乳腺癌T47D—tk细胞生长特性及移植瘤动物模型的建立

Growing characteristic of breast adenocarcinoma suicide gene cell line T47D-tk and construction of subcutaneous xenografs

目的进一步对已经获得的稳定表达自杀基因HSV1-tk的乳腺癌细胞株T47D—tk进行鉴定，观察其生长特性，建立移植瘤动物模型。方法提取细胞基因组、以HSV1-tk P1和HSV1-tkP2为引物，进行PCR反应，琼脂糖凝胶电泳基因鉴定。描绘生长曲线、镜下观察细胞形态。BALB／c nu／nu裸鼠前肢皮下接种T47D和T47D—tk细胞，并观察二者的成瘤特性。结果T47D-tk细胞基因组DNA进行PCR检测可见到大小约1131bp的片段，而正常的T47D细胞基因组则无此阳性条带出现。两组平均生长细胞数，第1天T47D：（5．58±1．04）×10^3，T47D-tk：（6．08±1．61）×10^3；第3天T47D：（10．00±1．30）×10^3；T47D—tk：（10．25±0．90）×10^3；第7天T47D：（19．25±0．66）×10^3，T47D—tk：（19．00±1．80）×10^3，两组间差异均无统计学意义（均P〉0．05），形态观察亦未见明显差别。接种后两组成瘤时间[T47D：（9．67±0．33）d；T47D—tk：（9．83±0．48）d]和肿瘤大小差异均无统计学意义（均P〉0．05）。结论T47D—tk细胞基因组中已整合人HSV1-tk基因，与T47D细胞相比生长特性无变化，成功建立了T47D和T47D—tk移植瘤动物模型，为进一步基因显像与基因治疗奠定了基础。

Objective To further identify the breast adenocarcinoma cell line T47D-tk stably expressing the suicide gene HSVI-tk, observe its growing characteristics, and establish an animal model of implanted breast adenocarcinoma. Methods Retroviral vector of HSVI-tk gene and breast carcinoma cell line T47D-tk stably expressing the HSVI-tk gene were established. Breast carcinoma cells of the lines T47D and T47D-tk were cultured, and observed by inverted microscope. Growth curve was drawn. Genomic DNA of T47D-tk cells was extracted, and amplified by PCR with HSVI-tk and HSVI-tk P2 as primers. Agarose gel electrophoresis was used to identify the HSVltk gene. Suspensions of T47D and T47D-tk cells were inoculated subcutaneously at bilateral roots of foreleg of female BALB/c nude mice respectively. The growth of tumor was observed every day. Results PCR showed 1131 bp fragment in the T47D-tk genome, but not in the T47D genome. The numbers of growing T47D cells on days 3, and 7 were （ 10.00 ± 1.30） × 10^3 and （19.25 ±0.66） × 10^3 respectively, not significantly different from those of the T47D-tk cells [ （10.25 ± 0.90） × 10^3 and （19.00 ± 1.80） × 10^3 respectively, both P 〉 0. 05]. The time needed for tumor formation after the inoculation of T47D cells was （9.67 ± 0.33 ）d, not significantly different from that of the T47D-tk ceils [ （9.83 ± 0.48） d, P 〉 0. 05 ]. The tumor size 19 days after inoculation of the T47D cells was （72.17±25.88） mm^3, not significantly different from that of the T47D-tk cells [ （70.66 ± 22. 16） mm^3, P 〉 0.05 ]. Conclusion The T47D-tk cells have integrated the HSVI-tk gene without changing its growing characteristics. An animal model of implanted breast cancer has been successfully established. The T47D and T47D-tk subcutaneous xenografts offer the foundation for further study of gene imaging and gene therapy.