【作者】 陈旭；

【导师】 刘彦；

【作者基本信息】 第二军医大学 ， 妇产科学， 2008， 博士

【摘要】 子宫内膜异位症（endometriosis,EMs）是临床妇科常见病之一,多发生于生育年龄妇女,临床上主要表现为经期出血、痛经、盆腔疼痛以及不孕。近年来,由于腹腔镜的广泛应用,该病的诊断率不断提高。腹腔镜是诊断的金标准,除可明确诊断、确定分期外,还可进行相应治疗。药物治疗的复发率较高,随不同临床分期、不同随访期限和不同的药物种类而产生不同的结果,3年内复发率最高可达61.5%。手术与药物联合治疗效果优于单用药物或手术治疗。采用药物治疗或保守性及半保守性手术（经腹腔镜或腹部手术）,对未生育及年轻妇女保留了生育功能及内分泌功能,复发却是棘手问题。子宫内膜异位症的发病机制目前仍未明确,分子生物学研究表明,子宫内膜异位症的发病具有粘附（Adhesion）、侵袭（Aggressive）和血管生成（Angiogenesis）三部曲组成,对于3A模式的研究已成为近年来的热点。不断加强基础研究,寻求更为完善的治疗对策,避免复发,仍需进一步研究。我们在子宫内膜异位症的临床治疗中发现,腹腔镜术后子宫内膜异位症的复发率显著低于其他研究报道的结果。除与异位症临床分期、腹腔镜手术技术与治疗的彻底性等因素相关外,腹腔镜手术时气腹对异位灶有无影响目前尚无相关研究。有较多研究显示,腹腔镜手术时,CO₂气腹可促进肿瘤细胞术后种植与转移,增加了术后复发的危险性。CO₂气腹作用于子宫内膜异位症病灶是否会产生一系列细胞与分子生物学改变,从而影响腹腔镜术后子宫内膜异位症的复发,目前尚无相关研究报道。本研究的目的在于建立子宫内膜异位症大鼠模型,观察不同气腹压力作用对子宫内膜异位症病灶的影响及相关细胞和分子机制研究,评价气腹压力对子宫内膜异位症术后复发的影响。第一部分CO₂气腹对子宫内膜异位症大鼠模型成模的影响目的:评价CO₂气腹对子宫内膜异位症造模成功率的影响及对EMs大鼠模型异位病灶和血清中相关细胞因子和分子生物学指标的变化。方法:按照诱发性子宫内膜异位症动物模型的建模方法建立子宫内膜异位症大鼠模型,雌性SD大鼠,体重250～300g,造模前1d肌注苯甲酸雌二醇0.1ml/kg使动物统一处于动情期。切除一侧子宫,取自体子宫内膜缝合于两侧腹膜。麻醉后随机分组,每组10只。分别于造模不同时间（A:造模术前、B:造模术后第4d、C:造模术后第10d）模拟CO₂气腹作用,压力15mmHg,时间1h,对照组无气腹干预,于造模4w后观察不同处理组与对照组的成模情况及异位灶和血清细胞因子、分子生物学等指标变化,评价CO₂气腹对子宫内膜异位症造模成功率的影响。结果:造模不同时期（造模术前、造模术后第4d、10d）CO₂气腹作用后,造模4w后对照组成模率100%（10/10）,CO₂气腹作用后的3组成模率分别为A组100%（10/10）、B组70%（7/10）、C组80%（8/10）。A组的侵袭、粘附和血管生成相关指标水平稍高于对照组（P＜0.05）或与对照组无显著差异（P＞0.05）。B、C组侵袭、粘附和血管生成相关的细胞因子水平多低于A组和对照组（P＜0.05）,并且两组间无显著差异（P＞0.05）。结论:CO₂气腹降低子宫内膜异位症造模成功率,对异位病灶的侵袭、粘附和血管生成等具有抑制作用。第二部分不同气腹压力对子宫内膜异位症大鼠模型异位病灶侵袭、粘附和血管生成等方面的影响目的:评估不同气腹压力对子宫内膜异位灶及血清与粘附、侵袭和血管生成相关因子的变化水平及其与术后复发相关因素分析。方法:造模成功后随机分组（每组10只）:对照组、10mmHg和20mmHg CO₂气腹组,作用时间1h,术毕时行右侧异位灶剥除术,放尽囊液后重新缝合于该侧腹膜处。气腹干预后1w、2w、6w检测气腹对子宫内膜异位灶侵袭（MMP2/TIMP-1）、粘附（CD44v6、ICAM-1）、血管生成相关因子（VEGF/ENS、TNF-α）的影响,及血清相应细胞因子变化水平,评估不同气腹压力对子宫内膜异位灶及血清相关因子的变化水平及其与术后复发相关因素分析。结果:与对照组比较术后早期（1周）10mmHg和20mmHg CO₂气腹组对子宫内膜异位灶侵袭、粘附和血管生成等相关因子水平均显著降低（P＜0.05）,且10mmHg组低于20mmHg组（P＜0.05）,这种影响在10mmHg组多可持续至术后2周以后。同组内行异位灶剥除术复种于腹膜处的异位灶仍能继续生长,相关细胞因子水平变化与另一侧未剥离的异位灶相似（P＞0.05）。随着气腹干预后时间变化,各组相关细胞因子水平逐渐升高,术后6周与对照组无差异（P＞0.05）。结论:气腹压力较低者对于异位病灶的侵袭、粘附和血管生成等抑制作用优于高气腹压力和对照组,可能是腹腔镜术后复发率低的因素之一。气腹作用后未剥除的异位灶与剥除后残留于腹腔内的异位灶具有相似的侵袭能力。第三部分不同气腹压力对子宫内膜异位症大鼠模型异位病灶细胞凋亡和细胞器超微结构的影响目的:观察异位病灶腺细胞细胞凋亡和其它细胞器超微结构改变,进一步评价CO₂气腹压力对子宫内膜异位灶的影响及术后复发的相关因素分析。方法:造模成功后随机分组（每组5只）:对照组、10mmHg和20mmHg CO₂气腹组,作用时间1h,手术结束后取子宫内膜异位灶及临近腹膜行电镜检查,观察异位病灶腺上皮细胞凋亡和其它细胞器超微结构改变,进一步评价CO₂气腹压力对子宫内膜异位灶的影响及术后复发的相关因素分析。结果:对照组异位灶腺细胞结构良好,基质细胞排列整齐;腹膜间皮细胞排列整齐,微绒毛正常。10mmHg和20mmHg CO₂气腹组均有异位灶腺细胞凋亡增加,线粒体嵴消失、线粒体肿胀明显;微绒毛消失,细胞连接缝隙增宽断裂,基底层细胞排列紊乱。与10mmHg比较,20mmHg气腹组异位灶细胞受损严重。结论:CO₂气腹可促进子宫内膜异位灶腺细胞凋亡及超微结构破坏,降低其侵袭能力,20mmHg组损伤作用稍大。但气腹压力过高可能导致腹膜间皮细胞及基底细胞受损更为严重,从而降低了腹膜的防御能力,故临床手术时气腹压力不宜过高。全文结论:1、CO₂气腹降低子宫内膜异位症造模成功率,对异位病灶的侵袭、粘附和血管生成等具有抑制作用。2、10mmHg和20mmHg CO₂气腹组均可对异位病灶的侵袭、粘附和血管生成等产生抑制作用,气腹压力较低者对于优于高气腹压力和对照组。3、CO₂气腹可促进子宫内膜异位灶腺细胞凋亡及超微结构破坏,降低其侵袭能力。CO₂气腹能对异位病灶的侵袭、粘附和血管生成等产生抑制作用,气腹压力较低者效果较好,同时对异位灶腺细胞凋亡及超微结构破坏,降低其侵袭能力,可能是腹腔镜手术后子宫内膜异位症复发率较低的因素之一。更多还原

【Abstract】 BackgroundEndometriosis（EMs） is a common gynecological disease which occurs more among in reproductive age women.The clinical symptoms contain menstruation haemorrhage, dysmenorrheal,pelvic pain and infertility.With the wide use of laprascopy recent years, the diagnostic rate of EMs becoming increased.Laparoscopy now is the gold standard in disease final diagnosis,staging and treatment.The recurrence rate of drug treatment is still higher,varying with the clinical stage,follow-up phase and medicine variety,with the highest recurrence rate to be 61.5 percent within three years（reported by Domowshi and Cohen）.The effect of therapeutic alliance of surgery and medicine is superior to medicine or surgery application alone.It can preserve the reproductive and endocrine function by drug treatment or conservative and semiconservative operation（laparoscopy or abdominal operation）.But the recurrence is still a troublesome problem,which lead us to emphasize the fundamental study,searching for more perfect treatment policies and recurrence prevention.In the clinical treatment of EMs,we find a low recurrence rate of laparoscopic treatment,which is lower than those results reported by other studies.Besides the clinical stage,laparoscopic techniques and treatment degree,there is still no any study on whether the CO₂ pneumoperitoneum（PNP） has an effect on EMs lesion during laparoscopic surgery.Many researches showed that CO₂ pneumoperitoneum stimulated the tumor cells implantation and metastases during laparoscopy,which increased the risk of recurrence. There has been no any correlated report on effect of CO₂ pneumoperitoneum on EMs lesion.Whether CO₂ pneumoperitoneum cause a series of cytological and molecular variation of EMs lesion that may affect the post-operative recurrence.The objective of our study is to set up the EMs rat model and to evaluate the pneumoperitoneum pressure on post-operative recurrence of EMs by determining the cytological and molecular variation of EMs lesion caused by CO₂ pneumoperitoneum.PartⅠEffects of CO2 pneumoperitoneum on achievement rate of foundation of EMs rat modelObjective:To evaluate the effect of CO₂ pneumoperitoneum on EMs model foundation.Methods:To erect the EMs rat model according to the method of induced EMs animal model.To select the female SD rats（mean weight 250～300g） and a intramuscular injection of estradiol benzoate（0.1ml/kg） to make the animals in estruation at the same time.The left uterus was resected and cut open,then endometrial tissue were sutured on both sides of the peritoneum.The rats were divided randomly into four groups（ten per group）:control group;group A:pre-operation;group B:4th day post-operation;group C:10th day post-operation of model erection.Then CO₂ pneumoperitoneum（15mmHg,1h） was simulated pre or post operation and the achievement rate of model foundation,cytokine and molecular biological parameter were determined to evaluate the effect of CO₂ pneumoperitoneum on EMs model foundation.Results:After four weeks of CO₂ pneumoperitoneum intervention pre and post operation, the achievement rate of model foundation is 100 percent in control group and group A,70 and percent in group B and C respectively.There is a slight increase or equal level of cytokines and molecular variation in group A compared with control group.And there is a significant decrease more often in group B and C than that of gourp A（P＜0.05）.Moreover there is no statistic difference between group B and C（P＞0.05）.Conclusion:The achievement rate was decreased by CO₂ pneumoperitoneum via inhibition of aggression,adhesion and angiogenesis.PartⅡEffects of CO2 pneumoperitoneum pressure on aggression,adhesion and angiogenesis of EMs lesionObjective:To evaluate the effect of different CO₂ pneumoperitoneum pressure on cytokines level of EMs lesion and serum and the correlation factors with post-operation recurrence.Methods:The rats were divided randomly into three groups（control group,10mmHg and 20mmHg CO₂ pneumoperitoneum,1h） after EMs model being founded successfully.After the CO₂ pneumoperitoneum was over,the right EMs lesion was resected and resutured on the situ peritoneum again.The correlated cytokines of aggression（MMP2/TIMP-1）, adhesion（CD44v6、ICAM-1） and angiogenesis（VEGF/ENS、TNF-α） in EMs lesion and serum were detected at 1w,2w and 6w after pneumoperitoneum intervention.Then to evaluate the effect of different CO₂ pneumoperitoneum pressure on cytokines level of EMs lesion and serum and the correlation factors with post-operation recurrence.Results:Compared with control group,the cytokine level correlating with aggression, adhesion and angiogenesis in the two CO₂ pneumoperitoneum groups decreased significantly in early post-operation（P＜0.05）.Moreover the cytokines level in 10mmHg group was lower than group with 20mmHg pneumoperitoneum（P＜0.05）,and this effect can be continued until two weeks after pneumoperitoneum intervention.The EMs lesion after resection can implant and grow sequently and has the similar cytokines level with EMs lesion on the other side of peritoneum（P＞0.05）.The level of cytokines increased gradually with time after CO₂ pneumoperitoneum intervention,but no difference compared with control group（P＞0.05）.Conclusion:The low CO₂ pneumoperitoneum pressure group is superior to the high pressure group in inhibition of aggression,adhesion and angiogenesis,which may be one of the factors that correlate with the low post-laparoscopic recurrence.There is a similar invasive capability of EMs lesions with or without resection. PartⅢEffects of CO2 pneumoperitoneum pressure on apoptosis and the Ultrastructure of cellular organelle of EMs lesionObjective:To evaluate the effect of CO₂ pneumoperitoneum pressure on EMs lesion and its correlation with post-operative recurrence.Methods:Thirty rats were divided randomly into three groups（five per group）,control group,10mmHg and 20mmHg CO₂ pneumoperitoneum groups,with duration lasting for 1h.EMs lesion and peritoneum nearby were sampled for electron microscope examination to determine the apoptosis and ultrasturcture variation of cell organelle of EMs gland cells and peritoneal mesothelium.To evaluate the effect of CO₂ pneumoperitoneum pressure on EMs lesion and its correlation with post-operative recurrence.Results:There is a well being structure of glandular cells with matrix cells and peritoneal mesothelium lining up in order and normal structure of microvilli（MV）.While in groups of 10mmHg and 20mmHg,the apoptosis of glandular cells increased with mitochondrial crista decreasing or vanishing and mitochondria swelling and moreover there is no significant difference between the two groups.But compared with the 10mmHg group, there is more severe damage of peritoneal mesothelium with microvilli vanishing, apoptosis increasing,cell junction widening and collapse and matrix cells lining up in disorder.Conclusion:There is a significant promotion of glandular cells apoptosis,which weakens the aggression capability of EMs lesion.But there is a little more worse damage of peritoneal mesothelium and matrix cells of EMs lesion,which reduces the defense capability of peritoneum.Therefore it is suggested that the pneumoperitoneum be selected with appropriate pressure.Summary1.The achievement rate was decreased by CO₂ pneumoperitoneum via inhibition of aggression,adhesion and angiogenesis.2.Both of the CO₂ pneumoperitoneum group with 10 and 20mmHg pressure can inhibit the aggression,adhesion and angiogenesis of EMs lesion and the low pressure are superior to the high pressure group,which may be one of the factors that correlate with the low post-laparoscopic recurrence.3.There is a significant promotion of glandular cells apoptosis and ultrastructure destruction,which weakens the aggression capability of EMs lesion.In conclusion,the aggression,adhesion and angiogenesis of EMs lesion can be inhibited by CO₂ pneumoperitoneum with the lower pressure having the better effects, and a significant apoptosis and ultrastructure destruction changing of glandular cells caused by CO₂ pneumoperitoneum,which may be one of the factors that lead to low post-laparoscopic surgical recurrence.更多还原