希佩尔林道（VHL）基因在人子宫内膜和输卵管的表达及甲基化初步研究

发布时间：2018-06-25 19:20

本文选题：希佩尔林道基因 + 甲基化　；参考：《暨南大学》2009年博士论文

【摘要】：目的 1.以育龄妇女子宫内膜组织标本为研究对象,探讨希佩尔林道(vonHippel-Lindau, VHL)基因和蛋白在人类子宫内膜的表达及其变化,分析VHL基因在人类胚胎着床及胚胎植入过程中的可能作用。 2.检测育龄妇女子宫内膜组织中乙酰基转移酶P300/CBP(P300 and cAMP responsive element-binding protein binding protein)和VHL基因甲基化的变化,探讨表观遗传学改变在子宫内膜生理功能中的可能作用。 3.以育龄妇女子宫内膜组织标本为研究对象,检测缺氧诱导因子-1α(hypoxia inducible factor 1α, HIF-1α)蛋白和基因的表达及变化,分析人类子宫内膜中HIF-1α表达变化的调控机制。 4.观察育龄妇女输卵管组织VHL蛋白和基因的表达及其变化,阐明人类输卵管中是否存在VHL基因及其表达状况,分析VHL蛋白在人类输卵管生理功能中的可能作用。材料与方法 1.VHL蛋白与VHL mRNA在人子宫内膜组织中的表达30例既往月经规律、有正常生育史的育龄妇女子宫内膜标本,按月经周期分为增生早期组7例,增生中晚期组8例,分泌早期组6例,分泌中晚期组9例。应用激光共聚焦显微镜法(laser cofocal scanning microscopy, LCSM)观察VHL蛋白在子宫内膜组织的表达(n=3)。应用免疫组化法检测人子宫内膜腺上皮细胞中VHL蛋白表达的平均灰度值(n=30)。采用逆转录聚合酶链反应法(reverse transcription polymerase chain reaction, RT-PCR)和蛋白印迹法(Western blotting)检测VHL在人子宫内膜组织中的蛋白和基因表达及变化,与内参照蛋白和内参照基因光密度比值表示VHL蛋白和基因的相对量(n=15)。 2.P300/CBP蛋白和VHL基因甲基化在人子宫内膜组织中的表达 15例既往月经规律、有正常生育史的育龄妇女子宫内膜标本,按月经周期分为增生期组7例,分泌期组8例。采用Western blotting法检测P300/CBP在人子宫内膜组织中的蛋白表达和变化。应用甲基化特异性聚合酶链反应法检测VHL基因在子宫内膜组织中的甲基化状态。 3.VHL、P300/CBP与HIF-1α蛋白在人子宫内膜组织中表达的相关性研究 30例既往月经规律、有正常生育史的育龄妇女子宫内膜标本,按月经周期分为增生早期组7例,增生中晚期组8例,分泌早期组6例,分泌中晚期组9例,应用免疫组织化学法检测人子宫内膜组织中的蛋白表达变化。采用RT-PCR法和Western blotting法检测人子宫内膜组织中的HIF-1α蛋白和基因的表达及其变化,应用Pearson相关分析进行统计分析VHL、P300/CBP蛋白与HIF-1α在子宫内膜组织中表达的相关性(n=15)。 4.VHL蛋白与VHL mRNA在人输卵管组织中的表达 27例既往月经规律、有正常生育史的育龄妇女输卵管标本,按月经周期分为增生早期组15例,增生中晚期组8例,分泌早期组4例,分泌中晚期组9例。采用LCSM法观察VHL蛋白的表达部位,RT-PCR法和Western blotting法检测人输卵管组织中VHLmRNA和蛋白的表达(n=3)。免疫组化法半定量分析VHL蛋白在人输卵管组织中的平均灰度值,单位以PU值表示(n=27)。 5.统计学分析免疫组化结果以阳性单位PU值表示,RT-PCR和蛋白印迹结果分别以目的基因与内参基因、目的蛋白与内参蛋白的光密度比值表示。实验数据以Mean±SD表示,采用统计软件SPSS11.5进行分析。结果 1. VHL mRNA与VHL蛋白在人子宫内膜组织中的表达 RT-PCR和Western blotting结果显示人子宫内膜组织中存在VHL mRNA及VHL蛋白的表达。VHL蛋白表达在子宫内膜上皮(包括腺上皮与腔上皮)细胞的胞浆中,呈弥散性分布。内膜间质细胞也可见阳性表达。随月经周期的变化,在增生早期子宫内膜腺上皮细胞中VHL蛋白表达的PU值最高(3.99±1.68),增生中晚期(3.68±1.31),分泌早期(2.29±0.73),分泌中晚期最低(2.02±0.59),增生期表达高于分泌期,差异有统计学意义(P0.05)。人子宫内膜组织中VHL mRNA表达也随月经周期发生变化,在增生期增高(1.26±0.46),分泌期降低(0.69±0.28),差异有统计学意义(P0.05)。 2.P300/CBP蛋白和VHL基因甲基化在人子宫内膜组织中的表达人子宫内膜组织中P300/CBP蛋白表达随月经周期变化,增生期增高(0.72±0.27),分泌期降低(0.24±0.19),差异有统计学意义(P0.05)。子宫内膜组织中VHL基因呈非甲基化状态,未检测出VHL基因甲基化变化。 3. VHL、P300/CBP与HIF-1α蛋白在人子宫内膜组织中表达的相关性研究 HIF-1α蛋白表达于子宫内膜上皮(包括腺上皮与腔上皮)细胞的胞浆中,内膜间质中也可见阳性表达。随月经周期的变化,上皮细胞中HIF-1α蛋白表达发生变化：HIF-1α表达增生中晚期最高(3.21±0.86),增生早期(2.81±1.01),分泌早期降低(1.83±0.73),分泌中晚期最低(1.63±0.53),增生期表达高于分泌期,差异有统计学意义(P0.05)。人子宫内膜组织中HIF-1αmRNA表达也随月经周期发生变化,增生期增高(0.86±0.43),分泌期降低(0.32±0.23),差异有统计学意义(P＜0.05)。 VHL蛋白与HIF-1α表达呈正相关(P0.05)。P300/CBP与HIF-1α表达呈正相关(P0.05)。 4. VHL mRNA与VHL蛋白在人输卵管组织中的表达 RT-PCR和Western blotting结果显示人输卵管组织中存在VHL mRNA及VHL蛋白表达。VHL蛋白表达于输卵管上皮细胞和间质细胞的胞浆中,间质细胞表达弱于上皮细胞。在月经周期的增生早期,输卵管峡部、壶腹部、伞部VHL蛋白表达的PU值分别是(5.21±4.17)、(4.27±2.26)、(3.58±1.31),增生中晚期分别是(2.99±1.00)、(2.98±0.83)、(4.26±1.03),分泌早期分别是(3.69±1.80)、(3.87±2.78)、(3.33±1.22),分泌中晚期分别是(3.44±1.66)、(3.84±2.52)、(2.88±1.26)。在月经周期的增生早期、增生中晚期、分泌早期和分泌中晚期,VHL蛋白在输卵管的峡部、壶腹部、伞部的表达差异均无统计学意义(P0.05)。在月经周期同一时期,输卵管峡部、壶腹部、伞部的表达差异无统计学意义(P＞0.05)。结论 1.人子宫内膜组织中存在VHL mRNA及VHL蛋白表达,且具有周期性变化,推测VHL基因可能参与调节增生期子宫内膜的血管新生平衡。 2.子宫内膜的乙酰基转移酶P300/CBP在月经周期中有周期性变化,可能是雌激素诱导正常子宫内膜效应的基础。子宫内膜VHL基因去甲基化状态,VHL基因转录表达,从而参与子宫内膜正常生理活动的调节。 3.人子宫内膜组织中存在HIF-1αmRNA及HIF-1α蛋白表达,且具有周期性变化,HIF-1α可能诱导其下游基因表达以维持子宫内膜基本的生理活动。P300/CBP和VHL蛋白调节子宫内膜中HIF-1α的表达。 4.人输卵管组织中存在VHL蛋白及VHL mRNA表达。VHL蛋白在输卵管组织的表达不具有周期性变化,VHL蛋白稳定表达可能参与输卵管上皮细胞之间的相互转化。
[Abstract]:objective
1. the expression and changes of the gene and protein of Hippel Lindau (vonHippel-Lindau, VHL) in the human endometrium were studied, and the possible role of VHL gene in the implantation and implantation of human embryo was analyzed.
2. the changes in the methylation of acetyltransferase P300/CBP (P300 and cAMP responsive element-binding protein binding protein) and VHL gene in endometrium of women of childbearing age were detected, and the possible role of epigenetic changes in the physiological function of the endometrium was explored.
3. the expression and changes of the protein and gene of hypoxia inducible factor -1 alpha (hypoxia inducible factor 1 alpha, HIF-1 alpha) were detected and the regulation mechanism of the expression of HIF-1 alpha in human endometrium was analyzed.
4. to observe the expression and change of VHL protein and gene in oviduct tissue of women of childbearing age, to clarify whether there is VHL gene and its expression in human fallopian tube, and to analyze the possible role of VHL protein in the physiological function of human fallopian tube.
Materials and methods
The expression of 1.VHL protein and VHL mRNA in the endometrium of human endometrium 30 cases of endometrium specimens of women of childbearing age with normal birth history were divided into 7 cases in early hyperplasia group, 8 cases in middle and late hyperplasia group, 6 in early secretory group and 9 in middle and late secretory group. Laser cofocal scanning m should be used. Icroscopy, LCSM) observation of the expression of VHL protein in endometrium (n=3). The average gray value of VHL protein expression in human endometrium epithelial cells was detected by immunohistochemistry (n=30). Reverse transcription polymerase chain reaction (reverse transcription polymerase chain reaction, RT-PCR) and Western blotting were used. The protein and gene expression and changes of VHL in human endometrium were measured, and the relative amount of VHL protein and gene (n=15) was expressed by the ratio of the light density of the internal reference protein and the internal reference gene.
Expression of 2.P300/CBP protein and VHL gene methylation in human endometrium
The endometrium specimens of 15 women of childbearing age with normal menstruation and normal reproductive history were divided into 7 cases and 8 secretory phase groups according to the menstrual cycle. The expression and changes of P300/CBP in human endometrium were detected by Western blotting. The VHL gene was detected by the methylation specific aggregation enzyme chain reaction method in endometrium. Methylation status in tissue.
Correlation between 3.VHL, P300/CBP and HIF-1 alpha protein expression in human endometrium
30 cases of endometrium specimens of women of childbearing age who had normal menstrual laws and normal reproductive history were divided into 7 cases in early hyperplasia group, 8 cases in middle and late hyperplasia group, 6 in early secretory group and 9 in secretory medium and late group. The changes of protein expression in human endometrium were detected by immunohistochemistry. RT-PCR method and Western blotting method were used to detect the changes of protein expression in human endometrium tissue. The expression and changes of HIF-1 alpha protein and gene in human endometrium were detected and the correlation between VHL, P300/CBP protein and HIF-1 alpha in endometrium was analyzed by Pearson correlation analysis (n=15).
Expression of 4.VHL protein and VHL mRNA in human oviductal tissue
27 cases of oviductal specimens of women of childbearing age with normal menstrual law and normal reproductive history were divided into 15 cases in early hyperplasia group, 8 cases in middle and late hyperplasia group, 4 in early secretory group and 9 in secretory medium and late group. The expression of VHL protein was observed by LCSM method. VHLmRNA and protein in human fallopian tube tissues were detected by RT-PCR method and Western blotting method. Expression (n=3). The mean gray value of VHL protein in human oviductal tissue was semi quantitatively analyzed by immunohistochemistry, and the unit was expressed by PU (n=27).
5. statistical analysis
The results of immunohistochemistry were expressed by the positive unit PU value. The results of RT-PCR and Western blot were expressed as the ratio of light density to the target gene and the internal reference gene, the target protein and the internal reference protein. The experimental data were expressed by Mean + SD, and the statistical software SPSS11.5 was used to analyze the results.
Result
Expression of 1. VHL mRNA and VHL protein in human endometrium
The results of RT-PCR and Western blotting showed that the expression of VHL mRNA and VHL protein expressed in the endometrium of the endometrium was dispersed in the cytoplasm of the epithelial cells of the endometrium (including the glandular epithelium and the cavity epithelium). The endometrial stromal cells also showed positive expression. With the changes of the menstrual cycle, the endometrium was on the early endometrium of the endometrium. The PU value of VHL protein expression in skin cells was the highest (3.99 + 1.68), middle and late hyperplasia (3.68 + 1.31), early secretion (2.29 + 0.73), the lowest (2.02 + 0.59) in the middle and late secretory period, and higher than the secretory phase in the stage of proliferation. The difference was statistically significant (P0.05).
The expression of VHL mRNA in human endometrium also changed with the menstrual cycle, and increased (1.26 + 0.46) during the proliferative period (0.69 + 0.28), and the difference was statistically significant (P0.05).
Expression of 2.P300/CBP protein and VHL gene methylation in human endometrium
The expression of P300/CBP protein in human endometrium changed with menstrual cycle, the proliferative phase increased (0.72 + 0.27) and the secretory phase decreased (0.24 + 0.19). The difference was statistically significant (P0.05). The VHL gene in endometrium was not methylation, and the change of methylation of VHL gene was not detected.
Correlation between the expression of 3. VHL, P300/CBP and HIF-1 alpha protein in human endometrium
The expression of HIF-1 alpha protein was expressed in the cytoplasm of the endometrium (including the glandular epithelium and the cavity epithelium). The expression of HIF-1 alpha protein in the epithelial cells changed with the changes of menstrual cycle. The expression of HIF-1 alpha was highest (3.21 + 0.86), early proliferation (2.81 + 1.01), and early secretion (1.83 + 0.7). 3), the lowest (1.63 + 0.53) in the middle and late stages of secretion, and the expression in proliferative phase was higher than that in secretory phase (P0.05).
The expression of HIF-1 alpha mRNA in human endometrium also changed with the menstrual cycle, the proliferation period increased (0.86 + 0.43) and the secretory phase decreased (0.32 + 0.23), and the difference was statistically significant (P < 0.05).
VHL protein was positively correlated with HIF-1 alpha expression (P0.05).P300/CBP and HIF-1 alpha expression was positively correlated (P0.05).
Expression of 4. VHL mRNA and VHL protein in human oviductal tissue
The results of RT-PCR and Western blotting showed that the expression of VHL mRNA and VHL protein expressed in the oviduct tissue was expressed in the cytoplasm of oviduct epithelial cells and interstitial cells, and the expression of interstitial cells was weaker than that of epithelial cells.
In the early period of menstrual cycle, the PU value of VHL protein expression in the isthmus, ampulla and parachute was (5.21 + 4.17), (4.27 + 2.26) and (3.58 + 1.31), respectively (2.99 + 1), (2.98 + 0.83), and (4.26 + 1.03) respectively. + 2.52), (2.88 + 1.26).
There was no significant difference in the expression of VHL protein in the isthmus, ampulla and parachute part of the oviduct (P0.05) in the early period of hyperplasia, middle and late secretion and middle and late secretory period (P0.05). There was no statistical difference between the oviduct isthmus, ampulla and parachute (P > 0.05) in the same period of the menstrual cycle.
conclusion
The expression of VHL mRNA and VHL protein exists in the endometrium of 1. people and has periodic changes. It is presumed that the VHL gene may be involved in regulating the angiogenesis balance in the endometrium of the hyperplastic period.
2. the endometrium acetyltransferase P300/CBP changes periodically during the menstrual cycle, which may be the basis of estrogen induced normal endometrial effect. The endometrial VHL gene demethylation state, the VHL gene transcriptional expression, and thus participate in the normal physiological activity of endometrium.
The expression of HIF-1 alpha mRNA and HIF-1 alpha protein is present in the endometrium of 3. people and has periodic changes. HIF-1 alpha may induce its downstream gene expression to maintain the basic physiological activity of endometrium.P300/CBP and VHL protein to regulate the expression of HIF-1 alpha in the endometrium.
The expression of VHL protein and VHL mRNA in the 4. human tubal tissue is not periodic in the expression of.VHL protein in oviduct tissue, and the stable expression of VHL protein may be involved in the transformation between oviduct epithelial cells.
【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R321

【引证文献】