人脐带间充质干细胞三维载体接种方法的研究

发布时间：2018-02-06 05:29

本文关键词： 脐带间充质干细胞组织工程接种方法循环灌注　出处：《兰州大学》2011年硕士论文　论文类型：学位论文

【摘要】：人脐带间充质干细胞在骨组织工程领域有广泛的应用前景。三维载体上细胞的接种方法对于骨组织构建有重要的影响。本文应用自制的循环灌注接种系统研究循环灌注接种,以及离心接种和静态接种这三种接种方法在人脐带间充质干细胞骨组织工程领域的应用。培养传代人脐带间充质干细胞,流式细胞仪检测细胞表面标记,将细胞用循环灌注方法,离心接种方法和静态接种方法接种至三维聚对苯二甲酸乙二醇酯(Polyethylene terephthalate, PET)无纺布片状载体,CCK-8细胞计数法计算接种效率,乳酸脱氢酶法测定细胞损伤,连续培养18天并绘制生长曲线。18天后使用成骨诱导分化培养液连续诱导分化20天,检测碱性磷酸酶活性比较不同接种方法的细胞成骨分化能力。结果显示传代细胞形态稳定、活力好,高表达CD90,CD105,不表达CD14,CD45。循环灌注方法组和离心接种方法组的接种效率,细胞密度,增殖能力均优于静态接种组。与离心接种方法和静态接种方法相比循环灌注组LDH偏高,并且细胞的延迟期长于静态接种。循环灌注方法组和离心方法组的碱性磷酸酶活性高于静态接种组。结果表明,循环灌注接种和离心接种方法组更适合间充质干细胞骨组织工程的应用,但进一步应用仍需对工程参数进行优化。
[Abstract]:Human umbilical cord mesenchymal stem cells have a wide application prospect in bone tissue engineering. The method of cell inoculation on three-dimensional carrier has an important effect on bone tissue construction. Ring perfusion inoculation. And the application of centrifugation and static inoculation in bone tissue engineering of human umbilical cord mesenchymal stem cells. Human umbilical cord mesenchymal stem cells were cultured and the surface markers were detected by flow cytometry. Centrifugal and static inoculation methods were used to inoculate the three-dimensional polyethylene terephthalate (PET) non-woven sheet carrier. CCK-8 cell count method was used to calculate the inoculation efficiency, and lactate dehydrogenase method was used to determine the cell damage. After 18 days of continuous culture, the growth curve was drawn and the osteogenic differentiation medium was used to induce differentiation for 20 days. The activity of alkaline phosphatase was compared with the osteogenic differentiation ability of different inoculation methods. The results showed that the passaged cells were stable in morphology and good in activity. CD90 CD105 was highly expressed and CD14 was not expressed. CD45. the inoculation efficiency and cell density of circulatory perfusion group and centrifugal inoculation group. The proliferative ability was superior to the static inoculation group, and the LDH of the circulatory perfusion group was higher than that of the centrifugal inoculation method and the static inoculation method. The activity of alkaline phosphatase in the circulatory perfusion group and centrifugation group was higher than that in the static inoculation group. The methods of circulatory perfusion inoculation and centrifugal inoculation were more suitable for the application of bone tissue engineering of mesenchymal stem cells, but further application still needed to optimize the engineering parameters.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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