成年大鼠嗅鞘细胞的纯化培养及其增殖剂的研究

发布时间：2018-01-01 13:23

本文关键词：成年大鼠嗅鞘细胞的纯化培养及其增殖剂的研究　出处：《苏州大学》2009年硕士论文　论文类型：学位论文

【摘要】： 目的:①运用改良差速贴壁法+无血清条件培养液来纯化培养嗅鞘细胞,以期建立一种新颖、经济、简单、实用的纯化培养成年大鼠嗅鞘细胞的方法。②全面探讨有血清条件培养液、Forskolin与碱性成纤维生长因子(bFGF)对嗅鞘细胞生长增殖的影响,以及多种增殖剂联合干预嗅鞘细胞的效果,以期解决基础与临床研究中嗅鞘细胞数量及来源有限的问题。方法:①将无菌条件下取材、消化的嗅鞘单细胞混悬液分为三份:单纯改良差速贴壁组、单纯无血清条件培养液纯化组和改良差速贴壁+无血清条件培养液纯化组。各组按要求分别行12 h +24 h差速贴壁纯化和预先制备的含同源嗅鞘培养上清液的无血清条件培养液孵育纯化36~48 h,运用倒置显微镜观察和细胞免疫荧光染色法评估和检测各组嗅鞘细胞的活力和纯度。②纯化的嗅鞘细胞按照随机化原则分为六组:空白对照组、有血清条件培养液组、Forskolin组、bFGF组、Forskolin+bFGF组和三联合干预组。应用BrdU (5-bromodeoxyuridine)掺入法和MTT活性检测法全面评估各增殖剂及其联合试剂对嗅鞘细胞生长增殖的影响效果。结果:①改良差速贴壁+无血清条件培养液可以使原代培养的嗅鞘细胞纯度达到88%~92%,明显优于其它两组纯化对照组。②有血清条件培养液、Forskolin较空白对照组可以促进嗅鞘细胞的活性(P0.05),其增值效应相对缓和。bFGF对嗅鞘细胞的增殖效果比较强烈,较空白对照组可以明显促进嗅鞘细胞活性(P0.001)。此外,Forskolin+bFGF和有血清条件培养液+Forskolin+bFGF联合干预组均可大幅度地提高嗅鞘细胞的数量(P0.001),三种增殖剂组合应用有明显的叠加效果。结论:①运用改良差速贴壁法+无血清条件培养液法可以建立起完善的成年大鼠嗅鞘细胞培养纯化新理论。②有血清条件培养液、Forskolin与bFGF对嗅鞘细胞均有增殖效应。三种干预因素的联合作用可以大幅度的提升嗅鞘细胞的数量,其叠加效果明显,可以作为常规扩增原代嗅鞘细胞的一种手段。
[Abstract]:Objective to purify and culture olfactory ensheathing cells by using modified differential adherent serum-free conditioned medium in order to establish a novel, economical and simple method. Practical method of purification and culture of olfactory ensheathing cells in adult rats. The effects of Forskolin and basic fibroblast growth factor (bFGF) on the growth and proliferation of olfactory ensheathing cells and the effects of multiple proliferators on olfactory ensheathing cells. To solve the problem of the limited number and source of olfactory ensheathing cells in basic and clinical studies. Methods the single cell suspensions of olfactory ensheathing were divided into three parts: simple modified differential adherent group. Pure serum-free conditioned medium purification group and modified differential adherent serum-free conditioned medium purification group. Each group was treated with 12 h 24 as required. H differential adherent purification and pre-prepared culture supernatant containing olfactory ensheathing culture supernatant were incubated with serum-free conditioned medium for 3648 h. The activity and purity of olfactory ensheathing cells in each group were evaluated and detected by inverted microscope and immunofluorescence staining. The purified olfactory ensheathing cells were divided into six groups according to randomization principle: blank control group. There were serum conditioned medium group and forskolin group and bFGF group. Forskolin bFGF group and three combined intervention groups. BrdU 5-bromodeoxyuridine). The effects of various proliferators and their combined reagents on the growth and proliferation of olfactory ensheathing cells were evaluated by incorporation and MTT assay. Results the purity of the primary cultured olfactory ensheathing cells reached 88 ~ 922a, which was better than that of the other two groups. 2. The serum-conditioned medium was better than that of the other two groups. Forskolin could promote the activity of olfactory ensheathing cells (P0.05), and its value-added effect was relatively mild. BFGF had stronger effect on the proliferation of olfactory ensheathing cells. Compared with the blank control group, the activity of olfactory ensheathing cells was significantly increased (P 0.001). The number of olfactory ensheathing cells was significantly increased in Forskolin bFGF group and Forskolin bFGF group (P 0.001). The combination of three proliferators has obvious superposition effect. Conclusion the modified differential adherent method can be used to establish a new theory of culture and purification of adult rat olfactory ensheathing cells with serum-conditioned medium. 2. Forskolin and bFGF have proliferative effect on olfactory ensheathing cells. The combined effect of three intervention factors can greatly increase the number of olfactory ensheathing cells, and the superposition effect is obvious. It can be used as a conventional method to amplify primary olfactory ensheathing cells.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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