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目的采用婴儿双歧杆菌为载体,探讨尿嘧啶磷酸核糖转移酶基因(UPRT)对胞嘧啶脱氨酶/5-氟胞嘧啶(CD/5-FC)自杀基因系统抗瘤作用的增强效应。方法构建原核表达质粒pGEX-UPRT,以电穿孔法将该质粒转化入婴儿双歧杆菌,筛选阳性重组菌并鉴定。采用M TT法检测表达的U PRT是否可以与CD产生抗瘤协同作用,并观察细胞形态学改变。结果重组婴儿双歧杆菌可以正确表达UPRT。体外M TT检测显示CD+UPRT组细胞存活率低于对照组(P<0.01),且可使B 16-F 10鼠黑色素瘤细胞对5-FC的杀伤敏感性(IC50=0.015μm o l/mL)提高,是CD组(IC50=0.127μm o l/mL)的8.5倍。形态学观察CD+UPRT组肿瘤细胞显示出明显的损伤性改变,细胞生长受到明显抑制,而UPRT组和CD组变化明显不如前者。结论婴儿双歧杆菌联合转导UPRT基因可明显增强CD/5-FC自杀基因系统对鼠黑色素瘤细胞B 16-F 10的杀伤作用。
Objective To investigate the anti-tumor effect of uracil phosphoribosyltransferase gene (UPRT) on cytosine deaminase / 5-fluorocytosine (CD / 5-FC) suicide gene system using Bifidobacterium infantis as carrier. Methods The prokaryotic expression plasmid pGEX-UPRT was constructed and transformed into Bifidobacterium infantis by electroporation. The positive recombinant strains were screened and identified. MTPT was used to detect the expression of UPRT can CD anti-tumor synergistic effect, and observe the morphological changes. Results Recombinant Bifidobacterium infantis could express UPRT correctly. In vitro MTT assay showed that the cell viability in CD + UPRT group was lower than that in control group (P <0.01), and the cytotoxicity against 5-FC in B 16-F 10 mouse melanoma cells was inhibited (IC50 = 0.015μmol / mL ) Increased 8.5 times that of the CD group (IC50 = 0.127 μm ol / mL). Morphological observation of tumor cells in CD + UPRT group showed obvious injury changes, cell growth was significantly inhibited, while the UPRT group and CD group changed significantly less than the former. Conclusion Bifidobacterium infantis combined with UPRT gene can significantly enhance the killing effect of CD / 5-FC suicide gene system on B 16 -F 10 in mouse melanoma cells.