本文選題：希佩爾林道基因 + 甲基化�。� 參考：《暨南大學(xué)》2009年博士論文

【摘要】：目的 1.以育齡婦女子宮內(nèi)膜組織標(biāo)本為研究對象,探討希佩爾林道(vonHippel-Lindau, VHL)基因和蛋白在人類子宮內(nèi)膜的表達及其變化,分析VHL基因在人類胚胎著床及胚胎植入過程中的可能作用。 2.檢測育齡婦女子宮內(nèi)膜組織中乙�；D(zhuǎn)移酶P300/CBP(P300 and cAMP responsive element-binding protein binding protein)和VHL基因甲基化的變化,探討表觀遺傳學(xué)改變在子宮內(nèi)膜生理功能中的可能作用。 3.以育齡婦女子宮內(nèi)膜組織標(biāo)本為研究對象,檢測缺氧誘導(dǎo)因子-1α(hypoxia inducible factor 1α, HIF-1α)蛋白和基因的表達及變化,分析人類子宮內(nèi)膜中HIF-1α表達變化的調(diào)控機制。 4.觀察育齡婦女輸卵管組織VHL蛋白和基因的表達及其變化,闡明人類輸卵管中是否存在VHL基因及其表達狀況,分析VHL蛋白在人類輸卵管生理功能中的可能作用。 材料與方法 1.VHL蛋白與VHL mRNA在人子宮內(nèi)膜組織中的表達30例既往月經(jīng)規(guī)律、有正常生育史的育齡婦女子宮內(nèi)膜標(biāo)本,按月經(jīng)周期分為增生早期組7例,增生中晚期組8例,分泌早期組6例,分泌中晚期組9例。應(yīng)用激光共聚焦顯微鏡法(laser cofocal scanning microscopy, LCSM)觀察VHL蛋白在子宮內(nèi)膜組織的表達(n=3)。應(yīng)用免疫組化法檢測人子宮內(nèi)膜腺上皮細胞中VHL蛋白表達的平均灰度值(n=30)。采用逆轉(zhuǎn)錄聚合酶鏈反應(yīng)法(reverse transcription polymerase chain reaction, RT-PCR)和蛋白印跡法(Western blotting)檢測VHL在人子宮內(nèi)膜組織中的蛋白和基因表達及變化,與內(nèi)參照蛋白和內(nèi)參照基因光密度比值表示VHL蛋白和基因的相對量(n=15)。 2.P300/CBP蛋白和VHL基因甲基化在人子宮內(nèi)膜組織中的表達 15例既往月經(jīng)規(guī)律、有正常生育史的育齡婦女子宮內(nèi)膜標(biāo)本,按月經(jīng)周期分為增生期組7例,分泌期組8例。采用Western blotting法檢測P300/CBP在人子宮內(nèi)膜組織中的蛋白表達和變化。應(yīng)用甲基化特異性聚合酶鏈反應(yīng)法檢測VHL基因在子宮內(nèi)膜組織中的甲基化狀態(tài)。 3.VHL、P300/CBP與HIF-1α蛋白在人子宮內(nèi)膜組織中表達的相關(guān)性研究 30例既往月經(jīng)規(guī)律、有正常生育史的育齡婦女子宮內(nèi)膜標(biāo)本,按月經(jīng)周期分為增生早期組7例,增生中晚期組8例,分泌早期組6例,分泌中晚期組9例,應(yīng)用免疫組織化學(xué)法檢測人子宮內(nèi)膜組織中的蛋白表達變化。采用RT-PCR法和Western blotting法檢測人子宮內(nèi)膜組織中的HIF-1α蛋白和基因的表達及其變化,應(yīng)用Pearson相關(guān)分析進行統(tǒng)計分析VHL、P300/CBP蛋白與HIF-1α在子宮內(nèi)膜組織中表達的相關(guān)性(n=15)。 4.VHL蛋白與VHL mRNA在人輸卵管組織中的表達 27例既往月經(jīng)規(guī)律、有正常生育史的育齡婦女輸卵管標(biāo)本,按月經(jīng)周期分為增生早期組15例,增生中晚期組8例,分泌早期組4例,分泌中晚期組9例。采用LCSM法觀察VHL蛋白的表達部位,RT-PCR法和Western blotting法檢測人輸卵管組織中VHLmRNA和蛋白的表達(n=3)。免疫組化法半定量分析VHL蛋白在人輸卵管組織中的平均灰度值,單位以PU值表示(n=27)。 5.統(tǒng)計學(xué)分析 免疫組化結(jié)果以陽性單位PU值表示,RT-PCR和蛋白印跡結(jié)果分別以目的基因與內(nèi)參基因、目的蛋白與內(nèi)參蛋白的光密度比值表示。實驗數(shù)據(jù)以Mean±SD表示,采用統(tǒng)計軟件SPSS11.5進行分析。 結(jié)果 1. VHL mRNA與VHL蛋白在人子宮內(nèi)膜組織中的表達 RT-PCR和Western blotting結(jié)果顯示人子宮內(nèi)膜組織中存在VHL mRNA及VHL蛋白的表達。VHL蛋白表達在子宮內(nèi)膜上皮(包括腺上皮與腔上皮)細胞的胞漿中,呈彌散性分布。內(nèi)膜間質(zhì)細胞也可見陽性表達。隨月經(jīng)周期的變化,在增生早期子宮內(nèi)膜腺上皮細胞中VHL蛋白表達的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),增生期表達高于分泌期,差異有統(tǒng)計學(xué)意義(P0.05)。 人子宮內(nèi)膜組織中VHL mRNA表達也隨月經(jīng)周期發(fā)生變化,在增生期增高(1.26±0.46),分泌期降低(0.69±0.28),差異有統(tǒng)計學(xué)意義(P0.05)。 2.P300/CBP蛋白和VHL基因甲基化在人子宮內(nèi)膜組織中的表達 人子宮內(nèi)膜組織中P300/CBP蛋白表達隨月經(jīng)周期變化,增生期增高(0.72±0.27),分泌期降低(0.24±0.19),差異有統(tǒng)計學(xué)意義(P0.05)。子宮內(nèi)膜組織中VHL基因呈非甲基化狀態(tài),未檢測出VHL基因甲基化變化。 3. VHL、P300/CBP與HIF-1α蛋白在人子宮內(nèi)膜組織中表達的相關(guān)性研究 HIF-1α蛋白表達于子宮內(nèi)膜上皮(包括腺上皮與腔上皮)細胞的胞漿中,內(nèi)膜間質(zhì)中也可見陽性表達。隨月經(jīng)周期的變化,上皮細胞中HIF-1α蛋白表達發(fā)生變化：HIF-1α表達增生中晚期最高(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),增生期表達高于分泌期,差異有統(tǒng)計學(xué)意義(P0.05)。 人子宮內(nèi)膜組織中HIF-1αmRNA表達也隨月經(jīng)周期發(fā)生變化,增生期增高(0.86±0.43),分泌期降低(0.32±0.23),差異有統(tǒng)計學(xué)意義(P＜0.05)。 VHL蛋白與HIF-1α表達呈正相關(guān)(P0.05)。P300/CBP與HIF-1α表達呈正相關(guān)(P0.05)。 4. VHL mRNA與VHL蛋白在人輸卵管組織中的表達 RT-PCR和Western blotting結(jié)果顯示人輸卵管組織中存在VHL mRNA及VHL蛋白表達。VHL蛋白表達于輸卵管上皮細胞和間質(zhì)細胞的胞漿中,間質(zhì)細胞表達弱于上皮細胞。 在月經(jīng)周期的增生早期,輸卵管峽部、壺腹部、傘部VHL蛋白表達的PU值分別是(5.21±4.17)、(4.27±2.26)、(3.58±1.31),增生中晚期分別是(2.99±1.00)、(2.98±0.83)、(4.26±1.03),分泌早期分別是(3.69±1.80)、(3.87±2.78)、(3.33±1.22),分泌中晚期分別是(3.44±1.66)、(3.84±2.52)、(2.88±1.26)。 在月經(jīng)周期的增生早期、增生中晚期、分泌早期和分泌中晚期,VHL蛋白在輸卵管的峽部、壺腹部、傘部的表達差異均無統(tǒng)計學(xué)意義(P0.05)。在月經(jīng)周期同一時期,輸卵管峽部、壺腹部、傘部的表達差異無統(tǒng)計學(xué)意義(P＞0.05)。 結(jié)論 1.人子宮內(nèi)膜組織中存在VHL mRNA及VHL蛋白表達,且具有周期性變化,推測VHL基因可能參與調(diào)節(jié)增生期子宮內(nèi)膜的血管新生平衡。 2.子宮內(nèi)膜的乙�；D(zhuǎn)移酶P300/CBP在月經(jīng)周期中有周期性變化,可能是雌激素誘導(dǎo)正常子宮內(nèi)膜效應(yīng)的基礎(chǔ)。子宮內(nèi)膜VHL基因去甲基化狀態(tài),VHL基因轉(zhuǎn)錄表達,從而參與子宮內(nèi)膜正常生理活動的調(diào)節(jié)。 3.人子宮內(nèi)膜組織中存在HIF-1αmRNA及HIF-1α蛋白表達,且具有周期性變化,HIF-1α可能誘導(dǎo)其下游基因表達以維持子宮內(nèi)膜基本的生理活動。P300/CBP和VHL蛋白調(diào)節(jié)子宮內(nèi)膜中HIF-1α的表達。 4.人輸卵管組織中存在VHL蛋白及VHL mRNA表達。VHL蛋白在輸卵管組織的表達不具有周期性變化,VHL蛋白穩(wěn)定表達可能參與輸卵管上皮細胞之間的相互轉(zhuǎn)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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R321

【引證文獻】

相關(guān)博士學(xué)位論文 前1條

1 徐楗熒;希佩爾林道（VHL）基因在人妊娠輸卵管及子宮內(nèi)膜惡變組織中表達的研究[D];暨南大學(xué);2011年

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本文編號：2067201