重组人白细胞介素-2活化的自然杀伤细胞对大鼠梗死心肌功能的影响

Restructuring human interleukin-2 activation of natural killer cells rat infarction myocardial function

目的探讨注射重组人白细胞介素-2(recombinant human interleukin-2,rhIL-2)活化后的自然杀伤细胞(natural killer cells,NKC)对大鼠梗死心肌功能的影响。方法体外提取大鼠NKC,实验组经rhIL-2活化,对照组未活化,比较2组NKC的杀伤能力;体外提取大鼠心肌微血管内皮细胞,分成3组,分别与rhIL-2-NKC、NKC和PBS液共培养0 d和10d,观察3组共培养液对内皮细胞增殖的影响;取大鼠梗死1h的心肌组织,分别注射rhIL-2-NKC、NKC和PBS(空白对照组),并于注射后0、1、2、4、8、16 d采用RTPCR检测各组单核细胞趋化蛋白-1(monocyte chemoattractant protein-1,MCP-1)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白细胞介素-2(interleukin-2,IL-2)的mRNA表达水平;取处理16 d的3组大鼠用于测定血液动力学指标:左心室收缩压(left ventricular systolic pressure,LVSP)、舒张末压(left ventricular end diastolic pressure,LVEDP)、平均动脉压(mean arterial pressure,MAP)、压力最大上升速率(+dp/dt)和压力最大下降速率(-dp/dt),并以免疫组织化学方法检测心肌梗死区域面积和微血管密度(MVD)及梗死边缘区域的血管密度以及血小板内皮细胞粘附分子(CD31)和血管内皮生长因子(vascular endothelial growth factor,VEGF)的蛋白表达水平。结果 rhIL-2-NKC杀伤力增加,且随效应细胞与靶细胞比例增加而增加;心肌微血管内皮细胞与rhIL-2-NKC共培养处理后,细胞增殖数量为空白对照组的1.28倍,差异有统计学意义(P<0.05);RT-PCR结果表明:rhIL-2-NKC处理组大鼠心肌组织MCP-1、TNF-α和IL-2 mRNA表达均上调,于2~8 d时显著高于NKC组和空白对照组(P<0.05);血液动力学指标检测表明:rhIL-2-NKC组大鼠LVEDP、MAP和+dp/dt均高于NKC组和空白对照组,但LVSP和-dp/dt均低于NKC组和空白对照组,rhIL-2-NKC组各指标与对照组相比差异均有统计学意义(P<0.05);免疫组化检测结果表明:rhIL-2-NKC组心肌梗死面积(19.23±5.61)最低,而MVD(15.29±1.77)最高,与其他2组差异均有统计学意义(P<0.05);rhIL-2-NKC组梗死边缘区域CD31(238.17±16.82)和VEGF(221.32±16.97)蛋白表达水平高于NKC(CD31和VEGF分别为66.81±11.84和87.33±10.18)和空白对照组(CD31和VEGF分别为29.23±5.57和76.21±9.23),差异均有统计学意义(P<0.05)。结论心肌注射rhIL-2-NKC能够促进梗死心肌的血管再生,具有改善大鼠心肌功能的作用。

Objective To investigate the effect of recombinant human interleukin-2( rhIL-2) activated natural killer cells( NKC) on infarcted myocardium. Methods NKC was isolated from mice and cultured in vitro,with one part of them was activated with rhIL-2. Then the lethality of two parts of NKC were determined. Cardiac microvascular endothelial cells in vitro were co-cultured with rhIL-2-NKC,NKC and PBS solution,respectively for 0d and 7d,and evaluate the effect of rhIL-2-NKC on endothelial cells proliferation,with NKC group as negative control and PBS group as blank control. Three groups of myocardial infarcted mice were injected with rhIL-2-NKC,NKC and PBS solution respectively,and mRNA expression levels of monocyte chemoattractant protein-1( MCP-1),tumor necrosis factor-α( TNF-α) and interleukin-2( IL-2) in myocardium were determined by RT-PCR method after 0,1,2,4,8,16 d treatment. After that,three groups of mice were used to measure left ventricular systolic pressure( LVSP),left ventricular end diastolic pressure( LVEDP),mean arterial pressure( MAP),maximum rate of pressure rise( + dp /dt) and maximum rate of pressure decrease(-dp /dt),and to determine the area and micro-vascular density( MVD) of myocardial infarction and protein levels of platelet endothelial cell adhesion molecules( CD31) and vascular endothelial growth factor( VEGF) of marginal area of myocardial infarction. Results The lethality of rhIL-2-NKC was increased,which was positively related with the ratio of effector cells to target cells. The cell proliferation of rhIL-2-NKC group was 1. 28 times of blank control group,and the difference was significant( P < 0. 05). RT-PCR results demonstrated an upregulation of MCP-1,TNF-α and IL-2 mRNA levels,which were significantly higher than NKC and blank control groups during 2 ~ 8d( P < 0. 05). Results Hemodynamic parameters results showed that compared to NKC and blank control groups,rhIL-2-NKC goup had a higher level of LVEDP,MAP and + dp /dt,but a lower level of LVSP and-dp /dt,the difference were all significant( P < 0. 05). Immunohistochemical results showed that rhIL-2-NKC group had a significant higher level of myocardial infarction area( 19. 23 ± 5. 61) and a significant lower level of MVD( 15. 29 ± 1. 77) than other groups( P < 0. 05). In the marginal area of myocardial infarction,the values of CD31 and VEGF protein levels of rhIL-2-NKC group were( 238. 17 ± 16. 82) and( 21. 32 ± 16. 97) respectively,which were significantly higher than that of NKC group( CD31 = 66. 81 ± 11. 84,VEGF = 87. 33 ± 10. 18) and blank control group( CD31 = 29. 23 ±5. 57,VEGF = 76. 21 ± 9. 23,P < 0. 05). Conclusion The myocardial injection of rhIL-2-NKC could promote angiogenesis and therefore improve myocardial function.