本文關(guān)鍵詞：成年大鼠嗅鞘細(xì)胞的純化培養(yǎng)及其增殖劑的研究　出處：《蘇州大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 嗅鞘細(xì)胞 脊髓損傷 條件培養(yǎng)液 Forskolin 堿性成纖維生長因子

【摘要】： 目的:①運用改良差速貼壁法+無血清條件培養(yǎng)液來純化培養(yǎng)嗅鞘細(xì)胞,以期建立一種新穎、經(jīng)濟、簡單、實用的純化培養(yǎng)成年大鼠嗅鞘細(xì)胞的方法。②全面探討有血清條件培養(yǎng)液、Forskolin與堿性成纖維生長因子(bFGF)對嗅鞘細(xì)胞生長增殖的影響,以及多種增殖劑聯(lián)合干預(yù)嗅鞘細(xì)胞的效果,以期解決基礎(chǔ)與臨床研究中嗅鞘細(xì)胞數(shù)量及來源有限的問題。 方法:①將無菌條件下取材、消化的嗅鞘單細(xì)胞混懸液分為三份:單純改良差速貼壁組、單純無血清條件培養(yǎng)液純化組和改良差速貼壁+無血清條件培養(yǎng)液純化組。各組按要求分別行12 h +24 h差速貼壁純化和預(yù)先制備的含同源嗅鞘培養(yǎng)上清液的無血清條件培養(yǎng)液孵育純化36~48 h,運用倒置顯微鏡觀察和細(xì)胞免疫熒光染色法評估和檢測各組嗅鞘細(xì)胞的活力和純度。②純化的嗅鞘細(xì)胞按照隨機化原則分為六組:空白對照組、有血清條件培養(yǎng)液組、Forskolin組、bFGF組、Forskolin+bFGF組和三聯(lián)合干預(yù)組。應(yīng)用BrdU (5-bromodeoxyuridine)摻入法和MTT活性檢測法全面評估各增殖劑及其聯(lián)合試劑對嗅鞘細(xì)胞生長增殖的影響效果。 結(jié)果:①改良差速貼壁+無血清條件培養(yǎng)液可以使原代培養(yǎng)的嗅鞘細(xì)胞純度達(dá)到88%~92%,明顯優(yōu)于其它兩組純化對照組。②有血清條件培養(yǎng)液、Forskolin較空白對照組可以促進嗅鞘細(xì)胞的活性(P0.05),其增值效應(yīng)相對緩和。bFGF對嗅鞘細(xì)胞的增殖效果比較強烈,較空白對照組可以明顯促進嗅鞘細(xì)胞活性(P0.001)。此外,Forskolin+bFGF和有血清條件培養(yǎng)液+Forskolin+bFGF聯(lián)合干預(yù)組均可大幅度地提高嗅鞘細(xì)胞的數(shù)量(P0.001),三種增殖劑組合應(yīng)用有明顯的疊加效果。 結(jié)論:①運用改良差速貼壁法+無血清條件培養(yǎng)液法可以建立起完善的成年大鼠嗅鞘細(xì)胞培養(yǎng)純化新理論。②有血清條件培養(yǎng)液、Forskolin與bFGF對嗅鞘細(xì)胞均有增殖效應(yīng)。三種干預(yù)因素的聯(lián)合作用可以大幅度的提升嗅鞘細(xì)胞的數(shù)量,其疊加效果明顯,可以作為常規(guī)擴增原代嗅鞘細(xì)胞的一種手段。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R329

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