胫骨注射和左心室注射乳腺癌细胞小鼠骨定植模型的研究

Tibial injection and left ventricular injection of breast cancer cells in a mouse model of bone colonization

目的:探讨胫骨注射和左心室注射乳腺癌细胞构建小鼠骨定植模型的方法。方法:通过对野生型MDA-MB-231乳腺癌细胞进行慢病毒感染,构建稳定表达绿色荧光蛋白(GFP)和荧光素酶(LUC)的细胞株。将GFP+/LUC+标记的处于对数生长期的MDA-MB-231乳腺癌细胞分别以6×10^(3)个/只和1×10^(5)个/只接种BALB/c-nu/nu小鼠和NOD/SCID小鼠,构建胫骨注射模型和左心室注射模型。采用活体成像观察肿瘤细胞在活体小鼠体内的定植部位及分布情况;X线检测观察骨损伤的位置及程度;苏木素-伊红(HE)染色和广谱角蛋白(Pan CK)免疫组化染色定位骨中的肿瘤细胞。结果:经荧光显微镜及活细胞成像检测,表达GFP和LUC的慢病毒对MDA-MB-231细胞感染效率高达95%,且继续传代后GFP和LUC表达效率无明显减退,可用于后续实验。胫骨注射4 h后进行活体成像观察,结果显示小鼠右下肢胫骨出现LUC信号;左心室注射后立即进行活体成像观察,发现肿瘤细胞已循环至全身,表明已成功构建胫骨注射模型及左心室注射模型。30 d进行活体成像观察发现,胫骨注射模型和左心室注射模型腿部出现LUC信号,表明腿部有定植灶形成。X线结果也表明存在骨损伤。HE染色和Pan CK免疫组化染色证实胫骨注射MDA-MB-231乳腺癌细胞小鼠模型100%出现骨定植灶,定植率高,而左心室注射MDA-MB-231乳腺癌细胞小鼠模型仅20%出现骨定植灶。结论:运用活体成像、X线等检测技术联合病理学分析评估胫骨注射及左心室注射乳腺癌细胞骨定植小鼠模型,可提高结果分析的准确性,能为乳腺癌转移机制研究、抗转移治疗等提供实验和理论依据。

Objective:To investigate the methodology of constructing bone colonization model in mice by tibial injection and left ventricular injection of breast cancer cells.Methods:Cell lines stably expressing green fluorescent protein(GFP)and luciferase(LUC)were constructed by lentiviral infection of wild type MDA-MB-231 breast cancer cells.The GFP+/LUC+-labeled MDA-MB-231 breast cancer cells in logarithmic growth phase were inoculated into BALB/c-nu/nu mouse and NOD/SCID mouse at 6×10^(3)/pc and 1×10^(5)/pc,respectively,to construct tibial injection model and left ventricular injection model.In vivo imaging was used to observe the colonization site and distribution of tumor cells in living mouse;X-ray detection was used to observe the location and extent of bone damage;HE staining and pancytokeratin(Pan CK)immunohistochemical staining were used to localize tumor cells in bone.Results:As detected by fluorescence microscopy and live cell imaging,the lentivirus expressing GFP and LUC had an infection efficiency of up to 95%on MDA-MB-231 cells,and there was no obvious decrease in the expression efficiency of GFP and LUC after continued passaging,which could be used for subsequent experiments.In vivo imaging 4 hours after tibial injection showed that LUC signals appeared in the right lower limb tibia of the mouse;in vivo imaging immediately after left ventricular injection showed that the tumor cells had circulated throughout the body,which indicated that the tibial injection model and the left ventricular injection model had been successfully constructed.In vivo imaging at 30 days showed that LUC signals appeared in the legs of the tibia-injected model and the left ventricle-injected model,which indicated that there was a foci of colonization formed in the legs.X-rays also showed bone damage.HE staining and Pan CK immunohistochemical staining confirmed the presence of foci of bone colonization in 100%of the tibial-injected MDA-MB-231 mouse model with a high rate of colonization,whereas only 20%of the left ventricle-injected MDA-MB-231 breast cancer cells mouse model showed foci of bone colonization.Conclusion:The use of in vivo imaging,X-ray and other detection techniques combined with pathological analysis to evaluate the tibial-injected and left ventricle-injected breast cancer cells bone colonization mouse models can improve the accuracy of the result analysis,and can provide experimental and theoretical basis for the study of metastatic mechanism and anti-metastatic treatment of breast cancer.