本文選題：人胚胎干細(xì)胞 + 胰島素分泌細(xì)胞��； 參考：《鄭州大學(xué)》2011年碩士論文

【摘要】：糖尿病(diabetes mellitus, DM)是一組以慢性血葡萄糖水平增高為特征的代謝疾病群。糖尿病的病因尚未完全闡明,主要認(rèn)為與遺傳因素和環(huán)境因素等有關(guān),口服降糖藥和外源性胰島素替代治療是臨床治療的主要方法,但卻無法防止晚期糖尿病并發(fā)癥的產(chǎn)生。近年來胰腺或胰島細(xì)胞移植作為目前治療Ⅰ型糖尿病和部分Ⅱ型糖尿病效果最理想的方法不斷受到關(guān)注。 人胚胎干細(xì)胞(human embryonic stem cells, hESCs)是一種具有分化發(fā)育成三 個胚層組織細(xì)胞潛能的全能性的細(xì)胞。利用hESCs的多向分化能力,將其定向誘導(dǎo)為胰島素分泌細(xì)胞(insulin producing cells, IPCs)為糖尿病的細(xì)胞移植治療提供了新的方向。 目前雖已有文獻(xiàn)報道hESCs成功誘導(dǎo)分化為胰島素分泌細(xì)胞,但誘導(dǎo)分化效率偏低,成為胰島素分泌細(xì)胞相關(guān)研究的主要障礙,故本研究通過經(jīng)由擬胚體方法和貼壁培養(yǎng)方法及添加不同濃度誘導(dǎo)因子誘導(dǎo)混合飼養(yǎng)層培養(yǎng)體系的人胚胎干細(xì)胞定向分化為胰島素分泌細(xì)胞進(jìn)行對比,通過優(yōu)化體外誘導(dǎo)條件,以期達(dá)到最佳的誘導(dǎo)分化效率。 目的 比較不同誘導(dǎo)分化方法對hESCs向胰島素分泌細(xì)胞分化效率的影響。 方法 1.實驗材料為鄭州大學(xué)第一附屬醫(yī)院生殖中心自主建立的hESCs系：ZZU(鄭州大學(xué))-hESC(人胚胎干細(xì)胞)-1,并在混合飼養(yǎng)層上培養(yǎng)。 2. hESCs誘導(dǎo)為限定性內(nèi)胚層過程中,根據(jù)加入活化素A的濃度(為25ng/ml、50ng/ml和100ng/ml)不同分為3組。 3.hESCs誘導(dǎo)為胰島素分泌細(xì)胞過程中,根據(jù)經(jīng)擬胚體法和貼壁培養(yǎng)直接誘導(dǎo)法分為2組,誘導(dǎo)分化主要分為4個階段。 4.采用Sox-17免疫熒光法測定限定性內(nèi)胚層的表達(dá),RT-PCR法檢測各個階段特異表達(dá)基因,誘導(dǎo)分化末行雙硫腙染色和胰島素釋放實驗鑒定分化成熟的胰島樣細(xì)胞團(tuán)。 5.通過比較不同誘導(dǎo)方案的誘導(dǎo)效率,得出較好的誘導(dǎo)方案。 結(jié)果 1.隨著誘導(dǎo)分化時間的延長,可見細(xì)胞形態(tài)的變化。 2.Sox-17免疫熒光結(jié)果示,活化素A的濃度為50ng/ml組和100ng/ml組誘導(dǎo)分化效率無明顯差異(P0.05),25ng/ml組誘導(dǎo)分化效率低于上述兩組.(P0.05)。 3.根據(jù)誘導(dǎo)分化末雙硫腙染色、RT-PCR鑒定和胰島素釋放試驗的結(jié)果估算出：采用貼壁培養(yǎng)細(xì)胞直接進(jìn)行誘導(dǎo)分化,可得到較多的胰島素分泌細(xì)胞。 結(jié)論 1、50ng/ml的活化素A同樣可獲得較多的限定性內(nèi)胚層細(xì)胞,為后續(xù)的研究提供細(xì)胞。 2、采用貼壁培養(yǎng)hESCs直接進(jìn)行4階段誘導(dǎo)分化得到胰島素分泌細(xì)胞的效率較高。
[Abstract]:Diabetes mellitus (DMM) is a group of metabolic diseases characterized by chronic elevated blood glucose levels. The etiology of diabetes has not been completely clarified, which is mainly related to genetic factors and environmental factors. Oral hypoglycemic drugs and exogenous insulin replacement therapy are the main methods of clinical treatment, but can not prevent the occurrence of complications in advanced diabetes mellitus. In recent years, pancreas or islet cell transplantation has attracted more and more attention as the most effective method for the treatment of type 1 diabetes and partial type 2 diabetes. Human embryonic stem cells (embryonic stem cells, hESCs) are totipotent cells with the potential to differentiate into three embryonic tissue cells. The induction of hESCs into insulin producing cells (IPCs) provides a new direction for the treatment of diabetes mellitus. Although it has been reported that hESCs have been successfully induced to differentiate into insulin-secreting cells, the efficiency of inducing differentiation is relatively low, which has become the main obstacle to the study of insulin secretory cells. In this study, human embryonic stem cells were induced to differentiate into insulin-secreting cells by embryoid method, adherent culture method and different concentration of induction factors, and the induction conditions in vitro were optimized. In order to achieve the best induction and differentiation efficiency. Objective to compare the effects of different differentiation induction methods on the differentiation efficiency of hESCs into insulin-secreting cells. Method 1. The experimental materials were the human embryonic stem cells (hESC) -1 and cultured on the mixed feeder layer. 2. The hESCs were induced to be limited endoderm in the process of inducing hESCs into the limited endoderm, which was established by the procreation Center of the first affiliated Hospital of Zhengzhou University. According to the concentration of activin A (25ng / ml 50ng / ml and 100ng / ml), the cells were divided into three groups. 3. During the induction of HESCs into insulin-secreting cells, they were divided into two groups according to the embryoid method and the direct induction method of adherent culture. Induction and differentiation are mainly divided into four stages. 4. Sox-17 immunofluorescence assay was used to detect the expression of restricted endoderm and RT-PCR was used to detect the specific expression genes in each stage. Dithizone staining and insulin release assay were used to identify the differentiated islet like cell clusters at the end of differentiation. 5. By comparing the induction efficiency of different induction schemes, a better induction scheme was obtained. Result 1. With the prolongation of induction and differentiation time, the changes of cell morphology were observed. 2. Sox-17 immunofluorescence results showed that, The concentration of activin A in 50ng/ml group and 100ng/ml group had no significant difference in inducing differentiation efficiency. The differentiation efficiency of 25 ng / ml group was lower than that of the above two groups. According to the results of RT-PCR identification and insulin release test, it was estimated that more insulin-secreting cells could be obtained by direct differentiation induced by adherent cells. Conclusion 1 50 ng / ml activin A can also obtain more limited endoderm cells. For the further study, the hESCs were directly induced to differentiate into insulin-secreting cells in four stages by adherent culture.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 何志旭,黃紹良,周其峰,李樹濃;體外定向誘導(dǎo)小鼠胚胎干細(xì)胞發(fā)育為造血干/祖細(xì)胞方法的初步研究[J];中國病理生理雜志;2003年04期

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