兔脂肪干细胞与骨髓间充质干细胞成软骨能力的比较

发布时间：2019-05-23 18:38

【摘要】：目的：比较兔脂肪来源和骨髓来源的两种间充质干细胞的成软骨能力。 方法：选取六月龄新西兰大白兔,取腹部脂肪,分离培养ADSCs并进行传代。取兔双侧股骨,采用全骨髓培养法分离培养BMSCs并进行传代。使用倒置显微镜观察两种间充质干细胞的形态及生长增殖情况。比较两种细胞的原代及2、3代的传代时间。绘制3代ADSCs和BMSCs的生长曲线,测定两种细胞的倍增时间并进行比较。选取两种间充质干细胞的3代细胞进行成软骨诱导,实验分为四组：ADSCs成软骨诱导组(A1),ADSCs一般DMEM培养基培养组(A2),BMSCs成软骨诱导组(B1), BMSCs一般DMEM培养基培养组(B2)。其中A1、B1为实验组,A2、B2为对照组。倒置显微镜下观察各组细胞形态及生长增殖情况。培养14天后,实验组行甲苯胺蓝染色和Ⅱ型胶原免疫组化染色,对照组行Ⅱ型胶原免疫组化染色。Ⅱ型胶原免疫细胞化学染色灰度值评分法比较实验组中A1、B1的成软骨能力。 结果：BMSCs原代细胞呈聚集样生长,而ADSCs原代细胞呈单个、散在生长。自第二代开始,BMSCs的生长方式与ADSCs一致,以单个、散在方式生长。单层培养时,这两种间充质干细胞增殖迅速且稳定,形态上均为成纤维细胞样,呈梭形,没有观察到明显的形态学差异。原代ADSCs传代时间为6天,第2代、第3代的ADSCs传代时间为3天。原代BMSCs传代时间为10天,第2代、第3代的BMSCs传代时间为5天。原代ADSCs传代时间较原代BMSCs快4天,而第2代、第3代的ADSCs传代时间较同代BMSCs快2天。生长曲线示ADSCs增殖速度要快于BMSCs, ADSCs倍增时间为26h,BMSCs为36h。实验组加入成软骨诱导液后,细胞的增殖缓慢,细胞的形态由长梭形逐渐变成了多角形,或者圆形。而对照组细胞的形态无变化,皆为梭形。成软骨诱导14天后,实验组A1和B1甲苯胺蓝染色及Ⅱ型胶原免疫组化染色皆为阳性表达。而对照组Ⅱ型胶原免疫组化染色阴性表达。Ⅱ型胶原免疫细胞化学染色灰度值评分示,B1组表达Ⅱ型胶原水平高于A1组(p0.05)。 结论：单层培养时,ADSCs与BMSCs形态上均为成纤维细胞样,呈梭形,两者之间没有明显的形态学差异。ADSCs与BMSCs增殖皆迅速且稳定,但是ADSCs的生长增殖速度更快。单层培养时,特定条件下,ADSCs与BMSCs均能向软骨细胞转化。两者成软骨分化时,BMSCs比ADSCs有更高的潜能。
[Abstract]:Objective: to compare the cartilage formation ability of rabbit adipose stem cells and bone marrow derived mesenchymal stem cells. Methods: six months old New Zealand white rabbits were selected, abdominal fat was taken, ADSCs was isolated and cultured and subcultured. The bilateral femurs of rabbits were isolated and cultured by whole bone marrow culture and subcultured. The morphology, growth and proliferation of two kinds of mesenchymal stem cells were observed by inverted microscope. The primary passage time and the passage time of 2 and 3 generations of the two kinds of cells were compared. The growth curves of ADSCs and BMSCs were drawn, and the doubling time of the two cells was measured and compared. Three generations of cells from two kinds of mesenchymal stem cells were selected for cartilage induction. The experiment was divided into four groups: ADSCs cartilage induction group (A1), ADSCs general DMEM medium culture group (A2), BMSCs cartilage induction group B1). BMSCs general DMEM medium culture group (B2). Among them, A1, B1 were the experimental group, A2 and B2 were the control groups. The morphology, growth and proliferation of cells in each group were observed under inverted microscope. After 14 days of culture, the experimental group was stained with toluene blue and type 鈪，

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