| Objective:Bone marrow mesenchymal stem cells(BMSCs)have a certain effect in the treatment of diseases,but the survival rate and viability of BMSCs in transplants are limited,and the improvement of patients' quality of life is not ideal.The apoptosis of transplanted stem cells may be one of the important reasons for the low survival rate of stem cell.The aim of this study is to investigate the effect and possible mechanism of Rspo1 on the apoptosis of BMSCs.BMSCs were extracted and cultured and then transfected with adenovirus encoding Rspo1.It was investigated whether Rspo1 has the effect on BMSCs apoptosis.Then,the relationship between the inhibition of BMSCs apoptosis and Wnt/?-catenin signaling pathway was further explored.Methods:1.BMSCs were extracted and cultured from SD rats by whole bone marrow adherence separation method.2.The differentiation of osteogenesis and adipogenesis was identified to detect the differentiation potential of BMSCs.3.BMSCs surface markers(CD29,CD90 and CD45)were detected using flow cytometry.4.BMSCs were transfected with an adenoviral vector encoding Rspo1,named BMSCs-Rspo1 group.BMSCs were divided into three groups: BMSCs group,BMSCs-GFP group and BMSCs-Rspo1 group.5.The expression of Rspo1 gene and Rspo1 protein was compared among the BMSCs-Rspo1 group and two control groups(untransfected BMSCs group and BMSCs-GFP group)by qPCR and Western blot.6.The apoptosis was detected by flow cytometry and AO-EB double dyeing in three groups.7.The expression of apoptosis-related proteins(Bax,Caspase3 and Cleavage-Caspase3)was detected by Western-blot.8.The activity of the Wnt/?-catenin signaling pathway was evaluated by measuring the key proteins(?-catenin,JNK and phospho-JNK)of this signaling pathway by qPCR and Western blot.9.After the addition of the LGK974,a blocker of the Wnt/?-catenin signaling pathway,the expression of apoptosis-related proteins(Bax,Caspase3 and Cleavage-Caspase3)was detected by Western blot and the key factors in the Wnt/?-catenin signaling pathway(?-catenin,JNK and phospho-JNK)were detected by qPCR and Western blot.Results:1.After the osteogenic and adipogenic differentiation of BMSCs,the reddish-brown round calcium nodules and orange-red round lipid droplets were shown.2.Flow cytometry analysis showed that BMSCs were positivity for CD29(97.1±0.76%)and CD90(95.83±0.76%),and negativity for the hematopoietic stem cell surface marker CD45(3.93±0.6%).3.The significantly increased expression of Rspo1 gene and Rspo1 protein was shown in the BMSCs-Rspo1 group,compared to the BMSCs group and the BMSCs-GFP group(p?0.01)by qPCR and Western blot.4.Flow cytometry analysis showed that the apoptotic rates of BMSCs,BMSCs-GFP and BMSCs-Rspo1 were(43.63±2.16)%,(44.56±2.04)% and(25.02±3.58)% respectively.5.The results of AO-EB double dyeing displayed that the condensed or beaded apoptotic cells which were green and orange-red in the BMSCs-Rspo1 group were significantly less than those in the BMSCs group and the BMSCs-GFP group.6.Western blot analysis demonstrated a significant decrease of Bax,Caspase3 and Cleavage-Caspase3 proteins in the BMSCs-Rspo1 group compared with the BMSCs group(p?0.01)and the BMSCs-GFP group(p?0.01).7.The expression of CTNNB1 and JNK genes and ?-catenin,JNK and phospho-JNK proteins in BMSCs-Rspo1 group were significantly higher than those in BMSCs group and BMSCs-GFP group(p<0.01).8.After adding LGK974,Western blot showed that the expression of CTNNB1 andJNK genes and ?-catenin,JNK and phospho-JNK proteins in BMSCs-Rspo1+LGK974group were significantly lower than those in BMSCs-Rspo1(p<0.01).The expression of Bax,Caspase3 and Cleavage-Caspase3 proteins in the BMSCs-Rspo1+LGK974 group was significantly higher than that in the BMSCs-Rspo1 group(p<0.01),and there was no difference between the BMSCs-Rspo1+LGK974 group and the BMSCs group.Conclusion:1.BMSCs in good condition were cultured by whole bone marrow adherent separation method.2.Rspo1 was successfully overexpressed in adenovirus-transfected BMSCs.3.Overexpression of Rspo1 could inhibit apoptosis of BMSCs.4.The Wnt/?-catenin pathway could play a vital role in the inhibition of Rspo1 on the apoptosis of BMSCs. |
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