CD47-TSP1通路在血管内皮细胞衰老中的促进作用

发布时间：2018-06-17 17:43

本文选题：CD47 + TSP1　；参考：《吉林大学》2016年博士论文

【摘要】：随着人口预期寿命的延长和出生率的不断降低,人口老龄化已成为日渐突出的社会现象。随之带来一系列社会和医疗相关的挑战,突出方面就是心血管疾病发生率的不断升高。流行病学调查已证实老龄是心血管疾病的高危因素,研究显示老龄人口血管功能受损,表现为血管顺应性下降,自我修复能力降低,最终导致动脉粥样硬化等血管疾病。其中的重要机制,就是内皮细胞的老化。细胞的衰老是各种内外环境压力共同作用的结果。衰老细胞内基因表达异于相对年轻细胞,导致细胞功能受损、分裂停止。内皮细胞的老化原因多样,主要是细胞不断分裂及血液内过氧化物的损伤,最终促进动脉粥样硬化及血栓的形成。因此,延缓内皮细胞的衰老可能在减少心血管疾病方面有着重大的意义。血小板反应蛋白(thrombospondin-1,TSP1)是一种细胞基质糖蛋白,最早发现以未活化形式储存于血小板中,也可来源于内皮细胞、成纤维细胞、平滑肌细胞等多种细胞类型。TSP1有多重结构域,可与多种细胞表面受体结合,例如CD47、CD36、低密度脂蛋白受体相关蛋白1(LRP1)等。正常人体循环系统内TSP1浓度仅有20?g/L,但对调节血管功能和内环境稳态却有重要意义。近年来研究发现,TSP1与配体CD47结合后可起到抑制内皮细胞NO通路的作用,从而抑制血管舒张。此外,TSP1可通过拮抗血管内皮生长因子(VEGF)作用抑制血管新生,还可通过上调TGF-?水平从而促进糖尿病相关动脉粥样硬化斑块形成。有研究发现,TSP1在衰老组织中表达上升,并推测可能与衰老生物体组织血供减少这一现象相关。CD47又称为整合素相关蛋白(IAP),广泛表达于多种组织细胞表面。CD47与巨噬细胞上的SIRPα结合后可保护细胞免于被巨噬细胞吞噬,与TSP1结合则可有效抑制内皮细胞NO通路,介导血管收缩;还有研究显示CD47与TSP1的多肽4N1K结合后可介导多种细胞死亡(包括人的脑微血管内皮细胞),但具体机制不详;最近有研究称CD47缺失的小鼠肺内皮细胞具有干细胞特性,可分化为多种组织细胞,具体机制仍不明了。我们在前期研究中发现CD47缺失的鼠脑微血管内皮细胞具有更强的传代能力,结合之前的文献报道,我们考虑CD47-TSP1通路对内皮细胞的衰老可能有一定的调控作用,而相关研究报道至今不明确,因此CD47-TSP1对内皮细胞衰老调控及其机制具有很大的研究价值。首先,我们提取8周龄雌性CD47缺失及WT小鼠的脑微血管内皮细胞作为研究对象,进行cell counting kit-8(cck-8)、CFSE、Brd U、内皮细胞成管等实验,比较CD47缺失和WT小鼠内皮细胞增殖能力及功能,从而判断细胞衰老情况。随后我们对细胞进行连续传代培养,使用?-半乳糖苷酶(?-gal)染色(衰老细胞可被绿染),更直观地对比细胞的衰老水平。进而我们对两组细胞TSP1表达水平进行分析,明确TSP1表达在衰老细胞中的改变。接下来,我们对两组细胞的细胞周期及其相关细胞周期抑制剂p53、p21、p16,细胞周期激酶CDK4、CDK6、Cyclin D1,下游E2F通路分子CDK1、CDK2等进行分析,探究CD47对细胞衰老调控可能的机制。最后,为研究CD47-TSP1通路在内皮细胞衰老调控中的作用,我们分别单独加入TSP1及TSP1抗体,使用?-半乳糖苷酶染色、荧光定量等方法明确CD47-TSP1通路在内皮细胞衰老中的作用。结果显示:1、对CD47缺失及WT小鼠的血管内皮细胞进行cck-8、Brd U、CFSE等实验发现,与WT相比,CD47缺失组内皮细胞在相同生长时间内细胞数目、增殖细胞数目和分裂次数均更多,说明CD47缺失的内皮细胞增殖能力更强,而增殖能力强弱是细胞衰老水平判断的重要指标,间接说明CD47缺失的内皮细胞抵抗衰老的能力更强。2、通过体外成管实验(tube formation assay)发现,CD47缺失组内皮细胞无论在细胞年轻(第2代)还是年老(第6代)时,成管能力均较WT组明显增强;将CD47缺失和WT组内皮细胞分别与matrigel混合后打入小鼠皮下,发现CD47缺失组内皮细胞与matrigel形成的团块含有更多的红细胞及血管管腔,体外和体内实验均表明CD47缺失组内皮细胞功能较WT组更强。功能强弱同样代表细胞的衰老程度,因此更进一步说明CD47缺失的内皮细胞抵抗衰老的能力更强。3、随着细胞传代,衰老细胞(?-gal染色阳性)比例不断增多,细胞周期分析显示S期细胞比例不断降低,与WT组相比,CD47缺失组内皮细胞?-gal染色阳性率较低,S期比例相对较高,说明CD47缺失组中衰老比例较低,表明在同等传代次数下,CD47缺失的内皮细胞衰老得到延缓;在两组细胞中,TSP1表达均随细胞传代逐渐增多,说明TSP1可能在内皮细胞衰老中起重要作用,而CD47缺失组衰老程度较低,结合CD47为TSP1的重要受体考虑,推测CD47-TSP1通路可能在内皮细胞衰老中起到促进作用,而这一作用可能通过细胞周期的调控来实现。进一步对细胞周期抑制剂p53、p21、p16,细胞周期激酶CDK4、CDK6、Cyclin D1,下游E2F通路分子CDK1、CDK2等进行分析,发现p53、p21、p16在WT组中高表达,CDK4、CDK6、Cyclin D1,下游E2F通路分子CDK1、CDK2等在WT中低表达,推测CD47-TSP1通路通过调控细胞周期中的关键分子来达到控制细胞周期的目的,从而实现对内皮细胞衰老的调节。4、为证实TSP1在内皮细胞衰老中具有促进作用,我们在细胞培养体系中分别单独添加TSP1蛋白及TSP1抗体,结果发现在年轻细胞的中(第2代),WT组添加TSP1蛋白可使?-gal染色阳性细胞比例增加,细胞成管能力变差,说明TSP1可促进细胞衰老;在CD47缺失组中未观察到此类现象,表明TSP1的作用是CD47介导实现的。而第2代细胞中,无论WT还是CD47缺失组,未发现TSP1抗体的添加与对照相比有显著改变,考虑为2代细胞尚年轻的缘故,TSP1-CD47通路尚未充分激发。对年老细胞(第4代)添加TSP1抗体后发现,WT组?-gal染色阳性细胞比例显著降低,p53、p21、p16表达下降,CDK4、CDK6、Cyclin D1,下游E2F通路分子CDK1、CDK2等表达上升,说明拮抗TSP1作用可延缓细胞衰老,而CD47缺失组中未观察到此类现象,更进一步证实CD47-TSP1通路在内皮细胞衰老中的促进作用。综上所述,我们研究得到的结论是:CD47-TSP1通路在内皮细胞衰老中起促进作用,这一作用可能通过对细胞周期的调控来实现。CD47-TSP1通路对细胞周期的调控是通过调节细胞周期抑制剂p53、p21、p16,细胞周期激酶CDK4、CDK6、Cyclin D1等细胞周期中的关键分子来实现,CD47-TSP1通路可促进细胞周期抑制剂并抑制细胞周期激酶表达,从而抑制细胞周期进行,最终促进细胞衰老。研究结果表明,TSP1水平对心血管系统衰老程度具有提示作用,调控TSP1的表达在防治心血管疾病中具有重要意义。
[Abstract]:With the prolongation of the life expectancy and the decreasing of the birth rate, the aging of the population has become an increasingly prominent social phenomenon. With a series of social and medical related challenges, the incidence of cardiovascular disease is increasing. Epidemiological investigation has confirmed that aging is a high risk factor for cardiovascular disease. The damage of vascular function in the aged population shows that the vascular compliance decreases, the ability of self repair is reduced, and the vascular diseases such as atherosclerosis are caused by the aging of endothelial cells. The aging of the cells is the result of the interaction of various internal and external environmental pressures. The cell function is damaged and the division stops. The aging causes of endothelial cells are various, mainly cell division and blood peroxide damage, which ultimately promote atherosclerosis and thrombosis. Therefore, the delay of endothelial cell aging may be of great significance in reducing cardiovascular disease. (thrombospondin-1, TSP1) is a cell matrix glycoprotein, which was first found in unactivated forms of platelets, or derived from endothelial cells, fibroblasts, smooth muscle cells, and many other cell types,.TSP1 with multiple domains, such as CD47, CD36, low density lipoprotein receptor related proteins. 1 (LRP1) and so on. The concentration of TSP1 in normal human circulation system is only 20? G/L, but it is important for regulating vascular function and homeostasis. In recent years, it has been found that TSP1 and ligand CD47 can inhibit the NO pathway of endothelial cells and inhibit vasodilatation. In addition, TSP1 can be used to antagonize vascular endothelial growth factor (VEGF). By inhibiting angiogenesis, it can also promote the formation of atherosclerotic plaques related to diabetes by increasing the TGF-? Levels. Studies have found that the expression of TSP1 in senescent tissues is increased, and that it may be associated with the decrease of blood supply in aging organisms..CD47 is also called integrin related protein (IAP), widely expressed in a variety of tissues. The binding of.CD47 on the cell surface and SIRP alpha on macrophages protects the cells from being phagocytic by macrophages. Binding to TSP1 can effectively inhibit the NO pathway of endothelial cells and mediate the vasoconstriction. There are also studies showing that CD47 and TSP1 polypeptide 4N1K can mediate multiple cell deaths (including human brain microvascular endothelial cells), but the specific mechanism is specific. We have recently found that CD47 missing mouse lung endothelial cells have the characteristics of stem cells and can differentiate into a variety of tissue cells. The specific mechanism is still unknown. In our previous study, we found that the CD47 deficient rat brain microvascular endothelial cells have a stronger generation ability. We consider the CD47-TSP1 pathway to the endothelium. Cell aging may have a certain regulatory role, and the related research reports are not clear so far, so CD47-TSP1 has great research value on the regulation and mechanism of endothelial cell aging. First, we extract 8 weeks old female CD47 deletion and WT mouse brain microvascular endothelial cells as the research object, and carry out cell counting kit-8 (CCK-8), CF SE, Brd U, endothelial cell formation and so on, compared CD47 deletion and proliferation and function of WT mouse endothelial cells to judge cell aging. Then we carried out continuous subculture of cells, using? - galactosidase (? -gal) staining (aging cells can be green), more intuitively compared cell senescence level. And then we to two groups The expression level of cell TSP1 was analyzed to identify the changes in TSP1 expression in senescent cells. Next, we analyzed the cell cycle of two groups and related cell cycle inhibitors p53, p21, p16, cell cycle kinase CDK4, CDK6, Cyclin D1, downstream E2F pathway molecule CDK1, CDK2 and so on. Finally, in order to study the role of the CD47-TSP1 pathway in the regulation of endothelial cell senescence, we separately added TSP1 and TSP1 antibodies, using galactosidase staining, and fluorescence quantitative methods to determine the role of CD47-TSP1 pathway in endothelial cell aging. Results: 1, CCK-8, Brd in CD47 deletion and WT mice endothelial cells. U, CFSE and other experiments found that compared with WT, the number of cells, the number of proliferating cells and the number of splits of the endothelial cells in the CD47 deletion group were more in the same time of growth, indicating that the proliferation ability of the endothelial cells with the CD47 deletion was stronger and the proliferation ability was the important index of the cell aging level, which indirectly indicated that the CD47 missing endothelial cells resisted and declined. The old ability of.2 was stronger. Through the tube formation assay test, it was found that the endothelial cells in the CD47 deletion group increased obviously in the young (second generation) or the old (sixth generation) groups. The CD47 deletion and the WT group endothelial cells were mixed with Matrigel in the mice subcutaneously, and the CD47 deletion group of endothelial cells was found. The group formed with Matrigel contained more red cells and vascular lumen. In vitro and in vivo, the function of endothelial cells in the CD47 deletion group was stronger than that of the WT group. The function strength also represented the senescence of the cells. Therefore, it further indicated that the endothelial cells with CD47 deletion were more capable of resisting senescence,.3, with cell passage, senescent cells ( The proportion of -gal staining positive) increased continuously. Cell cycle analysis showed that the proportion of S cells decreased continuously. Compared with group WT, the positive rate of -gal staining in CD47 deletion group was lower and the proportion of S phase was relatively high, indicating that the proportion of senescence in the CD47 deletion group was lower, indicating that under the same passages, the endothelial cell senescence missing CD47 was delayed; the aging of CD47 missing endothelial cells was delayed; In the two groups, the expression of TSP1 increased gradually with the cell passage, indicating that TSP1 may play an important role in the aging of endothelial cells, while the senescence of CD47 deletion group is low. Considering that CD47 is an important receptor for TSP1, it is presumed that the CD47-TSP1 pathway may play a role in the aging of endothelial cells, and this effect may be through the cell cycle. Further study the cell cycle inhibitors p53, p21, p16, cell cycle kinase CDK4, CDK6, Cyclin D1, and the downstream E2F pathway molecule CDK1, CDK2 and so on. In order to achieve the purpose of controlling cell cycle, the key molecules can realize the regulation of endothelial cell senescence. In order to confirm that TSP1 can promote the aging of endothelial cells, we added TSP1 protein and TSP1 antibody separately in the cell culture system. The results found that in the year light cells (second generations), the WT group added TSP1 protein. The proportion of -gal staining positive cells increased and the cell formation of cells became poor, indicating that TSP1 could promote cell senescence. No such phenomenon was observed in the CD47 deletion group, indicating that the effect of TSP1 was mediated by CD47. In the second generation cells, no matter the WT or the CD47 deletion group, the addition of the TSP1 anti body was significantly changed compared with the control, considering the 2 generation. After the cells were young, the TSP1-CD47 pathway was not fully stimulated. After adding TSP1 antibodies to the aged cells (fourth generation), the proportion of WT group -gal staining positive cells decreased significantly, the expression of p53, p21, p16 decreased, CDK4, CDK6, Cyclin D1, downstream E2F pathways and other expressions increased, indicating that the antagonistic effect could delay cell senescence. This phenomenon is not observed in the missing group and further confirms the role of the CD47-TSP1 pathway in the aging of endothelial cells. To sum up, we have concluded that the CD47-TSP1 pathway plays a role in the aging of endothelial cells, which may be mediated by the regulation of the cell cycle to achieve the cell cycle of the.CD47-TSP1 pathway. The regulation is realized by regulating key molecules in cell cycle, such as cell cycle inhibitor p53, p21, p16, cell cycle kinase CDK4, CDK6, Cyclin D1 and so on. CD47-TSP1 pathway can promote cell cycle inhibitors and inhibit cell cycle kinase expression, thus inhibiting cell cycle and eventually promoting cell senescence. The results show TSP1 water. Ping has a suggestive effect on the aging of the cardiovascular system. Regulating the expression of TSP1 is of great importance in the prevention and treatment of cardiovascular diseases.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R54

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