本文選題：心脈隆注射液 + 骨髓間充質(zhì)干細(xì)胞　； 參考：《山東大學(xué)》2012年碩士論文

【摘要】：研究目的 近年來,通過細(xì)胞工程技術(shù)體外分離和培養(yǎng)所需的靶細(xì)胞,已成為神經(jīng)退行性疾病細(xì)胞替代治療的研究熱點(diǎn)。其中骨髓間充質(zhì)干細(xì)胞(Bone marrow-derived mesenchvmal stem cells, BMSCs)由于來源豐富、取材簡便,易于體外分離、培養(yǎng)、純化,具有多向分化潛能等優(yōu)點(diǎn),已成為一種良好的替代治療的靶細(xì)胞,在神經(jīng)再生領(lǐng)域有著廣泛的應(yīng)用價值。心脈隆注射液是由美洲大蠊干品經(jīng)過浸漬、減壓濃縮、分離得到心脈隆浸膏再溶解后制得的,主要有擴(kuò)血管、利尿、改善微循環(huán)、糾正神經(jīng)內(nèi)分泌失衡等作用。本研究以大鼠骨髓間充質(zhì)干細(xì)胞為研究對象,心脈隆注射液為誘導(dǎo)劑,探討心脈隆注射液體外誘導(dǎo)大鼠骨髓間充質(zhì)干細(xì)胞(BMSCs)向神經(jīng)元樣細(xì)胞分化的可行性。 研究方法 1.貼壁篩選法培養(yǎng)大鼠骨髓間充質(zhì)干細(xì)胞(BMSCs),倒置顯微鏡下逐日觀察原代、傳代細(xì)胞的生長情況及形態(tài)特征。 2.流式細(xì)胞學(xué)檢測細(xì)胞表面標(biāo)志物CD29、CD90、CD34、CD45。 3.以心脈隆注射液為誘導(dǎo)劑,定向誘導(dǎo)第3代大鼠骨髓間充質(zhì)干細(xì)胞(BMSCs)向神經(jīng)元樣細(xì)胞轉(zhuǎn)化,每隔1h倒置相差顯微鏡下觀察細(xì)胞形態(tài)變化。 4.免疫細(xì)胞熒光法鑒定誘導(dǎo)4h、12h、36h后巢蛋白(Nestin)、神經(jīng)元特異性烯醇化酶(Neuron specific enolase, NSE)、微管相關(guān)蛋白2(Microtubule associated protein-2, MAP2)、膠質(zhì)纖維酸性蛋白(Glial fibrillary acidic protein, GFAP)的表達(dá)情況。 5. RT-PCR生檢測誘導(dǎo)36h后神經(jīng)元特異性烯醇化酶(NSE)、膠質(zhì)纖維酸性蛋白(GFAP)mRNA的表達(dá)。 結(jié)果 1.原代接種的細(xì)胞散在分布,折光性強(qiáng),混雜其他細(xì)胞,1d后可見少量單核圓形細(xì)胞貼壁生長,3d后細(xì)胞大部分貼壁,有聚集生長傾向；8-10d后細(xì)胞呈集簇狀生長,每個集簇細(xì)胞中心呈放射狀或螺旋狀,細(xì)胞鋪滿皿底并融合。傳代后細(xì)胞增殖速度加快,3-5代后已純化為全梭形細(xì)胞,呈魚群樣排列,6代后細(xì)胞生長減慢,部分開始變得寬大扁平。 2.流式細(xì)胞學(xué)檢測示CD29、CD90高表達(dá)(陽性細(xì)胞百分比分別為99.06%、99.61%),CD34、CD45低表達(dá)(陽性細(xì)胞百分比分別為2.03%、2.00%)。 3.誘導(dǎo)2h后實(shí)驗(yàn)組細(xì)胞形態(tài)即開始改變,部分長梭形細(xì)胞的胞體變小,變?yōu)椴灰?guī)則形或圓形。隨著誘導(dǎo)時間延長,突起不斷增長,呈雙極、多極形,36h誘導(dǎo)達(dá)到高峰,細(xì)胞形態(tài)明顯變化,每個細(xì)胞有兩個或多個突起并形成網(wǎng)狀結(jié)構(gòu),呈典型神經(jīng)元樣細(xì)胞。對照組細(xì)胞形態(tài)無明顯變化。 4.誘導(dǎo)4h、12h后,實(shí)驗(yàn)組Nestin陽性表達(dá)率分別為(81.0±1.6)%、(22.5±1.9)%,36h后,Nestin|陰性。誘導(dǎo)4h后,實(shí)驗(yàn)組NSE、MAP2陰性,12h、36h后,NSE陽性表達(dá)率分別為(73.5±2.2)%、(94.3±1.8)%,MAP2陽性表達(dá)率分別為(80.0±2.2)%、(96.4±2.8)%。誘導(dǎo)4h、12h、36h后實(shí)驗(yàn)組GFAP陰性,對照組Nestin、NSE、MAP2、GFAP均為陰性。 5.RT-PCR結(jié)果顯示,誘導(dǎo)36h后,實(shí)驗(yàn)組細(xì)胞表達(dá)NSE mRNA,不表達(dá)GFAP mRNA。對照組細(xì)胞NSE、GFAP mRNA均不表達(dá)。 結(jié)論 1.貼壁篩選法是一種簡單、快速分離及純化大鼠骨髓間充質(zhì)干細(xì)胞的方法。 2.心脈隆注射液可在體外快速、高效地誘導(dǎo)大鼠骨髓間充質(zhì)干細(xì)胞向神經(jīng)元樣細(xì)胞分化。 3.心脈隆注射液在體外誘導(dǎo)大鼠骨髓間充質(zhì)干細(xì)胞分化為神經(jīng)元樣細(xì)胞前,經(jīng)歷了短暫的神經(jīng)前體細(xì)胞階段。
[Abstract]:Purpose of study

In recent years , it has become a hot spot for the replacement therapy of neurodegeneration diseases by means of cell engineering technique in vitro isolation and culture . Bone marrow - derived mesenchymal stem cells ( MSCs ) are widely used in the field of nerve regeneration .

Research Methods

1 . The rat bone marrow mesenchymal stem cells were cultured with adherent screening method . The growth and morphological characteristics of primary and passaged cells were observed on a day - by - day basis under an inverted microscope .

2 . Flow cytometry was used to detect CD29 , CD90 , CD34 , CD45 in cell surface markers .

3 . In the third generation rat bone marrow mesenchymal stem cells were transformed into neuron - like cells , and the morphological changes of the cells were observed at every 1h inverted phase contrast microscope .

4 . The expression of Nestin , Neuron specific protein ( NSE ) , microtubules associated protein - 2 ( MAP2 ) and Glial glial acidic protein ( GFAP ) were identified by immunofluorescence .

5 . The expression of NSE and GFAP mRNA were detected by RT - PCR in 36 hours after induction .

Results

1 . The cells of primary inoculated cells were distributed , the refractive index was strong , and other cells were mixed . After 1d , a small number of single - core round cells were observed . After 3 days , most of the cells were adherent , and there was a tendency of aggregation and growth .
After 8 - 1days , the cells were clustered , and the centers of each cluster were radial or spiral , the cells were plated with the bottom of the dish and fused . After passage , the cell proliferation was accelerated . After 3 - 5 generations , the cells were purified into full spindle cells .

2 . Flow cytometry showed that CD29 and CD90 were highly expressed ( 99 . 06 % , 99 . 61 % , respectively ) .

3 . After 2 hours of induction , the cell morphology of the experimental group began to change , and the cells of some long spindle cells became irregular or round . As the induction time was prolonged , the growth of the cells became irregular or round . As the induction time was prolonged , the number of the cells increased , and the shape of the cells changed obviously . The cells had two or more protrusions and formed a net structure , showing the typical neuron - like cells .

4 . After 4 h , 12 h , the expression rates of Nestin positive in experimental group were ( 81.0 鹵 1.6 ) % , ( 22.5 鹵 1.9 ) % , 36h , Nestin immunoreactive expression rates were ( 73.5 鹵 2.2 ) % , ( 94.3 鹵 1.8 ) % , and MAP2 positive expression rates were ( 80.0 鹵 2.2 ) % , ( 96.4 鹵 2.8 ) % , respectively .

5 . RT - PCR showed that NSE mRNA and GFAP mRNA were not expressed in the experimental group after 36 hours of induction . The NSE and GFAP mRNA in the control group were not expressed .

Conclusion

1 . The adherent screening method is a simple and rapid method for the isolation and purification of rat bone marrow mesenchymal stem cells .

2 . Xinmailong injection can induce the differentiation of rat bone marrow mesenchymal stem cells into neuron - like cells rapidly and efficiently in vitro .

3 . After inducing the differentiation of bone marrow mesenchymal stem cells into neuron - like cells in vitro , Xinmailong injection has experienced a transient neural progenitor cell stage .
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329

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