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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态的维持。
Objective To observe the effect of “Yiqi Bushen Decoction” on foxp3mRNA and foxp3, STAT5 and NF-κB mRNA expression in splenic CD4 + CD25 + Treg cells of spontaneous abortion mice. Methods Female CBA / J mice were mated with male DBA / 2 or male BALB / c mice in 2: 1 cohorts to produce a spontaneous abortion model. CBA / J pregnant rats were divided into 5 groups: negative control group, positive control group, high-dose traditional Chinese medicine treatment group, middle-dose traditional Chinese medicine treatment group and traditional Chinese medicine low-dose treatment group, with 10 in each group. To the death day daily dose of 10mg / kg saline 1 times; positive control group on the 4th day of pregnancy at a dose of 10mg / kg cyclosporine A single dose of solution; Chinese medicine high, medium and low dose treatment group In the first day of pregnancy to the day of death were daily doses of 48,24,12 g / kg were fed Yiqi Bushen experimental side 1. The pregnant rats were killed on the 9th and 14th day of gestation, fresh spleen was taken for Treg cells extraction, and decidual tissue of pregnant mice was frozen at -80 ℃. The purity of CD4 + CD25 + was determined by flow cytometry. The expression of foxp3mRNA in CD4 + CD25 + cells was analyzed by RT-PCR. The expression of foxp3mRNA in CD4 + CD25 + The expression of foxp3, STAT 5A, STAT 5B and NF-κB mRNA in decidua was analyzed. Results The purity of CD4 + CD25 + Treg could reach over 88%, and the activity of CD4 + CD25 + Treg was greater than 95% by trypan blue assay. The average of CD4 + CD25 + / CD4 + before purification and purification was 13.20%. The average percentage of CD4 + CD25 + / CD4 + was 91.43% after purification. Compared with the negative control group, the expression levels of foxp3mRNA in Treg cells of the positive control group and the high-dose Chinese herbal medicine group were significantly increased (P <0.05) (P <0.05). Compared with the negative control group, the expressions of foxp3 and STAT5 mRNA in deciduas of the positive control group and the high and middle dose groups were significantly increased at 9 and 14 days (P <0.05); in the positive control group, 9 and 14 days of the traditional Chinese medicine high- The expression of STAT 5A mRNA in the decidua group was increased in the medium dose group (P <0.05, P <0.01). The expression of NF-κB mRNA in the deciduas of the positive control group and the traditional Chinese medicine groups was decreased (P <0.01). Conclusion Yiqi Bushen can up-regulate foxp3mRNA and foxp3, STAT5mRNA in splenic CD4 + CD25 + T cells of splenic spontaneous abortion mice and down-regulate the expression of NF-κB mRNA in the maternal fetal interface and promote the immune tolerance of pregnancy The maintenance.