發(fā)布時(shí)間：2018-10-18 10:45

【摘要】：目的：本課題首次將fBM-MSC用于DM動(dòng)物模型早期視網(wǎng)膜病變的治療，通過兩種間充質(zhì)干細(xì)胞、兩種途徑移植治療DM大鼠早期視網(wǎng)膜病變的療效觀察,旨在探索更為安全有效的供體細(xì)胞類型和移植方法，為MSC治療DR提供理論基礎(chǔ)和實(shí)驗(yàn)依據(jù)。 方法：全程無血清間充質(zhì)培養(yǎng)環(huán)境下體外分離、培養(yǎng)及鑒定hUC-MSC和fBM-MSC,利用HIV慢病毒表達(dá)載體轉(zhuǎn)染GFP，將表達(dá)GFP的P5代hUC-MSC和fBM-MSC通過玻璃體腔注射和尾靜脈注射兩種途徑移植到STZ誘導(dǎo)4w的糖尿病大鼠體內(nèi)，檢測各組移植前及移植后1w、4w、8w大鼠血糖、體重、FFA等大體情況，石蠟切片HE染色測量內(nèi)層視網(wǎng)膜和外核層厚度變化，，冰凍切片免疫熒光染色觀察供體細(xì)胞向視網(wǎng)膜的遷移定植及müller細(xì)胞的增生情況，western-blotting檢測視網(wǎng)膜GFAP和Rhodopsin的蛋白表達(dá)，F(xiàn)-ERG記錄各組視網(wǎng)膜功能的變化，統(tǒng)計(jì)對比后得出結(jié)論。結(jié)果：1.從人臍帶和胎兒骨髓中均易分離培養(yǎng)出MSC，細(xì)胞純度高，形態(tài) 均一且具有典型MSC形態(tài)特征，表達(dá)MSC特異抗原標(biāo)志，體外擴(kuò)增能力強(qiáng)且能保持其低分化狀態(tài)和多向分化潛能；2.hUC-BMC和fBM-BMC分別采取玻璃體腔注射和尾靜脈注射兩種途徑移植實(shí)驗(yàn)觀察期內(nèi)均無降血糖作用；3.hUC-MSC和fBM-MSC經(jīng)過同種途徑移植后表現(xiàn)出相似的生物學(xué)特點(diǎn)，經(jīng)玻璃體腔移植后均可移行定植于糖尿病4w的大鼠視網(wǎng)膜，移植后4w左右移行細(xì)胞數(shù)量較多，主要位于視網(wǎng)膜神經(jīng)纖維層和神經(jīng)節(jié)細(xì)胞層，8w內(nèi)細(xì)胞最遠(yuǎn)移行至視網(wǎng)膜外叢狀層；而尾靜脈注射移植組和正常對照組在實(shí)驗(yàn)觀察期視網(wǎng)膜內(nèi)未發(fā)現(xiàn)細(xì)胞。各組觀察期內(nèi)均未見成瘤或是其它不良病變；4.糖尿病大鼠4w左右就出現(xiàn)了視網(wǎng)膜組織結(jié)構(gòu)、功能及特異蛋白表達(dá)水平的改變;5.玻璃體腔注射和全身靜脈注射均是安全簡便有效的干細(xì)胞移植途徑，適用于臨床，其中玻璃體腔注射途徑更有利于供體細(xì)胞在病變視網(wǎng)膜內(nèi)的移行定植。本實(shí)驗(yàn)中兩種MSC通過兩種途徑移植于成模4w糖尿病大鼠后在8w觀察期內(nèi)均對視網(wǎng)膜結(jié)構(gòu)、功能及蛋白表達(dá)具有保護(hù)作用，4w時(shí)效果較明顯，其中hUC-MSC優(yōu)于fBM-MSC，玻璃體腔注射移植效果明顯好于尾靜脈。 結(jié)論：1.從人臍帶和胎兒骨髓中均易分離培養(yǎng)出MSC，細(xì)胞純度高，體外擴(kuò)增能力強(qiáng)且能保持其低分化狀態(tài)和多向分化潛能；2.hUC-MSC和fBM-MSC能夠感知和適應(yīng)DM早期視網(wǎng)膜病變微環(huán)境，經(jīng)玻璃體腔移植后均可移行定植于病變視網(wǎng)膜；3.兩種MSC通過兩種途徑移植對于DM早期視網(wǎng)膜病變均有治療效果，4w左右效果較明顯，其中hUC-MSC優(yōu)于fBM-MSC，玻璃體腔注射移植效果明顯好于尾靜脈；4.hUC-MSC在DR等視網(wǎng)膜變性疾病治療領(lǐng)域具有廣闊的應(yīng)用前景。
[Abstract]:Objective: in this study, fBM-MSC was used for the treatment of early retinopathy in DM animal model for the first time. Two kinds of mesenchymal stem cells (MSCs) and two ways of transplantation were used to treat early retinopathy in DM rats. The purpose of this study was to explore more safe and effective donor cell types and transplantation methods, and to provide theoretical and experimental basis for the treatment of DR by MSC. Methods: the whole process of serum-free mesenchymal culture was isolated in vitro. HUC-MSC and fBM-MSC, were cultured and identified. GFP, was transfected with HIV lentivirus expression vector to transfer hUC-MSC and fBM-MSC expressing GFP into STZ induced diabetic rats by vitreous injection and caudal vein injection. The blood glucose, body weight and FFA were measured before transplantation and 1 week and 8 weeks after transplantation. The thickness of inner retina and outer nuclear layer were measured by HE staining in paraffin sections. The migration and implantation of donor cells to the retina and the proliferation of m 眉 ller cells were observed by immunofluorescence staining in frozen sections. The expression of GFAP and Rhodopsin in the retina was detected by western-blotting, and the changes of retinal function in each group were recorded by F-ERG. The result is 1: 1. MSC, cells were easily isolated and cultured from human umbilical cord and fetal bone marrow with high purity, uniform morphology and typical MSC morphological characteristics, and expressed MSC specific antigen markers. 2.hUC-BMC and fBM-BMC had no hypoglycemic effect by vitreous injection and caudal vein injection respectively. 3.hUC-MSC and fBM-MSC showed similar biological characteristics after transplantation in the same way. After vitreous transplants, they could be transplanted to the retina of rats with diabetes mellitus for 4 weeks, and the number of transitional cells was large about 4 weeks after transplantation. The cells were mainly located in the retinal nerve fiber layer and ganglion cell layer, and the cells were furthest transferred to the outer plexiform layer of the retina within 8 weeks, but no cells were found in the retina in the caudal vein transplantation group and the normal control group. No tumor or other adverse lesions were found in each group during the observation period. 4. The changes of retinal tissue structure, function and specific protein expression were observed in diabetic rats at about 4 weeks. Vitreous injection and systemic intravenous injection are safe, simple and effective approaches for stem cell transplantation, which are suitable for clinical use. The vitreous injection approach is more conducive to donor cell transplantation in the diseased retina. In this experiment, two kinds of MSC were transplanted into the model of 4w diabetic rats through two ways, and both of them had protective effects on the structure, function and protein expression of retina during the observation period of 8 weeks, and the effects were obvious at 4 weeks. The effect of hUC-MSC was better than that of fBM-MSC,. Conclusion 1. MSC, cells were easily isolated and cultured from human umbilical cord and fetal bone marrow. 2.hUC-MSC and fBM-MSC could sense and adapt to the microenvironment of DM early retinopathy. After vitreous cavity transplantation, it can be transplanted to the lesion retina. Two kinds of MSC were transplanted through two ways to treat the early retinopathy of DM. The effect of hUC-MSC was better than that of the caudal vein in 4 weeks. The effect of hUC-MSC was better than that of fBM-MSC, by intravitreal injection. 4.hUC-MSC has a broad application prospect in the treatment of retinal degeneration such as DR.
【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R587.2;R774.1

【共引文獻(xiàn)】

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