大鼠肾小球系膜细胞表型的增龄性【摘要】目的 观察大鼠肾小球系膜细胞随增龄细胞表型及功能的变化特点 方法 取3、12、24月龄健康雄性wistar大鼠原代系膜细胞进行培养，mtt法检测细胞增殖能力，进行sa拨陋瞘al染色，采用rt瞤cr和western印迹法分别检测p21waf1/cip1/sdi1基因及蛋白质的表达变化，并用免疫荧光法检测系膜细胞中p21waf1/cip1/sdi1的表达与定位 结果 大鼠肾小球系膜细胞增殖能力随增龄逐渐减弱(p【关键词】系膜细胞；衰老；细胞表型；p21waf1/cip1/sdi1 the characteristic and significance of rat mesangial cells with advancing age zhuo li, cai guang瞴an, liu fu瞴ou, et al. department of nephrology, kidney institute & key lab of pla, chinese general hospital of pla, beijing 100853, china 【abstract】objectiveto investigate the characteristic of rat mesangial cells with advancing age. methodsmale wistar rats aged of 3, 12, 24 months were used. the rat mesangial cells were processed for mtt assay and senescence associated β瞘alactosidase (sa拨陋瞘al) staining. the expression and location of p21waf1/cip1/sdi1 in mesangial cells were studied by immunofluorescence. the mrna and protein levels of p21waf1/cip1/sdi1 were detected by western blot and/or rt瞤cr, respectively. resultsthe ability of proliferation was decreased in rat mesangial cells with advancing age(p＜0.05).the expression of senescence associated β瞘alactosidase activity was increased in mesangial cells with advancing age. percentages of sa拨陋瞘al staining positive cells were (11.9±3.6)% versus (39.0±4.0)% versus (86.9±7.4)% (p＜0.05) in young, middle and aging glomerular mesangial cells. the p21waf1/cip1/sdi1 protein showed localization in nucleus in mesangial cells, and the staining intensity of p21waf1/cip1/sdi1 in mesangial cells in old animals was markedly increased, as compared to that in young and middle age rats (p＜0.05).the mrna levels of p21waf1/cip1/sdi1 was significantly increased in mesangial cells in old animals, as compared to that at the age of 3 and 12 months, meanwhile the p21waf1/cip1/sdi1 proteins expression were dramatically increased with advancing age (p＜0.05). conclusionsthe cellular phenotype and function are changed in rat mesangial cells with advancing age. the rat mesangial cells appear replicative senescence with advancing age. 【key words】mesangial cells; senescence; phenotype；p21waf1/cip1/sdi1 多数学者认为，器官衰老是由于其固有细胞功能减退所导致，肾小球系膜细胞作为肾脏最重要的固有细胞之一，具有调节肾血流量，分泌细胞外基质、炎症介质等功能，其表型和功能的改变在肾脏衰老进程中必然发挥着重要的作用。
为了能客观真实地反映肾小球系膜细胞随衰老的变化，排除体外培养环境对细胞表型的影响，本研究应用自然衰老的24月龄大鼠，体外培养原代的肾小球系膜细胞，观察系膜细胞是否随机体增龄而衰老，以及老年大鼠系膜细胞p21waf1/cip1/sdi1表达的变化，为进一步研究系膜细胞衰老的作用机制奠定基础 1材料与方法 1.1原代大鼠系膜细胞培养不同鼠龄雄性wistar大鼠〔解放军总医院实验动物中心提供，scxk(京)20050013〕15只，分3月龄组、12月龄组及24月龄组，每组各5只，无菌条件下取双侧肾脏，剥离肾包膜，用剪刀把肾皮质剪成碎块，放于重叠的3层不锈钢筛网上研磨，镜下观察，若见98%以上的肾小球脱去包曼氏囊，视野中基本上无肾小管存在，即可停止冲洗收集第2层筛网上的组织移入离心管，v型胶原酶消化后，应用15％胎牛血清的rpmi 1640培养液，37℃、5％co2条件下培养原代的系膜细胞，约7 d后第1次传代，传代的细胞24 h全部贴壁，生长迅速，形态为梭形或不规则星形，3～4 d已汇合成片，纯度极高，经鉴定结蛋白(desmin)阳性、抗ⅷ因子抗体阴性，传至第3代，用于实验 1.2四甲基偶氮唑盐（mtt）法检测细胞增殖能力系膜细胞生长至80％融合，0.25％胰酶消化，计数后接种于96孔板，2 000个细胞/孔，细胞贴壁后，继续培养24、48、72、96、120 h，加入mtt(5 mg/ml)10 μl 孵育4 h后，加二甲基亚矾（dmso）200 μl/孔裂解细胞，避光10 min，于492 nm波长记录吸光度值。
1.3衰老相关半乳糖苷酶活性（sa拨陋瞘al）染色接种于6孔板中的各组细胞，待细胞生长至80％汇合，用磷酸盐缓冲液（pbs）清洗3次，于室温固定细胞5 min，pbs液清洗3次后，加上新配制的含1mg/ml 5蹭濯4策胚岐拨陋瞕半乳糖苷（x瞘al）（美国progrma公司）的sa拨陋瞘al染液，置37℃（无co2）温育12～16 h，pbs液清洗3次后，观察细胞胞浆中蓝色沉淀，用数码相机拍照 。