兔間充質(zhì)干細(xì)胞分離培養(yǎng)和體外向軟骨細(xì)胞分化的實(shí)驗(yàn)研究
發(fā)布時間：2018-10-29 20:36
【摘要】： 目的 1.建立一種分離培養(yǎng)兔骨髓間充質(zhì)干細(xì)胞(bone marrow derived mesenchymalstem cells,BMSCs)的有效方法,并觀察BMSCs在特定培養(yǎng)條件下向軟骨細(xì)胞分化的情況。 2.探討使用G-CSF動員兔外周血,分離培養(yǎng)外周血間充質(zhì)干細(xì)胞(peripheralblood mesenchymal stem cells,PBMSCs)的方法。 方法 1.以4月齡新西蘭大耳白兔為實(shí)驗(yàn)對象,采用密度梯度離心法、全血培養(yǎng)法、氯化胺紅細(xì)胞溶解法,分離培養(yǎng)BMSCs,對其細(xì)胞存活率、細(xì)胞克隆率、首次傳代時間、15d后細(xì)胞數(shù)及擴(kuò)增成功率進(jìn)行比較。 2.用含有骨形態(tài)發(fā)生蛋白(bone morphogenetic proteins,BMP)-2的條件培養(yǎng)液誘導(dǎo)BMSCs在體外單層培養(yǎng)模式下向軟骨細(xì)胞分化。采用甲苯胺藍(lán)染色、Masson染色、免疫組織化學(xué)染色、阿利新藍(lán)比色法測定糖胺聚糖(glycosaminoglycan,GAG)的含量來檢測BMSCs分化情況。統(tǒng)計學(xué)分析比較第2、4、7代(P_2、P_4、P_7)BMSCs誘導(dǎo)后增殖能力及向軟骨細(xì)胞分化能力的變化。 3.給新西蘭兔皮下注射粒細(xì)胞集落刺激因子30μg/(kg·d)4d,抽取動員前后兔外周血采用密度梯度離心法進(jìn)行分離培養(yǎng)。 結(jié)果 1.對于細(xì)胞存活率而言,密度梯度離心法和全血培養(yǎng)法均優(yōu)于氯化胺紅細(xì)胞溶解法。在其它指標(biāo)比較中,密度梯度離心法)全血培養(yǎng)法)氯化胺紅細(xì)胞溶解法。 2.BMSCs定向誘導(dǎo)后表現(xiàn)出軟骨細(xì)胞的特性,P_2、P_4間的細(xì)胞增殖能力和成軟骨細(xì)胞分化能力比較差異無統(tǒng)計學(xué)意義(P＞0.05);P_7與P_2、P_4相比,細(xì)胞增殖能力和成軟骨細(xì)胞分化能力均降低,差異有統(tǒng)計學(xué)意義(P＜0.01)。 3.未動員的外周血中未發(fā)現(xiàn)有梭形細(xì)胞貼壁;動員后的外周血中發(fā)現(xiàn)有梭形細(xì)胞貼壁,而且這些細(xì)胞可以形成小的集落,但是增殖緩慢,增殖能力有限,不能大量擴(kuò)增。 結(jié)論 1.利用成年兔骨髓,體外成功分離出骨髓間充質(zhì)干細(xì)胞,同時發(fā)現(xiàn)密度梯度離心法優(yōu)于全血培養(yǎng)法和氯化胺紅細(xì)胞溶解法。 2.兔BMSCs能在BMP-2為主要誘導(dǎo)因子的單層培養(yǎng)模式下在體外向軟骨細(xì)胞分化;細(xì)胞傳代對BMSCs增殖和向軟骨細(xì)胞分化有重要影響。 3.從動員后的外周血中,發(fā)現(xiàn)有梭形細(xì)胞但它們增殖能力有限,還不能說它們就是間充質(zhì)干細(xì)胞。PBMSCs分離培養(yǎng)有待于進(jìn)一步研究。
[Abstract]:Objective 1. To establish an effective method for isolation and culture of rabbit bone marrow mesenchymal stem cells (bone marrow derived mesenchymalstem cells,BMSCs) and to observe the differentiation of BMSCs into chondrocytes under specific culture conditions. 2. To explore the method of using G-CSF to mobilize rabbit peripheral blood and isolate and culture peripheral blood mesenchymal stem cells (peripheralblood mesenchymal stem cells,PBMSCs). Method 1. Four month old New Zealand white rabbits were isolated and cultured with BMSCs, by density gradient centrifugation, whole blood culture, erythrocytolysis of chloramines, cell viability, cell clone rate and first passage time. After 15 days, the number of cells and the success rate of amplification were compared. 2. BMSCs was induced to differentiate into chondrocytes in monolayer culture with conditioned medium containing bone morphogenetic protein (bone morphogenetic proteins,BMP)-2. Toluidine blue staining, Masson staining, immunohistochemical staining and alisin blue colorimetry were used to determine the content of glycosaminoglycan (glycosaminoglycan,GAG) to detect the differentiation of BMSCs. The ability of proliferation and differentiation into chondrocytes were compared after BMSCs induction. 3. Granulocyte colony stimulating factor 30 渭 g / (kg d) was injected subcutaneously into New Zealand rabbits for 4 days. The peripheral blood was isolated and cultured by density gradient centrifugation before and after mobilization. Result 1. For cell survival, density gradient centrifugation and whole blood culture were superior to chloramines in erythrocyte lysis. Among other indexes, density gradient centrifugation method) whole blood culture method) chloramine erythrocyte lysis method. 2.BMSCs showed the characteristics of chondrocytes after directed induction. There was no significant difference in cell proliferation and chondroblast differentiation among the four groups (P > 0. 05). The ability of proliferation and differentiation of chondroblasts decreased significantly (P < 0.01). 3. There were no fusiform cells adherent to the unmobilized peripheral blood, and fusiform cells adherent to the mobilized peripheral blood, and these cells could form small colonies, but the proliferation was slow and the ability of proliferation was limited and could not be expanded in large numbers. Conclusion 1. Bone marrow mesenchymal stem cells were successfully isolated from adult rabbit bone marrow in vitro. It was found that the density gradient centrifugation method was superior to the whole blood culture method and the chloride amine erythrocyte lysis method. 2. Rabbit BMSCs could differentiate into chondrocytes in vitro in the monolayer culture model with BMP-2 as the main inducer, and cell passage had an important effect on the proliferation and differentiation of BMSCs into chondrocytes. 3. From the mobilized peripheral blood, fusiform cells were found, but their proliferative ability was limited, which can not be said to be mesenchymal stem cells. The isolation and culture of PBMSCs need further study.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R329
本文編號：2298747
[Abstract]:Objective 1. To establish an effective method for isolation and culture of rabbit bone marrow mesenchymal stem cells (bone marrow derived mesenchymalstem cells,BMSCs) and to observe the differentiation of BMSCs into chondrocytes under specific culture conditions. 2. To explore the method of using G-CSF to mobilize rabbit peripheral blood and isolate and culture peripheral blood mesenchymal stem cells (peripheralblood mesenchymal stem cells,PBMSCs). Method 1. Four month old New Zealand white rabbits were isolated and cultured with BMSCs, by density gradient centrifugation, whole blood culture, erythrocytolysis of chloramines, cell viability, cell clone rate and first passage time. After 15 days, the number of cells and the success rate of amplification were compared. 2. BMSCs was induced to differentiate into chondrocytes in monolayer culture with conditioned medium containing bone morphogenetic protein (bone morphogenetic proteins,BMP)-2. Toluidine blue staining, Masson staining, immunohistochemical staining and alisin blue colorimetry were used to determine the content of glycosaminoglycan (glycosaminoglycan,GAG) to detect the differentiation of BMSCs. The ability of proliferation and differentiation into chondrocytes were compared after BMSCs induction. 3. Granulocyte colony stimulating factor 30 渭 g / (kg d) was injected subcutaneously into New Zealand rabbits for 4 days. The peripheral blood was isolated and cultured by density gradient centrifugation before and after mobilization. Result 1. For cell survival, density gradient centrifugation and whole blood culture were superior to chloramines in erythrocyte lysis. Among other indexes, density gradient centrifugation method) whole blood culture method) chloramine erythrocyte lysis method. 2.BMSCs showed the characteristics of chondrocytes after directed induction. There was no significant difference in cell proliferation and chondroblast differentiation among the four groups (P > 0. 05). The ability of proliferation and differentiation of chondroblasts decreased significantly (P < 0.01). 3. There were no fusiform cells adherent to the unmobilized peripheral blood, and fusiform cells adherent to the mobilized peripheral blood, and these cells could form small colonies, but the proliferation was slow and the ability of proliferation was limited and could not be expanded in large numbers. Conclusion 1. Bone marrow mesenchymal stem cells were successfully isolated from adult rabbit bone marrow in vitro. It was found that the density gradient centrifugation method was superior to the whole blood culture method and the chloride amine erythrocyte lysis method. 2. Rabbit BMSCs could differentiate into chondrocytes in vitro in the monolayer culture model with BMP-2 as the main inducer, and cell passage had an important effect on the proliferation and differentiation of BMSCs into chondrocytes. 3. From the mobilized peripheral blood, fusiform cells were found, but their proliferative ability was limited, which can not be said to be mesenchymal stem cells. The isolation and culture of PBMSCs need further study.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R329
【引證文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前2條
1 和小娥;兔骨髓間充質(zhì)干細(xì)胞分離培養(yǎng)及定向分化的研究[D];河南農(nóng)業(yè)大學(xué);2011年
2 閆穎穎;兔骨髓間充質(zhì)干細(xì)胞向心肌樣細(xì)胞的體外誘導(dǎo)分化研究[D];西北農(nóng)林科技大學(xué);2010年
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