【摘要】：人胚胎干細(xì)胞(Human embryonic stem cells,hESCs)是一種多潛能干細(xì)胞,具有在體外無(wú)限增殖、自我更新和向機(jī)體各種類型細(xì)胞分化的能力。hESCs在特定條件下可分化為胰島素分泌細(xì)胞(Insulin-producing cells,IPCs),可以用于移植治療Ⅰ型糖尿病,以解決臨床治療器官來(lái)源不足的問(wèn)題。MiR-375是胰腺發(fā)育和成熟過(guò)程中必不可少的轉(zhuǎn)錄調(diào)控因子,在胰島β細(xì)胞和非β細(xì)胞中均有表達(dá),能夠提高胰島素轉(zhuǎn)錄水平,促進(jìn)胰島β細(xì)胞增殖。因此,研究miR-375在hESCs向IPCs分化過(guò)程中的作用,對(duì)于提高h(yuǎn)ESCs向IPCs分化的效率,獲得單一表達(dá)Insulin的成熟胰島β細(xì)胞具有重要意義。本研究在特定條件下使hESCs向IPCs分化,分化過(guò)程包括終末內(nèi)胚層(Stage Ⅰ)、原腸(Stage Ⅱ)、內(nèi)分泌前體(Stage Ⅲ)和胰腺內(nèi)分泌細(xì)胞(Stage ⅣV)四個(gè)階段。先以分化體系中的維甲酸(Retinoic acid,RA)為變量?jī)?yōu)化分化條件,再分別采用慢病毒介導(dǎo)的穩(wěn)定過(guò)表達(dá)和脂質(zhì)體介導(dǎo)的瞬時(shí)過(guò)表達(dá)提高細(xì)胞miR-375的表達(dá)水平,通過(guò)實(shí)時(shí)定量PCR檢測(cè)miR-375和分化各階段標(biāo)志性基因Foxa2、Cxcr4、Ngn3、Pdx1、Insulin、Glucagon、Somatostatin的相對(duì)表達(dá)量,并通過(guò)免疫熒光染色和Elisa檢測(cè)胰島素合成和分泌情況,從而研究過(guò)表達(dá)miR-375對(duì)hESCs分化為IPCs的影響。1、為了優(yōu)化hESCs分化為IPCs的培養(yǎng)條件,在終末內(nèi)胚層向內(nèi)分泌前體細(xì)胞分化的不同時(shí)間段添加2μMRA對(duì)細(xì)胞進(jìn)行誘導(dǎo),分別為不添加RA、在Stage Ⅱ 添加 RA、在 Stage Ⅲ 添加 RA、在 Stage Ⅱ 和 Stage Ⅲ 都添加 RA。研究發(fā)現(xiàn)在沒(méi)有RA誘導(dǎo)的條件下,hESCs未能成功分化為IPCs;在Stage Ⅱ添加RA的條件下,胰向分化相關(guān)基因Foxa2、Cxcr4、Ngn2、Pdx1、Insulin、Glucagon和Somatoststin的相對(duì)表達(dá)量最高,并且在此條件下細(xì)胞一直保持良好的生長(zhǎng)狀態(tài)。因此在細(xì)胞分化Stage Ⅱ添加2 μMRA可作為分化培養(yǎng)的最佳條件。2、通過(guò)對(duì)hESCs進(jìn)行慢病毒介導(dǎo)的miR-375穩(wěn)定過(guò)表達(dá),發(fā)現(xiàn)慢病毒感染會(huì)對(duì)hESCs的生長(zhǎng)狀態(tài)造成極大影響,降低細(xì)胞增殖分化能力并增加細(xì)胞凋亡,使細(xì)胞在分化早期大量死亡,進(jìn)而導(dǎo)致hESCs無(wú)法分化產(chǎn)生IPCs。3、對(duì)分化不同時(shí)期的hESCs進(jìn)行脂質(zhì)體介導(dǎo)的miR-375 mimic轉(zhuǎn)染,發(fā)現(xiàn)在分化早期過(guò)表達(dá)miR-375會(huì)提高Foxa2和Cxcr4的相對(duì)表達(dá)量,在分化中期過(guò)表達(dá)miR-375會(huì)提高Ngn3和Pdx1的相對(duì)表達(dá)量,在分化末期過(guò)表達(dá)miR-375會(huì)提高Insulin的相對(duì)表達(dá)量同時(shí)降低Glucagon和Somatostatin的相對(duì)表達(dá)量。并且瞬時(shí)過(guò)表達(dá)miR-375可促進(jìn)胰島素的合成和分泌。因此,在hESCs向IPCs分化過(guò)程中過(guò)表達(dá)miR-375可提高胰島β細(xì)胞發(fā)育成熟相關(guān)基因的表達(dá)水平,降低分化末期細(xì)胞多激素共表達(dá)程度,促進(jìn)β細(xì)胞成熟和胰島素分泌。
[Abstract]:Human embryonic stem cell (Human embryonic stem cells,hESCs) is a multipotent stem cell with unlimited proliferation in vitro. The ability to self-renew and differentiate into various types of cells in the body. HESCs can differentiate into insulin-secreting cells (Insulin-producing cells,IPCs) under certain conditions and can be used for transplantation in the treatment of type 1 diabetes. In order to solve the problem of insufficient organ source in clinical treatment, MiR-375 is an essential transcriptional regulator in the development and maturation of pancreas. It is expressed in both 尾 cells and non-beta cells of pancreatic islets, and can improve the level of insulin transcription. Promote the proliferation of islet 尾 cells. Therefore, it is of great significance to study the role of miR-375 in the differentiation of hESCs into IPCs, in order to improve the differentiation efficiency of hESCs into IPCs and to obtain mature islet 尾 cells with single expression of Insulin. In this study, hESCs was differentiated into IPCs under certain conditions. The differentiation process consisted of four stages: Stage 鈪，

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