转化生长因子β_1诱导时间对骨髓基质细胞体外成软骨分化的影响  

Influence of transforming growth factor-β_1 inducing time on chondrogenesis of bone marrow stromal cells (BMSCs) :in vitro experiment with porcine BMSCs

作　　者：刘天一[1] 周广东[1] 魏娴[1] 武晓莉[1] 陈付国[1] 崔磊[1] 刘伟[1] 曹谊林[1] 

机构地区：[1]上海交通大学附属第九人民医院整复外科,上海市组织工程重点实验室200011

出　　处：《中华医学杂志》2007年第31期2218-2222,共5页National Medical Journal of China

基　　金：国家"973"计划资助项目(2005CB522702);国家"863"计划重大专项资助项目(2006AA02A126);国家自然科学基金(30300353)

摘　　要：目的：探讨转化生长因子β1（TGF-β1）诱导时间对骨髓基质细胞（BMSC）体外成软骨分化，及构建组织工程化软骨的影响。方法：将体外培养扩增的第二代猪BMSC按5．0×10^7个／ml的密度接种于聚羟基乙酸（PGA）制成的圆柱形支架材料上，第5天开始应用含TGF-β1（10μg/L）、IGF-I（50μg/L）和地塞米松（40μg/L）的软骨诱导液培养。按TGF-β1诱导时间的不同分为A（诱导2周）、B（诱导4周）、C（诱导6周）、D（诱导8周）和E（诱导10周）5组。体外培养10周后取材行大体观察、组织学和组织化学检测、聚合蛋白多糖（GAG）定量分析和生物力学测定，同时利用Western印迹和免疫组织化学进行Ⅱ型胶原表达的分析。结果：随诱导时间延长，软骨陷窝由周边开始逐渐向内部增多，排列渐趋均匀，蛋白聚糖等软骨特异性基质成分积聚。免疫组化染色及Western印迹结果均显示Ⅱ型胶原含量随诱导时间延长逐渐递增。这种情况至诱导6周后逐渐稳定，诱导6周和诱导10周间差异无统计学意义。诱导6周以上组弹性模量和抗压强度均明显高于A、B两组。结论：利用BMSC作为种子细胞构建组织工程化软骨，诱导持续时间与形成的软骨质量相关，6周的诱导时间基本可以形成具有良好组织结构、化学组成和力学性质的软骨组织。Objective To explore the influence of transforming growth factor （TGF）-β1 inducing time on the chondrngenesis of bone marrow stromal ceils （BMSC）, and on the construction of tissue engineering cartilage. Methods BMSCs were obtained from the greater trochanters of 3 pigs, cultured, seeded onto the cylindrical scaffolds made of polyglycolic acid at the density of 5.0 × 10^7 cells/ml, and then cultured with chondrngenetic media con .taining TGF-β1, （ 10 ng/ml）, insulin-like growth factor- Ⅰ （ 50 μg/ L）, and dexamethasone （40μg/L） to be induced by TGF-β1, for 2 weeks （Group A）, 4 weeks （Group B）, 6 weeks （Group C）, 8 weeks （Group D）, or 10 weeks （Group E） respectively. 10 weeks later the cylindrical scaffolds underwent gross observation and histological examination. Alcin blue method was used to examine the content of proteoglycan （GAG）. Immunochemistry and Western blotting were used to examine the type Ⅱ collagen. The cylindrical scaffolds underwent biomechanical analysis. Results HE staining showed cartilage lacunas increasing in number from the periphery to center of the cylindrical scaffolds with the extended inducing time, and were arranged more uniformly progressively. Histochemical staining showed GAG accumulation. Immunohistochemistry and Western blotting showed that the content of type Ⅱ collagen increased gradually, the amount of type Ⅱ collagen stabilized after 6 weeks＇ culture, and there was no significant difference in the content of type Ⅱ collagen between Group C and Group E. Biomechanical analysis showed that the modulus of dasticity and compression strength of the groups induced for more than 6 weeks （Groups C, D, and E） were all higher then those of Groups and B. Conclusion TGF-β1 inducing time is correlated with the cartilage engineering characteristics with BMSC as seed cells. Induction for 6 weeks helps construct tissue engineered cartilage with good tissue structure, chemical composition andbiomechanical properties.

关 键 词：生物医学工程 软骨 骨髓基质细胞 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]