徐丹丹，王建發(fā)，張旭，劉東宇，許小楠，王樂，陳嘉，單旭菲，王曉雅，武瑞，楊彬

（黑龍江八一農(nóng)墾大學(xué)動物科技學(xué)院，黑龍江大慶 163319）

胞壁酰二肽對體外奶牛乳腺上皮細胞生長及胞內(nèi)NOD2 mRNA表達的影響

徐丹丹，王建發(fā)，張旭，劉東宇，許小楠，王樂，陳嘉，單旭菲，王曉雅，武瑞，楊彬

（黑龍江八一農(nóng)墾大學(xué)動物科技學(xué)院，黑龍江大慶 163319）

【目的】奶牛乳房炎是奶牛養(yǎng)殖業(yè)中最為常見，同時也是帶來經(jīng)濟損失最為嚴重的疾病之一，其主要病因是細菌感染。奶牛乳腺的固有免疫是抵抗病原菌入侵的第一道防線。NOD2是機體固有免疫模式識別受體核苷酸結(jié)合寡聚域（nucleotide-binding oligomerization domain, NOD）蛋白家族中的重要一員，通過識別其特異性配體胞壁酰二肽（muramyl dipeptide, MDP）——一種廣泛存在于革蘭氏陽性菌和革蘭氏陰性菌細胞壁中的成分，而參與抵抗多種病原菌入侵。奶牛乳腺上皮細胞（bovine mammary epithelial cells, BMEC）除泌乳以外，還是奶牛乳腺的免疫屏障。本試驗欲探究MDP對BMEC體外生長狀態(tài)及胞內(nèi)NOD2表達量的影響?！痉椒ā窟x取健康泌乳中期荷斯坦奶牛乳腺腺泡為組織原材料，采用膠原酶Ⅰ消化法結(jié)合梯度濃度胰蛋白酶純化法分離BMEC；使用上皮細胞特異性表達的角蛋白-18（cytokeratin-18，CK-18）及成纖維細胞特異性表達的波形蛋白（Vimentin）抗體，通過免疫熒光技術(shù)對純化后獲得的細胞進行鑒定；將BMEC設(shè)為6個處理組，分別添加濃度為0（空白對照組）、1、5、10、15及20 μg·mL-1的MDP，24 h后顯微鏡下觀察細胞狀態(tài)，同時提取細胞RNA并反轉(zhuǎn)錄為cDNA，采用實時熒光定量PCR方法檢測BMEC中NOD2的表達量?！窘Y(jié)果】（1）膠原酶Ⅰ消化法結(jié)合梯度濃度胰蛋白酶純化法分離得到的細胞CK-18免疫熒光結(jié)果為陽性，而Vimentin反應(yīng)為陰性；細胞生長狀態(tài)良好。（2）空白對照組、1、5及10 μg·mL-1MDP刺激組的BMEC生長狀態(tài)良好，無任何肉眼可見變化；15 μg·mL-1MDP刺激組可見少量的BMEC脫落；而20 μg·mL-1MDP刺激組可見大量BMEC脫落，漂浮，且即使仍貼壁的細胞，其形態(tài)也發(fā)生了變化。（3）與空白對照組相比，各刺激組細胞NOD2 mRNA的表達量與MDP的刺激濃度呈正相關(guān)，即刺激時間為24 h時，隨著MDP濃度的增加，BMEC中NOD2受體mRNA的表達量逐漸增加（P ＜ 0.05）?！窘Y(jié)論】成功獲得了純度較好的BMEC，該細胞生長狀態(tài)良好，可以用于后續(xù)試驗；雖然BMEC中NOD2受體mRNA的表達量與MDP的刺激濃度呈正相關(guān)，但在保持BMEC生長狀態(tài)正常的前提下，MDP體外刺激濃度應(yīng)控制在10 μg·mL-1以下。這些結(jié)果提示我們：當病原菌入侵乳腺時，BMEC可以通過NOD2受體途徑參與免疫防御反應(yīng)，但這種防御能力受細菌數(shù)量或毒力強度的影響。即在一定的細菌數(shù)量或毒力范圍內(nèi)，隨著細菌數(shù)量的增加或毒力的增強，奶牛乳腺的免疫防御反應(yīng)也增強，進而清除外來病原菌；而當細菌數(shù)量或毒力強度超過一定范圍時，乳腺組織受到嚴重損傷，免疫防御屏障也隨之崩潰，奶牛乳腺局部甚至全身將呈現(xiàn)出明顯的臨床癥狀。

奶牛乳腺上皮細胞；MDP；NOD2

0 引言

【研究意義】奶牛乳房炎是奶牛養(yǎng)殖業(yè)中最為常見的疾病，其主要病因就是細菌感染，該病嚴重影響奶牛健康、牛奶質(zhì)量與產(chǎn)量，給奶牛養(yǎng)殖業(yè)造成了極大的經(jīng)濟損失。近年來，隨著耐藥菌群的不斷出現(xiàn)以及人們對抗生素殘留問題的逐漸重視，因此筆者逐漸將奶牛乳房炎的防治重點轉(zhuǎn)移到了提高奶牛機體以及乳腺組織局部免疫力的方向?！厩叭搜芯窟M展】NOD2是機體固有免疫模式識別受體核苷酸結(jié)合寡聚域（nucleotide-binding oligomerization domain，NOD）蛋白家族中的重要一員，其通過識別細菌細胞壁的固有成分胞壁酰二肽（muramyl dipeptide，MDP），進而激活下游的核轉(zhuǎn)錄因子-κB（nuclear transcription factor-κB，NF-κB）及促分裂素原活化蛋白激酶（mitogenactivated protein kinases，MAPK）信號通路，產(chǎn)生大量的炎性因子，從而啟動機體的免疫防御反應(yīng)[1-3]?！颈狙芯壳腥朦c】除了具有泌乳功能以外，奶牛乳腺上皮細胞（bovine mammary epithelial cells，BMEC）也是乳腺組織中重要的免疫細胞[4-5]。有研究表明，奶牛乳腺中存在NOD2受體，但MDP作為刺激劑對于體外BMEC的生長以及胞內(nèi)NOD2受體的表達有何影響未見報道[6]。本試驗采用膠原酶Ⅰ消化法體外培養(yǎng)BMEC，并以不同濃度的MDP刺激BMEC，觀察細胞狀態(tài)，同時采用實時熒光定量 PCR的方法檢測BMEC中NOD2的mRNA表達量?！緮M解決的關(guān)鍵問題】明確MDP作為BMEC體外刺激劑的安全濃度范圍，及其對BMEC中NOD2表達量的影響，為進一步揭示NOD2受體在奶牛乳腺組織局部免疫中的作用提供了理論基礎(chǔ)和試驗依據(jù)。

1 材料與方法

試驗于 2014—2015年在黑龍江八一農(nóng)墾大學(xué)獸醫(yī)臨床疾病診斷與治療實驗室完成。

1.1 材料

1.1.1 乳腺組織 取自黑龍江省某養(yǎng)殖場泌乳期健康荷斯坦奶牛。

1.1.2 主要試劑 DMEM F/12培養(yǎng)基購于 GIBCO公司；胎牛血清（FBS）購于 Hyclone公司；膠原酶Ⅰ及MDP購于Invivogen公司；胰蛋白酶購于碧云天公司；角蛋白-18（cytokeratin-18，CK-18）抗體購于Abcam公司；波形蛋白（Vimentin）抗體購于Neomarker公司；Mini BEST Universal RNA Extraction Kit及PrimeScript RT reagent Kit購于TaKaRa公司；SYBR?Green Master Mix購于Vazyme公司。

1.2 方法

1.2.1 BMEC的體外培養(yǎng)及分離純化 取新鮮乳腺組織，浸于75%酒精中2—3 min后轉(zhuǎn)至超凈臺中，用滅菌生理鹽水（含青霉素100 IU·mL-1，鏈霉素100 μg·mL-1）沖洗數(shù)次，將組織剪成小塊（約1 mm3）后加入Hank’s液洗滌數(shù)次，轉(zhuǎn)移組織塊至離心管，加入300 U·mL-1膠原酶Ⅰ，37℃水浴消化3—5 h，細胞篩過濾消化液后離心收集細胞，加入 DMEM F/12培養(yǎng)基（含10%FBS，青霉素100 IU·mL-1，鏈霉素100 μg·mL-1）混勻并移入培養(yǎng)瓶，置于CO2培養(yǎng)箱中培養(yǎng)。

待細胞初次長滿至70%—80%時，根據(jù)BMEC與成纖維細胞對胰酶的耐受能力不同，先用 0.05%的胰酶對成纖維細胞進行消化分離，PBS清洗后去除成纖維細胞；再用0.25%胰酶消化BMEC，離心收集BMEC加入培養(yǎng)基后繼續(xù)傳代培養(yǎng)，重復(fù)以上操作數(shù)次獲取試驗所需的BMEC，轉(zhuǎn)入含有細胞爬片的六孔板繼續(xù)培養(yǎng)。

1.2.2 免疫熒光方法對BMEC進行鑒定 將長滿至匯合的BMEC用PBS漂洗，而后加入預(yù)冷的4%多聚甲醛于4℃固定30 min，PBS漂洗；用0.5%的Trition處理10—15 min，PBS漂洗；用1%BSA封閉30 min，分別加入 CK-18（上皮細胞標志性蛋白）抗體、Vimentin（成纖維細胞標志性蛋白）抗體，4℃孵育過夜，PBS漂洗；加入相應(yīng)二抗，37℃作用 1 h，PBS漂洗；加入DAPI作用5—10 min；最后用抗猝滅劑封片，熒光顯微鏡下觀察結(jié)果。

1.2.3 實時熒光定量PCR檢測BMEC中NOD2的mRNA表達量 將BMEC分為5組，在培養(yǎng)基中加入MDP，使其濃度依次為1、5、10、15和20 μg·mL-1，另設(shè)空白對照組。24 h后觀察細胞狀態(tài)，并按照MiniBEST Universal RNA Extraction Kit說明書提取各組細胞總RNA，核酸測定儀檢測RNA的濃度及OD260/280值。按照PrimeScript RT reagent Kit說明書將RNA反轉(zhuǎn)為cDNA。實時熒光定量PCR所用引物由上海生工生物工程股份有限公司合成，序列詳見表1；反應(yīng)體系為：SYBR?Green Master Mix 10 μL，Primer1、Premer2各0.4 μL，cDNA 3 μL，滅菌水6.2 μL，總體系為20 μL；擴增程序見表2。

表1 引物序列Table 1 Primer sequences

表2 熒光定量PCR擴增程序Table 2 Quantitative real-time PCR amplification procedure

1.2.4 數(shù)據(jù)分析 采用 2－△△Ct法表示實時熒光定量PCR結(jié)果，數(shù)據(jù)應(yīng)用SPSS 19.0進行單因素方差分析，P＜0.05為具有統(tǒng)計學(xué)意義，以“*”表示。

2 結(jié)果

2.1 BMEC的體外培養(yǎng)及分離純化

酶消化法培養(yǎng)細胞數(shù)小時后就有少量的細胞先貼壁生長，培養(yǎng)至第3天時可見大片聚集生長的BMEC與形態(tài)不規(guī)則的成纖維細胞，但兩種細胞之間界限明顯并不混雜生長（圖1-a）。細胞純化后得到較為純凈的“鋪路石”或“島嶼狀”聚集生長的形態(tài)規(guī)則的BMEC，細胞核及核仁（多為多核仁）清晰可見，且部分細胞具有分泌乳汁的功能，形成空泡樣結(jié)構(gòu)（圖1-b）。

圖1 奶牛乳腺上皮細胞的顯微鏡下觀察Fig. 1 The observation of bovine mammary epithelial cells under the microscope (100×)

2.2 BMEC的免疫熒光鑒定

免疫熒光鑒定結(jié)果顯示，細胞CK-18反應(yīng)呈陽性，發(fā)出綠色熒光（圖2-a，b，c），而細胞Vimentin反應(yīng)呈陰性（圖2-d，e，f）。

2.3 MDP對BMEC生長狀態(tài)的影響

將添加了不同濃度MDP的BMEC于24h后置于倒置顯微鏡下觀察細胞狀態(tài)，結(jié)果如圖所示，空白對照組、添加1、5及10 μg·mL-1MDP組的BMEC生長狀態(tài)良好，無任何肉眼可見變化（圖3-a，b，c，d）；添加了15 μg·mL-1MDP組有少量的BMEC脫落（圖3-e）；而添加了20 μg·mL-1MDP組有大量的BMEC脫落、漂浮，且即使仍貼壁的細胞形態(tài)上也出現(xiàn)了變化（圖3-f）。

2.4 MDP對BMEC NOD2表達量的影響

實時熒光定量 PCR方法對各組細胞 NOD2 mRNA表達量檢測的結(jié)果顯示，與空白對照組相比，NOD2 mRNA表達量與MDP的刺激濃度呈正相關(guān)，即隨著MDP濃度的增加，BMEC中NOD2受體的mRNA表達量增加（圖4）。

3 討論

盡管有相關(guān)報道表明，酶消化法培養(yǎng)BMEC易破壞細胞結(jié)構(gòu)，不易成功獲得BMEC[7-8]；且常常添加多種外源激素及表皮生長因子[9]。而本試驗在盡可能減少外源激素及生長因子添加的情況下（只添加DMEM F/12，F(xiàn)BS，青、鏈霉素）采用膠原酶Ⅰ消化法成功獲得了BMEC，且細胞生長狀態(tài)良好，這提示要根據(jù)所使用酶的質(zhì)量及組織差異等實際情況掌握好消化時間。另外，本試驗所用膠原酶Ⅰ消化法相對于常用的組織塊培養(yǎng)法具有污染風險小、培養(yǎng)周期短、細胞純度高等優(yōu)點。分離純化及鑒定結(jié)果表明，本試驗獲得了生長狀態(tài)良好且較為純凈的BMEC，該細胞外源物質(zhì)干擾因素少，可以用于后續(xù)試驗。

NOD2是近年來才發(fā)現(xiàn)的一類細胞內(nèi)模式識別受體，存在于某些抗原提呈細胞和上皮細胞等細胞中[10-11]。其通過識別細菌固有成分 MDP進而參與機體固有免疫調(diào)節(jié)。據(jù)報道，NOD2參與機體抵抗葡萄球菌[12]、大腸桿菌[13]、沙門氏菌[14]、肺炎鏈球菌[15]、分支桿菌[16]、志賀氏菌[17]、李斯特菌[18-19]、嗜肺軍團菌[20]等多種細菌甚至是衣原體[21]、病毒[22]及胞內(nèi)原蟲[23]的侵襲。目前對于NOD2的研究多集中于其與克羅恩病、Blau綜合征、早發(fā)性結(jié)節(jié)及過敏性疾病等疾病的關(guān)系[13,24-28]。

圖2 奶牛乳腺上皮細胞的免疫熒光鑒定Fig. 2 Identification of bovine mammary epithelial cells by immuno- fluorescence

圖 3 MDP刺激奶牛乳腺上皮細胞24 h后的顯微鏡下觀察Fig. 3 Microscope observation of bovine mammary epithelial cells that stimulated by MDP after 24 h (200×)

圖4 不同濃度MDP刺激奶牛乳腺上皮細胞后NOD2 mRNA的相對表達量Fig. 4 The relative expression of mRNA NOD2 in bovine mammary epithelial cells that stimulated by different concentrations of MDP

引起奶牛乳房炎的主要病因為細菌感染，而BMEC是乳腺組織兼具泌乳及免疫功能的細胞[29]。相關(guān)研究表明NOD2在奶牛乳腺腺泡、乳導(dǎo)管、乳池及乳頭管組織以及BMEC中表達[6,30]。GILBERT等以金黃色葡萄球菌的培養(yǎng)上清液及LPS刺激BMEC，觀察不同刺激劑對BMEC免疫反應(yīng)的影響[31]。但至今MDP單獨作為體外刺激劑對 BMEC的生長狀態(tài)以及胞內(nèi)NOD2 mRNA表達量的影響仍沒有明確報道。本試驗結(jié)果顯示，MDP刺激BMEC后，細胞中NOD2受體mRNA表達量顯著增加。這說明，當奶牛乳腺受到外來病原菌入侵時，BMEC可以通過NOD2受體途徑啟動乳腺免疫防御機制，協(xié)同機體其他免疫途徑抵抗病原菌入侵。盡管BMEC中NOD2的表達量與MDP的刺激濃度呈正相關(guān)，但本試驗對MDP刺激后的BMEC進行形態(tài)學(xué)觀察的結(jié)果顯示，當MDP的刺激濃度達到15 μg·mL-1與20 μg·mL-1時，貼壁的BMEC出現(xiàn)了不同程度的死亡、脫落現(xiàn)象?？梢酝茰y，在高濃度的MDP作用下，BMEC中的NOD2受體及其下游通路被過度活化，過度的免疫應(yīng)答產(chǎn)生了大量的炎性因子，在免疫防御反應(yīng)的同時對細胞產(chǎn)生了損傷，但其中是否有其他通路參與，還有待進一步驗證。這一結(jié)果表明，如果試驗中以MDP作為BMEC模型的刺激劑，可以將 MDP的刺激濃度設(shè)在 10 μg·mL-1以下，避免因 BMEC過度損傷對試驗結(jié)果造成影響。這也提示，當奶牛乳腺受到外來病原菌入侵時，細菌數(shù)量或毒力強度與乳腺組織的免疫防御反應(yīng)有一定的關(guān)系，即細菌數(shù)量或毒力強度在一定范圍內(nèi)時隨著細菌數(shù)量的增加或毒力的增強，乳腺組織的免疫防御反應(yīng)也增強，進而清除外來病原菌入侵，而當細菌數(shù)量或毒力強度超過一定范圍時，乳腺的過度免疫應(yīng)答使組織受到嚴重損傷，免疫防御屏障隨之崩潰，奶牛乳腺局部甚至是全身將呈現(xiàn)出嚴重的臨床癥狀。

4 結(jié)論

本試驗采用膠原酶Ⅰ消化法，經(jīng)分離純化后成功獲得了奶牛乳腺上皮細胞；NOD2 mRNA的表達量與胞壁酰二肽的刺激濃度呈正相關(guān)；但在保持奶牛乳腺上皮細胞生長狀態(tài)正常的前提下，胞壁酰二肽的體外刺激濃度應(yīng)控制在10 μg·mL-1以下。

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（責任編輯 林鑒非）

Growth and Expression of NOD2 mRNA in Bovine Mammary Epithelial Cells Treated with Different Concentrations of MDP in Vitro

XU DanDan, WANG JianFa, ZHANG Xu, LIU DongYu, XU XiaoNan, WANG Le, CHEN Jia, SHAN XuFei, WANG XiaoYa, WU Rui, YANG Bin
(College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163319, Heilongjiang)

bovine mammary epithelial cell; MDP; NOD2

2016-04-13；接受日期：2016-11-22

國家自然科學(xué)基金（31472249，31402157）、黑龍江八一農(nóng)墾大學(xué)研究生創(chuàng)新項目（YJSCX2015-Y23）

聯(lián)系方式：徐丹丹，E-mail：xudandan19910520@163.com。通信作者武瑞，E-mail：fuhewu@126.com

Abstract:【Objective】 Dairy cow mastitis is one of the most common diseases causing serious economic losses in dairy-farming industry. Bacterial infection is the main cause of mastitis. Innate immunity is the first line of defense against the invasion of pathogenic bacteria in mammary gland. NOD2 is an important member of the innate immune pattern recognition receptor of nucleotide-binding oligomerization domain (NOD) family, which defenses against various microbial invasions by recognizing its specific ligand-muramyl dipeptide (MDP), a component widely existing in gram positive bacteria and gram negative bacteria cell wall. Bovine mammary epithelial cell (BMEC) is the immune barrier of dairy cow mammary gland other than secreting milk. Here, the effect of MDP on the in vitro growth state of BMEC and the expression of NOD2 in the BMEC was explored in this experiment.【Method】 Mammary gland tissue of healthy and lactating Holstein cows was chosen as raw materials. Collagenase digestion method combined with concentration gradient of trypsin was used to separate BMEC. Cytokeratin-18 specific expression in epithelial cells and vimentin specific expression in fibroblasts were used to identify the obtained cells by immunofluorescent techniques. BMEC was set to 6 treatment groups, including MDP stimulating concentrations of 0 (control group), 1, 5, 10, 15 and 20 μg·mL-1. Twenty-four hours of poststimulation, BMEC status were observed under a microscope, meanwhile total RNA was extracted from BMEC and reverse transcribed to cDNA. Real time fluorescent quantitative PCR method was used to detect the expression of NOD2 in BMEC.【Result】Those cells separated by collagenase digestion method combined with concentration gradient of trypsin, immunofluorescence results of CK-18 reaction was positive and vimentin reaction was negative. All cells were in a good growth condition. In the control group and in the groups of MDP stimulating concentration at 1, 5 and 10 μg·mL-1, BMEC grew well without any visible abnormalities. There was a small amount of BMEC detached from bottom in the group of MDP stimulating concentration at 15 μg·mL-1. However, the group of MDP stimulating concentration at 20 μg·mL-1showed a large number of BMEC detached and floated from bottom. Even though those BMEC were still attached to the bottom, their morphology had already changed. Compared with the control group, the expression of NOD2 mRNA in BMEC was positively correlated with the stimulating concentrations of MDP. In other words, 24 h of poststimulation, the expression of NOD2 mRNA in BMEC gradually increased along with the stimulating concentrations of MDP.【Conclusion】High purity BMEC was successfully obtained. The obtained cells grew well and could be used in the following experiments. Although the expression of NOD2 mRNA was positively correlated with the stimulating concentrations of MDP, the stimulating concentrations of MDP in vitro culture BMEC should be controlled below 10 μg·mL-1in order to maintain the normal growth condition. These results suggested that BMEC could participate in the immune defense response of bovine mammary gland through the NOD2 receptor pathway. But this defense capability was influenced by the number of bacteria or the intensity of bacterial virulence. In a certain number or virulence of bacteria, the immune defense response of bovine mammary gland was enhanced along with the increasing number of bacteria or the enhancement of virulence to eliminate intramammary pathogens. While the number or virulence of bacteria exceeded to a certain range, bovine mammary gland tissue would be seriously damaged, so the immune defense barrier would be collapsed. Under this condition, the local bovine mammary gland or even all over the body would present obvious clinical symptoms.