創(chuàng)傷后肘關(guān)節(jié)外源性攣縮形成中差異表達(dá)基因及相關(guān)通路研究

余寶富 曾紅 唐智明 廖新根 余歡 夏國(guó)明 黃黛翠 李洪波

. 論著 Original article .

創(chuàng)傷后肘關(guān)節(jié)外源性攣縮形成中差異表達(dá)基因及相關(guān)通路研究

余寶富 曾紅 唐智明 廖新根 余歡 夏國(guó)明 黃黛翠 李洪波

目的在分子水平研究創(chuàng)傷后肘關(guān)節(jié)外源性攣縮的機(jī)制。方法 建立新西蘭兔創(chuàng)傷后膝關(guān)節(jié)外源性攣縮模型，左膝經(jīng)手術(shù)導(dǎo)致關(guān)節(jié)內(nèi)骨折，并以克氏針固定 8 周為實(shí)驗(yàn)組，未經(jīng)手術(shù)的右膝為對(duì)照組，以模擬人肘關(guān)節(jié)外源性攣縮。使用 Agilent 兔全基因芯片技術(shù)檢測(cè)兔膝關(guān)節(jié)囊基因表達(dá)譜，通過比較實(shí)驗(yàn)組和對(duì)照組關(guān)節(jié)囊基因表達(dá)譜篩選攣縮關(guān)節(jié)的關(guān)節(jié)囊中差異表達(dá)基因，對(duì)差異基因進(jìn)行注釋及功能分析，并采用 Real-time RT-PCR 驗(yàn)證部分差異基因。結(jié)果 實(shí)驗(yàn)組與對(duì)照組基因表達(dá)譜相比較后篩選出差異表達(dá)基因90 個(gè)，其中表達(dá)上調(diào) 21 個(gè)，表達(dá)下調(diào) 69 個(gè)。這些差異表達(dá)基因在生化過程中主要參與調(diào)節(jié)外部的刺激反應(yīng)、調(diào)控脂多糖介導(dǎo)的信號(hào)轉(zhuǎn)導(dǎo)通路及正性調(diào)節(jié)生物刺激反應(yīng)，在分子功能中主要參與離子及 ATP 的結(jié)合，在細(xì)胞成分中主要參與構(gòu)成胞質(zhì)膜小泡。這些差異基因主要涉及的通路共有 31 個(gè)，主要包括風(fēng)濕性關(guān)節(jié)炎通路 ( rheumatoid arthritis )、吞噬小體通路 ( phagosome )、肌動(dòng)蛋白細(xì)胞骨架調(diào)節(jié)通路 ( regulation of actin cytoskeleton ) 等。Real-time RT-PCR 結(jié)果表明，Acta2、MMP2 及 KGF 3 個(gè)差異表達(dá)基因的定量結(jié)果與基因芯片檢測(cè)結(jié)果一致，均表達(dá)上調(diào)。結(jié)論 創(chuàng)傷后肘關(guān)節(jié)外源性攣縮的關(guān)節(jié)囊與正常關(guān)節(jié)囊相比存在差異表達(dá)基因，這些差異基因可能通過多種途徑在關(guān)節(jié)攣縮的形成中發(fā)揮作用。篩選出的差異表達(dá)基因及分析出的相關(guān)信號(hào)通路可為創(chuàng)傷后肘關(guān)節(jié)外源性攣縮機(jī)制的進(jìn)一步研究提供實(shí)驗(yàn)基礎(chǔ)。

關(guān)節(jié)攣縮；肘關(guān)節(jié)；創(chuàng)傷；基因；信號(hào)通路；基因芯片

肘關(guān)節(jié)攣縮是指肘關(guān)節(jié)主動(dòng)和 ( 或 ) 被動(dòng)活動(dòng)范圍減少或喪失，為肘部常見的并發(fā)癥之一。根據(jù)攣縮的原因可分為外源性、內(nèi)源性以及混合性三類。其中典型的外源性攣縮主要是指累及肘關(guān)節(jié)周圍軟組織結(jié)構(gòu)，包括關(guān)節(jié)周圍的關(guān)節(jié)囊、韌帶及肌肉等一系列病理改變。不同程度的關(guān)節(jié)周圍組織攣縮可直接導(dǎo)致肘關(guān)節(jié)屈伸障礙，嚴(yán)重影響患者的日常生活及工作。雖然近些年來對(duì)肘關(guān)節(jié)創(chuàng)傷的認(rèn)識(shí)不斷提高，治療方法不斷改進(jìn)，但是創(chuàng)傷后肘關(guān)節(jié)攣縮的問題仍普遍存在。因此，如何預(yù)防及治療創(chuàng)傷后肘關(guān)節(jié)攣縮已成為臨床工作及研究的重點(diǎn)，解決問題的關(guān)鍵在于了解攣縮發(fā)生的機(jī)制。相對(duì)于正常的關(guān)節(jié)囊，創(chuàng)傷后攣縮的關(guān)節(jié)囊中的基因差異表達(dá)是引起關(guān)節(jié)囊攣縮的根本原因。為此，本研究根據(jù)以往研究建立新西蘭兔創(chuàng)傷后膝關(guān)節(jié)攣縮模型[1-2]，以模擬人類創(chuàng)傷后肘關(guān)節(jié)外源性攣縮。使用基因芯片技術(shù)對(duì)新西蘭兔攣縮的膝關(guān)節(jié)囊進(jìn)行差異表達(dá)基因篩選，并對(duì)其功能及參與的通路進(jìn)行分析，試圖在分子水平上為肘關(guān)節(jié)外源性攣縮的形成機(jī)制提供依據(jù)。

材料與方法

一、兔膝關(guān)節(jié)外源性攣縮模型的建立

選用健康成年 ( 12～18 個(gè)月齡 ) 的新西蘭雌性大白兔 ( 3 只 )，體重約 3.5～3.8 kg。分開飼養(yǎng)。左膝為手術(shù)組，右膝為對(duì)照組。戊巴比妥鈉耳緣靜脈麻醉后，左膝處消毒，做左膝前方切口，先暴露股骨內(nèi)側(cè)髁，于側(cè)副韌帶止點(diǎn)近端用直徑 2.0 mm 的克氏針鉆孔并鉆出對(duì)側(cè)皮質(zhì)?？紫吨械臐B血流入膝關(guān)節(jié)以此模擬關(guān)節(jié)內(nèi)骨折。隨后以直徑 1.5 mm 的克氏針自脛骨前方往后穿入股骨，克氏針兩尾端彎曲，膝關(guān)節(jié)被固定于屈曲 150°，以此為膝關(guān)節(jié)制動(dòng)模型。

二、取材

兔膝關(guān)節(jié)制動(dòng)模型建立后 8 周，3 只兔克氏針無(wú)松脫，關(guān)節(jié)無(wú)感染表現(xiàn)，再次戊巴比妥鈉耳緣靜脈麻醉，左膝關(guān)節(jié)處消毒后，膝關(guān)節(jié)前方入路進(jìn)入，先暴露克氏針并取出，活動(dòng)膝關(guān)節(jié)可見左膝關(guān)節(jié)活動(dòng)范圍較右膝明顯減小，提示左膝關(guān)節(jié)明顯攣縮，然后切取膝關(guān)節(jié)后方關(guān)節(jié)囊組織，取下的標(biāo)本立即置入液氮保存。同樣方法取對(duì)側(cè)正常組標(biāo)本。

三、RNA 抽提和純化

四、差異基因基因芯片檢測(cè)

項(xiàng)目所用芯片為 Agilent 兔全基因 4×44K 芯片( design ID：020908 )，共有 6 個(gè)標(biāo)本，需要完成6 張上述芯片。

1. 樣品 RNA 的放大和標(biāo)記：實(shí)驗(yàn)樣品 RNA 采用 Agilent 表達(dá)譜芯片配套試劑盒，Low Input Quick Amp Labeling Kit，One-Color ( Cat#5190-2305，Agilent technologies，Santa Clara，CA，US ) 和標(biāo)準(zhǔn)操作流程對(duì)樣品 total RNA 中的 mRNA 進(jìn)行放大和標(biāo)記，并用 RNeasy mini kit ( Cat#74106，QIAGEN，GmBH，Germany ) 純化標(biāo)記后的 cRNA。

2. 芯片雜交：按照 Agilent 表達(dá)譜芯片配套提供的雜交標(biāo)準(zhǔn)流程和配套試劑盒，Gene Expression Hybridization Kit ( Cat#5188-5242，Agilent technologies，Santa Clara，CA，US )，在滾動(dòng)雜交爐 Hybridization Oven ( Cat#G2545A，Agilent technologies，Santa Clara，CA，US ) 中 65 ℃，10 rpm，滾動(dòng)雜交 17 h，雜交 cRNA 上樣量 1.65 μg，并在洗缸 staining dishes ( Cat#121，Thermo Shandon，Waltham，MA，US ) 中洗片，洗片所用的試劑為Gene Expression Wash Buffer Kit ( Cat#5188-5327，Agilent technologies，Santa Clara，CA，US )。

3. 芯片掃描：完成雜交的芯片采用 Agilent Microarray Scanner ( Cat#G2565CA，Agilent technologies，Santa Clara，CA，US ) 進(jìn)行掃描，軟件設(shè)置 Dye channel：Green，Scan resolution＝5 μm, PMT 100%，10%，16bit。用 Feature Extraction software 10.7 ( Agilent technologies，Santa Clara，CA，US ) 讀取數(shù)據(jù)，最后采用 Gene Spring Software 11.0 ( Agilent technologies，Santa Clara，CA，US ) 進(jìn)行歸一化處理，所用的算法為 Quantile。

4. 差異基因的篩選及分析：將掃描所得數(shù)據(jù)使用由上海伯豪有限公司提供的在線分析系統(tǒng) ( SAS )進(jìn)行分析：分別計(jì)算差異倍數(shù) ( fold change，F(xiàn)C ) 和每個(gè)探針點(diǎn)的 Flag / Call 值。采用配對(duì) t 檢驗(yàn)比較組間數(shù)據(jù)。差異基因篩選閾值設(shè)定包括以下 2 點(diǎn)：( 1 ) P＜0.05；( 2 ) 差異倍數(shù) ( FC ) ≥2 或者≤0.5，并過濾掉功能未知及無(wú)注釋的基因。

表1 提取的總 RNA 質(zhì)檢結(jié)果Tab.1 The quality inspection results of extracted total RNA

5. 數(shù)據(jù)分析：對(duì)差異基因行 GO ( gene ontology )功能分析、Pathway 分析等。GO 功能分析：由于暫時(shí)沒有找到直接支持新西蘭兔 EntrezGene ID 號(hào)檢索的 GO 功能的分析工具，本研究將 EntrezGene ID 號(hào)轉(zhuǎn)化成 UNIPROT 號(hào)，針對(duì)所有的 UNIPROT 蛋白GO 信息進(jìn)行了檢索，生成的 GO 注釋文件在 WEGO ( http://wego.genomics.org.cn/cgi-bin/wego/index.pl ) 進(jìn)行 GO 作圖。根據(jù)所有檢測(cè)到差異基因的 GO 數(shù)據(jù)和所有檢測(cè)到基因的 GO 數(shù)據(jù)進(jìn)行比對(duì)，分析 GO 富集數(shù)據(jù)，由于兔 GO 數(shù)據(jù)過少，本研究將篩選標(biāo)準(zhǔn)上調(diào)為 P＜0.10，在 REVIGO ( http://revigo.irb.hr/ ) 上作圖。KEGG Pathway 分析：使用 KOBAS 2.0 ( http:// kobas.cbi.pku.edu.cn/home.do ) 來做 KEGG Pathway 分析，篩選標(biāo)準(zhǔn)為 P＜0.05。

那是我最后一次看到萬(wàn)姐，背著一個(gè)比她身體還要大的包袱。老公要下樓幫她攔輛出租車，卻被她攔住了，一個(gè)人搖搖欲墜地向樓下走去。我這才發(fā)現(xiàn)，原來她住在我家時(shí)，竟然做了那么多的衣服。

五、Real-time RT-PCR 驗(yàn)證

查閱文獻(xiàn)并結(jié)合芯片的結(jié)果，初步選擇 Acta2、KGF、MMP2 基因行 Real-time RT-PCR 驗(yàn)證基因芯片結(jié)果。所用試劑：Real-time RT-PCR Takara kit。儀器：ABI stepone plus。實(shí)驗(yàn)步驟按照試劑盒進(jìn)行，以 Gapdh 為內(nèi)參，三復(fù)孔。所用引物：Acta2-forward ( 5’-GACAATGGCTCCGGGCTCTGT AA-3’ )，Acta2-reverse ( 5’-TGCGCTTCATCACCC ACGTA-3’ )，KGF-forward ( 5’-AAACGAGGCAAAG TAAAAGGGAC-3’ )，KGF-reverse ( 5’-CCATTTAGC TGATGCGTATGTGTTG-3’ )，MMP2-forward ( 5’-C ATGTCTACTATTGGCGGGAAC-3’ )，MMP2-reverse ( 5’-TTCTTCGTGTAGGTGTAAATGGG-3’ )，Gapdhforward ( 5’-GCACGGTCAAGGCTGAGAAC-3’ )，Gapdh-reverse ( 5’-TGGTGAAGACGCCAGTGG A-3’ )。采用 2-ΔΔCt法比較實(shí)驗(yàn)組和對(duì)照組間的差異[3]。使用配對(duì) t 檢驗(yàn)比較兩組間的差異，P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

表2 芯片實(shí)驗(yàn)質(zhì)控結(jié)果Tab.2 The quality inspection results of gene chip

結(jié) 果

一、提取的總 RNA 質(zhì)檢

共 6 份標(biāo)本，除 Trauma 2 以外，2100 RIN 均≥7.0，28 s / 18 s 均≥0.7，且 260 / 280 nm 的比值均＞1.8 ( 表1 )。Trauma 2 標(biāo)本雖然 2100 RIN ( 值為6.8 )＜7.0，但接近 7.0，提示 RNA 部分降解，但28 s / 18 s＞0.7，260 / 280 nm 比值＞1.8，考慮對(duì)芯片實(shí)驗(yàn)的表達(dá)檢測(cè)影響不大，因此仍進(jìn)行后續(xù)實(shí)驗(yàn)。

二、芯片實(shí)驗(yàn)質(zhì)控情況

基因芯片實(shí)驗(yàn)中，所有標(biāo)本 CV 值均＜10%，檢出率均＞60% ( 表2 )，提示芯片質(zhì)量合格。

三、差異基因篩選結(jié)果

篩選出兩組間差異表達(dá)基因 90 個(gè)，與對(duì)照組正常膝關(guān)節(jié)囊相比，創(chuàng)傷后的關(guān)節(jié)囊中表達(dá)上調(diào)的差異基因有 21 個(gè) ( 表3 )，表達(dá)下調(diào)的差異基因有69 個(gè) ( 表4 )。

四、GO 功能分析

GO 結(jié)果見圖1，2。在 REVIGO 上作圖結(jié)果顯示，這些差異基因在生化過程中主要參與調(diào)節(jié)外部的刺激反應(yīng)、調(diào)控脂多糖介導(dǎo)的信號(hào)轉(zhuǎn)導(dǎo)通路及正性調(diào)節(jié)生物刺激反應(yīng)，在分子功能中主要參與離子及 ATP 的結(jié)合，在細(xì)胞成分中主要參與構(gòu)成胞質(zhì)膜小泡。

五、KEGG Pathway 分析

結(jié)果顯示差異基因涉及到的通路有 31 個(gè)。主要包括風(fēng)濕性關(guān)節(jié)炎通路 ( rheumatoid arthritis )、吞噬小體通路 ( phagosome )、肌動(dòng)蛋白細(xì)胞骨架調(diào)節(jié)通路( regulation of actin cytoskeleton ) 等。31 條通路結(jié)果見表5 ( P≤0.05 )。

六、Real-time RT-PCR 對(duì)差異表達(dá)基因的驗(yàn)證結(jié)果

在差異基因中，選擇與創(chuàng)傷后關(guān)節(jié)囊攣縮明顯相關(guān)的 3 個(gè)基因進(jìn)行驗(yàn)證，Acta2、MMP2 及 KGF 均上調(diào)，與基因芯片結(jié)果一致。

表3 表達(dá)上調(diào)的差異基因Tab.3 The up-regulated differentially expressed genes

表4 表達(dá)下調(diào)的差異基因Tab.4 The down-regulated differentially expressed genes

描述ITI-HC2 Oryctolagus cuniculus inter-alpha-trypsin inhibitor heavy chain2 ( ITI-HC2 ), mRNA [ NM_001082647 ]NCF1 Oryctolagus cuniculus neutrophil cytosolic factor 1 ( NCF1 ), mRNA [ NM_001082102 ]CD4 Oryctolagus cuniculus CD4 molecule ( CD4 ), mRNA [ NM_001082313 ]AOAH Oryctolagus cuniculus acyloxyacyl hydrolase ( neutrophil ) ( AOAH ), mRNA [ NM_001082025 ]CCL2 Oryctolagus cuniculus chemokine ( C-C motif ) ligand 2 ( CCL2 ), mRNA [ NM_001082294 ]APOBEC1 Oryctolagus cuniculus apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1 ( APOBEC1 ), mRNA [ NM_001082341 ]CAPS Oryctolagus cuniculus calcyphosine ( CAPS ), mRNA [ NM_001082644 ]CD38 Oryctolagus cuniculus CD38 molecule ( CD38 ), mRNA [ NM_001082683 ]RLA-A3 Oryctolagus cuniculus MHC class I antigen-like ( RLA-A3 ), mRNA [ NM_001190434 ]LOC100101599 Oryctolagus cuniculus cathepsin W mRNA, partial cds [ EF472895 ]LOC100344030 Oryctolagus cuniculus cathepsin L1 ( LOC100344030 ), mRNA [ XM_002708256 ]SLC5A9 Oryctolagus cuniculus solute carrier family 5 ( sodium/glucose cotransporter ), member 9 ( SLC5A9 ), mRNA [ NM_001082699 ]CYBB Oryctolagus cuniculus cytochrome b-245 beta polypeptide ( CYBB ), mRNA [ NM_001082100 ]ACP5 Oryctolagus cuniculus acid phosphatase 5, tartrate resistant ( ACP5 ), mRNA [ NM_001081988 ]MYH11 Oryctolagus cuniculus myosin, heavy chain 11, smooth muscle ( MYH11 ), mRNA [ NM_001082308 ]IFNG Oryctolagus cuniculus interferon, gamma ( IFNG ), mRNA [ NM_001081991 ]IL8 Oryctolagus cuniculus interleukin 8 ( IL8 ), mRNA [ NM_ 001082293 ]NCF4 Oryctolagus cuniculus neutrophil cytosolic factor 4, 40 kDa ( NCF4 ), mRNA [ NM_001082654 ]FPR Oryctolagus cuniculus N-formyl peptide receptor ( FPR ), mRNA [ NM_001082314 ]SLC15A2 Oryctolagus cuniculus solute carrier family 15 ( H+/peptide transporter ), member 2 ( SLC15A2 ), mRNA [ NM_001082700 ]KCTD16 Oryctolagus cuniculus potassium channel tetramerisation domain containing 16 (LOC100357562), mRNA [XM_002710279]PLN Oryctolagus cuniculus phospholamban ( PLN ), mRNA [ NM_001082621 ]LOC100009317 Oryctolagus cuniculus heme carrier protein 1 ( LOC100009317 ), mRNA [ NM_001082630 ]FYB Oryctolagus cuniculus FYN binding protein ( FYB-120/130 ) ( LOC100350748 ), mRNA [ XM_002713989 ]LOC100008880 Oryctolagus cuniculus lipophilin AL2 ( LOC100008880 ), mRNA [ NM_001082139 ]LPXN Oryctolagus cuniculus leupaxin ( LPXN ), mRNA [ NM_ 001082048 ]OLFM3 Oryctolagus cuniculus olfactomedin 3 ( LOC100344242 ), mRNA [ XM_002715827 ]CX3CR1 Oryctolagus cuniculus chemokine ( C-X3-C motif ) receptor 1 ( CX3CR1 ), mRNA [ NM_001082134 ]ZAN Oryctolagus cuniculus zonadhesin ( ZAN ), mRNA [ NM_001082081 ]THBD Oryctolagus cuniculus thrombomodulin ( THBD ), mRNA [ NM_001082144 ]TNFAIP8L2 Oryctolagus cuniculus tumor necrosis factor, alpha-induced protein 8-like 2 ( TNFAIP8L2 ), mRNA [ NM_001171296 ]CTLA4 Oryctolagus cuniculus cytotoxic T-lymphocyte-associated protein 4 ( CTLA4 ), mRNA [ NM_001082685 ]CHM-I Oryctolagus cuniculus chondromodulin-I precursor ( CHM-I ), mRNA [ NM_001082040 ]LOC100101588 Oryctolagus cuniculus caspase 1 ( LOC100101588 ), mRNA [ XM_002708417 ]CASQ2 Oryctolagus cuniculus calsequestrin 2 ( cardiac muscle ) ( CASQ2 ), nuclear gene encoding mitochondrial protein, mRNA [ NM_001101691 ]CYP8B1 Oryctolagus cuniculus cytochrome P450, family 8, subfamily B, polypeptide 1 ( CYP8B1 ), mRNA [ NM_001082622 ]MSR1 Oryctolagus cuniculus macrophage scavenger receptor 1 ( MSR1 ), mRNA [ NM_001082248 ]LOC100009479 Oryctolagus cuniculus potassium channel subunit Kv 1.2 ( LOC100009479 ), mRNA [ NM_001082722 ]GRO-A Oryctolagus cuniculus GRO ( GRO-A ), mRNA [ NM_ 001082386 ]TACC3 Oryctolagus cuniculus transforming, acidic coiled-coil containing protein 3 ( TACC3 ), mRNA [ NM_001082146 ]基因符號(hào)

圖1 差異表達(dá)上調(diào)基因的 GO 分析結(jié)果Fig.1 The GO analysis results of the up-regulated expressed differential genes

圖2 差異表達(dá)下調(diào)基因的 GO 分析結(jié)果Fig.2 The GO analysis results of the down-regulated expressed differential genes

表5 差異基因主要參與的通路Tab.5 The main related pathways of differential genes

討 論

本研究建立模型研究創(chuàng)傷后關(guān)節(jié)外源性攣縮的機(jī)制，其中較多分子與人類的研究結(jié)果一致，證明了模型建立的合理性。如本研究中，攣縮的關(guān)節(jié)囊MMP1、MMP2 表達(dá)上調(diào)，而 Cohen 等[4]發(fā)現(xiàn)在人體中，創(chuàng)傷后攣縮的肘關(guān)節(jié)囊中 MMP1、MMP2 的表達(dá)水平也顯著上調(diào)。本研究使用 Agilent 兔全基因芯片技術(shù)篩選攣縮關(guān)節(jié)的關(guān)節(jié)囊中差異表達(dá)基因，篩選出兩組間差異表達(dá)基因 90 個(gè)，表達(dá)上調(diào)的差異基因有 21 個(gè)，表達(dá)下調(diào)的差異基因有 69 個(gè)，表明創(chuàng)傷后肘關(guān)節(jié)外源性攣縮的關(guān)節(jié)囊中存在差異表達(dá)基因。通過對(duì)差異基因的功能分析發(fā)現(xiàn)，它們參與對(duì)外部刺激反應(yīng)的調(diào)節(jié)、調(diào)控脂多糖介導(dǎo)的信號(hào)轉(zhuǎn)導(dǎo)通路、生物刺激反應(yīng)的正調(diào)節(jié)、離子結(jié)合、ATP結(jié)合及組成胞質(zhì)膜小泡等。這些過程與文獻(xiàn)報(bào)道一致[5-6]，如調(diào)節(jié)外部刺激反應(yīng)，機(jī)體的損傷和疼痛可引起神經(jīng)末梢釋放神經(jīng)肽 P 物質(zhì)及降鈣素 G 相關(guān)肽類物質(zhì)，從而引起肥大細(xì)胞脫顆粒。肥大細(xì)胞含有促纖維化顆粒，包括血小板生長(zhǎng)因子 A ( platelet derived growth factor A，PDGFA )，內(nèi)皮縮血管素 1( endothelin1，ET1 )，成纖維細(xì)胞生長(zhǎng)因子 ( fibroblast growth factor，F(xiàn)GF ) 和轉(zhuǎn)化生長(zhǎng)因子-β ( transforming growth factor-β，TGF-β1 ) 等，肥大細(xì)胞通過脫顆粒，釋放上述因子，從而促進(jìn)肌成纖維細(xì)胞的分化及增殖[7]。在動(dòng)物及人類的創(chuàng)傷后攣縮肘關(guān)節(jié)中，肥大細(xì)胞、含神經(jīng)肽的神經(jīng)纖維的數(shù)目 4 周內(nèi)便開始上升，而在慢性階段仍保持升高的狀態(tài)[8]。肥大細(xì)胞因此連接創(chuàng)傷后的急性炎癥期和隨后的攣縮期，也因此可能成為創(chuàng)傷后肘關(guān)節(jié)攣縮的干預(yù)靶點(diǎn)。酮替芬可以減少肥大細(xì)胞及肌成纖維細(xì)胞的數(shù)量，并且相對(duì)地可以減少關(guān)節(jié) 42%～52% 的攣縮程度。因此，酮替芬可能是人類皮膚損傷后減少傷口攣縮及纖維化的有效措施，并且不影響正常的愈合過程[9]。Hildebrand 等[8]發(fā)現(xiàn)兔子的創(chuàng)傷后膝關(guān)節(jié)攣縮的急、慢性階段及人類創(chuàng)傷后攣縮的肘關(guān)節(jié)慢性階段，關(guān)節(jié)囊中的肌成纖維細(xì)胞、肥大細(xì)胞、神經(jīng)肽的含量顯著上升，而上述過程必然伴隨著能量的代謝，與本研究結(jié)構(gòu)中參與 ATP 結(jié)合是一致的。差異基因的功能結(jié)果提示創(chuàng)傷后關(guān)節(jié)囊攣縮的發(fā)生、發(fā)展與多種基因差異表達(dá)、多個(gè)分子生物學(xué)過程共同所致。KEGG Pathway 分析顯示，差異基因涉及的通路有 31 個(gè)，主要包括風(fēng)濕性關(guān)節(jié)炎通路、吞噬小體通路、肌動(dòng)蛋白細(xì)胞骨架調(diào)節(jié)通路等。如α-平滑肌肌動(dòng)蛋白 ( α-SMA ) 是肌成纖維細(xì)胞胞內(nèi)表達(dá)的一種肌動(dòng)蛋白，α-SMA 可通過細(xì)胞膜上的整合素作用于細(xì)胞外基質(zhì)，從而影響細(xì)胞外基質(zhì)的構(gòu)成[10-11]。因此對(duì)肌動(dòng)蛋白細(xì)胞骨架調(diào)節(jié)通路的干預(yù)能夠預(yù)防關(guān)節(jié)的攣縮。KEGG Pathway 分析結(jié)果表明這些通路共同作用，構(gòu)成創(chuàng)傷后關(guān)節(jié)囊攣縮的發(fā)生發(fā)展中復(fù)雜的網(wǎng)絡(luò)調(diào)控系統(tǒng)。為了驗(yàn)證基因芯片的可靠性，本研究從所篩選的差異基因中挑選了Acta2、MMP2 及 KGF 3 個(gè)有意義的差異基因進(jìn)行Real-time RT-PCR 驗(yàn)證，其結(jié)果與基因芯片結(jié)果趨勢(shì)一致，從而確保了基因芯片結(jié)果的可靠性。

圖3 Real-time RT-PCR 結(jié)果顯示 Acta2 基因表達(dá)上調(diào) ( t = 6.092，P = 0.026 )圖4 Real-time RT-PCR 結(jié)果顯示 KGF 基因表達(dá)上調(diào) ( t = 8.199，P = 0.014 )圖5 Real-time RT-PCR 結(jié)果顯示 MMP2 基因表達(dá)上調(diào) ( t = 4.758，P = 0.041 )Fig.3 The results of Real-time RT-PCR showed Acta2 was up-regulated in expression ( t = 6.092, P = 0.026 )Fig.4 The results of Real-time RT-PCR showed KGF was up-regulated in expression ( t = 8.199, P = 0.014 )Fig.5 The results of Real-time RT-PCR showed that expression of MMP2 was up-regulated ( t = 4.758, P = 0.041 )

本研究也有一定的局限性，首先，本研究的樣本較少，增加兔子的數(shù)量可使研究結(jié)果更可靠。其次，攣縮的關(guān)節(jié)囊中，分子的表達(dá)水平會(huì)隨著時(shí)間的改變而改變[12]，因此可使用更多數(shù)量的兔子，分別固定后 8 周、16 周及 32 周再測(cè)定差異表達(dá)基因。再者，該研究是在 RNA 水平揭示基因表達(dá)，細(xì)胞調(diào)控蛋白的表達(dá)包括在轉(zhuǎn)錄水平和翻譯水平，RNA 水平未必與蛋白水平一致的，因此，有必要進(jìn)行蛋白水平的研究來證實(shí)本研究的結(jié)果。最后，可直接用來分析兔子基因的數(shù)據(jù)庫(kù)很少，對(duì)該研究數(shù)據(jù)的分析也有一定的影響。

創(chuàng)傷后關(guān)節(jié)攣縮發(fā)生的機(jī)理非常復(fù)雜，是多基因多通路共同作用的結(jié)果。創(chuàng)傷后肘關(guān)節(jié)攣縮仍是臨床中常見的難以預(yù)防及治療的問題，通過對(duì)創(chuàng)傷后肘關(guān)節(jié)攣縮的機(jī)制研究，可指導(dǎo)創(chuàng)傷后肘關(guān)節(jié)或者是肘關(guān)節(jié)松解后的靶向干預(yù)來預(yù)防肘關(guān)節(jié)攣縮或復(fù)發(fā)。目前主要觀點(diǎn)認(rèn)為，肌成纖維細(xì)胞－肥大細(xì)胞－神經(jīng)肽纖維化軸在創(chuàng)傷后肘關(guān)節(jié)攣縮的發(fā)生發(fā)展中起著最主要的作用，而對(duì)該軸的干預(yù)主要集中在酮替芬對(duì)肥大細(xì)胞的穩(wěn)定作用上[9,13-14]。本研究篩選出的差異基因及分析出的可能通路，將為今后研究創(chuàng)傷后肘關(guān)節(jié)外源性攣縮的預(yù)防及治療提供更多的可參考靶點(diǎn)。

[1]Hildebrand KA, Sutherland C, Zhang M. Rabbit knee model ofpost-traumatic joint contractures: the long-term natural history of motion loss and myofibroblasts[J]. J Orthop Res, 2004, 22(2):313-320.

[2]Hildebrand KA, Zhang M, Salo PT, et al. Joint capsule mast cells and neuropeptides are increased within four weeks of injury and remain elevated in chronic stages of posttraumatic contractures[J]. J Orthop Res, 2008, 26(10):1313-1319.

[3]Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C (T) method[J]. Nat Protoc, 2008, 3(6): 1101-1108.

[4]Cohen MS, Schimmel DR, Masuda K, et al. Structural and biochemical evaluation of the elbow capsule after trauma[J]. J Shoulder Elbow Surg, 2007, 16(4):484-490.

[5]Sch?ffer M, Beiter T, Becker HD, et al. Neuropeptides: mediators of inflammation and tissue repair[J]? Arch Surg, 1998, 133(10):1107-1116.

[6]Duggan AW, Morton CR, Zhao ZQ, et al. Noxious heating of the skin releases immunoreactive substance p in the substantia gelatinosa of the cat: a study with antibody microprobes[J]. Brain Res, 1987, 403(2):345-349.

[7]Gruber BL. Mast cells in the pathogenesis of fi brosis[J]. Curr Rheumatol Rep, 2003, 5(2):147-153.

[8]Hildebrand KA, Zhang M, Salo PT, et al. Joint capsule mast cells and neuropeptides are increased within four weeks of injury and remain elevated in chronic stages of posttraumatic contractures[J]. J Orthop Res, 2008, 26(10):1313-1319.

[9]Monument MJ, Hart DA, Befus AD, et al. The mast cell stabilizer ketotifen fumarate lessens contracture severity and myofibroblast hyperplasia: a study of a rabbit model of posttraumatic joint contractures[J]. J Bone Joint Surg Am, 2010, 92(6):1468-1477.

[10]Skalli O, Ropraz P, Trzeciak A, et al. A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation[J]. J Cell Biol, 1986, 103(6 Pt 2): 2787-2796.

[11]Hinz B. The myof i broblast: paradigm for a mechanically active cell[J]. J Biomech, 2010, 43(1):146-155.

[12]Hildebrand KA, Zhang M, Hart DA. Joint capsule matrix turnover in a rabbit model of chronic joint contractures: correlation with human contractures[J]. J Orthop Res, 2006, 24(5):1036-1043.

[13]Gottwald T, Coerper S, Sch?ffer M, et al. The mast cell-nerve axis in wound healing: a hypothesis[J]. Wound Repair Regen, 1998, 6(1):8-20.

[14]Gallant-Behm CL, Hildebrand KA, Hart DA. The mast cell stabilizer ketotifen prevents development of excessive skin wound contraction and fibrosis in red duroc pigs[J]. Wound Repair Regen, 2008, 16(2):226-233.

( 本文編輯：李慧文 )

Differentially expressed genes and related pathways of post-traumatic elbw joint extrinsic contractures

YU Bao-fu,ZENG Hong, TANG Zhi-ming, LIAO Xin-gen, YU Huan, XIA Guo-ming, HUANG Dai-cui, LI Hong-bo. The People’s Hospital of Jiangxi Province, Nanchang, Jiangxi, 330006, China

LI Hong-bo, Email: hongbolijx@163.com

ObjectiveTo study the mechanism of post-traumatic elbow joint extrinsic contracture at molecular level. Methods New Zealand rabbit model of post-traumatic knee extrinsic joint contracture was established to mimic post-traumatic elbow joint extrinsic contracture of human, of which left knees were operated on to result in intraarticular fractures and were fi xed for 8 weeks with K-wire as the experimental group. The right knees served as control group. The differentially expressed genes were screened with Agilent whole rabbit genome microarray technology by comparing gene expression prof i les of contracted joint capsules with normal ones. The functions of the differentially expressed genes were analyzed and annotated. Partial differentially expressed genes were conf i rmed by Real-time RTPCR. Results Ninety differentially expressed genes were detected between experimental group and control group, of which 21 were up-regulated and 69 down-regulated. In the analysis of differentially expressed genes, they were closely related to regulation of response to external stimulus, regulation of lipopolysaccharide-mediated signaling pathway and positive regulation of response to biotic stimulus in the function of biological process, ion binding and ATP binding in the molecular function, and cytoplasmic membrane-bounded vesicle in cellular component. There were mainly 31 pathways related to the differentially expressed genes, including rheumatoid arthritis, phagosome, regulation of actin cytoskeleton, etc. The results of Real-time RT- PCR were consistent with the results of the gene chip when tested for Acta2, MMP2 and KGF genes which were all up-regulated. Conclusions There are differentially expressed genes in the joint capsule of post-traumatic elbow joint extrinsic contracture, which may play a signif i cant role in the formation of joint contracture through multiple channels. These differentially expressed genes and related signal pathways mayprovide experimental basis for further research of post-traumatic elbow joint extrinsic contracture.

Arthrogryposis; Elbow joint; Trauma; Genes; Signal pathway; Gene chip

10.3969/j.issn.2095-252X.2017.03.007

R684

330006 南昌，江西省人民醫(yī)院

李洪波，Email: hongbolijx@163.com

2016-10-13 )