【作者】 冯建军；

【导师】 杨述华；

【作者基本信息】 华中科技大学 ， 骨外科， 2007， 博士

【摘要】 目的观察使用不同首次换液时间和不同获取骨髓间充质干细胞的方法对骨髓间充质干细胞分化增殖的影响,分析其部分表型特征,为其在组织工程的应用确立最佳培养方案;利用琼脂糖预先铺被培养皿的方法,使骨髓间充质干细胞悬浮,探讨在体外诱导骨髓间充质干细胞失巢凋亡过程中Caspase-3的作用;在体外模拟细胞离体后的悬浮状态,初步建立大鼠骨髓间充质干细胞体外悬浮培养模型,为骨髓基质干细胞的悬浮培养提供理论依据。方法以6w龄SPF级大鼠为实验对象,应用不同首次换液时间,对原代骨髓间充质干细胞进行培养,分离纯化大鼠骨髓MSCs,传代扩增,测定生长曲线,形态学观察,免疫细胞化学及图像分析测定细胞表面抗原表达情况;通过对全骨髓细胞贴壁培养法、改良全骨髓贴壁法、溶血纯化法和密度梯度离心法进行比较性研究,对大鼠骨髓间充质干细胞克隆形成率、贴壁细胞数测定、首次传代时间、培养成功率和台盼蓝染色等指标进行比较;应用Caspase-3活性检测试剂盒,荧光比色法和Western blotting法检测失巢凋亡中Caspase-3活性的改变;借助流式细胞仪分析骨髓间充质干细胞凋亡的变化。结果首次换液时间为6h时,骨髓间充质干细胞有效贴壁,并且可以加快骨髓间充质干细胞的纯化过程,其子代细胞生长曲线基本一致,增殖速度快。细胞呈均一的成纤维细胞样,均一表达CD44、CD71但是不表达CD34分子;全骨髓贴壁培养法组和改良骨髓细胞贴壁法组的细胞贴壁所需要的时间较其他两组最短;溶血纯化法组和密度梯度离心法组所获得的细胞较单一;全骨髓贴壁培养法组在原代培养第四天形成的平均克隆数为4.67;改良骨髓细胞贴壁法组为17.67;溶血纯化法组为3.67;密度梯度离心法组为4.33;改良骨髓细胞贴壁法组与其他组相比具有统计学显著性差异(p<0.05)。全骨髓贴壁培养法组和改良骨髓细胞贴壁法组在第1d的贴壁细胞数量明显高于其他两组,具有统计学显著性差异(p<0.05);第4d时各组之间贴壁细胞数的差别不大,在统计学方面没有显著性差异。第7d和第10d时改良骨髓细胞贴壁法组和密度梯度离心法组的贴壁细胞数明显高于溶血纯化法组(p<0.05)。在各实验组中改良骨髓细胞贴壁法组的首次传代所需时间最短,为6天,进行首次传代的平均时间为8.17天;溶血纯化法组首次传代的平均时间最长,11.75天。改良骨髓细胞贴壁法组和密度梯度离心法组的培养成功率为100%,溶血纯化法组的最低,为66.67%。全骨髓贴壁培养法组中被台盼蓝染料着色的细胞数平均有301.5个,细胞存活率为39.7%;改良骨髓细胞贴壁法组的着色细胞数平均为325个,细胞存活率为35%;溶血纯化法组的着色细胞数平均为306.5个,细胞存活率为38.7%;密度梯度离心法组的着色细胞数平均为333个,细胞存活率为33.4%,各组在统计学方面没有显著性差异;骨髓间充质干细胞在琼脂糖的作用下有效悬浮;流式细胞术显示诱导凋亡组出现了明显的凋亡峰,凋亡率随诱导时间的延长而明显增加;其它两组的凋亡率较低且较稳定。Western blotting检测和Caspase-3活性荧光检测显示,诱导凋亡组caspase-3的活性以及蛋白表达水平随诱导凋亡时间的延长而明显升高,且与细胞凋亡率相关,以后者更灵敏,其余两组其活性无明显变化;Caspase-3抑制剂可以有效抑制其活性,使悬浮细胞的凋亡率明显下降。结论本实验改良了一种体外分离纯化、培养扩增大鼠骨髓骨髓间充质干细胞的方法,经免疫组化细胞表型分析,所获细胞为纯化的骨髓间充质干细胞;应用琼脂糖可以使骨髓间充质干细胞有效悬浮;caspase-3蛋白在诱导骨髓间充质干细胞失巢凋亡的过程中发挥着重要作用;抑制Caspase-3的活性可以有效降低细胞失巢凋亡率,达到初步建立悬浮培养模型的目的。更多还原

【Abstract】 OBJECTIVETo observe the influence of the first replacing culture medium time and different methods to the MSCs.And identified its biologic characteristic.Providing an better project for organise engineering. To investigate the significance of Caspase-3 in anoikis induced by preventing mesenchymal stem cells(MSCs) from adherence. And to model MSCs suspending condition in vitro, in order to investigate MSCs anoikis phenomenon and the role of Caspase inhibitor in MSCs anoikis. And the model of MSCs will be investigated oreigienally.MATHODESWe choose specific pathogen free (SPF) rats, 6W, mean weight 150g, disassociate the femoral and shin bone, then wash the medullary cavity of bone with DMEM and resuspended cells in complete culture medium. Our experiment selects second filial generation MSCs. The cultured MSCs are randomly divided into different groups.And the influence of the first replacing culture medium time and different methods to the MSCs was observed with growth curves, morphologic, immunocyte chemistry, superficial expression, clone rate and filial generation. Before experiment, we coat dishes with 1.5% agarose liquor and waiting for its coagulation; in inhibitor group,DVED-CHO and cells are put into procoated dishes at the same time; control group has not any interventional measure. The cells are collected in three groups at 2h, 6h, 12h, 24h. The alteration of caspase-3 activity is evaluated by Caspase-3 Fluorometric Assay and Western blot analysis, and the apoptosis rates are detected by flow cytometry, repeat the experiment twice. RESULTSWhen the first replacing culture medium time is at 6 hour after resuspended, the MSCs was purified faster, and expressing CD44+ and CD71+, butCD34 was negative. Full marrow adherence culture had more clone rates, cells survival rates and shorter filial time than that of the others. Caspase-3 fluorometric assay shows that Caspase-3 activity in anoikis group at 2h, 6h, 12h, 24h are(9.2197, 7.8514), (12.3425, 16.2718), (24.3826, 25.7443), (22.7386, 24.2836); (2.1754, 2.3675), (2.7910, 2.3925), (3.7428, 3.2769), (4.7564, 5.7938 ) in inhibitor group; And(9.1632, 7.7438), (8.2463, 7.3524), (7.2673, 7.8294), (7.2783, 6.1245 ) in control group. The Caspase-3 activity in anoikis group increase significantly along with the apoptosis conditions going on, but they are lower and stable in the inhibitor group and the control group. Western blot analysis discovers that the Caspase-3 relative expression in anoikis group at 2h, 6h, 12h, 24h is (0.3640, 0.5352, 0.7124, 0.8456); (0.2375, 0.3527, 0.4743, 0.5123) inhibitor group; (0.3923, 0.4976, 0.5272, 0.5154) in control group. The flow cytometry indicates that apoptosis peaks appear distinctly in every group. And apoptosis rate increased dramaticly in the anoikis group and the apoptosis rates are(7.22%, 6.23%), (17.97%, 15.22%), (35.98%, 34.25%), (52.95%, 54.12%) at 2h, 6h, 12h, 24h relatively;In inhibitor and control group, they are(4.4%, 5.2%), (6.2%, 5.8%), (8.7%, 7.9%), (9.8%, 8.5%); and(5.3%, 5.5%), (6.7%, 6.3%), (9.6%, 8.5%), (11.3%, 10.4%). The flow cytometry analysis showd that apoptosis peak was appeared significantly in the induced group and increased dramaticly along with the apoptosis conditions going on.The apoptosis rate of the others were lower and stable.Western blotting and fluorometric assay showed the caspase-3 expressive level or activity in induced group was the highest and was related with its apoptosis rate. The fluorometric assay technique was sensitive to the western blotting.CONCLUSIONWe improved the measure of obtaining MSCs,and the MSCs was purified. Argorose can suspended MSCs efficiently. And MSCs will undergo anoikis in suspended condition if they are separated from extracellular matrix. Caspase-3 was play a vital part in suspended culture induced anoikis of MSCs in vitro. But with Caspase-3 activity suppressed, Caspase inhibitor should reduce the apoptosis rate significantly,and should set up the model of MSCs suspending culture.更多还原