去分化脂肪细胞与脂肪干细胞成骨及成软骨能力的比较研究

发布时间：2018-02-08 09:51

本文关键词： 骨科组织工程脂肪干细胞去分化脂肪细胞成骨能力成软骨能力　出处：《遵义医学院》2012年硕士论文　论文类型：学位论文

【摘要】：目的：比较兔去分化脂肪细胞(Dedifferentiated adipocytes, DA)和脂肪干细胞(Adipose-derived stem cells, ADSCs)在体外成骨及成软骨的能力,为骨科组织工程中种子细胞的选择提供依据。方法：①取4月龄新西兰大白兔腹股沟脂肪组织,机械分离和酶消化法提取成熟脂肪细胞(Mature adipocytes, MA)和ADSCs。②天花板贴壁培养法诱导MA去分化,获得DA。③将ADSCs和DA以2×104个/ml接种到24孔板中,每日倒置显微镜下观察细胞生长情况,并细胞计数法检测细胞增殖数量,取平均值通过Excel2007软件绘制原代至第三代的生长曲线图,计算两种细胞增殖倍增时间。④选取第三代细胞进行实验分组：ADSCs加入成骨诱导液(β-甘油磷酸钠、VitC、地塞米松和含10%FBS的基础培养基),设为Ao组,DA加入成骨诱导液,设为Do组；ADSCs加入成软骨诱导液(胰岛素、TGF-β1、VitC、转铁蛋白、地塞米松和含10%FBS的基础培养基),设为Ac组,DA加入成软骨诱导液,设为Dc组,各组都设立空白对照组。各组细胞诱导第14天时,Ao组和Do组行成骨细胞茜素红、Ⅰ型胶原免疫组化染色；Ac组和Dc组行软骨细胞甲苯胺蓝、Ⅱ型胶原免疫组化染色。⑤取各组培养第14天及第21的细胞,RT-PCR半定量检测Ao组和Do组中Ⅰ型胶原mRNA表达量,所得数据采用t检验行组间和组内比较；同法检测Ac组和Dc组中Ⅱ型胶原mRNA表达量及统计学分析。比较ADSCs和DA成骨及成软骨的能力。结果：①原代MA呈小圆形指环状、形态规则,聚集分布。天花板贴壁培养后,MA中脂质部分逐渐由细胞胞质内脱出,由小圆形变为椭圆形,最终变为长梭形成纤维细胞状；原代ADSCs呈长梭形或小多角形,大小均匀并散在分布。②传代培养后,两种细胞亦呈长梭形、密集分布,“漩涡样”生长。③原代培养时,细胞计数法检测ADSCs对数生长期终末细胞数为5.9±0.41,DA为6.3±0.36,t检验两者无统计学差异(p0.05)；传代培养后,第一代、第二代和第三代的ADSCs及DA对数生长期终末细胞数差异不大(p0.05)。两种细胞倍增时间在58-61小时之间(p0.05),说明两种细胞增殖能力相似。④Ao组和Do组茜素红和Ⅰ型胶原免疫组化染色阳性,对照组阴性；Ac组和Dc组甲苯胺蓝及Ⅱ型胶原免疫组化染色阳性,对照组阴性。⑤RT-PCR检测第14天和第21天Ao组中Ⅰ型胶原mRNA表达量为0.375±0.018、0.908±0.017,Do组Ⅰ型胶原表达量为0.246±0.014、0.821±0.012,相同时间点Ao组表达量明显高于Do组(p0.05),且Ⅰ型胶原表达量与培养时间成正比(p0.05)；Ac组和Dc组中Ⅱ型胶原mRNA表达量分别为0.316±0.027、0.458±0.020和0.204±0.024、0.377±0.021,Ac组中Ⅱ型胶原表达量也均明显较Dc组高(p0.05),随着培养时间的延长Ⅱ型胶原表达量增加(p《0.05)。两种细胞空白对照组中均无Ⅰ型胶原和Ⅱ型胶原mRNA的表达,与实验组相比具有统计学差异(p0.05)。结论：天花板贴壁培养法可诱导MA去分化,获得DA；体外培养时,DA与ADSCs形态上均呈长梭形成纤维细胞状,没有明显的形态学差异；DA和ADSCs在体外增殖能力相似；两种细胞都可通过体外定向诱导向成骨细胞及软骨细胞分化,且ADSCs成骨和成软骨分化能力均高于DA; ADSCs和DA都可以考虑作为骨科组织工程的种子细胞。
[Abstract]:Objective: To compare the ability of Dedifferentiated adipocytes (DA) and Adipose-derived stem cells (ADSCs) to induce osteogenesis and chondrogenesis in vitro, and to provide basis for selection of seed cells in tissue engineering of Department of orthopedics.
Methods: a total of 4 month old New Zealand rabbits inguinal adipose tissue from mature fat cells mechanical separation and enzymatic digestion method (Mature adipocytes, MA ADSCs.) and the ceiling adherent culture method to induce MA differentiation, DA. 3 and DA ADSCs with 2 * 104 /ml were inoculated into 24 well plate, to observe the growth of the cells under inverted microscope daily, and cell proliferation was detected by cell counting number, the average growth curve by Excel2007 software to draw the original to the third generation, calculation of two proliferating cell doubling time. The third generation cells were selected experimental groups: ADSCs with osteogenic medium (beta glycerophosphate, VitC the foundation, dexamethasone and 10%FBS containing medium), group Ao, DA with the osteogenic induction medium, group Do; ADSCs into chondrogenic media (insulin, TGF- beta 1, VitC, transferrin, dexamethasone and 10%FBS containing basic training Yang Ji), group Ac, DA joined the chondrogenic media, as group Dc, each group a blank control group. The cells were cultured for fourteenth days, Ao group and Do group for osteoblast alizarin red, type I collagen immunohistochemical staining; Ac group and Dc group for chondrocyte toluidine blue. Type II collagen immunohistochemical staining. 5 were taken and cultured for fourteenth days and 21 cells, the expression of RT-PCR semi quantitative detection of type Ao collagen in the mRNA group and Do group, the data obtained using the t test for comparison between and within group; analysis and statistics with type II was detected in Ac group and Dc group expression of collagen mRNA. Comparison of ADSCs and DA into the bone and cartilage.
Results: the primary MA were small round ring, regular shape, aggregation distribution. The ceiling after the adherent, MA lipid fraction gradually from the cytoplasm of prolapse, composed of small round into oval, eventually become spindle shaped fibroblast like; primary ADSCs were fusiform or small angle shape, uniform size and scattered. The passage, two cells were fusiform, densely distributed, the vortex like growth. The primary culture, detection of ADSCs cell count at the end of the logarithmic growth cells was 5.9 + 0.41, DA = 6.3 + 0.36, t test two statistical difference (P0.05); after subculture, the first generation, second generation and third generation of ADSCs and DA in the final at the end of the logarithmic growth cells had no difference (P0.05). Two kinds of cell doubling time in 58-61 hours (P0.05), shows two kinds of cell proliferation are similar. The Ao group and Do group alizarin red and collagen type I immune Positive staining, negative control group; Ac group and Dc group, toluidine blue staining and type II collagen immunohistochemical staining was positive and negative in the control group. The RT-PCR expression of type fourteenth and twenty-first days in group Ao, collagen mRNA was 0.375 + 0.018,0.908 + 0.017, Do group of type I collagen expression was 0.246 + 0.014,0.821 0.012, at the same time in Ao group was significantly higher than that in group Do (P0.05), and the expression of type I collagen and the amount of training time is proportional to the type II (P0.05); Ac group and Dc group in the expression of collagen mRNA were 0.316 + 0.027,0.458 + 0.020 and 0.204 + 0.024,0.377 + 0.021, type II in Ac group. The expression of collagen were significantly higher than group Dc (P0.05), with the extension of time the expression of type II collagen (p<0.05). The amount of training to increase the expression of two kinds of cells in the control group had no collagen type I and type II collagen mRNA, compared with the experimental group with significant difference (P0.05).
Conclusion: the ceiling adherent culture method to induce MA differentiation in vitro, DA; DA and ADSCs morphology were long spindle shaped fibroblast like morphology, no obvious difference; DA and ADSCs are similar in vitro proliferation ability; two kinds of cells can induce osteogenic and chondrogenic differentiation by in vitro, ADSCs and osteogenic and chondrogenic differentiation ability were higher than that of DA; ADSCs and DA can be considered as the seed cells for tissue engineering of the Department of orthopedics.

【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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