基于NKG2D在安多霖保护微波辐射致免疫损伤中的作用机制研究

发布时间：2018-09-02 10:06

【摘要】：目的及意义随着微波技术在军事、医疗、工业、通信及农业生产等方面的广泛应用，生物体不可避免地受到微波辐射的影响。研究表明，微波辐射对生物体的多个系统具有损伤作用，其对免疫系统的损伤及医学防护是近年来生物电磁学研究的热点之一。然而，微波辐射致免疫损伤的机制不明，且目前尚缺乏有效预防微波辐射致免疫损伤的药物。本文旨在研究中药复方制剂安多霖对微波辐射所致免疫损伤的预防作用及有效剂量，探讨NK细胞的重要活化性受体NKG2D在其中的作用，并对NKG2D/ERK信号通路在微波辐射致免疫损伤中的作用进行研究，为微波辐射致免疫损伤机制的阐明、有效防护药物的选择和应用提供理论基础，具有重要的现实意义。材料和方法本课题分为整体动物实验和离体细胞实验两部分。一、整体动物实验选用250只二级雄性Wistar大鼠，分为安多霖2w及4w预防实验。各实验大鼠125只，随机分为正常对照组、辐射对照组及0.75、1.5、3g/(kg d)药物组。大鼠连续给药2w或4w后，30mW/cm2微波辐射15min，于辐射（停药）后6h、7d、14d，应用血细胞分析仪检测外周血白细胞、淋巴细胞数量，放免法检测血清IL-2和IL-4浓度，流式细胞仪检测外周血CD3、CD4、CD8淋巴细胞亚群（计算CD4+/CD8+比值），光镜及电镜观察胸腺和脾脏组织结构及超微结构改变，Western Blot检测脾脏组织NKG2D蛋白表达。二、离体细胞实验1.将培养的NK92细胞随机分为假辐射组及10、30、50mW/cm2辐射组，分别应用10、30、50mW/cm2微波辐射细胞5min，于辐射后1h及24h，采用倒置相差显微镜观察细胞形态，流式细胞仪检测细胞凋亡、坏死和细胞周期，乳酸脱氢酶法检测NK92细胞对K562靶细胞的杀伤活性，建立微波辐射致NK92细胞损伤模型；2.采用30mW/cm2微波辐射NK92细胞5min，辐射后1h、6h及12h，应用Real-time PCR、Western Blot和图像分析技术检测细胞中NKG2D、perforin蛋白和mRNA以及ERK1/2、p-ERK1/2表达的改变；3.于辐射前30min加入50μmol/l U0126进行干预，Western Blot检测U0126干预及微波辐射后1h，ERK1/2、p-ERK1/2及perforin表达。实验结果一、大鼠外周血免疫功能指标变化：2w预防实验30mW/cm2微波辐射后14d，外周血白细胞数及CD4~+/CD8~+比值较正常对照组明显下降（p0.05或p0.01）；与辐射对照组相比，1.5、3g/(kg d)药物组在辐射后14d，淋巴细胞、白细胞数和CD4~+/CD8~+比值明显升高（p0.05或p0.01）。0.75g/(kg d)药物组外周血白细胞、淋巴细胞计数与辐射对照组相比无明显差异。IL-2和IL-4血清浓度各组间相比无明显差异。4w预防实验结果与2w预防实验结果相似。二、大鼠胸腺及脾脏组织学和超微结构改变：2w预防实验30mW/cm2微波辐射后6h，胸腺组织结构变化轻微，辐射后7d病变恢复；脾脏于微波辐射后6h和7d，白髓淋巴细胞核染色质浓缩、固缩、边移，红髓和白髓分界不清，红髓血窦充血；辐射后14d，病变基本恢复。辐射后7d超微结构观察见脾脏凋亡、坏死的淋巴细胞增多。0.75g/(kg d)药物组胸腺及脾脏组织学结构与辐射对照组相似，1.5、3g/(kg d)药物组组织学及超微结构损伤明显减轻。4w预防实验大鼠胸腺和脾脏组织学及超微结构变化规律与2w预防实验结果相似。三、大鼠脾脏组织NKG2D表达变化：30mW/cm~2微波辐射后6h，脾脏组织NKG2D表达较正常对照组明显降低（p0.01），1.5、3g/(kg d)药物组NKG2D表达较辐射对照组明显升高（p0.05或p0.01），0.75g/(kg d)药物组与辐射对照组相比无明显差异。四、NK92细胞形态、凋亡与坏死率、细胞周期改变：与假辐射组相比，辐射后1h，10、30、50mW/cm2组细胞轮廓不规则，折光性降低，G0/G1期细胞百分比明显增加（p0.01）， S期细胞百分比明显降低（p0.05或p0.01），细胞坏死率升高（p0.05或p0.01）；30、50mW/cm~2组G2/M期细胞百分比亦明显增加（p0.05或p0.01），细胞凋亡率升高（p0.05或p0.01）。辐射后24h，30、50mW/cm~2组细胞凋亡率较假辐射组升高（p0.05）。辐射后1h及24h，10mW/cm2组细胞凋亡率与假辐射组相比无明显差异。五、NK92细胞杀伤活性变化：辐射后1h，30、50mW/cm2组NK92细胞对K562细胞的杀伤活性较假辐射组明显降低（p0.01），10mW/cm~2组与假辐射组相比无明显差异。六、NK92细胞NKG2D蛋白及基因表达变化：30mW/cm~2微波辐射后6h，NK92细胞NKG2DmRNA及蛋白表达明显下降（p0.05或p0.01），辐射后12h与假辐射组相比无明显差异。七、NK92细胞NKG2D活化的效应分子perforin蛋白及基因表达变化：30mW/cm2微波辐射后1h，NK92细胞perforin mRNA表达明显下降（p0.05），辐射后1h及6h蛋白表达明显下降（p0.05或p0.01），辐射后12h与假辐射组相比无明显差异。八、NK92细胞p-ERK1/2表达变化：30mW/cm~2微波辐射后1h，NK92细胞p-ERK1/2表达明显低于假辐射组（p0.01），辐射后6h、12h，p-ERK1/2表达与假辐射组相比无明显差异。九、U0126干预后p-ERK1/2及perforin表达变化：U0126干预及30mW/cm~2微波辐射后，NK92细胞p-ERK1/2和perforin表达较单纯辐射组明显降低（p0.05或p0.01）。结论一、预防性给予1.5、3g/(kg d)安多霖2w或4w对30mW/cm~2微波辐射引起的免疫功能和免疫器官结构损伤有保护作用，预防性给予0.75g/(kg d)安多霖2w或4w保护作用不明显。二、连续给药2w或4w预防效果无明显差别；安多霖对微波辐射致免疫损伤的最佳有效预防剂量为1.5g/(kg d)；2w为最佳给药周期。三、30，，50mW/cm~2微波辐射可损伤NK92细胞，表现为细胞形态不规则、细胞凋亡及坏死率升高、增殖活性及杀伤活性降低，10mW/cm2微波辐射对NK92细胞凋亡及杀伤活性无明显影响。四、30mW/cm~2微波辐射可致NKG2D及perforin蛋白及基因表达下降。五、微波辐射后ERK信号通路活化对NK92细胞perforin表达有正向调控作用，微波辐射使NKG2D/ERK/perforin通路活化受到抑制。六、安多霖预防微波辐射致免疫损伤的作用可能通过促进NKG2D表达，活化NKG2D/ERK/perforin信号通路来实现。
[Abstract]:Purpose and significance
With the wide application of microwave technology in military, medical, industrial, communication and agricultural production, organisms are inevitably affected by microwave radiation. Studies have shown that microwave radiation can damage many systems of organisms, and its damage to the immune system and medical protection is one of the hotspots of bioelectromagnetism in recent years. 1. However, the mechanism of microwave-induced immune damage is unknown, and there is no effective drug to prevent microwave-induced immune damage at present. The purpose of this paper is to study the preventive effect and effective dosage of andolin on microwave-induced immune damage, and to explore the role of NKG2D, an important activator receptor of NK cells, in this process, and its effect on N. The role of KG2D/ERK signaling pathway in microwave-induced immune injury is studied, which provides theoretical basis for elucidating the mechanism of microwave-induced immune injury and for the selection and application of effective protective drugs.
Materials and methods
The research is divided into two parts: the whole animal experiment and the in vitro cell experiment.
Firstly, 250 male Wistar rats were divided into two weeks and four weeks prophylaxis test. 125 rats were randomly divided into normal control group, radiation control group and 0.75, 1.5, 3 g / (kg d) drug group. After 2 or 4 weeks of continuous administration, 30 mW / cm 2 microwave irradiation for 15 minutes, 6 h, 7 d, 14 d after radiation (withdrawal), blood cell analyzer was used. The number of white blood cells and lymphocytes in peripheral blood were detected. The serum levels of IL-2 and IL-4 were detected by radioimmunoassay. The peripheral blood CD3, CD4 and CD8 lymphocyte subsets (calculated CD4 +/CD8 +) were detected by flow cytometry. The histological and ultrastructural changes of thymus and spleen were observed by light and electron microscopy. The expression of NKG2D protein in spleen was detected by Western Blot.
2. In vitro cell experiment 1. NK92 cells were randomly divided into sham radiation group and 10,30,50 mW/cm 2 radiation group. NK92 cells were irradiated with 10,30,50 mW/cm 2 microwave for 5 minutes. The morphology of NK92 cells was observed by inverted phase contrast microscope at 1 h and 24 h after irradiation. Cell apoptosis, necrosis and cell cycle were detected by flow cytometry. NK92 cells were detected by lactate dehydrogenase method. NK92 cells were injured by microwave irradiation for 5 min, 1 h, 6 h and 12 h after irradiation. Real-time PCR, Western Blot and image analysis were used to detect the expression of NKG2D, perforin protein and mRNA, as well as ERK1/2, p-ERK1/2. The expression of ERK1/2, p-ERK1/2 and perforin was detected by Western Blot 1 hour after U0126 intervention and microwave irradiation.
experimental result
1. Changes of immune function in peripheral blood of rats: The number of white blood cells and the ratio of CD4~+/CD8~+ in peripheral blood were significantly decreased 14 days after 30 mW/cm2 microwave irradiation (p0.05 or p0.01), and the number of lymphocytes, white blood cells and the ratio of CD4~+/CD8~+ were significantly increased 14 days after irradiation (p0.05 or p0.01). There was no significant difference in peripheral blood leukocyte and lymphocyte counts between the two groups. There was no significant difference in serum IL-2 and IL-4 concentrations between the two groups.
2. Histological and ultrastructural changes of thymus and spleen in rats: 2 weeks after 30 mW/cm2 microwave irradiation, the histological changes of thymus were slight, and the pathological changes of thymus were restored 7 days after irradiation; 6 hours and 7 days after microwave irradiation, the chromatin of lymphocyte nucleus of white pulp was condensed, condensed, margined, the demarcation between red pulp and white pulp was unclear, and the blood sinus of red pulp was congested 14 days after irradiation. The histological structure of thymus and spleen in the drug group was similar to that in the radiation control group. The histological and ultrastructural damage of thymus and spleen in the 1.5 and 3 g / (kg d) drug group was significantly alleviated. The rule is similar to that of 2W preventive test.
Third, the expression of NKG2D in spleen tissue of rats: 6 hours after 30 mW/cm~2 microwave irradiation, the expression of NKG2D in spleen tissue was significantly lower than that of normal control group (p0.01), and the expression of NKG2D in 1.5, 3 g/(kg d) drug group was significantly higher than that of radiation control group (p0.05 or p0.01). There was no significant difference between 0.75 g/(kg d) drug group and radiation control group.
Fourthly, NK92 cell morphology, apoptosis and necrosis rate, cell cycle changes: compared with sham radiation group, 1 h, 10, 30, 50 mW / cm 2 group cells outline irregular, refractive decreased, G0 / G1 phase cell percentage significantly increased (p0.01), S phase cell percentage significantly decreased (p0.05 or p0.01), cell necrosis rate increased (p0.05 or p0.01); The percentage of apoptotic cells in phase I was also significantly increased (p0.05 or p0.01), and the apoptotic rate was increased (p0.05 or p0.01). The apoptotic rate in 30,50 mW/cm~2 group was higher than that in sham radiation group at 24 h, 30,50 mW/cm~2 group (p0.05).
Fifth, NK92 cell killing activity changes: 1 hour after radiation, 30,50 mW/cm 2 group NK92 cell killing activity to K562 cells was significantly lower than the sham radiation group (p0.01), 10 mW/cm~2 group and sham radiation group compared with no significant difference.
6. Changes of NKG2D protein and gene expression in NK92 cells: At 6 hours after 30 mW/cm~2 microwave irradiation, NKG2D mRNA and protein expression in NK92 cells decreased significantly (p0.05 or p0.01). There was no significant difference between the two groups at 12 hours after irradiation.
Seventh, the expression of NKG2D-activated effector molecule perforin protein and gene in NK92 cells: 1 hour after 30 mW/cm 2 microwave irradiation, the expression of perforin mRNA in NK92 cells decreased significantly (p0.05), 1 hour and 6 hours after irradiation (p0.05 or p0.01).
Eighth, the expression of p-ERK1/2 in NK92 cells: 1 hour after 30 mW/cm~2 microwave irradiation, the expression of p-ERK1/2 in NK92 cells was significantly lower than that in sham irradiation group (p0.01). The expression of p-ERK1/2 was not significantly different from that in sham irradiation group at 6 and 12 hours after irradiation.
Ninth, the expression of p-ERK1/2 and perforin after U0126 intervention: After U0126 intervention and 30 mW/cm~2 microwave irradiation, the expression of p-ERK1/2 and perforin in NK92 cells was significantly lower than that in the radiation group (p0.05 or p0.01).
conclusion
First, prophylactic administration of 1.5, 3 g / (kg d) andolin for 2 or 4 weeks has protective effects on immune function and structural damage of immune organs induced by 30 mW/cm~2 microwave irradiation, but prophylactic administration of 0.75 g / (kg d) andolin for 2 or 4 weeks has no obvious protective effects.
Secondly, there was no significant difference in the preventive effect between two weeks or four weeks of continuous administration; the best effective preventive dose of andolin for microwave-induced immune damage was 1.5 g / (kg d); and the best period of administration was two weeks.
Third, 30,50 mW/cm~2 microwave irradiation could damage NK92 cells, which showed irregular cell morphology, increased apoptosis and necrosis rate, decreased proliferation and killing activity. 10 mW/cm~2 microwave irradiation had no significant effect on apoptosis and killing activity of NK92 cells.
Four, 30mW/cm~2 microwave radiation can cause the decrease of NKG2D and perforin protein and gene expression.
Fifthly, the activation of ERK signaling pathway after microwave irradiation can positively regulate the expression of perforin in NK92 cells, and the activation of NKG2D/ERK/perforin pathway is inhibited by microwave irradiation.
Sixthly, the preventive effect of andolin on microwave-induced immune injury may be achieved by promoting NKG2D expression and activating NKG2D/ERK/perforin signaling pathway.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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