Establishment Of A Macrophage Cell Line From Hepatoma Tissue Of Hras12V Transgenic Female Mouse

Objective:To establish an immortalized macrophage cell line from hepatoma tissue of Hras12 V transgenic female mouse for providing an experimental tool to exploring the effect of tumor-related macrophages on the development of tumors.Methods:1.A 12-month-old Hras12 V transgenic female mouse was selected and killed by neck amputation and liver tumor tissues were collected aseptically.Cell clones were obtained by tissue culture method.After 60 consecutive passages,a macrophage cell line named FMHRL10 was successfully established.2.Genomic DNA was detected by PCR to identify the origin of FMHRL10,and the expression and activation level of signal molecules in FMHRL10 were detected by Western blotting.3.Cell Counting Kit-8 staining was used to detect the growth curve of FMHRL10.4.Cell cycle and apoptosis of FMHRL10 were detected by flow cytometry using cell cycle and apoptosis kit.5.Morphological analysis of FMHRL10 was performed by Giemsa.6.The macrophage type of FMHRL10 was detected by flow cytometry.7.Phagocytosis Assay Kit was used to detect the phagocytosis of FMHRL10.8.The similarities and differences between FMHRL10 and RAW264.7 were compared by Western blot and RT-qPCR.9.The in vivo tumorigenicity of FMHRL10 was detected in nude mice.10.The effect of secretions from FMHRL10 on the growth of Hep1-6 hepatoma cells was detected by flow cytometry using cell apoptosis kit.11.The effect of FMHRL10 on the in vivo tumorigenicity of H22 was detected by were subaxillary inoculating FMHRL10 and mouse hepatoma cell line H22(1:1)in C57BL/6J female mice.Results:1.After 60 passages,an immortalized macrophage cell line derived from hepatoma tissue of Hras12 V transgenic female mouse was obtained and named FMHRL10.2.The results from genomic DNA detected by PCR showed that FMHRL10 was derived from Hras12 V transgenic female mouse.The results of Western blotting assay showed that FMHRL10 was different from hepatoma in the activation levels of signal pathways.3.The growth curve of FMHRL10 showed that the growth curve of FMHRL10 was roughly "S" type and reached logarithmic growth stage on the 3rd to 6th day,indicating that the FMHRL10 had good proliferation ability.4.The cell cycle and apoptotic ratio of FMHRL10 detected by flow cytometry showed that the proportion of late apoptotic cells was 8.398±0.452% and that of early apoptotic cells was 2.070±0.414%).Cell cycle analysis showed that the percentage of cells in G1 phase was 62.85±2.61%,in G2/M phase was 4.658±0.415% and in S phase was 19.8±4.876%.It indicates that the apoptotic status of the cell cycle was in normal range.5.Morphological observation by Giemsa staining showed that the cell was adherent cell with small nucleus,round or elliptic shape,pseudopodia and typical macrophage characteristics.6.The results of flow cytometry showed that 77.1% of FMHRL10 was F4/80 positive cells,77.4% of RAW264.7 was F4/80 positive cells,which proved that FMHRL10 was a macrophage.In addition,both FMHRL10 and RAW264.7 were M1 activation type.7.Cell phagocytosis assay showed that FMHRL10 cell line has specific phagocytic function of macrophages which was significantly stronger than RAW264.7.8.The results of Western Blot and RT-qPCR showed that the activation levels of signaling molecules and the expression levels of genes in FMHRL10 were significantly different from RAW264.7.It indicates that FMHRL10 and RAW264.7 are two different macrophage cell lines.9.The results of in vivo tumorigenicity test in nude mice showed that FMHRL10 had no tumorigenicity.10.The secretions from FMHRL10 had no effect on the growth of Hep1-6 hepatoma cells.11.The results of subaxillary inoculation showed that FMHRL10 significantly inhibited the growth of H22 hepatoma in vivo.However,RAW264.7 did not.Conclusions:1.A macrophage cell line(FMHRL10)derived from hepatoma tissue of Hras12 V transgenic female mice was successfully established.The FMHRL10 can be stably passaged in vitro and is an immortalized tumor-derived macrophage cell line.2.The FMHRL10 belongs to M1 macrophage type and has inhibitory effect on the proliferation of hepatoma cells in vivo.