Ye Ping, Shu Hao-Xu, Lu Ying-Ye, Wang Huan-Huan, Ye Qian, Zhang Li
Zhongguo Zhong Xi Yi Jie He Za Zhi. 2016 Aug;36(8):946-952.
Objective To observe the effects of Yiqi Bushen Experimental Recipe (YBER, a rec- ipe 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-κB in decidua tissue of natural abortion (NA) model mice. Methods Female CBA/J mice were caged and mated to male DBA/2 mice or male BALB/c mice in 2:1 for NA model. Pregnant CBA/J mice were randomly divided into 5 groups, i.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. Preg- nant 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 °C 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 (P <0.05). Compared with the negative control group, mRNA expression levels of foxp3 and STAT5B in decidua tissue increased in the positive control group, high and middle dose YBER groups (P <0. 05). mRNA expression level of STAT5A increased in the positive control group and the high dose YBER group at day 9 and 14; as well as in the middle dose YBER group (P <0. 05, P <0. 01). NF-κB mR- NA expression level in decidua tissue was reduced in the positive control group and 3 dose YBER groups (P<0. 01). Conclusion YBER could up-regulate the expression of foxp3 mRNA in splenic CD4 + CD25 + Treg cells and mRNA expressions of foxp3 and STAT5 in decidua tissues of NA model mice, down-regulate NF-κB mRNA expression in maternal-fetal interface, and promote the maintenance of immune tolerance state.
目的 观察益气补肾实验方（YBER，一种益气补肾方剂）对自然流产（NA）模型小鼠脾脏CD4⁺CD25⁺调节性T细胞（Treg细胞）中foxp3 mRNA表达及蜕膜组织中foxp3、信号转导和转录激活因子5（STAT5）、核因子κB（NF-κB）mRNA表达的影响。方法 将雌性CBA/J小鼠与雄性DBA/2小鼠或雄性BALB/c小鼠按2∶1比例合笼交配建立NA模型。将妊娠CBA/J小鼠随机分为5组，即阴性对照组、阳性对照组、YBER高、中、低剂量组，每组10只。NS对照组小鼠于妊娠第0天至死亡每日经口灌胃给予生理盐水，剂量为10 mg/kg。阳性对照组小鼠于妊娠第4天经口灌胃给予环孢素A溶液。YBER高、中、低剂量组小鼠于妊娠第0天至死亡每日经口灌胃给予YBER（48、24、12 g/kg）。分别于妊娠第9天和第14天处死妊娠小鼠，取出新鲜脾脏提取Treg细胞。收集蜕膜组织并冻存于-80℃。采用磁珠法分离纯化脾脏CD4⁺细胞CD25⁺。磁珠前后通过流式细胞术（FCM）鉴定CD4⁺细胞CD25⁺的纯度。采用逆转录聚合酶链反应（RT-PCR）分析蜕膜组织中foxp3、STAT5A、STAT5B和NF-κB的mRNA表达。结果 CD4⁺Treg CD25⁺纯度可达88%以上。经台盼蓝检测其活性可超过95%。纯化分离前CD4⁺CD25⁺/CD4平均比例为13.20%，纯化分离后为91.43%。与阴性对照组比较，阳性对照组和YBER高剂量组Treg细胞中foxp3 mRNA表达水平明显升高（P<0.05）。YBER高剂量组Treg细胞中foxp3 mRNA表达水平升高明显高于YBER中剂量组和低剂量组（P<0.05）。与阴性对照组比较，阳性对照组、YBER高、中剂量组蜕膜组织中foxp3和STAT5B mRNA表达水平升高（P<0.05）。妊娠第9天和第14天，阳性对照组和YBER高剂量组以及YBER中剂量组STAT5A mRNA表达水平升高（P<0.05，P<0.01）。阳性对照组和YBER 3个剂量组蜕膜组织中NF-κB mRNA表达水平降低（P<0.01）。结论 YBER可上调NA模型小鼠脾脏CD4⁺CD25⁺Treg细胞中foxp3 mRNA表达及蜕膜组织中foxp3和STAT5 mRNA表达，下调母胎界面NF-κB mRNA表达，促进免疫耐受状态的维持。
