本文選題：豬 + iPSC　； 參考：《中南大學(xué)》2011年博士論文

【摘要】：目的：探討豬的誘導(dǎo)性多潛能干細(xì)胞(piPSC)在體外分化成視網(wǎng)膜神經(jīng)細(xì)胞的方法。研究piPSC移植治療豬的視網(wǎng)膜損傷的效果。 材料與方法：piPSC在放射處理后的SNL細(xì)胞上培養(yǎng)并傳代。取第28代、第40代和第43代piPSC分別懸浮培養(yǎng)3天后形成胚胎體,再將胚胎體轉(zhuǎn)至未稀釋基質(zhì)膠,1：10和1：20稀釋的基質(zhì)膠以及層粘連蛋白-纖維連接蛋白基質(zhì)上粘附培養(yǎng)以使其分化。用免疫熒光染色和實(shí)時(shí)PCR技術(shù)檢測(cè)piPSC以及分化細(xì)胞中干細(xì)胞標(biāo)志物OCT4、ABCG2、Nanog、Sox2,神經(jīng)細(xì)胞標(biāo)志物TUBB3,感光細(xì)胞標(biāo)志物RCVRN、NRL、RHO、IRBP、ROM1和ARR3以及視桿細(xì)胞型雙極細(xì)胞標(biāo)志物PRKCA的表達(dá),計(jì)數(shù)表達(dá)上述標(biāo)志物的細(xì)胞的比例以及在形態(tài)上具有外節(jié)結(jié)構(gòu)細(xì)胞的比例。用帶IRBP-GFP基因的慢病毒感染分化后的細(xì)胞,使視錐型和視桿型感光細(xì)胞表達(dá)綠色熒光蛋白(GFP)。碘乙酸(IAA)靜脈注射建立豬視桿細(xì)胞損傷的動(dòng)物模型,將感染GFP的piPSC分化細(xì)胞移植入的一側(cè)病眼視網(wǎng)膜下,對(duì)側(cè)病眼視網(wǎng)膜下注射DMEM/F12培養(yǎng)液作為對(duì)照。分別在用藥前及細(xì)胞移植術(shù)后三周檢測(cè)視網(wǎng)膜電生理反應(yīng)(ERG)。術(shù)后3周摘取視網(wǎng)膜,切片,通過(guò)免疫熒光染色觀察移植后的細(xì)胞在病變視網(wǎng)膜中的整合情況。 結(jié)果：實(shí)時(shí)PCR和免疫熒光染色顯示分化后的細(xì)胞失去干細(xì)胞特異性基因OCT4、Nanog和Sox2的表達(dá),轉(zhuǎn)而表達(dá)視網(wǎng)膜感光細(xì)胞基因RCVRN、NRL、RHO、IRBP、ROM1和cone-arrestin。其中有30.91±7.2%的分化細(xì)胞表達(dá)轉(zhuǎn)錄因子NRL,25.61±8.5%表達(dá)RCVRN,6.765±2.083%表達(dá)RHO,此外還有2.6±0.4%的細(xì)胞表達(dá)PRKCA。以層粘連蛋白—纖維連接蛋白為細(xì)胞外基質(zhì)可以分化出更多的RHO陽(yáng)性細(xì)胞,但其外形更類(lèi)似普通神經(jīng)細(xì)胞。以基質(zhì)膠為細(xì)胞外基質(zhì)可以分化出更多的具有視桿細(xì)胞形態(tài)的細(xì)胞,即有外節(jié)結(jié)構(gòu)的細(xì)胞。用攜帶IRBP-GFP基因的病毒感染分化細(xì)胞后,約44%的細(xì)胞表達(dá)GFP。這些細(xì)胞已經(jīng)定向分化成視網(wǎng)膜感光細(xì)胞前體細(xì)胞或成熟細(xì)胞。移植到視網(wǎng)膜下后可檢測(cè)到RHO陽(yáng)性的細(xì)胞在視網(wǎng)膜各層均有表達(dá),其主要表達(dá)部位在視網(wǎng)膜外核層,即正常的感光細(xì)胞所在的位置。在這些整合入視網(wǎng)膜的移植細(xì)胞中,有一部分細(xì)胞具有外節(jié)結(jié)構(gòu)。 結(jié)論：piPSC能在體外高效地分化成視網(wǎng)膜神經(jīng)細(xì)胞并且部分細(xì)胞具有與原代培養(yǎng)的視桿細(xì)胞類(lèi)似的外節(jié)樣結(jié)構(gòu)。將這些分化的細(xì)胞移植入視桿細(xì)胞損傷的豬視網(wǎng)膜下方,移植細(xì)胞可以整合入受損視網(wǎng)膜。部分移植細(xì)胞在眼內(nèi)分化出外節(jié)結(jié)構(gòu),這可能有助于視覺(jué)功能的恢復(fù)。
[Abstract]:Aim: to investigate the method of differentiation of porcine induced multipotential stem cells (piPSC) into retinal neurons in vitro. To study the effect of piPSC transplantation in the treatment of retinal injury in pigs. Materials and methods: PSC was cultured and subcultured on SNL cells after irradiation. In the 28th passage, the 40th generation and the 43rd generation of piPSC were cultured for 3 days, respectively, and the embryonic body was formed. The embryo body was then transferred to the undiluted matrix glue 1: 10 and 1:20 diluted matrix glue and laminin-fibronectin matrix adhesion culture to make it differentiate. Immunofluorescence staining and real-time PCR techniques were used to detect the expression of stem cell marker OCT4, ABCG2NogSox2, neuronal marker TUBB3, photoreceptor cell marker RCVRNNNRHORHON, IRBPROM1 and ARR3, and rod cell type bipolar cell marker PRKCA, respectively. The proportion of cells expressing these markers and the proportion of cells with outer ganglion structure were counted. The differentiated cells were infected with lentivirus with IRBP-GFP gene, and the green fluorescent protein (GFP) was expressed in the cone-type and rod type photoreceptor cells. PiPSC differentiated cells infected with GFP were transplanted into the subretinal of unilateral diseased eyes and DMEM- F12 culture medium was injected into the retina of the contralateral diseased eyes. Retinal electrophysiological response (ERG) was measured before medication and 3 weeks after cell transplantation. The retina was removed 3 weeks after operation and the integration of the transplanted cells in the diseased retina was observed by immunofluorescence staining. Results: Real-time PCR and immunofluorescence staining showed that the differentiated cells lost the expression of stem cell-specific genes OCT4Nanog and Sox2, and instead expressed the retinal photoreceptor cell genes RCVRN- RHOBPP-ROM1 and cone-arrestinin. Among them, 30.91 鹵7.2% of the differentiated cells expressed the transcription factor NRLN 25.61 鹵8.5%, and 6.765 鹵2.083% of the differentiated cells expressed RHO.In addition, 2.6 鹵0.4% of the cells expressed PRKCA. Using laminin-fibronectin as extracellular matrix, more Rho positive cells can be differentiated, but their appearance is more similar to normal nerve cells. Using matrix glue as extracellular matrix, more cells with the shape of rod cells, that is, cells with outer ganglia structure, can be differentiated. After infecting differentiated cells with IRBP-GFP, about 44% of the cells expressed GFP. These cells have been directed to differentiate into retinal photoreceptor cells, precursor cells or mature cells. The expression of RHO-positive cells was detected in all layers of the retina after transplantation to the retina, and the expression was mainly located in the outer nuclear layer of the retina, that is, the location of the normal photoreceptor cells. Some of the transplanted cells integrated into the retina have an outer ganglion structure. ConclusionTwo pipiPSC can effectively differentiate into retinal nerve cells in vitro, and some of the cells have an outer nodal structure similar to that of primary cultured rod cells. These differentiated cells are transplanted under the damaged porcine retina, which can be integrated into the damaged retina. Some of the transplanted cells differentiate into nodal structures in the eye, which may contribute to the restoration of visual function.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R774.1

【共引文獻(xiàn)】

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