中国医学前沿杂志（电子版）
中國醫學前沿雜誌（電子版）
중국의학전연잡지（전자판）
CHINESE JOURNAL OF THE FRONTIERS OF MEDICAL SCIENCE(ELECTRONIC VERSION)
2014年
9期
23-26
,共4页
食管鳞癌%Eca-109细胞%抑制效应%HIF-1α%RNA干扰%体外及体内表达
食管鱗癌%Eca-109細胞%抑製效應%HIF-1α%RNA榦擾%體外及體內錶達
식관린암%Eca-109세포%억제효응%HIF-1α%RNA간우%체외급체내표체
Esophageal squamous cell carcinoma%Eca-109 cells%Inhibition effect%HIF-1α%RNA interference%Expression in vitro and vivo
目的：探讨分析RNA干扰缺氧诱导因子-1α（HIF-1α）对食管鳞癌细胞Eca-109抑制效应的体外及体内的表达情况。方法选取研究用Eca-109细胞，设计RNA干扰片断h-EGFR构建重组质粒pGensil-1-EGFR转染目的基因片段。测定EGFR蛋白/mRNA表达，挑选转染成功的基因修饰细胞株分别植入裸鼠体内，构建移植瘤模型，且随机分为未转染组与实验组。运用免疫组化技术检测各组裸鼠HIF-1α蛋白表达，逆转录聚合酶链反应（RT-PCR）技术检测各组HIF-1αmRNA表达，最后测量裸鼠移植瘤的重量及体积。结果 Eca-109中HIF-1α蛋白伴随氯化钴浓度的升高而升高。并且HIF-1α蛋白水平在缺氧后12小时升高，24小时后降低，且持续减少。转染后EGFR mRNA的阳性表达率为26.55%、9.48%、4.60%，依次降低，P＜0.01。其中EGFR siRNA水平最低。转染后EGFR mRNA阳性表达率为24.30%、34.11%、34.05%，与未转染组（78.27%）相比显著降低（P＜0.01）。经RT-PCR分析，实验组裸鼠EIF-1αmRNA表达较少，与未转染组相比差异无显著性（P＞0.05）。实验组裸鼠HIF-1α蛋白表达的灰度值显著低于未转染组（P＜0.01）。接种1周后裸鼠均出现肿瘤，实验组裸鼠移植瘤体积为（0.20±0.13）cm3，重量为（0.21±0.05）g，均显著少于未转染组。结论 HIF-1α蛋白的表达与氯化钴浓度及缺氧有关，RNA干扰缺氧诱导的HIF-1α只发生在蛋白水平，而非基因水平， RNA干扰技术可以成功抑制食管鳞癌细胞HIF-1α基因，阻碍肿瘤细胞生长、增殖，降低肿瘤恶性程度，值得临床应用与推广。
目的：探討分析RNA榦擾缺氧誘導因子-1α（HIF-1α）對食管鱗癌細胞Eca-109抑製效應的體外及體內的錶達情況。方法選取研究用Eca-109細胞，設計RNA榦擾片斷h-EGFR構建重組質粒pGensil-1-EGFR轉染目的基因片段。測定EGFR蛋白/mRNA錶達，挑選轉染成功的基因脩飾細胞株分彆植入裸鼠體內，構建移植瘤模型，且隨機分為未轉染組與實驗組。運用免疫組化技術檢測各組裸鼠HIF-1α蛋白錶達，逆轉錄聚閤酶鏈反應（RT-PCR）技術檢測各組HIF-1αmRNA錶達，最後測量裸鼠移植瘤的重量及體積。結果 Eca-109中HIF-1α蛋白伴隨氯化鈷濃度的升高而升高。併且HIF-1α蛋白水平在缺氧後12小時升高，24小時後降低，且持續減少。轉染後EGFR mRNA的暘性錶達率為26.55%、9.48%、4.60%，依次降低，P＜0.01。其中EGFR siRNA水平最低。轉染後EGFR mRNA暘性錶達率為24.30%、34.11%、34.05%，與未轉染組（78.27%）相比顯著降低（P＜0.01）。經RT-PCR分析，實驗組裸鼠EIF-1αmRNA錶達較少，與未轉染組相比差異無顯著性（P＞0.05）。實驗組裸鼠HIF-1α蛋白錶達的灰度值顯著低于未轉染組（P＜0.01）。接種1週後裸鼠均齣現腫瘤，實驗組裸鼠移植瘤體積為（0.20±0.13）cm3，重量為（0.21±0.05）g，均顯著少于未轉染組。結論 HIF-1α蛋白的錶達與氯化鈷濃度及缺氧有關，RNA榦擾缺氧誘導的HIF-1α隻髮生在蛋白水平，而非基因水平， RNA榦擾技術可以成功抑製食管鱗癌細胞HIF-1α基因，阻礙腫瘤細胞生長、增殖，降低腫瘤噁性程度，值得臨床應用與推廣。
목적：탐토분석RNA간우결양유도인자-1α（HIF-1α）대식관린암세포Eca-109억제효응적체외급체내적표체정황。방법선취연구용Eca-109세포，설계RNA간우편단h-EGFR구건중조질립pGensil-1-EGFR전염목적기인편단。측정EGFR단백/mRNA표체，도선전염성공적기인수식세포주분별식입라서체내，구건이식류모형，차수궤분위미전염조여실험조。운용면역조화기술검측각조라서HIF-1α단백표체，역전록취합매련반응（RT-PCR）기술검측각조HIF-1αmRNA표체，최후측량라서이식류적중량급체적。결과 Eca-109중HIF-1α단백반수록화고농도적승고이승고。병차HIF-1α단백수평재결양후12소시승고，24소시후강저，차지속감소。전염후EGFR mRNA적양성표체솔위26.55%、9.48%、4.60%，의차강저，P＜0.01。기중EGFR siRNA수평최저。전염후EGFR mRNA양성표체솔위24.30%、34.11%、34.05%，여미전염조（78.27%）상비현저강저（P＜0.01）。경RT-PCR분석，실험조라서EIF-1αmRNA표체교소，여미전염조상비차이무현저성（P＞0.05）。실험조라서HIF-1α단백표체적회도치현저저우미전염조（P＜0.01）。접충1주후라서균출현종류，실험조라서이식류체적위（0.20±0.13）cm3，중량위（0.21±0.05）g，균현저소우미전염조。결론 HIF-1α단백적표체여록화고농도급결양유관，RNA간우결양유도적HIF-1α지발생재단백수평，이비기인수평， RNA간우기술가이성공억제식관린암세포HIF-1α기인，조애종류세포생장、증식，강저종류악성정도，치득림상응용여추엄。
Objective To study the interference of RNA hypoxia inducible factor-1α(HIF-1α) expression inhibition in vitro and in vivo effect of esophageal squamous cell carcinoma cell line Eca-109. Method Selected in esophageal squamous cancer cells (ESCC) to study the Eca-109, the design of RNA interference fragment h-EGFR to construct recombinant plasmid pGensil-1-EGFR gene transfection purpose. Determine EGFR protein/mRNA expression, pick out the transfection successful nude mice implanted with genetically modiifed cell lines respectively, transplantation tumor model was constructed, and randomly divided into untransfection group and experimental group. Using immunohistochemical techniques to detect alpha groups of HIF-1αprotein expression, reverse transcription polymerase chain reaction (RT-PCR) technology to detect each group HIF-1αmRNA expression, ifnally to measure the weight and volume of the nude mouse transplantation tumor. Result HIF-1αEca-109 cells protein increased along with the increase of the concentration of cobalt chloride. HIF-1αin 12 hours after hypoxia increases, reduced after 24 hours, and had been declining. After transfection EGFR mRNA positive expression rate was 26.55%, 9.48%, 4.60%, in turn (P < 0.01). With EGFR siRNA (small interference RNA) had the lowest levels. After transfection EGFR mRNA positive expression rate was 24.30%, 34.11%, 34.05%, compared with the blank control group (78.27%) (P<0.01). Through RT-PCR analysis, experimental mice HIF-1αmRNA expression was less, there was no signiifcant difference compared with not transfection group (P>0.05). Experimented group HIF-1αprotein expression of grey value signiifcantly lower than untransfection group (P<0.01). Vaccination in 1 week after nude mice tumor, experimental mice transplanted tumors for (0.20±0.13) cm3, weight (0.21±0.05) g, compared with untransfection group, experimental group mice transplanted tumor volume and weight were signiifcantly less than not transfection group. Conclusion HIF-1αprotein expression is associated with cobalt chloride concentration and lack of oxygen, oxygen to RNA interference of HIF-1αonly in protein level, rather than the genetic level, thus a technique called RNA interference can successfully suppress ESCC HIF-1αgene, hinder the growth of tumor cells, value-added, reduce tumor malignant degree, worthy of clinical application and promotion.
