本文選題：動脈培養(yǎng) + 高磷血癥　； 參考：《重慶醫(yī)科大學》2017年碩士論文

【摘要】：第一部分體外臍靜脈高磷培養(yǎng)血管鈣化模型目的:通過建立動脈體外高磷培養(yǎng)模型模擬體內高磷血癥致血管鈣化(vessels calcification,VC)過程。方法:臍靜脈M199培養(yǎng)基無血清培養(yǎng),分為正常組(Pi=1mmol/L)、高磷組(Pi=3mmol/L)。利用Von Kossa染色法和茜素紅染色法對臍靜脈切片進行染色,觀察并比較兩種方法的優(yōu)缺點,HE染色對臍靜脈組織形態(tài)進行評估。結果:體外血管鈣化模型建模成功,高磷組鈣化明顯高于正常組(P0.01)。體外培養(yǎng)在第二十天時組織結構清晰,細胞核染色清楚,胞漿紅染。對比兩種鈣化染色方法,Von kossa染色法對血管中層微鈣化的顯色比茜素紅更清晰。結論:20天體外高磷(Pi=3mmol/L)培養(yǎng)建立血管鈣化模型,選擇Von kossa染色鑒定動脈中層微鈣化。第二部分磷誘導動脈中層血管平滑肌細胞鈣化與SCAP表達的關系目的:通過慢病毒敲除臍靜脈SCAP,研究在高磷致動脈鈣化體外培養(yǎng)模型中,SCAP蛋白對鈣鹽沉積的影響。方法:臍靜脈自母體取出后,無血清培養(yǎng)2天,換液加慢病毒培養(yǎng)3天,加磷培養(yǎng)20天分為四組,對照組、高磷組、SCAP敲除組、高磷+SCAP敲除組。VK染色定量血管鈣化,WB檢測四組組織SCAP敲除效果和RUNX2表達情況。結果:Von kossa染色和血管鈣定量結果表明體外培養(yǎng)的臍靜脈鈣化高磷組高于正常組(P0.01),高磷+SCAP蛋白敲除組高于SCAP蛋白敲除組(P0.01);western blotting結果示RUNX2蛋白在高磷組表達量明顯高于對照組(P0.01),高磷+SCAP敲除組與SCAP敲除組比較RUNX2蛋白表達量顯著下降(P0.05)。結論:SCAP蛋白可能參與了磷致動脈中層鈣鹽沉積過程,并對鈣化起促進作用。第三部分血清磷通過調節(jié)SCAP蛋白過表達加速動脈粥樣硬化進程目的:通過臨床慢性腎病(CKD)透析患者的基本資料、橈動脈組織切片染色和臍靜脈體外模擬高磷血癥培養(yǎng),探討磷與動脈粥樣硬化性疾病的關系。方法:按照排除納入條件篩選CKD病例,分為動脈粥樣硬化(atherosclerosis,AS)組和非動脈粥樣硬化(NAS)組,用Logistic回歸分析檢驗兩組有統(tǒng)計學差異的指標。將收取橈動脈按照患者血清Pi水平分為高磷組和正常組,免疫熒光方法檢測橈動脈中SREBPs裂解激活蛋白(SREBP Cleavage Activating Protein,SCAP)、LDL受體((LDL receptor,LDLr)、膽固醇調節(jié)原件結合蛋白2(Sterol Regulatory Element Banding Protein 2,SREBP2)和羥甲基戊二酸單酰輔酶A還原酶(Hydroxy Methylglutaryl Coenzyme A Reductase,HMGCo AR)表達情況。WB和免疫組化檢測高磷培養(yǎng)臍靜脈SCAP、LDLr、SREBP2、HMGCo AR表達量。結果:logistic回歸分析結果表明,血清磷是AS的獨立致病因素。橈動脈免疫熒光染色顯示,高磷組SCAP、EREBP2、HMGCo AR、LDLr、RUNX2表達均高于正常組(P0.05)。WB示臍靜脈高磷組與對照組比較SCAP、SREBP2、HMGCo A、LDLr均上調(P0.05),免疫組化顯示結果與WB一致。結論:血清磷可能通過上調SCAP蛋白、打破SCAP-SREBP2通路的負反饋調節(jié),導致內膜細胞內脂質蓄積從而加速動脈粥樣硬化病的發(fā)生發(fā)展。
[Abstract]:Part one: vascular calcification model of umbilical vein hyperphosphorous culture in vitro objective: to simulate the process of vessel calcification induced by hyperphosphatemia in vivo by establishing an in vitro vascular calcification model. Methods: the umbilical vein M199 medium was serum-free and was divided into normal group (1 mmol / L) and high phosphorus group (3 mmol / L). Umbilical vein sections were stained with Von Kossa staining and alizarin red staining. The advantages and disadvantages of the two methods were observed and compared to evaluate the histological morphology of umbilical vein by HE staining. Results: the model of vascular calcification in vitro was successfully established, and the calcification in high phosphorus group was significantly higher than that in normal group (P 0.01). On the 20th day, the tissue structure was clear, the nucleus was stained clearly and the cytoplasm was red stained. Compared with two calcification staining methods, Von kossa staining was more clear than alizarin red in the development of microcalcification in vascular media. Conclusion the vascular calcification model was established by 20 days in vitro culture with high phosphorous content in vitro, and Von kossa staining was selected to identify the microcalcification in the arterial media. The second part is the relationship between phosphorus-induced calcification of vascular smooth muscle cells and the expression of SCAP. Objective: to investigate the effect of phosphorus-induced calcification of arterial smooth muscle cells on calcium deposition in vitro by lentivirus knockout of umbilical vein. Methods: after umbilical vein was removed from mother, serum free culture for 2 days, liquid exchange plus lentivirus culture for 3 days, phosphorus culture for 20 days were divided into four groups: control group, high phosphorus group, SCAP knockout group. High phosphorous SCAP knockout group. VK staining quantitative vascular calcification wideband detected SCAP knockout effect and RUNX2 expression in four groups. Results the expression of RUNX2 protein in high phosphorus group was significantly higher than that in SCAP protein knockout group and that in high phosphorous SCAP group was higher than that in SCAP protein knockout group. The results showed that the expression of RUNX2 protein in high phosphorus group was significantly higher than that in high phosphorus group. Compared with the SCAP knockout group, the expression of RUNX2 protein decreased significantly in the control group (P 0.01) and the high phosphorus SCAP knockout group (P 0.05). Conclusion the protein of: SCAP may be involved in the process of calcium deposition in the middle layer of artery induced by phosphorus, and may play a role in promoting calcification. The third part of serum phosphorus accelerates the process of atherosclerosis by regulating the overexpression of SCAP protein. Objective: to study the basic data of hemodialysis patients with chronic nephropathy, radial artery tissue section staining and umbilical vein culture in vitro to simulate hyperphosphatemia. To investigate the relationship between phosphorus and atherosclerotic diseases. Methods: the patients with CKD were selected according to the exclusion criteria and divided into two groups: atherosclerotic atherosclerosis group and non-atherosclerotic group. Logistic regression analysis was used to test the statistical difference between the two groups. The received radial artery was divided into high phosphorus group and normal group according to the serum Pi level of the patients. Detection of the expression of SREBPs lytic activator protein (SREBP Cleavage Activating protein), SREBPs receptor (2(Sterol Regulatory Element Banding Protein _ 2 SREBP2) and hydroxyxy Methylglutaryl Coenzyme A Reductase (HMG) in radial artery by immunofluorescence And immunohistochemistry was used to detect the expression of HMGCo AR in cultured umbilical vein SCAPP LDLrN SREBP 2 and HMGCo AR. Results the results of logistic regression analysis showed that serum phosphorus was an independent pathogenic factor of as. Radial artery immunofluorescence staining showed that the expression of RUNX2 in SCAPP EREBP2HMGCo ARN LDLrN RUNX2 in high phosphorus group was higher than that in normal control group (P 0.05). Compared with control group, SCAPP SREBP2HMGCo AMGCo ALDLr was up-regulated, and the immunohistochemical results were consistent with WB. Conclusion: serum phosphorus may accelerate the development of atherosclerotic disease by up-regulating the SCAP protein and breaking the negative feedback regulation of SCAP-SREBP2 pathway, resulting in the accumulation of lipid in intimal cells.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R692;R54

【參考文獻】

相關期刊論文 前1條

1 覃春美;歐三桃;;高磷血癥與慢性腎臟病血管鈣化研究進展[J];現(xiàn)代臨床醫(yī)學;2015年01期

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本文編號：1956311