骨形态发生蛋白9对人牙周膜干细胞增殖分化及信号通路的影响

Effect of bone morphogenetic proteins-9 on proliferation, differentiation and signaling pathway of human periodontal ligament stem cells

目的研究骨形态发生蛋白9(BMP9)对人牙周膜干细胞增殖分化及信号通路的影响。方法体外培养人牙周膜干细胞(hPDLSCs),将细胞分为空白组、绿色荧光蛋白(GFP)组、BMP9组、BMP9+5μmol·L-1SP组及BMP9+10μmol·L-1SP组;GFP组用携带绿色荧光蛋白的腺病毒(Ad-GFP)感染细胞,BMP9组、BMP9+5μmol·L-1SP组及BMP9+10μmol·L-1SP组则用携带BMP9的腺病毒(Ad-BMP9)感染;BMP9+5μmol·L-1SP组及BMP9+10μmol·L-1SP组在BMP9组的基础上分别加入5,10μmol·L-1抑制剂SP600125。用逆转录定量聚合酶链反应(RT-PCR)法检测Runx2、OPN和OCN基因的表达,用碱性磷酸酶(ALP)染色试剂盒法检测ALP活性,用蛋白免疫印迹法检测JNK信号通路相关蛋白表达情况。结果GFP组、BMP9组、BMP9+5μmol·L-1SP组、BMP9+10μmol·L-1SPALP活性水平分别为3.87±0.16,8.11±0.62,8.09±0.61,8.07±0.59;Runx2基因表达水平分别为1.07±0.01,2.13±0.02,1.87±0.02,1.85±0.02;OPN基因表达水平分别为1.06±0.01,4.72±0.24,3.86±0.22,3.69±0.25;OCN基因表达水平分别为1.01±0.01,4.26±0.27,3.84±0.23,3.66±0.17;BMP9组ALP活性显著高于其余3组,BMP9组Runx2、OPN、OCN基因表达水平显著高于其余3组。Westernblot检测显示,GFP组、BMP9组、BMP9+5μmol·L-1SP组、BMP9+10μmol·L-1SP组p-JNK蛋白相对表达量分别为0.24±0.03,0.32±0.04,0.21±0.03,0.19±0.02;JNK蛋白相对表达量分别为0.26±0.03,0.51±0.04,0.33±0.03,0.26±0.03;与BMP9组比较,随着SP600125浓度增加,JNK蛋白条颜色逐渐变浅。结论BMP9能够促进hPDLSCS细胞增殖、分化,对Runx2、OPN、OCN基因表达有促进作用,对JNK信号通路有促进作用。

Objective To investigate the effects of bone morphogenetic proteins-9(BMP9) on proliferation, differentiation and signaling pathways of human periodontal ligament stem cells. Methods Human periodontal ligament stem cells(hPDLSCs) were cultured in vitro. Adenovirus was used as a vector to mediate GFP and BMP9, and then hPDLSCs were infected. The expression of hPDLSCS was detected by immunofluorescence staining, the expression level of osteogenic differentiation-related genes was detected by RT-PCR, and the activity of alkaline phosphatase was detected by APL activity. After infection of hPDLSCs by Ad-BMP9, Western blot was used to detect the effect of BMP9 on JNK signaling pathway and the effect of BMP9-JNK signaling pathway on osteogenic differentiation of h PDLSCs after SP600125 intervention. Results The ALP activity levels of GFP group,BMP9 group,BMP9 + 5 μmol·L-1 SP group and BMP9 + 10 μmol·L-1 SP were 3. 87 ± 0. 16,8. 11 ± 0. 62,8. 09 ± 0. 61 and 8. 07 ± 0. 59,the expression levels of Runx2 gene were 1. 07 ± 0. 01,2. 13 ± 0. 02,1. 87 ± 0. 02,1. 85 ± 0. 02,the expression levels of OPN gene were1. 06 ± 0. 01,4. 72 ± 0. 24,3. 86 ± 0. 22,3. 69 ± 0. 25,the expression levels of OCN gene were 1. 01 ± 0. 01,4. 26 ± 0. 27,3. 84 ± 0. 23,3. 66 ± 0. 17;ALP activity in BMP9 group was higher than those in other 3 groups. The expression levels of Runx2,OPN and OCN in BMP9 group were higher than those in the other 3 groups. Western blot analysis showed that the relative expression levels of p-JNK protein in GFP group,BMP9 group,BMP9 + 5μmol·L-1 SP group and BMP9 + 10 μmol · L-1 SP group were 0. 24 ± 0. 03,0. 32 ± 0. 04,0. 21 ± 0. 03,0. 19 ± 0. 02 and the relative expression levels of JNK protein were 0. 26 ± 0. 03,0. 51 ± 0. 04,0. 33 ± 0. 03,0. 26 ± 0. 03. Compared with BMP9 + 5 μmol · L-1 SP group and BMP9 + 10 μmol · L-1 SP group,the expression levels of Runx2,OPN and OCN in BMP9 + DMSO group were significantly decreased in a dose-dependent manner.Conclusion BMP9 can promote the proliferation and differentiation of h PDLSCS cells,promote the expression of Runx2,OPN and OCN genes,and promote the JNK signaling pathway.