目的： 评价口服携带HPV16 E7 shRNA 和 IL-12 基因的重组短双歧杆菌在小鼠体内抗宫颈癌移植瘤的效果。方法： 将pMG36e-E7 shRNA、pMG36e-mIL-12D 质粒分别转化短双歧杆菌，经筛选鉴定并扩增获得携带HPV16 E7 shRNA和IL-12 基因的重组短双歧杆菌。通过小鼠皮下宫颈癌细胞移植建立荷瘤小鼠模型。口服重组短双歧杆菌1、7 d后，检测小鼠主要器官（心、肝、脾、肺、肾）和肿瘤组织匀浆液或血清在PYG培养基中形成的菌落数量，评价短双歧杆菌的肿瘤靶向性，以小鼠体内肿瘤生长曲线评估重组短双歧杆菌的抗肿瘤效果，通过主要器官切片H-E染色和检测荷瘤小鼠血清相关细胞因子水平评价口服重组短双歧杆菌的安全性。 结果： 成功制备重组短双歧杆菌和宫颈癌 TC-1 细胞移植瘤小鼠。7 d后，移植瘤组织匀浆液和血清的菌落数量证实短双歧杆菌具有靶向体内瘤组织的定殖能力，口服重组短双歧杆菌明显抑制荷瘤小鼠的肿瘤生长（ P<0.05或P<0.01），但联合使用携带HPV16 E7 shRNA和IL-12基因的重组双歧杆菌的肿瘤抑制率与单独使用的并无显著差异，治疗后未见对荷瘤小鼠主要器官的损伤和血清中IL-12及IFN-γ的水平明显变化。 结论： 短双歧杆菌可用作靶向肿瘤的治疗性基因分子递送载体，其对宫颈癌移植瘤的疗效明显且安全可控。

Objective: To evaluate the effect of oral administration of recombinant Bifidobacterium breve (B.breve) carrying HPV16 E7 shRNA and IL-12 gene in mice against cervical cancer xenografts. Methods: pMG36e-E7 shRNA and pMG36e-mIL-12D plasmids were used to modify the B.breve. After screening, verification and amplification, recombinant B.breve carrying HPV16 E7 shRNA and IL-12 gene were obtained. Tumor-bearing mouse model was established by subcutaneously injecting cervical cancer cells. On the 1st and 7th day after oral administration, the tumor-targeting property of recombinant B.breve were evaluated by counting the number of bacteria colonies formed in the homogenate of mouse major organs (heart, liver, spleen, lung and kidney) and tumor tissues or serum that cultured in PYG medium. The antitumor effect of recombinant B.breve was evaluated by in vivo tumor growth curve. The safety of oral administration of recombinant B.breve was evaluated by H-E staining of the tissue sections from major organs and determination of the levels of related cytokines in serum of tumor-bearing mice. Results: Recombinant B.breve and cervical TC-1 cell transplanted tumor bearing mice were successfully established. After 7 days, the number of colonies in the homogenate of tumor tissues and serum confirmed that recombinant B.breve had the property of targeting tumor tissues in vivo. Oral administration of recombinant B.breve significantly suppressed tumor growth in tumor-bearing mice (P<0.05 or P<0.01); however, there was no significant difference in tumor inhibition rate between the recombinant B.breve carrying both HPV16 E7 shRNA and IL-12 gene and those carrying single one. After the treatment, no major organ damage or significant changes in serum IL-12 and IFN-γ levels was observed in tumor-bearing mice. Conclusion: B. breve can be used as a safe and controllable therapeutic molecular delivery vehicle for targeting tumors, which exert significant treatment efficacy against cervical cancer xenografts.

Q789；R737.33；R730.54

新疆维吾尔自治区自然科学基金资助项目（No.2017D01C048