DDR2基因缺失对小鼠骨髓间充质干细胞生物学特性影响的研究

发布时间：2018-04-17 03:38

本文选题：DDR2 + 骨髓间充质干细胞　；参考：《第四军医大学》2014年硕士论文

【摘要】：骨髓间充质干细胞(Bone Mesenchymal Stem Cells,BMSCs)是现阶段研究广泛的一种重要的成体干细胞。因其具有良好的生长、分化能力以及向多种细胞分化的潜能,成为研究骨再生工程中重要的种子细胞,它对骨组织缺损的修复有着良好的效果。但是BMSCs向成骨细胞分化并修复骨组织缺损是一个复杂的过程,是由许多细胞外因子共同作用产生的结果。近年来的研究证实DDR2(Discoidin Domain Receptor2)作为I型胶原的特异性受体,在成骨细胞的分化中发挥重要的调节作用。而对于其在BMSCs向成骨细胞的分化过程中所起到的作用还鲜有研究,对其作用机理尚不明确。本实验通过DDR2基因缺失小鼠BMSCs的分离、培养和鉴定,初步探索DDR2基因缺失对小鼠BMSCs成骨分化能力的影响,为进一步研究BMSCs的成骨分化能力奠定理论基础。研究内容与方法:1.DDR2基因缺失小鼠BMSCs细胞的分离、培养和鉴定。2.DDR2对于BMSCs细胞特异性分化特性的影响作用。3.DDR2基因缺失对于BMSCs增殖、凋亡、分化的调控作用。4.DDR2基因缺失对于BMSCs成骨能力的影响。5.DDR2基因缺失对BMSCs在动物体内骨组织修复能力的影响。我们采用了利用改良型全骨髓贴壁法分离培养DDR2基因缺失小鼠及野生型小鼠的BMSCs,观察对比两种细胞形态;流式细胞技术检测两种细胞表面标记分子的表达;MTT分析检测细胞生长周期;流式细胞技术检测细胞凋亡情况,将两种细胞进行成骨、成脂诱导后,real time-PCR检测两种BMSCs成骨诱导3,7,14天后,其成骨相关基因的表达量变化;以及对成骨分化相关蛋白的检测;包括ALP、茜素红染色及OCN、ALP蛋白定量检测。最后采用小鼠颅骨缺损的模型,将BMSCs与支架材料HA/TCP复合后植入骨缺损区,在成骨6周后进行MicroCT扫描及HE染色,观察DDR2基因缺失对小鼠成骨能力的影响研究结果与结论:1.成功分离培养出DDR2基因缺失小鼠的BMSCs,其生长状态与正常野生小鼠的BMSCs相同,外形均呈长梭形,可见细胞成旋涡状紧密排列生长,其表面标记物的表达符合间充质干细胞的特点,细胞具有体外诱导成骨与成脂分化的能力,证明培养的细胞是BMSCs。2.DDR2基因缺失后,对BMSCs的凋亡略有抑制作用,但并不影响其正常的生长与传代。3.体外对两种BMSCs进行成骨和成脂诱导分化后发现,DDR2基因缺失后,BMSCs的成骨分化能力明显减弱,对其成脂分化能力略有减弱。4.两种BMSC s经成骨诱导以后,发现DDR2基因缺失后BMSCs的碱性磷酸酶活性降低,OCN分泌减少,对其成骨相关基因的表达量有明显的抑制作用。5.体内实验证实了DDR2基因缺失的BMSCs的骨修复能力明显低于正常野生型小鼠的BMSCs。本研究结果证实DDR2基因缺失后,对于BMSCs细胞形态特征生长特性没有明显影响。但对其向成骨细胞分化有明显的抑制作用,可以降低细胞碱性磷酸酶的活性,减少OCN分泌,抑制其成骨相关基因的表达。体内实验也证实DDR2基因缺失后,BMSCs修复骨组织缺损的能力明显减弱。
[Abstract]:Bone marrow mesenchymal stem cells (Bone Mesenchymal Stem Cells, BMSCs) is an extensive study of adult stem cell. Because of its good growth, differentiation ability and potential of multilineage differentiation, become the important seed cells of bone tissue engineering, bone defect repairing on it have a good effect. But BMSCs osteogenic differentiation and bone defect repair is a complex process that is produced by the interaction of many extracellular factors. Recent studies show that DDR2 (Discoidin Domain Receptor2) is a specific receptor of type I collagen, play an important role in the differentiation osteoblasts. And in the BMSCs to the differentiation of osteoblasts in the role but also little research, the mechanism is not clear. In this experiment, by separating the deletion of DDR2 gene in mouse BMSCs Culture and identification, and to explore the effect of DDR2 gene deletion on osteogenic differentiation of mouse BMSCs, which lays the theoretical foundation for the further study of BMSCs osteogenic differentiation ability. The research contents and methods: isolation of 1.DDR2 gene deletion in mouse BMSCs cells and influence on the differentiation of BMSCs cell specific effects of.3.DDR2 culture and identification of.2.DDR2 gene deletion for the BMSCs proliferation, apoptosis and regulation of.4.DDR2 gene deletion and differentiation for the BMSCs effect of.5.DDR2 gene deletion of BMSCs in the capacity of bone bone in vivo animal repair ability. We adopted the improved method of whole bone marrow adherent cultured DDR2 knockout mice and wild type mice BMSCs, observe and compare the two kinds of cells morphology; flow cytometry. The expression of two cell surface marker detection and analysis of MTT; cell cycle; flow cytometry The apoptosis of two cells osteogenic, adipogenic, real time-PCR detection of two BMSCs 3,7,14 days after osteogenic induction, the expression of bone related genes; and the detection of osteogenic differentiation related protein; including ALP, alizarin red staining and OCN protein, ALP quantitative detection finally. The mouse skull defect model, BMSCs and HA/TCP composite scaffold after implantation of bone defect, MicroCT scan and HE staining in the bone after 6 weeks of observation, to study the influence of DDR2 gene deletion on the osteogenesis of mice and conclusion: 1. successful separation of cultivated DDR2 gene deletion in mice BMSCs, its growth state and wild mice BMSCs same shape were fusiform cells arranged tightly into the whirlpool like growth, the expression of surface markers consistent with the characters of mesenchymal stem cells, cells with in vitro osteogenic and adipogenic differentiation can That is, the cultured cells of BMSCs.2.DDR2 gene deletion, apoptosis of BMSCs was slightly inhibited, but does not affect its normal growth and passage of two kinds of BMSCs.3. in vitro osteogenic and adipogenic differentiation showed that deletion of DDR2 gene, BMSCs osteogenic differentiation ability was significantly weakened, on the differentiation capacity of two kinds of BMSC s.4. decreased after osteogenic induction, alkaline phosphatase activity decreased BMSCs found DDR2 gene deletion, OCN secretion decreased the amount of bone related genes expression inhibition of.5. in vivo was confirmed by bone repair ability of DDR2 gene deletion of BMSCs was significantly lower than that in normal wild in BMSCs. the results of this study confirmed that the DDR2 gene deletion, the growth characteristics of BMSCs cell morphology had no obvious effect. But its to inhibit bone cell differentiation, can reduce the fine The activity of alkaline phosphatase reduces OCN secretion and inhibits the expression of osteogenic related genes. In vivo experiments also confirm that after DDR2 gene deletion, the ability of BMSCs to repair bone defects is significantly reduced.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R329.2

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