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1、锘?html浜哄壀鍒囦慨澶嶅熀鍥燲 PD瀵硅倽鐧岀粏鑳炵敓闀跨殑鎶戝埗浣滅敤浣滆 ?咃細榛勭瀹夛紝寰愭睙鏅讹紝寮犲悏缈旓紝鐔婄憶锛屽肮涓滐紝鏉庡啗浣滆 ?呭崟浣嶏細 1.330006 鍗楁槍澶 绗 簩闄勫睘鍖婚櫌娑堝寲鍐呯 锛?. 姹熻鐪佸垎瀛愬尰瀛噸鐐瑰疄楠屽銆愭憳瑕併 ?/strong 鐩 殑鎺 閲庣敓鍨嬩汉鍓 垏淇 鍩哄洜鐫 ?鑹叉 ? 共鐨 梾鍩哄洜 D(xeroderma pigmentosum D,XPD) 瀵硅倽鐧岀粏 鑳濻 MMC顎?721 澧炴畺鐨勫奖鍝嶏紝骞舵帰璁叾鏈哄埗銆傛柟娉曠敤鑴傝川 浣撹浆鏌撴硶鐬 椂杞 煋 SMMC顎?721 缁嗚優锛岃浆鏌撻噸缁勮川绮扻 PD顎
2、慛 2 鍜岀杞借川绮扤 2 锛屽苟鐢湭杞 煋鐨勪笌 XPD顎慛 2 銆丯 2 鍏锋湁 鐩稿悓閬椾紶鑳屾櫙鍜屼唬鏁扮殑 SMMC顎?721 缁嗚優浣滀负绌虹櫧瀵圭収銆 傝崸鍏夋樉寰 暅瑙傚療缁胯壊鑽 厜铔嬬櫧鎶憡鍩哄洜琛揪鎯呭喌锛 屾祦寮忕粏鑳炰华妫 ?娴嬬粏鑳炲懆鏈燂紝閫嗚浆褰曨 ?戣仛鍚堥叾閾惧弽搴 旓紙 RT顎慞 CR锛夈?乄 estern blot 娉曟 娴嬬粏鑳炰腑 XPD銆乧顎憁 yc 銆乧 dc25A 銆乧 dK2琛揪閲忓彉鍖栵紝 MTT娉曡 瀵熺粏鑳炲 娈栫殑娲诲 姏銆傜粨鏋滄彁鍙栧嚭鐨勯噸缁勮川绮抪 EGFP顎慛 2 顎慩 PD鐢叾鍒囬壌瀹 氾紝涓嶨 enebank 涓婄殑鐩哥
3、 銆傚湪鑽 厜鏄惧井闀滀笅锛屽彲浠湪 SMMC顎?721顎憄 EGFP顎慛 2銆丼 MMC顎?721顎憄 EGFP顎慛 2顎慩 PD缁嗚優 涓 瀵熷埌缁胯壊鑽 厜铔嬬櫧鐨勮杈撅紝璐矑鐨勮浆鏌撴晥鐜囦负 30%宸彸銆傛祦寮忕粏鑳炰华缁撴灉鏄剧锛宲 EGFP顎慛 2顎慩 PD閲嶇粍 璐矑杞 煋鍏粏鑳炲悗锛岃倽鐧岀粏鑳炶繘鍏 鏈熷彂鐢熼樆婊烇紝鍋滄 粸鍦1鏈熴?俁 T顎慞 CR銆乄 estern blot 妫?娴嬪彂鐜癝 MMC顎?721顎 憄 EGFP顎慛 2 顎慩 PD缁嗚優涓嶴 MMC顎 ?721 顎憄 EGFP顎慛 2 鍜孲 MMC 顎?721涓 鐓 粍鐩告瘮锛屽叾 XPD 琛揪鏄 ?br
4、/鏄惧 楂橈紙 P0.05锛夈?俢顎憁 yc 銆乧 dc25A銆乧 dK2鐩稿 琛揪閲忔 槑鏄惧噺灏戯紝宸 紓鏈夌粺璁 鎰忎箟锛圥 0.05 锛夈 ?備笌涓 鐓 粍鐩告瘮锛孲 MMC顎?721顎憄 EGFP顎慛 2顎慩 PD缁嗚優澧炴畺鐜囨槑鏄惧噺 寮憋紙 P 銆愬叧閿 瘝銆?/strong鑲濈檶锛 ?XPD锛沜顎憁 yc 锛沜 dc25A锛沜 dK2锛汥 NA鎹熶激妫 ?鎺 偣Xeroderma Pigmentosum D Gene InhibitsProliferation of HumanHepatocellular Carcinoma Cell Line SMMC顎 ?721HUANG
5、 Shen顎慳 n1, XU Jiang 顎慾 ing2, ZHANG Ji 顎憍 iang1,2, XIONG Ying1, YIN Dong2, LI Jun11 锛嶥 epartment of Gastroenterology, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, China锛?锛嶫 iangxi Provincial Key Laboratory of Molecular MedicineCorresponding Author:ZHANG Ji 顎憍 iang
6、,E 顎憁 ail: Abstract 锛歄 bjective The Xeroderma Pigmentosum D (XPD) gene plays an important role in the path to DNA repair. It has been reported that cells obtained high tumorsusceptibility whenmutation happened in XPD gene. The purpose of this study is to investigate the influence of XPD gene in the
7、growth and proliferation of hepatoma carcinoma cells (HCC).MethodspEGFP顎慛 2and pEGFP顎慛 2 顎慩 PD were transfected into SMMC 顎?721 cell lines by Lipofectamine2000 method. The expression of green fluorescent protein (GFP) was observed through fluorescence microscope. Cell cycle of the plasmid 顎憈 ransfec
8、ted SMMC 顎?721 cells was analyzed by Flow cytometry (FCM). The expressions of wild顎憈 ype XPD, c 顎憁 yc,cdc25A and cdK2 were detected by RT 顎慞 CR and Western blot. Cell proliferation was measured by MTT assay.ResultsThe recombinant plasmid pEGFP 顎慛 2 顎慩 PD was successfully transfected into SMMC 顎 ?721
9、 cells and green fluorescence in cells was observed by fluorescent microscopy. Around 30% transfection efficiency was obtained in this experiment. In addition, we have found that wild type XPD blocked the hepatoma cells from S stage to G1 stage The expression of XPD in SMMC 顎?721顎憄 EGFP顎慛 2顎慩 PD cel
10、ls was significantly higher than those in control group of SMMC顎?721 andSMMC顎?721顎憄 EGFP顎慛 2 cells (P0.05). The expressions of c顎憁yc, cdc25A and cdK2 in SMMC 顎?721顎憄 EGF顎慛 2顎慩 PD cells were significantly lower than those in control cells (all P0.05).Furthermore, SMMC 顎?721顎憄 EGFP顎慛 2顎慩 PD has reduce
11、d proliferation ability than empty plasmid顎憈 ransfected cells(P1.1 鏉愭枡1.1.1 缁嗚優鏍 拰璐矑鏉簮浜鸿倽鐧岀粏鑳濻 MMC顎?721 璐 嚜涓 浗鍏稿瀷鍩瑰吇鐗繚钘忎腑蹇 ?CCTCC銆） 傞噸缁勮川绮抪 EGFP顎慛 2 顎慩 PD鏈 疄楠屽 鏋勫缓銆 ?/p1.1.2 涓昏 璇曞墏鍜屼骇鍦癛 PMI顎?640璐 嚜缇庡浗 Gibco BRL 鍏 徃銆傝儙鐗涜 娓咃紙 FBS 锛夎喘鑷 崡闈濰 yclone 鍏 徃銆俆 rypsin 銆丮 onoclonal Antibody Against 顎慳 ctin 璐 嚜缇庡
12、浗 Sigma 鍏 徃 銆侺 ipofectamine2000TM 銆乀 rizolTM 璇曞墏璐 嚜缇庡浗 Invetrogen 鍏 徃銆俉 azard PureFection Plasmid DNA Pufiication System銆丮顎慚 LVReverse Transcriptase 銆丷 Nasin 銆乨 NTP銆丱 ligo（dT）15 璐 嚜缇庡浗 Promega鍏 徃銆侱 MSO銆丏 EPC璐 嚜缇庡浗 Amresco 鍏 徃銆侾 CR寮曠墿 璐 嚜涓婃捣鐢熺墿宸鎶 ?鏈 湇鍔湁闄愬叕鍙搞 ?俆 aq 閰惰喘鑷 棩鏈琓 aKaRa鍏 徃銆侱 NA 2000 Ladder M
13、arker 璐 嚜鍖椾含鍗庣編鐢熺墿 宸鍏 徃銆侻 TT璐 嚜涓婃捣鏅鐢熺墿鎶?鏈 湁闄愬叕鍙搞 ?傚崟鍏嬮殕鎶椾綋 XPD銆乧顎憁 yc 銆乧 dc25A 鍜宑 dK2 璐 嚜缇庡浗 Santa Cruz鍏 徃銆傚北缇婃姉榧營 gG顎戣荆鏍硅繃姘 寲鐗叾銆佸北缇婃姉鍏 擨 gG 顎戣荆鏍硅繃姘 寲鐗叾璐 嚜鍖椾含涓 潐閲戞鐢熺墿鎶 ?鏈 湁闄愬叕鍙搞 ?/p1.2 鏂规硶1.2.1 缁嗚優鍩瑰吇 SMMC顎?721缁嗚優鍦惈 15% FBS鐨凴 PMI 顎?640鍩瑰吇鍩轰腑锛 ?7 鈩冦?%CO2鐨勬潯浠朵笅瀛电 锛團 orma Scientific Inc,USA 锛変腑鍩瑰吇锛屼互 0
14、.25%鑳拌泲鐧介叾娑堝寲锛 ?锝?澶 紶浠竴娆 ?/p1.2.2 璐矑閴村畾鐢瑁傝 娉曟彁鍙栫杞借川绮抪 EGFP顎慛 2 鍜岄噸缁勮川绮抪 EGFP顎慛 2 顎慩 PD 锛屾彁鍙栧嚭鐨勮川绮掍互 KPN鈪犮 ?丅 GI 鈪拰 SPH鈪犻叾鍒囬壌瀹氥 ?/p1.2.3 璐矑杞 煋瀹為獙璁 ?缁勶紝姣忕粍 2 瀛旓紝鍒嗗埆涓烘棤杞 煋绌虹櫧瀵圭収缁勶紙 SMMC顎?721 锛夛紝绌鸿浇璐矑杞 煋缁嗚優缁勶 紙 SMMC顎?721顎憄 EGFP顎慛 2 锛変互鍙婇噸缁勮川绮掕浆鏌撶粏鑳炵粍 （SMMC7721顎憄 EGFP顎慛 2 顎慩 PD）銆備互 2105 涓 粏鑳 ?瀛旂殑瀵嗗害灏 哠MMC顎?721缁嗚優閾轰簬 6瀛旀澘鍐咃紝 24h缁嗚優閾?0%锛宲EGFP顎慛 2 銆乸 EGFP顎慛 2 顎慩 PD姣忓瓟 0.4 g锛岃剛璐綋姣忓瓟 10
