课题背景：课题为2007~2008度安徽省自然科学基金资助项目，编号070413083，课题名称“茶黄素对激素性股骨头缺血性坏死防治作用的基础研 究”，资助基金5万元，单位补贴5万元。 于2007年1~5月完成动物实验的一部分，在安徽医科大学学报上发表论文“茶黄素抑制激素性股骨头坏死的实验研究”。

创新要点：文章从细胞学角度探讨茶黄素对兔骨髓基质干细胞向脂肪细胞诱导分化的影响，未就其具体作用机制做更深一步的分析，后续试验将从基因水平观察茶黄素作用后兔骨髓基质干细胞基因表达的变化。

相关链接：茶黄素（Theaflavins，TFs）是一类具有苯骈卓酚酮结构的化合物的总称，由红茶中的多酚类及其衍生物酶促氧化缩合而来，是红茶中发挥生物学作用的主要物质，具有降低体内总胆固醇、三酰甘油和低密度脂蛋白的作用，还可抑制高脂饮食导致的血脂升高、减少食物摄入和脂肪体内的沉积。

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2×108 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1×105/cm2密度接种，加入含重组人胰岛素 10 mg/L、地塞米松10-6 mol/L、0.5 mmol/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1 mL；茶黄素组向1 mL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1 d为细胞适应期，3 d后为对数增长期，8 d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。
关键词：骨髓基质干细胞；茶黄素；脂肪细胞；诱导分化

李府，尚希福，谢涛.茶黄素对兔骨髓基质干细胞向脂肪细胞诱导分化的影响[J].中国组织工程研究与临床康复，2008，12(16):3061-3064 [www.zglckf.com/zglckf/ejournal/upfiles/08-16/16k-3061(ps).pdf]

Effect of theaflavins on the differentiation of rabbit bone marrow mesenchymal stem cells into adipocytes

Abstract

AIM：Theaflavins are the major constituent of black tea, and have a lot of pharmacological activities, for example, regulating blood fat, anti-oxygen, anti-tumor, resisting cerebrovascular disease, and so on. Previously, theaflavins' effect on human fat metabolism mostly belongs to macro-analysis, but this experiment try to find out the effect of theaflavins on the differentiation of rabbit mesenchymal stem cells (MSCs) into adipocytes in terms of cells.
METHODS: The experiment was completed in the center laboratory of Anhui Provincial Hospital Affiliated to Anhui Medical University from May to September in 2007.①Ten rabbits of cleaning grade and 4-8 weeks old were provided by the animal experiment center of Anhui Medical University. The disposal of animals in process of experiment referred to the ethical standard of animals.②The heparinized rabbits were sacrificed in drugged state, then bilateral femur, tibia and humerus were obtained and their soft tissues were removed, cutting epiphysis of two sides including epiphyseal plate. The MSCs were isolated and cultured with whole bone marrow culture method, inoculated at a density of 2×108 L-1. When the cells grew to the fusion of 80%-90%, digesting and passage culture were performed. Cells of the third generation were collected and inoculated at a density of 1×105/cm2 to be cultured for 2 weeks with DMEM adipocyte-induced liquid, which contained recombinant human insulin 10 mg/L, dexamethasone 10-6 mol/L, and IBMX 0.5 mmol/L. Three groups were set up: adipocytes control group in 1 mL adipocyte-induced liquid; theaflavins group with the 500 μg/L theaflavins in 1 mL adipocyte-induced liquid; blank group in equal amount of ordinary DMEM culture medium.③The cultured cells of the second generation were selected to draw growth curve; oil O staining was used to identify the induced adipocytes and calculate the differentiation efficiency of adipocytes.
RESULTS: ①Cell growth curve: MSCs had vigorous reproductive activity, 1 day of the culture was the cell adaptive phase, 3 days was the increased logarithmic phase, and 8 days was the platform phase. After 8 days, cell proliferation quickly stepped down and cells reduced.②Identification of adipocytes by oil O stain: Lipid droplets in adipocytes were stained into salmon pink, and the nucleus was stained into blue. Most of MSCs in adipocyte control group were induced into adipocytes, the differentiation efficiency was (64.8±4.8)%, while that in theaflavins group was only (32.0±3.4)%. No adipocytes obviously formed in blank control group.
CONCLUSION: Theaflavins can obviously inhibit differentiation from rabbit MSCs to adipocytes.

Li F, Shang XF, Xie T.Effect of theaflavins on the differentiation of rabbit bone marrow mesenchymal stem cells into adipocytes.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(16):3061-3064
[www.zglckf.com/zglckf/ejournal/upfiles/08-16/16k-3061(ps).pdf]