祛瘀解毒法对血瘀蕴毒型子宫内膜异位症卵泡液及颗粒细胞TNF-α、IL-6的研究
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摘要
目的:
观察祛瘀解毒颗粒结合辅助生殖技术(ART)治疗血瘀蕴毒型子宫内膜异位症不孕的疗效并探讨其作用机理,试图开辟中医药治疗EM不孕的新思路,为临床有效治疗该病提供依据。
方法:
临床研究:60例子宫内膜异位症(EM)不孕患者随机分为治疗组和对照组,每组30例。治疗组有20例口服祛瘀解毒颗粒结合氯米芬(CC)促排卵+宫腔内人工授精(IUI);另外10例口服祛瘀解毒颗粒同时行IVF-ET。对照组有20例单纯给CC促排卵+IUI,另外10例行IVF-ET。观察治疗前后血瘀蕴毒证候积分变化、妊娠率、免疫及内分泌学相关指标、血清和卵泡液TNF-α、IL-6含量以及卵巢颗粒细胞TNF-αmRNA、IL-6mRNA的表达情况。
实验研究一:40例EM模型大鼠随机分为试验组、观察组各20例,另取20只大鼠只牵拉子宫作为空白组。试验组每天给予祛瘀解毒颗粒溶于2ml生理盐水灌胃,观察组和空白组均给予2ml生理盐水,连续灌胃3周。末次给药后24h各组大鼠均给予人绝经后促性腺激素(HMG)50IU腹腔注射,48h后给予人绒毛膜促性腺激素(HCG)100IU/100g,14~16h后处死大鼠收集卵细胞及颗粒细胞团,观察优质卵细胞数目及优质卵细胞率,采用逆转录聚合酶链反应(RT-PCR)法检测卵巢颗粒细胞TNF-αmRNA、IL-6mRNA表达。
实验研究二:配制不同的培养液,对20只小鼠超排卵共收集189枚2-细胞鼠胚分别放入各组培养液进行培养。其中治疗组培养液为P-1胚胎培养液加10%SSS加10%治疗组EM患者卵泡液;对照组为培养液P-1胚胎培养液加10%SSS加10%对照组EM患者卵泡液;空白组培养液为P-1胚胎培养液加10%SSS加10%空白组人卵泡液。每日在显微镜下观察小鼠胚胎情况。
结果:
1.祛瘀解毒颗粒能够有效改善患者血瘀蕴毒证候,提高妊娠率,降低EMAb的阳性率及CA125水平,改善内分泌状态,降低患者血清及卵泡液TNF-α、IL-6的含量,降低卵巢颗粒细胞TNF-αmRNA、IL-6mRNA的表达,效果明显优于对照组(P<0.05)。
2.试验组大鼠优质卵细胞数目、优质卵细胞率明显高于观察组(P<0.05),颗粒细胞TNF-αmRNA、IL-6mRNA表达明显低于观察组(P<0.05)。
3.小鼠2-细胞胚胎在治疗组及空白组培养液中发育到8-细胞期、桑葚胚及囊胚及优质胚胎的比率均明显高于对照组(P<0.05)。
结论:祛瘀解毒颗粒可以改善血瘀蕴毒症候,提高EM不孕患者妊娠率,是治疗血瘀蕴毒型子宫内膜异位症的有效药物。推测其作用机理可能与调节免疫功能,改善内分泌状态,降低卵泡液TNF-α、IL-6水平及颗粒细胞TNF-αmRNA、IL-6mRNA表达等有关,通过旁分泌或(和)自分泌方式直接或间接参与卵巢功能的局部调节,改善卵子生存的内环境,提高卵细胞质量,减少对胚胎的毒性作用,从而提高EM患者妊娠率。
Objective:
EM-related infertility patients of blood stasis and toxin accumulation syndrome were treated with quyujiedu granule combined with ART. The efficacy was observed and the potential mechanism was explored.
Method:
Clinical research:60 cases of endometriosis(EM) related infertility patients were randomly divided into treatment group and control group with 30 cases in each. Quyujiedu granule was given orally accompanied by the routine ovarian hyperstimulation with clomiphene citrate(CC) and intra-uterine insemination(IUI)to 20 cases in the treatment group whereas IVF-ET combined with oral Quyujiedu granule was given to another 10 cases. 20 cases in the control group were given CC and IUI. 10 cases only had IVF-ET performed. The symptom scores,pregnancy rate,serum CA125,EMAb,E2,P,PRL and TNF-α、IL-6 in the serum and follicular fluid were measured.The expression of TNF–αmRNA、IL-6mRNA were observed.
Experiment 1:40 EM rats were randomly divided into experiment group and control group with 20 in each group. Another 20 rats were processed by only stretching the uterus as pseudo–operation group.Quyujiedu granule was given by gastric injection in experiment group daily for 3 weeks and saline was given in the other 2 groups in the same way. All the rats received peritoneal injection of 50IU of HMG and HCG (100IU/100g) 24 hours and 48 hours after the final gastric injection respectively. 14-16 hours later,the rats were killed and the occytes and granular cells were collected in order to observe the number and rate of quality ovum. The TNF-αmRNA and IL-6 mRNA expression in the granular cells were measured using RT-PCR.
Experiment 2:189 2-cell mouse embryos devided into treatment group,control group and blank group were cultured in medium respectively. As a supplement,follicular fluid from endometriosis patients treated with quyujiedufang granule was added to the medium of treatment group and follicular fluid from endometriosis patients to the medium of control group. Then the development of the mouse embryo were closely observed under microscope.
Result:
1.The symptoms was significantly improved and the pregnancy rate in the treatment group raised. The result also showed that Quyujiedu granule could significantly reduce the positive rate of EMAb and the level of CA125, TNF-α、IL-6 in serum and follicular fluid and their expression in granular cells compared to control group(P<0.05).
2.The quality ovum number and rate were significantly higher compared to control group(P<0.05). The expression of TNF-αmRNA and IL-6 mRNA in the granular cells were significantly lower than control group(P<0.05).
3.There was significant difference between treatment group and control group in the percent of8-cell stage,morula stage and blastocyst(P<0.05).
Conclusion:Quyujiedu granule could improve the symptoms and raise the pregnancy rate significantly. The mechanism might be raising the quality of the ovum through theregulation of the patients’immune state and improving of the ovarian microenvironment. Quyujiedu granule is an effective prescription for EM of blood stasis and toxin accumulation syndrome.
观察祛瘀解毒颗粒结合辅助生殖技术(ART)治疗血瘀蕴毒型子宫内膜异位症不孕的疗效并探讨其作用机理,试图开辟中医药治疗EM不孕的新思路,为临床有效治疗该病提供依据。
方法:
临床研究:60例子宫内膜异位症(EM)不孕患者随机分为治疗组和对照组,每组30例。治疗组有20例口服祛瘀解毒颗粒结合氯米芬(CC)促排卵+宫腔内人工授精(IUI);另外10例口服祛瘀解毒颗粒同时行IVF-ET。对照组有20例单纯给CC促排卵+IUI,另外10例行IVF-ET。观察治疗前后血瘀蕴毒证候积分变化、妊娠率、免疫及内分泌学相关指标、血清和卵泡液TNF-α、IL-6含量以及卵巢颗粒细胞TNF-αmRNA、IL-6mRNA的表达情况。
实验研究一:40例EM模型大鼠随机分为试验组、观察组各20例,另取20只大鼠只牵拉子宫作为空白组。试验组每天给予祛瘀解毒颗粒溶于2ml生理盐水灌胃,观察组和空白组均给予2ml生理盐水,连续灌胃3周。末次给药后24h各组大鼠均给予人绝经后促性腺激素(HMG)50IU腹腔注射,48h后给予人绒毛膜促性腺激素(HCG)100IU/100g,14~16h后处死大鼠收集卵细胞及颗粒细胞团,观察优质卵细胞数目及优质卵细胞率,采用逆转录聚合酶链反应(RT-PCR)法检测卵巢颗粒细胞TNF-αmRNA、IL-6mRNA表达。
实验研究二:配制不同的培养液,对20只小鼠超排卵共收集189枚2-细胞鼠胚分别放入各组培养液进行培养。其中治疗组培养液为P-1胚胎培养液加10%SSS加10%治疗组EM患者卵泡液;对照组为培养液P-1胚胎培养液加10%SSS加10%对照组EM患者卵泡液;空白组培养液为P-1胚胎培养液加10%SSS加10%空白组人卵泡液。每日在显微镜下观察小鼠胚胎情况。
结果:
1.祛瘀解毒颗粒能够有效改善患者血瘀蕴毒证候,提高妊娠率,降低EMAb的阳性率及CA125水平,改善内分泌状态,降低患者血清及卵泡液TNF-α、IL-6的含量,降低卵巢颗粒细胞TNF-αmRNA、IL-6mRNA的表达,效果明显优于对照组(P<0.05)。
2.试验组大鼠优质卵细胞数目、优质卵细胞率明显高于观察组(P<0.05),颗粒细胞TNF-αmRNA、IL-6mRNA表达明显低于观察组(P<0.05)。
3.小鼠2-细胞胚胎在治疗组及空白组培养液中发育到8-细胞期、桑葚胚及囊胚及优质胚胎的比率均明显高于对照组(P<0.05)。
结论:祛瘀解毒颗粒可以改善血瘀蕴毒症候,提高EM不孕患者妊娠率,是治疗血瘀蕴毒型子宫内膜异位症的有效药物。推测其作用机理可能与调节免疫功能,改善内分泌状态,降低卵泡液TNF-α、IL-6水平及颗粒细胞TNF-αmRNA、IL-6mRNA表达等有关,通过旁分泌或(和)自分泌方式直接或间接参与卵巢功能的局部调节,改善卵子生存的内环境,提高卵细胞质量,减少对胚胎的毒性作用,从而提高EM患者妊娠率。
Objective:
EM-related infertility patients of blood stasis and toxin accumulation syndrome were treated with quyujiedu granule combined with ART. The efficacy was observed and the potential mechanism was explored.
Method:
Clinical research:60 cases of endometriosis(EM) related infertility patients were randomly divided into treatment group and control group with 30 cases in each. Quyujiedu granule was given orally accompanied by the routine ovarian hyperstimulation with clomiphene citrate(CC) and intra-uterine insemination(IUI)to 20 cases in the treatment group whereas IVF-ET combined with oral Quyujiedu granule was given to another 10 cases. 20 cases in the control group were given CC and IUI. 10 cases only had IVF-ET performed. The symptom scores,pregnancy rate,serum CA125,EMAb,E2,P,PRL and TNF-α、IL-6 in the serum and follicular fluid were measured.The expression of TNF–αmRNA、IL-6mRNA were observed.
Experiment 1:40 EM rats were randomly divided into experiment group and control group with 20 in each group. Another 20 rats were processed by only stretching the uterus as pseudo–operation group.Quyujiedu granule was given by gastric injection in experiment group daily for 3 weeks and saline was given in the other 2 groups in the same way. All the rats received peritoneal injection of 50IU of HMG and HCG (100IU/100g) 24 hours and 48 hours after the final gastric injection respectively. 14-16 hours later,the rats were killed and the occytes and granular cells were collected in order to observe the number and rate of quality ovum. The TNF-αmRNA and IL-6 mRNA expression in the granular cells were measured using RT-PCR.
Experiment 2:189 2-cell mouse embryos devided into treatment group,control group and blank group were cultured in medium respectively. As a supplement,follicular fluid from endometriosis patients treated with quyujiedufang granule was added to the medium of treatment group and follicular fluid from endometriosis patients to the medium of control group. Then the development of the mouse embryo were closely observed under microscope.
Result:
1.The symptoms was significantly improved and the pregnancy rate in the treatment group raised. The result also showed that Quyujiedu granule could significantly reduce the positive rate of EMAb and the level of CA125, TNF-α、IL-6 in serum and follicular fluid and their expression in granular cells compared to control group(P<0.05).
2.The quality ovum number and rate were significantly higher compared to control group(P<0.05). The expression of TNF-αmRNA and IL-6 mRNA in the granular cells were significantly lower than control group(P<0.05).
3.There was significant difference between treatment group and control group in the percent of8-cell stage,morula stage and blastocyst(P<0.05).
Conclusion:Quyujiedu granule could improve the symptoms and raise the pregnancy rate significantly. The mechanism might be raising the quality of the ovum through theregulation of the patients’immune state and improving of the ovarian microenvironment. Quyujiedu granule is an effective prescription for EM of blood stasis and toxin accumulation syndrome.
引文
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