【作者】 陈珣；

【导师】 张绍芬； 胡卫国； 朱瑾； 王志军；

【作者基本信息】 复旦大学 ， 妇产科学， 2008， 博士

【摘要】 子宫内膜异位症是妇科常见疾病,并有逐年增加趋势,促性腺激素释放激素激动剂（Gonadotropin releasing hormone agonist,GnRHa）是目前子宫内膜异位症的主要治疗方案,但对反加治疗的系统研究不多。而免疫因素在子宫内膜异位症的发生发展中所起的作用一直倍受关注,但GnRHa联合反加治疗对T淋巴功能的影响研究较少。第一部分GnRHa联合反向添加疗法治疗子宫内膜异位症的临床疗效观察目的研究比较GnRHa联合经皮雌激素及口服安宫黄体酮反加治疗与单用GnRHa治疗子宫内膜异位症的临床疗效。方法选择2007年1月～7月在复旦大学附属妇产科医院就诊的子宫內膜异位症患者28例,均经腹腔镜手术确诊并手术治疗两个月内,随机分为单用GnRHa组（A组）和反加组（B组）。A组患者于月经周期的第2天或于术后3～5天予诺雷德（长效促性腺激素释放激素激动剂,戈舍瑞林）3.6mg皮下注射,隔28天注射1次,共3次;B组患者于月经周期的第2天或于术后3～5天予诺雷德3.6mg皮下注射,隔28天注射1次,共3次,于注射第1支时同时每周1/2片松奇贴剂（剂量为1.5mg/贴,活性成份释放为50μg雌二醇/24h）贴于腹部皮肤,并每晚口服安宫黄体酮6mg至治疗结束。于治疗前对所有入组患者行VAS评分及SF-36量表评分,并测量患者腰椎骨密度,同时抽取外周静脉血,采用全自动微粒子化学发光免疫分析系统测量FSH、E₂水平,采用酶联免疫吸附法（enzymelinked immunosorbent assay,ELISA）检测血清BGP水平;治疗3月后再次检测上述各项指标,并行Kupperman评分;月经来潮后再次行VAS评分。结果1、治疗3月末,两组患者疼痛评分总分、盆腔痛等各项评分均较治疗前显著下降,性交痛均消失,且差别均有统计学意义（P均＜0.01）,B组疼痛总分和盆腔痛评分高于A组,但差别均无统计学意义;月经来潮后两组患者疼痛总分、盆腔痛评分较治疗3月末时略有上升或不变,但均无统计学差异,痛经评分较治疗前均显著下降（P均＜0.01）,而B组疼痛总分、盆腔痛及痛经各项评分高于A组,但差异均无统计学意义。2、通过SF-36生活质量调查表比较,治疗后B组患者较A组患者有更好的生活质量,尤其表现在活力和躯体功能方面,A组患者在治疗末的分值较治疗前明显下降（P分别为＜0.05和＜0.01）;而在疼痛控制上,两组患者均有较好的满意度。3、治疗3月后,A、B两组的kupperman评分差别无统计学意义;但量表中各项比较,其中B组潮热出汗发生率显著低于A组（P＜0.05）。4、A组L1-L4骨密度治疗3月末较治疗前显著降低（P＜0.01）,而B组虽较治疗前也有降低趋势,但差别无统计学意义（P=0.201）;治疗末A组血清BPG较治疗前显著升高（P＜0.01）,而B组虽较治疗前也有升高趋势,但差别无统计学意义。5、治疗3月末,两组患者血清FSH及E₂水平均较治疗前显著降低（P＜0.01）,而B组E₂显著高于A组（P＜0.01）;而两组患者治疗末的FSH均处于卵泡期水平,但B组显著低于A组（P＜0.05）。结论1、反加组患者治疗3月末的疼痛评分总分、盆腔痛等各项评分均较治疗前显著下降,性交痛均消失,月经来潮后,痛经评分亦较治疗前显著下降,提示本研究的反加方案在改善术后子宫内膜异位症患者疼痛上,疗效与单用GnRHa相似。2、接受本研究反加方案的子宫内膜异位症患者较单用GnRHa组患者有更好的生活质量,尤其表现在机体疼痛、活力和躯体功能方面。3、本研究的反加方案对单用GnRHa造成的低雌激素症状不能全部缓解,但对潮热、出汗改善明显,推测与治疗后的雌激素水平有关。4、反加组L1-L4骨密度虽较治疗前也有降低趋势,但骨量丢失少于单用GnRHa组,血清BGP水平变化与腰椎骨密度变化呈负相关,提示本研究的反加方案对防止骨密度降低,减少骨量丢失有显著作用。5、本研究的反加方案能减少骨量的丢失,对骨密度起到一定的保护作用,随访中亦未刺激异位内膜的生长,提示本研究的反加方案疗效肯定、安全。第二部分GnRHa联合反加疗法治疗子宫内膜异位症对患者T淋巴细胞功能的影响目的研究GnRHa联合反加治疗对术后子宫内膜异位症患者T淋巴细胞功能的影响以及对患者外周血单个核细胞上ER亚型及GnRH受体表达的影响。方法本研究选取的子宫內膜异位症患者入组条件、分组情况及具体用药治疗等同第一部分,另选择同期就诊我院要求行输卵管吻合术患者10例为对照组（C组）。所有患者均在术前抽取静脉血,测定外周血T淋巴细胞亚群和T淋巴细胞分泌的细胞因子IL-2及IL-6,并在术中抽取腹腔液,检测IL-2及IL-6水平,以了解Th1/Th2之间平衡的关系,并测定T淋巴细胞的增殖,了解T细胞总的功能,同时检测子宫内膜异位症两治疗组患者外周血单个核细胞上ER-α、ER-β和GnRHRmRNA及蛋白质的表达。子宫内膜异位症患者分组治疗3月后,除腹腔液细胞因子外,再次检测上述各项指标。结果1、治疗前A、B两组患者外周血CD4⁺细胞、CD4/CD8比值均显著低于C组（P均＜0.05）;治疗3月后,A、B两组CD4⁺细胞均较治疗前显著升高,差别有统计学意义（P均＜0.05）,A、B两组CD4/CD8比值均较治疗前升高,但A组治疗前后差别无统计学意义（P=0.055）,而B组治疗前后差别有统计学意义（P＜0.05）。2、治疗前A、B两组T淋巴细胞的增殖均低于正常对照组（P＜0.05）;术后用药治疗3个月后,两组T淋巴细胞的增殖均高于治疗前（P＜0.01）。3、A、B两组患者术前PBMC培养上清液及腹腔液IL-6水平显著高于C组,IL-2/IL-6比值显著低于C组（P均＜0.05）;用药治疗3个月后,A、B两组PBMC培养上清液IL-6分泌较术前明显下降（P均＜0.01）,IL-2/IL-6比值较术前明显升高（P均＜0.05）。4、治疗后,A、B两组PBMC上ER-βmRNA及蛋白表达量均较用药前显著降低（P＜0.05）,而ER-α和GnRHRmRNA及蛋白表达量治疗前后无显著差异。结论1、治疗3月后,反加组患者外周血CD4⁺细胞及CD4/CD8比值均较治疗前升高,T淋巴细胞的增殖显著高于治疗前,与单用GnRHa组结果相似,提示每天经皮雌激素25μg及口服安宫黄体酮6mg的反加治疗与单用GnRHa在改善子宫内膜异位症患者细胞免疫异常上具有相似的效果。2、IL-6在反加治疗后分泌较术前均明显下降,IL-2/IL-6比值较术前显著升高,与单用GnRHa组相似,两治疗组均部分逆转了Th1/Th2平衡。3、反加组与单用GnRHa组在治疗后外周血单个核细胞上ER-β的表达均较用药前显著降低,推测GnRHa对子宫内膜异位症患者外周血T淋巴细胞的影响可能是通过低雌激素作用产生的,而非GnRHa的直接作用,提示这一低雌激素作用可能是通过ER-β发挥的,但及具体机制还不清楚,有待进一步研究。4、反加治疗与单用GnRHa对子宫内膜异位症患者免疫功能均有改善作用。更多还原

【Abstract】 Endometriosis is a common gynecologic disease,with a trend of increased incidence.Although gonadotropin releasing hormone agonist（GnRHa） is the major treatment of endometriosis,the study of add-back therapy is still not systemized.The effect of immune factors on the development of endometriosis has been focused on,but the influence of GnRHa combined with add-back therapy on endometriosis is still little to know.PartⅠThe effect of GnRH agonist combined with add-back therapy in the treatment of endometriosisObjectiveTo compare the effect of GnRH agonist combined with transdermal estrogen and medroxyprogesterone acetate as add-back therapy and GnRH agonist alone in the treatment of endometriosis.MethodsFrom January 1,2007 to July 31,2007,28 patients with endometriosis treated in our hospital and diagnosed by laparoscope were randomly divided into two groups, which were marked as GnRHa group（Group A） and GnRHa plus add-back therapy group（Group B）.Patients in group A received goserelin（Zoladx,AstraZeneca） every 28 days for 3 times.Patients in group B received goserelin every 28 days for 3 times plus transdermal E₂（50μg Fem7,Merk） one half of piece every week and daily oral medroxyprogesterone acetate 6mg.Before the treatment,the degree of pain was measured according to a visual analog scale（VAS）,the quality of life and health-related satisfaction were assessed with the Medical Outcomes Survey Short Form 36（SF-36） and bone mineral density（BMD） of the lumbar spine was measured by dual x-ray absorptiometry.At the same time venous blood was collected by venipuncture to detect the level of FSH,E2 and BGP in serum by fully auto microparticle chemiluminescence immune assay（ACCESS） and enzyme linked immunosorbent assay（ELISA） respectively.Besides the form of kupperman,the previous subjects were detected once after three months treatment.After the menstruation came back,the degree of pain was measured again.Results1、After three months treatment,the value of the total score of the degree of pain and pelvic pain was significantly decreased and the symptoms of dyspareunia disappeared in both groups（P＜0.01）.While the total scores of the degree of pain and the value of pelvic pain were higher in group B than those in group A.However, the difference was not significant.After the menstruation came back,the value of the total scores of the degree of pain,pelvic pain were a little incerased or remained unchanged in both groups compared with those of last stage of treatment,while the difference was of no significance.And the socre of dysmenorrhea was significantly decreased in both groups（P＜0.01）.Each score of those in group B was higher than that in group A;but the difference was also not significant.2、After treatment the patients in group B had better quality of life than group A, especially in vitality and physical function.The patients in both groups were satisfied with the control of pain,but the scores of vitality and physical function in group A were significantly descended（P＜0.05 and P＜0.01,respectively）.3、There was no difference in the value of the form of kupperman between two groups after three months treatment.But the incidence of hot flush and sweatness among the form of kupperman in group B was significantly lower than that of group A （P＜0.05）.4、Bone mineral density of the lumbar spine（L1-L4） was significantly decreased after three months treatment in group A（P＜0.01）;it was also decreased in group B,but the difference was not significant.A significant increase of the level of serum BGP was observed in group A（P＜0.01） after the treatment.Similar increase also occurred in group B,while the difference was of no significance.5、After the treatment,the level of serum FSH and E₂ in both group declined significantly compared with the level before treatment（P＜0.01）;the level of serum E₂ in group B was significantly higher than that in group A（P＜0.01）;and the level of serun FSH in group B was significantly lower than that in group A（P＜0.05）.Conclusions1、The significant decrease of the score of the degree of pain,pelvic pain and dysmenorrheal and the disappearance of dyspareunia in group B suggests that our project of add-back therapy could relieve the pain of women with endometriosis and could be the same with GnRHa alone.2、The patients receiving add-back therapy may have a better quality of life than GnRHa alone,especially in pain control,vitality and physical function.3、Not all the symptoms of hypoestrogenism induced by GnRHa could be eliminated by add-back therapy,while hot flush and sweatness was ameliorated remarkably.It is speculated that the level of serum estrogen could be the cause of that.4、Although there was a trend of decrease of BMD in add-back therapy group,the loss of bone mineral was much less than that in GnRHa alone group.And the change of the level of serum BGP had a negative relation with the BMD.It is concluded that our add-back therapy could preotet the patients treated with GnRHa from bone loss.5、The level of serum estrogen in our study could protect patients from bone loss and avoid stimulating the growth of ectopic endometrium.The effect of our add-back therapy is confirmed and safe.PartⅡEffect on T lymphocytes of patients with endometriosis after the treatment of GnRH agonist combined with add-back therapyObjectiveTo investigate the effect of GnRH agonist combined with add-back therapy on the expression of different estrogen receptors（ER） subtypes and gonadotropin releasing hormone receptor（GnRHR） of peripheral blood mononuclear cell（PBMC） and T lymphocytes function of patients with endometriosis. Materials and methodsThe inclusion criterion,allocation,and administration of patients with endometriosis were the same with partⅠ.During the same period,10 non-endometriosis cases were recruited as the control group（group C）.Venous blood was collected from all patients included by venipuncture to exame T lymphocyte subsets of peripheral blood with flow cytometry（FCM） and the specific cytokine（IL-2 and IL-6） secretion of PBMC induced by phytohemagglutinin（PHA） with ELISA and the proliferation of T lymphocytes with Methylthiazoletetrazolium（MTT） method.At the same time,ERα,ERβand GnRHR mRNA expression of PBMC were detected by real time RT-PCR and the ERα,ERβand GnRHR protein expression were detected by western-blot in patients with endometriosis.The peritoneal fluid from part of the patients was collected to mesure the level of IL-2 and IL-6 by ELISA.After 3 months treatment,all contents were detected again in patients with endometiosis except the cytokins from peritoneal fluid.Results1、Before treatment,the percentage of peripheral blood CD4⁺ lymphocytes and the ratio of CD4⁺/CD8⁺ of group A and B were significantly lower than that of group C （P＜0.05）.After three months treatment,the percentage of peripheral blood CD4⁺ lymphocytes was significantly increased in both group A and B（P＜0.05） and the ratio of CD4⁺/CD8⁺ of group B was increased significantly than the level before treatment（P＜0.05）.Similar increase also occurred in group A,but the difference was of no significance（P=0.055）.2、Before treatment,the proliferation of T lymphocytes in group A and B was significantly lower than that in group C（P＜0.05） and after three months treatment, the proliferation of T lymphocytes was increased in both groups（P＜0.01）.3、Compared with group C,the level of IL-6 in the supernatants of cultured PHA induced PBMC and peritoneal fluid of group A and B was significantly increased（P＜0.05）;the ratio of IL-2/IL-6 in the supernatants and peritoneal fluid of both group A and B was significantly lower than that of group C（P＜0.05）.After 3 months treatment,in both group A and B,the levels of IL-6 in the supernatants were significantly decerased,while the radio of IL-2/IL-6 was remarkably increased （P＜0.05,respectively）.4、The level of mRNA and protein of ERβof PBMC significantly decreased after three months treatment in group A and B（P＜0.05）,while the ERαand GnRHR expression made no obvious difference before and after treatment.Conclusions1、After three months treatment,the percentage of peripheral blood CD4⁺ lymphocytes and the ratio of CD4⁺/CD8⁺ was significantly increased and the proliferation of T lymphocytes was also increased in add-back group just like that of the group of GnRHa alone.It suggests that our add-back therapy with transdermal E₂ 25μg everyday and daily oral medroxyprogesterone acetate 6mg might have the same effect on the improvement of abnormity of cell immunity in patients with endometriosis as GnRHa alone.2、After treatment,the level of IL-6 secretion of T lymphocytes of add-back group decreased,the ratio of IL-2/IL-6 increased,just like that of the group of GnRHa alone and it is concluded that both GnRHa alone and GnRHa combined with add-back therapy could improve Th1/Th2 balance.3、In both endometriosis groups,the expression of ERβof PBMC was significantly decreased after three months treatment.It is speculated that the effect of GnRH agonist on peripheral T lymphocytes might not be the result of the direction effect of GnRH agonist.It might have the relations with the hypoestrogenism.The effect might exert through ERβof T lymphocyte,while the definite mechanism is still unclear and we need further investigation.4、Both GnRHa alone and GnRHa combined with add-back therapy might improve immune function of endometriosis.更多还原