本文選題：人臍帶間充質(zhì)干細(xì)胞 + 胰島素樣生長(zhǎng)因子-1��； 參考：《河北醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的：對(duì)人臍帶間充質(zhì)干細(xì)胞（human umbilical cord mesenchymalstem cells, hUMSCs）生物學(xué)特性進(jìn)行鑒定，探討胰島素樣生長(zhǎng)因子-1（insulin-like growth factor-1，IGF-1）在體外定向誘導(dǎo)hUMSCs向神經(jīng)細(xì)胞分化的作用和條件，為hUMSCs神經(jīng)分化以及臨床應(yīng)用提供理論和實(shí)驗(yàn)基礎(chǔ)。 方法：取足月妊娠剖宮產(chǎn)健康胎兒的臍帶，以D-Hank’s充分沖洗，剔除臍動(dòng)脈和臍靜脈，將剩余的臍帶間質(zhì)組織切割成1mm3大小的組織塊，0.2%膠原酶Ⅱ消化，在含有20%胎牛血清（FBS）、2ng/ml表皮細(xì)胞生長(zhǎng)因子（EGF）、25mM左旋谷氨酰胺（L-Glu）、100U/ml青霉素，100μg/ml鏈霉素的DMEM/F12中培養(yǎng)。觀察原代培養(yǎng)細(xì)胞的形態(tài)學(xué)變化，當(dāng)細(xì)胞達(dá)80％～90％匯合時(shí)，加入0.25%胰酶-1mM EDTA消化細(xì)胞，并傳代。 收集消化后的細(xì)胞，采用流式細(xì)胞術(shù)行細(xì)胞表型檢測(cè)，包括CD73、CD90和CD105、CD19、CD34、CD45、CD11和組織相容性抗原HLA-DR(MHC-II)，以抗鼠IgG1-PE和IgG1-FITC作為同型對(duì)照。 原代hUMSCs按1:1的比例傳代后是為P1代，倒置顯微鏡下觀察形態(tài)變化，細(xì)胞達(dá)到90%以上融合時(shí)按1:2或1:3比例進(jìn)行傳代，繪制細(xì)胞生長(zhǎng)曲線。 取P4代生長(zhǎng)狀態(tài)良好的hUCMSCs，分為四組：25ng/ml組、50ng/ml組、100ng/ml組和對(duì)照組。以3×104/孔的密度接種于4個(gè)多聚賴氨酸包被的六孔板中（每個(gè)六孔板為一組），待細(xì)胞融合達(dá)到70-80%時(shí)，三個(gè)六孔板分別換為含有25ng/ml、50ng/ml、100ng/ml IGF-1的無(wú)血清低糖DMEM/F12培養(yǎng)基的誘導(dǎo)液，為實(shí)驗(yàn)組；另一六孔板為對(duì)照組，除不加IGF-1外其余均同實(shí)驗(yàn)組。鏡下觀察細(xì)胞形態(tài)變化，每2小時(shí)在高倍顯微鏡視野（100倍）下進(jìn)行觀察12小時(shí)，隨機(jī)選取10個(gè)視野，拍照并計(jì)數(shù)典型神經(jīng)樣細(xì)胞形態(tài)（有明顯的兩個(gè)及以上突起形成，細(xì)胞體屈光性強(qiáng)，類似神經(jīng)元樣細(xì)胞）的細(xì)胞數(shù)及總細(xì)胞數(shù)，用以計(jì)算分化率，結(jié)果以均數(shù)±標(biāo)準(zhǔn)差表示。比較不同濃度IGF-1誘導(dǎo)hUCMSCs的分化率，選取分化率高且穩(wěn)定的一組誘導(dǎo)后8h用免疫細(xì)胞化學(xué)技術(shù)檢測(cè)星形膠質(zhì)細(xì)胞標(biāo)志（GFAP）、神經(jīng)元標(biāo)志（NSE）和神經(jīng)干細(xì)胞標(biāo)志（Nestin），拍照記錄計(jì)數(shù)陽(yáng)性細(xì)胞，計(jì)算陽(yáng)性細(xì)胞率結(jié)果以均數(shù)±標(biāo)準(zhǔn)差表示。 結(jié)果：原代細(xì)胞傳代24h后，大部分細(xì)胞逐漸貼壁，細(xì)胞呈菱形、三角形、橢圓形等形態(tài)；隨著培養(yǎng)時(shí)間的延長(zhǎng)，漸變?yōu)殚L(zhǎng)梭形，細(xì)胞數(shù)量逐漸增多。培養(yǎng)7-8d時(shí)細(xì)胞融合到90%左右，低倍鏡下可以看到hUCMSCs呈漩渦狀排列。按照1:2的比例進(jìn)行再傳代，連續(xù)傳代到P15代，hUCMSCs仍保持旺盛的生長(zhǎng)能力。流式細(xì)胞檢測(cè)P3、P5和P10代細(xì)胞的分子標(biāo)志物，結(jié)果顯示各代均表達(dá)CD73、CD90和CD105，而不表達(dá)CD34、CD45CD19、CD11b和HLA-DR。 加入IGF誘導(dǎo)液后30min，IGF-1為50ng/ml和100ng/ml組有個(gè)別細(xì)胞開(kāi)始發(fā)生形態(tài)改變，25ng/ml組細(xì)胞開(kāi)形態(tài)改變不明顯。光學(xué)顯微鏡下可見(jiàn)：胞體收縮變圓，伸出指狀突起，細(xì)胞體折光度增強(qiáng)；隨著誘導(dǎo)時(shí)間的延長(zhǎng)，，細(xì)胞進(jìn)一步收縮，突起較前增長(zhǎng)，1h后個(gè)別細(xì)胞形成雙極突起以100ng/ml組變化細(xì)胞數(shù)量最多同時(shí)也可以看到該組內(nèi)有少量細(xì)胞死亡漂浮于培養(yǎng)基上。4h后50ng/ml和100ng/ml組細(xì)胞折光度增大，突起較前更加細(xì)長(zhǎng)、增多，多在2至5條，有的細(xì)胞逐漸形成多級(jí)突起，25ng/ml組變化不明顯；8h后50ng/ml和100ng/ml組多個(gè)相臨的細(xì)胞逐漸連接形成樹(shù)突樣網(wǎng)絡(luò)，25ng/ml組細(xì)胞形態(tài)改變較前不明顯；隨誘導(dǎo)時(shí)間延長(zhǎng)，分化的細(xì)胞形態(tài)越來(lái)越典型，但死亡、漂浮的細(xì)胞也逐漸增多，以100ng/ml組死亡的細(xì)胞最多；24h后100ng/ml組細(xì)胞大量死亡，僅有少量細(xì)胞存活且細(xì)胞透亮度降低；50ng/ml組存活細(xì)胞表現(xiàn)出更為典型的神經(jīng)細(xì)胞樣改變，這些細(xì)胞的胞體折光度增大，細(xì)胞質(zhì)向細(xì)胞核收縮，形成雙極、多極樣突起；有的呈放射狀伸展，突起伸長(zhǎng)更加明顯，可以看到2、3級(jí)突起但同時(shí)死亡細(xì)胞也逐漸增多，漂浮于培養(yǎng)基表面。免疫組化染色可見(jiàn)大量GFAP、NSE和Nestin陽(yáng)性表達(dá)的神經(jīng)樣細(xì)胞，其中GFAP和NSE陽(yáng)細(xì)胞著色較深，Nestin陽(yáng)性細(xì)胞著色較淺；免疫熒光染色也顯示其GFAP、NSE，Nestin呈陽(yáng)性表達(dá)；對(duì)照組免疫組化和免疫熒光染色只有極少量的細(xì)胞表達(dá)GFAP，而不表達(dá)NSE和Nestin。 結(jié)論:hUCMSCs由臍帶中分離純化后可以在體外穩(wěn)定傳代，并保持較高的增殖能力；hUCMSCs表達(dá)間充質(zhì)干細(xì)胞表面標(biāo)志CD73、CD90和CD105，不表達(dá)造血細(xì)胞表面標(biāo)志CD34、CD45、CD19、CD11b和組織相容性抗原HLA-DR （MHC-Ⅱ）；IGF-1在體外可誘導(dǎo)hUCMSCs分化成神經(jīng)樣細(xì)胞，并表達(dá)神經(jīng)膠質(zhì)細(xì)胞表面標(biāo)志（GFAP）、神經(jīng)元標(biāo)志（NSE）神經(jīng)干細(xì)胞標(biāo)志（Nestin），以IGF-1濃度為50ng/ml組最為明顯。
[Abstract]:Objective: to identify the biological characteristics of human umbilical cord mesenchymal stem cells (human umbilical cord mesenchymalstem cells (hUMSCs) and hUMSCs), and to explore the use and conditions of insulin like growth factor -1 (insulin-like growth factor-1, IGF-1) to induce the differentiation of hUMSCs to neural cells in vitro. Theoretical and experimental basis.
Methods: the umbilical cord of the healthy fetus of the term pregnancy was taken, the umbilical artery and the umbilical vein were washed with D-Hank 's, the umbilical cord and umbilical veins were removed, the remaining umbilical cord interstitial tissue was cut into 1mm3 size tissue block, 0.2% collagenase II was digested, with 20% fetal bovine serum (FBS), 2ng/ml epidermal cell growth factor (EGF), 25mM L-glutamine (L-Glu), 100U/ml. Penicillin, cultured in DMEM/F 12 of 100 g/ml streptomycin, observed the morphological changes of the primary cultured cells. When the cells reached 80% to 90% confluence, 0.25% trypsin -1mM EDTA was added to the cells to digest the cells.
Cells after digestion were collected and cell phenotypes were detected by flow cytometry, including CD73, CD90 and CD105, CD19, CD34, CD45, CD11 and histocompatibility antigen HLA-DR (MHC-II), with anti mouse IgG1-PE and IgG1-FITC as the same type.
The primary hUMSCs was transmitted to the P1 generation after 1:1, and the morphological changes were observed under the inverted microscope. When the cells reached more than 90% fusion, the cell growth curve was plotted according to the proportion of 1:2 or 1:3.
HUCMSCs of good growth state of P4 generation was divided into four groups: group 25ng/ml, 50ng/ml group, 100ng/ml group and control group. The density of 3 x 104/ holes was inoculated in 4 polylysine coated six orifice plates (each group of six orifice plates), and when the cell fusion reached 70-80%, three six pore plates were replaced with 25ng/ml, 50ng/ml, 100ng/ml IGF-1, without blood. The induction fluid of DMEM/F12 culture medium with low glucose was used as the experimental group; the other 16 orifice plates were in the control group. The morphological changes of the cells were observed in the same group except without IGF-1. The morphological changes were observed under the microscope (100 times) for 12 hours at the high power microscope (100 times), and 10 fields were randomly selected to take pictures and count the typical neurolike cell morphology. Two or more protrusions were formed, cell body diopter was strong, the number of cells like neuron like cells and the total number of cells were used to calculate the differentiation rate. The results were expressed in the mean number of standard deviation. The differentiation rate of hUCMSCs induced by different concentrations of IGF-1 was compared, and a group of high and stable differentiation rates was selected to detect astrocytes by immunocytochemical technology. Cell markers (GFAP), neuron markers (NSE) and neural stem cell markers (Nestin) were taken to record counting positive cells. The results of the calculated positive cell rate were expressed as mean standard deviation.
Results: after the primary cell passage of 24h, most of the cells gradually adhered to the wall, and the cells were rhombic, triangular, oval and so on. With the prolongation of the culture time, the cells gradually changed into long spindle shape, and the number of cells gradually increased. The cells fused to about 90% when the 7-8d was cultivated, and the whirlpool arrangement could be seen under the low magnification mirror. The retransmission of the cells according to the proportion of 1:2 was retransmitted. Generation, continuous passage to P15 generation, hUCMSCs still maintained strong growth ability. Flow cytometry detected the molecular markers of P3, P5 and P10 generation cells. The results showed that all generations expressed CD73, CD90 and CD105, but did not express CD34, CD45CD19, CD11b, and HLA-DR..
In the group of 50ng/ml and 100ng/ml, there were some cells in the group of 50ng/ml and 100ng/ml after the addition of IGF. The morphological changes of the cells in the group of 50ng/ml and 100ng/ml were changed. The morphology of the cells in the 25ng/ml group was not obvious. The optical microscope showed that the contraction of the cell body became round, the finger shaped protuberance was extended, the cell body fold increased, and the cells further contracted with the time of induction, and the protuberance was increased by 1 After h, a few cells formed a bipolar protuberance to change the number of cells in the 100ng/ml group at the same time. At the same time, a small amount of cell death in the group was found to float on the medium of.4h. After.4h, the refraction degree of the cells in the 50ng/ml and 100ng/ml groups increased. The protuberances were more slender and more elongated than before, and the number of cells was 2 to 5. Some cells gradually formed multistage protuberances, and the 25ng/ml group changed unidentified. After 8h, the cells of 50ng/ml and 100ng/ml groups were connected gradually to form a dendrite like network, and the morphological changes of the 25ng/ml group were not obvious. With the prolongation of the induction time, the differentiated cells became more and more typical, but the cell died, the floating cells increased gradually, and the cells died in the 100ng/ml group were the most; the 100ng/ml group after 24h was larger than the 24h group. In the 50ng/ml group, the survival cells showed a more typical nerve cell like change, the cell body refraction of these cells increased, the cytoplasm contracted to the nucleus and formed a bipolar, multipolar protuberance; some were radially extended, the protuberance was more obvious, and the 2,3 protuberance could be seen. But at the same time, the number of dead cells increased gradually and floating on the surface of the culture medium. A large number of GFAP, NSE and Nestin positive neurons were found in the immunofluorescent staining cells, in which GFAP and NSE positive cells were coloured more deeply and the Nestin positive cells were coloured relatively shallow; immunofluorescence staining also showed that the positive expression of GFAP, NSE, Nestin was expressed in the immunofluorescence staining; and the immunofluorescence of the control group was positive. Immunofluorescence staining showed that only a few cells expressed GFAP but not NSE and Nestin..
Conclusion: after isolation and purification of hUCMSCs from the umbilical cord, it can be passaged in vitro and maintain high proliferation ability. HUCMSCs expressed CD73, CD90 and CD105 on the surface of mesenchymal stem cells, which do not express the hematopoietic cell surface markers CD34, CD45, CD19, CD11b and histocompatibility antigen HLA-DR (MHC- II), and IGF-1 can be induced to differentiate in vitro. Neurocyte like cells, and expression of glial cell surface markers (GFAP), neuron marker (NSE) neural stem cell marker (Nestin), the IGF-1 concentration is the most obvious in the 50ng/ml group.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 郭建新;孟凡超;;成人骨髓間充質(zhì)干細(xì)胞的體外培養(yǎng)和定向神經(jīng)分化[J];中國(guó)實(shí)用神經(jīng)疾病雜志;2009年09期

2 董立華;王勇;陸長(zhǎng)青;王凡;;黃芪誘導(dǎo)大鼠骨髓間充質(zhì)干細(xì)胞分化為神經(jīng)樣細(xì)胞的研究[J];四川大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2007年03期

3 胡琳燕;余加林;劉官信;陳波曼;李芳;李祿全;鐘海英;;丹參誘導(dǎo)鼠骨髓間充質(zhì)干細(xì)胞分化為神經(jīng)樣細(xì)胞優(yōu)化方案及電生理研究[J];細(xì)胞生物學(xué)雜志;2008年03期

4 項(xiàng)鵬,夏文杰,張麗蓉,陳振光,張秀明,李艷,李樹(shù)濃;胞內(nèi)cAMP濃度的增加可誘導(dǎo)骨髓間質(zhì)干細(xì)胞分化為神經(jīng)元樣細(xì)胞[J];中國(guó)病理生理雜志;2002年11期

5 夏文杰,項(xiàng)鵬,張麗蓉,陳振光,余偉華,張秀明,李艷,李樹(shù)濃;丹參酮ⅡA定向誘導(dǎo)骨髓間質(zhì)干細(xì)胞分化為神經(jīng)元樣細(xì)胞的研究[J];中國(guó)病理生理雜志;2003年07期

6 陳曉嵐;黃仁彬;莫艷秀;湯銀娟;陳繼華;;黃芩甙誘導(dǎo)人臍血間充質(zhì)干細(xì)胞分化為神經(jīng)元樣細(xì)胞[J];中風(fēng)與神經(jīng)疾病雜志;2007年03期

7 顏曉華;黃瑞濱;;黃芩苷體外誘導(dǎo)臍血間充質(zhì)干細(xì)胞向神經(jīng)元樣細(xì)胞分化的實(shí)驗(yàn)研究[J];中華兒科雜志;2006年03期

8 尹宗生,顧玉東,顧映紅,朱騰方;中藥治療對(duì)大鼠坐骨神經(jīng)損傷后神經(jīng)生長(zhǎng)因子蛋白表達(dá)的影響[J];中華手外科雜志;2003年01期

9 ;Human umbilical cord Wharton's Jelly-derived mesenchymal stem cells differentiation into nerve-like cells[J];Chinese Medical Journal;2005年23期

10 趙宗茂 ,張慶俊 ,劉海英 ,孫國(guó)柱 ,韓忠朝 ,李洪鈞 ,楊仁池 ,邱錄貴 ,蔡英林 ,朱慧芳 ,盧士紅;骨髓間質(zhì)干細(xì)胞移植對(duì)大鼠脊髓損傷神經(jīng)功能恢復(fù)的影響[J];中華神經(jīng)外科雜志;2003年01期

本文編號(hào)：2108916