CAJ | 학술논문

目的构建携带p53基因新型增殖性腺病毒CNHK600-p53,研究其对肝癌细胞株抑制效应是否优于Ad-p53.方法 PCR扩增p53基因,利用酶切连接方法将其插入CNHK600载体,PCR鉴定.经293细胞包装成病毒,抽提病毒DNA,PCR鉴定.氯化铯密度梯度离心法纯化病毒,TCID50 方法测病毒滴度.病毒增殖实验检测病毒在不同细胞增殖能力.四甲基偶氮唑盐(methyl-thiazolyl tetrazolium assay,MTT)法观察CNHK600-p53、Ad-p53两种病毒分别对肝癌细胞株的抑制率.结果成功构建新型增殖性腺病毒载体CNHK600-p53;293细胞包装成病毒,PCR方法鉴定无野生型病毒存在;病毒滴度为1.99×10~（10）pfu/ml;CNHK600-p53在肝癌细胞HepG2、SMMC-7721内复制能力明显高于正常肝细胞HEL-1和L02.对6种肝癌细胞(PLC/PRF5、SMMC7721、HepaG2、BEL-7402、BEL-7404、QGY-7703)而言,随着MOI值的不断增高.其对肝癌细胞的抑制作用也不断增强;在相同MOI情况下,CNHK600-p53组较Ad-p53组的细胞抑制率高(P＜0.05).当细胞抑制率达到80%以上时.两组之间差异无统计学意义(P＞0.05).结论利用新型增殖性腺病毒CNHK600一p53较 Ad-p53能更有效的抑制肝癌细胞,可能成为肝癌的基因治疗更有效的一种基因治疗手段.
목적 구건휴대p53기인신형증식성선병독CNHK600-p53,연구기대간암세포주억제효응시부우우Ad-p53.방법 PCR확증p53기인,이용매절련접방법 장기삽입CNHK600재체,PCR감정.경293세포포장성병독,추제병독DNA,PCR감정.록화색밀도제도리심법순화병독,TCID50 방법 측병독적도.병독증식실험검측병독재불동세포증식능력.사갑기우담서염(methyl-thiazolyl tetrazolium assay,MTT)법관찰CNHK600-p53、Ad-p53량충병독분별대간암세포주적억제솔.결과 성공구건신형증식성선병독재체CNHK600-p53;293세포포장성병독,PCR방법 감정무야생형병독존재;병독적도위1.99×10~（10）pfu/ml;CNHK600-p53재간암세포HepG2、SMMC-7721내복제능력명현고우정상간세포HEL-1화L02.대6충간암세포(PLC/PRF5、SMMC7721、HepaG2、BEL-7402、BEL-7404、QGY-7703)이언,수착MOI치적불단증고.기대간암세포적억제작용야불단증강;재상동MOI정황하,CNHK600-p53조교Ad-p53조적세포억제솔고(P＜0.05).당세포억제솔체도80%이상시.량조지간차이무통계학의의(P＞0.05).결론 이용신형증식성선병독CNHK600일p53교 Ad-p53능경유효적억제간암세포,가능성위간암적기인치료경유효적일충기인치료수단.
Objective To construct a new replicating adenovirus vector CNHK600-p53 which carried the anti-tumor gene p53 and determine whether its inhibitory effect on hepatoceltular carcinoma cell lines is better than Ad-p53 or not.Methods The p53 gene was amplified by PCR and inserted into plasmid CNHK600 genome to obtain the new replicating adenovirus vector CNHK600-p53 by enzymatic digestion and ligation methods.After packaging in 293 cells,the new replicating adenoviruses CNHK600-p53 DNA was extracted and identified by PCR.Viruses were purified by CsCl gradient purification and Viruses titer was estimated from TCID50.MTT method was used to observe the inhibition rates of hepatocellular carcinoma cells by CNHK600-p53 and Ad-p53 to.Results The new replicating adenovirus vector CNHK600-p53 was sucessfully constructed.There was no wild type virus after packaging in 293 cells and viruses titer was 1.99 × 10~(10) pfu/ml.The replication ability of CNHK600-p53 was better in hepatocellular carcinoma cell lines HepG2 and SMMC-7721 cells than in normal cell lines HEL-1and L02.To six hepatocellular carcinoma cell lines(PLC/PRF5,SMMC7721,HepaG2,BEL-7402,BEL-7404 and QGY-7703),MOI was higher and the inhibitory effect was stronger.To every cell line,the inhibitory rate of CNHK600-p53 was higher than that of Ad-p53 at the same MOI(P＜0.05).However,when inhibitory rate was larger than 80%,there are no significant difference between two groups(P＞0.05).Conclusion CNHK600-p53 is more effective to inhibit hepatocellular carcinoma cells than Ad-p53 and might serve as a new tool for hepatocellular carcinoma gene therapy.