【作者】 曲鑫建；

【导师】 孙世铎；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2006， 硕士

【摘要】 捐赠肝脏的缺乏、肝细胞体外培养增殖能力有限、没有合适的肝细胞冻存条件等,都给肝病的细胞治疗带来极大困难。肝干细胞具有自我更新和多潜能性,因此可能成为肝病细胞治疗以及组织工程理想的细胞来源。本文以大鼠肝干细胞系WB-F344为实验材料,采用RCCS系统模拟微重力效应,并以Cytodex-3为微载体对细胞进行培养（以平面培养的细胞为对照）,分析微重力三维培养条件下细胞形态、增殖、细胞内部显微结构及细胞粘附分子表达情况;检测了细胞膜流动性与肝干细胞标记分子甲胎蛋白（AFP）和白蛋白（ALB） RNA及蛋白水平表达变化;将两种培养条件下的细胞进行体外诱导和体内移植实验,分析模拟微重力三维培养条件下WB-F344细胞生长、体外及体内分化潜能等,旨在为肝组织工程及临床应用建立理想的体外细胞模型奠定基础。通过光学显微镜下细胞形态观察、透射电镜观察细胞显微结构变化、细胞计数分析细胞生长状态及流式细胞术测细胞周期等分析了微重力条件下WB-F344细胞的细胞生物学特点。结果表明,微重力三维培养条件下细胞贴壁于微载体上并且相互连接,细胞贴壁良好,折光性强,生长呈现三维立体排列方式;细胞纤维结构呈紧密多层排列、可见丰富的微绒毛和线粒体、胞间有桥粒和紧密连接形成,细胞粘着力加强、表现出良好的三维生长特征;与平面对照组相比,微重力三维条件下WB-F344细胞生长增殖速度快;在培养的6d,发现微重力三维细胞G₀/G₁和S期的比例增加,而G₂/M期细胞比例显著减少（P <0.05）。通过荧光偏振法检测细胞膜流动性变化,结果表明,与平面培养的细胞相比,微重力三维培养的细胞膜荧光偏振度与生物膜粘度下降,说明细胞膜流动性增加。采用半定量RT-PCR技术检测细胞各种粘附分子、流式细胞术检测钙粘蛋白（E-cadherin）的表达情况。结果微重力三维培养和平面培养下,各种粘附分子均可表达,而实验组纤粘连蛋白（Fn）和整合素-β1（integrin-β1） mRNA的表达明显上调,整合素-α5的表达也有所增加;微重力三维培养组E-cadherin的表达比对照组细胞增加了8.15 %,差异极显著（P<0.01）。通过RT-PCR和免疫细胞化学法检测了WB-F344细胞中AFP和ALB的表达。结果微重力三维培养细胞AFP mRNA表达强度明显高于对照组,而ALB mRNA的表达低于对照组;蛋白水平的免疫细胞化学AFP阳性细胞显著高于平面对照组且染色较深,平均阳性率比对照组高20.6%,统计学差异显著（P<0.05）,相反,ALB表达的平均阳性率实验组比对照组低27.9%,差异极显著（P<0.01）。说明微重力三维培养条件下,能较好的维持肝干细胞特性。为了研究细胞体外分化潜能,用RT-PCR检测了OSM诱导7 d后WB-F344细胞中ALB,AFP,desmin,TO,SMA,GGT基因表达情况。结果表明,WB-F344细胞诱导更多还原

【Abstract】 Because of the limited donor liver, lacked effectual approach to proliferiation of hepatocytes in vitro and the deficient cryodepression condition, make the liver diseases therapy still faced many difficulties. Hepatic stem cells have the characteristic of bipotential differentiation that would be make it a good cell resource for liver diseases’cell therapy and tissue engineering. In this paper, to use Cytodex-3 as microcarrier, we established Three-dimensional （3D） culture of rat WB-F344 cells with rotary cell culture system （RCCS） to simulate microgravity environment. 3D culture of cells could closely mimic the in vivo situation with regard to superority than plane monolayer cultured cells. In order to study the rotating 3D culture model affection on cells proliferation and differentiation we examined cells morphology, proliferation, microstructure, mRNA expression of adhesion molecules,E-cadherin protein,AFP and albumin,polarization of membranes and cell transplantation and so on, aim to establish a potential way for clinic liver diseases’cell therapy and constructe a virtual model for tissue engineering .cells morphology,microstructure and proliferation were examined by optical microscope, transmission electron microscope, count the number of cells and flow cytometry method to test the variety of cells character under two culture conditions. The results demonstrated that cells were take on polyhedron with lots of micovilli and mitochondria, which grow well and packed together densely to form irregular aggregates in 3D culture condition, adjacent cells were connected with desmosome and tight junction. cells proliferation was increased compared with control, In the same phase, the percent of WB-F344 cell’s cycle by flow cytometry demonstrated that G₀/G₁ and S phase increased, while G₂/M phase decreased（p<0.05）.The membrane fluidity and the adhesive molecules expression of the WB-F344 cells were determined to research the variation between two conditions environment. The results demonstrated the polarization of membrane fluorescence and viscousness decrease in 3D condition compared with monolayer cultured cells, that’s means the membrane fluidity increased（P﹤0.05）. To detected mRNA expression of adhesive molecules, E-cadherin protein quantity by reverse ranscriptase-polymerase chain reaction （RT-PCR） and更多还原