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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 effect of recombinant human IL-10 (rhIL-10) on apoptosis and Caspase-3 activity in human pulmonary microvascular endothelial cells (HPMVECs) under hypoxic conditions. Methods HPMVEC cell lines were routinely cultured and divided into five groups: normal control group (normal culture), hypoxic control group (50 mL·L-1CO2, 950 mL·L-1N2), hypoxia culture and rhIL-10 supplementation The rhIL-10 dose (10 × 103ng.L-1, 50 × 103ng.L-1,100 × 103ng.L-1) was divided into low dose rhIL-10 intervention group, rhIL-10 medium dose intervention group, rhIL-10 High-dose intervention group. The cells were harvested at 4 h, 12 h, 24 h and 48 h after centrifugation. The activity of Caspase-3 in the nucleus was detected by chemical colorimetry. The fluorescence intensity was observed by fluorescence microscopy after 24 h. The fluorescence intensity was observed under a fluorescence microscope and analyzed statistically. Results After 4 h, there was no significant difference in the relative activity of apoptotic protein Caspase-3 (P> 0.05). At 12 h, the expression of caspase-3 in hypoxia control group and rhIL-10 low and medium dose groups were significantly higher than those in normal control group (P <0.05). There was no significant difference between the rhIL-10 high dose intervention group and the normal control group (P> 0.05) Compared with the normal control group, the levels of rhIL-10 in the intervention group and the hypoxia control group were significantly decreased (P <0.05) Compared with the normal control group, the hypoxia control group and rhIL-10 low-dose intervention group were significantly increased (P <0.05), while the low, medium and high doses of rhIL-10 intervention group were significantly decreased compared with the hypoxia control group (Pa> 0.05) The peak of hypoxia apoptosis was found in 24 h. 24 h. Hoechst staining of apoptotic nuclei in each group was performed. The gray value of low, medium and high dose intervention groups of hypoxia control group and rhIL-10 group were significantly higher than that of the normal control group (P <0.05); rhIL-10 Low, medium and high doses of rhIL-10 intervention group and hypoxic control group was significantly lower (Pa <0.05). Conclusion Hypoxia can promote the apoptosis of HPMVEC and rhIL-10 can inhibit the apoptosis of HPMVEC to a certain extent. These changes may be closely related to the diseases of acute hypoxia.