發(fā)布時(shí)間：2018-02-26 17:10

  本文關(guān)鍵詞： 骨髓間充質(zhì)干細(xì)胞 細(xì)胞培養(yǎng)技術(shù) 神經(jīng)元 睫狀神經(jīng)節(jié)神經(jīng)營(yíng)養(yǎng)因子 堿性成纖維細(xì)胞生長(zhǎng)因子　出處：《南華大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的 采用體外分離、培養(yǎng)人骨髓間充質(zhì)干細(xì)胞(human Mesenchymal stem cells, h-MSCs),以及用堿性成纖維細(xì)胞生長(zhǎng)因子(basic fibroblast growth factor,bFGF)和不同濃度的睫狀神經(jīng)節(jié)神經(jīng)營(yíng)養(yǎng)因子(ciliary neurotrophic factor,CNTF)對(duì)其進(jìn)行預(yù)誘導(dǎo)及誘導(dǎo),通過觀察細(xì)胞形態(tài)學(xué)變化,及免疫細(xì)胞化學(xué)染色CD34、CD44、CD54、NSE和Nestin的表達(dá),探討建立一種較簡(jiǎn)便有效的體外擴(kuò)增培養(yǎng)h-MSCs的體系,以及CNTF對(duì)h-MSCs分化為神經(jīng)元樣細(xì)胞的誘導(dǎo)作用和適宜的誘導(dǎo)濃度。 方法 骨髓來源于南華大學(xué)附屬第二醫(yī)院和第一醫(yī)院血液內(nèi)科行骨髓穿刺檢查患者(經(jīng)患者同意),已排除腫瘤、傳染病及血液系統(tǒng)疾病,年齡18～55歲,性別不限,男39例,女29例,共68例。采用密度梯度離心法和全骨髓貼壁法相結(jié)合的方法,分離獲得h-MSCs,利用含血清的DMEM/F12培養(yǎng)基體外擴(kuò)增,以及細(xì)胞凍存、復(fù)蘇。相差倒置顯微鏡下觀察細(xì)胞形態(tài)學(xué)變化,并繪制第1、3、6代h-MSCs的生長(zhǎng)曲線。取傳至第3代的h-MSCs,分為4組進(jìn)行誘導(dǎo)分化:實(shí)驗(yàn)組、單純預(yù)誘導(dǎo)組、單純誘導(dǎo)組和空白對(duì)照組。實(shí)驗(yàn)組以bFGF預(yù)誘導(dǎo)24h后,再以5ng/mL、10ng/mL和20ng/mL濃度梯度的CNTF誘導(dǎo)12h;單純預(yù)誘導(dǎo)組僅行bFGF預(yù)誘導(dǎo)而無CNTF誘導(dǎo);單純誘導(dǎo)組不經(jīng)bFGF預(yù)誘導(dǎo)僅行CNTF誘導(dǎo),誘導(dǎo)液中的CNTF亦分為3個(gè)濃度梯度(5ng/mL、10ng/mL和20ng/mL);空白對(duì)照組為無處理組,既無bFGF預(yù)誘導(dǎo)亦無CNTF誘導(dǎo)。應(yīng)用免疫細(xì)胞化學(xué)技術(shù)對(duì)培養(yǎng)的h-MSCs進(jìn)行CD34、CD44和CD54鑒定及對(duì)誘導(dǎo)分化后的細(xì)胞進(jìn)行NSE和Nestin鑒定。 結(jié)果 1.h-MSCs在體外培養(yǎng)條件下,呈長(zhǎng)梭形,類似成纖維細(xì)胞,可穩(wěn)定增值傳代并可凍存復(fù)蘇,復(fù)蘇后細(xì)胞生長(zhǎng)特性與凍存前的細(xì)胞相似; 2.第1、3、6代h-MSCs生長(zhǎng)曲線呈“S”形。第1代與第3代的細(xì)胞濃度比較差異無統(tǒng)計(jì)學(xué)意義(P=0.704),第1代與第6代比較差異無統(tǒng)計(jì)學(xué)意義(P=0.132),第3代與第6代比較差異亦無統(tǒng)計(jì)學(xué)意義(P=0.256); 3.應(yīng)用免疫細(xì)胞化學(xué)技術(shù)檢測(cè)培養(yǎng)的h-MSCs,顯示CD44, CD54均呈陽性表達(dá),CD34表達(dá)呈陰性; 4.h-MSCs在bFGF預(yù)誘導(dǎo)24h后,細(xì)胞形態(tài)基本無改變。CNTF誘導(dǎo)5h后部分細(xì)胞體積變小,以細(xì)胞核為中心收縮,逐漸呈錐形,形成突起。CNTF誘導(dǎo)12h后,實(shí)驗(yàn)組中10ng/mLCNTF組和20ng/mLCNTF組細(xì)胞形態(tài)改變明顯,有2個(gè)或多個(gè)細(xì)長(zhǎng)突起,類似神經(jīng)元;5ng/mLCNTF亞組僅有少部分細(xì)胞形態(tài)稍發(fā)生變化�？瞻讓�(duì)照組、單純預(yù)誘導(dǎo)組和單純誘導(dǎo)組的細(xì)胞形態(tài)基本無改變,仍為寬大扁平的梭形。 5.免疫細(xì)胞化學(xué)檢測(cè)顯示誘導(dǎo)后的細(xì)胞神經(jīng)元特異性烯醇化酶(neuron specific enolase, NSE)和巢蛋白(Nestin)均呈陽性表達(dá)。各組陽性細(xì)胞率間比較,差異均有統(tǒng)計(jì)學(xué)意義(F=98.822,F=105.395,均P0.01);實(shí)驗(yàn)組與空白對(duì)照組、單純預(yù)誘導(dǎo)組及單純誘導(dǎo)組比較,差異均有統(tǒng)計(jì)學(xué)意義(均P0.01),但后三組之間比較差異無統(tǒng)計(jì)學(xué)意義(均P0.01);實(shí)驗(yàn)組內(nèi)兩兩之間比較顯示,20ng/mL組與10ng/mL組陽性細(xì)胞率差異無統(tǒng)計(jì)學(xué)意義(P=0.413,P=0.496),但均高于5ng/mL組(均P0.01)。10ng/mLCNTF實(shí)驗(yàn)組NSE陽性細(xì)胞率(46.38±4.99)%,Nestin陽性細(xì)胞率(45.76±4.46)%,為最多。 結(jié)論 1.梯度離心法和全骨髓貼壁法相結(jié)合的方法,是一個(gè)較簡(jiǎn)便有效的體外h-MSCs培養(yǎng)、純化體系; 2. h-MSCs可在體外培養(yǎng)擴(kuò)增,并可凍存、復(fù)蘇,復(fù)蘇后的細(xì)胞生長(zhǎng)特性與凍存前的細(xì)胞相似; 3.在本實(shí)驗(yàn)條件下,CNTF可誘導(dǎo)h-MSCs定向分化為神經(jīng)元樣細(xì)胞;且10ng/mLCNTF可能為較適宜的誘導(dǎo)濃度。
[Abstract]:Purpose The expression of CD34 , CD44 , CD54 , NSE and Nestin in human bone marrow mesenchymal stem cells ( hMSCs ) and the expression of CD34 , CD44 , CD54 , NSE and Nestin in vitro were investigated by using basic fibroblast growth factor ( bFGF ) and different concentrations of ciliary neurotrophic factor ( CNTF ) . method Bone marrow derived from bone marrow puncture examination patients ( consent of patients ) from the Second Affiliated Hospital of South China University and the First Affiliated Hospital of the First Affiliated Hospital of South China University . The growth curve of h - MSCs was observed by means of density gradient centrifugation and full bone marrow adherent method . The cells were divided into 4 groups : experimental group , pre - induction group , simple induction group and blank control group . Results 1 . h - MSCs can be cultured in vitro under the conditions of long shuttle shape and similar fibroblast , and can be stably added and recovered , and the growth characteristics of the cells after resuscitation are similar to those of the cells before freezing ; 2 . The growth curve of the 1st , 3rd , 6th generation h - MSCs was " S " . There was no significant difference between the 1st and 3rd generation cell concentrations ( P = 0.704 ) . There was no significant difference between the 1st and 6th generations ( P = 0.132 ) . There was no significant difference between the 3rd and 6th generations ( P = 0.256 ) . 3 . The expression of CD44 and CD54 was positive and CD34 was negative in cultured h - MSCs . After 12 hours of induction of CNTF , the morphological changes of the cells in the 10 ng / mLCNTF group and 20ng / mLCNTF group were significantly changed in the experimental group . 5 . The positive rates of NSE and Nestin in the cells were significantly higher than those in the control group ( F = 98.822 , F = 105.395 , P0.01 ) . There was no significant difference between the two groups ( P = 0.413 , P = 0.496 ) . The positive cell rates of NSE in the experimental group were 46.38 鹵 4.99 % and 45.76 鹵 4.46 % , respectively . Conclusion 1 . The method of combination of gradient centrifugation and full - bone marrow adherent method is a simple and effective method for culturing and purifying h - MSCs in vitro . 2 . h - MSCs can be cultured in vitro and can be frozen , recovered , and the cell growth after resuscitation is similar to that before freezing . 3 . Under the condition of this experiment , CNTF can induce the differentiation of h - MSCs into neuron - like cells , and 10 ng / mL CNTF may be a suitable induction concentration .

【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R329

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本文編號(hào)：1538932