新型干细胞iPSC-MSCs缓解ox-LDL诱导的HUVEC氧化应激损伤的研究

A study on the alleviate effect of iPSC-MSCs on oxidative stress injury in HUVEC induced by ox-LDL

目的探讨诱导性多能干细胞源性间充质干细胞(iPSC-MSCs)对氧化型低密度脂蛋白(ox-LDL)诱导的人脐静脉内皮细胞(HUVEC)氧化应激损伤的缓解作用及其机制。方法建立人脐静脉内皮细胞(HUVEC)的ox-LDL损伤模型,采取条件培养基预孵育和Transwell装置共培养2种方式发挥iPSC-MSCs的作用;细胞增殖-毒性检测(CCK8)法测定细胞活力,DCFH-ROS荧光探针法检测细胞内活性氧(ROS)水平,Western Blot法测定细胞内烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶4型(NOX4)的蛋白表达,荧光定量PCR检测核因子相关因子2(Nrf2)和血红素氧合酶1(HO-1)的mRNA表达。结果与iPSC-MSCs条件培养基(CM)共孵育后,ox-LDL-HUVEC模型产生的ROS水平明显下降(P<0.05),且下降幅度与iPSC-MSC-CM提取过程中的共孵育时间有关,在共孵育24 h CM(24 hcm)组达到最大(P<0.05);与iPSC-MSCs进行Transwell共培养后,ox-LDL-HUVEC模型细胞活力显著提高(P<0.05),NOX4蛋白表达显著下降(P<0.05),Nrf2和HO-1 mRNA表达明显增加(P<0.05);iPSC-MSCs-Transwell共培养对单HUVEC的NOX4表达无明显影响,但可显著上调其Nrf2和HO-1的mRNA表达水平(P<0.05)。结论 iPSCMSCs可缓解ox-LDL引起的HUVEC细胞活力损害,该过程可能与抑制ROS水平增高,下调NOX4表达及促进Nrf2和HO-1转录增多有关,提示iPSC-MSCs在动脉粥样硬化及氧化应激性疾病方面具有应用前景。

Objective To investigate the alleviative effect of mesenchymal stem cells derived from induced pluripotent stem cell(iPSC-MSCs)on oxidized low density lipoprotein(ox-LDL)induced oxidative stress injury in human umbilical vein endothelial cells(HUVEC)and its probable mechanism.Methods The ox-LDL-HUVEC injury model was developed,and iPSC-MSCs were applied to observe its effect on the model.The application of iPSC-MSCs to HUVECs was developed in two ways:conditioned medium(CM)pre-incubation and Transwell co-culture.Cell viability was measured using cell counting kit 8(CCK8).Intracellular reactive oxygen species(ROS)level was measured using DCFH-ROS probing method.The expression of nicotinamide adenine dinucleotide phosphate(NADPH)oxidase type 4(NOX4)protein was detected by Western Blot.Quantitive real-time PCR was applied to detect the mRNA expression of Nuclear Factor Erythroid 2-Related Factor 2(Nrf2)and heme oxygenase-1(HO-1).Results After pre-incubation with iPSC-MSC-CM,the ROS level produced by the ox-LDL-HUVEC model was significantly lowered(P<0.05).The extent of this effect was related to the coincubation time-length during the iPSC-MSC-CM extraction process and reached its maximum in the 24-hour co-incubation group(24 hcm,P<0.05).After the Transwell co-culture applied with iPSC-MSCs,the model demonstrated a significant higher level of cell viability(P<0.05),down regulation of NOX4 protein expression(P<0.05)and up regulation of Nrf2 and HO-1 transcription(P<0.05),compared to the model group that wasn’t applied with co-culture.And the iPSC-MSCsTranswell co-culture demonstrated no effect on the expression of NOX4 but a significant up regulation of Nrf2 and HO-1 mRNA expression(P<0.05)in HUVEC.Conclusion iPSC-MSCs were capable of alleviating the damage of cell viability induced by ox-LDL in HUVEC,which was probably related to the inhibition of intracellular ROS increase,the down regulation of NOX4 protein expression and up regulation of Nrf2 and HO-1 transcription.The iPSC-MSCs have a great prospect of application to atherosclerosis and oxidative stress injury-related diseases.