本文關(guān)鍵詞：甲基潑尼松龍對(duì)體外培養(yǎng)許旺細(xì)胞分泌神經(jīng)營養(yǎng)因子的研究　出處：《鄭州大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 甲基潑尼松龍 許旺細(xì)胞 神經(jīng)營養(yǎng)因子

【摘要】：目的: 研究甲基潑尼松龍對(duì)體外培養(yǎng)許旺細(xì)胞分泌神經(jīng)營養(yǎng)因子的促進(jìn)作用。 方法: 兩周齡新西蘭大白兔6只處死后,用75%酒精浸泡20min,無菌條件下取雙側(cè)坐骨神經(jīng)和臂叢神經(jīng),顯微鏡下仔細(xì)剝除神經(jīng)外膜后,將神經(jīng)剪成1 mm~3大小,間距1cm種植于預(yù)先鋪過明膠的培養(yǎng)瓶中;7d后傳代,繼續(xù)培養(yǎng)3d后應(yīng)用胰酶消化,聯(lián)合差速貼壁法純化,并S-100蛋白免疫組化染色檢查許旺細(xì)胞純度。鑒定過的許旺細(xì)胞配成5×10~4/ml單細(xì)胞懸液,每孔1ml加入4孔培養(yǎng)板中。 根據(jù)各孔所加入培養(yǎng)液中甲基潑尼松龍濃度的不同進(jìn)行分組:A孔為對(duì)照組,加入含20%小牛血清的雙抗高糖DMEM培養(yǎng)液2ml;B孔中加入含0.1mg/L甲基潑尼松龍的DMEM培養(yǎng)液2ml;C孔中加入含1.0mg/L甲基潑尼松龍的DMEM培養(yǎng)液2ml;D孔中加入含5.0mg/L甲基潑尼松龍的DMEM培養(yǎng)液2ml。將培養(yǎng)板置于37℃、CO_2體積分?jǐn)?shù)為5%的恒溫培養(yǎng)箱內(nèi)培養(yǎng),每天觀察細(xì)胞生長情況;每2d用隨即計(jì)數(shù)法估算細(xì)胞數(shù)。每2d吸出培養(yǎng)液分別存于離心管中并4℃冰箱保存;吸取干凈后,PBS反復(fù)沖洗培養(yǎng)孔,再加入相應(yīng)培養(yǎng)液。 將前后兩次計(jì)算的細(xì)胞數(shù)量分別相減,得出細(xì)胞數(shù)量的增加值,應(yīng)用單因素方差分析進(jìn)行統(tǒng)計(jì)學(xué)比較。將獲得的20管培養(yǎng)液混勻,三等分后,用ELISA法分別做NGF、CNTF、GDNF定量測定,將前后兩次的測量值分別相減,得出神經(jīng)營養(yǎng)因子分泌量的增加值,應(yīng)用單因素方差分析進(jìn)行統(tǒng)計(jì)學(xué)比較。實(shí)驗(yàn)數(shù)據(jù)采用spss13.0統(tǒng)計(jì)學(xué)軟件處理,取P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果: ①B、C、D組許旺細(xì)胞生長速度明顯快于A組,三種神經(jīng)營養(yǎng)因子分泌量明顯多于A組,統(tǒng)計(jì)學(xué)有顯著差異(P＜0.05),B、C、D組之間無顯著差異(P＞0.05); ②NGF的檢測結(jié)果有優(yōu)于CNTF、GDNF的趨勢(P＜0.01),CNTF和GDNF的促分泌情況無明顯差異(P＞0.05)。 結(jié)論: 甲基潑尼松龍能直接作用于許旺細(xì)胞來促進(jìn)體外培養(yǎng)許旺細(xì)胞的增殖和神經(jīng)營養(yǎng)因子的分泌。 甲基潑尼松龍的用量在促進(jìn)體外培養(yǎng)許旺細(xì)胞的增殖和神經(jīng)營養(yǎng)因子的分泌方面無明顯差異。 甲基潑尼松龍能通過促進(jìn)體外培養(yǎng)許旺細(xì)胞的增殖和神經(jīng)營養(yǎng)因子的分泌來促進(jìn)損傷周圍神經(jīng)的再生和修復(fù)。
[Abstract]:Objective:
To study the effect of methylprednisolone on the secretion of neurotrophic factors from Schwann cells in vitro.
Method:
Two week old New Zealand rabbits were sacrificed after 6, with 75% alcohol for 20min, under sterile conditions, take the sciatic nerve and brachial plexus, microscope stripping epineurium after the nerve was cut into 1 mm~3 size, spacing of 1cm grown in culture flask paved gelatin; 7d after subculture, continue to apply trypsin digestion after 3D culture, combined with differential centrifugation and purified S-100 protein immunohistochemical staining of Schwann cell purity. The identification of Schwann cells with 5 x 10~4/ml single cell suspension, each hole 1ml in 4 well culture plate.
According to the different groups of each hole by adding methyl prednisolone concentration in culture medium: A hole for the control group, containing 20% calf serum anti double high glucose DMEM medium 2ml medium containing 0.1mg/L 2ml; DMEM B methyllprednisolone hole; the medium 2ml containing 1.0mg/L methylprednisolone prednisolone DMEM C hole in the 2ml. of the medium; the culture plates were placed in the temperature of 37 DEG DMEM containing 5.0mg/L methylprednisolone in D hole, the volume fraction of CO_2 in the training of 5% incubation, cell growth was observed every day; every 2D by counting the number of cells. Then the estimation of each 2D aspirate culture liquid are stored in the centrifuge tube and 4 DEG C refrigerator; draw clean, PBS repeatedly flushing the culture hole, then add the appropriate medium.
The two times before and after the calculation of the number of cells were subtracted, that increase the value of the number of cells, one way ANOVA was used for statistical comparison. The 20 culture liquid mixing, is divided into three parts, NGF, CNTF and GDNF respectively by ELISA method, quantitative determination, the two values were measured before and after subtraction. The added value of neurotrophic factor secretion, one way ANOVA was used for statistical comparison. The experimental data using SPSS13.0 statistical software, P < 0.05, the difference was statistically significant.
Result:
(1) the growth rate of Schwann cells in group B, C and D was faster than that in group A, and the secretion of three neurotrophic factors was significantly higher than that in group A (P < 0.05). There was no significant difference between B, C and D group (P > 0.05).
The test results of NGF were better than that of CNTF and GDNF (P < 0.01), and there was no significant difference in the secretion of CNTF and GDNF (P > 0.05).
Conclusion:
Methylprednisolone can directly act on Schwann cells to promote the proliferation of Schwann cells in vitro and the secretion of neurotrophic factors.
The dosage of methylprednisolone has no significant difference in promoting the proliferation of Schwann cells in vitro and the secretion of neurotrophic factors in vitro.
Methylprednisolone can promote the regeneration and repair of injured peripheral nerves by promoting the proliferation of Schwann cells and the secretion of neurotrophic factors in vitro.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 蔡鴻敏;吳學(xué)建;;甲基潑尼松龍對(duì)體外培養(yǎng)兔雪旺細(xì)胞遷移及增殖的影響[J];鄭州大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2008年02期

2 朱浩;丁文龍;;施旺細(xì)胞發(fā)育及其調(diào)控的信號(hào)通路[J];解剖科學(xué)進(jìn)展;2006年04期

3 張繼東,李國棟,趙定麟;甲基強(qiáng)的松龍治療脊髓傷的機(jī)制[J];頸腰痛雜志;1997年03期

4 吳志偉;陳崢嶸;汪洋;程飚;;人胚雪旺細(xì)胞組織工程神經(jīng)修復(fù)坐骨神經(jīng)缺損的實(shí)驗(yàn)研究[J];中國臨床醫(yī)學(xué);2006年05期

5 程飚,陳崢嶸;成年兔坐骨神經(jīng)雪旺細(xì)胞的分離純化培養(yǎng)[J];復(fù)旦學(xué)報(bào)(醫(yī)學(xué)科學(xué)版);2001年03期

6 李金照,陳燕,夏另朝,邱蓉,張瑛,鄧巍;腦源神經(jīng)營養(yǎng)因子基因轉(zhuǎn)染神經(jīng)斷端促進(jìn)切斷坐骨神經(jīng)再生[J];生物物理學(xué)報(bào);1997年03期

7 張培訓(xùn),姜保國;骨髓間充質(zhì)干細(xì)胞的研究進(jìn)展與周圍神經(jīng)修復(fù)[J];中國臨床康復(fù);2004年02期

8 杜嬋;姜保國;;許旺細(xì)胞增殖和分化過程中的信號(hào)通路[J];中國組織工程研究與臨床康復(fù);2007年03期

9 劉婷;李世普;萬濤;袁琳;;體外原代培養(yǎng)成年大鼠許旺細(xì)胞的實(shí)驗(yàn)方法:組織塊反復(fù)種植+低濃度酶消化+差速貼壁[J];中國組織工程研究與臨床康復(fù);2007年28期

10 何繼銀,勞杰;周圍神經(jīng)損傷后雪旺細(xì)胞生存與凋亡的動(dòng)態(tài)平衡[J];中華創(chuàng)傷骨科雜志;2004年03期

，

本文編號(hào)：1379115