本文選題：鎂 + 血管平滑肌細胞��； 參考：《河北醫(yī)科大學》2017年碩士論文

【摘要】：目的:慢性腎臟病(Chronic Kidney disease,CKD)的發(fā)病率逐年升高,目前已成為一個世界性的問題,研究表明心血管疾病(Cardiovascular Disease,CVD)是CKD患者死亡的首位原因,其中血管鈣化是心血管疾病(Cardiovascular Disease,CVD)發(fā)病的獨立危險因素。血管中膜鈣化是CKD患者血管鈣化的主要特點。血管平滑肌細胞(Vascular Smooth Muscle Cells,VSMCs)是血管中膜的重要組成部分,研究發(fā)現(xiàn),鎂可以抑制血管中膜鈣化,但其具體機制及其復雜,目前尚不明確。本研究主要探討高鎂對慢性腎衰竭大鼠血管鈣化的影響及可能機制。方法:1大鼠VSMCs的原代培養(yǎng)及實驗分組貼壁法原代細胞培養(yǎng),3~4代細胞供實驗用,將細胞隨機分為A、正常對照組;B、高磷組(10mmol/Lβ-GP);C、高鎂組(10mmol/Lβ-GP+3mmol/L Mg SO4)D、鎂通道抑制劑(2-APB)干預組(10mmol/Lβ-GP+3mmol/L Mg SO4+10-4mol/L 2-APB)。各組VSMCs培養(yǎng)7天。2血管環(huán)的制備及分組1只SD雄性大鼠220-250 g,取其胸主動脈,去除內(nèi)、外膜后,并將血管切成約2mm-3mm長的血管環(huán)進行培養(yǎng),血管環(huán)被隨機分為4組:a、正常對照組;b、高磷組;c、高鎂組:d、鎂通道抑制劑干預組。各組培養(yǎng)基同細胞培養(yǎng)。各組血管環(huán)培養(yǎng)7天。3培養(yǎng)7天后測定各組VSMCs和血管環(huán)的鈣化水平各組VSMCs的鈣化染色應用茜素紅(Alizarin red stain)法,血管化的鈣化染色應用Von Kossa染色法;應用鄰甲酚酞絡合酮比色法測定各組VSMCs和血管環(huán)的鈣含量。4培養(yǎng)7天后各組VSMCs的堿性磷酸酶(ALP)活性測定各組細胞培養(yǎng)7天后棄上清,應用PBS洗3次,加入0.1%Triton X-100置于4℃環(huán)境中過夜,并依據(jù)ALP活性試劑盒說明測定其活性,每組實驗均重復3次。5反轉(zhuǎn)錄-PCR、Western Blot檢測各組VSMCs中RUNX2基因及蛋白的表達應用反轉(zhuǎn)錄-PCR方法檢測培養(yǎng)7天后各組VSMCs RUNX2基因的表達。應用Western Blot方法檢測各組VSMCs培養(yǎng)7天后RUNX2蛋白的表達。6免疫組織化學法檢測血管環(huán)Runx2表達應用免疫組織化學方法檢測各組血管環(huán)培養(yǎng)7天后RUNX2的表達。7反轉(zhuǎn)錄-PCR、Western Blot檢測各組VSMCs中目的基因及蛋白的表達應用反轉(zhuǎn)錄-PCR方法檢測培養(yǎng)7天后各組VSMCs LTCCα1c、β3基因的表達。應用Western Blot檢測培養(yǎng)7天后各組VSMCs LTCCα1c、β3蛋白的表達。8培養(yǎng)7天后各組VSMCs內(nèi)鈣離子濃度測定培養(yǎng)7天后各組VSMCs內(nèi)鈣離子濃度應用熒光探針Fluo-3/AM方法檢測。9統(tǒng)計學方法應用SPSS 17.0統(tǒng)計學軟件進行統(tǒng)計學處理,符合正態(tài)分布的計量資料用均數(shù)±標準差((?)±s)表示,多個樣本均數(shù)比較采用單因素方差分析,組間兩兩比較用S-N-K檢驗(Student-Newman-Keuls,SNK)。P0.05認為差異有統(tǒng)計學意義。結(jié)果:1高鎂抑制高磷誘導的VSMCs和血管環(huán)鈣化茜素紅染色結(jié)果示,與正常對照組相比,高磷組可見大量橘紅色鈣鹽沉積;而與高磷組相比,高鎂組VSMCs鈣鹽沉積顯著減少,鎂通道抑制劑干預組VSMCs鈣鹽沉積較鎂干預組明顯增加。與茜素紅染色相一致,鈣含量測定結(jié)果顯示高磷誘導的鈣鹽沉積可被鎂干預組明顯降低(P0.05),而2-APB可抑制這種作用(P0.05)。各組血管環(huán)Von Kossa染色結(jié)果示:與正常對照組相比,高磷組血管環(huán)中膜可見大量棕黑色顆粒沉積,高鎂可明顯減少棕黑色顆粒的沉積。鈣含量測定結(jié)果與血管環(huán)Von Kossa染色結(jié)果一致示:與正常對照組相比,高磷組血管環(huán)的鈣含量明顯升高(P0.05),而高鎂組血管環(huán)鈣含量明顯低于高磷組(P0.05)。2高鎂抑制高磷誘導的VSMCs ALP活性各組VSMCs ALP活性測定結(jié)果示:高磷組及鎂通道抑制劑干預組ALP活性明顯高于高鎂組(P0.05)。3高鎂抑制高磷誘導的VSMCs RUNX2的表達情況反轉(zhuǎn)錄-PCR和Western Blot結(jié)果顯示,各組VSMCs培養(yǎng)7天后,較高鎂組RUNX2表達水平相比,高磷組及鎂通道抑制劑干預組明顯增加(P0.05)。4高鎂抑制高磷誘導的血管環(huán)RUNX2的表達各組血管環(huán)培養(yǎng)給予不同干預7天后,免疫組織化學檢測結(jié)果示,高磷組血管細胞質(zhì)內(nèi)外可見棕黃色物質(zhì)沉積,明顯高于正常對照組,差異有統(tǒng)計學意義(P0.05);與高磷組相比,高鎂組血管細胞質(zhì)內(nèi)外棕黃色物質(zhì)變淡,著色區(qū)域面積明顯減少,Runx2表達明顯下降,差異有統(tǒng)計學意義(P0.05),與高鎂組相比,鎂通道抑制劑干預組血管細胞質(zhì)內(nèi)外棕黃色物質(zhì)增加,差異有統(tǒng)計學意義(P0.05)。5高鎂抑制高磷誘導的VSMCs LTCCα1c、β3的表達反轉(zhuǎn)錄-PCR和Western Blot結(jié)果示,各組VSMCs培養(yǎng)7天后,高鎂組LTCCα1c、β3的表達水平明顯低于高磷組及鎂通道抑制劑干預組(P0.05)。6高鎂抑制高磷誘導的VSMCs胞外鈣離子內(nèi)流效應各組VSMCs Fluo-3/AM熒光強度和熒光顯色結(jié)果顯示,與正常對照組相比,高磷組VSMCs熒光強度明顯增加(P0.05);與高磷組相比,高鎂組胞內(nèi)熒光強度下降(P0.05)。結(jié)論:1本研究結(jié)果顯示高鎂可抑制高磷誘導的VSMCs鈣化。2其可能機制之一是高鎂通過抑制VSMCs LTCCα1C、β3亞基的表達、阻滯鈣離子內(nèi)流、降低Runx2的表達,抑制VSMCs發(fā)生表型轉(zhuǎn)化,進而抑制了高磷誘導的鈣化過程的發(fā)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.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R692;R543

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本文編號：2110349