兔髓核细胞体外培养体系的建立及TGF-β1对其生物学行为的影响

发布时间：2018-04-08 12:01

本文选题：组织工程　切入点：组织再生　出处：《第三军医大学》2005年硕士论文

【摘要】： 目的:探讨兔髓核细胞体外单层培养的适宜条件,并观察一种自行构建的II型胶原支架上立体培养兔髓核细胞的变化和10ng/ml rhTGF-β1对其生物学行为的影响。方法: 单层培养:改变单层培养条件(培养瓶是否铺被PLL、培养液pH、胎牛血清浓度、rhTGF-β1浓度),利用MTT法和细胞计数法,决定兔髓核细胞体外单层培养的适宜条件。支架培养:将兔髓核细胞以5×10~6/ml接种到II型胶原支架上进行立体培养1w,利用倒置显微镜和扫描电镜观察细胞形态学变化;分别利用RT-PCR、3H-proline掺入法观察单层培养、立体培养和10 ng/ml rhTGF-β1刺激24小时后兔髓核细胞基因表达水平和总胶原合成的变化。结果: 单层培养: (1)兔髓核细胞对培养条件要求较高,其中培养介质是否铺被PLL、培养液的pH、胎牛血清浓度、rhTGF-β1对细胞影响较为明显。铺被PLL明显促进兔髓核细胞贴壁和生长(1.12倍,p0.01);pH在6.8～7.2之间,兔髓核细胞生长较好,pH7.0为较适宜培养液pH;rhTGF-β1在5～10 ng/ml范围内呈剂量依赖性促进细胞生长。在确立的适宜条件中培养时,细胞生长良好,电镜结果示细胞器增多。 (2)原代兔髓核细胞指数生长期是接种后的8～10d,第一代兔髓核细胞指数生长期是接种后的6～8d。兔髓核细胞体外培养时增殖能力较弱,第一代活性最高。 (3)兔髓核细胞单层培养时II型胶原和集聚蛋白聚糖免疫组化染色阳性。支架培养 (1)兔髓核细胞可以在II型胶原支架上黏附生长,细胞由单层培养时的多角形变为类圆形。 (2)兔髓核细胞在II型胶原支架上培养1周,其II型胶原、集聚蛋白聚糖基因表达水平和总胶原合成较单层培养增加(1.21、1.12、1.11倍,p0.05、p0.05、p0.01)。
[Abstract]:Aim: to investigate the suitable conditions for monolayer culture of rabbit nucleus pulposus cells in vitro, and to observe the changes of rabbit nucleus pulposus cells cultured on a self-constructed type II collagen scaffold and the effect of 10ng/ml rhTGF- 尾 1 on its biological behavior.Methods:Monolayer culture: changing the conditions of monolayer culture (whether the culture bottle was coated with PLL, the pH of culture medium, the concentration of rhTGF- 尾 1 in fetal bovine serum), the suitable conditions for monolayer culture of rabbit nucleus pulposus cells in vitro were determined by MTT method and cell counting method.Scaffold culture: rabbit nucleus pulposus cells were seeded on type II collagen scaffold with 5 脳 10~6/ml for 1 week. Morphological changes were observed by inverted microscope and scanning electron microscope, and monolayer culture was observed by RT-PCRN 3H-proline incorporation method.The changes of gene expression and total collagen synthesis in rabbit nucleus pulposus cells stimulated by 10 ng/ml rhTGF- 尾 1 and stereoscopic culture for 24 hours.Results:Monolayer culture:1) Rabbit nucleus pulposus cells require higher culture conditions, among them, whether the culture medium is covered with PLL, the pH of the culture medium, the concentration of fetal bovine serum and rhTGF- 尾 1 have obvious effects on the cells.When cultured in suitable conditions, the cells grew well and the organelles increased under electron microscope.(2) the growth period of primary rabbit nucleus pulposus cell index was 810 days after inoculation, and that of the first generation rabbit nucleus pulposus cell index was 6 days after inoculation.The proliferative ability of rabbit nucleus pulposus cells was weak in vitro and the first generation activity was the highest.Type II collagen and agglutinin were positive in monolayer culture of rabbit nucleus pulposus cells.Scaffold culture1) Rabbit nucleus pulposus cells could grow on type II collagen scaffold, and the cells changed from polygonal shape to round shape in monolayer culture.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329

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