氧浓度对蜕膜基质细胞成骨分化的影响

发布时间：2018-01-12 02:16

本文关键词：氧浓度对蜕膜基质细胞成骨分化的影响　出处：《山东大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的：研究发现,骨组织工程的种子细胞骨髓干细胞数量有限,取材及增殖分化困难,在临床应用中也受到病人经济条件的制约；而近期研究表明蜕膜基质细胞(Decidual Stromal Cells, DSC)可在一定条件下诱导为成骨细胞。本实验通过体外分离培养人早孕蜕膜基质细胞,并对其进行体外培养、扩增；将部分蜕膜细胞向成骨细胞(Osteoblast, OB)方向诱导；观察常氧、低氧条件下蜕膜细胞的增殖情况及体外成骨能力,探讨氧对蜕膜细胞诱导成骨能力的影响。方法：取人工流产6-8周妊娠蜕膜组织,采用机械研磨法分离蜕膜,复合酶消化,传代自然增殖法纯化细胞,进行体外培养扩增。倒置显微镜观察其大体形态,当细胞传至第五代以后性状较稳定,所以采用第六代蜕膜基质细胞进行成骨诱导分化培养。将细胞消化后吹打均匀,分为四组种入6孔板中：(1)DSC常氧对照组(2)DSC常氧成骨诱导培养组(3)DSC低氧对照组(4)DSC低氧成骨诱导培养组。将培养基更换为成骨诱导液,每隔两至三天换一次液,持续诱导28天,每7天用PNPP法检测各组细胞的碱性磷酸酶(ALP)活性的表达,酶标仪测定光密度值(OD值)及茜素红染色观察矿化结节形成情况。结果： 1.机械研磨及复合酶消化结合传代自然增殖法纯化细胞,获取高纯度的蜕膜基质细胞,且保持了较好的细胞活性。 2.蜕膜基质细胞在成骨诱导液的作用下,三天后明显形成集落,细胞明显变大,并呈明显的多边形、三角形、不规则形,细胞体积比加诱导液之前明显增大,胞浆透明,内含少量颗粒样或条索样高密度结构(附图1,2)；成骨诱导10天,细胞层叠生长,细胞密集,中央可见类似结节状结构(附图3)。经诱导后的细胞增殖较为缓慢,10～14天细胞才达到90%融合；对照组加入了常规培养基(1640培养基+10%FBS),细胞体积较小,呈长梭形,偶见多边形,细胞增殖较快,大约5-7天可达到90%的细胞融合。 3.酶标仪检测光密度值(OD值)、各组ALP活性表达显示：DSC常氧成骨诱导培养组的ALP活性表达明显高于其他三组(P0.05):DSC低氧成骨诱导培养组的ALP活性表达明显高于两个常规培养基培养组(P0.05); 4.四组样本培养7天,14天,21天,28天后茜素红染色,成骨诱导组细胞密集区有矿化结节形成(附图4),茜素红染色呈橘红色,常氧成骨诱导培养比低氧成骨诱导培养组明显,其余两组未见。结论： 1.蜕膜基质细胞在一定条件下可以诱导分化为成骨细胞； 2.在其他诱导条件相同的情况下,与低氧诱导相比,常氧诱导更有利于蜕膜基质细胞诱导成骨。
[Abstract]:Objective: It is found that the number of bone marrow stem cells in bone tissue engineering is limited, and it is difficult to obtain materials and proliferate and differentiate, which is also restricted by the economic conditions of patients in clinical application. Recent studies have shown that decidua stromal cells are Decidual Stromal Cells. Human decidual stromal cells were isolated and cultured in vitro. Some decidual cells were induced to osteoblast (OB). To observe the proliferation of decidual cells under normoxic and hypoxic conditions and the ability of osteogenesis in vitro, and to explore the effect of oxygen on the osteogenesis of decidual cells. Methods: The decidua tissue of pregnancy during 6-8 weeks of induced abortion was isolated by mechanical grinding, digested by complex enzyme, purified by natural proliferation method, cultured and amplified in vitro. The gross morphology of decidua was observed by inverted microscope. When the cells were transferred to the fifth generation, the characters were stable, so 6th passage decidual stromal cells were used for osteogenic differentiation culture. After digestion, the cells were blown evenly. Four groups were seeded into 6-well plate. The control group was divided into two groups: normal oxygen group (n = 2), osteogenesis induction group (n = 3) and hypoxic control group (n = 4). DSC hypoxic osteogenic culture group. The medium was replaced by osteogenic medium. The expression of alkaline phosphatase (ALP) activity was detected by PNPP assay every 7 days. Optical density value (OD value) and alizarin red staining were used to observe the formation of mineralized nodules. Results: 1. High purity decidual stromal cells were obtained by mechanical grinding and complex enzyme digestion combined with natural proliferation method. 2. Decidual stromal cells in osteoblast induction fluid, three days after the formation of colonies, the cells became significantly larger, and showed an obvious polygon, triangle, irregular shape. The cell volume increased obviously before the addition of inducer and the cytoplasm was transparent with a small amount of granular or strip-like high-density structure (Fig. 1). After induction of osteogenesis for 10 days, the cells were stacked, and the cells were dense, with nodular structure in the center (Fig. 3). After induction, the proliferation of the cells was slower than that of the cells at the end of 10 ~ 14 days before the cells reached 90% fusion. The control group was added to the conventional culture medium No. 1640. The cells were small, fusiform, occasionally polygonal, and the cells proliferated rapidly, and about 90% cells could be fused in 5-7 days. 3. The optical density value and OD value were detected by enzyme labeling instrument. The expression of ALP activity in each group was significantly higher than that in the other three groups (P 0.05). The expression of ALP activity in the osteogenic induction group was significantly higher than that in the two conventional culture medium groups. 4. Alizarin red staining was observed on the 7th day, 14th day, 21 day and 28 day after culture in the four groups. Mineralized nodules were formed in the dense areas of osteoblasts in the osteoblast induction group (Fig. 4, alizarin red staining was orange). Normogenic osteoblast induction culture was more obvious than hypoxia osteoblast induction culture group, but the other two groups were not seen. Conclusion: 1. Decidual stromal cells can be induced to differentiate into osteoblasts under certain conditions. 2. Under the same conditions, normoxic induction is more beneficial to osteogenesis of decidual stromal cells than hypoxia.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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