论文部分内容阅读
目的:探讨转化生长因子β2(TGF-β2)在低氧条件下诱导骨髓基质干细胞(BMSCs)向软骨细胞分化的作用。方法:无菌条件下分离Wistar大鼠股骨骨髓,采用全贴壁培养法纯化BMSCs。传6代后,将细胞随机分为3组,A组加入25 ng/mL TGF-β2在1%氧浓度条件下培养;B组加入25 ng/mL TGF-β2在21%氧浓度条件下培养;C组仅加入含10%胎牛血清的DMEM-α培养液在1%氧浓度条件下培养。3周后,通过甲苯胺蓝染色检测细胞糖胺多糖,聚合酶链反应检测Ⅱ型胶原和蛋白聚糖(Aggrecan)的表达水平。结果:骨髓细胞经换液后贴壁聚集生长,形态均一,连续传代后形态无明显改变。分组培养第1周,A、C组生长速度低于B组;第2周各组均出现不规则形态细胞,A、C组细胞形态小于B组;第3周各组均可见透明样基质,以A组最明显。3周后行甲苯胺蓝染色,A组细胞内外均可见丰富的蓝染颗粒,B、C组染色较A组略浅。A组Ⅱ型胶原的表达相对量(1.246±0.287)高于B组(0.973±0.365)、C组(0.802±0.196),差异有统计学意义(P<0.05);B、C组比较无明显差异(P>0.05)。A组Aggrecan的表达相对量(0.833±0.375)高于B组(0.724±0.173)、C组(0.602±0.091),差异有统计学意义(P<0.05);B、C组比较无明显差异(P>0.05)。结论:TGF-β2联合低氧环境可明显促进骨髓基质干细胞分化为软骨细胞。
Objective: To investigate the effect of transforming growth factor-β2 (TGF-β2) on the differentiation of bone marrow stromal stem cells (BMSCs) into chondrocytes under hypoxic conditions. Methods: Bone marrow was isolated from Wistar rats under aseptic conditions. BMSCs were purified using whole-mount culture. After passage 6, cells were randomly divided into three groups: group A was cultured with 25 ng / mL TGF-β2 at 1% oxygen concentration; group B was cultured with 25 ng / mL TGF-β2 under 21% oxygen concentration In group C, only DMEM-α medium containing 10% fetal bovine serum was cultured under the condition of 1% oxygen concentration. After 3 weeks, glycosaminoglycan was detected by toluidine blue staining, and the expression of type Ⅱ collagen and aggrecan was detected by polymerase chain reaction. RESULTS: Bone marrow cells adhered to the wall and grew in a fluid-like manner. The morphology of the bone marrow cells was uniform. There was no significant change in morphology after continuous passage. In the first week of grouping culture, the growth rate of group A and group C was lower than that of group B; irregular shaped cells appeared in each group in the second week, while the cell morphology in group A and group C was less than that in group B. In the third week, A group of the most obvious. Toluidine blue staining was performed after 3 weeks. A group of cells were observed rich in blue dye particles inside and outside the group, B, C group staining slightly lighter than the A group. The relative amount of type Ⅱ collagen expression in group A was significantly higher than that in group B (1.246 ± 0.287, 0.973 ± 0.365), and group C (0.802 ± 0.196, P <0.05). There was no significant difference between group B and C Difference (P> 0.05). The relative expression of Aggrecan in group A (0.833 ± 0.375) was higher than that in group B (0.724 ± 0.173) and group C (0.602 ± 0.091) (P <0.05). There was no significant difference between group B and C P> 0.05). Conclusion: TGF-β2 combined with hypoxic environment can significantly promote the differentiation of bone marrow stromal stem cells into chondrocytes.