| Objective: The cell line IL-23/MA-891 producing IL-23 protein was set up by transfection of IL-23 gene to mouse mammary cancer cell (MA-891), and the antitumor effects and immunomodulating mechanism of IL-23 were studied.Methods: 1 mIL-23 gene was transferred into two packing cell lines (ecotropicψ2 and amphotropic PA317) in sequence by a retrovirus vector (LXSN), and the positive cellular clones that can produce retroviruses carrying IL-23 gene were screened by G418. The retroviruses were used to transduce the mIL-23 gene into mouse mammary cancer cells (MA-891). After screening by G418, IL-23/MA-891 cells expressing IL-23 protein were obtained. Expression of mIL-23mRNA in IL-23/MA-891 cells was detected by RT-PCR. Expression of IL-23 protien in IL-23/MA-891 cells was detected by ELISA and immunocytochemical staining. Expression of MHCI, MHCII, CD80 and CD86 molecules on surface of IL-23/MA-891 cells was examined by flow cytometry. IFN-γsecreted by mouse splenocytes induced with the culture supernatant of IL-23/MA-891 cells was detected with ELISA. The proliferation of IL-23/MA-891, LXSN/MA-891 and MA-891 cells were detected by MTT colorimetry in vitro.2 IL-23/MA-891, LXSN/MA-891 and MA-891 cells were inoculated to TA-2 mice in the subcutaneous tissues respectively. The mouse models carrying tumor were used to observe the tumorigenic activity of the IL-23/MA-891 cells. Three mice of every group were killed on the thirtieth day after inoculation, the spleens and tumor tissues were obtained from mice. The other mice of every group were used to observe survival and tumor size. IFN-γ, TNF-α, IL-12 and IL-4 produced by splenocytes were detected with ELISA. Expression of the MHCI, MHCII, CD80 and CD86 molecules on the surface of tumor cells and the positive cells to expression of CD11c, CD4 and CD8 in splenocytes from three kinds of mice were detected with flow cytometry. Infiltration of CD4+ and CD8+ T cells in tumor tissues from three kinds of mice were detected by immunohistochemistry.Results: 1 Construction of IL-23 gene in MA-891 cells: IL-23 gene was transferred into two packing cells (ecotropicψ2 and amphotropic PA317) in sequence by a retrovirus vector (LXSN), and the positive cellular clones were screened by G418. The retroviruses were used to transduce the mIL-23 gene into MA-891 cells. IL-23/MA-891 cells which can express mIL-23mRNA and produce mIL-23(136.5±2.8ng/L)were obtained. This result suggested that mIL-23 gene had been integrated into the genome of IL-23/MA-891 cells.2 Biological character of IL-23/MA-891 cells: In the presence of Con A, the culture supernatant of IL-23/MA-891 cells can induce the production of IFN-γ(28.5±2.7ng/L) by murine splenocytes which was higher than that induced by LXSN/MA-891 cells and MA-891 cells supernatants(5.1±0.3 ng/L,4.7±0.6ng/L)respectively. The growth of IL-23/MA-891 cells was similar to that of LXSN/MA-891 and MA-891 cells in vitro. Expression levels of MHCI, MHCII, CD80 and CD86 molecules on surface of IL-23/MA-891, LXSN/MA-891 and MA-891 cells were showing no significant difference (P>0.05).3 Antitumor activity of IL-23 in vivo: After inoculation by s.c. injection, the tumor-forming rates of IL-23/MA-891, LXSN/MA-891 and MA-891 cells in mouse were all 100%, while the tumors inoculated with IL-23/MA-891 cells developed obviously slower than those inoculated with other two cells, and the mice survival time was more longer than 120 days. The tumor growth speed of the mice inoculated with LXSN/MA-891and MA-891 cells was similar, their mean survival times were 51±3 days and 50±3 days respectively.4 The splenocytes from the mice inoculated with IL-23/MA-891 cells can secret IFN-γ,TNF-αand IL-12 that were higher than those with other two cells(P<0.01), but the secretion of IL-4 has no difference in the three kinds of mice(P>0.05).5 In the splenocytes from the mice inoculated with IL-23/MA-891 cells, the ratio of CD4+, CD8+ T cells and CD11c positive cells was also obviously higher than those in the splenocytes from the mice inoculated with LXSN/MA-891 and MA-891 cells(P<0.01).6 Compared with the tumor tissues from LXSN/MA-891 and MA-891inoculated mice, the infiltration amounts of CD4+ and CD8+ T cells in the tumor tissues from IL-23/MA-891 inoculated mice was significantly increased(P<0.01).7 Expression levels of MHCI, MHCII, CD80 and CD86 molecules in the tumor tissues from IL-23/MA-891 inoculated mice were higher than those from other two kinds inoculated mice(P<0.01).Conclusion: IL-23/MA-891 cell secreting mIL-23 was obtained. Transfection of mIL-23 gene did not affect the growth of MA-891 cells in vitro. The culture supernatant of IL-23/MA-891 cells can induce murine splenocytes to produce higher level IFN-γ.The antitumor effect of IL-23 secreted by IL-23/MA-891 cells was obviously stronged in vivo, which enhanced the cellular immune function by increasing the infiltration amounts of CD4+ and CD8+ T cells in the tumor tissues and, promoting secretion of IFN-γ, TNF-αand IL-12 cytokines. IL-23 can also increase the amounts of DC in murine splenocytes, antigenicity of tumor tissues and co-stimulation of angtigen presentation. It suggests that IL-23 can prevent tumor escaping from immune system and play an important role in inhibiting the growth of tumor. |
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