摘要
目的:观察体外构建骨髓间充质干细胞、改良纤维蛋白胶及生长因子的组织工程软骨模块的可行性。方法:实验于2004-07/12在南方医科大学附属珠江医院中心实验室进行。选取新西兰兔10只,密度梯度法分离培养兔骨髓间充质干细胞,在培养系统中加入生长因子(含10μg/L转化生长因子β1,100mol/L地塞米松,50mg/L维生素C,以及1%ITS-A。培养第2,4,6,8天采用四甲基偶氮噻唑法在酶标仪上检测吸光度值表示细胞增殖。于培养7,14d行细胞爬片甲苯胺蓝染色及Ⅱ胶原免疫组化。诱导后骨髓间充质干细胞种植于改良纤维蛋白胶支架上,加入生长因子诱导培养,于培养7,14,21d行形态组织学及透射电镜检查。结果:实验兔10只均进入实验结果分析。①四甲基偶氮噻唑法测定培养第6和8天后,实验组骨髓间充质干细胞增殖的吸收度值高于对照组(实验组:0.4554±0.0206,0.5348±0.0169;对照组:0.4270±0.0255,0.5057±0.0368,P<0.05)。②细胞爬片甲苯胺蓝染色细胞周围有蓝色基质。Ⅱ型胶原免疫组化染色阳性。③诱导后骨髓间充质干细胞在改良纤维蛋白胶支架中培养,Masson染色可见胞浆及细胞周围有胶原的形成;Ⅱ型胶原免疫组化染色阳性。透射电镜可见细胞代谢旺盛。结论:骨髓间充质干细胞、改良纤维蛋白胶及生长因子的组织工程模块体外培养系统中,改良纤维蛋白胶支架相容性好,转化生长因子β等生长因子促进骨髓间充质干细胞在改良纤维蛋白胶支架增殖,诱导分化成软骨细胞表型。
AIM： To observe the feasibility of the reconstruction in vitro of cartilage tissue engineered compound by using rabbit bone mesenchymal stem cells （BMSCs） with growth factors in improved fibrin glue bracket. METHODS： The experiment was performed in the central laboratory of Zhujiang Hospital affiliated to Southern Medical University between June and December 2004. BMSCs were extracted from 10 New Zealand rabbits＇ bone marrow hy means of density gradient centrifugation. The culture system was added with growth factors [including 10 μg/L transforming growth factor-β1 （TGF-β1）, 100 mol/L dexamethasone, 50 mg/L vitamin C and 1% ITS-A]. The cells proliferation was detected by methyl-thiazoltetrazolium （MTF） reduction assay after 2, 4, 6 and 8 days. The cells morphology was ohserved and the cells were harvested at 7 and 14 days respectively and examined by histology and type Ⅱ collagen immunohistoehemical test. Then the cells were seeded in improved fibrin glue bracket to construct the compound in vitro with growth factor. The compounds were observed and were harvested at 7, 14 and 21 days respectively and examined by histology, electron microscopy and type Ⅱ collagen immunohistochemical test. RESULTS： All the 10 rabbits were involved in the analysis of results. ①The MTT assay indicated that the absnrbanee values of BMSCs proliferation at 6 and 8 days were higher in the experimental group than in the contrail group （experimental group： 0.455 4±0.020 6, 0.534 8±0.016 9; control group： 0.427 0±0.025 5, 0.505 7±0.036 8, P 〈 0.05）. ②There was blue matrix around the toluidine stained cells. The type Ⅱ collagen immunohistoehemical test was positive. ③ The BMSCs were cultured in the improved fibriu glue bracket after induction, Masson staining showed that collagen formed around cytoplasm and cells. The type Ⅱ collagen immunohistochemical test was positive. High productive cell metabolism couhl be observed under the electron microscopy. CONCLUSION： The improved fibrin glue b
出处
《中国临床康复》
CSCD
北大核心
2006年第5期4-6,i0001,共4页
Chinese Journal of Clinical Rehabilitation
基金
广东省自然科学基金资助项目(2002C31303)~~
