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  本文選題：孤雌胚胎干細胞 + 全能性�。� 參考：《西北大學》2011年碩士論文

【摘要】：在骨組織工程研究領域中,骨原細胞來源是制約骨組織工程研究與應用的關鍵問題,以往研究多采用骨髓基質干細胞、脂肪干細胞等作為種子細胞向成骨細胞誘導分化,但結果并不理想。因此需要為骨組織工程研究尋找一種理想的種子細胞。 目的：1.驗證KM×129／SV小鼠孤雌胚胎干細胞的全能性,證實其具有向成骨細胞定向分化的潛能；2.探索孤雌胚胎干細胞向成骨細胞定向誘導分化的條件,鑒定誘導的成骨細胞；3.觀察誘導的孤雌胚胎干細胞種植于三維支架珊瑚上的貼附和生長情況。 方法：將孤雌胚胎干細胞在體外穩(wěn)定培養(yǎng),通過堿性磷酸酶染色,全能性表面抗原SSEA-1、端粒酶和細胞因子的免疫組化染色,RT-PCR檢測全能性轉錄因子,孤雌胚胎干細胞體內分化檢測驗證KM×129/SV孤雌胚胎干細胞的全能性。通過向培養(yǎng)液中添加地塞米松、p-磷酸甘油、維生素C等成骨細胞誘導因子,直接誘導孤雌胚胎干細胞向成骨細胞定向分化,通過RT-PCR檢測成骨細胞相關基因、透射電鏡、茜素紅、von Kossa染色等方法檢測孤雌胚胎干細胞向成骨細胞定向分化的情況。并將誘導7天后的孤雌胚胎干細胞接種于三維支架材料珊瑚上,通過激光共聚焦顯微鏡和掃描電鏡觀察細胞在材料上的生長情況。 結果：實驗結果顯示：1.KM×129/SV小鼠孤雌胚胎干細胞在體外培養(yǎng)條件下能夠穩(wěn)定增殖,堿性磷酸酶染色呈陽性,表達全能性表面抗原SSAE-1和全能性轉錄因子Nanog、Oct4和Sox2,具有高水平的端粒酶活性,處于未分化狀態(tài)；在體內可以分化為三個胚層的細胞類型,具有全能性。2.用所建立的成骨細胞誘導條件誘導孤雌胚胎干細胞定向分化,誘導過程中細胞形態(tài)發(fā)生顯著變化；誘導28天時表達成骨細胞相關基因Ⅰ型膠原和骨鈣素,茜素紅、von Kossa染色均呈陽性,誘導的細胞發(fā)生礦化,形成鈣結節(jié)；線粒體、高爾基體增多,形成電子密度較高的礦化物。3.誘導7天的孤雌胚胎干細胞種植于珊瑚材料上,激光共聚焦顯微鏡和掃描電鏡觀察顯示細胞在珊瑚表面貼附良好,并能穩(wěn)定增殖。傳代,具有與胚胎干細胞相似的全能性,通過直接誘導的方法可以將孤雌胚胎干細胞定向誘導為成骨細胞,接種于三維支架材料珊瑚上生長良好。表明孤雌胚胎干細胞適宜作為骨組織工程的種子細胞,通過進一步研究能夠找到更為優(yōu)化的誘導條件,提高孤雌胚胎干細胞體外誘導成骨細胞的效率,使該方法最終應用于臨床治療中。
[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.
【學位授予單位】：西北大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329

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