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      亞精胺對鼠免疫器官指數(shù)及炎癥因子表達的影響

      2017-03-06 00:38:04徐麒麟姜冬梅魏春葉賈醫(yī)林易治鑫陳咨余
      浙江農業(yè)學報 2017年2期
      關鍵詞:精胺胸腺脾臟

      徐麒麟,康 波,姜冬梅,魏春葉,賈醫(yī)林,易治鑫,陳咨余

      (四川農業(yè)大學 動物科技學院/畜禽遺傳資源發(fā)掘與創(chuàng)新利用四川省重點實驗室, 四川 成都 611130)

      亞精胺對鼠免疫器官指數(shù)及炎癥因子表達的影響

      徐麒麟,康 波*,姜冬梅*,魏春葉,賈醫(yī)林,易治鑫,陳咨余

      (四川農業(yè)大學 動物科技學院/畜禽遺傳資源發(fā)掘與創(chuàng)新利用四川省重點實驗室, 四川 成都 611130)

      為闡明亞精胺對鼠免疫器官指數(shù)、胸腺和脾臟組織炎癥因子表達的影響,采用0、0.05、0.10和0.15 mg·g-1(體質量)亞精胺腹腔注射6周齡昆明小白鼠,測定鼠胸腺和脾臟指數(shù),并定量檢測胸腺和脾臟組織TNF-α、IL-1β、IL-6和IFN-γ表達量。結果表明:注射0.15 mg·g-1亞精胺組鼠胸腺指數(shù)和脾臟指數(shù)顯著(P<0.05)低于對照組;0.10 mg·g-1亞精胺組胸腺組織IFN-γ表達量顯著(P<0.05)高于對照組;0.05 mg·g-1亞精胺組鼠脾臟組織TNF-α和IL-1β表達量顯著(P<0.05)低于對照組,0.10 mg·g-1亞精胺組脾臟組織IL-6和IFN-γ表達量均顯著(P<0.05)高于對照組,0.15 mg·g-1組脾臟組織TNF-α表達量顯著(P<0.05)低于對照組。由此可見,亞精胺對炎癥因子表達具有組織特異性和劑量依賴性。低濃度亞精胺能抑制炎癥因子表達發(fā)揮抗炎作用,隨著亞精胺濃度增加則表現(xiàn)為誘導炎癥。

      亞精胺;胸腺指數(shù);脾臟指數(shù);炎癥因子

      多胺主要包括腐胺、亞精胺和精胺,在細胞生長、衰老、自噬和神經退行性疾病中扮演重要角色[1-2]。作為內源性的免疫調節(jié)分子,多胺與炎癥密切相關,炎癥組織中內源性多胺水平顯著升高表明多胺在炎癥反應中發(fā)揮直接作用[3]。創(chuàng)面壞死組織中大量產生的腐胺和尸胺經吸收入血后會顯著提高血清中炎癥因子TNF-α、IL-1β、IL-6的水平并誘導機體發(fā)生炎癥[4]。Soulet等[5]研究發(fā)現(xiàn)小鼠神經膠質細胞炎癥反應伴隨著多胺水平升高,進一步研究發(fā)現(xiàn)多胺水平降低后,炎癥因子TNF-α和TLR2表達也顯著降低。多胺具有重要的抗氧化和抗炎功能,外源性多胺能夠抑制炎癥介質,如促炎細胞因子和趨化因子的表達,進而發(fā)揮抗炎作用[6-7]。研究發(fā)現(xiàn),精胺能夠通過增加抗炎因子IL-10合成和抑制促炎因子IL-12和IFN-γ的表達進而保護巨噬細胞免受炎癥損傷[8]。

      胸腺和脾臟是機體重要的免疫器官,胸腺指數(shù)和脾臟指數(shù)可以反映機體的免疫水平,同時免疫器官又是機體免疫細胞來源的重要場所[9]。然而,目前尚未見亞精胺對小鼠免疫器官指數(shù)及免疫器官炎癥因子表達的影響的報道,鑒于此,本實驗通過研究不同濃度亞精胺對鼠免疫器官指數(shù)和胸腺脾臟中TNF-α、IL-1β、IL-6和IFN-γ基因表達的影響,為研究亞精胺對機體免疫功能的作用機制奠定基礎。

      1 材料與方法

      1.1 實驗動物分組與實驗設計

      選取同批昆明小白鼠32只,常規(guī)方式分籠飼養(yǎng)。6周齡時記錄小鼠體重并采用隨機分配編號法編號,將小鼠隨機分為4組,每組8只小鼠。參考周岳平等[10]腐胺安全性試驗的結果,設定注射劑量分別為0.05、0.10和0.15 mg·g-1的亞精胺低、中、高劑量組,對照組注射生理鹽水。試驗開始的當天上午9:00腹腔單次單點注射,注射劑量為300 μL。

      1.2 樣品采集及處理

      注射24 h后頸部脫臼處死小鼠,迅速采集小鼠的胸腺和脾臟組織,手術剪剪去表面脂肪和系膜后,用生理鹽水洗凈,濾紙吸干后電子天平稱量并記錄,胸腺和脾臟樣品置于-80 ℃冰箱保存?zhèn)溆谩?/p>

      1.3 免疫器官指數(shù)

      各組所有胸腺脾臟稱量記錄后計算免疫器官指數(shù),免疫器官指數(shù)計算公式如下:免疫器官指數(shù)(g·kg-1)=免疫器官質量(g)/體質量(kg)。

      1.4 實時熒光定量PCR

      按照Trizol試劑盒(TaKaRa,大連)操作說明提取所采集的胸腺和脾臟組織總RNA。參照反轉錄試劑盒(TaKaRa,大連)說明書將總RNA反轉錄成cDNA模板,反應體系20 μL:包括9.0 μL RNase Free dH2O,1.0 μL Oligo dT Primer,1.0 μL Random 6 mers,4.0 μL 5×PrimeScript Buffer,1.0 μL PrimeScript RT Enzyme Mix,4.0 μL總RNA。反應條件為:37 ℃ 15 min,85 ℃ 5 s和4 ℃ 5 min,反應結束后將cDNA模板置于-20 ℃冰箱中保存?zhèn)溆谩脤崟r熒光定量PCR檢測各處理組胸腺、脾臟樣品組織中TNF-α、IL-1β、IL-6和IFN-γ基因表達量(引物信息參見表1)。實時熒光定量PCR反應體系為10 μL∶SYBR Green 5.0 μL,上、下游引物(10 μmol·L-1)各0.2 μL,cDNA模板0.5 μL,RNase Free dH2O 4.1 μL。反應條件為:95 ℃預變性3 min;95 ℃變性10 s,58 ℃退火30 s(退火溫度如表1所示),72 ℃延伸30 s(采集熒光),39個循環(huán);95 ℃ 10 s,然后以0.05 ℃·s-1的速率從65 ℃緩慢升溫至95 ℃,繪制熔解曲線。

      1.5 統(tǒng)計分析

      實驗數(shù)據(jù)采用SAS 9.2軟件進行統(tǒng)計分析,結果用mean ± SE表示。熒光定量采用2-ΔΔCt法處理數(shù)據(jù),用GAPDH作為內參基因,每個樣品3個重復,以對照組胸腺和脾臟組織基因表達量為1,計算TNF-α、IL-1β、IL-6和IFN-γ基因的相對表達量,應用SAS 9.2統(tǒng)計分析軟件中的MEANS過程進行描述性統(tǒng)計分析、單因素方差分析和Duncan’s多重比較,P<0.05表示差異顯著。

      表1 鼠實時熒光定量PCR引物信息表

      Table 1 Real-time PCR information of primers for mice

      引物名稱Primername引物序列Primersequence(5'-3')退火溫度Annealingtemperature/℃片段大小Size/bpTNF-αF:CACGTCGTAGCAAACCACCAAGTGGR:GATAGCAAATCGGCTGACGGTGTGG65210IL-1βF:AATGCCTCGTGCTGTCTGACCR:TTGTCGTTGCTTGTCTCTCCTTG55118IL-6F:AGTTGCCTTCTTGGGACTGAR:CAGAATTGCCATTGCACAAC64191IFN-γF:TAACTCAAGTGGCATAGATGTG-GAAGR:GACGCTTATGTTGTTGCTGATGG64169GAPDHF:AACGACCCCTTCATTGACR:TCCACGACATACTCAGCAC58191

      2 結果與分析

      2.1 亞精胺對小鼠免疫器官指數(shù)的影響

      如圖1所示,注射0.15 mg·g-1亞精胺組鼠胸腺指數(shù)和脾臟指數(shù)顯著(P<0.05)低于對照組;0.05 mg·g-1和0.10 mg·g-1亞精胺組鼠的胸腺指數(shù)和脾臟指數(shù)與對照組相比均沒有明顯變化(P>0.05)。

      2.2 亞精胺對小鼠胸腺炎癥因子表達的影響

      如圖2所示,0.05 mg·g-1、0.10 mg·g-1和0.15 mg·g-1亞精胺組胸腺組織中TNF-α、IL-1β和IL-6基因的表達與對照組相比差異不顯著(P>0.05),0.10 mg·g-1亞精胺組胸腺組織中IFN-γ基因表達量顯著(P<0.05)高于對照組。

      同一免疫器官無相同小寫字母表示差異顯著(P <0.05);下圖同In the same immune organ, the bar graphs without the same lower letters indicated significant differences at P <0.05. The same as below圖1 亞精胺對小鼠免疫器官指數(shù)的影響Fig.1 Effects of spermidine on mice immune organ index

      2.3 亞精胺對小鼠脾臟炎癥因子表達的影響

      如圖3所示,0.05 mg·g-1亞精胺組鼠脾臟組織中TNF-α和IL-1β基因表達顯著(P<0.05)低于對照組,0.10 mg·g-1亞精胺組脾臟組織IL-6和IFN-γ基因的表達均顯著(P<0.05)高于對照組,0.15 mg·g-1組脾臟組織TNF-α基因的表達顯著(P<0.05)低于對照組。

      圖2 亞精胺對小鼠胸腺炎癥因子表達的影響Fig.2 Effects of spermidine on expression of mice thymus inflammatory cytokines

      圖3 亞精胺對小鼠脾臟炎癥因子表達的影響Fig.3 Effects of spermidine on expression of mice spleen inflammatory cytokines

      3 討論

      胸腺和脾臟是機體重要的免疫器官,免疫器官指數(shù)的高低可在一定程度上反映機體免疫水平的強弱[11]。一定濃度的多胺能夠促進動物機體細胞增殖、分化和生長,趙宏濤等[12]研究發(fā)現(xiàn),仔豬補充12和15 mg·kg-1外源性精胺能夠通過增加脾臟相對質量進而提高仔豬免疫能力,多胺過量或耗竭會影響DNA和蛋白質、蛋白質和蛋白質的相互作用及線粒體完整,最終導致細胞凋亡[13],同時,多胺代謝產生的過氧化氫和丙烯醛也能破壞細胞DNA、蛋白質,并最終誘導細胞凋亡[14]。本實驗發(fā)現(xiàn),0.15 mg·g-1濃度的亞精胺處理會降低小鼠的胸腺指數(shù)和脾臟指數(shù),致使小鼠發(fā)生免疫抑制,推測可能是由于亞精胺過量誘導產生活性氧并促進細胞凋亡,最終影響了免疫器官指數(shù),抑制了機體的免疫功能,但其仍有待進一步研究證實。

      機體攝入抗氧化物質可影響免疫反應和免疫狀態(tài)[15]。個體水平和細胞水平均發(fā)現(xiàn)多胺具有重要的抗氧化功能[16-17],同時也是免疫功能的重要調節(jié)劑。炎癥因子能夠反映機體的炎癥水平,炎癥因子水平越高,表明炎癥損傷越嚴重[18]。多胺能通過抑制炎癥因子表達發(fā)揮抗炎作用[17,19-20]。促炎細胞因子如TNF-α、IL-1β和IL-6等能夠活化NF-κB并激活炎癥和凋亡通路[21]。TNF-α是機體中重要的促炎細胞因子,其水平是評價機體免疫狀況的重要指標。IL-1β能夠促進成纖維細胞因子、趨化因子、一氧化氮合酶釋放增加并促進炎癥的發(fā)展[22]。IFN-γ是一種二聚體糖蛋白,在炎癥反應中扮演促炎因子角色[23]。本實驗發(fā)現(xiàn):各亞精胺組胸腺組織中TNF-α、IL-1β和IL-6基因的表達與對照組相比差異不顯著,0.10 mg·g-1亞精胺組胸腺組織中IFN-γ的表達顯著高于對照組,表明此劑量亞精胺能通過促進IFN-γ表達誘導胸腺組織發(fā)生炎癥反應。Choi等[24]研究發(fā)現(xiàn),外源性添加0.5和1 mmol·L-1的亞精胺可抑制LPS誘導的鼠小膠質細胞TNF-α和IL-6的轉錄,并降低培養(yǎng)液中炎癥因子TNF-α和IL-6水平。Merentie等[25]研究發(fā)現(xiàn),亞精胺能夠抑制大鼠急性胰腺炎模型中血清炎癥因子TNF-α和IL-6水平升高,本實驗發(fā)現(xiàn)脾臟組織中,0.05 mg·g-1亞精胺組鼠TNF-α和IL-1β基因表達顯著低于對照組,說明0.05 mg·g-1濃度亞精胺具有抗炎效果,此濃度下胸腺組織中TNF-α和IL-1β的表達與脾臟組織存在差異也說明亞精胺對小鼠炎癥因子的表達具有組織差異性。Jamwal等[26]研究發(fā)現(xiàn)灌胃5和10 mg·kg-1外源性亞精胺能夠抑制3-硝基丙酸誘導的鼠大腦紋狀體中炎癥因子IL-6的表達,同時該作者發(fā)現(xiàn)5和10 mg·kg-1外源性亞精胺預處理也能降低喹啉酸誘導的鼠大腦紋狀體中炎癥因子IL-6的表達[27],本實驗研究發(fā)現(xiàn),0.10 mg·g-1濃度亞精胺組IL-6表達顯著高于對照組,提示高濃度的亞精胺可能通過促進脾臟IL-6的表達誘發(fā)脾臟發(fā)生炎癥反應。IFN-γ等可通過一氧化氮合成酶誘導產生大量NO并最終誘導炎癥反應,多胺能夠通過降低NO水平進而保護巨噬細胞免受炎癥損傷[28]。本實驗研究發(fā)現(xiàn),0.10 mg·g-1濃度亞精胺組IFN-γ表達顯著高于對照組,提示0.10 mg·g-1亞精胺可能通過促進脾臟IFN-γ表達誘發(fā)脾臟發(fā)生炎癥反應。

      4 結論

      亞精胺可通過介導鼠胸腺、脾臟指數(shù)及炎癥因子表達來參與調節(jié)機體免疫功能。亞精胺對鼠胸腺和脾臟組織炎癥因子表達具有組織特異性和劑量依賴性。本實驗發(fā)現(xiàn):0.05 mg·g-1劑量亞精胺可通過抑制脾臟TNF-α和IL-1β表達發(fā)揮抗炎作用;0.10 mg·g-1劑量亞精胺能夠促進胸腺IFN-γ、脾臟IL-6和IFN-γ表達并最終誘導炎癥反應;0.15 mg·g-1劑量亞精胺則可抑制鼠免疫器官功能。

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      (責任編輯 盧福莊)

      Effects of spermidine on immune organ index and expression of inflammatory cytokines in mice

      XU Qilin, KANG Bo*, JIANG Dongmei*, WEI Chunye, JIA Yilin, YI Zhixin, CHEN Ziyu

      (CollegeofAnimalScienceandTechnology,SichuanAgriculturalUniversity/FarmAnimalGeneticResourcesExplorationandInnovationKeyLaboratoryofSichuanProvince,Chengdu611130,China)

      To clarify the effects of exogenous spermidine on mice immune organs index and expression of inflammatory cytokines in thymus and spleen, 0, 0.05, 0.10 and 0.15 mg·g-1(body weight) spermidine were intraperitoneal injected for the six-week Kunming mice. The thymus and spleen index were measured after 24 h and the expression ofTNF-α、IL-1β、IL-6 andIFN-γin thymus and spleen were assessed by quantitative real-time PCR. The results showed that, compared with the control group, intraperitoneal injection of 0.15 mg·g-1spermidine group significantly (P<0.05) decreased the thymus index and the spleen index. 0.1 mg·g-1spermidine group enhanced the thymusIFN-γexpression (P<0.05). In the spleen, the expressions ofTNF-αandIL-1βin 0.05 mg·g-1spermidine group were significantly (P<0.05) lower than those of the control group, but the expressions of theIL-6 andIFN-γgene in 0.1 mg·g-1spermidine group were significantly higher than those of the control group (P<0.05). The expression ofTNF-αin 0.15 mg·g-1spermidine group was significantly (P<0.05) lower than that of the control group. Thus, the spermidine showed tissue specific and dose dependent manner on the expression of inflammatory cytokines in mice. Low concentration of spermidine had anti-inflammatory effect by inhibiting the expression of inflammatory cytokines, but high-dose spermidine could also induce inflammation.

      spermidine; thymus index; spleen index; inflammatory cytokines

      10.3969/j.issn.1004-1524.2017.02.04

      2016-08-22

      國家自然科學基金資助項目(31201798);四川省教育綜合改革試點項目“構建服務現(xiàn)代農業(yè)發(fā)展的產學研協(xié)同創(chuàng)新體系”

      徐麒麟(1992—),男,四川崇州人,碩士研究生,主要從事動物卵泡發(fā)育機理研究。E-mail: albertxql@163.com

      *通信作者,康波,E-mail: bokang@sicau.edu.cn;姜冬梅,E-mail:jiangdm9277@163.com

      Q26

      A

      1004-1524(2017)02-0200-06

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