盐对内皮细胞肾上腺髓质素分泌和表达的影响及机制的研究

发布时间：2018-01-03 17:12

本文关键词：盐对内皮细胞肾上腺髓质素分泌和表达的影响及机制的研究　出处：《泸州医学院》2012年硕士论文　论文类型：学位论文

【摘要】：目的：（1）研究高盐对HUVECs增殖活性的影响。（2）研究高盐对HUVECsADM、ADMR mRNA表达的影响，探讨其可能的信号通路。(3)研究高盐对HVECs凋亡的影响及可能机制。方法：1.用含10%胎牛血清的RPMI1640培养基培养HUVECs，选3-9代对数生长期的细胞进行实验。2.用不同浓度NaCl（对照组、137mmol/L、142mmol/L、147mmol/L、152mmol/L、157mmol/L）干预HVECs24h后，用CCK-8试剂盒检测盐对HUVECs增殖活性的影响。3.将实验分两部分进行，实验1：实验共分六组，分别为对照组：正常培养HUVECs未加入任何药物；137mmol/L组、142mmol/L组、147mmol/L组、152mmol/L组、157mmol/L组分别为：培养24小时后加入不同浓度NaCl，使终浓度分别为137mmol/L、142mmol/L、147mmol/L、152mmol/L、157mmol/L，继续作用24小时后，用RT-PCR检测细胞中ADM及ADMR mRNA的表达；用酶联免疫吸附法(ELISA法)检测细胞培养液中ADM及ADMR浓度；用AnnexinV-FTTC/PI染色流式细胞仪检测细胞凋亡。实验2：通过实验1选出最佳盐浓度为152mmol/L，继续进行实验。实验分六组如下：152mmol/l组：培养48小时后加入NaCl，终浓度为152mmol/L，继续作用24小时。PD98059(ERK抑制剂)组、SP600125(JNK抑制剂）组、SB203508(P38抑制剂)组、Staurosporine(PKC抑制剂)组、LY-294002（PI3K抑制剂)组：培养24小时后，先分别用PD98059、SP600125、SB203508、Staurosporine、LY-294002作用细胞24h再加入NaCl（终浓度为152mmol/L），继续作用24小时。（其中除Staurosporine为10nmol/L外，其余均为10umol/L），并以与实验1相同方法检测ADM的浓度，ADM及ADMR mRNA的表达及细胞凋亡的影响。结果：1、对照组、137mmol/L组、142mmol/L组、147mmol/L组、152mmol/L组、157mmol/L组细胞增殖活力分别为（0.998±0.197)、（0.952±0.091）、（0.946±0.076）、（0.811±0.145）、（0.802±0.116）、（0.659±0.15）。137mmol/L组、142mmol/L组、147mmol/L组、152mmol/L组与对照组比较（P0.05），157mmol/L组与对照组比较（P0.05）。2、RT-PCR检测内皮细胞中ADM及ADMR mRNA的表达结果显示：对照组、137mmol/L组、142mmol/L组、147mmol/L组、152mmol/L组、157mmol/L组ADM mRNA的IODADM/IODGAPDH分别为0.03±0.01、0.10±0.01、0.20±0.01、0.21±0.02、0.43±0.02、0.40±0.04，各组与对照组相比（P0.05）；对照组、137mmol/L组、142mmol/L组、147mmol/L组、152mmol/L组、157mmol/L组ADMR mRNA的IODADMR/IODGAPDH分别为0.07±0.02、0.12±0.02、0.13±0.02、0.16±0.02、0.28±0.03、0.17±0.02，各组与对照组相比P均小于0.05；152mmol/L组、PD98059组、SP600125组、SB203508组、Staurospo rine组、LY-294002组ADM mRNA的IODADM/IODGAPDH分别为0.10±0.01、0.15±0.02、0.29±0.04、0.31±0.02、0.17±0.02、0.15±0.01， SP600125组、SB203508组与152mmol/L组相比（P0.05），，PD98059组、Staurosporine组、LY-294002组与152mmol/L组相比（P0.05）；152mmol/L组、PD98059组、SP600125组、SB203508组、Staurosporine组、LY-294002组ADMR mRNA的IODADMR/IODGAPDH分别为0.08±0.01、0.20±0.01、0.22±0.03、0.23±0.02、0.09±0.01、0.09±0.02，PD98059组、SP600125组、SB203508组与152mmol/L组相比（P0.05），Staurosporine组、LY-294002组与152mmol/L组相比(P0.05）。3、ELISA法检测ADM结果显示：对照组、137mmol/L组、142mmol/L组、147mmol/L组、152mmol/L组、157mmol/L组ADM的浓度（pg／ml）分别为20.13±0.15、34.91±0.59、41.15±0.79、41.30±1.13、43.16±1.04、34.68±0.27，各组与对照组比较(P0.05)，152mmol/L组与157mmol/L组相比(P0.05)。152mmol/L组、PD98059组、SP600125组、SB203508组、Staurosporine组、LY-294002组ADM的浓度（pg／ml）分别为43.16±1.04、51.50±8.28、72.23±2.23、75.33±2.93、44.95±3.33、36.33±3.78，SP600125组、SB203508组与152mmol/L组比较(P0.05)，PD98059组、Staurosporine组、LY-294002组与152mmol/L组比较(P0.05)。4、AnnexinV-FTTC/PI染色流式细胞仪检测细胞凋亡率结果显示：对照组、152mmol/L组、PD98059组、SP600125组、SB203508组、Staurosporine组、LY-294002组凋亡率分别为2.3±0.73%、30.07±2.13%、14.35±1.56%、13.47±0.99%、10.19±1.12%、35.7±2.01%、59.8±3.19%，各组与对照组相比(P0.05），PD98059组、SP600125组、SB203508组、LY-294002组与152mmol/L组相比（P0.05），Staurosporine组与152mmol/L组相比(P0.05)。结论：1、高盐抑制内皮细胞增殖。2、高盐通过MAPK通路抑制内皮细胞ADM、ADMR mRNA的表达。3、高盐能诱导内皮细胞ADM的分泌，而JNK、P38信号传导通路抑制盐诱导内皮细胞ADM的分泌， PKC、PI3K、ERKs信号传导通路与ADM分泌无关。4、盐呈剂量依赖性诱导内皮细胞凋亡，可能与P38、JNK、PI3K、ERK信号通路有关，与PKC信号通路无关，这可能是盐致高血压的机制之一。
[Abstract]:Objective: To study the effect of high salt (1) on proliferation of HUVECs. (2) research on high salt HUVECsADM, ADMR mRNA expression, to investigate the possible signal pathway. (3) effects of high salt on HVECs apoptosis and its possible mechanism. Methods: 1. with RPMI1640 containing 10% fetal bovine serum a medium HUVECs, 3-9 generation of the logarithmic phase cells were.2. with different concentrations of NaCl (control group, 137mmol/L, 142mmol/L, 147mmol/L, 152mmol/L, 157mmol/L) of HVECs24h treated with CCK-8 kit to detect the effect of salt on proliferation of HUVECs.3. experiment is divided into two parts, the experimental rats were divided into 1 the six group, including control group: normal cultured HUVECs without any drug; 137mmol/L group, 142mmol/L group, 147mmol/L group, 152mmol/L group, 157mmol/L group respectively: after 24 hours of incubation with different concentrations of NaCl, the final concentration were respectively 137mmol/L, 142mmol/L, 147mmol/L 152mmol/L, 157mmol/L, and continue to function after 24 hours, expression of RT-PCR was detected by ADM and ADMR mRNA cells; using enzyme-linked immunosorbent assay (ELISA) and ADM ADMR concentration detection cell culture; staining of apoptosis was detected by flow cytometry AnnexinV-FTTC/PI. Experiment 2: through the experiment 1 to choose the optimum salt concentration for 152mmol/L, to continue the experiment. The experiment was divided into six groups as follows: 152mmol/l group: cultured for 48 hours after joining NaCl, the final concentration of 152mmol/L, to 24 hours.PD98059 (ERK inhibitor) group, SP600125 (JNK inhibitor) group, SB203508 (P38 inhibitor) group, Staurosporine (PKC inhibitor) group, LY-294002 (PI3K inhibitor) group: after 24 hours of incubation, respectively with PD98059, SP600125, SB203508, Staurosporine, LY-294002, 24h cells before adding NaCl (final concentration 152mmol/L), to 24 hours. (in addition to the Staurosporine 10nmol/L, the I was 10umol/L), and to the same concentration and 1 experimental methods for detecting ADM, influence of ADM expression and cell apoptosis and ADMR mRNA. Results: 1, the control group, 137mmol/L group, 142mmol/L group, 147mmol/L group, 152mmol/L group, 157mmol/L group, the proliferation of the cells were (0.998 + 0.197), (0.952 + 0.091), (0.946 + 0.076), (0.811 + 0.145), (0.802 + 0.116), (0.659 + 0.15).137mmol/L group, 142mmol/L group, 147mmol/L group, 152mmol/L group compared with the control group (P0.05), 157mmol/L group compared with the control group (P0.05.2), the expression of endothelial cell detection in RT-PCR ADM and ADMR mRNA showed: control group, 137mmol/L group, 142mmol/L group, 147mmol/L group, 152mmol/L group, 157mmol/L group and ADM mRNA IODADM/IODGAPDH were 0.03 + 0.01,0.10 + 0.01,0.20 + 0.01,0.21 + 0.02,0.43 + 0.02,0.40 + 0.04, each group compared with the control group (P0.05); the control group, 137mmol/L group, 142mmol/L group, 147mm Ol/L group, 152mmol/L group, 157mmol/L group and ADMR mRNA IODADMR/IODGAPDH were 0.07 + 0.02,0.12 + 0.02,0.13 + 0.02,0.16 + 0.02,0.28 + 0.03,0.17 + 0.02, each group compared with the control group P was less than 0.05; 152mmol/L group, PD98059 group, SP600125 group, SB203508 group, Staurospo rine group, LY-294002 group and ADM mRNA IODADM/IODGAPDH respectively 0.10. 0.01,0.15 + 0.02,0.29 + 0.04,0.31 + 0.02,0.17 + 0.02,0.15 + 0.01, SP600125 group, SB203508 group compared with 152mmol/L group (P0.05), PD98059 group, Staurosporine group, LY-294002 group compared with 152mmol/L group (P0.05); 152mmol/L group, PD98059 group, SP600125 group, SB203508 group, Staurosporine group, LY-294002 group and ADMR mRNA IODADMR/IODGAPDH respectively. 0.08 + 0.01,0.20 + 0.01,0.22 + 0.03,0.23 + 0.02,0.09 + 0.01,0.09 + 0.02, PD98059 group, SP600125 group, SB203508 group and 152mmol/L group compared (P0.05), Staurosporine group, LY-294002 Compared with group 152mmol/L (P0.05).3, ELISA test results showed: ADM control group, 137mmol/L group, 142mmol/L group, 147mmol/L group, 152mmol/L group, 157mmol/L group, ADM concentration (PG / ml) were 20.13 + 0.15,34.91 + 0.59,41.15 + 0.79,41.30 + 1.13,43.16 + 1.04,34.68 + 0.27, compared with the control group (P0.05), group 152mmol/L compared with group 157mmol/L (P0.05).152mmol/L group, PD98059 group, SP600125 group, SB203508 group, Staurosporine group, LY-294002 group, ADM concentration (PG / ml) were 43.16 + 1.04,51.50 + 8.28,72.23 + 2.23,75.33 + 2.93,44.95 + 3.33,36.33 + 3.78, SP600125 group, SB203508 group and 152mmol/L group (P0.05), PD98059 group, Staurosporine group, LY-294002 group and 152mmol/L group (P0.05.4), AnnexinV-FTTC/PI staining and cell apoptosis rate were detected by flow cytometry. The results showed that the control group, 152mmol/L group, PD98059 group, SP600125 group, SB203508 group, S In group taurosporine, the apoptosis rate of LY-294002 group was 2.3 + 0.73%, 30.07 + 2.13%, 14.35 + 1.56%, 13.47 + 0.99%, 10.19 + 1.12%, 35.7 + 2.01%, 59.8 + 3.19%, each group compared with the control group (P0.05), PD98059 group, SP600125 group, SB203508 group, LY-294002 group compared with 152mmol/L group (P0.05), Staurosporine group compared with 152mmol/L group (P0.05). Conclusion: 1. High salt inhibit endothelial cell proliferation.2, high salt inhibit endothelial cell ADM through MAPK signaling pathway, the expression of.3 ADMR mRNA, high salt secretion, can induce endothelial cells ADM and JNK secretion, P38 signal transduction pathway inhibits salt induced endothelial cells ADM PKC, PI3K, ERKs signaling pathway and ADM secretion independent of.4, salt dose dependently induced apoptosis of endothelial cells, and P38, JNK, PI3K, ERK signal pathway, independent of PKC signaling pathway, which may be caused by salt mechanism of high blood pressure.

【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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