【摘要】：目的： 研究香菇多糖對(duì)輻射所致小鼠免疫應(yīng)激損傷的保護(hù)作用。從整體水平驗(yàn)證香菇多糖的防護(hù)干預(yù)作用,并從細(xì)胞及分子水平探討香菇多糖該作用的機(jī)制。 方法： 1.實(shí)驗(yàn)?zāi)Ｐ徒⑴c分組：①輻射損傷模型的建立：輻射條件：全身性輻射,吸收劑量為40cGy/d,吸收劑量率為300cGy/min,皮源距為150cm,照射野為20cm×20cm,連續(xù)照射30d。②實(shí)驗(yàn)分組：實(shí)驗(yàn)小鼠分為五組,分別為：輻射模型組、正常對(duì)照組、香菇多糖高劑量組、香菇多糖中劑量組和香菇多糖低劑量組。 2.T淋巴細(xì)胞提�。簯�(yīng)用尼龍毛柱法從小鼠脾臟提取T淋巴細(xì)胞。 3.NO含量測(cè)定：按照試劑盒說(shuō)明書(shū)要求進(jìn)行操作,測(cè)定不同組別小鼠T淋巴細(xì)胞分泌NO含量。 4.IL-2含量測(cè)定：按照劑盒說(shuō)明書(shū)要求進(jìn)行操作,測(cè)定不同組別小鼠T淋巴細(xì)胞分泌IL-2含量。 5.RNA提�。喊凑赵噭┖姓f(shuō)明書(shū)要求,從小鼠T淋巴細(xì)胞中提取mRNA. 6.RNA逆轉(zhuǎn)錄合成cDNA:按照試劑盒說(shuō)明書(shū)要求,從提取出的RNA合成cDNA。 7.cDNA擴(kuò)增：按照試劑盒說(shuō)明說(shuō)要求,采用熒光染料嵌合法,配制PCR反應(yīng)液,進(jìn)行PCR擴(kuò)增。 結(jié)果： 在整體水平,與正常組相比,慢性低劑量輻射可導(dǎo)致小鼠體重增加速率下降,脾臟指數(shù)和胸腺指數(shù)顯著下降,脾臟出現(xiàn)明顯的病理性改變。與輻射組相比,香菇多糖組可明顯改善上述改變。在細(xì)胞水平,與正常組相比,慢性低劑量輻射可導(dǎo)致小鼠T淋巴細(xì)胞活性降低,IL-2分泌減少,NO含量升高。與輻射組比較,香菇多糖組T淋巴細(xì)胞活性明顯增加,IL-2分泌增加,NO含量降低,且與正常組比較,無(wú)明顯差異。在分子水平,與正常組相比,慢性低劑量輻射可引起K+通道Kv1.3、Ca2+通道Orai1基因表達(dá)出現(xiàn)明顯上調(diào)。與正常組相比,香菇多糖低劑量組和高劑量組Kv1.3、Orai1基因表達(dá)無(wú)明顯差異。與輻射模型組相比,香菇多糖低劑量組Kv1.3基因表達(dá)下調(diào)、Orai1基因表達(dá)無(wú)顯著差異,香菇多糖高劑量組Kv1.3、Orail基因表達(dá)均明顯下調(diào)。 結(jié)論： 慢性低劑量輻射可導(dǎo)致小鼠整體及免疫系統(tǒng)功能改變,影響T淋巴細(xì)胞功能,引起T淋巴細(xì)胞膜上部分離子通道m(xù)RNA的表達(dá)改變,而香菇多糖可明顯干預(yù)輻射所致的上述改變。表明,香菇多糖作為一種免疫調(diào)節(jié)劑,對(duì)輻射所致小鼠免疫系統(tǒng)T淋巴細(xì)胞損傷具有明顯防護(hù)作用,而該作用可能與香菇多糖影響T淋巴細(xì)胞膜上的離子通道有一定關(guān)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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R818.74;R-332;R965

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