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颗粒体蛋白前体(progranulin,PGRN)在多种肿瘤中过表达。但PGRN在黑色素瘤发生发展中的作用尚无报道。为探究PGRN在黑色素肿瘤中的作用,本研究采用CRISPR-Cas9基因编辑技术建立了稳定敲低PGRN的小鼠黑色素瘤B16细胞株B16-PGRNlow。MTS法和Brd U掺入结合流式细胞(计量)术分析证明,敲低PGRN不影响B16细胞的细胞周期和增殖。将B16-ctrl(对照)和B16-PGRNlow细胞分别皮下接种野生型(WT)和PGRN敲除(KO)的C57BL/6J小鼠,比较观察黑色素移植瘤体积大小。移植瘤形成20 d后,与B16-ctrl细胞接种的移植瘤比较,无论在WT还是在KO荷瘤小鼠,B16-PGRNlow形成的移植瘤体积明显减小(WT鼠:P<0.05;KO鼠:P<0.01)。然而,比较B16-PGRNlow或B16-ctrl在WT鼠与KO鼠形成的移植瘤体积大小,并无显著差异,提示B16肿瘤细胞PGRN而非宿主PGRN影响移植瘤的生长。流式细胞术分析显示,在荷B16-PGRNlow移植瘤的WT型小鼠脾和淋巴结中,CD4+、CD8+T细胞数(百分比)比荷B16-ctrl移植瘤的WT鼠脾和淋巴结的CD4+、CD8+T细胞数明显增多(P<0.05,P<0.01),而在KO鼠却未见明显差异。上述结果证明,敲低肿瘤细胞PGRN可抑制黑色素移植瘤的生长。上述结果还提示,抑制PGRN在黑色瘤的表达可引起脾和淋巴结CD4+和CD8+T细胞增加,提高宿主的细胞免疫能力。其机制尚待进一步研究。本文的发现为PGRN作为黑色素瘤治疗的潜在靶点提供了新证据。
Progranulin (PGRN) is overexpressed in many tumors. However, the role of PGRN in the development of melanoma has not been reported yet. In order to explore the role of PGRN in melanoma, B16-PGRNlow was established in mouse melanoma B16 cell line stably knocked down by CRISPR-Cas9 gene editing technology. MTS and BrdU incorporation combined with flow cytometric analysis demonstrated that knockdown of PGRN did not affect the cell cycle and proliferation of B16 cells. The B16-ctrl (control) and B16-PGRNlow cells were subcutaneously inoculated with wild type (WT) and PGRN knockout (KO) C57BL / 6J mice respectively to compare the volume of melanoma xenografts. After transplanted tumors were established for 20 days, the volume of transplanted tumors formed by B16-PGRNlow was significantly reduced in both WT and KO-bearing mice (WT mice: P <0.05; KO mice : P <0.01). However, comparing the size of B16-PGRNlow or B16-ctrl in the size of xenografts in WT mice and KO mice, there was no significant difference, suggesting that PGRN in B16 tumor cells, but not host PGRN, affected the growth of xenografts. Flow cytometry analysis showed that the percentage of CD4 + and CD8 + T cells in WT and lymph nodes of B16-PGRNlow xenografts was higher than that in WT mice and lymph nodes of B16-ctrl xenografts CD8 + T cells were significantly increased (P <0.05, P <0.01), but no significant difference in KO rats. The above results demonstrate that knockdown of PGRN in tumor cells inhibits the growth of melanoma xenografts. The above results also suggest that inhibiting the expression of PGRN in melanoma can cause an increase in CD4 + and CD8 + T cells in the spleen and lymph nodes and increase host cellular immunity. The mechanism remains to be further studied. Our findings provide new evidence for PGRN as a potential target for the treatment of melanoma.