發(fā)布時(shí)間：2018-06-14 16:37

  本文選題：小鼠 + 肝臟干細(xì)胞��； 參考：《復(fù)旦大學(xué)》2008年博士論文

【摘要】： 目的 研究正常成體小鼠肝細(xì)胞的增殖能力和分化潛能,分離正常成體小鼠肝臟內(nèi)可能存在的干細(xì)胞或祖細(xì)胞并建立體外培養(yǎng)的細(xì)胞模型,研究其基本的生物學(xué)特性。 方法 應(yīng)用改良的Seglen二步法灌注和離心分離肝臟細(xì)胞,將肝細(xì)胞初步分為肝臟實(shí)質(zhì)細(xì)胞(parenchymal hepatocytes,PH)部分和富含AHPC(adult hepaticprogenitor cells,AHPC)部分,對(duì)兩部分的細(xì)胞用添加胎牛血清(FBS)的改良DMEM培養(yǎng)基進(jìn)行培養(yǎng),持續(xù)觀察超過(guò)60天,分析兩部分中肝細(xì)胞的形態(tài)學(xué)差異,以及通過(guò)細(xì)胞增殖和克隆的形成情況分析兩部分中肝細(xì)胞增殖能力的差異。應(yīng)用免疫熒光技術(shù)對(duì)具有高增殖能力細(xì)胞及其形成的克隆進(jìn)行Albumin、AFP、CK19、c-kit、CD45、CD34、Oct-4、Desmin、CD16、Thy-1和nestin等染色,分析細(xì)胞標(biāo)記物的表達(dá)和克隆內(nèi)細(xì)胞的成熟分化情況。利用形態(tài)學(xué)觀察、記錄細(xì)胞增殖狀況,以及免疫熒光染色技術(shù)初步分析肝臟非實(shí)質(zhì)細(xì)胞(nonparenchymalcell,NPC)的生長(zhǎng)對(duì)于AHPC活化、增殖和分化的影響。另外,嘗試了AHPC克隆的傳代培養(yǎng)。 結(jié)果 本研究中兩部分肝細(xì)胞均獲得較高的產(chǎn)量和活性(＞90%),完全滿足實(shí)驗(yàn)的需要。PH部分和富含AHPC部分的肝細(xì)胞大小分別為(37.03±6.65)μm和(22.63±2.04)μm,存在明顯的統(tǒng)計(jì)學(xué)差異(p＜0.05),但在大小分布比例上存在小部分的重疊。兩部分的貼壁細(xì)胞中均含有NPC污染,其中富含AHPC部分較多,占貼壁細(xì)胞總數(shù)的70.3%,NPC增殖后肝細(xì)胞活化并開(kāi)始增殖,所有的肝細(xì)胞克隆均表達(dá)肝星狀細(xì)胞標(biāo)記物Desmin。富含AHPC部分的肝細(xì)胞增殖能力明顯較PH部分高,兩部分的克隆形成率分別為21.45%±1.25%和0.28%±0.09%(p＜0.001)。富含AHPC部分中,約13.5%的貼壁肝細(xì)胞在接種后第2～3天活化并迅速增殖,第4～5天形成小的細(xì)胞克隆,極少數(shù)細(xì)胞(0.5%～1%)在接種后第3天即可形成克隆;培養(yǎng)30天后克隆內(nèi)出現(xiàn)類(lèi)似成熟的肝細(xì)胞,細(xì)胞克隆可持續(xù)擴(kuò)增超過(guò)60天,最大克隆面積達(dá)到0.64mm~2,細(xì)胞平均增殖超過(guò)10個(gè)周期。貼壁后24小時(shí),所有的肝細(xì)胞均強(qiáng)陽(yáng)性表達(dá)肝細(xì)胞標(biāo)記物Albumin,不表達(dá)AFP和CK19,培養(yǎng)第5天細(xì)胞克隆開(kāi)始表達(dá)Albumin和AFP,第30天克履誆糠窒赴鉤澩锏ü芟赴鉤曇俏榬K19,同時(shí)發(fā)現(xiàn)Albumin陰性細(xì)胞。通過(guò)免疫熒光雙染發(fā)現(xiàn),培養(yǎng)第30天,AHPC克隆內(nèi)同時(shí)存在Albumin陽(yáng)性和AFP陽(yáng)性、Albumin陽(yáng)性和AFP陰性、Albumin陽(yáng)性和CK19陰性、CK19陽(yáng)性和AFP陰性、CK19陽(yáng)性和AFP陽(yáng)性的細(xì)胞。另外,AHPC可以傳代培養(yǎng)超過(guò)60天,傳代培養(yǎng)中,貼壁的AHPC克隆內(nèi)細(xì)胞較小,核漿比率大,呈上皮樣細(xì)胞的形態(tài),部分細(xì)胞仍然具有獨(dú)立形成克隆的能力,但觀察發(fā)現(xiàn),AHPC克隆解離為細(xì)胞團(tuán)進(jìn)行傳代培養(yǎng)中細(xì)胞的增殖比解離為單細(xì)胞傳代好。 結(jié)論 1.在正常成體小鼠肝臟內(nèi)存在一種肝臟組織特異性的成體肝臟祖細(xì)胞(AHPC),并已成功建立了體外培養(yǎng)的細(xì)胞模型。 2.小鼠AHPC體外培養(yǎng)活化后可持續(xù)克隆性增殖超過(guò)60天,并可連續(xù)傳代培養(yǎng),具有向肝細(xì)胞和膽管細(xì)胞分化的雙向分化潛能。 3.肝臟非實(shí)質(zhì)細(xì)胞(NPC)的生長(zhǎng)可以促進(jìn)AHPC的增殖、成熟和分化。 4.與國(guó)外文獻(xiàn)報(bào)導(dǎo)的肝臟祖細(xì)胞相比,小鼠AHPC體外培養(yǎng)中細(xì)胞表型不同,具有較高的增殖能力和明顯的雙向分化潛能,為肝細(xì)胞移植、肝臟發(fā)育和肝病等研究提供了一種新的肝臟干細(xì)胞模型和研究工具。
[Abstract]:objective
To study the proliferation and differentiation potential of normal adult mouse hepatocytes, to isolate the possible stem cells or progenitor cells in normal adult mice and to establish a cell model in vitro, and to study their basic biological characteristics.
Method
Liver cells were separated by improved Seglen two step method and centrifugation, and hepatocytes were preliminarily divided into liver parenchyma cells (parenchymal hepatocytes, PH) and AHPC (adult hepaticprogenitor cells, AHPC). The two parts of the cells were cultured with a modified DMEM medium adding fetal bovine serum (FBS), and continued to observe more than 60. Analysis of the morphological differences in the two parts of the liver, and analysis of the difference in the proliferation of hepatocytes in the two part by cell proliferation and cloning. Immunofluorescence technique was used to carry out Albumin, AFP, CK19, c-kit, CD45, CD34, Oct-4, Desmin, CD16, Thy-1 and nestin in the Clones of high proliferative cells and their formation. Color, analysis of the expression of cell markers and the maturation and differentiation of cells in clones. The effects of the growth of nonparenchymalcell (NPC) on the activation, proliferation and differentiation of AHPC were preliminarily analyzed by morphological observation, and the immunofluorescence staining technique was used to analyze the proliferation and differentiation of non parenchymal cells in the liver. In addition, the transmission of AHPC clones was tried. Raise.
Result
In the two parts of this study, the two parts of the liver cells obtained high yield and activity (> 90%). They fully met the needs of the experiment and the size of the liver cells rich in the AHPC part were (37.03 + 6.65) m and (22.63 + 2.04) m respectively. There was a significant statistical difference (P < 0.05), but there was a small overlap in the proportion of the size distribution. The two part of the paste was attached. The parietal cells contained NPC pollution, which were rich in AHPC and accounted for 70.3% of the total number of adherent cells. After NPC proliferation, hepatocytes were activated and began to proliferate. All the hepatocyte clones expressed the hepatic stellate cell marker Desmin. rich in AHPC part of the hepatocytes, which was higher than that of PH, and the clone formation rate of the two parts was 21.45, respectively. % + 1.25% and 0.28% + 0.09% (P < 0.001). In the rich AHPC part, about 13.5% of the adherent hepatocytes were activated and proliferated at second to 3 days after inoculation, and small cell clones were formed on fourth to 5 days, and a few cells (0.5% ~ 1%) could form clones in 1.25% days after inoculation, and there were similar mature hepatocytes in clones and clones could be cloned after 30 days. For more than 60 days, the maximum clone area reached 0.64mm~2, and the cell proliferation was more than 10 cycles. After 24 hours of adherence, all liver cells strongly positive expression of liver cell marker Albumin, not AFP and CK19, and the cell clone began to express Albumin and AFP for fifth days. The thirtieth days of the thirtieth days At the same time, Albumin negative cells were found. By immunofluorescence double staining, it was found for thirtieth days that there were Albumin positive and AFP positive in AHPC clones, Albumin positive and AFP negative, Albumin positive and CK19 negative, CK19 positive and AFP negative, CK19 positive and AFP positive cells. The cells of the AHPC clones in the wall were small, the ratio of the nuclear plasma was large and the morphology of the epithelioid cells. Some cells still had the ability to form clones independently. However, it was found that the proliferation of AHPC clones in the subculture of cell groups was better than that of single cell subculture.
conclusion
1. in the liver of normal adult mice, a liver specific adult liver progenitor cell (AHPC) was established, and a cell model was established successfully in vitro.
2. the mouse AHPC was cultured and activated in vitro for more than 60 days, and could be cultured continuously, with the differentiation potential of hepatocyte and bile duct.
3. the growth of hepatic non parenchymal cells (NPC) can promote the proliferation, maturation and differentiation of AHPC.
4. compared with the liver progenitor cells reported in foreign literature, the cell phenotype of mouse AHPC in vitro is different. It has high proliferation ability and obvious bidirectional differentiation potential. It provides a new liver stem cell model and research tool for liver transplantation, liver development and liver disease.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R329

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本文編號(hào)：2018180