Objective To investigate the effect of PTGS2 knockdown on tumor-associated macrophage(TAM)M2 polarization in the treatment of nasopharyngeal carcinoma(NPC).Methods Adenovirus-mediated knockdown of PTGS2 was performed in primary NPC cells.A co-culture system of primary NPC cells and TAMs and a mixed three-dimensional(3D)co-culture of primary NPC cells,TAMs,and human CD8+T cells were established in vitro.The control,Adv-PTGS2 shRNA,and Adv-shRNA NT groups were subjected to in vitro and in vivo experiments.In the co-culture system of primary NPC cells and TAMs,IL-12,IL-10,and TGF-βlevels in the culture superna-tants were determined using the ELISA.MMP-2 and MMP-9 expression levels in primary NPC were determined using Western blotting.In the mixed culture system of the three types of cells,cell viability was measured using the CCK-8 method.IFN-γand GZMBmRNA levels were determined using qPCR.The three types of cell-mixed cultures were injected subcutaneously into xenografts,and tumor growth was monitored for 21 d.Results Compared with control group,IL-12 levels increased and IL-10 and TGF-βlevels decreased in the culture supernatant of Adv-PTGS2 shRNA cells(P<0.05).MMP-2 and MMP-9 expression levels in primary NPC decreased(P<0.05).Com-pared with the control group,CD8+T cell proliferation ability and relative IFN-γand GZMBmRNA expression levels increased(P<0.05 for both experiments)in the Adv-PTGS2 shRNA cells.Compared with the control group,the Adv-PTGS2 shRNA group showed slower tumor growth(P<0.05)and lower tumor mass(P<0.05)in in vivo xenografts.Conclusion PTGS2knockdown inhibited TAM M2 pola-rization,regulated the immune-hot state in the tumor microenvironment,and inhibited NPC growth.

Objective To investigate the effect of PTGS2 knockdown on tumor-associated macrophage(TAM)M2 polarization in the treatment of nasopharyngeal carcinoma(NPC).Methods Adenovirus-mediated knockdown of PTGS2 was performed in primary NPC cells.A co-culture system of primary NPC cells and TAMs and a mixed three-dimensional(3D)co-culture of primary NPC cells,TAMs,and human CD8+T cells were established in vitro.The control,Adv-PTGS2 shRNA,and Adv-shRNA NT groups were subjected to in vitro and in vivo experiments.In the co-culture system of primary NPC cells and TAMs,IL-12,IL-10,and TGF-βlevels in the culture superna-tants were determined using the ELISA.MMP-2 and MMP-9 expression levels in primary NPC were determined using Western blotting.In the mixed culture system of the three types of cells,cell viability was measured using the CCK-8 method.IFN-γand GZMBmRNA levels were determined using qPCR.The three types of cell-mixed cultures were injected subcutaneously into xenografts,and tumor growth was monitored for 21 d.Results Compared with control group,IL-12 levels increased and IL-10 and TGF-βlevels decreased in the culture supernatant of Adv-PTGS2 shRNA cells(P<0.05).MMP-2 and MMP-9 expression levels in primary NPC decreased(P<0.05).Com-pared with the control group,CD8+T cell proliferation ability and relative IFN-γand GZMBmRNA expression levels increased(P<0.05 for both experiments)in the Adv-PTGS2 shRNA cells.Compared with the control group,the Adv-PTGS2 shRNA group showed slower tumor growth(P<0.05)and lower tumor mass(P<0.05)in in vivo xenografts.Conclusion PTGS2knockdown inhibited TAM M2 pola-rization,regulated the immune-hot state in the tumor microenvironment,and inhibited NPC growth.

A promising genetic marker, sag5b, was cloned and expressed and the difference of the genes between highly virulent strain (RH) and less virulent strain(Prugniaud) of Toxoplasma gondii was compared. The PCR-generated product of sag5b was subcloned into T easy vector and plasmid pET28a consecutively. The fusion expression was induced by IPTG and identified by SDS-PAGE and Western blotting. The immunoreactivity of recombinant SAG5B was identical to that of native SAG5B on the membrane of tachyzoites of RH strain. The brains of mice infected with Prugniaud strain of T. gondii were homogenated. Sag1 was successully cloned by PCR from both RH strain tachyzoites and the homogenized brain tissues of mice infected with low virulent strain of Prugniaud,whereas sag5b was only detected in RH strain but not in Prugniaud strain, indicating that sag5b could be used as a genetic marker for differentiation of strain virulence. Expression and vaccination of the virulence-associated gene into mice failed to induce obvious protective immunity against the challenge of RH strain.