小鼠孤雌胚胎干细胞向成骨细胞定向诱导分化的研究

发布时间：2018-06-29 12:53

本文选题：孤雌胚胎干细胞 + 全能性　；参考：《西北大学》2011年硕士论文

【摘要】：在骨组织工程研究领域中,骨原细胞来源是制约骨组织工程研究与应用的关键问题,以往研究多采用骨髓基质干细胞、脂肪干细胞等作为种子细胞向成骨细胞诱导分化,但结果并不理想。因此需要为骨组织工程研究寻找一种理想的种子细胞。目的：1.验证KM×129／SV小鼠孤雌胚胎干细胞的全能性,证实其具有向成骨细胞定向分化的潜能；2.探索孤雌胚胎干细胞向成骨细胞定向诱导分化的条件,鉴定诱导的成骨细胞；3.观察诱导的孤雌胚胎干细胞种植于三维支架珊瑚上的贴附和生长情况。方法：将孤雌胚胎干细胞在体外稳定培养,通过碱性磷酸酶染色,全能性表面抗原SSEA-1、端粒酶和细胞因子的免疫组化染色,RT-PCR检测全能性转录因子,孤雌胚胎干细胞体内分化检测验证KM×129/SV孤雌胚胎干细胞的全能性。通过向培养液中添加地塞米松、p-磷酸甘油、维生素C等成骨细胞诱导因子,直接诱导孤雌胚胎干细胞向成骨细胞定向分化,通过RT-PCR检测成骨细胞相关基因、透射电镜、茜素红、von Kossa染色等方法检测孤雌胚胎干细胞向成骨细胞定向分化的情况。并将诱导7天后的孤雌胚胎干细胞接种于三维支架材料珊瑚上,通过激光共聚焦显微镜和扫描电镜观察细胞在材料上的生长情况。结果：实验结果显示：1.KM×129/SV小鼠孤雌胚胎干细胞在体外培养条件下能够稳定增殖,碱性磷酸酶染色呈阳性,表达全能性表面抗原SSAE-1和全能性转录因子Nanog、Oct4和Sox2,具有高水平的端粒酶活性,处于未分化状态；在体内可以分化为三个胚层的细胞类型,具有全能性。2.用所建立的成骨细胞诱导条件诱导孤雌胚胎干细胞定向分化,诱导过程中细胞形态发生显著变化；诱导28天时表达成骨细胞相关基因Ⅰ型胶原和骨钙素,茜素红、von Kossa染色均呈阳性,诱导的细胞发生矿化,形成钙结节；线粒体、高尔基体增多,形成电子密度较高的矿化物。3.诱导7天的孤雌胚胎干细胞种植于珊瑚材料上,激光共聚焦显微镜和扫描电镜观察显示细胞在珊瑚表面贴附良好,并能稳定增殖。传代,具有与胚胎干细胞相似的全能性,通过直接诱导的方法可以将孤雌胚胎干细胞定向诱导为成骨细胞,接种于三维支架材料珊瑚上生长良好。表明孤雌胚胎干细胞适宜作为骨组织工程的种子细胞,通过进一步研究能够找到更为优化的诱导条件,提高孤雌胚胎干细胞体外诱导成骨细胞的效率,使该方法最终应用于临床治疗中。
[Abstract]:In the research field of bone tissue engineering, the origin of osteogenic cells is the key problem restricting the research and application of bone tissue engineering. In the past, bone marrow stromal cells and adipose stem cells were used as seed cells to induce differentiation into osteoblasts. But the results were not satisfactory. Therefore, it is necessary to find an ideal seed cell for bone tissue engineering. Purpose 1. The totipotency of km 脳 129 / SV mouse parthenogenetic embryonic stem cells was verified. Objective: to explore the conditions for the differentiation of parthenogenetic embryonic stem cells into osteoblasts and identify the osteoblasts. To observe the adhesion and growth of induced parthenogenetic embryonic stem cells implanted on three-dimensional scaffold coral. Methods: parthenogenetic embryonic stem cells were cultured stably in vitro. Altipotent transcription factors were detected by alkaline phosphatase staining, immunocytochemical staining of human telomerase and cytokines, SSEA-1, immunocytochemical staining of telomerase and cytokines. The in vivo differentiation test of parthenogenetic embryonic stem cells verified the totipotency of km 脳 129% SV parthenogenetic embryonic stem cells. Osteoblast inducing factors such as dexamethasone pphosphate glycerol and vitamin C were added to the culture medium to directly induce the differentiation of parthenogenetic embryonic stem cells into osteoblasts. The osteoblast related genes were detected by RT-PCR and transmission electron microscopy (TEM). The differentiation of parthenogenetic embryonic stem cells into osteoblasts was detected by alizarin red von Kossa staining. After 7 days of induction, parthenogenetic embryonic stem cells were seeded on the three-dimensional scaffold coral, and the growth of the cells was observed by laser confocal microscope and scanning electron microscope. Results: the results showed that the parthenogenetic embryonic stem cells of W1. Km 脳 129 / SV mice could proliferate stably in vitro, and alkaline phosphatase staining was positive. The expression of totipotent surface antigen SSAE-1 and totipotent transcription factors Nanog-Oct4 and Sox2, with high telomerase activity and undifferentiated state, can differentiate into three cell types in vivo, with totipotency. The differentiation of parthenogenetic embryonic stem cells was induced by osteoblast induction condition, and the morphology of parthenogenetic embryonic stem cells was significantly changed during the induction, and the type I collagen and osteocalcin related genes were expressed at 28 days after induction. Alizarin red von Kossa staining was positive, and the cells were mineralized to form calcium nodules, mitochondria and Golgi bodies increased, forming mineralized compounds with high electron density. After 7 days of induction, parthenogenetic embryonic stem cells were implanted on coral materials. Laser confocal microscopy and scanning electron microscopy showed that the cells adhered well to the coral surface and could proliferate stably. Through direct induction, parthenogenetic embryonic stem cells can be induced into osteoblasts and grow well on three-dimensional scaffold coral. The results indicated that parthenogenetic embryonic stem cells were suitable for seed cells of bone tissue engineering. Through further study, we could find more optimal inducing conditions and improve the efficiency of inducing osteoblasts from parthenogenetic embryonic stem cells in vitro. Finally, the method can be used in clinical treatment.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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