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目的探讨转化生长因子-β_3(transforming growth factor-β_3,TGF-β_3)对体外培养的兔牙髓干细胞(dental pulp stem cells,DPSCs)增殖和分化的影响。方法采用酶解组织块法将兔DPSCs分离培养获得DPSCs,光镜下行形态学观察;将体外培养的第3代DPSCs分为5组,分别为对照组、20μg/L TGF-β_3组、40μg/L TGF-β_3组、80μg/L TGF-β_3组和100μg/L TGF-β_3组,分别加入0、20、40、80、100μg/L TGF-β_3;采用MTT法检测5组DPSCs A490值,采用免疫细胞化学法检测成骨细胞标记物骨钙素(osteocalcin,OC)、Ⅰ型胶原酶(collagenⅠ,Col-Ⅰ)、骨涎蛋白(bone sialoprotein,BSP)表达情况,采用茜素红染色检测出现矿化结节情况,并进行比较。结果兔DPSCs呈集落状生长,在体外有一定克隆形成能力;对照组及20、40、80、100μg/L TGF-β_3组兔DPSCs A490值分别为0.34±0.55、0.34±0.19、0.33±0.47、0.33±0.30、0.34±0.47,各组间两两比较差异均无统计学意义(P>0.05);80、100μg/L TGF-β_3组诱导后第3天出现Col-Ⅰ阳性表达,第7天开始消失,第5~7天出现BSP阳性表达,第7~14天出现OC阳性表达,第7天出现矿化结节;40μg/L TGF-β_3组第5~7天均有Col-Ⅰ阳性表达,第7天出现BSP阳性表达,第14天出现OC阳性表达,第14天出现矿化结节;20μg/L组第7天出现Col-Ⅰ阳性表达,第14天消失,第14天出现BSP阳性表达,第21天出现OC阳性表达,第21天出现矿化结节;对照组Col-Ⅰ、BSP和OC均为阴性,未出现矿化结节。结论TGF-β_3对体外培养的兔DPSCs增殖无影响,但对兔DPSCs向成骨细胞分化能力有一定促进作用,并在一定范围内呈浓度依赖关系。
Objective To investigate the effects of transforming growth factor-β_3 (TGF-β_3) on the proliferation and differentiation of dental pulp stem cells (DPSCs) cultured in vitro. Methods DPSCs were isolated from DPSCs by enzymatic method and observed under light microscope. The third generation DPSCs cultured in vitro were divided into 5 groups: control group, 20μg / L TGF-β 3 group, 40μg / L, TGF-β 3, 80 μg / L TGF-β 3 and 100 μg / L TGF-β 3, 0, 20, 40, 80 and 100 μg / The expression of osteocalcin (OC), collagenⅠ (Col-Ⅰ) and bone sialoprotein (BSP) were detected by immunocytochemistry. The expression of osteocalcin was detected by alizarin red staining Mineralized nodules, and compared. RESULTS: DPSCs in rabbit showed colony-like growth and had some colony-forming ability in vitro. The D90Cs A490 values in control, 20,40,80,100μg / L TGF-β 3 group were 0.34 ± 0.55,0.34 ± 0.19,0.33 ± 0.47, 0.33 ± 0.30,0.34 ± 0.47, there was no significant difference between every two groups (P> 0.05). The expression of Col-Ⅰ was observed on the 3rd day after the induction in 80,100μg / L TGF-β 3 group. On the 7th day Began to disappear, BSP positive expression appeared on the 5th to 7th day, OC positive expression appeared on the 7th to 14th day, and mineralized nodules appeared on the 7th day. Col-Ⅰ was positive on the 5th to 7th day in 40μg / L TGF-β_3 group The positive expression of BSP appeared on the 7th day, the OC expression appeared on the 14th day and the mineralized nodules appeared on the 14th day. The Col-Ⅰ positive expression appeared on the 7th day in the 20μg / L group, disappeared on the 14th day, and appeared on the 14th day BSP positive expression, positive expression of OC appeared on the 21st day and mineralized nodules on the 21st day. Col-Ⅰ, BSP and OC of the control group were negative, mineralized nodules did not appear. Conclusion TGF-β 3 has no effect on the proliferation of DPSCs cultured in vitro, but it can promote the differentiation of DPSCs into osteoblasts in a concentration-dependent manner.