1,25-二羥基維生素D3對腸黏膜屏障的保護作用及機制探討

趙紅偉, 韓華, 岳月紅

(河北省人民醫院, 石家莊050051)

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1,25-二羥基維生素D3對腸黏膜屏障的保護作用及機制探討

趙紅偉, 韓華, 岳月紅

(河北省人民醫院, 石家莊050051)

目的觀察1，25-二羥基維生素D3[1,25-(OH)2VitD3]對腸黏膜屏障的保護作用，并探討其可能的機制，為其在結腸炎治療中的應用提供依據。方法培養結腸癌Caco-2細胞，模擬人小腸上皮細胞單層細胞屏障；將細胞隨機分為3組，A組不處理，B組加2%右旋葡聚糖硫酸鈉(DSS)1 mL模擬結腸炎損傷，C組先用10-8mmol/L的1,25-(OH)2VitD3處理24 h再加入DSS 1 mL，干預后培養1 h。透射電鏡下觀察Caco-2單層細胞屏障超微結構變化，采用免疫熒光染色法檢測緊密連接蛋白claudin-1、zo-1和occludin的表達。結果透射電鏡下，A組細胞頂部出現排列整齊、分化良好的微絨毛狀結構,相鄰細胞間的頂端呈現緊密連接樣結構；B組細胞局部微絨毛消失或減少,緊密連接樣結構消失；C組細胞微絨毛分化較B組好，相鄰細胞間的局部微絨毛消失不明顯。免疫熒光染色顯示，A組3種緊密連接蛋白熒光條帶連續完整，熒光強度高；B組緊密連接蛋白熒光條帶斷續不完整,熒光強度弱；C組緊密連接蛋白熒光條帶較清晰連續，且熒光強度增強。結論1,25-(OH)2VitD3可通過增加細胞間緊密連接蛋白的表達發揮其對腸黏膜屏障的保護作用，為其在結腸炎中的應用提供了理論依據。

結腸炎；1,25-二羥基維生素D3；緊密連接；屏障功能

近年來結腸炎在我國呈高發趨勢，至今病因末明。腸黏膜屏障是機體最重要的免疫防御屏障系統，其中細胞間的緊密連接蛋白是黏膜屏障的核心組件[1～3]，例如occludin蛋白、claudin-1蛋白、zo-1蛋白等。研究表明，結腸炎時腸黏膜通透性增高[4～6]。有研究發現,1，25-二羥基維生素D3[1,25-(OH)2VitD3]可維持腸道黏膜表層的屏障完整性，提示其可能對結腸炎腸黏膜屏障具有一定的修復能力[7]。2010年11月～2011年9月,我們以結腸癌Caco-2細胞模擬人小腸上皮細胞單層細胞屏障,探討1,25-(OH)2VitD3對腸道黏膜屏障的保護機制。

1　材料與方法

1.1細胞培養與模型建立Caco-2細胞購自中科院上海生命科學研究細胞資源中心，從液氮中取出Caco-2細胞進行復蘇。將復蘇后的細胞接種在培養液中(含10 %的DMEM),于37 ℃、5% CO2條件的細胞培養箱中培養,每2 d更換培養液1次。第5天后細胞融合達85%～90%時進行細胞傳代,傳代比例為1∶3。將傳代細胞接種在24孔Millicell插入式培養皿(Transwell小室)中，接種密度為4×105/ mL，每孔200 μL；基底側加入培養液600 μL。接種24 h后更換培養液，隔天換液1次，細胞培養第21～30天體外腸上皮屏障模型形成。

1.2細胞分組與處理 將細胞分成3組，A組不處理，B組加2 %右旋葡聚糖硫酸鈉(DSS)1 mL模擬結腸炎損傷，C組先用10-8mmol/L的1,25-(OH)2VitD3處理24 h再加入DSS 1 mL。

1.3單層細胞的超微結構觀察取形成體外腸上皮屏障模型的細胞，分組處理同1.2，干預后培養1 h。用4 %戊二醛將單層Caco-2細胞進行固定,以二甲砷酸鈉緩沖液進行3次漂洗；漂洗后再用1 %餓酸固定約50 min,在蒸餾水中浸泡20 min；依次用50%、70%、95%、100 %丙酮進行脫水,把細胞浸透在EPons12環氧樹脂中過夜；包埋、切片后應用5%乙酰雙氧鈾鎂及檸檬酸鉛進行雙染,在透射電鏡下觀察單層Caco-2細胞與細胞之間緊密連接超微結構的變化。

1.4 細胞緊密連接蛋白claudin-1、occludin、zo-1檢測采用免疫熒光染色法。取1.1中的傳代細胞，分組處理同1.2，干預后培養1 h。用消毒過的PBS漂洗Caco-2細胞3次,每次約5 min；以4 %多聚甲醛固定15～20 min,用PBST漂洗5 min×3次；于5% BSA封閉1 h,用PBST洗5 min×3次；加入濃度為1∶100的鼠抗claudin-1單克隆抗體、occludin單克隆抗體及兔抗zo-1多克隆抗體后4 ℃過夜,用PBST漂洗5 min×3次；孵育對應的熒光二抗(濃度為1∶250的CY3標記的山羊抗小鼠lgG抗體及濃度為1∶50的FITC標記的山羊抗兔lgG抗體),室溫避光孵育1 h,PBST漂洗5 min×3次；最后加入DAPI復染,PBST漂洗10 min×3次；應用防淬滅封片劑封片,在熒光顯微鏡下進行觀察及拍照。claudin-1及occludin蛋白發紅光，而zo-1蛋白呈現綠光。

2　結果

2.11,25-(OH)2VitD3對單層細胞超微結構的影響在透射電鏡下，A組細胞頂部出現排列整齊、分化良好的微絨毛狀結構，相鄰細胞間的頂端呈現緊密連接樣結構；B組細胞局部的微絨毛消失或減少，緊密連接樣結構消失；C組細胞的微絨毛分化較B組好，相鄰細胞間的局部微絨毛消失不明顯,但與A組的緊密連接形態有所不同。

2.21,25-(OH)2VitD3對單層細胞claudin-1、zo-1及occludin表達的影響A組細胞緊密連接蛋白熒光沿細胞胞膜分布,且細胞邊緣光滑,呈鋪路石形狀；細胞之間緊密連接,無明顯間隙,強熒光表現。與A組比較,B組細胞緊密連接蛋白熒光強度減弱,分布較分散,邊緣粗糙呈鋸齒狀；細胞之間出現間隙,呈現不連續的線狀熒光染色,環斷裂甚至崩解。C組細胞緊密連接蛋白的熒光沿胞膜分布,較A組熒光強度稍減弱,無明顯間隙,條帶較清晰且連續；與B組比較有明顯的好轉,但與A組比較仍可見細胞間的間隙。見圖1。

注：A為A組，B為B組，C為C組。

圖1各組Caco-2細胞緊密連接蛋白表達(免疫熒光染色法)

3　討論

結腸炎是一種慢性非特異性腸道炎癥性疾病,嚴重威脅人們的健康。目前,關于結腸炎發病機制的基本共識是：易感個體在某些環境因素的作用下,腸道黏膜屏障削弱,腸道致病菌群及其有害成分穿過黏膜屏障,與機體免疫系統發生接觸并產生過度的級聯免疫反應,使炎癥反復發生。由此可見，腸道黏膜屏障的損傷與該病的發生有著緊密的聯系,故從結腸黏膜屏障保護角度出發為本病的治療提供了一種新的思路[8～11]。

正常情況下,腸道上皮細胞之間連接組成一道完整的黏膜屏障，可阻止微生物及毒性大分子通過。細胞間的緊密連接是細胞間的重要連接方式,對維持黏膜屏障的完整性起決定作用。因此,腸上皮細胞間緊密連接的破壞及其所致的腸黏膜屏障功能受損在一系列腸道受損疾病的發病機制中起著關鍵作用[12]。細胞之間的緊密連接是由很多種相關聯的蛋白組成,主要包括occludin蛋白、claudin-1蛋白、zo-1蛋白等。其中,occludin蛋白、claudin-1蛋白是構成選擇性黏膜屏障的結構蛋白,在腸黏膜上皮細胞的黏膜屏障中起著非常重要的作用。如果這些緊密連接蛋白失活則會引起腸道屏障功能損傷,使腸道黏膜上皮細胞之間的通透性增加[13～15]；一些致病細菌穿透腸黏膜屏障,調控上述緊密連接蛋白的表達,破壞細胞間緊密連接結構,損害腸道黏膜屏障功能,從而引起腸道炎癥。

1,25-(OH)2VitD3是一種脂溶性維生素,是機體內維生素D的活性代謝產物。其作為類固醇激素參與調節機體的多種生理功能,其中以調節鈣磷代謝、促進骨骼生長為人所熟知。近年研究表明,維生素D缺乏會增加患炎癥性腸病的風險。維生素D受體(VDR)缺陷小鼠表現為結腸黏膜嚴重潰瘍、傷口愈合不良及腸黏膜單層細胞電阻降低。

Caco-2細胞的結構和功能類似于分化的人小腸上皮細胞,在形態學上具有相同的細胞極性和緊密連接,并可分泌與人體相同的酶類、轉化因子等。此細胞來源于人體結直腸癌細胞株,在生長至融合后有柱狀突起,是目前較理想的體外腸屏障模型,在緊密連接蛋白表達研究中廣泛應用。本研究結果發現,B組Caco-2單層細胞緊密連接樣結構消失,claudin-1、zo-1和occludin緊密連接蛋白表達斷續不完整且熒光強度弱；1,25-(OH)2VitD3處理的細胞緊密連接樣結構有不同程度的恢復，緊密連接蛋白表達條帶較清晰連續且熒光強度增強。因此，我們認為，DSS可降低緊密連接蛋白表達，可增加腸上皮屏障的通透性；而1,25-(OH)2VitD3可增加緊密連接蛋白表達，降低腸上皮屏障的通透性,在屏障功能和緊密連接形態的維持中起重要作用，可為臨床用藥提供理論依據。

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Protective effect of 1,25-dihydroxyvitamin D3on intestinal mucosa barrier and its mechanism

ZHAOHongwei,HANHua,YUEYuehong

(HebeiGeneralHospital,Shijiazhuang050051,China)

ObjectiveTo observe the protective effect and the mechanism of 1,25-dihydroxyvitamin D3[1,25-(OH)2VitD3] on intestinal mucosa barrier and to provide the basis for its application in the treatment of colitis. Methods Caco-2 cells were cultivated for human intestinal epithelial cell monolayer cell barrier. These cells were randomly assigned to three group: groups A, B and C. Group A was not treated, group B was treated with 2% DSS to establish colitis model and group C first was treated with 1,25-(OH)2VitD3(10-8mmol/L) for 24 hours, then was incubated with 1 mL DSS for one hour. Caco-2 cell monolayers were observed in ultrastructural features by a transmission electron microscope (TEM); and the expression levels of zo-1, claudin-1 and occludin proteins in Caco-2 cells were detected by immunofluorescence. ResultsThere were well-differentiated microvilli structures which were also in good order at the top of cells in the group A, and there was also closely connection between adjacent cells at the top of the structure; while part of the microvilli disappeared or reduced in the group B; the microvilli of cells in the group C were more well-differentiated that those of group B, but part of microvilli disappeared. Immunofluorescence staining showed that in the group A, three kinds of tight junction protein fluorescence strips were complete, and had high fluorescence intensity; those in the group B were incomplete, and the fluorescence intensity was weak; and those in the group C were clear and continuous, and the fluorescence intensity was enhanced. Conclusion1,25-(OH)2VitD3may protect the intestinal mucosa barrier by increasing intercellular tight junction protein expression which also provides the basis for its application in the treatment of colitis.

colitis; 1, 25-dihydroxyvitamin D3; tight junction; barrier function

趙紅偉(1979-),女,博士研究生,主治醫師,主要研究方向為消化系統疾病的診斷與治療。E-mail: hongweizhao788@126.com

10.3969/j.issn.1002-266X.2016.34.005

R977.24

A

1002-266X(2016)34-0014-03

2016-03-18)