Inhibitory Effects of VAP33 on Malignant Behaviors of Murine Dendritic Cell Sarcoma
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摘要:
目的 探讨囊泡膜蛋白相关蛋白（VAP33）过表达对小鼠树突状肉瘤细胞DG6增殖及侵袭转移的影响。 方法 反转录获得VAP33 cDNA，利用基因重组技术构建VAP33-GFP融合基因慢病毒表达载体（pWPXL-VAP33），在脂质体Lipofectamine 2000介导下与辅助质粒（PsPAX2、MD2G）共转染293T细胞包装生产重组慢病毒。取病毒上清感染内源性低表达VAP33的DG6细胞，通过荧光显微镜挑选、Western-blot鉴定获得VAP33稳定过表达的阳性单克隆细胞株（DG6-VAP33）。MTT法检测细胞增殖情况，Transwell和集落形成检测肿瘤细胞体外侵袭、成瘤能力。同时建立小鼠皮下移植瘤模型，连续6周观察VAP33过表达对活体内肿瘤细胞生长、成瘤、转移能力的影响。 结果 重组慢病毒表达载体pWPXL-VAP33构建成功，包装滴度达6×107IU/mL，感染DG6细胞后筛选到VAP33稳定过表达细胞株（DG6-VAP33）。与DG6细胞相比，DG6-VAP33细胞体外增殖减慢，每孔集落数分别为（52±8）个和（36±5）个（P < 0.05）；Transwell方法观察到，DG6-VAP33细胞体外侵袭能力显著下降，穿膜细胞数（14±11）个少于DG6（36±17）个（P < 0.01）。通过6周的连续观察，DG6-VAP33组肿瘤生长速度显著慢于DG6组，成瘤率50%（5/10）、肺转移率（0）也明显降低（DG6组成瘤率100%、肺转移率80%）。 结论 VAP33过表达可抑制肿瘤细胞的增殖、成瘤及侵袭转移能力。 Abstract:Objective This study aims to investigate the effect of VAP33 on the proliferation, invasion, and metastasis of murine dendritic cell sarcoma. Methods VAP33 cDNA was cloned, and gene recombinant technology was employed to construct a recombinant plasmid (pWPXL - VAP33) carrying the VAP33-GFP fusion gene. 293T cells were cotransfected with the recombinant plasmid and the helper plasmids PsPAX2 and MD2G using Lipofectamine 2000 to produce lentiviral particles. The recombinant lentiviruses were then used to infect DG6 cells with minimal endogenous VAP33 expression. Positive clones (DG6 - VAP33) were screened using fluorescence microscopy and Western blot analysis. The proliferation, invasion, metastasis, and cloning formation efficiency of DG6 - VAP33 cells were compared with those of DG6 through in vitro MTT, Transwell, and colony formation assays, respectively. Tumor models in KM mice were established to determine the effects of VAP33 on the behavior of malignant tumors in vivo. DG6 cells were used as the control in all experiments. Results The recombinant plasmid expressing VAP33 - GFP fusion gene was successfully constructed with a titer of 6 × 107 IU/mL. After transfection, the VAP33 gene was highly expressed in the transfected DG6 cells. An in vitro assay showed that VAP33 overexpression decreased the proliferation of DG6 cells; the number of colonies formed by DG6 - VAP33 cells (52 ± 8 colonies / well) was lower than that in DG6 cells (36 ± 5 colonies / well; P < 0.05). The Transwell assay results showed that DG6-VAP33 had fewer membrane-penetrating cells than DG6 cells (36 ± 17 vs. 14 ± 11, P < 0.01), which indicated a decrease in invasiveness. When the DG6-VAP33 cells were subcutaneously transplanted into KM mice, the DG6-VAP33 cells exhibited a decreased proliferative ability. The tumorigenesis rate was 50 % (5 / 10), significantly lower than that of DG6 cells (100 %, 10 / 10; P < 0.05). Furthermore, the lung metastasis rate was 0 %, compared with 80 % for DG6. Conclusion VAP33 overexpression decreased proliferation, tumorigenesis, invasion, and metastasis of murine dendritic cell sarcoma. -
Key words:
- Dendritic sarcoma cells /
- VAP33 /
- Proliferation /
- Invasion /
- Metastasis
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图 1 Western blot检测DG6-VAP33细胞中VAP33蛋白表达水平
1：DG6细胞；2：DG6-VAP33细胞
Figure 1. VAP33 protein expression in DG6-VAP33 cells detected by Western blot analysis
图 2 DG6-VAP33细胞中VAP33-GFP融合蛋白（×600）
Figure 2. VAP33-GFP fusion proteins in DG6-VAP33 cells(×600)
图 3 VAP33过表达抑制DG6细胞的体外增殖能力
DG6与DG6-VAP33对应时间点比*P<0.05，**P<0.01
Figure 3. VAP33 overexpression decreased the proliferative ability of DG6 cells
图 4 Transwell小室穿膜细胞（×100）
A：DG6细胞；B：DG6-VAP33细胞
Figure 4. Transmembrane cells in Transwell chambers
图 5 VAP33过表达对DG6细胞移植瘤生长能力的抑制
Figure 5. VAP33 overexpression decreased the in vivo proliferative ability of DG6 cells
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