肌酐代谢产物对人肾小管上皮细胞毒性作用的比较研究

The study of cytotoxic effect to creatinine metabolite on HK-2 cells in vitro

目的比较观察甲基胍、1。甲脲乙醇酸酐对人肾小管上皮细胞的毒性作用。方法HK．2细胞作为研究对象，分为三组培养：正常对照（A）组，甲基胍（B）组、1－甲脲乙醇酸酐（C）组，用MTr比色试验法检测细胞增殖，NAG酶释放检测对细胞的毒性，Hoechst染色及AnnexinV—FITC／PI流式细胞术检测凋亡。结果HK－2细胞加入甲基胍、1－甲脲乙醇酸酐后，较正常对照组吸光度值显著下降（0．188±0．011，0．176±0．010VS0．545±0．021，F=1557．74，P〈0．01），NAG酶浓度显著上升（20．488±0．473，22．225±0．565VS5．1254-0．198，F：3848．22，P〈0．01）。Hoechst33258染色显示凋亡细胞呈现核固缩，染色加深，并出现凋亡小体。流式细胞术显示B组、C组HK-2细胞凋亡率显著高于A组（18．23±1．1581，20．22±1．1433VS2．4734-0．321，F=526．06，P〈0．01）。C组吸光度值显著低于B组（0．176±0．010VS0．1884-0．011，t=2．26，P〈0．05），NAG酶浓度显著高于B组（22．225±0．565VS20．488±0．473，t=－6．67．P〈0．01），凋亡率显著高于B组（20．224-1．1433VS18．23±1．1581，t=－2．762，P〈0．05）。结论1．甲脲乙醇酸酐对肾小管上皮细胞的毒性作用强于甲基胍。

Objective To investigate the effect of methylguanidine and 1-methylhydantoin on cells cytotoxicity, apoptosis in human renal tubular epithelial cell line （HK-2）. Methods Human PTEC cell line HK-2 was used in this study. HK-2 was cultured and divided into 3 groups： Normal control group （A） , methylguanidine group（B） and 1-methylhydantoin group （C）. The cell inhibitory rate of HK-2 was detected by MTF method. The cytotoxicity of methylguanidine to HK-2 was determined by NAG release test. Cell apoptosis was evaluated by using Hoechst stain and FACS with Annexin-V/PI. Results The OD value and NAG concentration of ereatinine, methylguanidine and 1-methylhydantoin group were compared with normal control group. OD value decreased and NAG concentration significantly increased（0. 188 ± 0. 011, 0. 176±0. 010 vs 0. 545±0. 021, F = 1557.74, P 〈0. 01 ; 20. 488 ±0.473, 22. 225 ±0. 565 vs 5. 125 ± 0. 198, F = 3848.22, P 〈 0. 01 ）. By Hoechst stain, pycnosis and apoptotic body could be found when HK-2 was cultivated in methylguanidine 1-methylhydantoin group. In methylguanidine, 1-methylhydantoin group apoptotic HK-2 apparently increased, compared with that in control group （ 18.23 ± 1. 1581, 20. 22 ±1. 1433 vs 2. 473 ±0. 321, F =526. 06, P 〈0. 01）. Compared with group B, the OD value in group C decreased significantly （0. 176 ± 0. 010 vs 0. 188 ± 0. 011, t = 2. 26, P 〈 0.05 ） ,＂ NAG concentration increased significantly （22. 225 ± 0. 565 vs 20. 488 ± 0. 473, t = - 6. 67, P 〈 0. 01 ）, and apoptotic rate increased significantly （20. 22 ＋ 1. 1433 vs 18.23±1. 1581, t = - 2. 762, P 〈 O. 05 ）. Conclusions 1- methylhydantoin has more powerful cytotoxic effect to renal tubular epithelial cells than that of Methylguanidine.