CAJ | 학술논문

目的：构建共同表达人miR-200c基因和荧光素酶基因的慢病毒（Lentivious）表达载体质粒，实现该重组体在人结肠癌多药耐药细胞中的稳定整合以及动物模型的建立，为进一步研究miR-200c调控大肠癌多药耐药提供实验基础。方法将miR-200c基因克隆至慢病毒pGC-Lentivirus表达载体中，测序鉴定后，包装重组慢病毒表达质粒，并进行滴度测试。利用细胞转染技术，将携带miR-200c过表达的慢病毒载体质粒转染到人结肠癌多药耐药细胞株HCT-116／L-OHP中，并接种于裸鼠皮下，以空病毒载体作为对照。待肿瘤生长2周后，随机分为4组，分别给予生理盐水和奥沙利铂（ L-OHP），4周后处死裸鼠，剥取肿瘤，测量肿瘤体积。结果病毒滴度测试最佳滴度为2×108 TU／mL。 qPCR结果显示，转染miR-200c-Lentivirus病毒质粒的人结肠癌耐药细胞中miR-200c表达高于转染空载体组以及空白对照组，P均＜0．01。活体成像观察证实，带有荧光素酶的HCT-116／L-OHP-miR-200c-luc细胞在动物体内可被快速而灵敏的检测到，可以准确地观察肿瘤的生长情况。与HCT-116／L-OHP-luc组相比，HCT-116／L-OHP-miR-200c-luc组对L-OHP的敏感性更强，P＜0．05。结论成功构建携带人miR-200c基因慢病毒载体质粒，实现重组体在人结肠癌HCT-116／L-OHP多药耐药细胞中的稳定整合，并建立了带有荧光素酶miR-200c过表达的人结肠癌多药耐药动物模型。
목적：구건공동표체인miR-200c기인화형광소매기인적만병독（Lentivious）표체재체질립，실현해중조체재인결장암다약내약세포중적은정정합이급동물모형적건립，위진일보연구miR-200c조공대장암다약내약제공실험기출。방법장miR-200c기인극륭지만병독pGC-Lentivirus표체재체중，측서감정후，포장중조만병독표체질립，병진행적도측시。이용세포전염기술，장휴대miR-200c과표체적만병독재체질립전염도인결장암다약내약세포주HCT-116／L-OHP중，병접충우라서피하，이공병독재체작위대조。대종류생장2주후，수궤분위4조，분별급여생리염수화오사리박（ L-OHP），4주후처사라서，박취종류，측량종류체적。결과병독적도측시최가적도위2×108 TU／mL。 qPCR결과현시，전염miR-200c-Lentivirus병독질립적인결장암내약세포중miR-200c표체고우전염공재체조이급공백대조조，P균＜0．01。활체성상관찰증실，대유형광소매적HCT-116／L-OHP-miR-200c-luc세포재동물체내가피쾌속이령민적검측도，가이준학지관찰종류적생장정황。여HCT-116／L-OHP-luc조상비，HCT-116／L-OHP-miR-200c-luc조대L-OHP적민감성경강，P＜0．05。결론성공구건휴대인miR-200c기인만병독재체질립，실현중조체재인결장암HCT-116／L-OHP다약내약세포중적은정정합，병건립료대유형광소매miR-200c과표체적인결장암다약내약동물모형。
Objective To construct the lentiviral vector co-expressing human miR-200c gene and luciferase , and es-tablish a stable colorectal cancer multidrug resistance ( MDR) cell model , in order to lay an experiment foundation for ex-ploring the function of miR-200c in MDR.Methods miR-200c gene was cloned into the lentiviral vector (pGC-Lentivir-us), which was verified by PCR and sequencing analysis .After sequencing, we made lentiviral vector and titer test .Using cell transfection technique , miR-200c lentiviral vector was infected into MDR cell line HCT-116/L-OHP.A nude mouse model of colorectal MDR cancer was established by subcutaneous inoculation of this kind of cell ; meanwhile , the empty vector was taken as the control group .Experimental nude mice were randomly divided into 4 groups after 2-week tumor-growth.Mice in the control group were administered with normal saline daily .Mice in the experiment group were adminis-tered with oxaliplatin (L-OHP).After treatment for 4 weeks, mice in each group were sacrificed to measure tumor volume . Results The best titer was 2 ×108 TU/mL.The qPCR showed that the levels of miR-200c mRNA expression in the resist-ant cells of human colon cancer which were transfected by miR-200c-Lentivirus were significantly higher than those in the empty vector group and the blank control group ( all P<0.01).In vivo imaging confirmed that HCT-116/L-OHP-miR-200c-luc cells with luciferase could be tested quickly and sensitively , and could be used to watch the tumor growth .Com-pared with HCT-116/L-OHP-luc group, HCT-116/L-OHP-miR-200c-luc group showed more sensitive to L-OHP ( P <0.05).Conclusion The lentiviral screening vector with human miR-200c gene is successfully constructed , which realizes the stable integration of the recombinant in the human colon cancer HCT-116/L-OHP MDR cells;meanwhile, its MDR co-lon cancer animal models which have miR-200c overexpression with luciferase are successfully constructed .