富含半胱氨酸酸性分泌糖蛋白及其肽段对人系膜细胞增殖和凋亡的作用

目的 研究富含半胱氨酸酸性分泌糖蛋白（SPARC）的全长蛋白及其2．1肽段对人肾小球系膜细胞（HMC）增殖、凋亡及细胞周期的影响，初步探讨SPARE全长蛋白及其肽段对HMC作用的可能机制。方法 采用体外传代培养的HMC，用不同浓度的SPARC全长蛋白及其肽段分别作用不同时间。采用噻唑蓝（MTT）法检测细胞增殖；流式细胞术检测细胞周期及细胞凋亡；应用Western印迹检测SPARC全长蛋白及肽段对HMC细胞周期调控蛋白cyclin D1、p21^Wafl表达的影响。结果 （1）与对照组比较，不同浓度（12．5、25、50μg／ml）SPARC全长蛋白及其肽段作用48h都能明显抑制HMC的增殖，并呈剂量和时间依赖性，96h达到高峰。（2）12．5μg／ml SPARC全长蛋白及其肽段作用96h，能明显影响细胞周期，使G0／G1期细胞增加，S期细胞减少，可使HMC的细胞周期阻滞在G0／G1期。（3）SPARC全长蛋白及其肽段可在体外诱导HMC细胞凋亡。（4）cyclin D1的表达明显减弱、而p21^Wafl的表达明显增强。结论 SPARC全长蛋白及其肽段可剂量依赖性地抑制HMC的增殖，其作用可能通过抑制cyclin D1的表达、上调p21^Wafl的表达，使细胞周期阻滞在G0／G1期。这为临床治疗原发性、继发性系膜增生性肾小球肾炎提供了线索。

Effects of SPARC and its peptide on proliferation and apoptosis of human mesangial cells cultured in vitro

Objective To investigate the effects of SPARC (secreated protein acidic and rich in cysteine) and its peptide on proliferation, apoptosis and cell cycle of human mesangial cells cultured in vitro, and explore the possible mechanism. Methods Mesangial cells were incubated in the media with various concentrations of SPARC and its peptide cultured in vitro. Cell proliferation was assessed by MTT colorimetric assay. Cell cycle and apoptosis index were analyzed by flow cytometry. The expression of cyclinD1 and p21Wafl proteins in response to SPARC and its peptide in HMC was determined by Western blot. Results Various concentrations of SPARC and its peptide could significantly inhibit the proliferation of mesangial cells in dose- and time-dependent manner, regulate the cell cycle at phrase G-0/G1 increased while cells phrase S reduced, and could also induce apoptosis. Under the stimulation of SPARC and its peptide, the expression of cyclinDl in HMC decreased markedly meanwhile the expression of p21Wafl increased significantly. Conclusions SPARC and its peptide can effectively inhibit HMC proliferation and regulate cell cycle progression. The mechanism may be mediated by inhibiting cyclinDl and stimulating p21Wafl expression, subsequently blocking cells passing through G-S check point, which will be useful for treating mesangial proliferative glomerulonephritis.