Clonal Analysis And Protein Expression, Tie2 Gene Mutation Differences Study In Vertebral Vascular Anomalies

Objective:(1) To clear whether vertebral vascular anomalies are monoclonal vascular tumor or polyclonal vascular malformations(VMs),the clonility nature will be confirmed by PCR through analysing the X-chromosome inactivation status and the polymorphisms of AR.(2) To measure the different expression of Glut-1, VEGF, Ki-67,VEGF, bFGF, uPA, MMP2 and Tie2 pathway related proteins in hemangioma and VMs,to explore the immunohistochemical features of hemangioma and VMs, and provide immunohistochemical basis to identify vertebral vascular anomalies.(3) Tie2 gene mutations were closely related to VMs,to detect Tie2 gene mutations and mRNA in hemangioma and VMs, observe their genetic change characteristics, and explore the pathogenic role of Tie2 gene mutations in vertebral vascular anomalies,and to provide a theoretical basis for the correct diagnosis and treatment of vertebral vascular anomalies.Methods:(1) Using laser microdissection techniques to capture vascular anomalies tissue,and extracted DNA of vascular abnormalities tissue, made polymerase chain reaction(PCR), according to X chromosome inactivation chimerism and AR gene polymorphism to make clonal analysis of vertebral vascular anomalies, to clear vertebral artery abnormalities being neoplastic lesions or VMs.(2) The method of immunohistochemistry was used to compare the expression differences of GLUT1, Ki-67, VEGF, bFGF, uPA,MMP2 proteins and Tie2 gene pathways related proteins(Tie2, Ang1, Ang2, Akt, PI3K)in the vascular tumor and malformations, looked for specific immune markers to identify them, and to explore the role of Tie2 gene pathway related proteins in the pathogenesis of vascular abnormalities.(3) We extracted DNA and RNA of vascular anomalies tissue,taked sequencing after PCR reaction to evaluate the Tie2 gene mutations points in the vascular anomalies, and to explore the relationship of vertebral VMs and Tie2 gene mutations.(4) RT-PCR was used to detect Tie2 mRNA in hemangioma and VMs, tocompare their Tie2 mRNA expression level differences, and understand whether there are differences in Tie2 mRNA expression of mutant and wild-type VMs. Results:(1) Clonal analysis showed that AR digested amplified two bands in vertebral and soft tissue VMs,which may be polyclonal non-neoplastic lesions, AR digested amplified a band weaken or disappeared in hemangioma,which may be monoclonal neoplastic lesions.(2) The immunohistochemical results showed that the expression of GLUT1, Ki-67, VEGF, bFGF,uPA, MMP2, PI3 K and Akt protein in vascular tumors were significantly higher than the VMs(P<0.05); the expression of GLUT1, Ki-67 and VEGF protein in vertebral vascular tumors were also significantly higher than vertebral VMs(P<0.05); the difference had statistical significance(P<0.05).(3) In hemangiomas and VMs, the expression of Tie2 and Ang2 were high, the expression of Ang1 was low, the expressing of Tie2, Ang1 and Ang2 were unbalanced.(4) We identified four types of Tie2 mutations in 60 patients with soft tissue VMs by PCR including the previously detected missense mutations 2690A>G(Y897C), 2740C>T(L914F), and two nonsense mutations 2763G>A, 2688C>T, we identified Tie2 mutation 2743C>T(R915C) in primary vertebral VMs for the first time,and we didnot detect Tie2 gene mutation in DNA samples of all vascular abnormalities and hemangiomas.(5) Tie2 mRNA expression level was significantly lower in the VMs than hemangioma, the difference had statistical significance(P < 0.01). Tie2 m RNA expression level was higher in mutant VMs than the wild type VMs(P < 0.05). Tie2 mRNA expression level in vertebral and soft tissue VMs had not significant difference(P>0.05).Conclusion:(1) Most of vertebral vascular abnormalities may be polyclona VMs,were not real tumor.(2) The expressions of GLUT1, Ki-67, VEGF, bFGF, uPA, MMP2,PI3 K, Akt proteins were higher in hemangioma than VMs, which can help to identify vertebral hemangiomas and VMs, but GLUT1 and Ki67 have better specificity,(3) The expression imbalance of Tie2, Ang1 and Ang2 protein may have regulation role in the formation process of vertebral hemangiomas and VMs.(4) VMs had Tie2 gene mutations,but had not been detected in hemangioma, and their Tie2 mRNA expression levels had difference, they may be two kinds of disease with different genetic change.(5) Vertebral and soft tissue VMs all had Tie2 gene mutations, and the expression level of Tie2 mRNA in hemangioma and VMs had not obvious difference, vertebral and soft tissue VMs may be the same disease in different organizations lesions.The role of Tie2 gene in the pathogenesis of vertebral VMs needs to be further studied.