结缔组织生长因子反义寡核苷酸抑制血管紧张素Ⅱ诱导人近曲肾小管上皮细胞肥大  

Effect of connective tissue growth factor antisense oligonucleotide on angiotensin Ⅱ induced human proximal tubular cell hypertrophy

作　　者：陈珑[1] 刘必成[1] 马坤岭[1] 刘宏[1] 罗冬冬[1] 

机构地区：[1]东南大学肾脏病研究所附属中大医院肾脏科,南京210009

出　　处：《肾脏病与透析肾移植杂志》2005年第4期318-322,共5页Chinese Journal of Nephrology,Dialysis & Transplantation

基　　金：江苏省自然科学基金(NO:BK2002052);江苏省医学重点人才基金(NO:RC2002072)

摘　　要：目的:进一步探讨结缔组织生长因子(connectivetissuegrowthfactor,CTGF)反义寡核苷酸对血管紧张素Ⅱ(AngⅡ)诱导的肾小管细胞肥大的影响。方法:采用体外培养的人肾近端小管上皮细胞株(HK2),分别采用考马斯亮蓝法、流式细胞分析技术及扫描电镜和透射电镜,观察CTGF反义寡核苷酸对AngⅡ诱导的细胞内总蛋白含量、细胞周期分布改变及细胞直径和超微结构改变的影响。结果:AngⅡ干预HK2细胞48h后,细胞内总蛋白含量显著增加(P<0·01);这种作用可被CTGF反义寡核苷酸显著抑制,且呈时间和浓度依赖性。AngⅡ干预HK2细胞后,细胞大部分阻滞在G0-G1期(P<0·01),而CTGF反义寡核苷酸可逆转这种周期阻滞现象(P<0·05)。经AngⅡ干预的HK2细胞,细胞平均直径显著增加,细胞超微结构显示表面微绒毛减少、细胞内粗面内质网增多、线粒体减少、高尔基体增加,这些作用均可被CTGF反义寡核苷酸所抑制。结论:CTGF反义寡核苷酸对AngⅡ诱导HK2细胞肥大具有显著的抑制作用,提示CTGF可能介导了AngⅡ诱导的肾小管上皮细胞肥大。Objective： Proximal tubular cell hypertrophy is an important pathological feature in remnant kidney and diabetic nephropathy. Our previous work suggested that connective tissue growth factor （CTGF） is involved in Ang Ⅱ induced human proximal tubular cell line （HK2） hypertrophy. This study was designed to further investigate the effect of CTGF antisense oligonueleotide （CTGF AS） on Ang Ⅱ induced tubular cell hypertrophy. Methodology： HK2 cells were grown in DMEM containing 10% heat inactivated fetal calf serum （FCS）. After rested in serum-free medium for 24 hours, the influence of CTGF AS（20＆micro;g/ml） on Ang Ⅱ （10^-7 M） induced CTGF mRNA and protein expression were observed by RT-PCR and confocal microscopy respectively. The effect of CTGF AS on Ang Ⅱ induced total cellular protein content （Coomassie brilliant blue G250 method）, change of cellular size （determined by scanning electronic microscope, SEM） were observed respectively. The influence of CTGF AS on cell cycle was analyzed by fluorescence-activated cell sorter （FACS） flow cytometer. The control group was treated with scrambled oligonucleotide. Results： It was shown that Ang Ⅱ signficantly induced the increased expression of CTGF mRNA and protein （P〈0.01, respectively）. After stimulating HK2 cells with Ang Ⅱ for 48h, the total protein content was significantly increased （P〈0.01, respectively）, which were significantly attenuated by treatment with CTGF AS （P〈0.05 and 0.01, respectively）. Furthermore, flow cytometer study showed that Ang Ⅱ arrested most of the cell cycle at G0-G1 phase, which was significantly attenuated by treatment with CTGF AS （P〈0.01 and 0.05, respectively）. SEM showed that the diameter of cells was significantly enlarged by treatment with Ang Ⅱ, while CTGF AS could significantly reduced the enlargement of Ang Ⅱ treated cells, Conclusion： Ang Ⅱ could induce the hypertrophy of tubular epithelial cells, which could be significantly attenuated by tre

关 键 词：人近端肾小管上皮细胞 血管紧张素Ⅱ 结缔组织生长因子 细胞肥大 肾小管上皮细胞肥大 反义寡核苷酸 酸抑制 流式细胞分析技术 超微结构改变 CTGF 

分 类 号：R692.6[医药卫生—泌尿科学] R692[医药卫生—外科学]