血管生成素对肾小球内皮细胞衰老的影响及其机制探讨
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摘要
第一部分过氧化氢诱导肾小球内皮细胞衰老模型的建立
目的：
在体外培养条件下,正常体细胞在经过有限次数的增殖分裂后即进入不可逆转的分裂停滞状态,这种由于在连续的细胞分裂中端粒逐渐缩短所引发的现象称为细胞复制性衰老。除此以外,氧化应激、某些癌基因过度表达、紫外线、化学诱变剂等应激因素亦可诱导细胞进入一种应激性早衰状态。细胞早衰不依赖端粒的缩短,并且与复制性衰老细胞在形态、功能及生物学特性上具有许多相同的特征。细胞衰老是生物整体衰老的基础,以细胞为模型的衰老生物学研究是当前老年医学研究的重要内容。H202是一种广泛使用的致细胞早衰的诱导剂,通过DNA损伤机理诱导细胞衰老,且外源性上调端粒酶的活性不能抑制其诱导衰老作用。因此本研究首先通过过氧化氢诱导,探讨可否在体外建立成功而稳定的肾小球内皮细胞(MGECs)衰老模型,从而为进一步的实验奠定良好模型基础。
方法：
应用体外细胞培养技术,培养小鼠肾小球内皮细胞,选择过氧化氢(H2O2)作为诱导物。设立正常对照组、H2O2组进行实验。应用p-半乳糖苷酶染色(SA-β-Gal)方法检测衰老细胞阳性率,流式细胞术检测细胞周期,Western blot免疫印迹法检测p16蛋白的表达,通过分析H202对细胞衰老指标的影响鉴定其作用后是否可引起MGECs衰老。同时通过Hoechst-33342染色法及流式细胞术检测H2O2对细胞凋亡的影响。
结果：
1. H2O2(50μmol/L)诱导7天后,在倒置相差显微镜下观察可见肾小球内皮细胞形态发生明显改变,细胞体积增大,胞体扁平,呈现衰老细胞形态；
2. SA-β-Gal染色实验结果显示：正常细胞SA-β-Gal染色几乎无阳性表达(0.76±0.24%),随H2O2作用时间延长阳性细胞数逐渐增多；H202作用3天即可观察到少量SA-β-Gal染色阳性衰老细胞(13.12±1.81%)的出现,继续予以H2O2作用7天后SA-β-Gal染色阳性细胞可达70.47%；
3.对细胞周期的分析发现,与正常对照组相比,H2O2具有阻止肾小球内皮细胞进入增殖期(S期),而将细胞周期阻滞于静止期(Gl期)的作用,差别具有统计学意义(P<0.05)；
4. Western blot实验结果显示,与正常对照组相比H202组p16蛋白的表达显著增高(Control：21.09±0.67 vs. H2O2:69.39±4.10),差别具有统计学意义(P<0.05)；
5. hoechest-33342着染后H2O2组并未出现明显凋亡样细胞改变,同样流式细胞术检测显示正常对照组及H2O2组细胞G1峰前均无凋亡峰出现,细胞凋亡率无显著性差异(3.19±0.49%vs 3.26±0.39%,P>0.05)。
结论：
过氧化氢在体外能成功诱导肾小球内皮细胞衰老。H2O2(50μmol/L)诱导7天后衰老相关SA-β-Gal染色阳性,细胞周期阻滞于G1期,p16蛋白表达上调。且H2O2作用后细胞无异常凋亡发生。过氧化氢诱导肾小球内皮细胞衰老的模型成功、可信。
第二部分血管生成素对过氧化氢诱导的肾小球内皮细胞衰老的作用研究
目的：
衰老是一个自然进程,在所有物种中都有此现象,并导致很多器官出现退行性改变。肾脏是受衰老影响最明显的器官之一,肾脏功能随衰老进程逐渐减退。肾脏衰老时存在血管形成异常,表现为局灶性肾小管周围及肾小球毛细血管密度减少；随着年龄的增长,肾小球出现内皮细胞进行性减少和丢失。但目前有关肾脏毛细血管改变与衰老相关肾脏病变的直接关系尚未明了。血管生成素(Ang)是一组内皮细胞特异性生长因子,可以有效促进内皮细胞的增殖、分化、介导新生血管管腔形成。Angl可促使Tie2磷酸化,提高内皮细胞的存活能力,促进血管生成,而Ang2则作用相反,能抑制Angl介导的Tie2活化。本部分研究拟探讨血管生成素系统是否能够调控肾小球内皮细胞的衰老,进而影响肾小球内皮细胞的功能及肾脏新生血管的生成,并对其作用机制进行初步探讨。
方法：
体外培养小鼠肾小球内皮细胞,设立正常对照组、H2O2组、Angl组、Ang2组进行实验。应用H2O2诱导MGECs衰老,通过p-半乳糖苷酶染色(SA-β-Gal)、流式细胞术检测细胞周期、Western blot检测p16蛋白表达评估细胞衰老指标的变化。应用硝酸还原酶法检测内皮细胞NO的分泌量,ELISA方法检测内皮细胞vWF的分泌量,Matrigel凝胶实验检测内皮细胞血管生成能力,探讨血管生成素对内皮细胞功能的影响。比色法测定细胞裂解液中的丙二醛(MDA)、谷胱甘肽(GSH)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)的水平。Western blot方法检测Tie2、ERK1／2信号通路的变化。
结果：
1.血管生成素对肾小球内皮细胞衰老指标的影响：H2O2组SA-β-Gal阳性率增高,Angl作用后细胞SA-β-Gal着色变浅、阳性率降低,与H2O2组相比其阳性率下降达到51.80%(P<0.05),而Ang2组和H2O2组之间SA-β-Gal阳性率无显著性差异(P>0.05)；H2O2诱导衰老后细胞周期阻滞于Gl期,Angl组Gl期细胞比例下降至74.50+0.16%,S期细胞比例上升至18.89±0.68%,与H202组相比差别有统计学意义(P<0.05),但Ang2组Gl期及S期细胞数与H2O2组无明显差异(P>0.05)；H202组p16蛋白表达增高,Angl作用后p16表达水平较H2O2组下降44.30%(P<0.05),但Ang2对p16蛋白表达无影响(P>0.05)。
2.血管生成素对肾小球内皮细胞功能的影响：H2O2组肾小球内皮细胞NO分泌量明显减少,Angl组细胞NO分泌显著增加(H202：10.26±0.69vs.Angl：15.61±1.61,P<0.05),Ang2组NO分泌量与H2O2组无明显差异(Ang2：10.15±0.42,P>0.05)；H2O2组vWF含量明显升高(P<0.05),Angl作用后vWF含量显著降低(Angl：5.13±0.69 vs H2O2：9.88±0.27,P<0.05),而Ang2对vWF分泌无明显影响(Ang2：10.02±0.31,P>0.05)；H2O2组MGECs管状结构形成能力显著抑制,Angl组细胞管状结构形成明显增加(P<0.05),而Ang2组则与衰老组无明显差异。
3.血管生成素对肾小球内皮细胞氧化应激指标的影响：H2O2组MDA含量增加,与H2O2组相比,Angl组MDA含量降低(H2O2；10.31±0.22 vs.Angl；5.02±0.47,P<0.05),而Ang2组MDA含量则无显著变化(Ang2；9.13±0.81,P>0.05)；与正常对照组相比,H2O2组GSH、SOD、CAT活性均明显降低,与H2O2组相比,Angl作用后GSH、SOD、CAT活性均显著升高(P<0.05),但Ang2组GSH、SOD、CAT活性则无显著性差异。
4.血管生成素对Tie2-ERK1/2通路的影响：Westen blot结果显示Angl组P-Tie2水平明显增高(H2O2：4.12±2.06 vs.Angl：71.09±2.15,P<0.05),Ang2组P-Tie2无明显变化(Ang2：10.07±3.16,P>0.05)；Angl能显著提高细胞P-ERK1/2水平(P<0.05),但Ang2作用未能引起P-ERK1/2的明显变化(P>0.05)。
结论：
Angl对过氧化氢诱导的肾小球内皮细胞衰老有保护作用,可以有效减少细胞的衰老程度,保护肾小球内皮细胞的正常功能,增强细胞抗氧化酶活性,拮抗H2O2引起的细胞氧化损伤。同时,Angl可以激活Tie2、ERK1/2的磷酸化进程。然而Ang2对内皮细胞无上述保护作用。
第三部分血管生成素1对肾小球内皮细胞衰老保护作用的机制探讨
目的：
Angl能与Tie2受体结合并活化Tie2受体,诱导磷酸化的ERK1/2表达升高,进一步发挥其对内皮细胞的增殖、粘附等的作用。在第二部分的研究中,我们的研究结果也提示Angl的确可以启动内皮细胞Tie2及ERK1/2的磷酸化过程。Angl对H2O2所诱导的肾小球内皮细胞衰老的保护作用是否是通过激活Tie2-ERK1/2信号通路所致?为了更进一步探讨Angl对肾小球内皮细胞衰老保护作用的机制,我们用Angl的天然拮抗剂Ang2、Tie2受体的特异性抑制剂sTie2-Fc及MAPK信号通路MEK特异性抑制剂PD98059预处理肾小球内皮细胞,以明确Angl对肾小球内皮细胞衰老的保护作用的信号通路。
方法：
应用体外细胞培养技术,培养小鼠肾小球内皮细胞,设立正常对照组、H2O2组、Angl组、Ang2+Angl组、sTie2-Fc组、PD98059组进行实验。应用H2O2诱导MGECs衰老,通过p-半乳糖苷酶染色(SA-β-Gal)、流式细胞术检测细胞周期、Western blot检测p16蛋白表达评估细胞衰老指标的变化。应用硝酸还原酶法检测内皮细胞NO的分泌量,ELISA方法检测内皮细胞vWF的分泌量,Matrigel凝胶实验检测内皮细胞血管生成能力,探讨血管生成素对内皮细胞功能的影响。Western blot方法检测Tie2、ERK1/2信号通路的变化。
结果：
1. Tie2-ERK1/2通路抑制剂对肾小球内皮细胞衰老指标的影响：与Angl组比较,Ang2+Ang1组、sTie2-Fc组及PD98059组SA-β-Gal阳性细胞数显著增加(Ang2:63.94±2.05, sTie2-Fc:72.56±1.51, PD98059:73.31±2.73 vs.Ang1:18.67±2.73, P<0.05); Ang2、sTie2-Fc及PD98059作用后,细胞周期分布比例仍呈现衰老细胞特征,细胞周期被阻滞于G1期,S期细胞较少,与Angl组相比,差别有统计学意义(P<0.05)；Ang2、sTie2-Fc及PD98059能阻断Angl对p16蛋白表达降低的作用(Ang2：58.39±2.02,sTie2-Fc: 61.08±3.10, PD98059: 62.19±3.72vs. Ang1: 21.16±1.11, P＜0.05);
2. Tie2-ERK1/2通路抑制剂对肾小球内皮细胞功能的影响：Ang2、sTie2-Fc及PD98059可以下调Angl对内皮细胞NO分泌的促进作用(Ang2：11.92±0.44, sTie2-Fc: 10.22±0.36,PD98059:10.87±0.66 vs. Ang1: 16.38±0.35,P<0.05)；与Angl组相比,予以Ang2、sTie2-Fc及PD98059作用后细胞vWF分泌量显著增加(P<0.05)；Ang2+Angl组、sTie2-Fc组及PD98059组细胞血管腔形成能力下降(Ang2: 23.11±2.29, sTie2-Fc: 15.21±3.31,PD98059：18.56±2.36 vs.Angl：57.66±3.17,P<0.05)；
3. Tie2-ERK1/2通路抑制剂对Tie2-ERK1/2通路的影响：Westen blot结果显示Ang2及sTie2-Fc能显著抑制Angl引起的肾小球内皮细胞P-ERK1/2水平的提高(Ang2: 47.12±2.89, sTie2-Fc: 35.62±3.71 vs. Ang1:135.16±2.27,P<0.05)；PD98059不能抑制Angl引起的Tie2磷酸化(PD98059：63.72±2.87 vs.Angl：61.25±1.91,P>0.05)
结论：
血管生成素样1可能通过Tie2-ERK1/2信号通路而调控过氧化氢诱导的肾小球内皮细胞衰老。
PART I The established model of mouse glomerular endothelial cell senescence induced by hydrogen peroxide
OBJECTIVE:
Normal primary cells proliferate a limited number of times in vitro and activate senescence process due to progressive telomere shortening. Furthermore, cells may also undergo acute stress-induced senescence, which could be triggered by cumulative stimuli involving oxidation, ultraviolet(UV) light, toxic compounds, as well as oncogens. Stress induces senescence through telomere-independent mechanisms, and displays all the major characteristics of replicatively senescent cells. The molecular basis of aging in organs is not well established, but organ aging may reflect aspects of cellular senescence involved with molecular pathway changes. In recent years, much attention has been devoted to studying the relevance of cellular senescence to aging and disease. H2O2, a common causative agent of cellular senescence, can produce various types of DNA damage, which represents the type of stress-induced senescence via DNA damage and cannot be rescued by over-expression of telomerase reverse transcriptase(TERT).The purpose of the study is to investigate whether H2O2 can induce mouse glomerular endothelial cells(MGECs) senescence in vitro.
METHODS:
The MGECs stimulated with hydrogen peroxide(H2O2) were cultured in vitro,and were devided into control group、H2O2 group. The senescent status of cells were verified by senescence-associated P-Galactosidase (SA-β-Gal) staining. The cell cycle of mouse glomerular endothelial cells was analyzed by flow cytometry. The expression of p16 was detected by Western Blot. The senescent of MGECs which was induced by H2O2, was evaluated according above detection. Furthermore, the apoptosis characteristic of MGECs was observed by Hoechst-33342 staining and flow cytometry.
RESULTS:
1. MGECs treated with H2O2 (50μM) for 1 week exhibited an enlarged size and flattened morphology, characteristic of the senescent phenotype of MGECs.
2. The staining of SA-β-Gal assay show that:there was almost negative expression of SA-P-Gal activity in control group; The effect that H2O2 increased SA-β-gal-positive cells was increased with longer incubating time.With H2O2 treatment, the percentage of SA-β-gal-positive MGECs was increased from 13.12%at 3 days to 70.47%at 7 days.
3. Through analyzing from cell cycle result, we could conclude that:compared with control group, H2O2-induced senescent cells presented with the classical senescence-related cell cycle dysfunction. H2O2-induced senescence group mitosis is delayed in G1 phase and the cells have a limited capacity to enter S phase (P＜0.05).
4. Western blot data demonstrated that H2O2 induced the significant increasing in induction of p16INK4a(Control:21.09±0.67 vs. H2O2:69.39±4.10).
5. After hoechest-33342 staining, we could see no apoptosis morphology changes. Furthermore,the result of flow cytometry didn't show typical sub-G1 apoptosis peak. H2O2 did not induce apoptosis (apoptosis rate:3.19±0.49% vs 3.26±0.39%, P>0.05).
CONCLUSIONS:
H2O2 can induce mouse glomerular endothelial cells senescence in vitro.MGECs treated with H2O2 (50μM) for 1 week exhibited characteristic of the senescent phenotype of MGECs, increased SA-β-gal-positive cells and the expression of p16INK4a presented with cell cycle G1 arrest. Furthermore, H2O2 did not induce apoptosis. These results indicate that H2O2 can mimic the induction of the cell senescent phenotype.
PART II Effects of Angiopoietins on the senescence of mouse glomerular endothelial cell induced by hydrogen peroxide
OBJECTIVE:
Aging is a normal biological process defined as a form of permanent and irreversible proliferation arrest, with progressive organ modification and ultimately the loss of organ function. The kidney also participates in the aging process of the whole body, and age-associated changes have many implications for nephrology, including the slightly lower level of renal function with normal aging. The vasculature plays a key role in the progression of renal damage in aging, with reduction in glomerular and peritubular capillary density and decreased endothelial proliferative response. However, a direct link between changes in renal capillary endothelium and aging-associated renal disease has not been yet elucidated. The angiopoietins, which regulate the transition between a mature stable vasculature and angiogenic or remodeling vessels, play an important role in vascularization. Angl acts primarily as an agonist ligand of Tie2, which induces Tie2 phosphorylation, stabilizes vascular branch networks, and promotes endothelial cell survival. In contrast, Ang2 is a natural antagonist ligand of Tie2, promoting vascular destabilization by opposing the effects of Ang1. In the second part of this study, we investigated the role of Angl in protecting mouse glomerular endothelial cells from H2O2-induced cellular senescence.,and discuss its preliminary mechanisms.
METHODS:
The MGECs were cultured in vitro and devided into control group、H2O2 group、Ang1 group Ang2 group. MGECs were subjected to H2O2 induced senescence, which was evaluated by senescence-associated (3-Galactosidase (SA-P-Gal) staining, cell-cycle analysis and expression of p16. Endothelial cell function was assessed by NO, vWF secretion and capillary-like structure formation. The assays of malonaldehyde (MDA),glutathione(GSH),superoxide dismutase (SOD) and catalase (CAT) in cell lysis were measured by colorimetric method. Western blot were used to locate and quantitate the signal activity of Tie2、ERK1/2.
RESULTS:
1. Senescent phenotypes:The percentage of SA-β-gal-positive MGECs was increased with H2O2 treatment. Co-incubation with Angl markedly inhibited the induction of senescence by 51.80%(P<0.05). However, SA-β-Gal activity of MGECs treated with H2O2 in the presence of Ang2 showed no significant change. H2O2 group mitosis is delayed and the cells have a limited capacity to enter S phase. In contrast, Angl treatment induced a dramatic change in this pattern, the G1 population reduced from 84.10%incubated with H2O2 to 74.50%, whereas S populations were increased from 10.88% to 18.89%. No significant difference in this pattern of distribution was observed in cells treated with only Ang2. H2O2 induced the increased induction of p16INK4a, Angl showed 44.30% down-regulated p16INK4a levels in MGECs under H2O2 stimulation. However, under Ang2 treatment conditions, high expression of p16INK4a was also noticeable.
2. Endothelial cell functions:The mean NO level decreased dramatically in senescence cells in contrast to control. Whereas incubation of MGECs with Angl resulted in a significant increase in the release of NO(15.61±1.61 vs 10.26±0.69, P<0.05). However, there was no significant difference in NO concentration between Ang2 and H2O2 group. Elevated levels of vWF were detected in the culture medium of senescent cells.Treatment of endothelial cells with Angl had a decreased effect on release of vWF into the culture medium(5.13±0.69 vs 9.88±0.53, P<0.05). In contrast, when endothelial cells were treated with Ang2, no difference was detected in the amount of vWF secreted by the senescent cells (Ang2:10.02±0.31, P>0.05) Angl-treated cells formed capillary-like structures more efficiently than senescent cells at base line. In contrast, no significant changes were observed with Ang2.
3. Oxidative stress:MDA of H2O2 group was significantly higher than that of control group. Whereas incubation of MGECs with Angl resulted in a significant decrease in the release of MDA(H2O2; 10.31±0.22 vs. Ang1; 5.02±0.47, P＜0.05). In contrast, when endothelial cells were treated with Ang2, no difference was detected in the amount of MDA. SOD、GSH and CAT of H2O2 group were notably lower in contrast to control. Treatment of endothelial cells with Angl had a increase effect on activity of SOD、GSH and CAT(P<0.05). No significant difference was observed in cells treated with only Ang2(P>0.05).
4. Tie2-ERKl/2 signaling:Western blot data demonstrated that a dramatic increase of Tie2 tyrosine phosphorylation was detected exposure to Angl, while total Tie2 level was not affected(H2O2:4.12±2.06 vs. Ang1: 71.09±2.15, P<0.05). In contrast, in the H2O2 plus Ang2 group, there was no significant elevation of Tie2 phosphorylation level (Ang2:16.07±3.16, P>0.05). Angl caused significant elevation of phosphorylated but not total-ERK levels(P<0.05). However, Ang2 could not elicit ERK activation.
CONCLUSIONS:
The present studies suggest that Angl inhibits H2O2-induced senescence in mouse glomerular endothelial cells。Moreover, Angl regulated the secretion and capillary-like structure formation of endothelial cells with aging. Improvement of oxidative stress may also be involved. Angl causes significant elevation of phosphorylated Tie2 and ERK1/2 levels. However, under Ang2 treatment conditions, no significant difference was observed in cells treated with H2O2.
PARTⅢThe mechanisms of angiopoietin-1 inhibits mouse glomerular endothelial cell senescence
METHODS:
The MGECs were cultured in vitro and devided into control group、H2O2 group、Ang1 group、Ang2+Ang1 group、sTie2-Fc group、PD98059 group. MGECs were subjected to H2O2 induced senescence, which was evaluated by senescence-associatedβ-Galactosidase (SA-β-Gal) staining, cell-cycle analysis and expression of p16. Endothelial cell function was assessed by NO, vWF secretion and capillary-like structure formation. Western blot were used to locate and quantitate the signal activity of Tie2、ERK1/2.
OBJECTIVE:
Angl acts primarily as an agonist ligand of Tie2, which induces ERK1/2 phosphorylation, stabilizes vascular branch networks, and promotes endothelial cell survival. In the second part of this study, we have found Ang1could significant elevated phosphorylation of Tie2 and ERK1/2 levels. However the question arose as to whether Tie2-ERK1/2 signal was involved in the inhibition of cellular senescence induced by Ang1 in MGECs. To further investigated the signaling pathway of Tie2 in mediating the effect of ERK1/2 in mouse glomerular endothelial cells during aging, cells were pretreated with Ang2, Tie2 antagonist (sTie2-Fc) or a specific ERK1/2 inhibitor(PD98059) before adding Ang1.
RESULTS:
1. Senescent phenotypes:Compaired with Ang1, the percentage of SA-β-gal-positive MGECs was increased with Ang2、sTie2-Fc and PD98059 treatment(Ang2:63.94±2.05, sTie2-Fc:72.56±1.51, PD98059:73.31±2.73 vs. Ang1:18.67±2.73, P＜0.05). MGECs treated with sTie2-Fc, Ang2 and PD98059 subsequently exhibited the same senescence-associated morphologies as H2O2-induced senescence cells. Cell cyle is delayed and the cells have a limited capacity to enter S phase. Angl showed down-regulated p16INK4a levels in MGECs under H2O2 stimulation. However, such effects could be completely eliminated either by sTie2-Fc, Ang2 or by PD98059(Ang2:58.39±2.02, sTie2-Fc:61.08±3.10, PD98059:62.19±3.72vs. Ang1:21.16±1.11, P<0.05).
2. Endothelial cell functions:Angl resulted in a significant increase in the release of NO. However, this change was completely blocked by treatment with Ang2, sTie2-Fc and PD98059(Ang2:11.92±0.44, sTie2-Fc:10.22±0.36, PD98059:10.87±0.66 vs. Ang1:16.38±0.35, P＜0.05). Treatment of endothelial cells with Ang2, sTie2-Fc and PD98059 had a increased effect on release of vWF into the culture medium (P<0.05). Cells incubated with Ang2, sTie2-Fc or PD98059 plus Angl failed to stimulate capillary-like structure formation(Ang2:23.11±2.29, sTie2-Fc:15.21±3.31, PD98059:18.56±2.36 vs.Ang1:57.66±3.17, P<0.05).
3. Tie2-ERK1/2 signaling:Western blot data demonstrated that sequestering Angl by Ang2 and sTie2-Fc blocked ERK phosphorylation(Ang2: 47.12±2.89, sTie2-Fc:35.62±3.71 vs. Angl:135.16±2.27, P<0.05). Furthermore, when a specific ERK inhibitor PD98059 was used in combination with Ang1, the same intense bands corresponding to phosphorylation of Tie2 tyrosine were observed as cells treated with Ang1(Ang1:61.25±1.91, vs.PD98059:63.72±2.87, P>0.05).
CONCLUSIONS:
The present studies suggest that Angl inhibits H2O2-induced senescence in mouse glomerular endothelial cells via the Angl-Tie2-ERKl/2 signaling pathway.
目的：
在体外培养条件下,正常体细胞在经过有限次数的增殖分裂后即进入不可逆转的分裂停滞状态,这种由于在连续的细胞分裂中端粒逐渐缩短所引发的现象称为细胞复制性衰老。除此以外,氧化应激、某些癌基因过度表达、紫外线、化学诱变剂等应激因素亦可诱导细胞进入一种应激性早衰状态。细胞早衰不依赖端粒的缩短,并且与复制性衰老细胞在形态、功能及生物学特性上具有许多相同的特征。细胞衰老是生物整体衰老的基础,以细胞为模型的衰老生物学研究是当前老年医学研究的重要内容。H202是一种广泛使用的致细胞早衰的诱导剂,通过DNA损伤机理诱导细胞衰老,且外源性上调端粒酶的活性不能抑制其诱导衰老作用。因此本研究首先通过过氧化氢诱导,探讨可否在体外建立成功而稳定的肾小球内皮细胞(MGECs)衰老模型,从而为进一步的实验奠定良好模型基础。
方法：
应用体外细胞培养技术,培养小鼠肾小球内皮细胞,选择过氧化氢(H2O2)作为诱导物。设立正常对照组、H2O2组进行实验。应用p-半乳糖苷酶染色(SA-β-Gal)方法检测衰老细胞阳性率,流式细胞术检测细胞周期,Western blot免疫印迹法检测p16蛋白的表达,通过分析H202对细胞衰老指标的影响鉴定其作用后是否可引起MGECs衰老。同时通过Hoechst-33342染色法及流式细胞术检测H2O2对细胞凋亡的影响。
结果：
1. H2O2(50μmol/L)诱导7天后,在倒置相差显微镜下观察可见肾小球内皮细胞形态发生明显改变,细胞体积增大,胞体扁平,呈现衰老细胞形态；
2. SA-β-Gal染色实验结果显示：正常细胞SA-β-Gal染色几乎无阳性表达(0.76±0.24%),随H2O2作用时间延长阳性细胞数逐渐增多；H202作用3天即可观察到少量SA-β-Gal染色阳性衰老细胞(13.12±1.81%)的出现,继续予以H2O2作用7天后SA-β-Gal染色阳性细胞可达70.47%；
3.对细胞周期的分析发现,与正常对照组相比,H2O2具有阻止肾小球内皮细胞进入增殖期(S期),而将细胞周期阻滞于静止期(Gl期)的作用,差别具有统计学意义(P<0.05)；
4. Western blot实验结果显示,与正常对照组相比H202组p16蛋白的表达显著增高(Control：21.09±0.67 vs. H2O2:69.39±4.10),差别具有统计学意义(P<0.05)；
5. hoechest-33342着染后H2O2组并未出现明显凋亡样细胞改变,同样流式细胞术检测显示正常对照组及H2O2组细胞G1峰前均无凋亡峰出现,细胞凋亡率无显著性差异(3.19±0.49%vs 3.26±0.39%,P>0.05)。
结论：
过氧化氢在体外能成功诱导肾小球内皮细胞衰老。H2O2(50μmol/L)诱导7天后衰老相关SA-β-Gal染色阳性,细胞周期阻滞于G1期,p16蛋白表达上调。且H2O2作用后细胞无异常凋亡发生。过氧化氢诱导肾小球内皮细胞衰老的模型成功、可信。
第二部分血管生成素对过氧化氢诱导的肾小球内皮细胞衰老的作用研究
目的：
衰老是一个自然进程,在所有物种中都有此现象,并导致很多器官出现退行性改变。肾脏是受衰老影响最明显的器官之一,肾脏功能随衰老进程逐渐减退。肾脏衰老时存在血管形成异常,表现为局灶性肾小管周围及肾小球毛细血管密度减少；随着年龄的增长,肾小球出现内皮细胞进行性减少和丢失。但目前有关肾脏毛细血管改变与衰老相关肾脏病变的直接关系尚未明了。血管生成素(Ang)是一组内皮细胞特异性生长因子,可以有效促进内皮细胞的增殖、分化、介导新生血管管腔形成。Angl可促使Tie2磷酸化,提高内皮细胞的存活能力,促进血管生成,而Ang2则作用相反,能抑制Angl介导的Tie2活化。本部分研究拟探讨血管生成素系统是否能够调控肾小球内皮细胞的衰老,进而影响肾小球内皮细胞的功能及肾脏新生血管的生成,并对其作用机制进行初步探讨。
方法：
体外培养小鼠肾小球内皮细胞,设立正常对照组、H2O2组、Angl组、Ang2组进行实验。应用H2O2诱导MGECs衰老,通过p-半乳糖苷酶染色(SA-β-Gal)、流式细胞术检测细胞周期、Western blot检测p16蛋白表达评估细胞衰老指标的变化。应用硝酸还原酶法检测内皮细胞NO的分泌量,ELISA方法检测内皮细胞vWF的分泌量,Matrigel凝胶实验检测内皮细胞血管生成能力,探讨血管生成素对内皮细胞功能的影响。比色法测定细胞裂解液中的丙二醛(MDA)、谷胱甘肽(GSH)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)的水平。Western blot方法检测Tie2、ERK1／2信号通路的变化。
结果：
1.血管生成素对肾小球内皮细胞衰老指标的影响：H2O2组SA-β-Gal阳性率增高,Angl作用后细胞SA-β-Gal着色变浅、阳性率降低,与H2O2组相比其阳性率下降达到51.80%(P<0.05),而Ang2组和H2O2组之间SA-β-Gal阳性率无显著性差异(P>0.05)；H2O2诱导衰老后细胞周期阻滞于Gl期,Angl组Gl期细胞比例下降至74.50+0.16%,S期细胞比例上升至18.89±0.68%,与H202组相比差别有统计学意义(P<0.05),但Ang2组Gl期及S期细胞数与H2O2组无明显差异(P>0.05)；H202组p16蛋白表达增高,Angl作用后p16表达水平较H2O2组下降44.30%(P<0.05),但Ang2对p16蛋白表达无影响(P>0.05)。
2.血管生成素对肾小球内皮细胞功能的影响：H2O2组肾小球内皮细胞NO分泌量明显减少,Angl组细胞NO分泌显著增加(H202：10.26±0.69vs.Angl：15.61±1.61,P<0.05),Ang2组NO分泌量与H2O2组无明显差异(Ang2：10.15±0.42,P>0.05)；H2O2组vWF含量明显升高(P<0.05),Angl作用后vWF含量显著降低(Angl：5.13±0.69 vs H2O2：9.88±0.27,P<0.05),而Ang2对vWF分泌无明显影响(Ang2：10.02±0.31,P>0.05)；H2O2组MGECs管状结构形成能力显著抑制,Angl组细胞管状结构形成明显增加(P<0.05),而Ang2组则与衰老组无明显差异。
3.血管生成素对肾小球内皮细胞氧化应激指标的影响：H2O2组MDA含量增加,与H2O2组相比,Angl组MDA含量降低(H2O2；10.31±0.22 vs.Angl；5.02±0.47,P<0.05),而Ang2组MDA含量则无显著变化(Ang2；9.13±0.81,P>0.05)；与正常对照组相比,H2O2组GSH、SOD、CAT活性均明显降低,与H2O2组相比,Angl作用后GSH、SOD、CAT活性均显著升高(P<0.05),但Ang2组GSH、SOD、CAT活性则无显著性差异。
4.血管生成素对Tie2-ERK1/2通路的影响：Westen blot结果显示Angl组P-Tie2水平明显增高(H2O2：4.12±2.06 vs.Angl：71.09±2.15,P<0.05),Ang2组P-Tie2无明显变化(Ang2：10.07±3.16,P>0.05)；Angl能显著提高细胞P-ERK1/2水平(P<0.05),但Ang2作用未能引起P-ERK1/2的明显变化(P>0.05)。
结论：
Angl对过氧化氢诱导的肾小球内皮细胞衰老有保护作用,可以有效减少细胞的衰老程度,保护肾小球内皮细胞的正常功能,增强细胞抗氧化酶活性,拮抗H2O2引起的细胞氧化损伤。同时,Angl可以激活Tie2、ERK1/2的磷酸化进程。然而Ang2对内皮细胞无上述保护作用。
第三部分血管生成素1对肾小球内皮细胞衰老保护作用的机制探讨
目的：
Angl能与Tie2受体结合并活化Tie2受体,诱导磷酸化的ERK1/2表达升高,进一步发挥其对内皮细胞的增殖、粘附等的作用。在第二部分的研究中,我们的研究结果也提示Angl的确可以启动内皮细胞Tie2及ERK1/2的磷酸化过程。Angl对H2O2所诱导的肾小球内皮细胞衰老的保护作用是否是通过激活Tie2-ERK1/2信号通路所致?为了更进一步探讨Angl对肾小球内皮细胞衰老保护作用的机制,我们用Angl的天然拮抗剂Ang2、Tie2受体的特异性抑制剂sTie2-Fc及MAPK信号通路MEK特异性抑制剂PD98059预处理肾小球内皮细胞,以明确Angl对肾小球内皮细胞衰老的保护作用的信号通路。
方法：
应用体外细胞培养技术,培养小鼠肾小球内皮细胞,设立正常对照组、H2O2组、Angl组、Ang2+Angl组、sTie2-Fc组、PD98059组进行实验。应用H2O2诱导MGECs衰老,通过p-半乳糖苷酶染色(SA-β-Gal)、流式细胞术检测细胞周期、Western blot检测p16蛋白表达评估细胞衰老指标的变化。应用硝酸还原酶法检测内皮细胞NO的分泌量,ELISA方法检测内皮细胞vWF的分泌量,Matrigel凝胶实验检测内皮细胞血管生成能力,探讨血管生成素对内皮细胞功能的影响。Western blot方法检测Tie2、ERK1/2信号通路的变化。
结果：
1. Tie2-ERK1/2通路抑制剂对肾小球内皮细胞衰老指标的影响：与Angl组比较,Ang2+Ang1组、sTie2-Fc组及PD98059组SA-β-Gal阳性细胞数显著增加(Ang2:63.94±2.05, sTie2-Fc:72.56±1.51, PD98059:73.31±2.73 vs.Ang1:18.67±2.73, P<0.05); Ang2、sTie2-Fc及PD98059作用后,细胞周期分布比例仍呈现衰老细胞特征,细胞周期被阻滞于G1期,S期细胞较少,与Angl组相比,差别有统计学意义(P<0.05)；Ang2、sTie2-Fc及PD98059能阻断Angl对p16蛋白表达降低的作用(Ang2：58.39±2.02,sTie2-Fc: 61.08±3.10, PD98059: 62.19±3.72vs. Ang1: 21.16±1.11, P＜0.05);
2. Tie2-ERK1/2通路抑制剂对肾小球内皮细胞功能的影响：Ang2、sTie2-Fc及PD98059可以下调Angl对内皮细胞NO分泌的促进作用(Ang2：11.92±0.44, sTie2-Fc: 10.22±0.36,PD98059:10.87±0.66 vs. Ang1: 16.38±0.35,P<0.05)；与Angl组相比,予以Ang2、sTie2-Fc及PD98059作用后细胞vWF分泌量显著增加(P<0.05)；Ang2+Angl组、sTie2-Fc组及PD98059组细胞血管腔形成能力下降(Ang2: 23.11±2.29, sTie2-Fc: 15.21±3.31,PD98059：18.56±2.36 vs.Angl：57.66±3.17,P<0.05)；
3. Tie2-ERK1/2通路抑制剂对Tie2-ERK1/2通路的影响：Westen blot结果显示Ang2及sTie2-Fc能显著抑制Angl引起的肾小球内皮细胞P-ERK1/2水平的提高(Ang2: 47.12±2.89, sTie2-Fc: 35.62±3.71 vs. Ang1:135.16±2.27,P<0.05)；PD98059不能抑制Angl引起的Tie2磷酸化(PD98059：63.72±2.87 vs.Angl：61.25±1.91,P>0.05)
结论：
血管生成素样1可能通过Tie2-ERK1/2信号通路而调控过氧化氢诱导的肾小球内皮细胞衰老。
PART I The established model of mouse glomerular endothelial cell senescence induced by hydrogen peroxide
OBJECTIVE:
Normal primary cells proliferate a limited number of times in vitro and activate senescence process due to progressive telomere shortening. Furthermore, cells may also undergo acute stress-induced senescence, which could be triggered by cumulative stimuli involving oxidation, ultraviolet(UV) light, toxic compounds, as well as oncogens. Stress induces senescence through telomere-independent mechanisms, and displays all the major characteristics of replicatively senescent cells. The molecular basis of aging in organs is not well established, but organ aging may reflect aspects of cellular senescence involved with molecular pathway changes. In recent years, much attention has been devoted to studying the relevance of cellular senescence to aging and disease. H2O2, a common causative agent of cellular senescence, can produce various types of DNA damage, which represents the type of stress-induced senescence via DNA damage and cannot be rescued by over-expression of telomerase reverse transcriptase(TERT).The purpose of the study is to investigate whether H2O2 can induce mouse glomerular endothelial cells(MGECs) senescence in vitro.
METHODS:
The MGECs stimulated with hydrogen peroxide(H2O2) were cultured in vitro,and were devided into control group、H2O2 group. The senescent status of cells were verified by senescence-associated P-Galactosidase (SA-β-Gal) staining. The cell cycle of mouse glomerular endothelial cells was analyzed by flow cytometry. The expression of p16 was detected by Western Blot. The senescent of MGECs which was induced by H2O2, was evaluated according above detection. Furthermore, the apoptosis characteristic of MGECs was observed by Hoechst-33342 staining and flow cytometry.
RESULTS:
1. MGECs treated with H2O2 (50μM) for 1 week exhibited an enlarged size and flattened morphology, characteristic of the senescent phenotype of MGECs.
2. The staining of SA-β-Gal assay show that:there was almost negative expression of SA-P-Gal activity in control group; The effect that H2O2 increased SA-β-gal-positive cells was increased with longer incubating time.With H2O2 treatment, the percentage of SA-β-gal-positive MGECs was increased from 13.12%at 3 days to 70.47%at 7 days.
3. Through analyzing from cell cycle result, we could conclude that:compared with control group, H2O2-induced senescent cells presented with the classical senescence-related cell cycle dysfunction. H2O2-induced senescence group mitosis is delayed in G1 phase and the cells have a limited capacity to enter S phase (P＜0.05).
4. Western blot data demonstrated that H2O2 induced the significant increasing in induction of p16INK4a(Control:21.09±0.67 vs. H2O2:69.39±4.10).
5. After hoechest-33342 staining, we could see no apoptosis morphology changes. Furthermore,the result of flow cytometry didn't show typical sub-G1 apoptosis peak. H2O2 did not induce apoptosis (apoptosis rate:3.19±0.49% vs 3.26±0.39%, P>0.05).
CONCLUSIONS:
H2O2 can induce mouse glomerular endothelial cells senescence in vitro.MGECs treated with H2O2 (50μM) for 1 week exhibited characteristic of the senescent phenotype of MGECs, increased SA-β-gal-positive cells and the expression of p16INK4a presented with cell cycle G1 arrest. Furthermore, H2O2 did not induce apoptosis. These results indicate that H2O2 can mimic the induction of the cell senescent phenotype.
PART II Effects of Angiopoietins on the senescence of mouse glomerular endothelial cell induced by hydrogen peroxide
OBJECTIVE:
Aging is a normal biological process defined as a form of permanent and irreversible proliferation arrest, with progressive organ modification and ultimately the loss of organ function. The kidney also participates in the aging process of the whole body, and age-associated changes have many implications for nephrology, including the slightly lower level of renal function with normal aging. The vasculature plays a key role in the progression of renal damage in aging, with reduction in glomerular and peritubular capillary density and decreased endothelial proliferative response. However, a direct link between changes in renal capillary endothelium and aging-associated renal disease has not been yet elucidated. The angiopoietins, which regulate the transition between a mature stable vasculature and angiogenic or remodeling vessels, play an important role in vascularization. Angl acts primarily as an agonist ligand of Tie2, which induces Tie2 phosphorylation, stabilizes vascular branch networks, and promotes endothelial cell survival. In contrast, Ang2 is a natural antagonist ligand of Tie2, promoting vascular destabilization by opposing the effects of Ang1. In the second part of this study, we investigated the role of Angl in protecting mouse glomerular endothelial cells from H2O2-induced cellular senescence.,and discuss its preliminary mechanisms.
METHODS:
The MGECs were cultured in vitro and devided into control group、H2O2 group、Ang1 group Ang2 group. MGECs were subjected to H2O2 induced senescence, which was evaluated by senescence-associated (3-Galactosidase (SA-P-Gal) staining, cell-cycle analysis and expression of p16. Endothelial cell function was assessed by NO, vWF secretion and capillary-like structure formation. The assays of malonaldehyde (MDA),glutathione(GSH),superoxide dismutase (SOD) and catalase (CAT) in cell lysis were measured by colorimetric method. Western blot were used to locate and quantitate the signal activity of Tie2、ERK1/2.
RESULTS:
1. Senescent phenotypes:The percentage of SA-β-gal-positive MGECs was increased with H2O2 treatment. Co-incubation with Angl markedly inhibited the induction of senescence by 51.80%(P<0.05). However, SA-β-Gal activity of MGECs treated with H2O2 in the presence of Ang2 showed no significant change. H2O2 group mitosis is delayed and the cells have a limited capacity to enter S phase. In contrast, Angl treatment induced a dramatic change in this pattern, the G1 population reduced from 84.10%incubated with H2O2 to 74.50%, whereas S populations were increased from 10.88% to 18.89%. No significant difference in this pattern of distribution was observed in cells treated with only Ang2. H2O2 induced the increased induction of p16INK4a, Angl showed 44.30% down-regulated p16INK4a levels in MGECs under H2O2 stimulation. However, under Ang2 treatment conditions, high expression of p16INK4a was also noticeable.
2. Endothelial cell functions:The mean NO level decreased dramatically in senescence cells in contrast to control. Whereas incubation of MGECs with Angl resulted in a significant increase in the release of NO(15.61±1.61 vs 10.26±0.69, P<0.05). However, there was no significant difference in NO concentration between Ang2 and H2O2 group. Elevated levels of vWF were detected in the culture medium of senescent cells.Treatment of endothelial cells with Angl had a decreased effect on release of vWF into the culture medium(5.13±0.69 vs 9.88±0.53, P<0.05). In contrast, when endothelial cells were treated with Ang2, no difference was detected in the amount of vWF secreted by the senescent cells (Ang2:10.02±0.31, P>0.05) Angl-treated cells formed capillary-like structures more efficiently than senescent cells at base line. In contrast, no significant changes were observed with Ang2.
3. Oxidative stress:MDA of H2O2 group was significantly higher than that of control group. Whereas incubation of MGECs with Angl resulted in a significant decrease in the release of MDA(H2O2; 10.31±0.22 vs. Ang1; 5.02±0.47, P＜0.05). In contrast, when endothelial cells were treated with Ang2, no difference was detected in the amount of MDA. SOD、GSH and CAT of H2O2 group were notably lower in contrast to control. Treatment of endothelial cells with Angl had a increase effect on activity of SOD、GSH and CAT(P<0.05). No significant difference was observed in cells treated with only Ang2(P>0.05).
4. Tie2-ERKl/2 signaling:Western blot data demonstrated that a dramatic increase of Tie2 tyrosine phosphorylation was detected exposure to Angl, while total Tie2 level was not affected(H2O2:4.12±2.06 vs. Ang1: 71.09±2.15, P<0.05). In contrast, in the H2O2 plus Ang2 group, there was no significant elevation of Tie2 phosphorylation level (Ang2:16.07±3.16, P>0.05). Angl caused significant elevation of phosphorylated but not total-ERK levels(P<0.05). However, Ang2 could not elicit ERK activation.
CONCLUSIONS:
The present studies suggest that Angl inhibits H2O2-induced senescence in mouse glomerular endothelial cells。Moreover, Angl regulated the secretion and capillary-like structure formation of endothelial cells with aging. Improvement of oxidative stress may also be involved. Angl causes significant elevation of phosphorylated Tie2 and ERK1/2 levels. However, under Ang2 treatment conditions, no significant difference was observed in cells treated with H2O2.
PARTⅢThe mechanisms of angiopoietin-1 inhibits mouse glomerular endothelial cell senescence
METHODS:
The MGECs were cultured in vitro and devided into control group、H2O2 group、Ang1 group、Ang2+Ang1 group、sTie2-Fc group、PD98059 group. MGECs were subjected to H2O2 induced senescence, which was evaluated by senescence-associatedβ-Galactosidase (SA-β-Gal) staining, cell-cycle analysis and expression of p16. Endothelial cell function was assessed by NO, vWF secretion and capillary-like structure formation. Western blot were used to locate and quantitate the signal activity of Tie2、ERK1/2.
OBJECTIVE:
Angl acts primarily as an agonist ligand of Tie2, which induces ERK1/2 phosphorylation, stabilizes vascular branch networks, and promotes endothelial cell survival. In the second part of this study, we have found Ang1could significant elevated phosphorylation of Tie2 and ERK1/2 levels. However the question arose as to whether Tie2-ERK1/2 signal was involved in the inhibition of cellular senescence induced by Ang1 in MGECs. To further investigated the signaling pathway of Tie2 in mediating the effect of ERK1/2 in mouse glomerular endothelial cells during aging, cells were pretreated with Ang2, Tie2 antagonist (sTie2-Fc) or a specific ERK1/2 inhibitor(PD98059) before adding Ang1.
RESULTS:
1. Senescent phenotypes:Compaired with Ang1, the percentage of SA-β-gal-positive MGECs was increased with Ang2、sTie2-Fc and PD98059 treatment(Ang2:63.94±2.05, sTie2-Fc:72.56±1.51, PD98059:73.31±2.73 vs. Ang1:18.67±2.73, P＜0.05). MGECs treated with sTie2-Fc, Ang2 and PD98059 subsequently exhibited the same senescence-associated morphologies as H2O2-induced senescence cells. Cell cyle is delayed and the cells have a limited capacity to enter S phase. Angl showed down-regulated p16INK4a levels in MGECs under H2O2 stimulation. However, such effects could be completely eliminated either by sTie2-Fc, Ang2 or by PD98059(Ang2:58.39±2.02, sTie2-Fc:61.08±3.10, PD98059:62.19±3.72vs. Ang1:21.16±1.11, P<0.05).
2. Endothelial cell functions:Angl resulted in a significant increase in the release of NO. However, this change was completely blocked by treatment with Ang2, sTie2-Fc and PD98059(Ang2:11.92±0.44, sTie2-Fc:10.22±0.36, PD98059:10.87±0.66 vs. Ang1:16.38±0.35, P＜0.05). Treatment of endothelial cells with Ang2, sTie2-Fc and PD98059 had a increased effect on release of vWF into the culture medium (P<0.05). Cells incubated with Ang2, sTie2-Fc or PD98059 plus Angl failed to stimulate capillary-like structure formation(Ang2:23.11±2.29, sTie2-Fc:15.21±3.31, PD98059:18.56±2.36 vs.Ang1:57.66±3.17, P<0.05).
3. Tie2-ERK1/2 signaling:Western blot data demonstrated that sequestering Angl by Ang2 and sTie2-Fc blocked ERK phosphorylation(Ang2: 47.12±2.89, sTie2-Fc:35.62±3.71 vs. Angl:135.16±2.27, P<0.05). Furthermore, when a specific ERK inhibitor PD98059 was used in combination with Ang1, the same intense bands corresponding to phosphorylation of Tie2 tyrosine were observed as cells treated with Ang1(Ang1:61.25±1.91, vs.PD98059:63.72±2.87, P>0.05).
CONCLUSIONS:
The present studies suggest that Angl inhibits H2O2-induced senescence in mouse glomerular endothelial cells via the Angl-Tie2-ERKl/2 signaling pathway.
引文
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