辐射应激对小鼠T淋巴细胞的作用及香菇多糖的干预机制研究

发布时间：2018-09-17 19:18

【摘要】：目的：研究香菇多糖对辐射所致小鼠免疫应激损伤的保护作用。从整体水平验证香菇多糖的防护干预作用,并从细胞及分子水平探讨香菇多糖该作用的机制。方法： 1.实验模型建立与分组：①辐射损伤模型的建立：辐射条件：全身性辐射,吸收剂量为40cGy/d,吸收剂量率为300cGy/min,皮源距为150cm,照射野为20cm×20cm,连续照射30d。②实验分组：实验小鼠分为五组,分别为：辐射模型组、正常对照组、香菇多糖高剂量组、香菇多糖中剂量组和香菇多糖低剂量组。 2.T淋巴细胞提取：应用尼龙毛柱法从小鼠脾脏提取T淋巴细胞。 3.NO含量测定：按照试剂盒说明书要求进行操作,测定不同组别小鼠T淋巴细胞分泌NO含量。 4.IL-2含量测定：按照剂盒说明书要求进行操作,测定不同组别小鼠T淋巴细胞分泌IL-2含量。 5.RNA提取：按照试剂盒说明书要求,从小鼠T淋巴细胞中提取mRNA. 6.RNA逆转录合成cDNA:按照试剂盒说明书要求,从提取出的RNA合成cDNA。 7.cDNA扩增：按照试剂盒说明说要求,采用荧光染料嵌合法,配制PCR反应液,进行PCR扩增。结果：在整体水平,与正常组相比,慢性低剂量辐射可导致小鼠体重增加速率下降,脾脏指数和胸腺指数显著下降,脾脏出现明显的病理性改变。与辐射组相比,香菇多糖组可明显改善上述改变。在细胞水平,与正常组相比,慢性低剂量辐射可导致小鼠T淋巴细胞活性降低,IL-2分泌减少,NO含量升高。与辐射组比较,香菇多糖组T淋巴细胞活性明显增加,IL-2分泌增加,NO含量降低,且与正常组比较,无明显差异。在分子水平,与正常组相比,慢性低剂量辐射可引起K+通道Kv1.3、Ca2+通道Orai1基因表达出现明显上调。与正常组相比,香菇多糖低剂量组和高剂量组Kv1.3、Orai1基因表达无明显差异。与辐射模型组相比,香菇多糖低剂量组Kv1.3基因表达下调、Orai1基因表达无显著差异,香菇多糖高剂量组Kv1.3、Orail基因表达均明显下调。结论：慢性低剂量辐射可导致小鼠整体及免疫系统功能改变,影响T淋巴细胞功能,引起T淋巴细胞膜上部分离子通道mRNA的表达改变,而香菇多糖可明显干预辐射所致的上述改变。表明,香菇多糖作为一种免疫调节剂,对辐射所致小鼠免疫系统T淋巴细胞损伤具有明显防护作用,而该作用可能与香菇多糖影响T淋巴细胞膜上的离子通道有一定关系。
[Abstract]:Objective: to study the protective effect of lentinan on immune stress injury induced by radiation in mice. The protective effect of Lentinus edodes polysaccharides (Lentinus edodes) was verified from the whole level, and the mechanism of Lentinus edodes polysaccharides was discussed from cell and molecular level. Methods: 1. The establishment of experimental model and group 1 radiation injury model: radiation condition: systemic radiation, absorbed dose was 40cGy / d, absorbed dose rate was 300cGy / min, skin source distance was 150cm, irradiation field was 20cm 脳 20cm, 30d.2 experiment group: experiment mice were divided into five groups. Respectively: radiation model group, normal control group, lentinan high dose group, Lentinus edodes polysaccharide medium dose group and lentinan low dose group. 2. T lymphocyte extraction: extraction of T lymphocytes from mouse spleen by nylon hair column method. Determination of 3.NO content. The content of NO secreted by T lymphocytes in different groups of mice was determined. The content of 4.IL-2 was determined. The content of IL-2 secreted by T lymphocytes from different groups of mice was determined. 5.RNA extraction: according to the requirements of kit instructions, mRNA. 6.RNA reverse transcription synthesis cDNA: was extracted from T lymphocytes of mice. Synthetic cDNA. 7.cDNA amplification from extracted RNA: according to the requirements of the kit, the PCR reaction solution was prepared by fluorescence dye embedding method, and the PCR amplification was carried out. Results: compared with the normal group, chronic low dose radiation could decrease the weight gain rate, spleen index and thymus index, and induce pathological changes in the spleen. Compared with radiation group, lentinan group could significantly improve the above changes. At the cell level, chronic low dose radiation could reduce the activity of T lymphocytes and decrease the secretion of IL-2 and increase the content of no. Compared with the radiation group, the activity of T lymphocytes in the lentinan group was significantly increased and IL-2 secretion was increased, the no content was decreased, and there was no significant difference between the Lentinus edodes group and the normal group. At the molecular level, chronic low dose radiation induced a significant up-regulation of K channel Kv1.3,Ca2 channel Orai1 gene expression. There was no significant difference in Kv1.3,Orai1 gene expression between low dose and high dose groups of Lentinus edodes polysaccharides compared with normal group. Compared with the radiation model group, there was no significant difference in the down-regulation of Kv1.3 gene expression in the low dose group of Lentinus edodes polysaccharides, but the down-regulation of Kv1.3,Orail gene expression in the high dose group of Lentinus edodes polysaccharide. Conclusion: chronic low dose radiation can change the whole and immune system function of mice, affect the function of T lymphocytes and change the expression of mRNA in some ion channels on T lymphocyte membrane. Lentinus edodes polysaccharides can significantly interfere with the radiation-induced changes. The results showed that Lentinus edodes polysaccharide, as an immunomodulator, had an obvious protective effect on T lymphocyte damage induced by radiation in mice, which might be related to the effect of lentinan on the ion channels on T lymphocyte membrane.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R818.74;R-332;R965

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