摘要
背景:p38丝氨酸蛋白激酶信号激活引起细胞生长、增殖、分化,可能是糖尿病血管并发症发生发展的共同通路。目的:观察阿托伐他汀对抗p38丝氨酸蛋白激酶信号通路的激活,及其对氧化修饰低密度脂蛋白作用下人肾小球系膜细胞增殖与转化生长因子β1表达的干预。设计:随机、平行对照、开放性实验。单位:中国医科大学附属第一医院内分泌科,解放军沈阳军区总医院内分泌科、呼吸科及泌尿外科。材料:实验于2004-05/2005-05先后在中国医科大学药理教研室、沈阳军区总医院呼吸科实验室完成。以肾肿瘤行单侧肾切除患者正常部分的肾皮质(由沈阳军区总医院泌尿外科向军主任提供,征得患者同意)作为实验用人肾小球系膜细胞的来源。氧化修饰低密度脂蛋白浓度为(5.3±1.0)nmol/100μg(购自协和医科大学生化研究所,批号20040711);阿托伐他汀(辉瑞制药,批号45837088);p38丝氨酸蛋白激酶单克隆抗体(Santa Cruz)。方法:①取肾肿瘤行单侧肾切除患者的正常部分肾皮质6.0～8.0cm3,分离制备肾小球系膜细胞,待生长单层达80%培养面积后进行消化传代。传至第2代时,观察细胞形态,行DAB染色,胞浆内出现棕黄色团块或泥沙样颗粒为DAB染色阳性。油红“O”染色检测细胞吞噬氧化修饰低密度脂蛋白的情况。取传至4～10代的细胞用于实验。②按5×103个细胞接种于96孔板,200μL/孔。实验共分5组:阿托伐他汀1.2,6,12mg/L浓度组分别加入对应浓度的阿托伐他汀溶液,氧化低密度脂蛋白组和空白对照组此时仅加入培养基,6个平行孔/组。各组预处理30min后,除空白对照组外均暴露于终浓度为80mg/L的氧化修饰低密度脂蛋白中,24h后每孔加入四甲基偶氮唑蓝,在492nm波长的酶标仪上检测吸光度值,计算细胞增殖抑制率。③将1×106～3×106细胞接种于6个200mL培养瓶中,随机数字表法选1瓶作为空白对照组,第2～4号瓶均加入适量氧化修饰低密度脂蛋白使其终浓度分别为10,40,80mg/L,第5号瓶加入阿托伐他汀12mg/L。各组预处理30min,然后加入氧化修饰低密度脂蛋白使其终浓度为80mg/L常规培养24h。所获各组细胞用半定量反转录多聚酶链法检测细胞转化生长因子β1mRNA的表达,Westernblot法检测细胞p38丝氨酸蛋白激酶信号通路激活情况。主要观察指标:①人肾小球系膜细胞鉴定结果。②人肾小球系膜细胞的增殖情况检测。③人肾小球系膜细胞转化生长因子β1mRNA的表达。④人肾小球系膜细胞p38丝氨酸蛋白激酶信号通路激活情况。结果:①细胞传至第2代时,细胞体积大,多数为梭形、不规则的星形或树枝状,细胞内有大量的平行于中轴的微丝,密集处细胞可重叠生长。DAB染色后胞浆内肌动蛋白、波形蛋白为阳性,角蛋白为阴性。油红“O”染色后细胞内可见红色颗粒,为人肾小球系膜细胞吞噬的氧化修饰低密度脂蛋白。证明培养传代的细胞为人肾小球系膜细胞。②与空白对照组比较,氧化修饰低密度脂蛋白组细胞增殖抑制率明显降低,阿托伐他汀1.2,6,12mg/L浓度组细胞增殖抑制率均明显升高(0,-17.4%,6.4%,22.5%,61.5%,P<0.05或0.01),并呈剂量依赖性。③与空白对照组比较,氧化修饰低密度脂蛋白10,40,80mg/L组以浓度依赖的方式增加转化生长因子β1mRNA表达和激活p38丝氨酸蛋白激酶信号通路,其中80mg/L浓度组最为显著(P均<0.01);阿托伐他汀则可明显抑制在氧化修饰低密度脂蛋白刺激作用下转化生长因子β1的表达增加与p38丝氨酸蛋白激酶信号通路蛋白活性,与氧化修饰低密度脂蛋白80mg/L浓度组比较差异有显著性意义(P均<0.01)。结论:阿托伐他汀可能通过对抗p38丝氨酸蛋白激酶信号通路蛋白活性、减少转化生长因子β1分泌,抑制氧化修饰低密度脂蛋白引起的肾小球系膜细胞增殖,从而在预防和治疗伴有血脂异常的糖尿病肾脏病变的发生过程中起一定作用。
BACKGROUND： The cell growth, proliferation and differentiation caused by p38 mitogen-activated protein kinase （p38MAPK） might act as the common pathway in the onset and development of diabetic vascular complication.
OBJECTIVE： To investigate the effect of atorvastatin on p38MAPK signal pathway and the influence of atorvastatin on cell proliferation and expression of transforming growth factor-β1 （TGF-β1） at transcriptional level in human glomerular mesangial cells （HGMCs） cultured with oxidative modification of low-density lipoprotein （ox-LDL）.
DESIGN ： A randomized, parallelized, controlled and open tria.
SETTING： Endocrinology Department, First Hospital Affiliated to China Medical University; Endocrinology Department, Respiratory Department, Urology Department, the General Hospital of Shenyang Military Area Command of Chinese PLA.
MATERIALS： The experiment had been done in the laboratories for Pharmaceutical Department of China Medical University and Respiratory Department of Shenyang Military Area Command of Chinese PLA from May 2004 to May 2005. The sample was cut from renal cortex from the healthy segment of nephroectomy from a tumor patient （Provided by Xiang Jun, Urology Department, the General Hospital of Shenyang Military Area Command of Chinese PLA; Informed consent was obtained）. OX-LDL was purchased from biochemistry institute of Peking Union Medical College （Batch No. 20040711 ）. ox-LDL was 5.3±1.0 nmol in 100μg protein. Atorvastatin was purchased from Pfizer Pharmaceutical Co. Ltd （No. 45837088）; p38MAPK monocloncal antibody was purchased from Santa Cruz.
METHODS ： （1） 6.0-8.0 cm^3 blocks of cortex were cut from renal cortex from the healthy segment of nephroectomy from a tumor patient, glomerular mesangial cells were isolated. When grew into 80% confluent monolayer, the cells were digested and performed passage. After the first two passages, the cells were pure based on morphology and characterized by DAB staining for Vimentin antigen and actin antigen was positive, whereas cytokeratin antigen was negative. Oil red ＂O＂ staining confirmed that ox-LDL was intaken by HGMCs. The 4-8^th passages of cells were used to study. （2）HGMCs were seeded into 96-well plates with 5×10^3 cells per well and grown in 200 μL culture medium. The study was divided into 5 groups （6 wells each group）： 1.2, 6,12 mg/L atorvastatin group, ox-LDL group and blank control group, The cells were pre-incubated with atorvastatin for 30 minutes, then exposed to 80 mg/L ox-LDL. The cells in blank control group were untouched. After 24 hours, MTT was added. The absorbance of each sample at the wavelength of 492 nm was measured with immunosorbant assay system. The inhibitory rate of cell proliferation was calculated. （3）1×10^6 to 3×10^6 cells were seeded into six 200 mL flasks. The trial was divided into 5 groups randomly： control group, 10, 40, 80 mg/L ox-LDL groups and atorvastatin group （12 mL/g）. The cells in each group were pre-incubated for 30 minutes, then exposed to 80 mg/L ox-LDL for 24-routine culture. The expressions of TGF-β1 mRNA of harvested cells were detected with semi-quantitative reverse transcription-polymerase chain reaction and p38MAPK signal pathway activation was detected by Western blot.
MAIN OUTCOME MEASURES： （1）Identification results of HGMCs. （2）Proliferation of HGMCs. （3） TGF-β1 expression of HGMCs. （4）p38MAPK signal pathway activation of HGMCs.
RESULTS ： （1）When the cells were sub-cultured to the second generation, cell volume was big. Most of the cells were spindle-shaped, irregular stellate or branch-like, filled with microfilaments which paralleled axis. Cells overlapped in the intensive area. After DAB staining, cytoplastic actin and vimentin were positive and keratin was negative. Oil red ＂O＂ staining confirmed that ox-LDL was intaken by HGMCs with red granules in the cytoplasma, while control group did not. It was proved that the cells cultured for passage were HGMCs. （2） As compared with control group, the inhibitory rate of cell proliferation in ox-LDL group was significantly decreased, but that in atorvastatin 1.2, 6 and 12 mg/L groups was significantly increased （0, -17.4%, 6.4%, 22.5%, 61.5%, respectively, P 〈 0.05 or 0.01） on concentration-dependent manner. （3） As compared with control group, ox-LDL （10, 40, 80 mg/L） increased the expression of TGF-β1 and activation of p38MAPK in concentration-dependent manner, the effect of 80 mg/L ox-LDL group was the most significantly （P 〈 0.01）. Atorvastatin decreased the increment of TGF-β1 expression and the activation of p38MAPK pathway induced by ox-LDL significantly. There was significant difference when compared with 80 mg/L ox-LDL group （P 〈 0.01）.
CONCLUSION.： Atorvastatin can antagonize the activation of p38MAPK pathway, decrease the secretion of TGF-β1 and inhibit mesangial cell proliferation induced by ox-LDL, suggesting that it may exert beneficial effect in the prevention and treatment of diabetic nephropathy with dyslipidemia.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第16期3184-3188,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
辽宁省科技攻关项目(2001225004)~~
