体外转录OVA mRNA抑制小鼠结肠癌和三阴型乳腺癌移植瘤的研究  

Inhibition of mouse colon cancer and triple negative breast cancer by in vitro-transcribed OVA mRNA

作　　者：张瑾宬 刘雨 冯娜 何梦梦 张洁 刘丽娜 ZHANG Jincheng;LIU Yu;FENG Na;HE Mengmeng;ZHANG Jie;LIU Li'na(School of Biology and Engineering&College of Modern Industry of Health Medicine,Guizhou Medical University,Guiyang 550025,Guizhou,China)

机构地区：[1]贵州医科大学生物与工程学院&健康医药现代产业学院,贵州贵阳550025

出　　处：《贵州医科大学学报》2025年第3期399-404,共6页Journal of Guizhou Medical University

基　　金：贵州省科技计划项目(黔科合支撑[2020]4Y233);贵州省大学生创新创业训练计划项目(S202210660079)。

摘　　要：目的比较瘤内注射自制体外转录鸡卵清蛋白(ovalbumin,OVA)mRNA和市售OVA mRNA对小鼠CT26结肠癌和4T1三阴型乳腺癌移植瘤细胞的免疫治疗效果。方法将合成的OVA基因片段插入真核表达载体pcDNA3.1(+)构建重组表达质粒pcDNA3.1-OVA,经酶切和测序鉴定正确后,以重组质粒为模板、PCR扩增包含T7启动子和OVA基因片段的PCR产物作为体外转录模板,通过体外转录、加poly(A)尾、纯化后获得修饰的OVA mRNA;分别在小鼠右后侧皮下接种结肠癌CT26细胞和三阴型乳腺癌4T1细胞、在小鼠乳腺脂肪垫注射三阴型乳腺癌4T1细胞,第6天和第13天时进行两次瘤内注射自制体外转录OVA mRNA或市售OVA mRNA,观察各组小鼠肿瘤生长抑制情况。结果酶切鉴定和测序分析结果证实重组质粒pcDNA3.1-OVA构建成功;体外转录OVA mRNA和市售OVA mRNA都能抑制小鼠结肠癌和乳腺癌皮下肿瘤生长(P<0.05),治疗效果比较,差异无统计学意义(P>0.05),但不能抑制乳腺原位移植瘤生长(P>0.05)。结论瘤内注射自制体外转录OVA mRNA能抑制小鼠结肠癌和三阴型乳腺癌皮下肿瘤生长,不能抑制三阴型乳腺癌原位瘤生长,治疗效果与市售OVA mRNA相当。Objective To compare immunotherapeutic effect of intratumoral injection on mouse CT26 colon cancer and 4T1 triple negative breast cancer cells between self-made in vitro-transcribed OVA mRNA and commercial OVA mRNA.Methods Synthesized OVA gene fragment was cloned into the eukaryotic expression vector pcDNA3.1(+)to construct a recombinant expression plasmid pcDNA3.1-OVA.After verification by restriction enzyme digestion and DNA sequencing,the recombinant plasmid was used as a PCR template for amplifying a PCR product containing T7 promoter and OVA gene fragment as an in vitro transcribed template.The modified OVA mRNA was obtained by in vitro transcription,poly(A)tailing and purification.Mouse CT26 colon cancer cells and 4T1 triple negative breast cancer cells were subcutaneously injected into mouse right rear flank,respectively.Additionally,4T1 triple negative breast cancer cells were also subcutaneously injected into mouse mammary fat pad.On the 6 th and 13 rd day,tumor-bearing mice were intratumorally injected with self-made in vitro-transcribed OVA mRNA or commercial OVA mRNA twice to observe the tumor growth inhibition in each group.Results Restriction enzyme digestion and sequencing analysis confirmed that recombinant plasmid pcDNA3.1-OVA was successfully constructed.Both in vitro-transcribed OVA mRNA and commercial OVA mRNA could inhibit the growth of subcutaneous colon cancer and breast cancer(P<0.05),with no significant difference in therapeutic effect(P>0.05),but could not inhibit the growth of orthotopic breast cancer(P>0.05).Conclusion Intratumoral injection with self-made in vitro-transcribed OVA mRNA can inhibit the growth of subcutaneous colon cancer and breast cancer,but cannot inhibit the growth of orthotopic breast cancer.The therapeutic effect is equivalent to that of commercial OVA mRNA.

关 键 词：结肠肿瘤 三阴型乳腺癌移植瘤 瘤内治疗 体外转录mRNA 卵清蛋白 

分 类 号：R735.35[医药卫生—肿瘤] R737.9[医药卫生—临床医学]