miR-21对慢性睡眠剥夺诱导的青少年晕厥发作血管内皮源性硫化氢调控机制研究

Sleep deprivation increases the risk of syncope in adolescents. The increased plasma hydrogen sulfide (H2S) concentration as well as the vascular endothelial dysfunction were found in the patient, but the reason is unclear by now. This project planned to explore the H2S generation derived from vascular endothelial cells and the way of its regulation. Elucidate the mechanism of vasoconstriction induced by endogenous H2S in adolescents with syncope. First, Population-based case-control study: plasma H2S concentration and miR-21 expression were detected among the sleep deprivation-induced syncope patients and the normal control group. Determine the relationship between the syncope and H2S or miR-21. Second, the model of chronic sleep deprivation-vasovagal rat was induced by sleep deprivation and sinusoidal galvanic vestibular stimulation (sGVS). The H2S derived from endothelium, the expression of miR-21, SP1 mRNA, CSE mRNA and the protein were detected and compared between groups. Third, the 3'UTR amplification of target genes, vector construction, cell transfection and luciferase activity analysis proved SP1 to be the target gene of miR-21. Fourth, miR-21 transiently overexpressed or specifically inhibited were set up by transfection of miR-21 mimics or miR-21 inhibitor into the vascular endothelial cell. Then the effect of miR-21 on CSE mRNA was observed after SP1 mRNA were knocked down by SP1-siRNA. Finally, the mechanism of miR-21 regulates the concentration of vascular endogenous H2S by regulating the expression of CSE mRNA in vascular endothelium via the target gene SP1 were clear. For the first time, this study elucidates the pathways of vascular endothelium H2S production and its regulation way in sleep deprivation and sinusoidal galvanic vestibular stimulation syncope rat model. Explores the mechanism of peripheral vasomotor abnormalities in syncope caused by chronic sleep deprivation, which provides a theoretical basis for the prevention and treatment of syncope in adolescents.

睡眠剥夺增加青少年晕厥发作机率，且患者外周血H2S浓度升高、血管内皮舒张异常，迄今原因未明。本项目深入探索血管内皮源性H2S生成与调节，揭示青少年晕厥发作外周血管扩张的分子基础。通过人群病例对照研究，对比晕厥患者和正常对照组外周血H2S浓度和miR-21表达，确定其与晕厥相关性；建立慢性睡眠剥夺后正弦电流前庭刺激致晕厥大鼠模型，检测血管内皮H2S，miR-21，SP1、CSE转录和蛋白表达；通过荧光素酶活性分析验证SP1是miR-21的靶基因；通过miR-21mimic/inhibitor转染血管内皮细胞，再以SP1siRNA敲减SP1mRNA，最终明确血管内皮miR-21/SP1/CSE/H2S轴的调控通路。本研究将首次阐明睡眠剥夺后正弦电流前庭刺激致晕厥大鼠血管内皮H2S生成与调节通路，探索慢性睡眠剥夺致青少年晕厥外周血管舒缩异常的分子机制，为青少年晕厥病因研究和防治提供理论依据。

晕厥患者血浆硫化氢（Hydrogen sulfide, H2S）浓度显著升高，但原因未知。本项目通过人群研究及动物细胞试验探索晕厥患儿血浆H2S升高的原因及机制。（1）人群研究：研究发现晕厥患儿以POTS、VVS居多，POTS患儿以心率增加为主但血压变化不大，VVS组以血压下降为主，提示疑似POTS患儿可先行直立试验。部分筛检阴性的患儿不排除VVS的可能性，HUTT试验中若患儿耐受可适当增加观察时间，以辅助亚型的鉴别诊断。此外，当BMI＜19.30kg/m2时，晕厥患儿更可能患POTS。综合利用BMI值、病史、体格检查及其他检查方法，对临床医生更快区别POTS和VVS具有一定的意义。对比POTS患者与正常对照组的全血MicroRNA (miRNA)谱，发现POTS患者血浆H2S水平较高，全血miR-21表达水平较高，且miR-21对POTS患者的识别敏感性为92.5%，特异性为100%。全血miR-21水平升高可以作为POTS的一个指标，并可能解释POTS患者血浆H2S水平升高的原因。（2）动物及细胞试验：成功构建正弦电流前庭刺激诱导大鼠血压下降模型，以 1~4mA，0.008~0.5Hz正弦电流前庭刺激可诱发大鼠血压下降、心率减慢和脑血流量降低，同时，可以观察到大鼠经正弦电流前庭刺激后产生晕厥样表现。通过细胞实验，确定miR-21调控CSE产生H2S，HUVEC（人脐静脉内皮细胞）中CSE表达量为3pg/ml。通过miR-21 mimic或miR-21inhibitor转染HUVEC，并以SP1-siRNA敲减SP1 mRNA观察miR-21对CSE mRNA的调控作用。初步预实验证实miR-21通过靶基因SP1调节血管内皮CSE mRNA的表达进而调节血管内皮源性H2S的浓度。（3）在探索晕厥患儿治疗方面，本课题研究发现CGRP在预测盐酸米多君治疗小儿VVS疗效中的价值，治疗前测量CGRP水平，可以快速预测患儿对该药物治疗的疗效，实现个体化治疗的目标，治疗敏感性可提升至97.7%。此外，POTS患儿77.5%存在睡眠质量问题，且唾液皮质醇能反映POTS临床症状的严重程度，应用睡眠治疗法可改善POTS患儿的临床症状，有效的敏感性为91.7%，特异性为43.1%。