应用组织工程技术提高脂肪前体细胞存活率的研究

发布时间：2018-01-25 00:57

  本文关键词： 应用 组织 工程技术 提高 脂肪 体细胞 存活率 研究　出处：《浙江大学》2011年硕士论文　论文类型：学位论文

【摘要】：背景：自体脂肪组织作为人体组织的填充材料被广泛的运用。虽然脂肪组织在人体内来源相对丰富,但大量的脂肪组织移植来源仍然受限。所以具有分化、增殖潜能的脂肪干细胞和脂肪前体细胞作为脂肪组织工程的种子细胞引起了关注。脂肪前体细胞是从脂肪组织分离出的脂肪干细胞分化出的早期脂肪细胞,通过诱导具有分化增殖的潜能。外源性血管内皮细胞生长因子(vascular endothelial growth factor, VEGF)可促进移植组织内的血管增生和脂肪细胞分化增殖,但外源性VEGF需要持续作用于组织才能起到较好效果。透明质酸钠(HA)是葡糖醛酸-N-乙酰氨基葡糖为双糖单位组成的直链高分子多糖,广泛存在于组织细胞基质中；具有优良的生物相容性和生物可降解性。HA的大分子网状结构通过与水形成氢键可结合大量的水,可以维持局部VEGF浓度,同时其独特的三维结构使之成为理想的种子细胞的支架材料。 目的：以透明质酸支架为载体,复合小鼠脂肪前体细胞、VEGF植入小鼠体内,通过检测组织血管形成脂肪分化情况,评价该复合载体材料对脂肪前体细胞移植存活率的影响。 方法：将小鼠3T3-L1脂肪前体细胞复苏、传代培养后,以脂肪分化诱导剂诱导8天。然后分为4组注射入小鼠皮下：实验组(诱导后脂肪细胞、PBS缓冲液、透明质酸、VEGF)、HA对照组(诱导后脂肪细胞、PBS缓冲液、透明质酸)、VEGF对照组(诱导后脂肪细胞、PBS缓冲液、VEGF)和空白组(诱导后脂肪细胞、PBS缓冲液)。术后2,4,8周取材,仔细剥离取出移植物,观察色泽、质地。电子天平测量质量。标本分别进行HE组织染色和苏丹Ⅲ染色。体外PCR实验检测。 结果：8周取材见小鼠背部皮下脂肪组织淡黄色,质软,容易剥离。实验组、,HA对照组均取到标本。HE染色切片表现为移植物外周为纤维包膜,空白对照组可见部分坏死脂肪细胞,炎性细胞浸润。脂肪细胞大小不一,存活较少。实验组存活脂肪细胞较多,坏死灶较少,细胞较大排列均一,可见微血管生长。苏丹Ⅲ染色可见对照组红染脂肪较少,排列不规则,大小不一,有局部纤维化。实验组红染然脂肪较多,坏死灶较少,细胞较大排列均一。应用SPSS 16.0软件对2周,4周,8周各组重量进行方差分析,2周、4周VEGF对照组与空白组无统计学差异,其余各组均有统计学差异。8周实验组与其他三组有统计学差异,三组间没有统计学差异。体外PCR实验结果提示各组PPAR-γ、C-EBPα条带密度均相近。说明在体外各组表达无明显差异。 结论：应用组织工程技术,以透明质酸支架复合小鼠3T3-L1脂肪前体细胞、VEGF注射充填可以延长VEGF持续作用时间,促进新生脂肪组织血管形成,提高小鼠脂肪前提细胞诱导后移植的存活率。
[Abstract]:Background: autogenous adipose tissue is widely used as the filling material of human body tissue. Although adipose tissue is relatively abundant in human body, a large number of adipose tissue transplantation sources are still limited. The proliferation potential of adipose stem cells and adipose progenitor cells as seed cells for adipose tissue engineering have attracted much attention. Adipose progenitor cells are early adipocytes derived from adipose stem cells isolated from adipose tissue. Exogenous vascular endothelial growth factor (VEGF) and vascular endothelial growth factor were induced to have the potential of differentiation and proliferation. VEGF can promote vascular proliferation and adipocyte differentiation and proliferation in grafts. Hyaluronic acid sodium hyaluronate (HA) is a straight-chain polymer polysaccharide composed of glucuronic acid -N-acetyl glucosamine as a disaccharide unit. It is widely found in tissue and cell matrix. The macromolecular reticular structure with excellent biocompatibility and biodegradability. Ha can maintain local VEGF concentration by forming hydrogen bonds with water. At the same time, its unique three-dimensional structure makes it an ideal scaffold for seed cells. Objective: using hyaluronic acid scaffold as carrier, combined with mouse adipocyte precursor cells (VEGF) were implanted into mice, and adipose differentiation was detected by tissue angiogenesis. To evaluate the effect of the composite carrier material on the survival rate of fat precursor cell transplantation. Methods: the 3T3-L1 adipose progenitor cells were resuscitated and cultured for 8 days, then divided into 4 groups: experimental group (adipocytes). PBS buffer, hyaluronic acid VEGFU HA control group (induced adipocyte buffer), hyaluronic acid (hyaluronic acid) PBS buffer group (induced adipocyte buffer solution). VEGF) and blank group (induced adipocyte PBS buffer). The grafts were removed and the color was observed 8 weeks after operation. Texture. Measurement of quality by electronic balance. Specimens were stained by HE and Sudan 鈪，

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