本文關(guān)鍵詞：小鼠胰島干細(xì)胞的分離培養(yǎng)、鑒定及其生物學(xué)功能研究　出處：《吉林大學(xué)》2013年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 胰島 胰腺干/祖細(xì)胞 nestin~+細(xì)胞 胰島樣細(xì)胞團(tuán) ICR小鼠

【摘要】：利用胰腺干/祖細(xì)胞治療糖尿病受到科研人員的高度關(guān)注，目前雖然已經(jīng)發(fā)現(xiàn)了幾種具有胰腺干/祖細(xì)胞特性的細(xì)胞，但是還沒(méi)有一種真正意義上的胰腺干/祖細(xì)胞被大家所公認(rèn)，因此確定胰腺干/祖細(xì)胞所特有的標(biāo)記成為發(fā)現(xiàn)胰腺干/祖細(xì)胞的主要研究方向。小鼠的Nestin基因是由5945bp組成的，表達(dá)一種中間絲蛋白，與Desmin和Vimentin等同源，最早被發(fā)現(xiàn)在神經(jīng)干細(xì)胞中表達(dá)，并被認(rèn)為是神經(jīng)干細(xì)胞的分子標(biāo)記，將從大腦分離出來(lái)的nestin~+細(xì)胞在體外培養(yǎng)，其可以分化為神經(jīng)元、膠質(zhì)細(xì)胞等，說(shuō)明了其具有干細(xì)胞特性。在胚胎發(fā)育早期，神經(jīng)細(xì)胞和胰腺細(xì)胞共同表達(dá)許多基因，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/祖細(xì)胞的標(biāo)記分子。 本實(shí)驗(yàn)利用膽總管膠原酶灌注法分離出ICR小鼠的胰島細(xì)胞團(tuán)，，并研究胰島細(xì)胞團(tuán)內(nèi)nestin~+細(xì)胞的特性，結(jié)果如下：（1）在本實(shí)驗(yàn)室條件下，膽總管膠原酶灌注法分離ICR小鼠胰島的最佳靜止消化條件是，膠原酶濃度0.8mg/ml、37℃、水浴靜止消化20min；（2）利用RT-PCR技術(shù)和免疫熒光染色技術(shù)證實(shí)了ICR小鼠胰島內(nèi)存在nestin~+細(xì)胞，并且這類(lèi)nestin~+細(xì)胞不表達(dá)胰島素；（3）將胰島內(nèi)的nestin~+細(xì)胞體外分離、擴(kuò)大培養(yǎng)，在低糖、低血清的培養(yǎng)條件下可以誘導(dǎo)其形成胰島樣細(xì)胞團(tuán)，經(jīng)雙硫腙（DTZ）染色后呈褐紅色，用RT-PCR技術(shù)檢測(cè)出胰島素基因的表達(dá)。 以上結(jié)果表明，在ICR小鼠胰島內(nèi)存在著nestin~+細(xì)胞，并且通過(guò)體外實(shí)驗(yàn)證明了這類(lèi)細(xì)胞具有分化為胰島素分泌細(xì)胞的能力，表現(xiàn)出一定的胰腺干/祖細(xì)胞的特性。但是這類(lèi)nestin~+細(xì)胞在體內(nèi)是否同樣具有這一特性，還需要進(jìn)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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