Study On The Effect Of Hlx In Th1/Th2Balance And Occurrence And Development Of Brain/Spinal Cord Injury

ObjectiveHlx plays an important role in embryonic development, gastrointestinal function and the nerve growth of spinal cord, it is also the important transcription factor involved in the differentiation of CD4positive T cells. In view of the roles of Hlx both in growth and development of associated tissue cells, as well as the regulating immune cells, this study focused on the regulation of Hlx on immune function of dendritic cells, and the function of Hlx on the type of immune response in inflammatory induced by brain and spinal cord injury, as well as the effect on immune state of patients with gastric cancer.(1) To investigate the role of transcription factor Hlx in dendritic cells in vitro, we constructed a eukaryotic expression vector of mouse Hlx gene, transferred to the mouse dendritic cell line DC2.4, established a stable Hlx over-expressed dendritic cell line, investigated its biological behavior and functional changes, as well as set up the cell line as a tool to lay the foundation for further study.(2) By using the animal model of cerebral spinal cord injury, we observed the relationship between the expression levels of transcription factor T-bet or GATA3and Th1/Th2which associated cytokines expression in cerebral spinal cord injury in rats, and also the relationship between spinal cord inflammation and T-bet/IFN-y, trying to understand the effects of Hlx, T-bet/IFN-γ on the inflammation induced by cerebral spinal cord injury.(3) By using quantitative PCR, we studied the expression of Hlx mRNA in peripheral blood mononuclear cells of gastric cancer patients, and the relationship between Hlx gene expression level and Th1/Th2balance in patients with gastric cancer, as well as the relation of Hlx expression and the development of gastric cancer.Methods (1) Construction of pIRES2-mHlx-EGFP:The PCR method was used to amplify mHlx gene by PGEM-T plasmids as a template which containing mHlx gene, the mHlx gene was cloned into the eukaryotic expression vector pIRES2-EGFP, and the positive clones were selected and sequenced by PCR, double enzyme digestion.(2)The Screening of DC2.4cell line with Hlx high expression:using liposome method to transfer pIRES2-mHlx-EGFP into DC2.4cells, then treated with600μg/ml G418screening, cloning the DC2.4cells which contained pIRES2-mHlx-EGFP, we obtained the G418-resistant DC2.4cell line which has the stable expression of EGFP. Followed by RT-PCR, Western-blot screening, the DC2.4clone with high expression of Hlx was obtained.(3) The qRT-PCR was used to detect the mRNA of T-bet, Hlx, GATA3, IFN-y, IL-4, IL-12p40, IL-12p35and IL-6in peripheral blood mononuclear cells (PBMC) from patients with gastric cancer or DC2.4/Hlx cells, and the results were compared with healthy control. Meanwhile, the relationship between the expression of Hlx and T-bet in gastric cancer patients was analyzed to understand whether the Thl/Th2imbalance exist in gastric cancer patients was through the level of transcription factor.(4) The expression level of IFN-γ, IL-12P70、TNF-α、IL-10and other related cytokines cell culture supernatant and PBMC were detected by ELISA method.(5) The cell surface molecules were analyzed by flow cytometry:using PE-labeled monoclonal antibody to MHC-I, MHC-II, CD40, CD80, CD86, TLR4and CCR7for cell staining, we analysed the molecules on cell surface.(6) Analysis of dendritic cell phagocytosis:The Escherichia coli carrying red fluorescent protein HcRed (fixed with3%paraformaldehyde) and Hlx high-expressed DC2.4cell line were co-incubated at37℃for2h, we analyzed the phagocytic function of dendritic cells (LPS stimulation group as positive control).(7) Antigen presenting analysis of dendritic cell:Antigen-presenting function of DC was assessed by allogeneic T cell activation responding to a specific antigen. The DC2.4, DC2.4/EGFP and DC2.4/Hlx were cultured with100mg/L ovalbumin (OVA; Sigma) for24h. Spleens from C57BL/6mice immunized by OVA were aseptically obtained and the single cell suspensions were prepared. Purified CD4+T cells were prepared by negative selection with magnetic activated cell sorting (MACS). T cells were stained using3mmol/L fluorescein diacetate succinimidyl ester (CFSE; santa cruz biotechnology, inc) for10minute at37℃and washed three times by complete RPMI medium with10%FBS. In a24-well plate,2×106CD4+T cell per well were co-cultured72h with2×105different lines of DC that were pre-treated by50μg/ml mitomycin C at37℃for20min, respectively. The cells were harvested and green fluorescence from CFSE was analyzed by a FACSCalibur.(8) Detection of cell proliferation by MTT:The method of MTT was used to detect the ability of cellular proliferation. Briefly,2×05CD4+T cells were cocultured with2×104DCs as described above in96-well flat-bottom plates for72h, and then20ul5mg/ml MTT3-(4,5)-dimethylthiahiazo (-z-yl)-3,5-di-phenytetrazoliumro-mide solution (Y-S Biotechnology, China) was added to each well and incubated another4hours, the untransformed MTT was removed carefully by pipetting and150ul DMSO was added.10minutes later, the optical density (OD) was determined in an ELISA reader at570nm and the value of optical density reflected the number of living cells.(9) Preparation of the animal model with brain/spinal cord injury:The physical damage and LPS damage model were used. After intraperitoneal anesthesia with0.3%pentobarbital sodium (10ml/kg), the T12vertebral spinous dorsal midline incision was performed, and the skin and muscle were separated to expose Ti1～L1spinous process and lamina, and then hemostatic forceps bite the T12spinous process and lamina to expose the dorsal spinal cord transection of the spinal cord and sides, thoroughly. The rat limb symmetry convulsion was found, followed by paralysis, and the muscle layer and skin were sutured, the physical damage animal model was established. LPS damage model were achieved by subarachnoid injection of LPS after fully exposed the dorsal and bilateral spinal cord.Results(1) The mHlx high-expressed DC2.4cell line (DC2.4/mHlx) was established:PIRES2-mHlx-EGFP and PIRES2-EGFP were transferred into dendritic cell line DC2.4, the resistant cell lines were screened by G418resistance selection, and the characteristic of expression of homogeneous mHlx in DC2.4/mHlx cell line was confirmed through FACS, RT-PCR, Western-blot identification.(2) The proficiency of DC2.4/mHlx line in promoting Thl cell differentiation was showed. RT-PCR results showed that the expression of Hlx, T-bet, IL-12p35, IL-12p40, IFN-y IL-6mRNA were increased in DC2.4/mHlx line, and the transcription factor GATA3showed no obvious change, and no expression of IL-4. ELISA results showed that the secretion of cytokines IFN-γ and I1-12p70were increased in the supernatant of cell culture.The APC function of DC2.4/Hlx was enhanced:the surface molecular analysis showed that the expression of MHC-I, MHC-II, CD40, CD80, CD86were increased significantly, the phagocytosis was decreased, and the antigen presenting capacities was enhanced.(3) LPS stimulation in brain and spinal cord injury model, The expression levels of T-bet, Hlx or IFN-γin injuried brain and spinal cord tissues were significantly higher than those in control group, There was no significant change of GATA3and IL-4. In addition, ELISA test for TNF-a and IL-10in injury model shows that TNF-α increased at the beginning of brain and spinal cord injury, however, IL-10expression was low at the early stage of the injury and gradually increased at the mid-late stage. These indicate that Thl differentiation occurs at the early stage of brain and spinal cord injury, which is consistent with the increased expression of IFN-γ.(4) The result of Hlx from brain and spinal cord injury suggests that upon LPS stimulation, the increase of Hlx significantly accompanied by the increasing of T-bet and IFN-γ,but GATA3and IL-4did not change obviously. The correlation analysis showed there was high correlation between the expression of Hlx and those of T-bet and IFN-γ.IFN-γ controlled by Hlx, and the T-bet was also closely related to the injury intensity of brain and spinal cord, which indicates that IFN-γ as Th1cell specific cytokines, plays an important role in the brain and spinal cord injury.However, in the brain tissue of the build-off model of spinal cord, the expression of transcription factor T-bet, Hlx and GATA3didn’t show obvious change although the expression of IFN-γ was increased, it was suggested that Thl cells and IFN-γ cytokines from other sources might be released into the damaged brain tissue, and Th1or Th2transcription factors, T-bet or GATA3in the inner nuclear was expressed at low level in brain tissue due to T cells delay in immerse brain tissue or infiltration dipped less brain tissue (5) Hlx expression levels in patients PBMC with gastric cancer by real-time quantitative PCR assay:Our results showed that the mRNA expression levels of Hlx were significantly lower than that of the healthy control group, and was positively correlated with T-bet expression levels, suggesting that the down-regulation of both T-bet and Hlx expression in the gastric cancer patients might be the tumor specific Th1cell function.ConclusionsIn immature dendritic cell line DC2.4, over-expression of the transcription factor Hlx can promote the transcription and expression of IFN-y, and promote the maturation of dendritic cells and secretion of IL-12as well as antigen presenting ability or CD4+T cell proliferation function, but the phagocytosis of DC2.4/Hlx was decreased.Hlx expression levels have been suggested in relation with the immune inflammation by the brain and spinal cord injury, which can promote the damaged body to the advantages of Thl cell differentiation state, immune and inflammatory spinal cord injury plays a secondary role in fueling. Presence of difference distribution of the cytokine IFN-y and Thl cells characteristic of transcription factor T-bet, Hlx in the spinal cord and brain injury, possibly because of T cells immersed in spinal cord injury occurred so characteristic of transcription factors in increased expression of the spinal cord; and T cells delay in immerse brain tissue or infiltration dipped less brain tissue, making the transcription factor T-bet. Hlx expression levels in brain tissue did not change significantly, but Thl cells characteristic cytokine IFN-y still showed up-regulation in the damaged brain tissue.We also investigated the H1x change expression levels which plays the similar important role in gastrointestinal tissue development, and found that the polarization state of Th2cells in gastric cancer patients of gastric cancer showed decreased expression levels with Hlx relevant, Hlx impede the expression of Thl-mediated low anti-tumor immune response.