母胎界面维生素D3通路调控Th1/Th2因子预防自然流产的机制研究

One of the causes of spotaneous miscarriage (SM) is associated with a up-regulation of Th1 cytokine activating and down-regulation of Th2 cytokine activity. The treatment with anti-Th1 therapy at the time of conception may be associated with an increased risk of congenital malformations. It is very important to find new ways to avoid SM by maintaining Th1/Th2 cytokine balance. Vitamin D3 has been known to be effective in the regulation of Th1/Th2 cytokines and treatment of diseases, which are associated with overactivity of Th1 cytokine. But there is no reportion how this vitamin exerts its modulatory action in the fetomaternal interface of women with URSA. Based on the research of Th1 cytokines in the fetomaternal interface of the miscarriage before, the production of vitamin D3 receptor (VDR), 1α-hydroxylase and Th1/Th2 cytokine pro?le by whole chorionic villus cells and decidual cells are measured in patients with SM and healthy controls in order to explore the relationship between pathway of VDR and 1α-hydroxylase and SM and Th1/Th2 cytokines in the fetomaternal interface. The trophoblast cells and decidual stromal cells from healthy controls and SM are cultured and transfected with small interfering RNA for VDR & 1α-hydroxylase. Or concentrations of 1,25(OH)2D3 are used in the cell culture. Levels of Th1/Th2 cytokine pro?le and the proliferation of cells in the presence of 1,25(OH)2D3 and absence of VDR & 1α-hydroxylase activity of these cell populations are measured in order to evaluate the gene function of VDR & 1α-hydroxylase and regulation of 1,25(OH)2D3 pathway on cytokine production and the proliferation by trophoblast and decidual stromal cells. It will validate the effects of vitamin D3 in the prevention of SM and look forward to provide new ideas for the prevention and treatment of SM.

Th1/Th2因子比例增高是导致自然流产的原因之一，Th1因子拮抗剂的应用又与胎儿畸形发生有关，寻找新的方法维持Th1/Th2因子平衡预防自然流产至关重要。维生素D3具有调节Th1/Th2因子的功能，已用于Th1因子所致疾病的治疗，但其对自然流产母胎界面的调节作用尚未见报道。我们在对早期自然流产母胎界面Th1因子研究的基础上，采集临床标本检测母胎界面维生素D3受体（VDR）、维生素D3代谢的关键酶（1α羟化酶）及Th1/Th2因子表达，确定VDR和1α羟化酶与自然流产及Th1/Th2因子的关系；并取新鲜绒毛和蜕膜组织原代培养滋养细胞和蜕膜间质细胞，构建VDR或1α羟化酶基因沉默细胞模型，或用外源性1,25（OH）2D3后，检测Th1/Th2因子代谢及滋养和蜕膜间质细胞活性，探讨VDR和1α羟化酶基因功能及调控机制，证明维生素D3在预防自然流产中的作用，期待能为自然流产的防治提供新的思路。

本课题依据自然流产母胎界面Th1细胞因子及其受体表达增高、维生素D3可以调控Th1 /Th2免疫趋势的事实，探讨了维生素D3及其代谢的关键酶25-羟维生素D3-1α-羟化酶（1α羟化酶）和维生素D3受体（VDR）在正常早孕（NP）和早期复发自然流产（RM）妇女功能表达及与细胞因子的相关性。主要内容（1）检测RM和NP妇女母胎界面VDR和1α羟化酶mRNA和蛋白的表达水平。（2）正常育龄、有自然流产史的育龄、NP及RM妇女血清VD3、VDR及1α羟化酶的浓度。（3）RM和NP妇女母胎界面VDR和1α羟化酶与Th1/Th2细胞因子表达。（4）1,25(OH)2D3对原代培养的绒毛滋养及蜕膜细胞活性及细胞因子表达的影响。（5）VDR或1α羟化酶静默后，对滋养细胞株细胞活性的影响及细胞因子及VDR靶基因SLC37A2表达水平变化。重要结果及其科学意义：（1）绒毛和蜕膜组织RM 妇女VDR mRNA水平（0.31和0.34）及蛋白表达量（0.77和0.54）低于NP妇女（1.09和1.01；1.42和1.12，P均<0.01）。（2）RM妇女绒毛和蜕膜组织1α羟化酶mRNA水平（0.40和0.44）及蛋白表达量（0.90和1.11）低于NP妇女（1.20 和1.35；1.45 和1.58，p均<0.05）。(3)RM妇女血清VD（34.49μg/L）、VDR（57.55pg/mL）和1α羟化酶（37.87pg/mL）水平低于NP妇女（49.32μg/L，87.07 pg/mL，82.00 pg/mL，P均<0.01）；RM和有流产史的育龄妇女低VD的发生率(43.3%，96.7%)明显增高。上述结果证明VD、VDR和1α羟化酶表达降低与RM有关。（4）RM妇女 VDR和1α羟化酶在间质、滋养层和蜕膜腺上皮细胞表达明显低于NP妇女，提示在不同的细胞VDR和1α羟化酶表达水平不一致。（5）VDR和和1α羟化酶与TNF-α，TNFR1，IFN-γ，IL-2，IL-4, IL-6、IL-10共表达在绒毛和蜕膜细胞。原代培养的滋养细胞经1,25(OH)2D3干预后，D3对TNF-α、TNFR1表达有抑制作用，对IL6、IL-10有促进作用。滋养细胞株实验也证实了VD3对细胞因子有调节作用，同时发现D3对SLC37A2的调节作用，但试验还需进一步重复以确定VD3对调控相关基因表达的机制。