摘要
目的:观察富含半胱氨酸酸性分泌型糖蛋白(SPARC)2.1肽段对体外培养人肾小球系膜细胞(HMC)超微结构及其分泌细胞外基质的影响。方法:50μg/mlSPARC2.1肽段作用48h、96h后,倒置显微镜下观察HMC形态变化,透射电镜观察其超微结构,以正常培养的HMC作空白对照组。荧光定量RT-PCR法检测空白对照组和50μg/mlSPARC2.1肽段作用96h后HMC分泌细胞外基质[Ⅰ型和Ⅳ型胶原(ColⅠ、ColⅣ)、层粘连蛋白(LN)、纤维连接蛋白(FN)]相关基因mRNA表达的变化;ELISA法比较两组HMC培养上清中细胞外基质(ColⅠ、ColⅣ、LN、FN)的蛋白含量。结果:空白对照组HMC无明显变化;50μg/mlSPARC2.1肽段作用48h电镜可见少数HMC发生凋亡早期改变;96h可见较多细胞发生典型凋亡改变,电镜下可见典型的凋亡小体。与空白对照组相比,SPARC2.1肽段作用96h后,HMC的ColⅠ、ColⅣ、FNmRNA和蛋白含量显著下调(P<0.01,P<0.05),LNmRNA及其蛋白含量无明显变化。结论:SPARC2.1肽段可在体外诱导HMC细胞的凋亡,超微结构可见典型凋亡小体,并抑制其分泌细胞外基质。
Objective： To investigate the effects of secreted protein acidic and rich in cysteine （SPARC） peptide on ultramicrostructure and extracellular matrix secretion of human mesangial cells cultured in vitro. Methods： Human mesangial cells （HMC） were incubated at the presence of SPARC peptide （50μg/ml） for 48 h and 96 h separately; HMC cultured without SPARC peptide was taken as control. The cell ultramicrostructure was observed by transmission electron microscope. Real-time fluorescent quantitative PCR （RT-PCR） was used to detect mRNA levels of collagen typeⅠ （ColⅠ ）, collagen type Ⅳ （ColⅣ）, fibronectin （FN）, and laminin （LN） 96 hours after culture, and the results were compared between the 2 groups. The secreted levels of ColⅠ , ColⅣ, FN, and LN protein were measured and compared by enzyme-linked immunosorbent assay （ELISA） in the 2 groups. Results： There was no obvious change in HMC in the control group. After cultured for 48 hours, a few HMC in experimental group showed initial appearance of apoptosis; after cultured for 96 h, a great number of HMC had a typical apoptotic appearance and there were typical apoptotic bodies under electron microscope. RT-PCR showed that, compared with the control group, the experimental group had significantly decreased levels of ColⅠ , ColⅣ and FN mRNA （P〈0.05, P〈0. 01） ; ELISA results showed that the secretions of ColⅠ , ColⅣ, and FN protein in the experimental group were greatly lower than those in the control garoup （P〈0.05, P〈0.01）. Conclusion： SPARC peptide can effectively induce apoptosis of human mesangial cells and down regulate the secretion of extracellular matrix in vitro.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2007年第1期36-39,共4页
Academic Journal of Second Military Medical University
