高镁通过L型钙通道调控高磷诱导的大鼠血管平滑肌细胞钙化发生的研究

发布时间：2018-07-09 18:59

本文选题：镁 + 血管平滑肌细胞　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:慢性肾脏病(Chronic Kidney disease,CKD)的发病率逐年升高,目前已成为一个世界性的问题,研究表明心血管疾病(Cardiovascular Disease,CVD)是CKD患者死亡的首位原因,其中血管钙化是心血管疾病(Cardiovascular Disease,CVD)发病的独立危险因素。血管中膜钙化是CKD患者血管钙化的主要特点。血管平滑肌细胞(Vascular Smooth Muscle Cells,VSMCs)是血管中膜的重要组成部分,研究发现,镁可以抑制血管中膜钙化,但其具体机制及其复杂,目前尚不明确。本研究主要探讨高镁对慢性肾衰竭大鼠血管钙化的影响及可能机制。方法:1大鼠VSMCs的原代培养及实验分组贴壁法原代细胞培养,3~4代细胞供实验用,将细胞随机分为A、正常对照组;B、高磷组(10mmol/Lβ-GP);C、高镁组(10mmol/Lβ-GP+3mmol/L Mg SO4)D、镁通道抑制剂(2-APB)干预组(10mmol/Lβ-GP+3mmol/L Mg SO4+10-4mol/L 2-APB)。各组VSMCs培养7天。2血管环的制备及分组1只SD雄性大鼠220-250 g,取其胸主动脉,去除内、外膜后,并将血管切成约2mm-3mm长的血管环进行培养,血管环被随机分为4组:a、正常对照组;b、高磷组;c、高镁组:d、镁通道抑制剂干预组。各组培养基同细胞培养。各组血管环培养7天。3培养7天后测定各组VSMCs和血管环的钙化水平各组VSMCs的钙化染色应用茜素红(Alizarin red stain)法,血管化的钙化染色应用Von Kossa染色法;应用邻甲酚酞络合酮比色法测定各组VSMCs和血管环的钙含量。4培养7天后各组VSMCs的碱性磷酸酶(ALP)活性测定各组细胞培养7天后弃上清,应用PBS洗3次,加入0.1%Triton X-100置于4℃环境中过夜,并依据ALP活性试剂盒说明测定其活性,每组实验均重复3次。5反转录-PCR、Western Blot检测各组VSMCs中RUNX2基因及蛋白的表达应用反转录-PCR方法检测培养7天后各组VSMCs RUNX2基因的表达。应用Western Blot方法检测各组VSMCs培养7天后RUNX2蛋白的表达。6免疫组织化学法检测血管环Runx2表达应用免疫组织化学方法检测各组血管环培养7天后RUNX2的表达。7反转录-PCR、Western Blot检测各组VSMCs中目的基因及蛋白的表达应用反转录-PCR方法检测培养7天后各组VSMCs LTCCα1c、β3基因的表达。应用Western Blot检测培养7天后各组VSMCs LTCCα1c、β3蛋白的表达。8培养7天后各组VSMCs内钙离子浓度测定培养7天后各组VSMCs内钙离子浓度应用荧光探针Fluo-3/AM方法检测。9统计学方法应用SPSS 17.0统计学软件进行统计学处理,符合正态分布的计量资料用均数±标准差((?)±s)表示,多个样本均数比较采用单因素方差分析,组间两两比较用S-N-K检验(Student-Newman-Keuls,SNK)。P0.05认为差异有统计学意义。结果:1高镁抑制高磷诱导的VSMCs和血管环钙化茜素红染色结果示,与正常对照组相比,高磷组可见大量橘红色钙盐沉积;而与高磷组相比,高镁组VSMCs钙盐沉积显著减少,镁通道抑制剂干预组VSMCs钙盐沉积较镁干预组明显增加。与茜素红染色相一致,钙含量测定结果显示高磷诱导的钙盐沉积可被镁干预组明显降低(P0.05),而2-APB可抑制这种作用(P0.05)。各组血管环Von Kossa染色结果示:与正常对照组相比,高磷组血管环中膜可见大量棕黑色颗粒沉积,高镁可明显减少棕黑色颗粒的沉积。钙含量测定结果与血管环Von Kossa染色结果一致示:与正常对照组相比,高磷组血管环的钙含量明显升高(P0.05),而高镁组血管环钙含量明显低于高磷组(P0.05)。2高镁抑制高磷诱导的VSMCs ALP活性各组VSMCs ALP活性测定结果示:高磷组及镁通道抑制剂干预组ALP活性明显高于高镁组(P0.05)。3高镁抑制高磷诱导的VSMCs RUNX2的表达情况反转录-PCR和Western Blot结果显示,各组VSMCs培养7天后,较高镁组RUNX2表达水平相比,高磷组及镁通道抑制剂干预组明显增加(P0.05)。4高镁抑制高磷诱导的血管环RUNX2的表达各组血管环培养给予不同干预7天后,免疫组织化学检测结果示,高磷组血管细胞质内外可见棕黄色物质沉积,明显高于正常对照组,差异有统计学意义(P0.05);与高磷组相比,高镁组血管细胞质内外棕黄色物质变淡,着色区域面积明显减少,Runx2表达明显下降,差异有统计学意义(P0.05),与高镁组相比,镁通道抑制剂干预组血管细胞质内外棕黄色物质增加,差异有统计学意义(P0.05)。5高镁抑制高磷诱导的VSMCs LTCCα1c、β3的表达反转录-PCR和Western Blot结果示,各组VSMCs培养7天后,高镁组LTCCα1c、β3的表达水平明显低于高磷组及镁通道抑制剂干预组(P0.05)。6高镁抑制高磷诱导的VSMCs胞外钙离子内流效应各组VSMCs Fluo-3/AM荧光强度和荧光显色结果显示,与正常对照组相比,高磷组VSMCs荧光强度明显增加(P0.05);与高磷组相比,高镁组胞内荧光强度下降(P0.05)。结论:1本研究结果显示高镁可抑制高磷诱导的VSMCs钙化。2其可能机制之一是高镁通过抑制VSMCs LTCCα1C、β3亚基的表达、阻滞钙离子内流、降低Runx2的表达,抑制VSMCs发生表型转化,进而抑制了高磷诱导的钙化过程的发生。
[Abstract]:Objective: the incidence of Chronic Kidney disease (CKD) is increasing year by year, and it has become a worldwide problem. The study shows that cardiovascular disease (Cardiovascular Disease, CVD) is the first cause of death in CKD patients, and vascular calcification is an independent risk factor for the pathogenesis of cardiovascular disease (Cardiovascular Disease, CVD). Vascular calcification is the main feature of vascular calcification in CKD patients. Vascular smooth muscle cell (Vascular Smooth Muscle Cells, VSMCs) is an important part of the vascular membrane. It is found that magnesium can inhibit the calcification of the membrane in the blood vessels, but its specific mechanism and complexity is not clear. This study mainly deals with high magnesium in rats with chronic renal failure. The effect and possible mechanism of vascular calcification. Methods: the primary culture of VSMCs in 1 rats and experimental group adherence method were used for cell culture and 3~4 generation of cells for experimental use. The cells were randomly divided into A, normal control group, B, high phosphorus group (10mmol/L beta -GP), C, high magnesium group (10mmol/L beta -GP+ 3mmol/L Mg SO4), magnesium channel inhibitor intervention group Mol/L Mg SO4+10-4mol/L 2-APB). Each group of VSMCs culture 7 days.2 vascular ring preparation and group 1 SD male rats 220-250 g, take the thoracic aorta, remove the inner and outer membrane, and cut the vessel into about 2mm-3mm long blood vessel ring, and the vascular ring is randomly divided into 4 groups: A, normal control group; B, high phosphorus group; C, magnesium channel inhibitor dry Group culture medium and cell culture. Each group of vascular rings was cultured for 7 days and 7 days after.3 culture, the calcification levels of VSMCs and vascular rings were measured in each group. Alizarin red (Alizarin red stain) method was applied to the calcification staining of VSMCs, and Von Kossa staining was used for calcification of vascular calcification, and the determination of VSMCs and blood vessels in each group by Phthalo phthalein complexing colorimetry. VSMCs alkaline phosphatase (ALP) activity of each group was determined for 7 days after 7 days of culture. The cell culture of each group abandoned supernatant after 7 days. PBS was washed 3 times and 0.1%Triton X-100 was placed in the environment for the night, and the activity was determined according to the ALP Activity Kit. 3 times.5 reverse transcriptional -PCR was repeated in each group. Western Blot was used to detect the VSMCs in VSMCs. Expression of UNX2 gene and protein using reverse transcription -PCR method to detect the expression of VSMCs RUNX2 gene in each group after 7 days. Western Blot method was used to detect the expression of RUNX2 protein in each group of VSMCs culture for 7 days. The expression of Runx2 expression of vascular ring Runx2 was detected by immunohistochemistry The expression of.7 reverse transcription -PCR, Western Blot was used to detect the expression of target gene and protein in each group of VSMCs, and the expression of VSMCs LTCC a 1C, beta 3 gene was detected by reverse transcription method for 7 days. Western Blot was detected and cultured for 7 days, and the expression of beta 3 protein was determined for 7 days, and the concentration of calcium ion in each group was determined for 7 days. 7 days after culture, the concentration of calcium ion in VSMCs was detected by the fluorescence probe Fluo-3/AM method, and the statistical method of.9 was detected by SPSS 17 statistics software, which was in accordance with the normal distribution of the mean number + + standard deviation ((?) + s), and the ratio of the number of samples was compared with the single factor analysis of variance, and 22 of the groups was compared with S-N-K. Student-Newman-Keuls, SNK.P0.05 thought the difference was statistically significant. Results: 1 high phosphorus inhibition of high phosphorus induced VSMCs and calcified alizarin red staining of vascular ring showed that a large number of orange red calcium salts were deposited in the high phosphorus group compared with the normal control group; compared with the high phosphorus group, the deposition of VSMCs calcium salt in the high magnesium group decreased significantly, and the magnesium channel inhibitor dry was significantly lower than that in the high phosphorus group. The pre group VSMCs calcium salt deposition was significantly higher than that of the magnesium intervention group. The calcium content assay showed that the calcium content induced by high phosphorus could be significantly reduced by the magnesium intervention group (P0.05), while 2-APB could inhibit the effect of calcium salt (P0.05). The Von Kossa staining results in each group of vascular rings showed that the middle membrane of the high phosphorus group was compared with the normal control group. A large number of brown black particles were deposited, and high magnesium could obviously reduce the deposition of brown black particles. The results of calcium content determination and vascular ring Von Kossa staining results showed that the calcium content of the vascular ring in the high phosphorus group was significantly higher than that in the normal control group (P0.05), while the calcium content in the high magnesium group was significantly lower than the high phosphorus group (P0.05).2 with high magnesium and high phosphorus inhibition. The results of VSMCs ALP activity of the induced VSMCs ALP activity showed that the ALP activity of the high phosphorus group and the magnesium channel inhibitor group was significantly higher than the high magnesium group (P0.05).3 high magnesium inhibition of the high phosphorus induced VSMCs RUNX2 expression, the reverse transcription -PCR and Western Blot results showed that the higher magnesium group was cultured for 7 days, and the higher magnesium group expression level was higher than that of the higher magnesium group. Phosphorus group and magnesium channel inhibitor intervention group significantly increased (P0.05).4 high magnesium inhibition of high phosphorus induced vascular ring RUNX2 expression in each group of vascular ring culture for 7 days after different intervention, immunohistochemical detection results showed that the cytoplasm and cytoplasm of high phosphorus group inside and outside the cytoplasm of brown and yellow material deposition, obviously higher than the normal control group, the difference was statistically significant ( P0.05): compared with the high phosphorus group, the brown and yellow substance inside and outside the cytoplasm of the high magnesium group became pale, the area of the coloring area decreased obviously, and the expression of Runx2 decreased significantly (P0.05). Compared with the high magnesium group, the brown and yellow substance inside and outside the cytoplasm and cytoplasm of the magnesium channel inhibitor group increased, and the difference was statistically significant (P0.05).5 high magnesium inhibition The high phosphorus induced VSMCs LTCC alpha 1C, the expression of -PCR and Western Blot in the expression of beta 3, the expression of LTCC a 1c in the high magnesium group for 7 days, the expression level of the beta 3 was significantly lower than that of the high phosphorus group and the magnesium channel inhibitor intervention group (P0.05).6 high magnesium inhibition of the extracellular calcium ion internal flow effect induced by high phosphorus and high phosphorus. The light coloration results showed that the fluorescence intensity of VSMCs in the high phosphorus group was significantly increased (P0.05) compared with the normal control group. Compared with the high phosphorus group, the intracellular fluorescence intensity of high magnesium group decreased (P0.05). Conclusion: 1 the results showed that high magnesium could inhibit the high phosphorus induced VSMCs calcification.2, one of the possible mechanisms was the high magnesium table by inhibiting the VSMCs LTCC a 1C, the beta 3 subunit. It can block the calcium influx, reduce the expression of Runx2, inhibit the phenotypic transformation of VSMCs, and inhibit the occurrence of high phosphorus induced calcification.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692;R543

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