血管瘤中血管内皮生长因子受体KDR基因异常甲基化的研究

发布时间：2018-03-17 01:00

本文选题：血管瘤　切入点：血管内皮生长因子受体　出处：《泸州医学院》2011年硕士论文　论文类型：学位论文

【摘要】：目的:本研究拟通过建立甲基化敏感性高分辨率溶解曲线法(MS- HRM)检测血管内皮生长因子VEGF受体KDR基因启动子区域在不同时期血管瘤、血管畸形及正常皮肤组织等中的甲基化状态,初步探讨基因甲基化在血管瘤形成、增生、退化过程中的作用。方法:选取不同时期血管瘤石蜡标本48例、血管畸形石蜡标本15例、正常包皮皮肤组织标本8例,分别提取DNA,经亚硫酸氢盐甲基化修饰、纯化、回收DNA,以QIAGEN公司的全基因组甲基化DNA作为100%甲基化标准品,与100%非甲基化健康孕妇脐带血DNA按比例混合,稀释制成0%、5%、25%、50%、75%、100%系列浓度甲基化DNA标准品,将甲基化标准品经过PCR扩增后进行MS HRM溶解曲线检测获得标准曲线,并进行重复性和灵敏度评价。然后用甲基化敏感性高分辨率溶解曲线法(MS-HRM)定量检测增生期血管瘤24例、消退期血管瘤24例,血管畸形15例,正常包皮皮肤组织8例等标本中血管内皮生长因子受体KDR甲基化水平。结果:成功制成的100%、75%、50%、25%、5%、0%甲基化标准曲线依次从右往左排列,经过三次重复检测,曲线基本重叠一致;经过MS HRM检测,48例血管瘤标本共检出32例不同程度KDR基因启动子区域甲基化(66.67%),其中24例增殖期血管瘤标本中检出21例(21/24,87.50?%)不同程度甲基化,1例甲基化程度为0?%～5%,?11例甲基化为25?%～50%,5例甲基化为75?%,4例甲基化为100%;24例消退期血管瘤标本中检出11例(11/24,45.83?%)不同程度甲基化,其中,1例甲基化程度为75?%,8例甲基化为25?%～50?%,2例甲基化为0%～5%,增殖期血管瘤与消退期血管瘤标本中KDR的甲基化程度比较差异显著,具有统计学意义(χ2=8.889,P0.05);15例血管畸形标本中仅检出甲基化程度为0%～25%2例(2/15,13.?33%),8例正常包皮皮肤组织中检测到1例甲基化0%～5%(1/8,12.50%)。与血管瘤相比,差异均具有统计学意义(P0.05,Fisher’确切概率法);消退期血管瘤与血管畸形中KDR的甲基化程度比较有显著差异,具有统计学意义(p=0.45,Fisher’确切概率法)。结论:①血管瘤中血管内皮生长因子受体KDR基因启动子序列CpG岛存在异常甲基化,血管内皮生长因子受体KDR基因异常甲基化可能与血管瘤增生、退化等有关;②MS HRM技术定量检测血管瘤中KDR甲基化程度的方法操作简单,灵敏度高,成本低,可重复性强,为进一步研究血管瘤组织中基因异常甲基化建立了一种检测方法。
[Abstract]:Objective: to establish a methylation sensitive high resolution dissolution curve (MSHRM) to detect the promoter region of vascular endothelial growth factor (VEGF) receptor KDR gene in different stages of hemangioma. The role of gene methylation in the formation, proliferation and degeneration of hemangioma was studied. Methods: DNA was extracted from 48 cases of hemangioma, 15 cases of vascular malformation and 8 cases of normal prepuce skin tissue. The DNA was modified and purified by hydrogen sulfite methylation. The whole genome methylated DNA of QIAGEN Company was used as the 100% methylation standard, and then mixed with 100% unmethylated healthy pregnant women's umbilical cord blood DNA according to the proportion, diluted to make 0 ~ (5) and 25 ~ (25) ~ (25)% series of methylated DNA standard material. After the methylation standard product was amplified by PCR, the standard curve was determined by MS HRM dissolution curve, and the reproducibility and sensitivity were evaluated. Then 24 cases of proliferative hemangioma were quantitatively detected by methylation sensitivity high resolution dissolving curve method (MS-HRM). Vascular endothelial growth factor receptor (KDR) methylation was detected in 24 cases of involuted hemangioma, 15 cases of vascular malformation and 8 cases of normal prepuce skin tissue. Results: the standard curve of 0% methylation was arranged from right to left. A total of 32 patients with different degrees of methylation of KDR gene promoter region were detected by MS HRM in 48 hemangioma specimens, of which 21 were detected in 21 / 2487.50% of 24 proliferative hemangioma specimens. The degree of methylation in 1 case with different degrees of methylation was 0? What is it? 11 cases methylated to 25? 5 cases were methylated into 75? Of the 24 cases of hemangioma in the receding phase, 11 cases were detected in 11 / 24 cases, and 45.83 cases were found in 4 cases of hypermethylation and 100 cases of cytomeglytic hemangioma. The degree of methylation in one case was 75? Methylation in 8 cases? 50? There were significant differences in the methylation of KDR between proliferative hemangioma and receding hemangioma specimens (蠂 ~ 2 / 8.889 / P 0.05). One case of methylation was detected in 8 cases of normal prepuce skin tissue. There was a significant difference in the methylation level of KDR between hemangioma and vascular malformation in comparison with hemangioma, the difference was statistically significant (P 0.05) and the degree of KDR methylation in vascular malformation was significantly higher than that in hemangioma. It is statistically significant that 0.45% Fisher' exact probability method. Conclusion abnormal methylation of vascular endothelial growth factor receptor (KDR) gene promoter CpG island in vascular endothelial growth factor receptor (KDR) gene may be associated with vascular endothelial growth factor receptor (KDR) gene hypermethylation in vascular endothelial growth factor receptor (VEGFR) hemangioma. The method of quantitative detection of KDR methylation in hemangioma by degenerative 2MS / HRM technique is simple, high sensitivity, low cost and strong reproducibility. A new method is established for further study of abnormal methylation of gene in hemangioma tissue.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.5

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