小鼠胰岛干细胞的分离培养、鉴定及其生物学功能研究

发布时间：2018-01-07 19:00

  本文关键词：小鼠胰岛干细胞的分离培养、鉴定及其生物学功能研究　出处：《吉林大学》2013年硕士论文　论文类型：学位论文

  更多相关文章： 胰岛 胰腺干/祖细胞 nestin~+细胞 胰岛样细胞团 ICR小鼠

【摘要】：利用胰腺干/祖细胞治疗糖尿病受到科研人员的高度关注，目前虽然已经发现了几种具有胰腺干/祖细胞特性的细胞，但是还没有一种真正意义上的胰腺干/祖细胞被大家所公认，因此确定胰腺干/祖细胞所特有的标记成为发现胰腺干/祖细胞的主要研究方向。小鼠的Nestin基因是由5945bp组成的，表达一种中间丝蛋白，与Desmin和Vimentin等同源，最早被发现在神经干细胞中表达，并被认为是神经干细胞的分子标记，将从大脑分离出来的nestin~+细胞在体外培养，其可以分化为神经元、胶质细胞等，说明了其具有干细胞特性。在胚胎发育早期，神经细胞和胰腺细胞共同表达许多基因，其中包括Isl-1，Brain-4，Pax4，Beta2/NeuroD和IDX-1等。因此nestin可能也是胰腺干/祖细胞的标记分子。 本实验利用胆总管胶原酶灌注法分离出ICR小鼠的胰岛细胞团，，并研究胰岛细胞团内nestin~+细胞的特性，结果如下：（1）在本实验室条件下，胆总管胶原酶灌注法分离ICR小鼠胰岛的最佳静止消化条件是，胶原酶浓度0.8mg/ml、37℃、水浴静止消化20min；（2）利用RT-PCR技术和免疫荧光染色技术证实了ICR小鼠胰岛内存在nestin~+细胞，并且这类nestin~+细胞不表达胰岛素；（3）将胰岛内的nestin~+细胞体外分离、扩大培养，在低糖、低血清的培养条件下可以诱导其形成胰岛样细胞团，经双硫腙（DTZ）染色后呈褐红色，用RT-PCR技术检测出胰岛素基因的表达。 以上结果表明，在ICR小鼠胰岛内存在着nestin~+细胞，并且通过体外实验证明了这类细胞具有分化为胰岛素分泌细胞的能力，表现出一定的胰腺干/祖细胞的特性。但是这类nestin~+细胞在体内是否同样具有这一特性，还需要进一步的研究。
[Abstract]:The use of pancreatic stem / progenitor cells in the treatment of diabetes has attracted great attention, although several types of pancreatic stem / progenitor cells have been identified. But none of the true pancreatic stem / progenitor cells have been recognized. Therefore, the identification of pancreatic stem / progenitor cells is the main research direction for the discovery of pancreatic stem / progenitor cells. The mouse Nestin gene is composed of 5945bp and expresses a kind of intermediate filament protein. Homologous to Desmin and Vimentin, it was first found to be expressed in neural stem cells and considered to be a molecular marker of neural stem cells. Neisin ~ cells isolated from the brain were cultured in vitro, which could differentiate into neurons, glial cells and so on, indicating that it has the characteristics of stem cells in the early stage of embryonic development. Nerve cells and pancreatic cells co-express a number of genes, including Isl-1, Brain-4 and Pax4. Therefore, nestin may also be a marker of pancreatic stem / progenitor cells. The islet cell clusters of ICR mice were isolated by choledochal collagenase perfusion and the characteristics of Neisin ~ cells in islet cell clusters were studied. The results are as follows: 1. The optimal conditions for static digestion of islets isolated from ICR mice by choledochus collagenase perfusion were as follows: the concentration of collagenase was 0.8mg / ml 37 鈩

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