间充质干细胞跨越分化肝细胞及其参与肝脏损伤修复的探索研究
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摘要
间充质干细胞(Mesenchymal stem cells,MSCs)来源于中胚层,能够分化为成骨、软骨和脂肪,为原始造血干/祖细胞在骨髓微环境中的生长和分化提供支持。近年来研究发现MSCs的分化潜能远超过最初的预期,甚至可以跨越分化为其他胚层来源的细胞,MSCs也因此迅速成为干细胞领域的一个热点。本研究重点研究了小鼠骨髓MSCs向肝细胞的跨越分化能力,以及在体内情况下能否参与肝脏损伤修复。
研究的第一部分,我们首先建立了三套条件培养液诱导体系,成功将小鼠骨髓来源MSCs诱导分化为肝样细胞。诱导体系包括肝损伤条件培养液、肝细胞条件培养液和胎肝条件培养液。诱导得到的细胞具备典型的肝细胞形态,表达肝系细胞特异性基因,具备肝细胞的正常功能,如ICG吞噬、白蛋白合成和糖原储存等。随后的研究发现HGF、FGF-4和OSM是条件培养液诱导体系中发挥作用的关键因子,组合上述三种细胞因子即可将小鼠骨髓MSCs诱导成典型的肝样细胞,但诱导效率较低。此外,本研究发现使用丙戊酸钠(VPA)预处理MSCs,可以显著提高MSCs向肝细胞的分化效率。实验发现MSCs在VPA处理过程中伴随着细胞周期阻滞、组蛋白乙酰化程度增加、染色质结构疏松。表观遗传修饰导致的FGF受体和HGF受体基因表达上调可能是肝细胞分化效率提高的关键因素。
研究的第二部分,我们从肝脏损伤小鼠中分离得到了外周血间充质干细胞,发现该细胞与骨髓来源的MSCs具备类似的细胞表面标记,可以向成骨、脂肪和软骨诱导分化,但是增殖能力较弱,易衰老。本研究还通过活体动物分子成像等技术对骨髓腔移植的eGFP~+MSCs进行追踪,证明MSCs在特定状态下可以从骨髓迁移到外周血继而迁移到损伤肝脏中,提示病理状态下分离得到的外周血间充质干细胞可能来源于骨髓。此外,通过免疫荧光染色等检测方法,发现迁移到损伤肝脏的MSCs可以通过多种方式参与组织修复,包括直接分化为多种细胞类型,包括AFP阳性、ALB阳性的肝系细胞、CK19阳性的胆管类细胞、CD146阳性的血管内皮类细胞等,同时MSCs还能通过分泌细胞因子调控星形细胞的活性来改善肝脏胶原沉积和纤维化。
综上所述,本研究证明小鼠骨髓MSCs不仅在体外可以跨越分化为肝实质细胞,而且体内状态能够在肝脏损伤条件下,自发的迁移出骨髓,并通过血液循环向肝脏迁移,通过跨越分化和细胞因子分泌等方式参与肝脏的损伤修复。
Mesenchymal stem cells (MSCs) were initially characterized as plastic adherent, fibroblastoid cells. These cells can differentiate into osteogenic, adipogenic and chondrogenic lineages under appropriate conditions. In recent years, a number of reports have also indicated that these cells possess the capacity to trans-differentiate into epithelial cells and lineages derived from the neuro-ectoderm.
In this study, we mainly focus on the potential of MSCs for liver repair, which includes two parts: in vitro trans-differentiation of mouse bone marrow mesenchymal stem cell (mBM-MSCs) into hepatocytes, and in vivo recruiting and homing of mBM-MSCs towards injured liver. In the 1~(st) part of the project, three original protocols for directing mBM-MSCs trans-differentiate into functional hepatocyte-like cells were established by conditional medium of injured liver tissues, normal hepatocytes and fetal livers separately. Further studies by microarray analysis and antibody blocking experiments indicated that FGF4, HGF and OSM were crucial for these conditional media-induced hepatic differentiations, and mBM-MSC derived hepatocytes can also be acquired by treatment of these cytokines. Furthermore, we demonstrated that the differentiation efficacy could be considerably enhanced by pre-treatment of VPA, and regulation of FGFRs and c-Met gene expression through post translational modification of core histones might be the primary initiating event for these effects. In the 2nd part of the project, we successfully isolated and characterized circulating MSCs in the peripheral blood of liver-injured mice with expansion in culture, and provided direct evidence that mBM-MSCs were mobilized into the circulation and recruited into livers after stimulation of liver-injury. CCR9, CXCR4 and c-Met were essential for directing these cell migrate to injured liver. The recruited mBM-MSCs may play different roles, including hepatic fate specification and down-regulation of the activity hepatic satellite cells, which inhibits of over-accumulation of collagen and development of fibrosis. Our results provide new insights into liver repair involving endogenous BM-MSCs and add new information for consideration when developing clinical protocols involving the MSCs.
研究的第一部分,我们首先建立了三套条件培养液诱导体系,成功将小鼠骨髓来源MSCs诱导分化为肝样细胞。诱导体系包括肝损伤条件培养液、肝细胞条件培养液和胎肝条件培养液。诱导得到的细胞具备典型的肝细胞形态,表达肝系细胞特异性基因,具备肝细胞的正常功能,如ICG吞噬、白蛋白合成和糖原储存等。随后的研究发现HGF、FGF-4和OSM是条件培养液诱导体系中发挥作用的关键因子,组合上述三种细胞因子即可将小鼠骨髓MSCs诱导成典型的肝样细胞,但诱导效率较低。此外,本研究发现使用丙戊酸钠(VPA)预处理MSCs,可以显著提高MSCs向肝细胞的分化效率。实验发现MSCs在VPA处理过程中伴随着细胞周期阻滞、组蛋白乙酰化程度增加、染色质结构疏松。表观遗传修饰导致的FGF受体和HGF受体基因表达上调可能是肝细胞分化效率提高的关键因素。
研究的第二部分,我们从肝脏损伤小鼠中分离得到了外周血间充质干细胞,发现该细胞与骨髓来源的MSCs具备类似的细胞表面标记,可以向成骨、脂肪和软骨诱导分化,但是增殖能力较弱,易衰老。本研究还通过活体动物分子成像等技术对骨髓腔移植的eGFP~+MSCs进行追踪,证明MSCs在特定状态下可以从骨髓迁移到外周血继而迁移到损伤肝脏中,提示病理状态下分离得到的外周血间充质干细胞可能来源于骨髓。此外,通过免疫荧光染色等检测方法,发现迁移到损伤肝脏的MSCs可以通过多种方式参与组织修复,包括直接分化为多种细胞类型,包括AFP阳性、ALB阳性的肝系细胞、CK19阳性的胆管类细胞、CD146阳性的血管内皮类细胞等,同时MSCs还能通过分泌细胞因子调控星形细胞的活性来改善肝脏胶原沉积和纤维化。
综上所述,本研究证明小鼠骨髓MSCs不仅在体外可以跨越分化为肝实质细胞,而且体内状态能够在肝脏损伤条件下,自发的迁移出骨髓,并通过血液循环向肝脏迁移,通过跨越分化和细胞因子分泌等方式参与肝脏的损伤修复。
Mesenchymal stem cells (MSCs) were initially characterized as plastic adherent, fibroblastoid cells. These cells can differentiate into osteogenic, adipogenic and chondrogenic lineages under appropriate conditions. In recent years, a number of reports have also indicated that these cells possess the capacity to trans-differentiate into epithelial cells and lineages derived from the neuro-ectoderm.
In this study, we mainly focus on the potential of MSCs for liver repair, which includes two parts: in vitro trans-differentiation of mouse bone marrow mesenchymal stem cell (mBM-MSCs) into hepatocytes, and in vivo recruiting and homing of mBM-MSCs towards injured liver. In the 1~(st) part of the project, three original protocols for directing mBM-MSCs trans-differentiate into functional hepatocyte-like cells were established by conditional medium of injured liver tissues, normal hepatocytes and fetal livers separately. Further studies by microarray analysis and antibody blocking experiments indicated that FGF4, HGF and OSM were crucial for these conditional media-induced hepatic differentiations, and mBM-MSC derived hepatocytes can also be acquired by treatment of these cytokines. Furthermore, we demonstrated that the differentiation efficacy could be considerably enhanced by pre-treatment of VPA, and regulation of FGFRs and c-Met gene expression through post translational modification of core histones might be the primary initiating event for these effects. In the 2nd part of the project, we successfully isolated and characterized circulating MSCs in the peripheral blood of liver-injured mice with expansion in culture, and provided direct evidence that mBM-MSCs were mobilized into the circulation and recruited into livers after stimulation of liver-injury. CCR9, CXCR4 and c-Met were essential for directing these cell migrate to injured liver. The recruited mBM-MSCs may play different roles, including hepatic fate specification and down-regulation of the activity hepatic satellite cells, which inhibits of over-accumulation of collagen and development of fibrosis. Our results provide new insights into liver repair involving endogenous BM-MSCs and add new information for consideration when developing clinical protocols involving the MSCs.
引文
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