本文關鍵詞：鹽對內皮細胞腎上腺髓質素分泌和表達的影響及機制的研究　出處：《瀘州醫(yī)學院》2012年碩士論文　論文類型：學位論文

  更多相關文章： 鹽 腎上腺髓質素 凋亡 信號通路

【摘要】：目的：（1）研究高鹽對HUVECs增殖活性的影響。（2）研究高鹽對HUVECsADM、ADMR mRNA表達的影響，探討其可能的信號通路。(3)研究高鹽對HVECs凋亡的影響及可能機制。方法：1.用含10%胎牛血清的RPMI1640培養(yǎng)基培養(yǎng)HUVECs，選3-9代對數(shù)生長期的細胞進行實驗。2.用不同濃度NaCl（對照組、137mmol/L、142mmol/L、147mmol/L、152mmol/L、157mmol/L）干預HVECs24h后，用CCK-8試劑盒檢測鹽對HUVECs增殖活性的影響。3.將實驗分兩部分進行，實驗1：實驗共分六組，分別為對照組：正常培養(yǎng)HUVECs未加入任何藥物；137mmol/L組、142mmol/L組、147mmol/L組、152mmol/L組、157mmol/L組分別為：培養(yǎng)24小時后加入不同濃度NaCl，使終濃度分別為137mmol/L、142mmol/L、147mmol/L、152mmol/L、157mmol/L，繼續(xù)作用24小時后，用RT-PCR檢測細胞中ADM及ADMR mRNA的表達；用酶聯(lián)免疫吸附法(ELISA法)檢測細胞培養(yǎng)液中ADM及ADMR濃度；用AnnexinV-FTTC/PI染色流式細胞儀檢測細胞凋亡。實驗2：通過實驗1選出最佳鹽濃度為152mmol/L，繼續(xù)進行實驗。實驗分六組如下：152mmol/l組：培養(yǎng)48小時后加入NaCl，終濃度為152mmol/L，繼續(xù)作用24小時。PD98059(ERK抑制劑)組、SP600125(JNK抑制劑）組、SB203508(P38抑制劑)組、Staurosporine(PKC抑制劑)組、LY-294002（PI3K抑制劑)組：培養(yǎng)24小時后，先分別用PD98059、SP600125、SB203508、Staurosporine、LY-294002作用細胞24h再加入NaCl（終濃度為152mmol/L），繼續(xù)作用24小時。（其中除Staurosporine為10nmol/L外，其余均為10umol/L），并以與實驗1相同方法檢測ADM的濃度，ADM及ADMR mRNA的表達及細胞凋亡的影響。結果：1、對照組、137mmol/L組、142mmol/L組、147mmol/L組、152mmol/L組、157mmol/L組細胞增殖活力分別為（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組、142mmol/L組、147mmol/L組、152mmol/L組與對照組比較（P0.05），157mmol/L組與對照組比較（P0.05）。2、RT-PCR檢測內皮細胞中ADM及ADMR mRNA的表達結果顯示：對照組、137mmol/L組、142mmol/L組、147mmol/L組、152mmol/L組、157mmol/L組ADM mRNA的IODADM/IODGAPDH分別為0.03±0.01、0.10±0.01、0.20±0.01、0.21±0.02、0.43±0.02、0.40±0.04，各組與對照組相比（P0.05）；對照組、137mmol/L組、142mmol/L組、147mmol/L組、152mmol/L組、157mmol/L組ADMR mRNA的IODADMR/IODGAPDH分別為0.07±0.02、0.12±0.02、0.13±0.02、0.16±0.02、0.28±0.03、0.17±0.02，各組與對照組相比P均小于0.05；152mmol/L組、PD98059組、SP600125組、SB203508組、Staurospo rine組、LY-294002組ADM mRNA的IODADM/IODGAPDH分別為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組、SB203508組與152mmol/L組相比（P0.05），，PD98059組、Staurosporine組、LY-294002組與152mmol/L組相比（P0.05）；152mmol/L組、PD98059組、SP600125組、SB203508組、Staurosporine組、LY-294002組ADMR mRNA的IODADMR/IODGAPDH分別為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組、SP600125組、SB203508組與152mmol/L組相比（P0.05），Staurosporine組、LY-294002組與152mmol/L組相比(P0.05）。3、ELISA法檢測ADM結果顯示：對照組、137mmol/L組、142mmol/L組、147mmol/L組、152mmol/L組、157mmol/L組ADM的濃度（pg／ml）分別為20.13±0.15、34.91±0.59、41.15±0.79、41.30±1.13、43.16±1.04、34.68±0.27，各組與對照組比較(P0.05)，152mmol/L組與157mmol/L組相比(P0.05)。152mmol/L組、PD98059組、SP600125組、SB203508組、Staurosporine組、LY-294002組ADM的濃度（pg／ml）分別為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組、SB203508組與152mmol/L組比較(P0.05)，PD98059組、Staurosporine組、LY-294002組與152mmol/L組比較(P0.05)。4、AnnexinV-FTTC/PI染色流式細胞儀檢測細胞凋亡率結果顯示：對照組、152mmol/L組、PD98059組、SP600125組、SB203508組、Staurosporine組、LY-294002組凋亡率分別為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%，各組與對照組相比(P0.05），PD98059組、SP600125組、SB203508組、LY-294002組與152mmol/L組相比（P0.05），Staurosporine組與152mmol/L組相比(P0.05)。結論：1、高鹽抑制內皮細胞增殖。2、高鹽通過MAPK通路抑制內皮細胞ADM、ADMR mRNA的表達。3、高鹽能誘導內皮細胞ADM的分泌，而JNK、P38信號傳導通路抑制鹽誘導內皮細胞ADM的分泌， PKC、PI3K、ERKs信號傳導通路與ADM分泌無關。4、鹽呈劑量依賴性誘導內皮細胞凋亡，可能與P38、JNK、PI3K、ERK信號通路有關，與PKC信號通路無關，這可能是鹽致高血壓的機制之一。
[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.

【學位授予單位】：瀘州醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R363

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