肾小球系膜细胞金属蛋白酶1组织抑制剂对血管内皮生长因子及转化生长因子β_1表达的影响

Effect of TIMP-1 on Expression of VEGF and TGF-β_1 in Rat Mesangial Cells

目的:探讨高糖(葡萄糖25mmol/L)条件下大鼠肾小球系膜细胞(RMC)金属蛋白酶1组织抑制剂(TIMP-1)对血管内皮生长因子(VEGF)及转化生长因子β1(TGF-β1)表达的影响。方法:用高糖培养基体外培养未转染及分别用pcDNA3空载体、人反义TIMP-1基因重组真核表达载体转染的RMC24h,将细胞分为未转染组、空载体组及反义组,并设正常培养条件下的RMC作为正常对照组。RT-PCR检测各组RMC TIMP-1、VEGF及TGF-β1mRNA的表达。Western印迹检测胞质中TIMP-1、VEGF及TGF-β1蛋白的表达。免疫荧光检测各组RMC胞质中VEGF的表达。结果:高糖培养条件下未转染组及空载体组RMC大鼠TIMP-1、VEGF及TGF-β1mRNA表达均较对照组明显增加(P<0.01),而此两组间相应指标表达差异无统计学意义;反义组大鼠TIMP-1、VEGF mRNA表达较未转染组显著降低(P<0.01),而TGF-β1 mRNA表达则无变化。Western印迹也得到相同的结果。免疫荧光检测发现,未转染组及空载体组VEGF荧光表达较对照组显著增强,反义组荧光则较未转染组明显减弱。结论:高糖可促进RMC TIMP-1、VEGF及TGF-β1表达增加。在高糖条件下,TIMP-1可能位于VEGF上游,促进其表达;而TGF-β1表达并不受TIMP-1调控,高糖可能通过其他途径上调其表达。

Objective ：To investigate the effect of the tissue inhibitor of metalloproteinase - 1 （TIMP - 1 ） on the expression of vascular endothelial growth factor（VEGF） and transforming growth factor βl （TGF - 61 ） in rat mesangial cells （RMC）under the condition of high glucose（ Glucose 25 mmol/L）. Methods：The human antisense TIMP- 1 recombinant plasmid was transfected into RMC through liposome. After RMC was stimulated with high glucose for 24 hours in vitro, the expression of TIMP - 1, VEGF and TGF - βl mRNAs was assayed by RT - PCR. The expression of TIMP - 1 ,VEGF and TGF - 61 proteins was analysed by Western blot. The expression of VEGF protein was furtherly measured by immunolluorescence. Results：The expression of TIMP - 1, VEGF arid TGF - 6, mRNAs was higher in non- transfected and pcDNA3 plasmid groups than control one（ P 〈 0.01 ）, however, there weren＇t statistical differences between pcDNA3 plasmid and non - transfected groups. The expression of TIMP - 1 and VEGF mRNAs was lower in anti- sense group than non- transfected one （P 〈 0. 01 ） ,while the expression of TGF -β1 mRNA didn＇t appeared lower in antisense one. Above results were also observed by western blot. The fluorescent of VEGF was stronger in non - transfected and pcDNA3 plasmid groups than contron one, and the fluorescent of VEGF was weaker in antisense group than non - transfected one. Conclusion： High glu- cose could enhance the expression of TIMP - 1 ,VEGF and TGF -β1 in rat mesangial ceils. TIMP - 1 could be located in the upper reaches of VEGF and promote the expression of it; but the expression of TGF - β1 wasn＇ t regulated by TIMP - 1 in rat mesangial cells under the condition of high glucose. High glucose could upregulate the expression of TGF - β by other means in rat mesangial cells.