本文關(guān)鍵詞： 胎肝干細(xì)胞 誘導(dǎo) 分化 胰島素　出處：《第一軍醫(yī)大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

【摘要】： 本文研究了胎肝干細(xì)胞的特性，并誘導(dǎo)其分化為胰島素分泌細(xì)胞，著重進(jìn)行了胎肝干細(xì)胞的體外擴(kuò)增培養(yǎng)，探討胎肝干細(xì)胞的生物學(xué)特性和性質(zhì)，以及胚胎發(fā)育中胎肝前體細(xì)胞的類型；采用階段性化學(xué)誘導(dǎo)、生物誘導(dǎo)以及二者相結(jié)合的方法，啟動(dòng)胰腺發(fā)育的相關(guān)基因，使胎肝干細(xì)胞分化為胰島素分泌細(xì)胞。 全文共分四個(gè)部分。 第一部分，胎肝干細(xì)胞的分離、培養(yǎng)與鑒定。 通過體外分離培養(yǎng)并擴(kuò)增大鼠胎肝干細(xì)胞，研究其形態(tài)、生物學(xué)特性，采用免疫細(xì)胞化學(xué)分析胎肝干細(xì)胞表面標(biāo)志物的表達(dá)情況，初步研究胎肝干細(xì)胞體外擴(kuò)增培養(yǎng)的實(shí)驗(yàn)條件，并探討肝臟的發(fā)生發(fā)育及肝干細(xì)胞的性質(zhì)及增殖潛能。 本實(shí)驗(yàn)選取胎齡12～16d SD大鼠，先用機(jī)械分離結(jié)合酶消化法分離培養(yǎng)胎肝細(xì)胞，繼而用特異的干細(xì)胞培養(yǎng)基進(jìn)行克隆篩選法獲得了數(shù)量較多、較純化的胎肝干細(xì)胞克隆。SABC法檢測原代、傳代后及細(xì)胞克隆中的肝干細(xì)胞特異表面標(biāo)志物OV-6、CK-19及胰腺前體細(xì)胞特異標(biāo)志蛋白Nestin的表達(dá)。 以上研究表明，胎肝干細(xì)胞可通過克隆篩選法進(jìn)行體外擴(kuò)增，含有特殊細(xì)胞因子的干細(xì)胞培養(yǎng)基有助于肝干細(xì)胞的擴(kuò)增，可獲得較好的效果。培養(yǎng)3d左右開始出現(xiàn)小細(xì)胞團(tuán)，1個(gè)月即形成肉眼可見的細(xì)胞集落，5～7d傳代一次。原代、傳代培養(yǎng)的胎肝細(xì)胞部分表達(dá)OV-6、CK-19及Nestin；細(xì)胞克隆幾乎全部為干細(xì)胞標(biāo)志陽性細(xì)胞，表明胎肝干細(xì)胞具有較強(qiáng)的分裂增殖能力。胎肝內(nèi)存在Nestin陽性干細(xì)胞，我們稱之為肝源性Nestin陽性祖細(xì)胞(nestin-positive hepatic-derived progenitor，NHP cells)，可能是一種更為原始的干細(xì)胞在胚胎發(fā)育中起重要作用。 第二部分，胎肝組織細(xì)胞的鑒定及分類。 本部分實(shí)驗(yàn)旨在研究胎肝干細(xì)胞性質(zhì)的同時(shí)也為肝干細(xì)胞進(jìn)行較系統(tǒng)的分類提供實(shí)驗(yàn)依據(jù)，探討肝臟中干／祖細(xì)胞的類型并篩選出易于向胰腺方向分化的細(xì)胞類型，為下一步的誘導(dǎo)獲取較佳的種子細(xì)胞。 采用免疫熒光雙標(biāo)記法，針對OV-6、CK-19及Nestin三種抗體中二種抗體的不同組合，F(xiàn)ITC和Cy3分別標(biāo)記，DAPI染核，針對胎肝組織和原代、傳代及克隆培養(yǎng)的細(xì)胞中不同的前體細(xì)胞類型進(jìn)行初步的定位及分類。 胎肝中包含了從原始到成熟不同發(fā)育程度的各種肝臟細(xì)胞：(1)多能前體細(xì)胞：OV-6~+nestin~+CK-19~-細(xì)胞，可能具有肝胰多向分化潛能；(2)雙能前體細(xì)胞：OV-6~+CK-19~+nestin~-細(xì)胞，是肝臟的雙能干細(xì)胞，可分化為肝細(xì)胞系和膽管細(xì)胞系；(3)定向前體細(xì)胞或過渡細(xì)胞：僅表達(dá)OV-6或僅表達(dá)CK-19，可能分別是僅有向肝臟方向分化能力的肝前體細(xì)胞和向膽管方向分化的細(xì)胞，或是正在分化為成熟細(xì)胞的過渡階段；(4)較成熟細(xì)胞。體外獲得的nestin~+細(xì)胞數(shù)量較少，大多是與OV-6一同表達(dá)，僅表達(dá)nestin的細(xì)胞數(shù)量很少。OV-6~+nestin~+細(xì)胞和nestin~+細(xì)胞可能具有向胰腺細(xì)胞分化的潛能，從胰島發(fā)育的過程推測β細(xì)胞與肝細(xì)胞有共同的來源，它們共同的祖細(xì)胞是胰腺導(dǎo)管上皮內(nèi)和肝內(nèi)的nestin陽性細(xì)胞。因此我們選取此類肝源性nestin陽性祖細(xì)胞作為下一步誘導(dǎo)分化的種子細(xì)胞。 第三部分，EGFP-PDX-1融合表達(dá)載體電穿孔轉(zhuǎn)染大鼠胎肝干細(xì)胞的研究。 本室構(gòu)建了PDX-1綠色熒光蛋白融合表達(dá)載體，通過電穿孔轉(zhuǎn)染入大鼠胎肝干細(xì)胞以得到較穩(wěn)定的表達(dá)，期望借助于外源性生物因子啟動(dòng)胎肝干細(xì)胞中PDX-1基因的表達(dá)，從生物誘導(dǎo)方面研究胰腺內(nèi)分泌發(fā)育的關(guān)鍵基因PDX-1在肝干細(xì)胞定向分化中的調(diào)控機(jī)制，為今后定向分化的深入研究提供物質(zhì)平臺(tái)。 由PDX-1插入pEGFP-C1的多克隆位點(diǎn)中構(gòu)建的重組質(zhì)粒pEGFP-C1-PDX-1用電穿孔法轉(zhuǎn)染體外培養(yǎng)的大鼠胎肝干細(xì)胞，分析轉(zhuǎn)染前后細(xì)胞的生長曲線，熒光顯微鏡下觀察GFP發(fā)光情況，RT-PCR鑒定PDX-1基因的表達(dá)情況。 經(jīng)電穿孔轉(zhuǎn)染后GFP和PDX-1的融合蛋白能在胎肝干細(xì)胞中一起表達(dá)，并維持較長的時(shí)間，，對胎肝干細(xì)胞的生長增殖無顯著影響(P＞0.05)。構(gòu)建的PDX-1綠色熒光蛋白融合表達(dá)載體能在胎肝干細(xì)胞中較持續(xù)地表達(dá)，為研究PDX-1在干細(xì)胞定向分化為胰島素分泌細(xì)胞中的調(diào)控作用提供了物質(zhì)基礎(chǔ)。 第四部分，胎肝干細(xì)胞誘導(dǎo)分化為胰島素分泌細(xì)胞的研究。 采用階段特異性的化學(xué)誘導(dǎo)、生物誘導(dǎo)以及二者相結(jié)合的方法，模擬胰腺發(fā)育的內(nèi)外環(huán)境，使胎肝干細(xì)胞分化為胰島素分泌細(xì)胞，并探討肝干細(xì)胞誘導(dǎo)分化為胰島素分泌細(xì)胞的內(nèi)源性及外源性調(diào)控機(jī)制。 將篩選出的nestin陽性胎肝細(xì)胞分組誘導(dǎo)，分別進(jìn)行分階段的以Nicotinamide為誘導(dǎo)劑的化學(xué)誘導(dǎo)、電穿孔轉(zhuǎn)染PDX-1基因的生物誘導(dǎo)、生物誘導(dǎo)后化學(xué)誘導(dǎo)以及未誘導(dǎo)組。ELISA檢測細(xì)胞上清液中胰島素的含量，繪制標(biāo)準(zhǔn)曲線后換算出各組細(xì)胞釋放的胰島素量并比較；RT-PCR檢測與胰腺發(fā)育相關(guān)的基因和肝細(xì)胞特異基因在各組的表達(dá)情況。 Nestin~+胎肝細(xì)胞經(jīng)生物、化學(xué)及二者結(jié)合法誘導(dǎo)后分泌的胰島素量分別為0.539、0.943和5.58ng／ml上清液(1×10~5細(xì)胞)，與陰性對照組相比均有顯著性差異(P=0.000)，其中任意兩組相比也有統(tǒng)計(jì)學(xué)意義(P=0.000)。各誘導(dǎo)組均表達(dá)胰島素Ⅰ、GLUT-2及肝干細(xì)胞標(biāo)志HNF-1、AFP和c-met，而不表達(dá)胰高血糖素、生長抑素和ALB。生物誘導(dǎo)組和生化誘導(dǎo)組表達(dá)PDX-1和胰島素Ⅱ，化學(xué)誘導(dǎo)組弱表達(dá)這兩個(gè)基因。生化誘導(dǎo)法通過模擬細(xì)胞內(nèi)外的微環(huán)境能獲得較高的胰島素分泌水平，并表達(dá)一系列胰腺發(fā)育相關(guān)的基因，但是，nestin~+胎肝細(xì)胞誘導(dǎo)分化為胰島素分泌細(xì)胞的機(jī)制還需更深入的研究，以實(shí)現(xiàn)胰島素的生理性分泌調(diào)節(jié)。
[Abstract]:This paper studied the characteristics of fetal liver stem cells, and induce their differentiation into insulin secreting cells, focus on the proliferation of fetal liver stem cells in vitro and explore the biological characteristics of fetal liver stem cells and the nature, and the type of embryo fetal liver progenitor cells; the stage of chemical induction, induction and biological method the combination of the two, start the related gene of pancreas. The fetal liver stem cells to differentiate into insulin secreting cells.
The full text is divided into four parts.
The first part, the isolation, culture and identification of fetal liver stem cells.
By in vitro culture and amplification of rat fetal liver stem cells and study its morphology, biological characteristics, analysis of cell surface marker expression of fetal liver stem cells by chemical amplification, experimental conditions of cultured cells in vitro study of fetal liver stem, and investigate the occurrence of liver development and liver stem cell proliferation properties and potential.
This experiment selects the gestational age of 12 ~ 16d SD rats by mechanical separation and enzyme digestion method cultured fetal liver cells, then use specific stem cell medium to obtain a large number of clone screening method, compared with the purified fetal liver stem cell clone.SABC was used to detect the primary cell specific surface marker OV-6. And cell clone in hepatic stem CK-19 and pancreatic precursor cells specific marker protein Nestin expression.
The above research results show that the cells were amplified in vitro by cloning and screening of fetal liver stem cells, containing special factors in stem cell medium contribute to liver stem cell expansion, good results can be obtained. 3D culture began to appear around the small cell groups, 1 months to form a visible cell colony, 5 ~ 7d passaged. Primary cultured fetal liver cells, the expression of OV-6 and CK-19 part, Nestin; cell clones for almost all stem cell marker positive cells showed that fetal liver stem cells with high proliferation ability. Nestin positive stem cells exist in fetal liver, we called hepatic Nestin positive progenitor cells (nestin-positive hepatic-derived progenitor, NHP cells), may be a more primitive stem cells play an important role in embryonic development.
The second part, the identification and classification of fetal liver tissue cells.
This experiment aims to study the properties of fetal liver stem cells and provide experimental basis for liver stem cells are classified systematically, discusses the types of liver stem / progenitor cells were selected to differentiate into pancreatic cell type direction, to obtain better seed cells for the induction of the next step.
Double immunofluorescence labeling for OV-6, different combinations of two kinds of CK-19 antibody and Nestin three antibodies, FITC and Cy3 were labeled, DAPI staining, the fetal liver tissue and cultured in different cell types were cloned and precursor cells cultured in the localization and classification of.
Fetal liver contains from original to mature in different developmental stages of hepatic cells: (1) multipotent progenitor cells: OV-6~+nestin~+CK-19~- cells, liver and pancreas may have multiple differentiation potential; (2) two precursor cells: OV-6~+CK-19~+nestin~- cells, the liver is the double stem cells, can differentiate into hepatic cells and bile duct cell line; (3) directional precursor cells or transitional cells expressing only OV-6 or only CK-19 expression may be cells and somatic cells to differentiate into hepatic bile duct only differentiation ability of the liver, or is the transitional stage differentiation into mature cells; (4) mature cells in vitro. The number of nestin~+ cells are mostly expressed with the OV-6, only the expression of the number of nestin cells rarely.OV-6~+nestin~+ cells and nestin~+ cells may have potential to differentiate into pancreatic cells, from pancreatic island development push Beta cells and hepatocytes share a common source. Their common progenitor cells are nestin positive cells in the pancreatic ductal epithelium and liver. Therefore, we select this hepatocyte derived nestin positive progenitor cells as the next seed cells to induce differentiation.
The third part, the transfection of rat fetal liver stem cells by electroporation with EGFP-PDX-1 fusion expression vector.
The room was constructed PDX-1 fusion expression vector of green fluorescent protein by electroporation into rat fetal liver stem cells to obtain stable expression, with expectations to exogenous biological factors start fetal liver stem cells PDX-1 gene expression and induction of key gene PDX-1 from biological research in the development of pancreatic secretion in liver stem regulation the mechanism of cell differentiation, to provide material platform for future in-depth study of directed differentiation.
The recombinant plasmid pEGFP-C1-PDX-1 was constructed by inserting pEGFP-C1 multiple cloning sites in PDX-1 by electroporation transfection of cultured rat fetal liver stem cells, growth curve analysis before and after transfection, fluorescence microscope GFP light, the expression of RT-PCR PDX-1 gene was identified.
By GFP after electroporation and PDX-1 fusion protein in fetal liver stem cells expressed together, and maintain a longer period of time, no significant effect on the proliferation of fetal liver stem cells (P > 0.05). The expression vector is stably expressed in fetal liver stem cell fusion PDX-1 green fluorescent protein construct. For the study of PDX-1 in the differentiation of stem cells into insulin secreting cells in the regulation provides a material basis.
The fourth part is the study of the induced differentiation of fetal liver stem cells into insulin secreting cells.
Stage specific induced by chemical, biological induction method and the combination of the two, the simulation of pancreatic development internal and external environment, the fetal liver stem cells to differentiate into insulin secreting cells, and to explore the induction and differentiation of hepatic stem cells into insulin secreting cells of endogenous and exogenous control mechanism.
Nestin positive fetal liver cell group screened were induced in phases induced by Nicotinamide chemical inducers, electroporation of PDX-1 gene induced by biological, biological and chemical induced induced content did not induce insulin group.ELISA cell supernatants were measured in the standard curve after the calculated amount of insulin cells were released and compared; RT-PCR detection and development of pancreas and liver cell specific gene related gene expression in each group.
Nestin~+ fetal liver cells by biological, chemical and the combination of the two amount of insulin induced secretion were 0.539,0.943 and 5.58ng / ml supernatant (1 x 10~5 cells), compared with the negative control group were significantly different (P=0.000), which compared to any two groups had statistical significance (P=0.000). The induction group both the expression of insulin I, HNF-1 and hepatic stem cell marker GLUT-2, AFP and c-Met, but the expression of glucagon, somatostatin and ALB. biological induction group and biochemical induction group PDX-1 and insulin expression induced by chemical group II, weak expression of these two genes. The biochemical induced secretion of insulin can be obtained through the simulation of micro environment the higher the inside and outside of the cells, and the expression of related genes, a series of pancreatic development but the mechanism of nestin~+ induced differentiation of fetal liver cells into insulin secreting cells also need more in-depth research, to achieve physiological insulin secretion Adjust.

【學(xué)位授予單位】：第一軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

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2 孫曉艷,安靚;胎肝中肝干細(xì)胞的免疫組織化學(xué)研究[J];中國組織化學(xué)與細(xì)胞化學(xué)雜志;2003年04期

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本文編號(hào)：1454518