本文選題：胚胎干細(xì)胞　切入點(diǎn)：間充質(zhì)干細(xì)胞　出處：《浙江大學(xué)》2010年博士論文　論文類型：學(xué)位論文

【摘要】：胚胎干細(xì)胞(ES)具有無限增殖能力,定向分化后可成為細(xì)胞移植的重要來源。ES定向分化后的細(xì)胞在體內(nèi)尤其是病理模型體內(nèi)的歸巢研究對(duì)ES的醫(yī)療應(yīng)用具有重要的理論和實(shí)踐參考價(jià)值。在本研究中,通過ROCK抑制劑Y-27632處理并在有血清體系下單層誘導(dǎo)人胚胎干細(xì)胞分化為MSC(hES-MSC),在體外生物素標(biāo)記hES-MSC后,將其異種移植到ICR小鼠體內(nèi)。36h后,通過流式細(xì)胞術(shù)檢測(cè)和組織冰凍切片的免疫組織化學(xué)(IHC)鑒定,發(fā)現(xiàn)了hES-MSC在ICR小鼠體內(nèi)早期歸巢的規(guī)律：在正常ICR小鼠體內(nèi),hES-MSC傾向于歸巢到脾臟、骨髓和肺中,而幾乎不在肝臟中駐留；在CCl4誘導(dǎo)急性肝損傷的ICR小鼠中,hES-MSC在骨髓、脾臟歸巢的平均數(shù)量分別減少了64.6%和74.5%,而在損傷肝部位能發(fā)現(xiàn)大量歸巢的hES-MSC。同時(shí)還發(fā)現(xiàn)在正常ICR小鼠體內(nèi)隨著傳代次數(shù)增加,hES-MSC向脾臟、肝臟早期歸巢的能力會(huì)顯著性減弱,但是hES-MSC向損傷肝歸巢的能力卻不會(huì)受細(xì)胞代數(shù)變化的顯著性影響。隨后,通過RT-PCR和冰凍切片的IHC檢測(cè)證明CXCR4在hES-MSC向損傷肝歸巢過程中具有重要作用,而損傷肝環(huán)境會(huì)促進(jìn)hES-MSC表達(dá)CXCR4和CXCR7,其中CXCR7會(huì)影響CXCR4與其配體SDF-1的結(jié)合。通過AFP和biotin的共定位,我們發(fā)現(xiàn)損傷肝環(huán)境還能在細(xì)胞移植早期就誘導(dǎo)hES-MSC表達(dá)肝向分化的指標(biāo)AFP,說明hES-MSC對(duì)損傷肝的治療作用與hES-MSC的原位肝向分化有關(guān)。在檢測(cè)了TNF-α對(duì)hES-MSC歸巢的影響后,發(fā)現(xiàn)在體外TNF-α可以促進(jìn)hES-MSC表達(dá)CXCR4和CXCR7, TNF-α預(yù)處理的hES-MSC向ICR小鼠損傷肝臟早期歸巢的平均數(shù)量比未處理組提高了1.4倍。綜合以上的結(jié)果,可以得出這樣的結(jié)論：hES-MSC不僅能定向歸巢到ICR小鼠的損傷肝臟,而且還能在損傷部位進(jìn)行原位肝向分化,因此hES-MSC是理想的治療急性肝損傷的種子細(xì)胞,TNF-α預(yù)處理hES-MSC可以提高其向損傷肝早期歸巢的效率從而提高治療效果。 小鼠胚胎干細(xì)胞(mES)表面分子與細(xì)胞外信號(hào)系統(tǒng)和胞間通訊密切相關(guān),對(duì)mES的鑒定、自我更新及分化機(jī)理的研究具有重要意義。通過全細(xì)胞免疫獲得針對(duì)mES特異性表面分子的兔單克隆抗體,利用改進(jìn)的免疫共沉淀方法純化細(xì)胞膜蛋白抗原,再結(jié)合LC-LTQ質(zhì)譜鑒定和商業(yè)化抗體的western blot驗(yàn)證,獲得了針對(duì)Glut3在mES細(xì)胞膜表面的構(gòu)象表位的抗體。利用該抗體封閉Glut3可以抑制mES增殖,說明Glut3在維持mES自我更新中具有重要作用。
[Abstract]:Embryonic stem cells (es) have unlimited proliferative ability. The homing study of es cells in vivo, especially in pathological model, has important theoretical and practical reference value for the medical application of es. ROCK inhibitor Y-27632 was used to induce human embryonic stem cells to differentiate into MSCHES-MSCG in serum system. After biotin labeled hES-MSC in vitro, xenografts were transplanted into ICR mice for .36h. The early homing rule of hES-MSC in ICR mice was found by flow cytometry and immunohistochemistry identification of frozen sections. In normal ICR mice, hES-MSC tended to homing to spleen, bone marrow and lung. In ICR mice with acute liver injury induced by CCl4, HES-MSC was found in bone marrow. The average number of homing spleen decreased by 64.6% and 74.5 respectively, but a large number of homing cells were found in the injured liver. It was also found that in normal ICR mice, the ability of homing in the early stage of liver homing was significantly decreased with the increase of passage of hES-MSC to the spleen. However, the ability of hES-MSC to homing to injured liver was not affected by the significant changes of cell algebra. Subsequently, RT-PCR and frozen sections of IHC showed that CXCR4 played an important role in the homing process of hES-MSC to damaged liver. The expression of CXCR4 and CXCR7 was promoted by liver environment injury, where CXCR7 affected the binding of CXCR4 to its ligand SDF-1. We found that the injured liver environment could also induce the expression of hES-MSC in liver differentiation at the early stage of cell transplantation, indicating that the therapeutic effect of hES-MSC on the injured liver was related to the in situ liver differentiation of hES-MSC. After detecting the effect of TNF- 偽 on homing of hES-MSC, It was found that TNF- 偽 could promote the expression of CXCR4 and CXCR7 in hES-MSC in vitro. The average number of early homing of hES-MSC pretreated with TNF- 偽 to ICR mice was 1.4-fold higher than that of untreated group. It can be concluded that: HES-MSC can not only homing to the injured liver of ICR mice, but also differentiating into the liver in situ at the injured site. Therefore, hES-MSC is an ideal seed cell for the treatment of acute liver injury. TNF- 偽 pretreatment hES-MSC can improve the efficiency of homing to the early stage of liver injury and improve the therapeutic effect. The surface molecules of mouse embryonic stem cell mESs are closely related to the extracellular signaling system and intercellular communication. It is of great significance to study the mechanism of self-renewal and differentiation. Rabbit monoclonal antibodies against specific surface molecules of mES were obtained by whole-cell immunization, and cell membrane protein antigens were purified by modified co-precipitation method. Combined with LC-LTQ mass spectrometry and western blot verification of commercial antibody, the antibody against conformational epitope of Glut3 on the surface of mES cell membrane was obtained. Using this antibody to block Glut3 could inhibit mES proliferation, indicating that Glut3 plays an important role in maintaining mES self-renewal.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

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