俞小衛(wèi), 楊明夏, 鄭建洲, 高瑞辰,

賀　琛1, 鄢新民2, 龐　勝2

(1. 江蘇省常州市第二人民醫(yī)院 呼吸科, 江蘇 常州, 213003; 2. 美國(guó)加州大學(xué)洛杉磯分校醫(yī)學(xué)院)

肺癌患者唾液中細(xì)菌構(gòu)成分析

俞小衛(wèi)1, 楊明夏1, 鄭建洲1, 高瑞辰1,

賀琛1, 鄢新民2, 龐勝2

(1. 江蘇省常州市第二人民醫(yī)院 呼吸科, 江蘇 常州, 213003; 2. 美國(guó)加州大學(xué)洛杉磯分校醫(yī)學(xué)院)

摘要:目的探討唾液中細(xì)菌構(gòu)成與肺癌的關(guān)系。方法收集鱗癌和腺癌以及非腫瘤對(duì)照組唾液標(biāo)本，每組10例。對(duì)唾液中大量的16 s rDNA進(jìn)行測(cè)序分析。結(jié)果經(jīng)定量PCR(qPCR)證實(shí)，5個(gè)細(xì)菌集群在癌癥和對(duì)照組樣本間有顯著差異。結(jié)論16 s測(cè)序和PCR相結(jié)合的研究顯示，有兩個(gè)細(xì)菌屬(二氧化碳噬纖維菌屬和韋永氏球菌屬)水平癌癥組要顯著高于非癌癥組。

關(guān)鍵詞:肺癌; 16 s rDNA測(cè)序; 二氧化碳噬纖維菌屬; 韋永氏球菌屬; 唾液微生物群

肺癌5年存活率約11%[1],在北美和全球范圍是癌癥相關(guān)死亡最常見(jiàn)原因[2-3]。吸煙被認(rèn)為是肺癌的一個(gè)重要病因，原因是煙草中含有可能會(huì)誘發(fā)細(xì)胞轉(zhuǎn)換的致癌物質(zhì)[4]。然而,有報(bào)告表明肺癌涉及免疫應(yīng)答[5]、病毒性感染[6]以及其他與煙草不相關(guān)的因素[6-7]。最近的研究[8-13]報(bào)告唾液菌群與幾種癌癥有關(guān)。先前的報(bào)道[14-15]表明衣原體感染與肺癌有關(guān)。大多數(shù)唾液細(xì)菌來(lái)自口腔,有些也可能來(lái)自食道和上呼吸道?？谇焕锏募?xì)菌生存和發(fā)展依賴于口腔環(huán)境，而口腔環(huán)境可以受到飲食、蛋白質(zhì)或唾液中其他營(yíng)養(yǎng)物質(zhì)、痰液的成分以及習(xí)慣譬如吸煙的影響。這些因素與肺癌的發(fā)生和發(fā)展有關(guān)。基于這些考慮，以及之前報(bào)道的細(xì)菌參與肺癌中腫瘤發(fā)生[14-16], 作者調(diào)查了肺癌患者的唾液細(xì)菌水平。作者使用Illumina公司HiSeq 2000, 通過(guò)測(cè)序16 s rDNA的V3和V6來(lái)量化肺癌和對(duì)照組唾液樣本中菌群組成。這種方法可以識(shí)別超過(guò)500種流行的人類細(xì)菌種類和高達(dá)每樣本100 000細(xì)菌序列。這種高靈敏度和綜合分析提供一種新的方法來(lái)研究肺癌和細(xì)菌組成之間的關(guān)系。

非小細(xì)胞肺癌(NSCLC)在肺癌中所占比例超過(guò)80%，其中絕大多數(shù)是鱗狀細(xì)胞癌(SCC, 30%～35%)或腺癌(AC～50%)。在第一輪的研究中,作者選擇鱗癌或腺癌患者和非腫瘤對(duì)照組，每組10個(gè)樣本，確定了能證明肺癌患者和非腫瘤對(duì)照組之間存在顯著差異的細(xì)菌綱/屬。隨后作者使用qPCR量化豐富的細(xì)菌群證實(shí)了這些結(jié)果。在第二輪的研究中,所選的細(xì)菌集群被用來(lái)研究新的一群患者樣本，進(jìn)一步評(píng)估這些細(xì)菌集群和肺癌之間的關(guān)系。

1材料與方法

1.1　唾液樣本收集

唾液樣本收集是根據(jù)常州第二人民醫(yī)院(CSPH)倫理委員會(huì)批準(zhǔn)的協(xié)議。所有對(duì)象的招募皆來(lái)自常州市第二人民醫(yī)院。因?yàn)槲鼰熆赡苡绊懣谇焕锛?xì)菌的增長(zhǎng),只有有著10年以上吸煙史的受試者被選擇。唾液樣本采集是在癌癥診斷之后和治療之前。沒(méi)有一個(gè)對(duì)象，包括病人和對(duì)照者，證明有與唾液細(xì)菌相關(guān)的其他疾病，譬如糖尿病、免疫功能紊亂、皰疹病毒感染、口腔黏膜潰瘍?；颊咝畔⒁?jiàn)表1。

表1　肺癌和非癌癥對(duì)照組唾液樣本比較

年齡在兩癌癥組間及每癌癥組與對(duì)照組間無(wú)顯著差異。

1.2　唾液樣本菌群的測(cè)序分析

唾液中DNA含量通過(guò)SDS溶菌、苯酚萃取去除唾液中蛋白質(zhì)后被分離。乙醇沉淀恢復(fù)樣本中的DNA，其含量通過(guò)PCR擴(kuò)增,正如前面所描述的[17]。作者利用IlluminaHiSeq2000對(duì)擴(kuò)增的樣本進(jìn)行測(cè)序，并對(duì)16 s rDNA的V3和V6 進(jìn)行分析。為確保高質(zhì)量的數(shù)據(jù),作者采用嚴(yán)格的條件來(lái)處理樣本序列,并因此能夠產(chǎn)生有效的結(jié)果。按以下操作步驟進(jìn)行: ① 所有讀數(shù)通過(guò)允許一個(gè)不匹配的樣本條形碼和兩個(gè)不匹配相鄰PCR引物被分配給對(duì)應(yīng)的樣本; ② 讀數(shù)隨后通過(guò)PyroNoise算法消除干擾[18]; ③ 讀數(shù)包含模糊的核苷酸或一個(gè)被移除超過(guò)8個(gè)堿基對(duì)(bp)的均聚物，是序列短于200個(gè)bp或超過(guò)1 000個(gè)bp; ④ 這些讀數(shù)使用最近的空間終止對(duì)齊方式對(duì)齊(NAST)，基于序列同步自定義，基于席爾瓦對(duì)齊參考[19], 并將沒(méi)有預(yù)期的對(duì)齊參考比對(duì)區(qū)域的序列丟棄; ⑤ 通過(guò)UCHIME算法確定的嵌合序列被移除[20]; ⑥ 讀數(shù)通過(guò)貝葉斯分類法和核糖體數(shù)據(jù)庫(kù)項(xiàng)目(RDP)進(jìn)行分類。線粒體序列或未知(這些數(shù)據(jù)不能在國(guó)家級(jí)水平上分類)被移除。最后，所有有效的讀數(shù)聚集到操作分類單位(OTUs)，基于97%序列相似性，使用MOTHUR程序[21]。樣本在不同分類水平的分類分析(門、綱、目、科、屬)通過(guò)使用QIIME被創(chuàng)建[22]。任何指定的綱/屬的水平被算作序列獲得總額的百分比。

定量聚合酶鏈反應(yīng):從發(fā)現(xiàn)階段細(xì)菌測(cè)序所確定細(xì)菌集群將被qPCR進(jìn)一步檢測(cè)。具體檢測(cè)16 s rDNA PCR引物的設(shè)計(jì)(表2)。引物是選擇從先前出版的文獻(xiàn)[23]或通過(guò)搜索RDP數(shù)據(jù)庫(kù)。

表2　16S rDNA定量PCR引物

用于qPCR寡核苷酸引物: F, forward PCR primers;

R, reverse PCR primers。

1.3　統(tǒng)計(jì)血分析

根據(jù)已發(fā)表的文獻(xiàn)[24-29],作者使用接收機(jī)操作特征(ROC)曲線分析,這是MedCalc提供的軟件包(MedCalc Software, Acacialaan 22, B-8400 Ostend, Belgium)。為了更好的呈現(xiàn)數(shù)據(jù),線的顏色和厚度和字體的大小修改。

2結(jié)果

2.1　肺癌患者與非腫瘤對(duì)照組間不同唾液微生物剖面圖

基于作者的測(cè)序結(jié)果,一個(gè)類桿菌綱和4個(gè)細(xì)菌屬(奈瑟氏菌屬, 二氧化碳噬纖維菌屬, 月形單胞菌屬和韋永氏球菌屬)在腫瘤和非腫瘤病人之間具有顯著差異(P<0.05)。類桿菌屬和奈瑟氏菌屬在癌癥患者的水平低于非腫瘤對(duì)照組，而二氧化碳噬纖維菌屬, 月形單胞菌屬和韋永氏球菌屬水平高于非腫瘤對(duì)照組。

2.2　細(xì)菌定量PCR檢測(cè)

作者采用qPCR來(lái)量化所選擇細(xì)菌綱/屬的水平。利用可以擴(kuò)增大多數(shù)細(xì)菌16 s rDNA 一對(duì)PCR引物標(biāo)準(zhǔn)化癌癥和對(duì)照組rDNA水平。qPCR結(jié)果與測(cè)序結(jié)果比較，癌癥樣本的qPCR顯示二氧化碳噬纖維菌屬和韋永氏球菌屬的水平顯著增高，而類桿菌屬和奈瑟氏菌屬水平較低， 支持使用qPCR量化所選擇細(xì)菌集群水平的可行性。月形單胞菌屬的水平在一些標(biāo)本中沒(méi)有被檢測(cè)到，所以作者從進(jìn)一步qPCR研究剔除了這個(gè)細(xì)菌生物標(biāo)志物。qPCR的總體結(jié)果跟測(cè)序研究結(jié)果類似。

經(jīng)16 s rDNA定量PCR而確定。因此, 作者反復(fù)qPCR來(lái)評(píng)估實(shí)驗(yàn)之間的變化，每組包含20個(gè)數(shù)據(jù)點(diǎn)(樣標(biāo)100倍)

2.3　利用所選擇細(xì)菌生物標(biāo)志物對(duì)標(biāo)本量化

評(píng)估選擇的細(xì)菌集群,作者使用qPCR量化附加的樣本,包括41個(gè)癌癥樣本(13 SCC，28 AC)和15個(gè)非腫瘤對(duì)照。作者使用qPCR量化類桿菌屬和奈瑟氏菌屬、二氧化碳噬纖維菌屬、韋永氏球菌屬水平。這些附加的樣本的結(jié)果表明二氧化碳噬纖維菌屬和韋永氏球菌屬癌癥患者的鱗癌和腺癌亞型水平顯著增高，與作者先前30例樣本分析一致。

然而，類桿菌屬和奈瑟氏菌屬 并沒(méi)有象在第一輪研究中 那么顯著增高。雖然在非癌癥對(duì)照組中奈瑟氏菌屬的水平明顯高于那些腺癌患者,ROC曲線下面積(AUC)值為0.77, 但非腫瘤對(duì)照組跟鱗癌比較沒(méi)有顯著差異,AUC低于0.70。對(duì)于類桿菌屬,通過(guò)比較鱗狀細(xì)胞癌、腺癌患者與非腫瘤患者樣本所獲得的AUC值小于0.70, 提示二氧化碳噬纖維菌屬和韋永氏球菌屬與肺癌有關(guān)，而類桿菌屬和奈瑟氏菌屬可能需要更多的研究來(lái)說(shuō)明這兩個(gè)細(xì)菌集群是否與肺癌有關(guān)。

3討論

作者集中的測(cè)序證明類桿菌屬和四個(gè)菌屬(奈瑟氏菌屬, 二氧化碳噬纖維菌屬, 月形單胞菌屬和韋永氏球菌屬)水平肺癌和非腫瘤對(duì)照樣本中顯著不同?；谶@些結(jié)果,作者進(jìn)行第二輪的研究,這些結(jié)果表明,至少五個(gè)細(xì)菌集群中有二個(gè), 二氧化碳噬纖維菌屬和韋永氏球菌屬，其水平癌癥患者的唾液樣本顯著高于非癌癥的對(duì)照組。

唾液微生物群與肺癌之間的內(nèi)在關(guān)系正處在調(diào)查中。細(xì)菌產(chǎn)生的毒素可以干擾細(xì)胞周期,從而改變細(xì)胞生長(zhǎng)[15, 30-31]。這是由于改變的基因控制正常細(xì)胞分裂和凋亡[32-33]。對(duì)肺癌特定細(xì)菌群水平較高的另一種解釋是與肺癌相關(guān)的飲食可能會(huì)偏愛(ài)一些細(xì)菌，而抑制一些其他細(xì)菌。因此,唾液中細(xì)菌組成的模式可能間接與肺癌有關(guān)。正如前面報(bào)道的,大豆和茶抑制肺癌的風(fēng)險(xiǎn)[34-36]和癌癥進(jìn)展[37], 而紅肉會(huì)增加肺癌的風(fēng)險(xiǎn)[38], 提示飲食與肺癌的發(fā)生有關(guān)。

由于飲食也與口腔細(xì)菌組成有關(guān)[39]，菌群在一定程度上可能反映肺癌的風(fēng)險(xiǎn)。唾液微生物群也可能通過(guò)誘導(dǎo)長(zhǎng)期免疫反應(yīng)影響肺部細(xì)胞。眾所周知,腸道微生物群由于免疫耐受的破壞參與炎癥性腸病(IBD)的發(fā)病機(jī)制[40]。

作者注意到鱗癌和腺癌之間有著不同。鱗癌樣本中韋永氏球菌屬水平要高于腺癌樣本中(圖1 e、2 d、3 d)，當(dāng)與非癌癥對(duì)照組樣本比較，證明存在顯著的差異。小韋榮氏球菌是從肺癌患者下呼吸道分離出來(lái)的細(xì)菌，提示這一屬跟肺癌有關(guān)[41]。然而,這一屬在肺癌發(fā)生發(fā)展的發(fā)生中精確的作用還不清楚。

二氧化碳噬纖維菌種通常在口咽道發(fā)現(xiàn)，該菌種已報(bào)道參與肺癌，因?yàn)樗麄円呀?jīng)被證明參與肺膿腫形成[42]和下呼吸道感染[43]，表明癌癥增長(zhǎng)偏愛(ài)這些細(xì)菌的生長(zhǎng)。Neisseria在癌癥患者被發(fā)現(xiàn)其水平顯著低于二氧化碳噬纖維菌屬和韋永氏球菌屬。胰腺癌樣本中也發(fā)現(xiàn)了相似的結(jié)果[12]，表明奈瑟氏菌屬可以抑制癌細(xì)胞。一個(gè)眾所周知的例子是使用卡介苗(BCG)對(duì)膀胱癌治療[44]。

關(guān)于吸煙,所有的受試者在這項(xiàng)研究中已經(jīng)抽了10年了。盡管患者集中的一個(gè)城市,超過(guò)一半的人從許多不同的地方搬到常州。因此,作者認(rèn)為在此描述的細(xì)菌集群是全球分布的。然而,正如前面所報(bào)道的，唾液細(xì)菌也與飲食有關(guān)[39], 因?yàn)閬?lái)自不同地區(qū)的人有著不同的飲食，有必要擴(kuò)大研究來(lái)說(shuō)明不同種族,宗教,或飲食限制中飲食所產(chǎn)生的影響。

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Analysis in saliva bacterial composition

in patients with lung cancer

YU Xiaowei1, YANG Mingxia1, ZHENG Jianzhou1, GAO Ruichen1,

HE Chen1, YAN Xinmin2, PANG Shen2

(1.DepartmentofRespiratory,ChangzhouSecondPeople′sHospital,Changzhou,Jiangsu, 213003;

2.DepartmentofOrthopaedicSurgeryandtheOrthopaedicHospitalResearchCenter,

DavidGeffenSchoolofMedicineatUniversityofCalifornia,LosAngeles,USA, 90095)

ABSTRACT:ObjectiveTo investigate the relationship between saliva bacteria composition and lung cancer. MethodsSalivary samples were collected in squamous cell carcinoma, adenocarcinoma and normal people without tumors, 10 cases for each group. Extensive 16S rDNA sequencing was analyzed. ResultsBy quantitative PCR (qPCR), there were significant differences between five bacterial clusters and control samples. ConclusionThe combination of the 16S sequencing study and the PCR study reveal that the levels of two bacterial genera (capnocytophaga and veillonella) are significantly higher in the saliva samples than those in people without cancer.

KEYWORDS:lung cancer; 16S rDNA sequencing; capnocytophaga; veillonella; saliva microbiota

通信作者:龐勝, E-mail: spang@ucla.edu

基金項(xiàng)目:江蘇省常州市中外合作科技基金(CZ20110021)

收稿日期:2014-12-16

中圖分類號(hào):R 734.2

文獻(xiàn)標(biāo)志碼:A

文章編號(hào):1672-2353(2015)15-029-05

DOI:10.7619/jcmp.201515009