【作者】 王飞；

【导师】 温泽清；

【作者基本信息】 山东大学 ， 妇产科学， 2008， 博士

【摘要】 第一部分HLA-G在子宫腺肌病中的表达及意义目的:证明免疫耐受相关分子HLA-G在子宫腺肌病患者的在位内膜和异位灶中的表达情况;比较其在子宫腺肌病患者和正常子宫内膜人群中的差异表达,以期说明HLA-G分子在子宫腺肌病患者免疫耐受状态建立中的作用和在本病发生发展中的意义。方法:34例经病理证实为子宫腺肌病标本为观测组,同时收集30例正常子宫内膜人群的手术切除标本作为对照,采用免疫组织化学染色技术和原位杂交技术检测正常子宫内膜和腺肌病组的在位内膜和异位灶中的HLA-G蛋白和mRNA差异表达情况。采用Hscore评分系统对染色结果进行定量分析,应用SPSS13.0软件进行统计学分析。结果:1.HLA-G蛋白在正常增殖期和分泌期内膜中几乎均不表达,不论是腺上皮细胞还是间质细胞。2.在34例分泌期和增殖期腺肌病在位内膜和异位灶中均发现HLA-G蛋白的表达。腺肌病在位内膜和异位灶中的上皮细胞的染色强度及阳性率均高于间质细胞。上皮细胞和间质细胞的染色均主要位于细胞浆和细胞膜,未见胞核有表达。在腺肌病在位内膜中,增殖期和分泌期腺上皮均存在HLA-G蛋白的表达,增殖期与分泌期内膜Hscore相比无显著差异(P＞0.05),未发现周期性变化。增殖期和分泌期内膜的间质细胞间Hscore相比也无显著差异(P＞0.05)。腺肌病的异位内膜腺上皮HLA-G蛋白表达Hscore显著高于在位内膜腺上皮(P＜0.05);而异位内膜间质细胞HLA-G蛋白表达则低于在位内膜间质细胞,差异有显著性(P＜0.05)。3.腺上皮中的HLA-G蛋白表达在子宫腺肌病患者的异位内膜和在位内膜之间存在正相关,r=0.89(P＜0.0001);间质细胞中的HLA-G蛋白表达在子宫腺肌病患者的异位内膜和在位内膜之间也存在正相关,r=0.78(P＜0.0001)。4.HLA-G mRNA在正常内膜、在位内膜与异位内膜组织中的表达阳性率分别为1/30、28/34、31/34。其中,阳性信号主要位于腺上皮细胞胞浆,间质细胞阳性细胞率较低。结论:1.子宫腺肌病患者在位内膜和异位灶中均存在HLA-GmRNA和HLA-G蛋白的表达,而正常子宫内膜中则几乎未发现该分子的存在,说明HLA-G可能通过建立局部的免疫耐受和免疫抑制的微环境而参与子宫腺肌病的发生发展。2.子宫腺肌病患者在位内膜和异位灶中的HLA-G表达存在正相关性,提示在位内膜的生物学特质在子宫腺肌病的发病中起重要作用。第二部分IL-10在子宫腺肌病中的表达及意义目的:证明免疫耐受相关分子IL-10在子宫腺肌病患者的在位内膜和异位灶中的表达情况;比较其在子宫腺肌病患者和正常子宫内膜人群中的差异表达,以期说明IL-10分子在子宫腺肌病患者免疫耐受状态建立中的作用和在本病发生发展中的意义。方法:34例经病理证实为子宫腺肌病标本为观测组,同时收集30例正常子宫内膜人群的手术切除标本作为对照,采用免疫组织化学染色技术和原位杂交技术检测正常子宫内膜和腺肌病组的在位内膜和异位灶中的IL-10蛋白和mRNA差异表达情况。采用Hscore评分系统对染色结果进行定量分析,应用SPSS13.0软件进行统计学分析。结果:1.在30例(分泌期和增殖期)正常子宫内膜中均发现IL-10的表达。不论是增殖期内膜还是分泌期内膜,上皮细胞的染色强度均高于间质细胞。二者的染色均主要位于细胞浆中。分泌期上皮细胞的Hscore显著高于增殖期上皮细胞(P＜0.01),而间质细胞的染色则未发现周期性变化(P=0.62)。2.在34例分泌期和增殖期在位内膜和异位灶中均发现IL-10的表达。与正常内膜相似,腺肌病在位内膜和异位灶中的上皮细胞的染色强度高于间质细胞。上皮细胞和间质细胞的染色均主要位于细胞浆中。腺肌病的在位内膜和异位灶腺上皮Hscore均显著高于正常内膜(均P＜0.01)。然而,在位内膜和腺肌病灶之间相比,腺上皮Hscore差异不显著(P=0.08)。此外,腺肌病灶、在位内膜和正常内膜之间两两比较,间质细胞Hscore无显著差异(均P＞0.05)。腺肌病在位内膜中,IL-10分泌期腺上皮染色Hscore明显高于增殖期(P＜0.01),呈现周期性变化。然而在间质细胞中未发现这种周期性变化(P=0.22)。3.腺上皮中的IL-10蛋白表达在子宫腺肌病患者的异位内膜和在位内膜之间存在正相关,r=0.55(P＜0.001);间质细胞中的IL-10蛋白表达在子宫腺肌病患者的异位内膜和在位内膜之间未发现相关性,r=0.02(P＞0.05)。4.IL-10 mRNA在正常内膜、在位内膜与异位内膜组织中的表达阳性率分别为28/30、33/34、34/34。其中,阳性信号主要位于腺上皮细胞胞浆,间质细胞阳性细胞率较低。结论:1.子宫腺肌病患者在位内膜和异位灶中均存在较高水平的IL-10的表达,而正常子宫内膜中该分子表达水平较低,说明IL-10可能通过建立局部的免疫耐受和免疫抑制的微环境而参与了子宫腺肌病的发生发展。2.子宫腺肌病患者在位内膜和异位灶中的IL-10表达存在正相关性,提示在位内膜的生物学特质子宫腺肌病的发病中起重要作用。第三部分HLA-G和IL-10在卵巢子宫内膜异位囊肿液中的表达及相关性目的:证明免疫耐受相关分子HLA-G和IL-10在卵巢子宫内膜异位囊肿液中的表达情况;以期说明HLA-G和IL-10在子宫内膜异位症患者免疫耐受状态建立中的作用和在本病发生发展中的意义并探讨这两种分子之间的相关性。方法:收集15例子宫内膜异位症患者的卵巢子宫内膜异位囊肿液和腹腔液,收集15例子宫肌瘤患者的腹腔液,采用Western blot技术检测其中的HLA-G和IL-10分子。应用SPSS13.0软件进行统计学分析,采用t检验对两种分子进行相关性分析。结果:1.在15例卵巢子宫内膜异位囊肿患者的腹腔液中,2例HLA-G蛋白阳性,其余13例均未发现该分子;在15例子宫肌瘤患者的腹腔液中均未发现HLA-G蛋白的表达;而在卵巢子宫内膜异位囊肿液中则发现该分子高表达。2.在15例卵巢子宫内膜异位囊肿患者的腹腔液中均发现IL-10蛋白的表达.在15例子宫肌瘤患者的腹腔液中4例发现IL-10蛋白的表达,其余11例均为阴性;而在卵巢子宫内膜异位囊肿液中则发现该分子高表达。3.IL-10和HLA-G在卵巢子宫内膜异位囊肿液中的表达存在相关性,(r=0.78,P＜0.0001)。4.IL-10和HLA-G在子宫腺肌病患者异位内膜上皮细胞中的表达存在相关性(r=0.66,P＜0.001);在位内膜皮细胞中的表达也存在相关性(r=0.60,P＜0.001)。结论:1.子宫内膜异位囊肿液中存在较高水平的HLA-G和IL-10的表达,说明这两种分子可能通过建立局部的免疫耐受和免疫抑制的微环境而参与了子宫内膜异位症的发生发展。2.IL-10和HLA-G在子宫内膜异位症的表达存在相关性。3.IL-10和HLA-G在子宫腺肌病患者异位内膜上皮细胞中的表达存在相关性;在位内膜上皮细胞中的表达也存在相关性。更多还原

【Abstract】 PARTⅠExpression of HLA-G in Patients with AdenomyosisObjective:To demonstrate the expression of HLA-G,an immunotolerance related molecule,in the eutopic and ectopic endometrium of patients with adenomyosis;to compare the different levels of HLA-G in endometrium between normal women and patients with adenomyosis.To illustrate the role of HLA-G in establishing the state of immunotolerance and the possible contribution of HLA-G to understanding the pathogenesis of adenomyosis.Methods:The surgical biopses from 34 patients with adenomyosis were collected as experimental group,30 biopses normal endometrium collected as control groups.Immunohistological staining and hybridization in situ were used to detect the protein and mRNA of HLA-G in normal endometrium,eutopic and ectopic endometrium of adenomyosis.Quantitative analysis of HLA-G expression was performed according to H score system.The software of SPSS13.0 was used to analyze the experimental data.Results:1.In all the normal endometrial samples,neither epithelial nor stromal HLA-G expression was observed throughout the menstrual cycle.2.Epithelial and stromal HLA-G immunoreactivity was observed in all eutopic endometrium and adenomyosis foci of women with adenomyosis in both proliferative and secretory phases.Similar to the normal endometrial samples,the epithelial cells of the eutopic endometrium and adenomyosis foci showed higher staining intensity than the stromal cells.The epithelial and stromal staining was mostly cytoplasmic and not in nuclear. In the eutopic endometrium of women with adenomyosis,both proliferative and secretory epithelial cells showed HLA-G positive and no cyclic variance was found with no statistical difference between the two phases(P>0.05).The Hscore for the proliferative and secretory stomal cells was not found statistically different(P>0.05).The ectopic epithelial cells of adenomyosis have statistically higher Hscore than the eutopic epithelial cells(P<0.05);however,the ectopic stromal cells have lower Hscore than the eutopic stromal cells(P<0.05).3.A good correlationship was found between HLA-G expression in ectopic and eutopic endometrium in patients with adenomyosis,both in glands(r=0.89,P<0.0001)and in stroma(r=0.78,P<0.0001).4. The positive rate for HLA-G mRNA in normal endometrium,eutopic endometrium and ectopic endometrium is 1/30、28/34、31/34,respectively.The positive signal mostly localized in epithelial cells and stromal cells have weak signal.Conclusions:1.HLA-GmRNA and HLA-G protein are found in the eutopic and ectopic endometrium of patients with adenomyosis,whereas not found in normal endometrium.These findings indicate that HLA-G may involve in the pathogenesis of adenomyosis by establishing the local microenviroment of immunotolerance and immunosuppression.2.The good correlationship between HLA-G expression in eutopic and ectopic endometrium in patients with adenomyosis indicates that the characterism of eutopic endometrium may play an important role in the pathogenesis of adenomyosis. PARTⅡExpression of IL-10 in Patients with AdenomyosisObjective:To demonstrate the expression of IL-10,an immunotolerance related molecule,in the eutopic and ectopic endometrium of patients with adenomyosis;to compare the different levels of IL-10 in endometrium between normal women and patients with adenomyosis.To illustrate the role of IL-10 in establishing the state of immunotolerance and the possible contribution of IL-10 to understanding the pathogenesis of adenomyosis.Methods:The surgical biopses from 34 patients with adenomyosis were collected as experimental group,30 biopses normal endometrium collected as control groups,Immunohistological staining and hybridization in situ were used to detect the protein and mRNA of HLA-G in normal endometrium,eutopic and ectopic endometrium of adenomyosis.Quantitative analysis of IL-10 expression was performed according to H score system.The software of SPSS13.0 was used to analyze the experimental data.Results:1.In 30 cases normal endometrium,epithelial and stromal IL-10 expression was observed throughout the menstrual cycle.The epithelial cells of the endometrium showed higher staining intensity than the stromal cells in both proliferative and secretory phase samples.The epithelial and stromal staining was mostly cytoplasmic.The H score values of epithelial IL-10 immunostaining were significantly higher in the secretory phase than in the proliferative phase(P<0.01), whereas no difference was observed in stromal 1L-10 immunoreactivity throughout the menstrual cycle(P=0.62).2.Epithelial and stromal IL-10 immunoreactivity was observed in 34 eutopic endometrium and adenomyosis foci of women with adenomyosis in both proliferative and secretory phases.Similar to the normal endometrial samples,the epithelial cells of the eutopic endometrium and adenomyosis foci showed higher staining intensity than the stromal cells.The epithelial and stromal staining was mostly cytoplasmic.In the eutopic and ectopic endometrium of women with adenomyosis,epithelial cells showed higher staining intensity than those of the normal controls(both P<0.01).However,no significant difference was observed in the epithelial IL-10 mmunostainingH score values between the eutopic endometrium and adenomyosis foci(P=0.08).In addition,no differences were observed in stromal IL-10 expression among adenomyosis foci,their eutopic endometrium,and normal controls(all P>0.05).We observed a cyclic variation in eutopic epithelial IL-10 immunoreactivity throughout the menstrual cycle,with higher H score values in the secretory phase than in the proliferative phase(P<0.01).However,no significant difference was observed in eutopic stromal IL-10 immunoreactivity throughout the menstrual cycle in eutopic endometrium(P=0.22).3.A good correlationship was found between epithelial IL-10 expression in ectopic and eutopic endometrium in patients with adenomyosis(r=0.55,P<0.0001),however,no similar correlationship was found in stromal cells(r=0.02,P>0.05).4.The positive rate for IL-10 mRNA in normal endometrium,eutopic endometrium and ectopic endometrium is 28/30、33/34、34/34,respectively.The positive signal mostly localized in epithelial cells and stromal cells have weak signal.Conclusions:1.The higher level of IL-10 protein is found in the eutopic and ectopic endometrium of patients with adenomyosis,whereas the lower level was found in normal endometrium.These findings indicate that IL-10 may involve in the pathogenesis of adenomyosis by establishing the local microenviroment of immunotolerance and immunosuppression.2.The good correlationship between IL-10 expression in eutopic and ectopic endometrium in patients with adenomyosis indicates that the characterism of eutopic endometrium may play an important role in the pathogenesis of adenomyosis. PARTⅢExpression and Correlation of HLA-G and IL-10 in Ovarian Endometriotic Cyst FluidObjective:To demonstrate the expression of HLA-G and IL-10,the immunotolerance related molecule,in ovarian endometriotic cyst fluid.To illustrate the role of HLA-G and IL-10 in establishing the state of immunotolerance and the possible contribution of HLA-G and IL-10 to understanding the pathogenesis of endometriosis.To illustrate the correlation between the two molecules.Methods:The ovarian endometriotic cyst fluid and peritoneal fluid from 15 patients with endometriosis were collected as experimental group,15 cases of peritoneal fluid from patients with myoma as control group and Western blotting was used to analyze the level of HLA-G and IL-10 in the cyst fluid and peritoneal fluid. The software of SPSS13.0 was used to analyze the experimental data and t test was used to analyze the correlationship between the two molecules.Results:1.The peritoneal HLA-G was found neither in 15 patients with myoma nor in 13 patients with endometriosis and 2 cases HLA-G positive;whereas this molecule was found in high level in the ovarian endometriotic cyst fluid.2.The peritoneal IL-10 was found in patients with endometriosis but not in patients with myoma;whereas this molecule was found in high level in the ovarian endometriotic cyst fluid.3.A good correlationship was found between HLA-G and IL-10 expression in ovarian endometriotic cyst fluid(r=0.78,P<0.0001).4.A good correlationship was found between HLA-G and IL-10 expression not only in eutopic epithelial cells of adenomyosis(r=0.66,P<0.001),but also in ectopic epithelial cells of adenomyosis (r=0.60,P<0.001).Conclusions:1.The high levels of HLA-G and IL-10 in ovarian endometriotic cyst fluid indicate that the two molecules may involve in the pathogenesis of endometriosis by establishing the local microenviroment of immunotolerance and immunosuppression.2.The expression of HLA-G correlated with the expression of IL-10 in ovarian endometriotic cyst fluid.3.The expression of HLA-G correlated with the expression of IL-10 in adenomyosis.更多还原