本文選題：心脈隆注射液 + 骨髓間充質干細胞��； 參考：《山東大學》2012年碩士論文

【摘要】：研究目的 近年來,通過細胞工程技術體外分離和培養(yǎng)所需的靶細胞,已成為神經退行性疾病細胞替代治療的研究熱點。其中骨髓間充質干細胞(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,已成為一種良好的替代治療的靶細胞,在神經再生領域有著廣泛的應用價值。心脈隆注射液是由美洲大蠊干品經過浸漬、減壓濃縮、分離得到心脈隆浸膏再溶解后制得的,主要有擴血管、利尿、改善微循環(huán)、糾正神經內分泌失衡等作用。本研究以大鼠骨髓間充質干細胞為研究對象,心脈隆注射液為誘導劑,探討心脈隆注射液體外誘導大鼠骨髓間充質干細胞(BMSCs)向神經元樣細胞分化的可行性。 研究方法 1.貼壁篩選法培養(yǎng)大鼠骨髓間充質干細胞(BMSCs),倒置顯微鏡下逐日觀察原代、傳代細胞的生長情況及形態(tài)特征。 2.流式細胞學檢測細胞表面標志物CD29、CD90、CD34、CD45。 3.以心脈隆注射液為誘導劑,定向誘導第3代大鼠骨髓間充質干細胞(BMSCs)向神經元樣細胞轉化,每隔1h倒置相差顯微鏡下觀察細胞形態(tài)變化。 4.免疫細胞熒光法鑒定誘導4h、12h、36h后巢蛋白(Nestin)、神經元特異性烯醇化酶(Neuron specific enolase, NSE)、微管相關蛋白2(Microtubule associated protein-2, MAP2)、膠質纖維酸性蛋白(Glial fibrillary acidic protein, GFAP)的表達情況。 5. RT-PCR生檢測誘導36h后神經元特異性烯醇化酶(NSE)、膠質纖維酸性蛋白(GFAP)mRNA的表達。 結果 1.原代接種的細胞散在分布,折光性強,混雜其他細胞,1d后可見少量單核圓形細胞貼壁生長,3d后細胞大部分貼壁,有聚集生長傾向；8-10d后細胞呈集簇狀生長,每個集簇細胞中心呈放射狀或螺旋狀,細胞鋪滿皿底并融合。傳代后細胞增殖速度加快,3-5代后已純化為全梭形細胞,呈魚群樣排列,6代后細胞生長減慢,部分開始變得寬大扁平。 2.流式細胞學檢測示CD29、CD90高表達(陽性細胞百分比分別為99.06%、99.61%),CD34、CD45低表達(陽性細胞百分比分別為2.03%、2.00%)。 3.誘導2h后實驗組細胞形態(tài)即開始改變,部分長梭形細胞的胞體變小,變?yōu)椴灰?guī)則形或圓形。隨著誘導時間延長,突起不斷增長,呈雙極、多極形,36h誘導達到高峰,細胞形態(tài)明顯變化,每個細胞有兩個或多個突起并形成網(wǎng)狀結構,呈典型神經元樣細胞。對照組細胞形態(tài)無明顯變化。 4.誘導4h、12h后,實驗組Nestin陽性表達率分別為(81.0±1.6)%、(22.5±1.9)%,36h后,Nestin|陰性。誘導4h后,實驗組NSE、MAP2陰性,12h、36h后,NSE陽性表達率分別為(73.5±2.2)%、(94.3±1.8)%,MAP2陽性表達率分別為(80.0±2.2)%、(96.4±2.8)%。誘導4h、12h、36h后實驗組GFAP陰性,對照組Nestin、NSE、MAP2、GFAP均為陰性。 5.RT-PCR結果顯示,誘導36h后,實驗組細胞表達NSE mRNA,不表達GFAP mRNA。對照組細胞NSE、GFAP mRNA均不表達。 結論 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干細胞的方法。 2.心脈隆注射液可在體外快速、高效地誘導大鼠骨髓間充質干細胞向神經元樣細胞分化。 3.心脈隆注射液在體外誘導大鼠骨髓間充質干細胞分化為神經元樣細胞前,經歷了短暫的神經前體細胞階段。
[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 .
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329

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