大鼠白色和棕色脂肪间充质干细胞的成脂分化趋向

发布时间：2018-03-20 06:32

本文选题：解偶联蛋白1　切入点：成脂诱导　出处：《新乡医学院》2016年硕士论文　论文类型：学位论文

【摘要】：背景哺乳动物体内存在两种脂肪组织:白色脂肪组织(white adipose tissue,WAT)和棕色脂肪组织(brown adipose tissue,BAT),前者是储能器官,后者是耗能器官。两种脂肪组织均含有间充质干细胞,目前尚不知两种脂肪间充质干细胞在向脂肪分化过程中是否存在差异,是本文研究的重点。目的比较大鼠腹股沟区WAT和肩胛间区BAT的结构及组成的差异,探讨白色脂肪间充质干细胞(white adipose-derived mesenchymal stem cells,WADMSCs)和棕色脂肪间充质干细胞(brown adipose-derived mesenchymal stem cells,BADMSCs)的形态学差异及成脂分化特性的差异。方法1.体外分离腹股沟区WAT和肩胛间区BAT,大体形态观察两种脂肪组织之间的差别,石蜡切片,HE染色观察两者的组织学差异,透射电镜观察两者的超微结构差异。2.石蜡包埋切片,利用解偶联蛋白1(uncoupling protein 1,UCP1)免疫组织化学染色比较两种脂肪组织差异。3.酶消化法体外分离培养腹股沟WADMSCs和肩胛间区BADMSCs,比较两种脂肪间充质干细胞的形态学差异,并利用UCP1免疫荧光技术对两种脂肪间充质干细胞进行鉴定。4.对所得的第3代间充质干细胞进行成脂诱导,利用油红O染色检测两种间充质干细胞的成脂诱导率并行统计学分析,利用免疫荧光技术检测成脂诱导、分化后的细胞是棕色脂肪还是白色脂肪细胞。5.DAPI标记两种脂肪间充质干细胞后移植至大鼠腹股沟区,分别在1w、2w、3w取移植区域的脂肪组织,OCT包埋后切片,利用UCP1免疫荧光技术检测植入细胞的分化趋向。结果1.大体观腹股沟区WAT呈亮白色,肩胛间区BAT呈棕褐色。HE染色显示腹股沟区WAT内的细胞多为空泡型且体积较大;肩胛间区BAT内的细胞多为小而分散的脂滴,细胞较小,其间也分布少量单房脂肪细胞。透射电镜显示WAT细胞器较少,BAT含有丰富的线粒体。2.免疫组化显示WAT不表达UCP1,BAT大量表达UCP1。3.BADMSCs胞体明显比WADMSCs透亮,且细胞突起更加细长。两种3代细胞均表达CD73、CD90、CD105等间充质干细胞标记物。4.WADMSCs的成脂诱导率为0.205±0.069,BADMSCs的成脂诱导率为0.165±0.053,两种间充质干细胞的成脂诱导率无统计学意义(P0.05)。成脂诱导后的细胞均表达棕色脂肪的特异性标记物UCP1。5.两种间充质干细胞分别植入大鼠腹股沟区后,在1w、2w、3w发现,两种间充质干细胞在体内微环境的诱导下均表达棕色脂肪特异性蛋白UCP1。结论WAT和BAT组织结构有差异;BADMSCs和WADMSCs在形态学特征和成脂诱导率无明显差异,在体外及体内诱导成脂后均分化为棕色脂肪细胞。
[Abstract]:Background there are two kinds of adipose tissue in mammals: White adipose tissue (white adipose tissue) and brown adipose tissue (BATA). The former is an energy storage organ, the latter is an energy consuming organ, and both adipose tissues contain mesenchymal stem cells. It is not known whether there is a difference between the two kinds of adipose mesenchymal stem cells in adipose differentiation, which is the focus of this study. Objective to compare the structure and composition of WAT and BAT in rat inguinal region and interscapular region. To investigate the morphological differences and adipogenic differentiation of white adipose-derived mesenchymal stem cells (WADMSCs) and brown adipose-derived mesenchymal stem cells (BAD MSCs). Methods 1. Isolation of WAT and BATs from the inguinal and interscapular regions in vitro. Morphological observation of the difference between the two kinds of adipose tissue, The histological difference was observed by HE staining in paraffin sections, and the ultrastructural difference was observed by transmission electron microscope. 2. Paraffin embedded sections, The difference of adipose tissue between the two adipose tissues was compared by immunohistochemical staining with uncoupling protein 1 (UCP1). 3. The morphological differences of adipose mesenchymal stem cells (MSCs) were compared between groin WADMSCs and scapular BAD MSCs by enzyme digestion in vitro. UCP1 immunofluorescence technique was used to identify two kinds of adipose mesenchymal stem cells. 4. The third generation of mesenchymal stem cells were induced by adipogenesis, and the lipid induction rates of two kinds of mesenchymal stem cells were detected by oil red O staining and statistical analysis. Immunofluorescence technique was used to detect whether the differentiated cells were brown fat or white adipocytes. 5. DAPI labeled two kinds of adipose mesenchymal stem cells were transplanted into the groin region of rats. The grafted adipose tissue of the grafted area was grafted with Oct at 1 and 2 wand for 3 weeks, and the differentiation tendency of the implanted cells was detected by UCP1 immunofluorescence technique. Results 1.The WAT in the inguinal area was bright white in general. The BAT of interscapular area was brown. He staining showed that the cells in WAT of inguinal area were mostly vacuolar and large, and the cells in BAT of interscapular area were mostly small and dispersed lipid droplets, and the cells were small. A small number of monocytic adipocytes were also distributed. Transmission electron microscopy showed that WAT organelles were less abundant in mitochondria than bat. Immunohistochemical staining showed that WAT did not express UCP1BAT in a large number of UCP1.3.BADMSCs cells and the body was significantly brighter than that of WADMSCs. The cell processes were more slender. Both of them expressed CD73, CD90, CD105 and other mesenchymal stem cell markers. 4. The adipogenic induction rate of WADMSCs was 0.205 卤0.069 BADMSCs (0.165 卤0.053), while the adipogenic induction rate of the two mesenchymal stem cells was not significant (P0.05). UCP 1.5. two kinds of mesenchymal stem cells were implanted into the groin region of rats. It was found that the two mesenchymal stem cells expressed brown fat specific protein UCP1.Conclusion there is no significant difference between WAT and BAT in the morphological characteristics and fat-forming induction rate of BADMSCs and WADMSCs. In vitro and in vivo, adipose cells were evenly divided into brown adipocytes.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R589.2

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