碱性成纤维细胞生长因子和转化生长因子-β1对表皮干细胞增殖和分化的作用

Effects of basic fibroblast growth factor and transforming growth factor-β1 on the proliferation and differentiation of epidermal stem cell

目的观察碱性成纤维细胞生长因子(b FGF)和转化生长因子-β1(TGF-β1)对体外培养的表皮干细胞(ESC)增殖和分化的作用。方法从10只1~3 d龄SD大鼠提取ESC,实验选用第2代细胞。按随机数字表法将细胞分为bFGF组、TGF-β1组、bFGF+SU5402组和TGF-β1+SB505124组和对照组,各干预组角质形成细胞无血清培养基中分别加入10 ng/mLbFGF、10 ng/mLTGF-β1、10 ng/mLbFGF+10μM-SU5402(bFGF受体抑制剂)、10 ng/mLTGF-β1+1 uM-SB505124(TGF-β1受体抑制剂),对照组不做处理,于处理后即刻、1、3、7、10 d 5个时间点显微镜下观察各组细胞形态,以MTS法检测各组ESC增殖情况,取各组处理后10 d细胞以Transwell法检测迁移能力,并采用实时荧光定量RT-PCR法及免疫印迹法检测各组基质金属蛋白酶-1(MMP-1)、α-平滑肌肌动蛋白(α-SMA)及Ⅰ、Ⅲ型胶原的相对表达量。结果 bFGF组ESC较对照组增殖能力(t=6.65,P<0.01)及迁移能力(t=7.50,P<0.01)显著增强,MMP-1表达(t=12.90,P<0.01)明显增高,而α-SMA(t=-30.31,P<0.01)、Ⅰ型胶原(t=-10.61,P<0.01)和Ⅲ型胶原的表达量明显偏低(t=-7.91,P<0.01);TGF-β1组ESC与对照组相比增殖能力(t=-3.36,P<0.05)及迁移能力(t=-3.96,P=0.01)显著减弱,MMP-1表达(t=-8.81,P<0.01)明显减少,而α-SMA(t=15.92,P<0.01)、Ⅰ型胶原(t=-16.47,P<0.01)和Ⅲ型胶原(t=22.80,P<0.01)的表达量较对照组明显偏高;bFGF+SU5402组ESC增殖迁移能力及分化程度较对照组均无明显差别(P>0.05),而TGF-β1+SB505124组ESC迁移能力较对照组显著增强(t=9.81,P<0.01)。结论 bFGF可以显著促进ESC增殖和迁移,进而减少Ⅰ、Ⅲ型胶原的形成;而TGF-β1在抑制ESC增殖及迁移,进而促进Ⅰ、Ⅲ型胶原的形成。

Objective To investigate the effects of b FGF and TGF-β1 on the proliferation and differentiation of epidermal stem cells( ESC) cultured in vitro. Methods ESC extracted from 10 neonatal SD rats( 1-3 d old) were cultured in vitro,The 2 nd passage ESC were used in the study. Cells were redivided into groups bFGF,bFGF + M-SU5402,TGF-β1,TGF-β1 + M-SB505124 and control,which were treated with k-SFM medium containing 10 ng / mLbFGF,10 ng / mLbFGF + 10 uM-SU5402,10 ng / mLTGF-β1,the 10 ng /mLTGF-β1 + 1uM-SB505124,the control group not for processing. The morphology and the number of cells were observed by the microscope in immediately,0,1,3,7,10 d after processing. The cell proliferation was detected by MTS method,and Transwell assay was used to measured the migration ability of10 d cells. The mRNA and protein relative expressions of MMP1,α-SMA,Collagen I,and Collagen Ⅲ were tested by RT-qPCR and Western Blotting respectively. Results The proliferation( t = 6. 65,P = 0. 01) and the migration( t = 7. 50,P < 0. 01) ability of bFGF group increased significantly compared to the control group. The differentiation of ESC and the expressions of α-SMA( t =- 30. 31,P < 0. 01),Collagen I( t =- 10. 61,P < 0. 01) and Collagen Ⅲ( t = 22. 80,P < 0. 01) were significantly lower than the control group,the difference being statistically significant( P < 0. 01). On the contrary,the cell proliferation( t =- 3. 36,P < 0. 05) and migration( t =- 3. 96,P = 0. 01) ability of TGF-β1 group was decreased obviously than the control group,meanwhile,the differentiation of ESC and the expression of α-SMA( t = 15. 92,P <0. 01),Collagen I( t =- 16. 47,P < 0. 01) and Collagen Ⅲ( t = 22. 80,P < 0. 01) were much higher than the control group. However,the bFGF + SU5402 group made no obvious difference with the control group( P > 0. 05),while the migration ability of TGF-β1 + SB505124 group increased significantly compared to the control group( t = 9. 81,P < 0. 01). Conclusion b FGF can not only promote the proliferation and migration ability of ESC significantly,but aslo inhibit their differentiation to myofibroblasts.While TGF-β1 contributes to decrease the proliferation and migration ability of the ESC and accelerate their differentiation to the myofibroblasts.