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目的 Wnt信号异常激活与肿瘤发生有关,观察Wnt信号的天然拮抗剂FrzB对Wnt信号转导的影响和对肿瘤细胞增殖抑制。方法采用AdEasy系统构建重组腺病毒AdFrzB;卡那霉素抗性筛选、酶切鉴定及荧光显微镜和Western blot检测标记基因GFP和His表达等方法鉴定AdFrzB。AdFrzB与AdWnt3A共感染人骨肉瘤细胞株143B,AdGFP与AdWnt3A共感染为对照,β-catenin/Tcf荧光素酶反应系统检测FrzB对Wnt信号转导的影响;MTT实验检测FrzB对细胞增殖的抑制。结果卡那霉素抗性筛选及酶切鉴定获得pAdFrzB-His;检测到报告基因GFP和His表达,确证AdFrzB构建成功。FrzB抑制了Wnt信号转导并抑制了143B细胞增殖。结论 AdEasy系统有效构建了AdFrzB;外源性FrzB拮抗Wnt信号转导并抑制143B细胞增殖,FrzB可能是一种天然抗肿瘤因子。
Purpose Wnt signaling abnormal activation is associated with tumorigenesis. To observe the effect of FrzB, a natural antagonist of Wnt signaling, on Wnt signal transduction and the inhibition of tumor cell proliferation. Methods Recombinant adenovirus AdFrzB was constructed by AdEasy system. AdFrzB was identified by Kanamycin resistance screening, restriction enzyme digestion and fluorescent microscopy and Western blotting to detect the expression of GFP and His. AdFrzB and AdWnt3A were co-infected with human osteosarcoma cell line 143B and co-infected with AdGFP and AdWnt3A. The effect of FrzB on Wnt signal transduction was detected by β-catenin / Tcf luciferase assay. The inhibition of FrzB on cell proliferation was detected by MTT assay. Results kanamycin resistance screening and restriction enzyme digestion obtained pAdFrzB-His; reporter gene GFP and His expression was confirmed to confirm the successful construction of AdFrzB. FrzB inhibited Wnt signaling and inhibited 143B cell proliferation. Conclusion AdEasy system effectively constructs AdFrzB; exogenous FrzB antagonizes Wnt signaling and inhibits the proliferation of 143B cells. FrzB may be a natural anti-tumor factor.