劉莉，楊永姣，陳業(yè)剛，王尚任，劉曉強(qiáng)，陳少峰，孫光

(1天津醫(yī)科大學(xué)第二醫(yī)院，天津300211；2天津市泌尿外科研究所)

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過表達(dá)及沉默細(xì)胞黏附分子1基因膀胱癌細(xì)胞株的構(gòu)建

劉莉1,2，楊永姣1,2，陳業(yè)剛1,2，王尚任1,2，劉曉強(qiáng)1,2，陳少峰1,2，孫光1,2

(1天津醫(yī)科大學(xué)第二醫(yī)院，天津300211；2天津市泌尿外科研究所)

摘要：目的構(gòu)建過表達(dá)及沉默細(xì)胞黏附分子1(CADM1)基因的膀胱癌細(xì)胞株。方法 培養(yǎng)并收集膀胱癌細(xì)胞株T24。①過表達(dá)CADM1基因T24細(xì)胞構(gòu)建：將T24細(xì)胞分為1、2、3組，real-timePCR擴(kuò)增CADM1基因全長，與慢病毒載體pHBLV-IRES -ZsGreen-PGK-puro進(jìn)行連接(1組)，并設(shè)載體對照組(2組)和空白對照(3組)；經(jīng)酶切及測序鑒定正確后在293T細(xì)胞中包裝病毒，病毒濃縮后感染T24細(xì)胞。②沉默CADM1基因T24細(xì)胞構(gòu)建：將T24細(xì)胞分為A、B、C、D、E組，設(shè)計3條針對CADM1基因的干擾序列(siRNA1/2/3)分別與載體pHBLV-U6-shRNA-ZsGreen-Puro進(jìn)行連接(A、B、C組)，D組與pHBLV-U6-ZsGreen-Puro進(jìn)行連接，E組為空白對照；經(jīng)雙酶切、PCR及測序鑒定連接載體，在293T細(xì)胞中包裝病毒，病毒濃縮后感染T24細(xì)胞。采用real-time PCR檢測T24細(xì)胞中的CADM1 mRNA，Western blotting法檢測CADM1蛋白。結(jié)果過表達(dá)和沉默CADM1基因的慢病毒載體及細(xì)胞株均正確構(gòu)建。1、2、3組CADM1 mRNA相對表達(dá)量分別為4.84±0.02、1.12±0.03、1.00±0.00，CADM1蛋白相對表達(dá)量分別為4.53±0.03、1.05±0.04、1.00±0.01，1組CADM1 mRNA及蛋白相對表達(dá)量高于2、3組(P均<0.05)。A、B、C、D、E組CADM1 mRNA相對表達(dá)量分別為0.28±0.05、0.98±0.02、0.74±0.01、0.99±0.01、1.00±0.00，CADM1蛋白相對表達(dá)量分別為0.33±0.04、0.86±0.12、0.67±0.07、1.00±0.04、1.00±0.03，A組CADM1 mRNA及蛋白相對表達(dá)量低于B、C、D、E組(P均<0.05)。結(jié)論 成功構(gòu)建了過表達(dá)及沉默CADM1基因的膀胱癌細(xì)胞株T24。

關(guān)鍵詞：膀胱腫瘤；膀胱癌細(xì)胞株；細(xì)胞黏附分子1；基因沉默

膀胱癌是我國最常見的泌尿系統(tǒng)腫瘤，膀胱癌的預(yù)后與多種因素相關(guān)[1，2]。近年來，許多腫瘤標(biāo)記物相繼被發(fā)現(xiàn)可用于膀胱癌的預(yù)后判斷。細(xì)胞黏附分子1(CADM1)是2001年由Kuramochi等[3]在人類非小細(xì)胞肺癌(NSCLC)中發(fā)現(xiàn)的一種抑癌基因。大量研究顯示，CADM1在食管癌[4]、黑色素瘤[5]、肝癌[6]、卵巢癌[7]、乳腺癌[8]、肺癌[9]、喉鱗癌[10]、結(jié)腸癌[11]等人類多種腫瘤組織和細(xì)胞系中呈低表達(dá)或表達(dá)缺失，且其低表達(dá)與腫瘤較早發(fā)生侵襲、轉(zhuǎn)移有關(guān)。本課題組前期研究[12]發(fā)現(xiàn)，膀胱尿路上皮癌組織中CADM1蛋白表達(dá)下調(diào)，其主要機(jī)制是啟動子區(qū)CpG島甲基化。為進(jìn)一步研究CADM1在膀胱癌發(fā)病中的作用及機(jī)制，我們采用慢病毒載體構(gòu)建穩(wěn)定過表達(dá)和沉默CADM1基因的膀胱癌細(xì)胞株T24，現(xiàn)將結(jié)果報告如下。

1材料與方法

1.1實驗細(xì)胞人膀胱尿路上皮癌細(xì)胞株T24由天津市泌尿外科研究所饋贈。待細(xì)胞生長穩(wěn)定后進(jìn)行后續(xù)實驗。收集膀胱癌細(xì)胞，分別用于穩(wěn)定過表達(dá)和干擾CADM1基因慢病毒載體的構(gòu)建實驗。

1.2過表達(dá)CADM1基因的T24細(xì)胞的構(gòu)建

1.2.1CADM1基因擴(kuò)增以CADM1 cDNA為模板，進(jìn)行PCR擴(kuò)增。CADM1基因上游引物序列為5′-AATCTCGAGATGGCGAGTGTAGTGCTGCC-3′，下游引物序列為5′-ATAGCGGCCGCCTAGATGAAGTACTCTTTCT-3′。 PCR反應(yīng)體系50 μL共包括10×buffer 5 μL、dNTP 5 μL、MgSO43 μL、上下游引物各1 μL、KOD Plus酶1 μL、模板DNA 0.5 μL、ddH2O 33.5 μL。PCR反應(yīng)條件：94 ℃ 5 min，1個循環(huán)；94 ℃ 45 s，55 ℃ 1 min，72 ℃ 90 s，共35個循環(huán)；72 ℃ 10 min，1個循環(huán)。PCR產(chǎn)物經(jīng)1%瓊脂糖凝膠電泳后進(jìn)行膠回收。

1.2.2慢病毒載體的構(gòu)建及鑒定PCR產(chǎn)物回收后與慢病毒載體pHBLV-IRES-ZsGreen-PGK-puro用Xho Ⅰ、Not Ⅰ雙酶切。酶切完成后再次膠回收，處理好的目的片段與載體在T4連接酶16 ℃連接過夜。連接產(chǎn)物轉(zhuǎn)化感受態(tài)E.coli，將轉(zhuǎn)化后的感受態(tài)E.coli 100 μL涂布于含Amp抗性(100 mg/L)的LB平板培養(yǎng)過夜。挑取中等大小、飽滿且未與其他克隆相接觸的單克隆接種到卡那霉素選擇性(100 mg/L)LB培養(yǎng)基(3 mL)，置于37 ℃恒溫?fù)u床培養(yǎng)過夜。質(zhì)粒小提，PCR鑒定正確后，將陽性菌液送上海桑尼生物技術(shù)有限公司測序。

1.2.3慢病毒包裝將構(gòu)建好的慢病毒載體與輔助質(zhì)粒進(jìn)行大量抽提，當(dāng)濃度大于1 μg/μL、A260/A280在1.7~1.8方可用以包裝。將處于對數(shù)生長期的293T細(xì)胞接種于直徑10 cm的培養(yǎng)皿中，細(xì)胞融合達(dá)80%~90%時進(jìn)行轉(zhuǎn)染。慢病毒包裝系統(tǒng)3質(zhì)粒(pSPAX2、pMD2G、pHBLVTM系列載體各10 μg)共轉(zhuǎn)染293T細(xì)胞，轉(zhuǎn)染后6 h換液。分別于轉(zhuǎn)染后48、72 h分兩次收集病毒上清(其間置換新鮮培養(yǎng)液)，收集后濾器過濾，4 ℃下72 000 r/min離心120 min。新鮮培養(yǎng)液500 μL重懸濃縮病毒沉淀，置于-80 ℃冰箱或液氮保存。

1.2.4濃縮病毒感染T24細(xì)胞將細(xì)胞分為1、2、3組，鋪盤于6孔板，每孔約5×105個，1組加入pHBLV-CADM1-IRES-ZsGreen-PGK-puro，2組加入pHBLV-IRES-ZsGreen-PGK-puro，3組不做任何處理，作為空白對照，24 h后換液。48 h后，將培養(yǎng)液完全更換為加有嘌呤霉素(2 μg/mL)的培養(yǎng)液，2 d更換1次培養(yǎng)液，待細(xì)胞生長穩(wěn)定后，即可進(jìn)行細(xì)胞傳代，2代之后無需加嘌呤霉素培養(yǎng)，建系完成。感染3~4 d后熒光顯微鏡下觀察綠色熒光蛋白(GFP)表達(dá)情況。

1.3沉默CADM1基因的T24細(xì)胞的構(gòu)建設(shè)計針對CADM1的三條siRNA序列，siRNA1序列為5′-CAGATGACTTATCCTCTACAA-3′；siRNA2序列為5′-CCAGACATAAAGGTACATACT-3′；siRNA3序列為5′-AGACGCAGACACAGCTATAAT-3′。將T24細(xì)胞分為A、B、C、D、E組，其中A、B、C組分別轉(zhuǎn)染搭載siRNA1、2、3的慢病毒載體(pHBLV-U6-ZsGreen-Puro)；D組僅轉(zhuǎn)染pHBLV-U6-ZsGreen-Puro，作為空載體對照；E組作為空白對照。詳細(xì)操作步驟參考“1.2”。

1.4CADM1 mRNA及蛋白檢測

1.4.1CADM1 mRNA檢測采用real-time PCR法。CADM1 mRNA上游引物序列為5′-ATGGCGAGTGTAGTGCTGC-3′，下游引物序列為5′-GATCACTGTCACGTCTTTCG-3′；內(nèi)參GAPDH mRNA上游引物序列為5′-TGTGGGCATCAATGGATTTGG-3′，下游引物序列為5′-ACACCATGTATTCCGGGTCAAT-3′。 PCR反應(yīng)結(jié)束后進(jìn)行分析，以2-ΔΔCt代表CADM1 mRNA相對表達(dá)量。

1.4.2CADM1 蛋白檢測采用Western blotting法。按照組織蛋白抽提試劑盒說明書提取蛋白，繪制標(biāo)準(zhǔn)曲線并檢測樣本蛋白濃度；制備SDS-PAGE凝膠，取20 μg蛋白經(jīng)沸水煮5 min后上樣，設(shè)置電壓80 V，電泳1.5 h；將電泳分離的樣品從凝膠轉(zhuǎn)移到固相載體PVDF膜上，5%脫脂奶粉封閉1 h，相應(yīng)一抗4 ℃孵育過夜、二抗孵育2 h，以GAPDH為內(nèi)參；ECL化學(xué)發(fā)光試劑加于PVDF膜上反應(yīng)5 min，凝膠成像分析系統(tǒng)下進(jìn)行顯影、定影。采用Image Pro Plus5.0軟件分析目的蛋白相對表達(dá)量。

2結(jié)果

2.1過表達(dá)CADM1基因細(xì)胞株構(gòu)建情況PCR擴(kuò)增產(chǎn)物與重組質(zhì)粒pHBLV-CADM1-IRES-ZsGreen-PGK-puro轉(zhuǎn)化DH5α感受態(tài)細(xì)胞后，挑取單克隆菌株進(jìn)行PCR鑒定，經(jīng)雙酶切后得到片段大小約為1.3 kb，說明目的基因CADM1成功插入到載體pHBLV-IRES-ZsGreen-PGK-puro中。重組質(zhì)粒pHBLV-CADM1-IRES-ZsGreen-PGK-puro測序結(jié)果與Genbank上公布的序列同源性為100%。病毒濃縮感染T24細(xì)胞72 h后，GFP在T24中表達(dá)，說明細(xì)胞株構(gòu)建成功。1、2、3組CADM1 mRNA相對表達(dá)量分別為4.84±0.02、1.12±0.03、1.00±0.00，CADM1蛋白相對表達(dá)量分別為4.53±0.03、1.05±0.04、1.00±0.01，1組CADM1 mRNA及蛋白相對表達(dá)量高于2、3組(P均<0.05)。

2.2沉默CADM1基因細(xì)胞株構(gòu)建情況重組質(zhì)粒pHBLV-U6-shRNA1/2/3-ZsGreen-Puro轉(zhuǎn)化DH5α感受態(tài)細(xì)胞后，挑取單克隆菌株進(jìn)行測序，測序結(jié)果與設(shè)計序列完全一致，說明siRNA1/2/3成功插入載體pHBLV-U6-shRNA-ZsGreen-Puro中。病毒濃縮感染T24細(xì)胞72 h后，GFP在細(xì)胞中表達(dá)，說明細(xì)胞株構(gòu)建成功。A、B、C、D、E組CADM1 mRNA相對表達(dá)量分別為0.28±0.05、0.98±0.02、0.74±0.01、0.99±0.01、1.00±0.00，CADM1蛋白相對表達(dá)量分別為0.33±0.04、0.86±0.12、0.67±0.07、1.00±0.04、1.00±0.03，A組CADM1 mRNA及蛋白相對表達(dá)量低于B、C、D、E組(P均<0.05)。

3討論

CADM1屬于細(xì)胞黏附分子中免疫球蛋白超家族成員，是參與細(xì)胞間相互作用的一種膜蛋白[3]，最早是Murakami等[13]在NSCLC患者分析染色體11q23.2區(qū)域時發(fā)現(xiàn)。CADM1可參與細(xì)胞間黏附、細(xì)胞運(yùn)動、信號轉(zhuǎn)導(dǎo)、介導(dǎo)免疫監(jiān)視、抑制上皮細(xì)胞向間質(zhì)細(xì)胞轉(zhuǎn)變、調(diào)節(jié)細(xì)胞周期進(jìn)展、誘導(dǎo)細(xì)胞凋亡等過程。

大量研究[10~15]顯示，CADM1在腫瘤發(fā)生發(fā)展過程中扮演重要角色。Mao等[14]構(gòu)建了TSLC1重組腺病毒載體(Ad-TSLC1)并轉(zhuǎn)染至小細(xì)胞肺癌A549細(xì)胞株，發(fā)現(xiàn)Ad-TSLC1可明顯抑制A549細(xì)胞增殖并促使其凋亡；將轉(zhuǎn)染Ad-TSLC1的A549細(xì)胞株移植到裸鼠皮下，發(fā)現(xiàn)TSLC1過表達(dá)后可使腫瘤體積縮小70%~80%，且TSLC1過表達(dá)后還可激活Caspase-3活性，促使腫瘤細(xì)胞凋亡。Lu等[10]研究發(fā)現(xiàn)，喉癌組織中TSLC1 mRNA和蛋白表達(dá)明顯降低，并與腫瘤TNM分期和淋巴結(jié)轉(zhuǎn)移有關(guān)；TSLC1過表達(dá)后可抑制腫瘤細(xì)胞增殖并減弱其侵襲能力。我們前期研究也發(fā)現(xiàn)，膀胱尿路上皮癌組織中CADM1蛋白表達(dá)降低[12]。為進(jìn)一步研究CADM1在膀胱癌中的作用及機(jī)制，我們分別構(gòu)建了穩(wěn)定過表達(dá)和沉默CADM1基因的膀胱癌細(xì)胞株T24。

本研究使用的過表達(dá)慢病毒包裝系統(tǒng)含有GFP和嘌呤霉素抗性兩個標(biāo)記基因。GFP有特異性高、易識別定位、不需要底物和輔助因子、不影響細(xì)胞功能等優(yōu)點(diǎn)，目前已作為報告基因[16]和示蹤標(biāo)記[17]在多領(lǐng)域得到應(yīng)用。我們將慢病毒載體pHBLV-IRES-ZsGreen-PGK-puro(Ad-GFP1)及CADM1過表達(dá)慢病毒(Ad-CADM1)分別感染T24細(xì)胞，發(fā)現(xiàn)GFP在T24細(xì)胞中穩(wěn)定表達(dá)，且1組CADM1 mRNA及蛋白相對表達(dá)量高于2、3組，說明過表達(dá)CADM1基因的慢病毒載體成功感染T24細(xì)胞，且CADM1得到穩(wěn)定表達(dá)，成功獲得Ad-CADM1-T24和Ad-GFP1-T24細(xì)胞株。

本研究中使用的沉默慢病毒包裝系統(tǒng)同樣為3質(zhì)粒系統(tǒng)，組成為pSPAX2、pMD2G和pHBLV-U6-ZsGreen-Puro。將pHBLV-U6-ZsGreen-Puro(Ad-GFP2)及對應(yīng)沉默載體分別感染T24細(xì)胞，結(jié)果GFP在T24細(xì)胞中穩(wěn)定表達(dá)，A組CADM1 mRNA和蛋白表達(dá)量較B、C、D、E組明顯下調(diào)，成功獲得Ad-si1-T24和Ad-GFP2-T24細(xì)胞株。

穩(wěn)定過表達(dá)和沉默CADM1基因的膀胱癌細(xì)胞株的成功建立，為研究CADM1對膀胱癌侵襲、轉(zhuǎn)移的影響提供了良好基礎(chǔ)。CADM1在膀胱癌生長、侵襲、轉(zhuǎn)移過程中的作用及機(jī)制尚需進(jìn)一步研究證實。

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Establishment of bladder cancer cell line overexpressing and silencing cell adhesion molecule 1

LIULi1,YANGYongjiao,CHENYegang,WANGShangren,LIUXiaoqiang,CHENShaofeng,SUNGuang

(1TheSecondHospitalofTianjinMedicalUniversity,Tianjin300211,China)

Abstract:Objective To establish the bladder cancer cell subline stably expressing and silencing cell adhesion molecule 1 (CADM1). MethodsWe cultured and collected the bladder cancer cell line T24. ① Establishment of T24 overexpressing CADM1. T24 cells were divided into the groups 1, 2 and 3. The full length of CADM1 was amplified by real-time PCR and was connected with pHBLV-IRES -ZsGreen-PGK-puro lentiviral vector (group 1), a vector control group (group 2) and a blank group (group 3). Virus was packaged in 293T cells after enzyme digestion and the sequencing was correct, and viruses after being concentrated were used to infect T24 cells. ②Establishment of T24 stably silencing CADM1. T24 cells were divided into the groups A, B, C, D and E. Three sequences (siRNA1/2/3) interfering CADM1 gene were designed and were connected with pHBLV-U6-shRNA-ZsGreen-Puro lentiviral vector, respectively (groups A, B and C), pHBLV-U6-ZsGreen-Puro (group D) and a blank group (group E). The connected vector was identified using double-enzyme digestion, virus was packaged in 293T cells, and virus of enrichment was used to infect T24 cells. CADM1 mRNA and protein were assessed by real-time PCR and Western blotting. ResultsThe recombinant lentiviral vectors and cell lines of expressing and silencing of CADM1 were correctly established. The relative expression levels of CADM1 mRNA in the groups 1, 2, and 3 were 4.84±0.02, 1.12±0.03, 1.00±0.00, and the relative expression levels of CADM1 protein in the groups 1, 2, and 3 were 4.53±0.03, 1.05±0.04 and 1.00±0.01, respectively. CADM1 mRNA and protein in the group 1 was enhanced effectively as compared with that of the groups 2 and 3 (all P<0.05). The relative expression levels of CADM1 mRNA in the groups A, B, C, D and E were 0.28±0.05, 0.98±0.02, 0.74±0.01, 0.99±0.01, 1.00±0.00, and the relative expression levels of CADM1 protein in the groups A, B, C, D and E were 0.33±0.04, 0.86±0.12, 0.67±0.07, 1.00±0.04 and 1.00±0.03, respectively. CADM1 mRNA and protein in the group A was decreased as compared with that of groups B, C, D and E (all P<0.05). ConclusionThe stable bladder cancer cell lines T24 overexpressing or silencing CADM1 were successfully established.

Key words:urinary bladder neoplasms; urinary bladder carcinoma cell line; cell adhesion molecule 1; gene silencing

(收稿日期：2015-11-04)

中圖分類號：R737.14

文獻(xiàn)標(biāo)志碼：A

文章編號：1002-266X(2016)07-0020-04

doi：10.3969/j.issn.1002-266X.2016.07.007

通信作者簡介：劉曉強(qiáng)(1971-)，男，教授，博士生導(dǎo)師，主任醫(yī)師，主要研究方向為泌尿系腫瘤學(xué)和男科學(xué)。E-mail： xiaoqiangliu1@163.com

作者簡介：第一劉莉(1981-)，女，主管技師，主要研究方向為泌尿系腫瘤學(xué)和男科學(xué)。E-mail： 18622208373@163.com

基金項目：天津市自然科學(xué)基金重點(diǎn)項目(12JCZDJC23700)。