脂肪成体干细胞定向诱导分化为软骨细胞的实验研究

发布时间：2018-03-14 00:35

本文选题：组织工程　切入点：脂肪成体干细胞　出处：《第四军医大学》2007年硕士论文　论文类型：学位论文

【摘要】： 关节软骨的损伤和缺损是骨科常见的问题之一。关节软骨再生及自我修复能力极为有限,而传统的治疗方法如磨削术、钻孔微骨折术等均存在一定的缺陷,远期疗效不甚理想。近年来组织工程技术的发展为解决这一难题带来了新的希望,有望成为治疗软骨损伤的新方法。软骨组织工程就是将种子细胞接种到合适的生物支架材料上,在体外诱导后或者直接植入体内修复软骨缺损。因此,种子细胞及生物支架材料在组织工程软骨的构建中有着举足轻重的地位。目的:从兔脂肪组织中分离出脂肪成体干细胞(ADASCs),体外经原代及传代培养后,在转化生长因子-β1(TGF-β1)存在的条件下,对比观察不同浓度的血清对ADASCs向软骨细胞增殖和分化的影响,探索较为合适的血清浓度;再将稳定传代后的ADASCs种植于纤维蛋白胶/透明质酸(FG/HA)复合支架上,在含TGF-β1的软骨诱导液中诱导培养,体外构建ADASCs、纤维蛋白胶/透明质酸及转化生长因子的组织工程软骨模块,探讨FG/HA作为软骨组织工程支架材料的可行性。方法:1、ADASCs在不同的血清浓度下向软骨细胞分化:取成年新西兰兔颈部脂肪组织,剪碎后通过I型胶原酶消化,得到脂肪成体干细胞;稳定传代后,分别用含1%、10% FBS的软骨诱导液干预ADASCs 2周,采用MTT检测方法对细胞增殖活性进行比较,应用甲苯胺蓝染色及Ⅱ型胶原免疫组化染色进行软骨细胞鉴定,利用Leica病理图像软件分析两组间Ⅱ型胶原免疫组化染色后的灰度,比较两组细胞分化的效果。 2、TGF-β1诱导ADASCs体外构建改良纤维蛋白胶软骨模块:取传3代的ADASCs,消化、离心后,弃上清液,实验组(FG/HA组)加入100μL纤维蛋白胶(FG)催化剂液,将细胞与之混匀,再分别加入200μL透明质酸(HA)及100μL FG主体胶液,充分混匀,待其在室温下成凝胶状后移入24孔培养板中,加入诱导液培养。对照组(FG组)中不加HA,只加入200μL FG催化剂液和200μL主体胶液,其它操作同实验组。倒置显微镜逐日观察两组细胞生长情况以及支架降解情况;第14 d取出标本,2.5%戊二醛固定,扫描电镜下分别观察FG组及FG/HA组细胞与支架材料复合情况;10%中性甲醛溶液固定其余标本,石蜡包埋,切片,行HE染色、甲苯胺蓝染色,免疫组化法检测Ⅱ型胶原的表达。结果:MTT检测显示10%FBS组细胞增殖活性高于1%FBS组(P0.05),两组细胞的甲苯胺蓝染色及Ⅱ型胶原免疫组化染色均为阳性,并以10%FBS组更为显著;灰度分析提示10%FBS组Ⅱ型胶原表达量高于1%FBS组(P0.05)。脂肪成体干细胞在支架内呈多层的三维生长,形态为圆形,增殖活性高。诱导培养后,两组支架材料均有降解,但实验组较对照组的降解速度明显减慢。扫描电镜可见FG/HA复合支架呈三维立体多孔结构,孔隙率较高,细胞与支架材料附着情况良好。石蜡切片HE染色可见类似正常软骨的组织学特征,甲苯胺蓝染色及Ⅱ型胶原免疫组化染色阳性,实验组的阳性染色程度较高。结论:在体外单层培养的条件下,ADSASCs可以定向诱导分化为软骨细胞,含10%FBS的软骨诱导液更有利于脂肪成体干细胞向软骨细胞方向增殖和分化。体外培养系统中,纤维蛋白胶/透明质酸(FG/HA)复合支架生物相容性好,孔隙率较高,且有效解决了FG降解速度过快的问题,是一种较为理想的软骨组织工程载体材料。三维环境下培养更有利于干细胞向软骨细胞方向分化,而且有利于软骨细胞表型的维持。
[Abstract]:The injury and defect of articular cartilage is one of the common problems. Department of orthopedics, articular cartilage regeneration and self repair ability is very limited, and the traditional treatment methods such as drilling and grinding operation, micro fracture have some defects, the long-term curative effect is not ideal. In recent years the development of tissue engineering technology in order to solve this problem brought new hope, is expected to become a new method for the treatment of cartilage injury. Cartilage tissue engineering areinoculated seed cells into suitable biomaterial scaffolds and induced in vitro directly or after implantation to repair cartilage defect in vivo. Therefore, seed cells and scaffold materials plays an important role in the construction of tissue engineered cartilage.
Objective: to isolate adipose derived adult stem cells from rabbit adipose tissue (ADASCs), in vitro and cultured after transforming growth factor beta 1 (TGF- beta 1) in the presence of comparing the effects of different concentrations of serum on the proliferation and differentiation of ADASCs to chondrocytes, explore the serum concentration appropriate; then passaged after ADASCs transplantation on fibrin glue / hyaluronic acid (FG/HA) composite scaffold in cartilage, containing TGF- beta 1 induced cultured fluid, in vitro ADASCs, fibrin glue / hyaluronic acid and transforming growth factor for cartilage tissue engineering module, as of FG/HA the feasibility of scaffold materials for cartilage tissue engineering.
Methods: 1, ADASCs in the serum of different concentrations of chondrogenic differentiation: adult New Zealand rabbits neck fat tissue, cut by type I collagenase digestion, obtained adipose derived adult stem cells; stable after passage, respectively with 1%, 10% FBS of cartilage induced liquid ADASCs 2 week intervention, using MTT detection method to compare the activity of cell proliferation, toluidine blue staining and type II collagen immunohistochemical staining of cartilage cell identification, analysis of two groups of type II collagen immunohistochemical staining using Leica pathological image gray software, compared with two groups of cell differentiation.
2, TGF- beta 1 induced by ADASCs in vitro improved fibrin glue bracket: the 3 generation of ADASCs, digestion, after centrifugation, the supernatant was removed, the experimental group (group FG/HA) with 100 L fibrin glue (FG) catalyst liquid, mixing with the cells, then adding 200 L transparent hyaluronic acid (HA) and 100 L FG body glue, mixing, to the gel at room temperature after moving into the 24 hole culture plate, adding induction medium. The control group (FG group) without HA, adding only 200 L FG and 200 L the main catalyst liquid glue the other solution, the same as the experimental group. The inverted microscope daily observation two groups of cells and scaffold degradation of growth; fourteenth D specimens were fixed in 2.5% glutaraldehyde and were observed in FG group and FG/HA group of cells and scaffold material composite by scanning electron microscope; 10% neutral Formaldehyde Solution other specimens were fixed, paraffin embedded, sliced, HE staining, toluidine blue staining, free The expression of type II collagen was detected by the Phytophthora histochemical method.
Results: MTT showed that 10%FBS group cell proliferation activity is higher than that of 1%FBS group (P0.05), toluidine blue staining and type II collagen immunohistochemical staining of cells in two groups were positive, and the 10%FBS group was more significant; gray analysis showed that in 10%FBS group, the expression of type II collagen was higher than that of group 1%FBS (P0.05) adipose derived adult stem. The growth of three-dimensional cell layers within the scaffold, shape is round, the proliferation activity is high. After induction, two groups of scaffold materials were degraded, but the experimental group than in the control group decreased significantly. The degradation speed of scanning electron microscopy showed that the FG/HA composite scaffold showed a three-dimensional porous structure, high porosity, good adhesion of cells and scaffold material features. HE staining of the paraffin sections showed similar to normal cartilage tissue, toluidine blue staining and type II collagen immunohistochemical staining, the experimental group of the high degree of positive expression.
Conclusion: in vitro monolayer culture conditions, ADSASCs can differentiate into cartilage cells containing 10%FBS cartilage induced liquid is more conducive to the proliferation of adipose derived adult stem cell differentiation and chondrogenic. The in vitro culture system, fibrin glue / hyaluronic acid (FG/HA) composite scaffold has good biocompatibility, high porosity, and effectively solves the problem of excessive degradation rate of FG, which is an ideal carrier material for cartilage tissue engineering. More conducive to the differentiation of stem cells into chondrocytes cultured in 3D environment, but also beneficial to maintain the phenotype of chondrocytes.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

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