重组人白细胞介素-10对缺氧条件下人肺微血管内皮细胞凋亡的影响

Effect of Recombinant Human Interleukin-10 on Hypoxia-Induced Apoptosis in Human Pulmonary Microvascular Endothelial Cells

目的研究低氧条件下,重组人IL-10(rhIL-10)对人肺微血管内皮细胞(HPMVEC)凋亡和半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)活性的影响。方法常规培养HPMVEC细胞株,分为5组:正常对照组(常规培养);缺氧对照组(50 mL·L-1CO2,950mL·L-1N2);缺氧培养并加入rhIL-10,并按rhIL-10剂量(10×103ng.L-1、50×103ng.L-1、100×103ng.L-1)分为rhIL-10低剂量干预组、rhIL-10中剂量干预组、rhIL-10高剂量干预组。培养4 h、12 h、24 h、48 h离心收集细胞,化学比色法检测细胞核内Caspase-3活性,24 h核荧光染色并在荧光显微镜下观察荧光强度,并进行统计学分析。结果 4 h,凋亡蛋白Caspase-3相对活性各组间无明显差异(P>0.05);12 h,缺氧对照组,rhIL-10低、中剂量干预组与正常对照组比较显著升高(Pa<0.05),rhIL-10高剂量干预组与正常对照组比较无明显差异(P>0.05),rhIL-10各剂量干预组与缺氧对照组比较显著降低(Pa<0.05);24 h,缺氧对照组、rhIL-10各剂量干预组与正常对照组比较显著升高(Pa<0.05),rhIL-10各剂量干预组与缺氧对照组比较明显降低(Pa<0.05);48 h,缺氧对照组和rhIL-10低剂量干预组与正常对照组比较显著升高(Pa<0.05),低、中、高剂量rhIL-10干预组与缺氧对照组比较明显降低(Pa>0.05);缺氧凋亡峰值见于24 h。24 h,各组行凋亡细胞核Hoechst荧光染色,缺氧对照组和rhIL-10低、中、高剂量干预组灰度值与正常对照组比较均显著升高(Pa<0.05);rhIL-10低、中、高剂量rhIL-10干预组与缺氧对照组比较显著降低(Pa<0.05)。结论缺氧可促进HPMVEC凋亡,rhIL-10可一定程度地抑制HPMVEC凋亡,这些变化可能与急性缺氧性各器官疾病密切相关。

Objective To investigate the ameliorative mechanism of recombinant human interleukin - 10 （ rhIL - 10） through inhibiting the apoptosis and Caspase - 3 activity in hypoxia - induced human pulmonary microvascular endothelial cells （ HPMVEC ）. Methods The cultured HPMVEC were randomly divided into normal control group,hypoxia control group,the low- rhIL- 10 interveation group,the middle- rhlL - 10 intervention group and the high - dose rhlL - 10 intervention group（ 10 ×10^3 ng·L-1 ,50×103 ng · L-l, 100×103 ng· L-1 ）. The normal group was cultured with the 37 ℃ and 50 mL· L-1COz and 210 mL· L-I O2 condition,and the hypoxis group was cultured with 50 mL· L -1 CO2 ,950 mL · L-I N2 condition. The culture time lasted for 4 h, 12 h ,24 h and 48 h ,respectively. The relative Caspase - 3 activity in all groups was detected with chromatometry at the end of 4 h, 12 h ,24 h and 48 h. The apoptosis fluorescence intensity was analyzed by using the fluorescent microscope at the end of 24 h with statistical method. Results There was no significant diffennce in the apoptosis and Caspase -3 activity among all the groups at the end of 4 h（ P 〉 0.05 ）. At the end of 12 h, the relative Caspase -3 activity was significantly increased in the hypoxia control group,the low dose and the middle dose rhIL- 10 intervention groups,compared to the normal control group （Pa 〈0.05）. The relative Caspase -3 activity in the high dose rhIL- 10 intervention groups was not obviously different from that in the nor- mal group （Pa 〉 0.05 ）, while it was relatively lower than that in the other groups （P 〈 0.05 ）. At the end of 24 h, the relative Caspase -3 activity in the hypoxia control group, the low dose, the middle dose and the high dose rhlL - 10 intervention groups were higher than that in the normal group （Pa 〈 0.05 ） ,while the relative Caspase -3 activity in the low ,the medial and the high dose rhIL- 10 intervention groups were lower than that in the hypoxia control group （P, 〈 0.05 ）. At the end of 48 h, the relative Caspase -3 activity in the hypoxia control group and the low dose rhfL - 10 intervention groups were higher than that in the normal group （ P 〈 0.05 ）, and there was no significant difference among all the other groups （ P 〉 0.05 ）. The peak value of hypoxia appeared at the end of 24 h. The apoptosis fluorecence intensity increased in the hypoxia control group ,the low ,the medial and the high dose rhIL- 10 intervention groups ,compared to the normal control group （Pa 〈 0.05 ）,while the apoptosis fluorescence intensity in all the rhIL- 10 intervention groups decreased, compared to the hypoxia control group （P 〈 0.05 ）. Conclusions Hypoxia can promote the apoptosis of HPMVEC, and the rhlL - 10 may play an impor：ant role in inhibiting the apoptosis of the hypoxia - induced HPMVEC by down - regulating the Caspase - 3 activity. These changes can be closely related to the acute hypoxia diseases of various organs.