CACNA1D基因变异与妊娠期高血压疾病免疫耐受异常的关联研究与机制探讨

Hypertensive disorder complicating pregnancy, especially preeclampsia (PE) affected maternal-fetal health and increased cardiovascular disease risk in pregnant women, so an effective risk prediction index is needed. Our previous study found that: (1) progesterone-induced blocker (PIBF) mediated immune tolerance imbalance associated with severe PE. (2) The exon mutation of CACNA1D gene in PE patients was significantly higher than that in normal pregnant women. As the CACNA1D gene encodes a Ca2+ transmembrane pore subunit, we hypothesized that this gene mutation might involve in intracellular Ca2+ concentration, Ca2+/NFAT activation and/or PIBF associated IL-4R/JAK1/STAT6 pathway, then affect Th2 cytokine secretion. In this study, we will construct CACNA1D site-directed mutagenesis Jurkat lymphocyte model, and compare the above indexes in different treatment jurkat lymphocyte groups. We will observe the effect of this site-directed mutated Jurkat lymphocyte on human extravillous trophoblast cells and explore the potential role of the CACNA1D gene mutation in abnormal immune tolerance of PE. Then we will verify the association between CACNA1D gene mutation and PE in PE patients and normal maternal cohorts. We aim to provide a new idea for finding the genetic risk index and treatment of PE.

妊娠期高血压疾病，尤其子痫前期（PE）影响母胎健康，增加孕妇高血压等心血管病发病及死亡风险，但仍缺乏风险预测指标。我们前期研究发现：（1）孕酮诱导封闭因子（PIBF）介导的免疫耐受失衡与重度PE相关。（2）PE患者CACNA1D基因外显子变异明显高于正常孕妇。CACNA1D基因编码钙离子跨膜孔道亚基，提示该基因变异可能影响细胞内Ca2+浓度、Ca2+/NFAT活化和/或PIBF相关IL-4R/JAK1/STAT6通路，影响Th2细胞因子分泌。本研究拟构建CACNA1D基因定点突变Jurkat淋巴细胞模型，比较不同处理组上述指标变化；并观察此定点突变Jurkat淋巴细胞株对人绒毛外滋养层细胞株的影响，探讨CACNA1D基因突变触发PE免疫耐受异常的可能机制；并在PE患者与正常产妇队列中，验证CACNA1D基因变异与PE的关联性，为寻找预测PE的遗传风险指标和治疗提供新思路。

妊娠期高血压疾病是一种特殊类型的高血压。CACNA1D（CaV1.3）基因是LTCC（L型电压门控钙通道)α-1D亚单位，也是一个新发现的血压易感区。本课题研究了子痫前期患者和正常产妇CACNA1D基因的变异情况, 探讨PE患者产前血压与产后3个月蛋白尿的相关性。研究了胎盘中CACNA1D基因定位和表达及可能作用的分子在胎盘中的表达。探索了基因变异对子痫前期患者胎盘微血管生成和外周T细胞亚型的影响。观察了LTCCs蛋白各亚基在人脐静脉内皮细胞上的表达情况，初步探索其通道激动剂及CACNA1D过表达后对HUVECs炎症反应的影响。应用CRISPR-Cas9技术构建了CACNA1D外显子胚系突变大鼠模型，连续测试32周大鼠的收缩压值，评估大鼠组织器官形态改变和血清生化参数等。再使用L型钙通道阻滞剂伊拉地平和内皮素-1抑制剂 BQ-123测试双杂合突变大鼠（CACNA1D p.D307G/p.R1561Q）。再研究CACNA1D 基因突变对人脐静脉内皮细胞基因表达的研究 (HUVEC) 和血管平滑肌细胞 (VSMC)的影响，从而探讨CACNA1D基因变异影响血压的机制。得出结论1.产前高SBP是PE患者产后3个月蛋白尿的独立危险因素。2.CACNA1D变异可能与子痫前期相关。3.LTCCs在内皮细胞中有阳性表达，且可能在介导血管内皮细Ca2+内流中发挥了重要的作用；初步验证LTCCS激动可上调内皮细胞ICAM-1、VCAM-1的表达，并与Ca2+/PKC/Erk/NF-κB信号轴的激活可能相关；CACNA1D过表达后HUVECs分子表达谱有明显变化，其中众多黏附分子表达及白细胞渗出相关信号通路呈现上调和激活趋势，表明Cav1.3过表达后可能参与内皮细胞的炎症反应过程。4.CACNA1D基因是血压调节的关键基因。5.CACNA1D基因变异大鼠可能成为新的高血压大鼠模型，为高血压的诊治提供新思路。