不同负压对干细胞表皮分化影响的实验研究

发布时间：2018-04-27 16:27

  本文选题：负压 + 骨髓间充质干细胞　； 参考：《石河子大学》2017年硕士论文

【摘要】：目的:探讨体外不同负压对于骨髓间充质干细胞(Bone mesenchymal stem cells,BMSCs)表皮分化的影响。方法:1.采用密度梯度离心法分离培养4-6月龄新西兰大白兔BMSCs;2.培养至第三代BMSCs进行鉴定:倒置显微镜对细胞进行形态学观察,间充质干细胞的三系分化鉴定,流式细胞仪鉴定细胞表面标志物;3.设置实验组,分别是0mmhg组(对照组)、50mmhg组、100mmhg组、200mmhg组,诱导时间为30min/h;4.负压下常规培养第三代BMSCs,分别于1d、3d、5d和7d进行细胞计数绘制细胞生长曲线,于3d、5d和7d使用CCK8检测细胞增殖情况;5.诱导BMSCs向表皮细胞分化,使用免疫荧光法检测BMSCs向表皮细胞分化诱导的结果;6.负压条件下诱导BMSCs向表皮细胞分化14d后,RT-PCR检测CK5、CK10m RNA的表达,western-blot检测广谱角蛋白的表达情况。结果:1.倒置显微镜下可见原代BMSCs呈鱼群样、漩涡样生长,另可见大量淋巴细胞与血细胞,第三代BMSCs呈规则长梭形,杂细胞较少;2.第三代BMSCs可向骨细胞、脂肪细胞、软骨细胞分化;3.第三代BMSCs高表达骨髓间充质干细胞表面抗原CD29、CD44,不表达骨髓造血干细胞表面抗原CD34;4.随着负压的逐渐增高,细胞的增殖能力逐渐受抑制,细胞生长曲线及CCK8实验在第7d时,四个组比较两两比较均有统计学意义;5.随着负压的逐渐增高,表皮样细胞CK5、CK10m RNA的表达逐渐增高;6.随着负压的逐渐增高,表皮样细胞广谱角蛋白的表达逐渐增高。结论:随着负压增高,BMSCs向表皮分化的能力逐渐增强,但是细胞增殖能力逐渐受抑制。
[Abstract]:Aim: to investigate the effect of different negative pressure on epidermal differentiation of bone mesenchymal stem cells (BMSCs) from bone marrow mesenchymal stem cells (BMSCs) in vitro. Method 1: 1. BMSCs 2 was isolated from 4-6 month old New Zealand white rabbits by density gradient centrifugation. Culture to the third generation of BMSCs for identification: inverted microscope to observe the morphology of mesenchymal stem cells, identification of the three line differentiation, flow cytometry identification of the cell surface marker. The experimental group was divided into 0mmhg group (control group, 50mmhg group) and 100mmhg group (200mmhg group). The third generation BMSCs were cultured under negative pressure, and the cell growth curves were plotted at 1 d ~ 3 d ~ 5 d and 7 d, respectively. At 3 d ~ 5 d and 7 d, CCK8 was used to detect the proliferation of BMSCs. BMSCs was induced to differentiate into epidermal cells. Immunofluorescence assay was used to detect the differentiation of BMSCs into epidermal cells. The expression of CK5CK10m RNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) after 14 days of differentiation of BMSCs into epidermal cells induced by negative pressure. Western blot was used to detect the expression of broad-spectrum keratin. The result is 1: 1. Under the inverted microscope, the primary BMSCs appeared as a fish group, swirl like growth, and a large number of lymphocytes and blood cells could be seen. The third generation of BMSCs had a regular long spindle shape and less miscellaneous cells. The third generation of BMSCs could differentiate into osteocytes, adipocytes and chondrocytes. The third generation of BMSCs overexpressed bone marrow mesenchymal stem cell surface antigen CD29 and CD44, but did not express bone marrow hematopoietic stem cell surface antigen CD344. With the increase of negative pressure, the cell proliferation was inhibited gradually. At the 7th day, the cell growth curve and CCK8 test showed significant difference between the four groups. With the increase of negative pressure, the expression of CK5 + CK10 m RNA in epidermoid cells increased gradually. With the increase of negative pressure, the expression of broad spectrum keratin in epidermoid cells increased gradually. Conclusion: with the increase of negative pressure, the ability of BMSCs to differentiate into epidermis is gradually enhanced, but the ability of cell proliferation is gradually inhibited.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R61

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