摘要
目的 探究粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因修饰的穿梭质粒能否对BALB/c小鼠结肠癌有疗效。方法 构建表达鼠粒细胞-巨噬细胞集落刺激因子(mGM-CSF)的穿梭质粒。实验分为pT7 mGM-CSF组(质粒组)、空载体对照组(pT7 GFP组)和溶剂对照组(PBS组)。Elisa检测pT7 mGM-CSF体外转染真核细胞后的表达量;选取雌性BALB/c小鼠,用表达近红外荧光蛋白(IRFP)的鼠结肠癌细胞株(CT26-IRFP)皮下植瘤;采用瘤内注射方式给药,游标卡尺测量实验组BALB/c小鼠肿瘤生长的情况;用小动物活体成像仪观察BALB/c小鼠体内肿瘤IRFP荧光强度表达情况并记录存活率;IHC法检测瘤组织内免疫细胞浸润情况。结果 与pT7 GFP组和PBS组比较,质粒组的表达在转染pT7 mGM-CSF质粒的293T细胞上清中的含量达到600pg/mL;和溶剂PBS组相比,质粒组与pT7 GFP组动物肿瘤生长趋势更为平缓,给药第21d,与PBS组比较,质粒组治疗效果明显;在治疗28d后质粒组小鼠存活率为40%,与pT7 GFP和PBS组相比有显著性差异;携带mGM-CSF基因的穿梭质粒组IRFP基因表达的荧光信号与pT7 GFP组和PBS组相比有显著性差异(P<0.05);IHC检测结果显示质粒组瘤组织内有大量的T淋巴细胞、NK细胞浸润聚集。结论 mGM-CSF基因修饰的穿梭质粒对鼠结肠癌具有明显的抑瘤效应,可能与质粒pT7 mGM-CSF有效刺激免疫细胞浸润有关。
Objective To study whether the shuttle plasmid modified by granulocyte-macrophage colony-stimulating factor(GM-CSF)gene has curative effect on colon cancer in BALB/c mice.Methods The shuttle plasmid expressing murine granulocyte-macrophage colony-stimulating factor(mGM-CSF)was constructed.The experiments were divided into pT7 mGM-CSF plasmid group,empty vector control group(pT7 GFP)and solvent control group(PBS).Elisa detected the expression of pT7 mGM-CSF in eukaryotic cells transfected in vitro.Female BALB/c mice were selected and subcutaneously transplanted with mouse colon cancer cell line(CT26-IRFP)expressing near-infrared fluorescent protein(IRFP).The tumor growth of BALB/c mice in the experimental groups were measured with a vernier caliper.The expression of IRFP fluorescence intensity of tumor in BALB/c mice was observed with a small animal in vivo imager,and the survival rate was recorded.Immune cell infiltration in tumor tissues was detected by IHC method.Results Compared with the pT7 GFP and PBS groups,the expression level of pT7 mGM-CSF plasmid in the supernatant of 293T cells transfected with pT7 mGM-CSF plasmid reached 600 pg/ml.Compared with the pT7 GFP group,the tumor growth trend of the CSF plasmid and pT7 GFP groups were more gradual.On the 21st day of administration,the pT7 mGM-CSF shuttle plasmid group had a significant therapeutic effect,when compared with the control group.After 28 days,the survival rate of mice in the pT7 mGM-CSF plasmid group was 40%,which was significantly different with the pT7 GFP and PBS groups.The fluorescence signal of IRFP gene expression in the shuttle plasmid group carrying the mGM-CSF gene was significantly different(P<0.05)with the pT7 GFP and PBS groups.IHC test results showed a large number of T lymphocytes,NK cells infiltrating and aggregating in the tumor tissue of the combined application group.Conclusion The shuttle plasmid modified by mGM-CSF gene has obvious anti-tumor effect on colon cancer in mouse,which may be related to the effective stimulation of i
作者
苏红霞
蔡林康
刘滨磊
李炎坤
SU Hong-xia;LIU Bin-lei;LI Yan-kun(Hubei Engineering Research Center of Traditional Chinese Medicine of South Hubei Province,School of Pharmacy,Hubei University of Science and Technology,Xianning Hubei 437100,China)
出处
《湖北科技学院学报（医学版）》
2023年第3期200-204,共5页
Journal of Hubei University of Science and Technology(Medical Sciences)
基金
国家自然科学基金项目(32170906)。
