本文選題：血管瘤　切入點：血管內(nèi)皮生長因子受體　出處：《瀘州醫(yī)學院》2011年碩士論文　論文類型：學位論文

【摘要】：目的:本研究擬通過建立甲基化敏感性高分辨率溶解曲線法(MS- HRM)檢測血管內(nèi)皮生長因子VEGF受體KDR基因啟動子區(qū)域在不同時期血管瘤、血管畸形及正常皮膚組織等中的甲基化狀態(tài),初步探討基因甲基化在血管瘤形成、增生、退化過程中的作用。 方法:選取不同時期血管瘤石蠟標本48例、血管畸形石蠟標本15例、正常包皮皮膚組織標本8例,分別提取DNA,經(jīng)亞硫酸氫鹽甲基化修飾、純化、回收DNA,以QIAGEN公司的全基因組甲基化DNA作為100%甲基化標準品,與100%非甲基化健康孕婦臍帶血DNA按比例混合,稀釋制成0%、5%、25%、50%、75%、100%系列濃度甲基化DNA標準品,將甲基化標準品經(jīng)過PCR擴增后進行MS HRM溶解曲線檢測獲得標準曲線,并進行重復性和靈敏度評價。然后用甲基化敏感性高分辨率溶解曲線法(MS-HRM)定量檢測增生期血管瘤24例、消退期血管瘤24例,血管畸形15例,正常包皮皮膚組織8例等標本中血管內(nèi)皮生長因子受體KDR甲基化水平。 結果:成功制成的100%、75%、50%、25%、5%、0%甲基化標準曲線依次從右往左排列,經(jīng)過三次重復檢測,曲線基本重疊一致;經(jīng)過MS HRM檢測,48例血管瘤標本共檢出32例不同程度KDR基因啟動子區(qū)域甲基化(66.67%),其中24例增殖期血管瘤標本中檢出21例(21/24,87.50?%)不同程度甲基化,1例甲基化程度為0?%～5%,?11例甲基化為25?%～50%,5例甲基化為75?%,4例甲基化為100%;24例消退期血管瘤標本中檢出11例(11/24,45.83?%)不同程度甲基化,其中,1例甲基化程度為75?%,8例甲基化為25?%～50?%,2例甲基化為0%～5%,增殖期血管瘤與消退期血管瘤標本中KDR的甲基化程度比較差異顯著,具有統(tǒng)計學意義(χ2=8.889,P0.05);15例血管畸形標本中僅檢出甲基化程度為0%～25%2例(2/15,13.?33%),8例正常包皮皮膚組織中檢測到1例甲基化0%～5%(1/8,12.50%)。與血管瘤相比,差異均具有統(tǒng)計學意義(P0.05,Fisher’確切概率法);消退期血管瘤與血管畸形中KDR的甲基化程度比較有顯著差異,具有統(tǒng)計學意義(p=0.45,Fisher’確切概率法)。 結論:①血管瘤中血管內(nèi)皮生長因子受體KDR基因啟動子序列CpG島存在異常甲基化,血管內(nèi)皮生長因子受體KDR基因異常甲基化可能與血管瘤增生、退化等有關;②MS HRM技術定量檢測血管瘤中KDR甲基化程度的方法操作簡單,靈敏度高,成本低,可重復性強,為進一步研究血管瘤組織中基因異常甲基化建立了一種檢測方法。
[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.
【學位授予單位】：瀘州醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R739.5

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