益气补肾实验方对自然流产模型小鼠foxp3、STAT5、NF-κB mRNA表达的影响  

Effects of Yiqi Bushen Experimental Recipe on mRNA Expressions of foxp3,STAT5,and NF-κB in Decidua Tissue of Natural Abortion M odel M ice

作　　者：叶平[1] 舒昊旭 鲁颖晔[3] 王欢欢[1] 叶骞[1] 张丽[4] 

机构地区：[1]浙江省立同德医院生殖免疫科,杭州310012 [2]阿尔伯塔大学数学与统计科学学部 [3]浙江中医药大学第一临床医学院,杭州310053 [4]浙江省中医院妇科,杭州310006

出　　处：《中国中西医结合杂志》2016年第8期946-952,共7页Chinese Journal of Integrated Traditional and Western Medicine

基　　金：浙江省中医药重点研究计划项目(No.2012ZZ004)

摘　　要：目的观察益气补肾实验方对自然流产模型小鼠脾脏CD4+CD25+Treg细胞中foxp3mRNA表达及蜕膜组织foxp3、STAT 5、NF-κB mRNA表达的影响。方法将雌性CBA/J与雄性DBA/2或雄性BALB/c小鼠以2∶1合笼交配,制备自然流产模型。将CBA/J孕鼠分为5组:阴性对照组、阳性对照组、中药高剂量治疗组、中药中剂量治疗组、中药低剂量治疗组,每组10只;阴性对照组于妊娠第0天至处死日每天以10mg/kg的剂量灌服生理盐水1次;阳性对照组于妊娠第4天以10mg/kg的剂量单次灌服环孢素A溶液;中药高、中、低剂量治疗组于妊娠第0天至处死日每日分别按48、24、12g/kg的剂量灌服益气补肾实验方1次。将孕鼠于妊娠第9、14天处死,取新鲜脾脏用于提取Treg细胞,并取孕鼠蜕膜组织-80℃冻存。采用磁珠分选的方法分离和纯化脾脏CD4+CD25+细胞,采用流式细胞术鉴定分选前后CD4+CD25+的纯度,运用RT-PCR分析CD4+CD25+细胞中foxp3mRNA的表达情况;运用RT-PCR分析蜕膜组织中foxp3、STAT 5A、STAT 5B、NF-κB mRNA的表达情况。结果 CD4+CD25+Treg的纯度可达到88%以上,经台盼蓝检测其活性大于95%。纯化分离前CD4+CD25+/CD4+平均为13.20%;纯化分离后CD4+CD25+/CD4+平均为91.43%。与阴性对照组比较,阳性对照组、中药高剂量治疗组Treg细胞中foxp3mRNA表达水平明显升高(P<0.05);中药高剂量治疗组较中、低剂量组小鼠脾脏Treg中foxp3mRNA表达升高(P<0.05)。与阴性对照组比较,阳性对照组、中药高、中剂量组9、14天蜕膜组织foxp3、STAT 5BmRNA表达升高(P<0.05);阳性对照组、中药高剂量组9、14天及中药中剂量组蜕膜组织STAT 5A mRNA表达升高(P<0.05,P<0.01);阳性对照组及中药各剂量组蜕膜组织NF-κB mRNA表达降低(P<0.01)。结论益气补肾实验方可上调自然流产模型小鼠脾脏CD4+CD25+T细胞foxp3mRNA及蜕膜组织中foxp3、STAT 5mRNA表达,下调母胎界面组织中NF-κB mRNA的表达,促进妊娠免疫耐受状Objective To observe the effects of Yiqi Bushen Experimental Recipe （YBER, a recipe for benefiting qi and Shen supplementing） on mRNA expression of foxp3 in splenic CD4＋CD25＋ Treg cells and mRNA expressions of foxp3, STAT5, and NF-~B in decidua tissue of natural abortion （NA） model mice. Methods Female CBA/J mice were caged and mated to male DBAf2 mice mice in 2：1 for NA model. Pregnant CBA/J mice were randomly divided into 5 groups, or male BALB/c e., the negative control group, the positive control group, high, middle, and low dose YBER groups, 10 in each group. Mice in the NS control group were administered with normal saline by gastrogavage from day 0 to their death, 10 mg/kg, once per day. Mice in the positive control group were administered with Cyclosporine A solution by gastrogavage on the 4th day of pregnancy. YBER （48,24, 12 g/kg） was respectively administered to mice in the 3 dose YBER groups by gastrogavage from day 0 to their death, once per day. Pregnant mice were sacrificed at day 9 and 14, and fresh spleens were taken out for extracting Treg cells. Dcidua tissues were collected and stored in -80 ℃ for frozen. Splenic CD4 ＋ cells CD25 ＋ were isolated and purified by magnetic bead. The purity of CD4 ＋ cells CD25 ＋ was identified by flow cytometry （FCM） before and after magnetic bead. mRNA expressions of foxp3, STAT5A, STAT5B, and NF-KB in decidua tissue were analyzed by RT-PCR. Results The purity of CD4 ＋Treg CD25 ＋ could arrived at 88% plus. Its activity could be over 95% after trypan blue test. The average ratio of CD4 ＋ CD25＋/CD4＋ was 13.20% before purified isolation, while it was 91.43% after purified isolation. Compared with the negative control group, foxp3 mRNA expression level in Treg cells was obviously elevated in the positive control group and the high dose YBER group （P 〈0.05）. foxp3 mRNA expression level in Treg cells was obviously ele- vated more in the high dose YBER group than in the middle dose YBER group and the low dose YBER group �

关 键 词：自然流产模型 转录因子FOXP3 转录信号转导子与激活子5 核转录因子-ΚB 益气补肾实验方 

分 类 号：R285.5[医药卫生—中药学] R-332[医药卫生—中医学]