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      中西醫說蟲草
      冬蟲夏草對大鼠小腸黏膜機械屏障功能影響的研究
      發布時間:2016-01-28
      摘要:近年來,腸功能已作為判斷危重癥病人預后的重要條件之一,腸道被認為是機體應激時的中心器官之一。完整的腸上皮、上皮間緊密連接(TJ)和基膜等構成腸道機械屏障,是阻擋微生物入侵的第一道防線,在腸屏障功能中起了很重要的作用。
             作者:顧國勝 任建安 李寧 黎介壽
             摘要 目的:探討冬蟲夏草對大鼠小腸黏膜機械屏障功能的影響。 方法:實驗分為正常對照組、內毒素灌胃組和內毒素灌胃+冬蟲夏草組。內毒素灌胃組于實驗開始第1天給予內毒素5 EU/kg灌胃,內毒素灌胃+冬蟲夏草組每天給予冬蟲夏草5g(kg·d)混懸液灌胃,對照組給予等量的等滲鹽水灌胃,三組均自由進食和飲水。分別于灌胃第4、7、l0天取8只大鼠,取小腸黏膜行流式細胞儀檢測黏膜細胞的增殖指數(PI),透射電鏡檢測黏膜上皮細胞間緊密連接寬度,免疫組化染色檢測黏膜增殖細胞核抗原(PCNA)。 結果:內毒素灌胃+冬蟲夏草組大鼠腸黏膜細胞的PI、PCNA比例和腸黏膜上皮細胞間的緊密連接第7天即恢復至正常水平,與內毒素灌胃組相比,有顯著性差異。 結論:冬蟲夏草可促進大鼠小腸黏膜細胞的增殖,維持小腸黏膜上皮細胞間緊密連接的完整性,保護腸黏膜的機械屏障功能。
             近年來,腸功能已作為判斷危重癥病人預后的重要條件之一,腸道被認為是機體應激時的中心器官之一。完整的腸上皮、上皮間緊密連接(TJ)和基膜等構成腸道機械屏障,是阻擋微生物入侵的第一道防線,在腸屏障功能中起了很重要的作用。以往的研究表明,冬蟲夏草可促進細胞增殖,但其對小腸黏膜機械屏障的研究較少。為此,我們探討了冬蟲夏草對大鼠小腸黏膜機械屏障功能的影響。
             1 材料和方法
             1.1 動物和材料56只健康SD大鼠,體重150~200g,雌雄各半,由本院解放軍動物醫學實驗中心提供。冬蟲夏草粉由杭州中美華東制藥有限公司提供,每1g冬蟲夏草粉溶于5ml等滲鹽水中,制成冬蟲夏草混懸液。碘化丙啶溶液試劑盒由聯科生物技術工程公司提供。細菌內毒素和增殖細胞核抗原(PCNA)免疫組化試劑盒由深圳晶美公司提供。
             1.2 動物分組和處理 將大鼠隨機分為正常對照組(A組,8只)、內毒素灌胃組(B組,24只)、內毒素灌胃+冬蟲夏草組(C組,24只)。B組于實驗開始第1天給予內毒素5EU/kg灌胃,C組第1天給予內毒素5EU/kg灌胃,同時給予冬蟲夏草5g(kg·d)混懸液灌胃,連續10d;對照組給予等量的等滲鹽水灌胃,三組大鼠均自由進食和飲水。分別于灌胃當天和第4、7、10天取8只大鼠作相關指標測定。
             1.3 指標檢測
             1.3.1 小腸黏膜細胞增殖指數(PI) 剖腹取空回腸交界處小腸5cm,機械法刮取腸黏膜,經Medima—chine系統制備成單細胞懸液后,加入熒光染料碘化丙啶溶液,經流式細胞術(FCM)測量,可獲得DNA含量分布組方圖或細胞凋亡比例。采用專門的計算機擬合程序(DNA細胞周期分析程序),快速計算出各期細胞的分布百分比,用S+G2M期或S期細胞百分比,以表示細胞群體的增殖狀態和DNA合成速度。
             1.3.2 細胞間緊密連接取大鼠小腸切成1mm×1mm×2mm,迅速投人4℃的3%戊二醛-1.5%多聚甲醛-0.1mol/L磷酸鹽緩沖液(pH7.2),前固定2h以上。經緩沖液漂洗后,用1%餓酸4℃下固定1.5h,再漂洗后用系列乙醇、丙酮脫水,環氧樹脂618包埋,經半薄切片定位。60nm超薄切片,用醋酸鈾、枸橡酸鉛染色。置透射電鏡下觀察攝片。
             1.3.3 小腸黏膜增殖細胞核抗原檢測 石蠟切片按常規脫蠟至水,在體積分數為3%的H202中孵育10min,以消除內源性過氧化物酶活性。PBS沖洗,置于0.O1mol/L、pH6.0的枸櫞酸鹽緩沖液中行抗原熱修復后,滴加1:100稀釋一抗,37℃下孵育40min。PBS沖洗,滴加辣根過氧化物酶(HRP)標記二抗,37℃下孵育20min,PBS漂洗后DAB顯色,蘇木精對比染,常規脫水、透明、封固,在顯微鏡下觀察。結果以細胞質和(或)細胞核著棕色者為陽性染色。另用PBS代替一抗為陰性對照。結果判定與分析:在光鏡下觀察陽性細胞數和在組織中的分布。在目鏡為10倍、物鏡為40倍的條件下,選擇具有良好隱窩腔的縱切面,從隱窩底部中央細胞開始,依次向上計數陽性細胞和陰性細胞,直至隱窩與絨毛交界處。每張切片需計數10個隱窩,計算陽性細胞的比例。
             1.4 統計學方法 所有數據運用SPSS13.0軟件完成統計學的各項處理,以x±s表示。試驗過程中得到的數據均輸入本實驗的數據庫,多組間資料比較采用One-way ANOVA單因素方差分析,兩組間比較運用Least-significant difference法。P≤0.05為差異有顯著性統計學意義。
             2 結 果
             2.1 小腸黏膜細胞增殖指數 C組及B組第4天腸黏膜細胞的PI與A組比明顯下降。至第7天,B組仍低于A組,而C組恢復至A組水平。至第10天,C組較A組比明顯增高,而B組才恢復至正常水平,見表l。
             2.2 小腸黏膜上皮細胞間的緊密連接 C組和B組第4天黏膜上皮細胞間的PCNA與A組比出現明顯變化,至第7天,B組恢復至正常水平,而C組變化更為明顯,且與B組有顯著性差異,見表2。
             2.3 小腸黏膜PCNA的變化 C組和B組第4天黏膜PCNA與A組比出現明顯變化;至第7天,B組恢復至正常水平,而C組變化更為明顯,且較B組有顯著性差異,見表3。
             3 討 論
             近來研究表明,腸道在感染、應激所致全身性炎癥反應綜合征(SIRS)和多器官功能障礙綜合征(MODS)的發展中居重要地位。腸黏膜是代謝活躍的組織細胞群,更易受缺血、缺氧和炎癥反應等因素的傷害。感染、創傷等應激和長期禁食后使腸黏膜蛋白質合成能力下降,腸黏膜上皮細胞修復、更新受損,嚴重者出現腸黏膜萎縮。腸黏膜結構和功能的損害,影響腸黏膜屏障功能。在正常生理狀況下,細菌和毒素并不對機體產生危害。這與機體各系統之間的相互協調和腸道特有的屏障功能有關。完整的腸上皮、上皮間TJ和基膜等構成腸道機械屏障,是阻擋微生物入侵的第一道防線。一旦機械屏障遭到破壞,細菌和毒素可穿過腸壁,侵入腸系膜淋巴結、血液、肝、脾等器官,即發生腸道細菌易位(BT)。腸道BT是內源性感染發生率難以降低的主要原因之一。同時,腸道損害是許多疾病或損傷后多器官功能障礙的中心器官和始動因素。因此,如何促進腸道屏障功能的恢復,維持微生態平衡是急待解決的問題。
             以往的研究表明,冬蟲夏草能促進細胞的增殖。蟲草水提液可使小鼠脾的吞噬細胞增殖,增加肝的重量,肝內肝巨噬細胞功能增強;蟲草多糖不僅可增加脾的重量、脾中漿細胞增殖、使脾營養性血流量增加,而且還能拮抗可的松或環磷酰胺所致脾萎縮的作用;蟲草提取物不僅能促進正常小鼠脾活化T細胞的增生,而且還能使受環磷酰胺和氫化可的松抑制的小鼠脾活化T細胞恢復正常。蟲草多糖脂質體對體外培養的外周血淋巴細胞,可單獨或協同外源凝集素(PHA)誘導IL-22受體的表達,促進可溶性IL-22的生成。
             以往的實驗還證明,蟲草具有明顯的促腎小管上皮細胞增殖的作用,且在一定范圍內隨劑量增大而增強;蟲草還能促進人類單核巨噬細胞的分化和增殖。口服冬蟲夏草液對輻射誘發的小腸急性損傷有一定的保護作用。對于輻射所引起的骨髓抑制,蟲草也有很好的保護作用。
             本實驗結果顯示,給予內毒素灌胃后第4天,腸黏膜機械屏障明顯受損,表現為腸黏膜PI明顯下降,細胞問TJ增大,腸道通透性增加,細胞PCNA的比例明顯下降;至第7天時,兩組均恢復至正常水平,但C組變化更為明顯。本研究證實冬蟲夏草不僅具有促進腸黏膜增殖的作用,而且還有修復內毒素破壞的腸黏膜TJ作用。

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