【作者】 周梅；

【导师】 白靖平；

【作者基本信息】 新疆医科大学 ， 外科学， 2015， 博士

【摘要】 目的:(1)基于X染色体失活的嵌合性和X连锁雄激素受体基因的多态性,运用巢式PCR技术进行克隆性分析,明确椎体血管异常的病变性质为单克隆性的血管肿瘤亦或多克隆性的血管畸形。(2)检测血管瘤、血管畸形GLUT1、Ki-67、VEGF、bFGF、uPA、MMP2及Tie2基因通路相关蛋白表达差别,探讨椎体血管瘤和血管畸形的免疫组织化学特征,为难以鉴别的椎体血管异常提供免疫组织化学依据。(3)Tie2基因突变与血管畸形关系密切,本研究运用RT-PCR技术,对血管瘤和血管畸形进行Tie2基因突变及mRNA检测,比较两者的基因变化特点,探讨Tie2基因突变在椎体血管畸形发病中的作用及机制。通过上述研究为椎体血管异常的正确诊断和治疗方法的选择提供理论依据。方法:(1)运用激光显微切割技术捕获血管异常组织,提取血管异常组织的DNA,进行聚合酶链反应(PCR),根据女性X染色体失活的嵌合性及与X染色体连锁的雄激素受体(AR)基因位点的多态性,对血管异常进行克隆性分析,明确椎体血管异常为单克隆性的血管肿瘤亦或多克隆性的血管畸形。(2)用免疫组化的方法,比较GLUT1、Ki-67、VEGF、bFGF、uPA、MMP2及Tie2基因通路相关蛋白(Tie2、Ang1、Ang2、Akt、PI3K)在血管瘤和血管畸形中表达的差异,寻找用于鉴别椎体血管瘤和血管畸形的特异性免疫标记物,探索Tie2基因通路相关蛋白在椎体血管异常发病中的作用。(3)提取血管瘤和血管畸形的DNA和RNA,设计引物,进行PCR反应后测序,检测Tie2基因突变点,探索椎体血管畸形与Tie2基因突变的关系。(4)运用RT-PCR方法检测血管瘤及血管畸形Tie2基因mRNA,比较血管瘤及血管畸形Tie2基因mRNA表达水平的差异,并进一步了解Tie2基因突变是否影响Tie2 mRNA的表达水平。结果:(1)经克隆性分析结果显示,椎体及软组织血管畸形AR基因酶切后扩增出两条带,可能为多克隆性的非肿瘤性病变;增殖期血管瘤AR基因经酶切后两条带中一条带明显减弱或消失,可能为单克隆性的肿瘤性病变。(2)用免疫组化的方法进行检测,结果显示GLUT1、Ki-67、VEGF、bFGF、uPA、MMP2、PI3K和Akt在增殖期血管瘤中的表达均显著高于血管畸形,差异有统计学意义(P均<0.05);GLUT1、Ki-67和VEGF在椎体血管瘤中的表达均高于椎体血管畸形,差异也具有统计学意义(P<0.05)。(3)Tie2和Ang2在血管瘤和血管畸形中的表达率较高,而Ang1在血管瘤和血管畸形中的表达率较低,Tie2、Ang1和Ang2的表达失衡。(4)在60例软组织血管畸形中检测到了2种Tie2基因有意义突变,2690A>G(Y897C)、2740C>T(L914F),2种无意义突变,2763G>A、2688C>T;并且我们首次在椎体血管畸形中检测到了Tie2基因突变2743C>T(R915C);在所有血管异常组织的DNA样本及血管瘤中未检测到Tie2基因突变。(5)Tie2 mRNA在血管畸形中的表达水平显著低于血管瘤,差异具有统计学意义(P<0.01);在血管畸形中突变型Tie2mRNA的表达水平显著高于野生型(P<0.05);Tie2 mRNA在椎体血管畸形和软组织血管畸形中的表达水平差异无统计学意义(P>0.05)。结论:(1)绝大多数椎体血管异常为多克隆性的血管畸形,而非真性肿瘤。(2)GLUT1、Ki-67、VEGF、bFGF、uPA、MMP2、PI3K、Akt蛋白在增殖期血管瘤中表达较高,在血管畸形中表达较低,这些蛋白可能有助于椎体血管瘤和血管畸形的鉴别,其中GLUT1和Ki67特异性更好。(3)Tie2、Ang1和Ang2表达失衡可能在椎体血管瘤及血管畸形的形成过程中具有一定的调节作用。(4)血管畸形具有Tie2基因突变,而在血管瘤中未检测到,且两者的的Tie2 mRNA表达水平具有差异,提示血管瘤和血管畸形为基因学改变不同的两种病变。(5)椎体及软组织血管畸形均具有Tie2基因突变,且两者的Tie2 mRNA表达水平无明显差异,提示两者可能为不同部位的同种病变,Tie2基因在椎体血管畸形发病中的作用有待于进一步研究。更多还原

【Abstract】 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.更多还原