本文選題：內(nèi)皮祖細胞 + 維生素D��； 參考：《中南大學(xué)》2014年碩士論文

【摘要】：背景：腎臟是機體供血量最豐富的器官,其多種功能均通過血管得到實現(xiàn),如腎小球濾過、腎小管重吸收等重要生理過程,且目前研究證實血管損害的嚴重程度與腎臟疾病的預(yù)后關(guān)系密切,因此,維持血管正常結(jié)構(gòu)及功能對實現(xiàn)腎臟功能至關(guān)重要。早期血管損害表現(xiàn)為VEC受損、平滑肌細胞遷移及基質(zhì)增生等,其中VEC受損是始動環(huán)節(jié),而晚期血管損害則表現(xiàn)為解剖結(jié)構(gòu)的改變?nèi)缪芫植炕巍ⅹM窄、斷裂甚至完全阻塞進而影響血供。血管損傷治療方法包括病因治療、藥物治療和手術(shù)治療等。藥物主要有抗氧化劑、降壓藥、血管緊張素轉(zhuǎn)換酶抑制劑、血管緊張素受體抑制劑等,但藥物療效甚微僅能夠緩解血管損害進展而并不能從根本上修復(fù)血管。臨床上可通過支架置入、血管成形術(shù)等外科手段治療血管晚期損害,但手術(shù)治療創(chuàng)傷大、風(fēng)險高,且干預(yù)時機晚。研究者發(fā)現(xiàn)機體內(nèi)VEC一旦受損,骨髓中EPCs就可被動員、遷移、歸巢、分化為成熟VEC,以取代損傷的VEC進而修復(fù)血管。研究者提出EPCs移植治療法,且多項動物和人體干預(yù)實驗均證實EPCs移植對組織缺血療效良好。因其療效好,創(chuàng)傷小,干預(yù)時機早,EPCs移植治療已成為血管性疾病的新希望。但該技術(shù)目前仍面臨一些問題如血管修復(fù)需大量EPCs,而骨髓中數(shù)量少,體外培養(yǎng)擴增不能滿足需求,且體外成血管能力不穩(wěn)定。因此,關(guān)于EPCs增殖和成血管能力的研究必將受到關(guān)注。 研究顯示VD缺乏與心血管疾病明顯關(guān)聯(lián),VD干預(yù)對心血管相關(guān)疾病有一定益處,但作用機制尚不清楚。EPCs中也存在VDR表達,利用VD干預(yù)可促進EPCs增殖及成血管能力,并呈VDR數(shù)量依賴性,提示VDR與EPCs增殖及成血管能力有關(guān)。VDR作為核受體與配體-VD結(jié)合可調(diào)節(jié)細胞周期,影響EPCs的增殖、凋亡及衰老,但近期有研究表明VDR可作為第二信使對細胞發(fā)揮調(diào)控作用。那么,VDR在沒有VD干預(yù)的情況下,能否獨立地對EPCs的生物學(xué)功能進行調(diào)控?VDR基因提前轉(zhuǎn)染至EPCs中是否會有利于EPCs移植的治療效果?目前并無相關(guān)研究。 目的：初步研究不同水平VDR基因?qū)PCs增殖、凋亡、衰老、細胞周期及遷移的影響,為EPCs移植治療提供新的視角和理論基礎(chǔ)。 方法：(1)分離大鼠骨髓源單核細胞,定向誘導(dǎo)分化EPCs,并應(yīng)用免疫熒光染色鑒定。(2)構(gòu)建Ad-VDR和Ad-VDR shRNA。分別轉(zhuǎn)染Ad-VDR/Ad-VDR shRNA至EPCs中,以未處理EPCs為對照組,檢測各組VDR mRNA(RT-PCR法)和VDR蛋白質(zhì)(WB法)相對表達水平。(3)設(shè)立6個組：正常水平組即EPCs未處理組、高水平組即轉(zhuǎn)染Ad-VDR至EPCs中、低水平組即轉(zhuǎn)染Ad-VDR shRNA至EPCs中、正常水平+Rapamycin組、高水平+Rapamycin組及低水平-+Rapamycin組。分別檢測各組EPCs增殖(MTT法和BrdU法)、凋亡(EACS法和WB法)、衰老(p-半乳糖苷酶染色法)、細胞周期(FACS法)和遷移(侵襲小室法)。 結(jié)果：(1)成功分離大鼠骨髓源單核細胞,經(jīng)定向誘導(dǎo)分化,免疫熒光染色鑒定細胞為CD34+/CD133+/VEGFR2+細胞,是目前公認的EPCs。(2)轉(zhuǎn)染Ad-VDR至EPCs組的VDR mRNA及VDR蛋白質(zhì)表達水平均明顯高于對照組,而轉(zhuǎn)染Ad-VDR shRNA至EPCs組的VDR mRNA及VDR蛋白質(zhì)表達量均明顯低于對照組。(3)相對于對照組別,高水平組別OD值和BrdU LI均增高,而低水平組別OD值和BrdU LI均降低。高水平組別Caspase3表達量降低,而低水平組別FACS所檢測凋亡率和Caspase3表達量均增高。在衰老檢測中,低水平組別中衰老細胞數(shù)增多,高水平組別和正常水平組別對比無顯著差異。高水平組別中G1期細胞比例降低,S期細胞比例增高,而低水平組別S期細胞比例降低,甚至在低水平+Rapamycin組無S期細胞,G2期細胞比例增高。在遷移檢測中,高水平組別中EPCs遷移細胞數(shù)明顯增多,低水平組別與正常水平組別對比無顯著差異。 結(jié)論：一定水平的VDR基因?qū)S持EPCs正常功能是必需的；VDR基因呈低表達水平時,EPCs的增殖受抑、凋亡和衰老增加、G2/M期過渡受阻。高表達水平的VDR基因可促進EPCs增殖,抑制凋亡,促進G1/S期過渡,對EPCs的遷移有顯著促進作用,但對衰老無明顯影響。
[Abstract]:Background: the kidney is the most abundant organ supplying blood in the body, and its various functions are realized through blood vessels, such as glomerular filtration, reabsorption of renal tubules and other important physiological processes, and the present study confirms that the severity of vascular damage is closely related to the prognosis of renal diseases. Therefore, the normal structure and function of the blood vessels are maintained to achieve the function of the kidney. The early vascular damage is VEC damage, smooth muscle cell migration and matrix hyperplasia, in which VEC damage is the starting link, and late vascular damage is manifested by changes in anatomical structure such as vascular local malformation, stenosis, fracture and even complete obstruction that affect blood supply. The treatment of vascular injury includes etiological treatment, drugs The drugs mainly include antioxidants, antihypertensive drugs, angiotensin converting enzyme inhibitors, angiotensin receptor inhibitors, but the drug has little effect on vascular damage and can not be radically repaired. However, the researchers found that once the VEC was damaged, the EPCs in the bone marrow could be mobilized, migrated, and returned to mature VEC to replace the damaged VEC and then repair the blood vessels. The researchers proposed a EPCs transplantation therapy, and a number of animal and human intervention tests confirmed EPCs transplant. The effect of tissue ischemia is good. Because of its good curative effect, small trauma and early intervention, EPCs transplantation has become a new hope for vascular diseases. However, the technology still faces some problems, such as a large number of EPCs in vascular repair, while the number of bone marrow is few, in vitro culture expansion can not meet the requirement, and in vitro the vascular ability is unstable. Therefore, about EPCs The study of proliferation and angiogenesis will be concerned.
Studies have shown that VD deficiency is significantly associated with cardiovascular disease, VD intervention has certain benefits for cardiovascular related diseases. However, the mechanism of action is still unclear in the presence of VDR expression in.EPCs. VD intervention can promote EPCs proliferation and angiogenesis, and the VDR quantitative dependence. It suggests that VDR is associated with EPCs proliferation and vascular ability as a nuclear receptor and a nuclear receptor. Ligand -VD binding can regulate cell cycle and influence the proliferation, apoptosis and senescence of EPCs, but recent studies have shown that VDR can play a role in regulating cells as second messengers. Then, can VDR regulate the biological function of EPCs independently without VD intervention? Whether the VDR gene is transfected to EPCs in advance will be beneficial to the EPCs shift. The effect of plant therapy? There is no related research at present.
Objective: To study the effects of different levels of VDR gene on the proliferation, apoptosis, senescence, cell cycle and migration of EPCs, and provide a new perspective and theoretical basis for the treatment of EPCs transplantation.
Methods: (1) isolated rat bone marrow mononuclear cells were isolated and induced to differentiate EPCs. (2) Ad-VDR and Ad-VDR shRNA. were transfected into Ad-VDR/Ad-VDR shRNA to EPCs respectively, and the untreated EPCs as the control group, and the relative expression level of VDR mRNA (RT-PCR method) and VDR protein was detected in each group. (3) 6 groups were set up: positive The normal level group was EPCs untreated group, and the high level group was transfected into Ad-VDR to EPCs, and the low level group was transfected into Ad-VDR shRNA to EPCs, normal level +Rapamycin group, high level +Rapamycin group and low level -+Rapamycin group. The EPCs proliferation of EPCs (MTT method and BrdU method), the dying method and the aging (galactosidase staining method) were detected in each group. Cell cycle (FACS) and migration (invasive chamber method).
Results: (1) the mononuclear cells of bone marrow derived from rats were successfully separated and differentiated by directed induction. The immunofluorescence staining was used to identify the CD34+/CD133+/VEGFR2+ cells. It was recognized that the expression level of VDR mRNA and VDR protein in the EPCs. (2) transfected Ad-VDR to EPCs group was significantly higher than that of the control group, while the VDR mRNA and protein transfected from the Ad-VDR shRNA to EPCs group were transfected. The quantity of qualitative expression was significantly lower than that of the control group. (3) the high level group O value and BrdU LI were all higher than the control group, while the low level group O and BrdU LI decreased. The high level group Caspase3 expression decreased, while the low level group FACS detected the apoptosis rate and the Caspase3 expression. In the aging test, the low level group senescence. There was no significant difference in the number of cells in the high level group and the normal level group. The proportion of G1 cells in the high level group decreased, the proportion of S cells increased, while the proportion of S phase cells in the low level group decreased, even in the low level +Rapamycin group, there was no S phase cells, and the proportion of cells in the G2 phase increased. In the migration detection, the EPCs migration in the high-level group was fine. EPCs migration was fine in the high level group. The number of cells increased significantly, and there was no significant difference between low level group and normal group.
Conclusion: a certain level of VDR gene is necessary for the maintenance of normal function of EPCs. When the VDR gene is low expression level, the proliferation of EPCs is inhibited, apoptosis and senescence are increased, and the transition of G2/M phase is blocked. The high expression of VDR gene can promote EPCs proliferation, inhibit apoptosis, promote the transition of G1/S phase, and promote the migration of EPCs, but not to senescence. Obviously.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R692

【共引文獻】

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1 夏晶;DDAH2/ADMA通路在高糖誘導(dǎo)內(nèi)皮祖細胞線粒體功能障礙中的作用及機制[D];中南大學(xué);2013年

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