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人脂肪间充质干细胞对裸鼠急性肝损伤的治疗作用
时间:2011-02-28 浏览次数:1795次 无忧论文网
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外科学
干细胞分化诱导的研究干细胞分化诱导的研究
    背景和目的 脂肪间充质干细胞作为一种优质的成体干细胞,以来源广泛、易于获取、低免疫原性等良好的生物学特性受到研究者的重视。我们的研究旨在探讨人脂肪间充质干细胞在体内外分化为肝样细胞细胞的机制,并且研究其对裸鼠肝损伤潜在的治疗作用。
    研究方法 脂肪颗粒Ⅰ型胶原酶消化、贴壁分选法分离获得AT-MSCs;相差显微镜观察细胞形态;CCK-8检测细胞生长动力学;流式细胞仪鉴定CD分子;透射电镜内部结构扫描;成脂、成骨定向诱导。体外诱导AT-MSCs向类肝样细胞分化:以HGF、OSM、EFG、FGF、地塞米松为基础诱导基,基础诱导基中加或者不加血清,通过形态学观察、PAS染色检测血清对诱导分化的影响;基础诱导基中加入TSA为成熟诱导基,通过检测ALB、AFP的表达,LDL吸收实验检测TSA对类肝样细胞成熟的影响;体内诱导:10%四氯化碳腹腔注射(100 μl/20 g体重)诱导裸鼠急性肝损伤模型,血清检查ALT、AST、DBIL等指标,检测AT-MSCs对裸鼠急性肝损伤的肝功能影响。细胞移植一个月后,处死裸鼠,观察移植细胞在裸鼠肝脏内的分布、向类肝样细胞分化的情况。
    研究结果 分离获得的细胞呈长梭形贴壁生长;流式细胞仪检测低表达CD11b、CD34、CD45、HLA-DR、CD271,高表达CD73、CD90、CD105;细胞器幼稚,具多项分化潜能。体外分化实验:脂肪间充质干细胞在不加血清的细胞诱导基中诱导更易向类肝样细胞转化;同时添加TSA能增促进类肝样细胞的成熟。体内实验:四氯化碳腹腔注射致裸鼠肝功能损伤1天后达到高峰,肝脏病理学改变与转氨酶以及直接胆红素升高平行。尾静脉输注脂肪间充质干细胞能明显改善肝功能,降低转氨酶。处死裸鼠,在裸鼠肝脏内找到阳性表达人ALB的细胞。
    研究结论 分离获得的AT-MSCs具有典型的间充质干细胞表型,在诱导AT-MSCs向类肝细胞转化的过程中,血清起着重要作用,并且TSA能促使类肝样细胞的成熟。体内实验发现,AT-MSCs能在裸鼠肝脏内生存、改善裸鼠肝功能,并且可以分化为成熟的肝样细胞,表达ALB。
     [英文摘要]:     Background and objective Cell-based therapy is a potential alternative to liver transplantation. The goal of the present study was to examine the in vitro and in vivo hepatic differentiation potential of adipose tissue-derived mesenchymal stem cells (AT-MSCs) and to explore its therapeutic use. With a sufficient and easily obtained supply of adipose tissue as the source of adult stem cells, AT-MSCs are regarded as a hopeful tool for cell-based therapy.
    Method Human adipose tissue was collected after liposuction surgery from the cosmetic and plastic surgery center. The raw lipoaspirates (10–20 ml) were then washed with phosphate-buffered saline twice and digested with 0.075% collagenase I ; Proliferation of AT-MSCs was measured using a CCK-8 kit; The cells were analyzed with a FACSscan flow cytometer with a 488 nm wavelength for cell cycle. Transmission electron microscope was used for detection of ultramicrostructure; cell surface makers was analyzed on a FACSscan flow cytometer ; osteogenic and adipogenic differentiatio assay were performed using the Human Mesenchymal Stem Osteogenic and or Adipogenic differentiation Medium Kit. To induce hepatogenic differentiation, AT-MSCs were plated on 5-㎜ culture dishes coated with collagen in the expansion medium. After reaching confluency, the cells were were cultured in basic hepatic differentiation medium After 2 weeks of culture, the medium was replaced with maturing hepatic differentiation medium with addition of a higher concentration of dexamethasone at 10-5 M and/or 1 μM TSA. Furthermore, the differentiation medium was used with or without serum to detect the effect of the serum on the AT-MSCs. RT-PCR, immunofluorescence, PAS, uptake of Low-Density Lipoprotein staining were used for biological detection to hepatogenic differentiated cells. mice were administered a single abdominal injection of olive oil containing 10% carbon tetrachloride at a dose of 100 μL/20 g body weight. simultaneously, cells for transplantation or control solutions were injected in the tail vein. the concentration of ammonia, ALT, AST and DBIL in serum of the sacrificed mice were detected at days 1, 3, 7. To evaluate the engraftment of the AT-MSCs in the mice livers. the mice were sacrificed after one month, and the livers were removed and fixed for further study. Histological analysis of liver tissues was conducted by serial tissue sectioning and stained with hematoxylin and eosin (H&E) or immunohistochemically examined for human specific ALB expression.
    Result The AT-MSCs express specific markers including CD29, CD73, CD90 and CD105, but are negative for the hematopoietic lineage markers such as CD11b, CD34, CD45, HLA-DR, and CD271. In this study, we isolated, identified, and cultured AT-MSCs with hepatic differentiation medium to evaluate their potential to differentiate into hepatic cells. After treatment, the morphologies of differentiated AT-MSCs changed into polygonal epithelial cells in serum-free hepatic differentiation medium but not in a similar medium containing 2% fetal bovine serum. The differentiated cells cultured without serum showed hepatocyte-like cell morphologies and hepatocyte-specific markers including Albumin (ALB) and α-fetoprotein (AFP). The bioactivity assays revealed that these hepatocyte-like cells could uptake low-density lipoprotein (LDL) and store glycogen. Furthermore, trichostatin A (TSA) enhanced ALB production and LDL uptake by the hepatocyte-like cells, similar to the functions of human liver cells. Importantly, transplantation of the human AT-MSCs could relieve the impairment of acute CCl4 injured liver in nude mice, and ALB could be detected in the liver of the injured mice one month post-transplantation.
    Conclusion Adipose tissue is a source of multipotent stem cells that can differentiate into mature, transplantable hepatocyte-like cells in vitro and in vivo, and TSA is essential to promote differentiation of human MSC towards functional hepatocyte-like cells. The relief of liver injury after treatment with adipose tissue-derived mesenchymal stem cells suggests that AT-MSCs might be a novel therapeutic method for liver disorders or injury.
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