本文選題：人臍帶間充質(zhì)干細胞 + 視網(wǎng)膜色素上皮細胞　； 參考：《河北醫(yī)科大學》2014年碩士論文

【摘要】：視網(wǎng)膜色素上皮(Retinalpigmentepithelium,RPE)，位于視網(wǎng)膜的最外層，為單層立方或矮柱狀細胞。色素上皮來源于胚胎時期的神經(jīng)外胚層，為光感受器細胞提供必需的營養(yǎng)并起到支持和保護的作用，同時為維持視網(wǎng)膜和感光細胞的正常生理功能提供多種營養(yǎng)因子。視網(wǎng)膜變性等損傷性疾病將導致視覺功能的損傷，尤其是視細胞的丟失將導致視覺功能的永久喪失。再生醫(yī)學的發(fā)展使移植恢復視覺功能成為希望。近年來，學者將研究重點放在了視網(wǎng)膜細胞的移植上，特別是視細胞和視網(wǎng)膜色素上皮細胞的移植。各國學者先后應用胚胎干細胞（embryonic stem cells，ES)、誘導性多能干細胞（induced pluripotent stem cells，iPS)和成體干細胞中的脂肪來源干細胞誘導分化形成視網(wǎng)膜色素上皮樣細胞，由于在培養(yǎng)過程中應用到了動物來源的血清、飼養(yǎng)層細胞、重組蛋白和營養(yǎng)因子，容易引起免疫反應，因此這些細胞的臨床應用受到限制。在前期的研究中，我們在將ES誘導分化為視細胞的過程中外源性的添加了小分子化合物，沒有使用動物來源的血清、生長因子和胚胎成纖維細胞飼養(yǎng)層，將此種方法誘導形成的視細胞進行移植實驗，未發(fā)現(xiàn)有移植排斥反應，，并且細胞與宿主細胞的融合較好。這種誘導方法使視細胞移植的臨床應用成為可能。 人臍帶Wharton膠來源的間充質(zhì)干細胞（human Wharton’sjelly-derived mesenchymal stem cells，HWJ-MSCs）又稱為臍帶間充質(zhì)干細胞(human umbilical cord mesenchymal stem cells，huc-MSCs)，多項研究顯示，該細胞增殖和誘導分化能力很強；來源廣泛且采集方便；不受道德倫理制約，是自體細胞移植和異體細胞移植治療的理想種子細胞。與同樣屬于成體干細胞范疇的骨髓間充質(zhì)干細胞（bone marrow mesenchymal stemcells，BM-MSC）相比，臍帶間充質(zhì)干細胞在取材上不受疾病、年齡等限制。臍帶間充質(zhì)干細胞在細胞移植的臨床應用中具有更大優(yōu)勢。 因此，本研究通過實驗研究旨在探索相對成熟、穩(wěn)定的體外分離培養(yǎng)、誘導臍帶間充質(zhì)干細胞向視網(wǎng)膜色素上皮樣細胞分化的方法，為其最終應用于臨床治療提供實驗依據(jù)。 目的：在直接共培養(yǎng)體系中共培養(yǎng)SD大鼠睪丸支持細胞和人臍帶間充質(zhì)干細胞，探討huc-MSCs向視網(wǎng)膜色素上皮細胞誘導分化的可行性，并對誘導分化的視網(wǎng)膜色素上皮細胞進行生物學特性鑒定，為后續(xù)的視細胞分化研究提供技術方法和實驗基礎。 方法： 1已鑒定huc-MSCs的傳代培養(yǎng)：常規(guī)培養(yǎng)經(jīng)過鑒定的huc-MSCs。倒置顯微鏡觀察細胞形態(tài)，選取第三代細胞進行共培養(yǎng)。 2SD乳鼠睪丸支持細胞（sertoli cells，SCs）的原代培養(yǎng)：選取19~21天齡的SD大鼠分離睪丸,低滲處理培養(yǎng)細胞進行純化；倒置相差顯微鏡下觀察純化過程中細胞形態(tài)變化；吉姆薩染色、吖啶橙熒光染色、Feulgen染色鑒定SCs。 3huc-MSCs與SCs直接共培養(yǎng)：單層培養(yǎng)SCs至融合率80%；接種huc-MSCs并加入視黃酸進行共培養(yǎng)，形態(tài)學觀察細胞生長情況；免疫熒光檢測培養(yǎng)細胞中Pax6蛋白的表達；RT-PCR檢測培養(yǎng)細胞中酪氨酸酶相關蛋白(tyrosinase-related protein2,TRP-2)，視網(wǎng)膜色素上皮細胞蛋白(Retinal pigment epithelium-specific65kDa protein，RPE65), Mer受體酪氨酸激酶（Proto-oncogene tyrosine-protein kinase MER, MERTK）和細胞視黃醛結(jié)合蛋白(cellular retinaldehyde-binding protein,CRALBP)的mRNA的表達。 結(jié)果： 1第三代huc-MSCs呈長梭形，形態(tài)均一，生長狀態(tài)良好。 2原代培養(yǎng)的SCs于第二天開始貼壁，培養(yǎng)七天后SCs純度達95%以上；細胞形態(tài)結(jié)構(gòu)特征與用伊紅染色、吖啶橙熒光染色、Feulgen染色鑒定為SCs的形態(tài)結(jié)構(gòu)特征一致。 3在huc-MSCs與SCs直接共培養(yǎng)體系中，培養(yǎng)一周后鏡下觀察細胞呈棕褐色，培養(yǎng)二周后細胞密度顯著增高;免疫熒光法檢測到細胞中有Pax6蛋白的表達；RT-PCR檢測細胞內(nèi)有Trp2、RPE65、Mertk和CRALBP的mRNA的表達。 結(jié)論：huc-MSCs以睪丸支持細胞作為共培養(yǎng)細胞，加入視黃酸可以誘導分化為視網(wǎng)膜色素上皮細胞，方法簡便可行。
[Abstract]:Retinal pigment epithelium ( RPE ) , which is located at the outermost layer of retina , is a single - layer cubic or low - columnar cell . The pigment epithelium is derived from neuroectoderm in embryonic period . It can provide the necessary nutrients for photoreceptor cells and play a role of support and protection . In recent years , we have focused on the transplantation of retinal cells . In recent years , scholars have focused on the transplantation of retinal cells , such as embryonic stem cells ( ES ) , induced pluripotent stem cells ( ES ) and embryonic fibroblast feeder layers .

Human umbilical cord - derived mesenchymal stem cells ( HWJ - MSCs ) are also known as human umbilical cord mesenchymal stem cells ( huc - MSCs ) .
wide sources and convenient acquisition ;
Compared with bone marrow mesenchymal stem cells ( BM - MSCs ) , umbilical cord mesenchymal stem cells are not limited by disease , age and so on .

Therefore , the aim of this study was to explore the method of differentiation of mesenchymal stem cells into retinal pigment epithelium - like cells in vitro and to provide experimental basis for clinical treatment .

Objective : To investigate the feasibility of cultured human umbilical cord mesenchymal stem cells ( MSCs ) and human umbilical cord mesenchymal stem cells ( MSCs ) in direct co - culture system , and to investigate the feasibility of huc - MSCs in inducing differentiation of retinal pigment epithelial cells , and to provide a technical and experimental basis for the subsequent study of the differentiation of retinal pigment epithelial cells .

Method :

1 . The culture of huc - MSCs has been identified : conventionally cultured huc - MSCs were cultured . The morphology of cells was observed by reversed microscope and the third generation cells were selected for co - culture .

The primary culture of the testis - supporting cells ( SCs ) of 2SD rats was carried out : the cells were isolated from 19 to 21 days old SD rats , and the cells were purified by hypotonic treatment .
The morphological changes of cells were observed under reversed phase contrast microscope .
The SCs were identified by fluorescence staining of acridine orange and Feulgen staining .

3huc - MSCs were co - cultured with SCs : single - layer cultured SCs to fusion rate of 80 % ;
huc - MSCs were inoculated and retinoic acid was added for co - culture and morphological observation of cell growth ;
Immunofluorescence was used to detect the expression of pa6 protein in cultured cells .
RT - PCR was used to detect the mRNA expression of tyrosinase - related protein ( RPE65 ) , retinal pigment epithelium - specific 65 kDa protein ( RPE65 ) , Mer receptor tyrosine kinase ( RPE65 ) , Mer receptor tyrosine kinase ( MERTK ) and cellular retinal aldehyde - binding protein ( CRAB ) in cultured cells .

Results :

The third generation of huc - MSCs had long shuttle shape , uniform morphology and good growth state .

2 primary cultured SCs began to adherent on the second day , and the purity of SCs reached over 95 % after seven days .
The morphological and structural features of the cells were stained with eosin , acridine orange fluorescence staining and Feulgen staining to identify the morphological characteristics of SCs .

3 In the direct co - culture system of huc - MSCs and SCs , the observed cells were brown - brown under the mirror of one week , and the density of cells increased significantly after two weeks .
The expression of Trp2 , RPE65 , Mertk , and CRACKI mRNA in the cells was detected by RT - PCR .

Conclusion : huc - MSCs can induce differentiation into retinal pigment epithelial cells by using testicular support cells as co - cultured cells , and the method is simple and feasible .

【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R774.1

【參考文獻】

相關期刊論文 前1條

1 張德迎;何大維;魏光輝;宋曉峰;李旭良;林濤;;精原干細胞在支持細胞飼養(yǎng)層上的長期培養(yǎng)(英文)[J];四川大學學報(醫(yī)學版);2008年01期

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