AngⅡ诱导血管内皮细胞衰老相关基因表达的研究

目的 探讨血管紧张索Ⅱ(AngⅡ)诱导人脐静脉内皮细胞(HUVECs)衰老相关基因p16INK4a、p21cip1的表达.方法 体外培养HU-VECs并予AngⅡ(10-6mol/L)干预,采用光镜观察细胞形态学改变,β-半乳糖苷酶(β-gal)染色和流式细胞术鉴定细胞衰老,并利用免疫细胞化学染色法分析Ang Ⅱ诱导HUVECs 0、12、24、36、48 h的p16INK4a阳性细胞表达率,Western印迹法分析AngⅡ诱导HUVECs 0、12、24、36、48 h的p16INK4a、p21cip1蛋白表达的时间效应关系.结果 AngⅡ诱导组HUVECs出现典型的细胞体积增大,形态不规则;约80%的细胞呈现β-gal阳性染色(80.10±6.81)%,流式细胞仪检测细胞周期停滞于G0/G1(91.36±6.45)%,证实细胞衰老;AngⅡ呈时间依赖性上调p16IK4a、p21cip1蛋白表达.结论 AngⅡ诱导血管内皮细胞衰老的分子机制之一可能与上调衰老细胞内细胞周期蛋白p16INK4a、p21cip1的表达,使细胞周期停滞于G1期有关.     

Valsartan延缓血管内皮细胞衰老及p16INK4a表达变化的研究

目的 探讨Valsartan对血管内皮细胞衰老与p16INK4a表达变化的影响,为寻求廷缓内皮细胞衰老途径提供理论和实验依据.方法 体外培养人脐静脉内皮细胞,子血管紧张素Ⅱ及Valsartan干预,实验分为空白对照组、血管紧张素Ⅱ诱导组及Valsartan组,采用β-半乳糖苷酶(β-gal)染色鉴定细胞衰老;流式细胞术分析细胞周期变化;免疫细胞化学染色法、Western blot分析各组细胞p16INK4a蛋白的表达.结果 与对照组比较,血管紧张素Ⅱ诱导组β-半乳糖苷酶阳性染色率显著增多81.24％±6.46％,细胞周期停滞于G0-G1(88.36％±6.45％),p16INK4a 蛋白表达水平上调(P＜0.05);予以Valsartan干预后,β-半乳糖苷酶阳性细胞染色率减少,G0-G1细胞减少,p16INK4a蛋白表达水平下调(P＜0.05).结论 血管内皮细胞衰老分子机制可能通过下调p16INK4a的表达,使细胞周期停滞于G1期有关,Valsartan对血管内皮细胞衰老有一定保护作用,可能通过调控p16INK4a的表达发挥其延缓3血管内皮细胞衰老的作用.     

Shc相关磷酸化酪氨酸适配蛋白在大鼠血管衰老中的调控作用

目的 探讨Shc相关磷酸化酪氨酸适配蛋白p66shc在血管衰老表达的变化,对阐明血管衰老对动脉粥样硬化的诊治具有重要意义.方法 将Wistar大鼠分为4、10、16、24月龄组,测定各组血浆丙二醛(MDA)、超氧化物歧化酶(SOD)水平,同时采用恒速注入流体方法测定大鼠颈动脉血管的顺应性,并利用蛋白免疫印迹法分析各组p66shc、半胱氨酸天门冬氨酸蛋白酶-3水平.结果 随增龄,大鼠主动脉管壁增厚,纤维化程度增高,MDA浓度明显升高(与4月龄和10月龄相比,均P<0.01),SOD浓度明显降低(与4月龄相比,均P<0.01;与10月龄相比,均P<0.05),颈动脉血管的顺应性增高、弹性速率和弹性面积的差异有统计学意义(与4月龄相比,均P<0.01;与10月龄相比,均P<0.05),p66shc、Caspase-3蛋白表达呈时间依赖性上调.结论 血管衰老性重塑的分子机制之一可能与上调p66shc、Caspase-3蛋白的表达有关,进一步阐明其调控机制可为延缓血管衰老、防治动脉硬化提供理论依据.     

松花粉抗成纤维细胞复制性衰老的机制

目的 研究松花粉对衰老成纤维单细胞面积、β-半乳糖苷酶染色(SA-β-gal)阳性率、p16INK4A和p21CIP-1表达以及细胞周期的影响.方法 以二倍体成纤维(2BS)细胞建立衰老细胞模型.生物体视学法分析单细胞面积变化;免疫组化法测SA-β-gal阳性率;流式细胞术分析细胞周期;RT-PCR法测定p16INK4A、p21CIP-1基因mRNA表达量.结果 56代细胞出现典型的衰老细胞形态改变,同时SA-β-gal染色阳性率与G1期细胞比例均增高.经松花粉处理后,衰老细胞的单细胞面积、SA-β-gal染色阳性率与G1期细胞比例均显著减少(P＜0.05);p16INK4A与p21CIP-1mRNA的表达量均较衰老模型组明显下调(P＜0.05). 结论松花粉具有改善细胞复制性衰老的作用,其分子机制可能与下调p16INK4A及p21CIP-1基因mRNA的表达从而改善衰老细胞G1期阻滞有关.     

血管紧张素Ⅱ和NADPH氧化酶与血管衰老的相关性研究

目的探讨血管紧张素Ⅱ（AngⅡ）和NADPH氧化酶在血管衰老中的地位及作用机理.方法健康Wistar大鼠分为青年组、老龄组、Valsantan组,分析各组大鼠主动脉形态结构及功能;测定血浆和主动脉AngⅡ水平、主动脉活性氧水平;分别应用RT-PCR和Western bolt检测各组大鼠AngⅡ1型和2型受体（AT1R和AT2R）、NADPH氧化酶p22phox的mRNA及蛋白表达.结果随增龄大鼠主动脉管壁增厚,纤维化程度增高,内皮功能受损,活性氧产生增加;主动脉AngⅡ含量增高,AT2R、p22phox的mRNA及蛋白表达上调,AT1R表达下降;Valsantan（AngⅡ1型受体特异性阻断剂）干预后,p22phox表达下降,活性氧水平降低,衰老血管形态结构和功能异常有所改善.结论衰老血管有其特征性结构和功能改变;AngⅡ经由AT1R上调NADPH氧化酶的基因表达可能是血管衰老的重要机制之一.     

C反应蛋白和白细胞介素6在自然衰老大鼠血管中的表达研究

目的:研究炎性因子C反应蛋白(CRP)和白细胞介素(IL)-6在不同年龄组大鼠主动脉组织中的表达差异,及其可能在血管衰老机制中的作用。方法:清洁级雄性SD大鼠24只,其中12只青年大鼠(青年组,2月龄)和12只老年大鼠(老年组,20月龄),经适应性喂养1周后麻醉采静脉血待测,处死后取胸主动脉组织,石蜡包埋切片HE染色后光镜下观察血管组织形态改变,Western blot检测主动脉p16和p21蛋白水平,化学发光酶免疫分析法和酶联免疫吸附试验(ELISA)分别测定大鼠血清CRP和IL-6的含量。结果:老年组大鼠主动脉组织HE染色血管中膜层增厚、血管平滑肌细胞排列紊乱,Western blot检测p16和p21蛋白表达阳性;青年组大鼠主动脉组织HE染色血管中膜层正常、血管平滑肌细胞排列整齐,p16和p21蛋白表达阴性。与青年组大鼠相比,老年组血清CRP和IL-6含量均明显升高(均P0.05)。结论:炎症可能是导致血管衰老的原因之一。     

睾酮对衰老心肌细胞及细胞周期调控因子的作用

目的 探讨不同剂量睾酮对心肌细胞衰老的干预作用及其可能机制.方法 用1 μmol/L、 100 nmol/L、10 nmol/L三种剂量睾酮干预自然衰老的小鼠心肌细胞,应用流式细胞仪检测各组细胞周期分布,用RT-PCR及Western印迹检测各组细胞p16INK4a、cyclinD1 mRNA、蛋白及去磷酸化RB蛋白的表达.结果 与衰老心肌细胞相比,1 μmol/L、100 nmol/L、10 nmol/L睾酮干预细胞G0/G1期比例明显降低(P＜0.05),这一作用具有剂量依赖性.睾酮干预可上调cyclinD1mRNA及蛋白表达,下调p16INK4a mRNA及蛋白表达,并使去磷酸化RB蛋白表达下降(P＜0.05),而这些作用均可被雄激素受体阻断剂而不被雌激素受体阻断剂所阻断(P＜0.05).结论 睾酮可剂量依赖性的抑制小鼠心肌细胞衰老,这一作用部分是通过睾酮上调cyclinD1表达,下调p16INK4a及去磷酸化RB蛋白表达而实现的.     

睾酮对亚急性衰老小鼠下丘脑的影响及其机制

目的观察低刘量睾酮对小鼠下丘脑衰老的影响及其可能的调控机制。方法将24只健康雄性C57小鼠随机分为3组:D-半乳糖+睾酮治疗组(治疗组)、D-半乳糖组和正常对照组,每组8只。用药5个月后分离小鼠下丘脑组织,制备石蜡切片,测定小鼠下丘脑衰老相关性β-半乳糖苷酶(SA-β-Gal)染色情况,用免疫组织化学法检测p16~(INK4a)蛋白的表达水平。结果与正常对照组比较,D-半乳糖组小鼠体重降低(P<0.05),下丘脑SA-β-Gal染色阳性细胞率和p16~(INK4a)蛋白阳性细胞率明显升高(P<0.05);与D-半乳糖组比较,治疗组小鼠体重增加(P<0.05).下丘脑SA-β-Gal染色阳性细胞率和p16~(INK4a)蛋白阳性细胞率明显降低(P<0.05)。结论低剂量睾酮可能通过改变p16~(INK4a)的表达而发挥抗下丘脑细胞衰老的作用。     

哈蟆油对雌性衰老大鼠子宫组织p16和cyclinD1蛋白表达的影响

目的 探讨哈蟆油(OR)对D-半乳糖所致雌性衰老大鼠子宫组织细胞增殖负性调控因子p16和正性调控因子cyclinD1蛋白表达的影响,进一步探讨OR延缓雌性大鼠生殖器官衰老机制.方法 SPF级SD雌性青年大鼠40只随机分为模型组(D-gal组)、维生素E(VE组)、哈蟆油高剂量组(OR-H组)、中剂量组(OR-M)、低剂量组(OR-L组),每组8只,D-半乳糖颈背部皮下注射42 d,建立亚急性衰老模型.另取雌性青年大鼠8只,同样部位每日注射生理盐水,作为空白组.第15天开始灌胃给药,给药时间28 d.给药结束后,免疫组化法检测衰老大鼠子宫组织p16和免疫印迹法检测cyclinD1蛋白的表达情况.结果 雌性衰老大鼠子宫组织免疫组化结果表明,子宫组织p16阳性染色多为胞浆着色,见于子宫内膜、上皮细胞胞浆、子宫间质腺体腺上皮细胞胞浆,外膜上皮也有表达,弥漫性、灶性分布均有.D-gal组p16阳性细胞积分与空白组比较升高(P＜0.01).OR各剂量组与D-gal组相比,p16阳性细胞积分降低,差异有显著性(P均＜0.01).免疫印迹法结果表明D-gal组子宫组织cyclinD1蛋白表达与空白组比较降低,差异有显著性(p＜0.01).OR-H、M、L组cyclin D1蛋白表达与D-gal组比较,表达均升高(P值均＜0.01),OR-H组尤为明显.结论 应用OR可以减缓衰老子宫组织结构的损伤,改善衰老子宫萎缩和衰老程度,OR可降低雌性衰老大鼠子宫组织细胞增殖负性调控因子p16蛋白的高表达,同时显著提高细胞增殖正性调控因子cyclinD1蛋白的表达,促进衰老雌性大鼠子宫细胞增殖.哈蟆油延缓雌性生殖器官衰老作用可能通过调控子宫p16-cyclinD1信号通路来促进有关增殖调控蛋白表达发挥延缓衰老作用.     

慢性低氧对肺动脉高压大鼠肺血管ERK 1/2、p38MAPK表达的影响

目的：通过建立慢性低氧性肺动脉高压大鼠模型,研究慢性低氧对大鼠肺血管细胞外信号调节蛋白激酶(ERK1/2)、p38MAPK蛋白表达的影响。方法建立慢性常压低氧肺动脉高压大鼠模型,将雄性SD大鼠随机分为正常对照组、低氧1d、3d、7d、14d和21d组,应用免疫组织化学技术检测肺动脉高压形成过程中大鼠肺血管 ERK1/2、p38MAPK 蛋白表达水平。结果①RVSP 和 RV/(LV+S)比值较正常对照组明显增加(P<0.05),低氧后3 d、7 d、14 d和21 d后大鼠肺血管明显增厚;②ERK1/2、p38MAPK蛋白广泛分布于肺血管内皮细胞、平滑肌细胞和成纤维细胞中,且随着低氧时间的延长,ERK1/2、p38MAPK蛋白表达量增加。结论 ERK1/2、p38MAPK 蛋白表达量的上调可能参与了慢性低氧诱导的大鼠肺动脉高压肺血管重塑的发生、发展过程。     

Alveolar cell senescence in patients with pulmonary emphysema

RATIONALE AND OBJECTIVES: The prevalence of chronic obstructive pulmonary disease (COPD) is age-dependent, suggesting an intimate relationship between the pathogenesis of COPD and aging. In this study we investigated whether the senescence of alveolar epithelial and endothelial cells is accelerated in emphysematous lungs. METHODS: Samples of lung tissue were obtained from patients with emphysema, asymptomatic smokers, and asymptomatic nonsmokers. Paraffin-embedded lung tissue sections were evaluated for cellular senescence by quantitative fluorescence in situ hybridization to assess telomere shortening, and by immunohistochemistry to assess the expression of senescence-associated cyclin-dependent kinase inhibitors. Tissue sections were also immunostained for proliferating cell nuclear antigen (PCNA), surfactant protein A, and CD31. MAIN RESULTS: The patients with emphysema had significantly higher percentages of type II cells positive for p16INK4a and p21CIP1/WAF1/Sdi1 than the asymptomatic smokers and nonsmokers. They had also significantly higher percentages of endothelial cells positive for p16INK4a than the asymptomatic smokers and nonsmokers, and higher percentages of endothelial cells positive for p21CIP1/WAF1/Sdi1 than the asymptomatic nonsmokers. Telomere length in alveolar type II cells and endothelial cells was significantly shorter in the patients with emphysema than in the asymptomatic nonsmokers. The level of p16INK4a expression was negatively correlated with the level of PCNA expression. The level of alveolar cell senescence was positively correlated with airflow limitation. CONCLUSIONS: These results suggest that the senescence of alveolar epithelial and endothelial cells is accelerated in patients with emphysema. Cellular senescence may explain the abnormal cell turnover that promotes the loss of alveolar cells in emphysematous lungs.     

Hypertension induces somatic cellular senescence in rats and humans by induction of cell cycle inhibitor p16INK4a

There is increasing evidence for a role of somatic cellular senescence in physiological aging but also in injury and disease. Cell cycle inhibitor p16(INK4a) is the key mediator for stress and aberrant signaling induced senescence. Here we report that elevated blood pressure markedly induced p16(INK4a) expression in rat kidneys and hearts, as well as in human kidneys. In kidneys from deoxycorticosterone acetate-salt-treated rats, p16(INK4a) induction was found in tubular, glomerular, interstitial, and vascular cells and correlated with the typical histopathologic features of hypertensive target organ damage. p16(INK4a) expression also correlated with phospho-p38, a positive upstream regulator of p16(INK4a) expression. In left ventricles, increased p16(INK4a) expression was found in myocardium and cardiac arteries. Antihypertensive medication consistent of hydrochlorothiazide, hydralazine, and reserpine ameliorated the histopathologic changes and attenuated p16(INK4a) expression in kidneys of deoxycorticosterone acetate-salt-treated rats. Nonantihypertensive administration of spironolactone also reduced kidney damage and p16(INK4a) expression. p16(INK4a) induction was further observed in kidneys from hypertensive transgenic rats heterozygous for the mouse Ren-2 gene and was prevented by the angiotensin II type 1 receptor blocker losartan. In human kidney biopsies showing hypertensive nephrosclerosis, increased p16(INK4a) expression was found compared with age-matched normotensive control subjects. Thus, hypertension induces cellular senescence via p16(INK4a), possibly through p38, thereby contributing to hypertensive target organ damage. This detrimental effect can be overcome by different therapeutic drug strategies.     

Senescence-like changes induced by hydroxyurea in human diploid fibroblasts

Hydroxyurea was found to inhibit the growth of human diploid fibroblasts, which resulted in senescence-like changes both in morphology and replicative potential similar to the replicative senescence. SA-beta-gal activity, a typical characteristic of the replicative senescence was also induced through a long-term treatment of the presenescent cells with 400-800 microgM of hydroxyurea for about 3 weeks. In addition, we determined the levels of cyclin-dependent kinase inhibitors, p21(Waf1) and p16(INK4a), and the p53 tumor suppressor in order to monitor its effect on cell cycle and stress responses. We observed a great induction of both p53 and p21(Waf1), but not of p16(INK4a) in the premature senescent cells. UV-irradiation of the premature senescent cells showed a decreased level of DNA fragmentation presumably ascribed to the reduced activation of stress-activated protein kinases. These results suggest that a chronic hydroxyurea treatment induces the cellular senescence in association with the induction of p53 and p21(Waf1).     

Correlations between age,functional status,and the senescence-associated proteins HMGB2 and p16<Superscript>INK4a</Superscript>

Cellular senescence is a central component of the aging process. This cellular response has been found to be induced by multiple forms of molecular damage and senescent cells increase in number with age in all tissues examined to date. We have examined the correlation with age of two key proteins involved in the senescence program, p16^INK4a and HMGB2. These proteins are involved in cell cycle arrest and chromatin remodeling during senescence. Circulating levels of these markers increases with age and correlates with functional status. The levels of HMGB2 appear to be significantly correlated with functional status, whereas p16^INK4a levels are more weakly associated. Interestingly, there is a strong correlation between the two proteins independent of age. In particular, a single high-functioning individual over 90 years of age displays a disproportionately low level of HGMB2. The results suggest that with improved testing methodology, it may be possible to monitor circulating protein markers of senescence in human populations.     

增龄与心力衰竭的研究进展

心力衰竭(简称心衰)发病率及患病率随年龄增加而逐渐升高,研究显示增龄可独立于其他疾病造成心肌损伤,最终导致心衰发生。本文综述了增龄导致心脏的结构、功能及表型变化,着重叙述了增龄导致心衰的机制研究进展,包括心肌细胞衰老、血管衰老、细胞外基质重塑、神经内分泌失衡及非编码RNA改变等,并介绍了几种针对增龄性心衰的新兴治疗措施。     

Telomere shortening in the damaged small bile ducts in primary biliary cirrhosis reflects ongoing cellular senescence

Telomere shortening is a trigger of cellular senescence. Biliary epithelial cells in damaged small bile ducts in primary biliary cirrhosis (PBC) show senescent features such as the expression of senescence-associated beta-galactosidase and the increased expression of p16(INK4a) and p21(WAF1/Cip1). We investigated whether the telomere shortening is involved in the pathogenesis of biliary cellular senescence in PBC. We analyzed the telomere length of biliary epithelial cells using quantitative fluorescence in situ hybridization in livers taken from the patients with PBC (n = 13) and control livers (n = 13). We also assessed immunohistochemically the prevalence of DNA damage and the expression of p16(INK4a) and p21(WAF1/Cip1). The study showed a significant decrease in telomere length in biliary epithelial cells in the damaged small bile ducts and bile ductules in PBC compared with normal-looking bile ducts and bile ductules in PBC, chronic viral hepatitis, and normal livers (P < 0.01). gammaH2AX-DNA-damage-foci were detected in biliary epithelial cells in damaged small bile ducts and bile ductules in PBC but were absent in biliary epithelial cells in chronic viral hepatitis and normal livers. The expression of p16(INK4a) and p21(WAF1/Cip1) was increased corresponding to telomere shortening and gammaH2AX-DNA-damage-foci in the damaged small bile ducts in PBC. CONCLUSION: Telomere shortening and an accumulation of DNA damage coincide with increased expression of p16(INK4a) and p21(WAF1/Cip1) in the damaged bile ducts, characterize biliary cellular senescence, and may play a role in the following progressive bile duct loss in PBC.     

人乳头状瘤病毒在食管癌中的表达及其作用

背景：人乳头状瘤病毒（HPV）感染与宫颈癌和口腔鳞状细胞癌的发生密切相关，但其与食管癌的关系及其所致相关基因表达的变化尚不十分清楚。目的：研究HPV在食管癌中的表达，探讨其对相关基因蛋白表达的影响。方法：以DNA原位杂交法检测HPV16基因的表达，以免疫组化方法检测HPV16E。突变型p53、p21WAF1、pRb和p16INK4a蛋白的表达。结果：食管癌组织中HPV16DNA的阳性率显著高于正常对照组（65．6％对10．0％，P〈0．001），HPV16E。蛋白的表达率亦显著高于正常对照组（58．9％对15．0％，P〈0．001）；突变型p53蛋白的表达率显著高于正常对照组（53．3％对45．0％，P〈0．05），p21WAF1、pRb和P16INK4a蛋白的表达率则显著低于正常对照组（22．2％对65．0％、23-3％对55．0％和35．6％对85．0％，P〈0．001）。食管癌组织中HPV16E。蛋白与突变型p53蛋白的表达呈正相关，与p21WAF1、pRb和p16INK4a蛋白的表达呈负相关（P〈0．01）。结论：HPV感染是食管癌发生的重要危险因素之一，其可能通过p53突变和p21WAF1、pRb、p16INK4a失活参与了食管癌的发生。