future generation.We inoculate cells of the third generation according to the density of 1×10~5/cm~2 ,and add the DMEM adipocyte-induced liquid contained rh-insulin 10mg/L,dexamethasone 10-6mol/L,and IBMX 0.5 mmol/L to be cultured for 2 weeks.Establishing 3 groups:Adipocyts control group puts in 1 ml adipocyte-induced liquid;theaflavin group put the 500 ug/L Theaflavin in 1 ml adipocyte-induced liquid;blank group only put in equal amount of ordinary DMEM culture medium.③Experical evaluation:We take the cultured cells of the second group to draw growth curve;oil O stain is used to identify the induced adipocytes,and calculate the transformation efficiency of adipocytes.
Result:①Cell growth curve:MSCs have vigorous reproductive activity,1 days to be cultured is in the cell adaptive phase,afer 3 days is in the increased logarithmic phase,8 days in the platform phase,and after 8 days ,cell proliferation quickly steps down.②Identification result of adipocytes oil O stain: lipid droplets in adipocyte are stained into salmon pink,and the nucleus is stained into blue.Most MSCs in adipocyte control group are induced into adipocytes,the transformation efficiency is(64.8±4.8)%,theaflavin group only has (32.0±3.4)% ,blank group hasn’t obviously the formation of adipocytes.
Conclusion:The theaflavins obviously inhibit diffrentiation from rabbit MSCs to adipocytes.
引文
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