The Study On The Effect Of RNF213 Gene To Pathogenic Factors Related To Moyamoya Disease

Objective To explore the influences of RNF213 gene on VEGF?b FGF?TGF-1?MMP-9 in rat bone marrow mesenchymal stem cells. To clear the role of RNF213 gene quantitative change in the process of the pathogenesis of moyamoya disease.Methods The levels of VEGF?b FGF?TGF-?1?MMP-9 were detected in the serum with Elisa method and the differences between the two groups were compared and analyzed by SPSS19.0.The isolated r BMSCs were cultured with whole bone marrow adherent method and identified by flow cytometry and osteogenesis,adipogenic differentiation.Third-generation r BMSCs were chosen as the cell subjects and transfected respectively by RNF213-sh RNA and Negative-sh RNA lentivirus.The transfection efficiency of lentivirus was detected by RT-q PCR.The experiment was divided into three groups : A(r BMSCs group transfected by RNF213-sh RNA lentivirus), B(r BMSCs group transfected by Negative-sh RNA lentivirus) and C(normal r BMSCs group).VEGF, b FGF, TGF-?1, MMP-9 m RNA expression in the three groups were detected on seventh day and fifteenth day respectively by RTq PCR.Results 1. Concentration of VEGF?b FGF?TGF-?1?MMP-9 in the serum of patients with MMD were significantly increased. 2. Flow cytometry identification results show that the third-generation r BMSCs cell surface expressed CD90(95.15%) and CD44(99.23%) at a high level, but CD45(0.74%) at low level.The third-generation r BMSCs inducted to osteogenesis after 21 days appeared clearly visible red density calcified nodules with alizarin red staining. The third-generation r BMSCs inducted to adipogenic after 14 days appeared numerous red lipid droplets with oil red O staining. 3. RNF213-sh RNA lentivirus transfection efficiency results showed that the quantitation expression of RNF213 m RNA in the group A and group B are(0.028±0.002) and(0.976±0.021) respectively relative to group C. The difference between group A and group B is statistically significant(P<0.01). 4. 7th day after transfection: The quantitation expression of VEGF and b FGF m RNA in group A and group B were(0.915±0.112),(0.949±0.012) and(0.964±0.023),(0.958±0.035) relative to group C. The difference of VEGF m RNA and b FGF m RNA between group A and group B has no statistical significance(P>0.05). The quantitation expression of TGF-?1 and MMP-9 m RNA in group A and group B were(3.269±0.968),(0.996±0.005) and(3.147±0.320),( 0.946±0.052) relative to group C. The difference of TGF-?1 and MMP-9 m RNA between group A and group B is statistically significant(P<0.01). 5. 15 th day after transfection: The quantitation expression of VEGF m RNA in group A and group B were(0.973±0.019),( 0.966±0.026) relative to group C. The difference of VEGF m RNA and b FGF m RNA between group A and group B has no statistical significance(P>0.05). The quantitation expression of b FGF,TGF-?1 and MMP-9 m RNA in group A and group B were(1.582±0.272),( 0.966±0.039) and(2.370±0.068),( 0.974±0.019) and(2.741±0.374),( 0.976±0.044) relative to group C. The difference of b FGF, TGF-?1 and MMP-9 m RNA between group A and group B is statistically significant(P<0.01).Conclusion 1. Concentration of VEGF, bFGF, TGF-?1, MMP-9 in the serum of patients with MMD increased significantly compared with healthy people,indicating that VEGF, b FGF, TGF-?1, MMP-9 may play an important role in the occurence and development of MMD. 2. SD rat bone marrow mesenchymal stem cells were isolated, cultured and identified successfully with the whole bone marrow adherence method. 3. RNF213-sh RNA can effectively inhibit RNF213 m RNA expression in the r BMSCs and induce r BMSCs to express b FGF, TGF-?1 and MMP-9 m RNA increasingly. 4. RNF213 gene may affect b FGF, TGF-?1 and MMP-9 genes in some gene pathway so as to induce b FGF, TGF-?1 and MMP-9 abnormal expression,indicting that RNF213 gene quantitative change may play an important role in the process of the pathogenesis of moyamoya disease.