补肾活血方对子宫自然杀伤细胞与子宫间质细胞旁分泌基因表达谱的影响

Effect of Bushen Huoxue Recipe on Paracrine Gene Expression Profiling of Uterine Natural Killer Cells and Uterine Stromal Cells

目的观察补肾活血方对子宫自然杀伤(uterine natural kil er,u NK)细胞与子宫间质细胞旁分泌基因表达谱的影响。方法从育龄期妇女的增殖期子宫内膜提取人间质细胞,分为空白组、对照组和中药组。中药组加入DMEM/F12稀释至终浓度为2 mg/m L的补肾活血方药液,空白组和对照组加入等体积的DMEM/F12,对照组和中药组培养24 h后再加入20%无血清DMEM加80%u NK细胞分泌提取液,于37℃,5%CO2培养6 h,提取3组细胞RNA。运用基因芯片技术检测各组间质细胞的基因表达谱,同时应用q RT-PCR和ELISA法检测筛选基因粒细胞趋化蛋白1(CXCL1)]、细胞间黏附分子-(ICAM-1)、IL-8和白血病抑制因子(leukocyte inhibitor factor,LIF)m RNA和蛋白表达。结果与空白组比较,对照组和中药组上升4倍的差异基因表达谱基本一致,共63个基因。与对照组比较,中药组IL-15受体α(IL-15RA)上调1.27倍,血管内皮生长因子(VEGF)上调1.55倍,LIF上调1.45倍,IL-8上调1.10倍,IL-11上调1.23倍,转化生长因子-β(TGF-β)上调1.40倍,表皮生长因子(EGF)上调1.10倍,单核细胞趋化蛋白8(CCL8)上调1.13倍,运输蛋白1(TAP 1)上调1.02倍,细胞表面趋化因子受体2(CXCR2)上调1.22倍,ICAM-1上调1.15倍(P<0.05)。结论 u NK细胞旁分泌作用对提高子宫内膜容受性和种植妊娠率发挥着重要的作用,补肾活血方可改善并提高此旁分泌系统的功能。

Objective To observe the effect of Bushen Huoxue Recipe （BHR） on paracrine gene expression profiling of uterine natural killer cells （uNK cells） and uterine stromal cells. Methods Human stromal cells were extracted from proliferative phase endometrium of child-bearing age females, which were then divided into the blank group, the control group, and the BHR group. DMEM/F12 was added in cells of the BHR group to dilute into final concentration of 2 mg/mL herbal liquor. Equal volume of DMEM/ F12 was added to cells in the normal group and the control group. Cells in the control group and the BHR group were cultured for 24 h, with 20% serum-free DMEM plus 80% uNK cell secretion extracting solution added. Then they were cultured in 5% CO2 at 37 ℃ for 6 h. Total RNAs were extracted after culture. The gene expression profile of stromal cells was detected using gene chip technology. At the same time mR- NA and protein expressions of chemokine （C-X-C motif） ligand 1 （CXCL1）, intercellular cell adhesion molecule-1 （ICAM-1）, IL-8, and leukocyte inhibitor factor （LIF） were screened and detected using qRT- PCR and ELISA. Results Compared with the blank group, profiles of differentiated genes with 4-fold in- crease （a total of 63 genes） were basically agreeable in the control group and the BHR group. Compared with the control group, IL-15 receptor alpha （IL-15RA） was up-regulated by 1.27 times, vascular endotheli-al growth factor （VEGF） up-regulated by 1.55 times, LIF 1.10 times, IL-11 up-regulated by 1.23 times, transformi up-regulated by 1.45 times, IL-8 up-regulated by ng growth factor-β （TGF-β） up-regulated by 1.40 times, epidermal growth factor （EGF） up-regulated by 1.10 times, chemokine （C-C motif） ligand 8 （CCL8） up-regulated by 1.13 times, transporter 1 （TAP1） up-regulated by 1.02 times, chemokine （C-X-C motif） receptor 2 （CXCR2） up-regulated by 1.22 times, ICAM-1 up-regulated by 1.15 times （P 〈0.05） in the BHR group. Conclusion uNK paracrine played an important role in elevating endometrial receptivity and embryo implantation, and BHR could improve and elevate the function of this paracrine system.