【作者】 郭文菁；

【导师】 孔北华；

【作者基本信息】 山东大学 ， 妇产科学， 2008， 博士

【摘要】 第一部分骨桥蛋白在母胎界面的表达及与uNK细胞选择性聚集关系的研究目的:研究骨桥蛋白（Osteopontin,OPN）在人正常妊娠早期和不明原因的反复自然流产（unexplained recurrent spontaneous abortion,URSA）患者母胎界面组织中的表达及蜕膜组织中CD56⁺NK细胞的聚集情况;分析蜕膜组织中OPN的表达与CD56⁺NK细胞募集之间的相关性;并观察人妊娠早期蜕膜基质细胞中OPN的表达及孕酮对其表达的调节作用;探讨OPN表达在URSA发病中可能的作用。方法:（1）选择2005年1月-2006年10月来齐鲁医院计划生育科就诊的妊娠4-10周的正常妊娠人工流产妇女及不明原因的反复自然流产患者各45例,分别按妊娠周数分为3组(组1:4-5^6/7w;组2:6-7^6/7w;组3:8-10w;n=15/每组),无菌条件下分离其绒毛及蜕膜组织。（2）RT-PCR、免疫组织化学法检测不同孕周正常妊娠早期和不明原因反复自然流产患者蜕膜及绒毛组织中OPN的表达情况。（3）免疫组织化学法检测不同孕周正常妊娠早期和不明原因反复自然流产患者蜕膜组织中CD56⁺NK细胞的动态变化情况。（4）Spearman等级相关法分析蜕膜组织中OPN表达与CD56⁺NK细胞募集之间的相关性。（5）选择正常妊娠4-10周行人工流产术的妇女5例,无菌收集蜕膜组织,采用酶消化法对新鲜蜕膜基质细胞进行分离及培养,倒置显微镜观察细胞形态,免疫组化检测波形蛋白的表达。（6）传代后的细胞分别接种在放有盖玻片的6孔培养板和培养瓶中培养,对传代后的蜕膜基质细胞进行分组,用5×10^-7mol/L的黄体酮作用48、72、96小时,采用RT-PCR和免疫组织化学法检测黄体酮对蜕膜基质细胞中OPN表达的影响。结果:（1）RT-PCR结果显示正常妊娠早期和不明原因反复自然流产患者的蜕膜及绒毛组织中都能检测到OPN的表达;但是OPN在URSA患者蜕膜及绒毛组织中的表达水平显著低于正常妊娠组（P＜0.05）;在正常妊娠早期随着孕周的增加,OPN在蜕膜及绒毛组织中的表达呈升高趋势（P＜0.05）。（2）免疫组化染色结果显示在蜕膜及绒毛组织中,OPN主要表达于细胞胞浆内胞核周围,阳性细胞呈局灶或弥漫性分布;在子宫蜕膜组织中的上皮细胞、蜕膜基质细胞都有表达,尤以蜕膜基质细胞OPN蛋白表达最强;在绒毛组织的细胞滋养层细胞中呈强表达、合体滋养细胞层弱表达或不表达;但是在URSA患者蜕膜及绒毛组织中OPN蛋白的表达均低于正常妊娠组（P＜0.05）。（3）蜕膜组织中CD56⁺NK细胞的免疫组化染色结果显示:CD56⁺NK细胞局灶性或弥漫性的分布在蜕膜基质细胞中间;与相同孕周的正常妊娠组相比,CD56⁺NK细胞在URSA组患者蜕膜组织中的表达明显减弱（P＜0.05）;正常早孕期间,随着妊娠周数的增加,CD56⁺NK细胞的数量呈增加趋势（P＜0.05）。（4）Spearman等级相关分析结果显示:蜕膜组织中OPN的表达与CD56⁺NK细胞的聚集呈正相关（r_s=0.87,P＜0.05）。（5）消化后的蜕膜基质细胞在接种过夜后大部分已贴壁;贴壁生长的细胞为纤维母细胞状,呈较细长的梭形或不规则的星形,核呈卵圆形,一般培养5-7天后细胞融合成单层。波形蛋白免疫组化结果显示体外培养传代后的细胞95%以上为蜕膜基质细胞。（6）5×10^-7mol/L的黄体酮对蜕膜基质细胞中OPN表达的刺激作用存在作用时间依赖性,药物作用时间越长,OPN表达越强;对照组基质细胞中OPN的表达不随作用时间延长而增强。结论:（1）OPN与正常妊娠维持中的胚胎植入、胎盘形成有关,人妊娠早期母胎界面OPN的表达异常与URSA的发生有关。（2）蜕膜组织中OPN的表达对组织中CD56⁺NK细胞的募集起调控作用,与正常妊娠的维持密切相关。（3）黄体酮调控蜕膜基质细胞中OPN的表达,提示孕激素参与妊娠早期母胎界面OPN表达的调控及uNK细胞的聚集。第二部分B细胞刺激因子在母胎界面的表达及其生物学意义研究目的:研究B细胞刺激因子（B cell-activating factor of TNF family,BAFF）在正常早孕人工流产妇女和不明原因的反复自然流产（unexplained recurrentspontaneous abortion,URSA）患者母胎界面组织中的表达,探讨BAFF在URSA发病中的生物学意义。方法:（1）选择2005年1月-2006年10月来齐鲁医院计划生育科就诊的妊娠4-10周的正常早孕人工流产妇女及不明原因的反复自然流产患者各45例,分别按妊娠周数分为3组(组1:4-5^6/7w;组2:6-7^6/7w;组3:8-10w;n=15/每组),无菌条件下分离其绒毛及蜕膜组织。（2）分别提取不同孕周正常妊娠早期和不明原因的反复自然流产患者蜕膜及绒毛组织中的总RNA和蛋白,采用RT-PCR和Western blotting技术分别从mRNA水平和蛋白水平检测绒毛组织及蜕膜组织中BAFF及BAFF受体（BAFF-R）的表达。（3）将获取的新鲜绒毛和蜕膜组织用10%福尔马林固定,石蜡包埋,免疫组织化学染色的方法检测BAFF蛋白在早孕人工流产妇女及不明原因的反复自然流产患者绒毛及蜕膜组织中的表达分布。结果:（1）RT-PCR和Western blotting结果显示正常早孕人工流产妇女及不明原因的反复自然流产患者的蜕膜及绒毛组织中都有BAFF的表达;但是在相同孕周的情况下,BAFF在URSA患者蜕膜及绒毛组织中的表达水平均弱于正常妊娠组（P＜0.05）。（2）在正常早孕人工流产妇女及不明原因的反复自然流产患者的标本中皆未检测到BAFF-R的表达。（3）免疫组化染色结果显示BAFF蛋白在全部标本绒毛组织中的细胞滋养层细胞和合体滋养细胞层均表达;但在URSA患者绒毛组织中的表达弱于正常妊娠组。BAFF蛋白在正常早孕人工流产妇女蜕膜组织的基质细胞、血管内皮细胞中表达,在URSA患者蜕膜组织中基质细胞中亦有表达,但表达水平明显低于正常早孕人工流产妇女,在血管内皮细胞中则未见其表达。结论:BAFF作为一种非凋亡诱导的、胎盘起源的TNF超家族细胞因子,在胚泡植入和早期妊娠的维持中起重要作用,其表达水平的降低与URSA的发生有关。更多还原

【Abstract】 PARTⅠTHE EXPRESSION OF OSTEOPONTIN AT THE MATERNAL-FETAL INTERFACE AND IT’S EFFECT ON THE RECRUITMENT OF uNK CELLOBJECTIVE:To study the expression of osteopontin（OPN）in trophoblasts and deciduas of normal early pregnant women and unexplained recurrent spontaneous abortion（URSA）patients and the relationship between the expression of OPN and the the number of CD56⁺NK cell in deciduas.To demonstrate the effect of OPN on migration of CD56⁺NK cell to the uterine.To explore the effect of progesterone on the expression of OPN in early pregnancy decidual stromal cells in vitro.METHODS:（1）45 clinically elective normal pregnancy termination and 45 unexplained recurrent spontaneous abortion patients（gestational age,4-10w）were included at the Department of Obstetrics and Gynecology,Qi Lu Hospital of Shandong University from Jan 2005 to Oct 2006.The 45 patients were allotted to three groups according to gestational weeks(group1:4-5^6/7w;group2:6-7^6/7w,group3: 8-10w;n=15 each group),respectively.The first-trimester human trophoblasts and deciduas were obtained from the above patients.（2）Expression of OPN in the trophoblasts and deciduas of clinically elective normal pregnancy termination and unexplained recurrent spontaneous abortion patients was studied by reverse transcriptase-polymerase chain reaction（RT-PCR）and immunohistochemistry.（3）The number of CD56⁺NK cell was detected in URSA deciduas and artificial abortion deciduas by immunohistochemistry.（4）Correlations between the expression of OPN and the number of CD56⁺NK cell in decidual tissues were analyzed by Spearman’s correlation coefficient by rank test.（5）Early pregnancy decidual stromal cell was isolated by enzymolysis method and cultured in vitro.Decidual stromal cell was investigated under the inverted microscope and identified by immunohistochemical analysis.（6）Passaged decidual stromal cells in 6-well plates and plastic flasks were cultured in serum-free media supplemented with 5×10^-7mol/L of progesterone dissolved in ethanol for 48,72 and 96h.Ethanol was also used as a vehicle control. Expression of OPN in cells was studied by RT-PCR and immunohistochemistry.RESULTS:（1）OPN were clearly detected in total RNA acquired from first-trimester trophoblasts and deciduas of normal early pregnant women and unexplained recurrent spontaneous abortion patients by RT-PCR.There was significantly higher expression of OPN in the trophoblasts and deciduas of normal early pregnant women compared to that of unexplained recurrent spontaneous abortion patients under the same gestational weeks（p＜0.05）.The expression of OPN was increaded as gestation progressed（p＜0.05）.（2）OPN protein were expressed in the cytoplasm of epithelia and stroma cell of human deciduas.In trophoblasts,OPN protein was localized to the cytotrophoblast,and almost no staining in the syncytiotrophoblast cells.The expression of OPN was decreased in the trophoblasts and deciduas of unexplained recurrent spontaneous abortion patients compared to that of normal early pregnant women（p＜0.05）.This was consistent with the results of RT-PCR.（3）CD56 was expressed in the cytoplasm of NK cells.The location of CD56⁺NK cells in the decidual stroma was focally and diffusely.There was dramatic increase of CD56⁺ NK cell accompanied with first-trimester gestation progression （P＜0.05）.The number of CD56⁺ NK cell in the deciduas of normal early pregnant women was more than that of unexplained recurrent spontaneous abortion patients （P＜0.05）.（4）Expression of OPN in deciduas has positive-correlation with number of CD56⁺ cells（r_s=0.87,P＜0.05）.（5）The isolated decidual stromal cells attached after overnight and were positive for vimentin staining.（6）We tested the effect of 5×10^-7mol/L progesterone on the expression of OPN in decidual stromal cells for 48, 72,96h and found that the stimulating effect was hormone time-dependent.The expressions of OPN in the control cells didn’t changed with the culture time prolonged.CONCLUSIONS:（1）OPN probably is orchestrated to ensure establishment and maintenance of a successful pregnancy.The lower expression of OPN may be involved in the pathogenesis of URSA.（2）OPN expressed at the maternal-fetus interface and may relate with the recruitment of the CD56⁺ NK cell to the uterus.（3） Progesterone may affect CD56⁺ NK cell recruitment through influencing the expression of OPN. PARTⅡTHE EXPRESSION AND FUNCTION OF BAFF AT THE MATERNAL-FETAL INTERFACEOBJECTIVE:To study the expression and the effect of B cell-activating factor of TNF family（BAFF）in trophoblasts and deciduas of normal early pregnant women and unexplained recurrent spontaneous abortion（URSA）patients.METHODS:（1）45 clinically elective normal pregnancy termination and 45 unexplained recurrent spontaneous abortionpatients（gestational age,4-10w）were included at the Department of Obstetrics and Gynecology,Qi Lu Hospital of Shandong University from Jan 2005 to Oct 2006.The 45 patients were allotted to three groups according to gestational weeks(group1:4-5^6/7w;group2:6-7^6/7w;group3: 8-10w;n=15 each group),respectively.The trophoblasts and deciduas were gotten in the asepsis operations.（2）The expression of BAFF and BAFF-R in trophoblasts and deciduas of normal early pregnant women and URSA patients was detected by reverse transcriptase-polymerase chain reaction（RT-PCR）and Western blotting.（3）The expression and distribution of BAFF protein at the maternal-fetal interface of normal early pregnant women and URSA patients was observed with technique of immunohistochemical staining.RESULTS:（1）The expression of BAFF were clearly detected at RNA and protein level in first-trimester trophoblasts and deciduas of normal early pregnant women and unexplained spontaneous abortion patients by RT-PCR and Western blotting.There was significantly higher expression of BAFF in the trophoblasts and deciduas of normal early pregnant women compared to that of unexplained spontaneous abortion patients under the same gestational weeks（p＜0.05）.（2）The expression of BAFF-R was not observed in the trophoblasts and deciduas of all the samples.（3）BAFF protein was localized to the cytotrophoblast and syncytiotrophoblast cells.In the same cells of unexplained spontaneous abortion patients,immunostaining was weaker than that of normal early pregnant women. BAFF in deciduas of normal early pregnant women was localized to stromal cells and blood vessel endothelium cells.As in first--trimester trophoblasts,BAFF declined as immunohistochemistry was performed in decidual cells of unexplained spontaneous abortion patients.We failed to find the localization of BAFF protein in decidual blood vessel endothelium cells of unexplained spontaneous abortion patients.CONCLUSIONS:BAFF,a member of the placenta-derived, nonapoptosis-inducing TNF superfamily members,is an important factor in the implantation of blastocyst and maintenance of pregnancy.The lower expression of BAFF may be involved in the pathogenesis of URSA.更多还原