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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SPARC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cyclinD1、p21Wafl 表达的影响。结果 (1)与对照组比较,不同浓度(12．5、25、50μαg／ml)SPARC全长蛋白及其肽段作用48 h都能明显抑制HMC的增殖,并呈剂量和时间依赖性,96 h达到高峰。(2)12．5μg／ml SPARC全长蛋白及其肽段作用96 h,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)cyclinD1的表达明显减弱、而p21Wafl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cyclinD1的表达、上调p21Wafl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。
Objective To investigate the effects of full-length protein rich in cysteine acidic secreted glycoprotein (SPARC) and its 2.1 peptide on the proliferation, apoptosis and cell cycle of human mesangial cells (HMC) Possible mechanism of the effect of full-length protein and its peptide on HMC. Methods The HMCs cultured in vitro were treated with different concentrations of SPARC full-length protein and its peptides for different time. Cell proliferation was detected by MTT assay. Cell cycle and apoptosis were detected by flow cytometry. The expression of cyclinD1 and p21Wafl proteins in HMC was detected by Western blotting. Results (1) Compared with the control group, the proliferation and proliferation of HMCs were significantly inhibited at different concentrations (12.5, 25 and 50μαg / ml) of SPARC full-length protein and its peptide for 48 h in a dose- and time- h reached its peak. (2) 12.5μg / ml SPARC full-length protein and its peptide function 96 h, can significantly affect the cell cycle, G0 / G1 phase cells increased, S phase cells decreased, HMC cell cycle arrest in the G0 / G1 phase. (3) SPARC full-length protein and its peptide can induce HMC cell apoptosis in vitro. (4) CyclinD1 expression was significantly reduced, while p21Wafl expression was significantly enhanced. Conclusion The SPARC full-length protein and its peptide can inhibit the proliferation of HMC in a dose-dependent manner. Its function may up-regulate the expression of p21Wafl and block the cell cycle in G0 / G1 phase by inhibiting the expression of cyclinD1. This provides a clue for clinical treatment of primary, secondary mesangial proliferative glomerulonephritis.