小鼠骨髓间充质干细胞联合IL-12重组质粒对小鼠乳腺癌的治疗作用

The therapeutic effect of mouse bone marrow-derived mesenchymal stem cells combined with IL-12 recombinant plasmid on mice bearing breast cancer

目的探讨小鼠骨髓间充质干细胞(BMSC)对小鼠乳腺癌EMT6细胞在体内外的抑制作用以及BMSC与IL-12真核表达质粒(pcDNA6/IL-12)联合应用对荷瘤小鼠的治疗作用。方法全骨髓贴壁法培养BMSC并鉴定;CCK-8法检测BMSC条件培养基对EMT6细胞增殖的影响;Annexin V-FITC/7-AAD双染色结合流式细胞术检测BMSC条件培养基对EMT6细胞凋亡的影响。建立小鼠乳腺癌荷瘤模型,分别为对照组(瘤内注射PBS)、BMSC组(瘤内注射BMSC)、IL-12组(瘤内注射pcDNA6/IL-12质粒)、BMSC联合IL-12组(瘤内注射BMSC和pcDNA6/IL-12质粒)。于致瘤后第17天处死小鼠,取出肿瘤和脾脏并称其质量,计算脾指数;MTT法检测脾淋巴细胞增殖活性,LDH法检测脾淋巴细胞杀伤活性,HE染色观察肿瘤组织的病理学改变。结果 BMSC条件培养基在体外可显著抑制EMT6的增殖并促进其凋亡(P<0.05﹚。BMSC单独及其与pcDNA6/IL-12联合应用可导致荷瘤小鼠肿瘤的体积缩小(P<0.01﹚,脾淋巴细胞增生反应及杀伤活性增强(P<0.05﹚,肿瘤组织坏死区扩大、炎性细胞浸润增多。结论 BMSC在体内外均具有良好的抗肿瘤作用,与pcDNA6/IL-12在荷瘤小鼠体内联合应用可获得更强的抗肿瘤活性。

This study was designed to investigate the inhibitory effect of murine bone marrow-derived mesenchymal stem cells （BMSC） on EMT6 breast cancer cells, and the therapeutic effect of BMSC combined with IL-12 recombinant plasmid （pcDNA6/IL-12） on mice bearing breast cancer. Firstly, primary BMSC were isolated and cultured by whole bone marrow cell attachment method and identified. The effects of the BMSC conditioned medium on the proliferation and apoptosis of EMT6 were respectively detected by flow cytometry using CCK-8 and Annexin V-FITC/7-AAD double staining in vitro. Mouse models of breast cancer were prepared and assigned into four groups： control group （intra-tumor injection of PBS）, BMSC group （intra-tumor injection of BMSC）, IL-12 group （intra-tumor injection of pcDNA6/IL-12）, BMSC＋IL-12 group （intra-tumor co-injection of BMSC and pcDNA6/IL- 12）. All of the mice were sacrificed on day 17. The tumor and spleen were taken out and weighed, spleen index was calculated and pathologic changes of tumor tissue were observed by HE staining. The proliferative and cytotoxic activities of spleen lymphocytes were measured by means of MTT and LDH release, respectively. We found BMSC- conditioned medium significantly inhibited the proliferation and accelerated the apoptosis of EMT6. In the tumor bearing mice, BMSC treatment or BMSC＋pcDNA6/IL-12 treatment evidently reduced tumor volume, expand tumor necrosis area, increased inflammatory cell infiltration, and enhanced the functions of anti-tumor immunity （proliferation and cytotoxicity） of spleen lymphocytes. These results demonstrated that BMSC possess expected anti- tumor effect both in vitro and in vivo, and a synergistic effect could be acquired when combined with pcDNA6/IL- 12 in mice with breast cancer.