SRC kinase family inhibitor PP2 promotes DMSO-induced cardiac differentiation of P19 cells and inhibits proliferation.

BACKGROUND: It has been reported recently that PP2, a Src family kinase inhibitor, promotes selective cardiogenesis in embryonic stem cells. However, there is no other research proved pro-cardiogenic characteristic of PP2 so far. In this study, we explored the potential cardiogenic effect of PP2 on P19 cells differentiation. METHODS: P19-αMHC-EGFP cell line was established by transfecting P19 cells with αMHC-EGFP vector in order to evaluate cardiogenesis with EGFP. P19-αMHC-EGFP cells and P19 cells were induced to differentiate into cardiomyocytes with 1%DMSO, 5 µmol/L PP2, or both 1%DMSO and 5 µmol/L PP2. Differentiated cells from P19-αMHC-EGFP cells were then assessed under confocal microscope. Western-blot and RT-PCR were also performed to detect expression of cardiac troponin I and cardiac transcription factors respectively. In addition, the effects of PP2 on proliferation of P19 cells were further examined using Cell Counting Kit-8. RESULTS: EGFP positive cells were firstly detected on day 7 and PP2 alone cannot induce efficient cardiac differentiation of P19-αMHC-EGFP cells. However PP2 supplementation dramatically increases DMSO induced cardiac differentiation than DMSO alone. It was also found that PP2 inhibit proliferation of P19 cells in both a dose-dependent manner and a time-dependent manner. CONCLUSION: PP2 alone cannot substitute DMSO to induce cardiac differentiation, however, PP2 supplementation drastically promotes DMSO-induced cardiac differentiation of P19 cells. The increased percentages of differentiated cardiac myocytes is partly resulting from cell proliferative inhibit effect of PP2 in undifferentiated P19 cells. P19-αMHC-EGFP cell line has the potential to be used for regenerative therapies in experimental models of heart repair.

Correlation between circulating myeloid-derived suppressor cells and Th17 cells in esophageal cancer.

AIM: To perform a comprehensive investigation into the potential correlation between circulating myeloid-derived suppressor cells (MDSCs) and Th17 cells in esophageal cancer (ECA). METHODS: A total of 31 patients newly diagnosed with ECA and 26 healthy subjects were included in the current study. The frequencies of MDSCs and Th17 cells in peripheral blood were determined by flow cytometry. The mRNA expression of cytokines, arginase 1 (Arg1) and inducible NO synthase (iNOS) in peripheral blood mononuclear cells (PBMCs) and plasma Arg1 were assessed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS: There was an increased prevalence of MDSCs in the peripheral blood from ECA patients (15.21% ± 2.25%) when compared with healthy control (HC) (1.10% ± 0.12%, P < 0.0001). The plasma levels of Arg1 in ECA patients were significantly higher than those in HC (28.28 ± 4.10 ng/mL vs 9.57 ± 1.51 ng/mL, P = 0.0003). iNOS mRNA levels in the peripheral blood of ECA patients also showed a threefold increase compared with HC (P = 0.0162). The frequencies of Th17 cells (CD4⁺IL-17A⁺) were significantly elevated in ECA patients versus HC (3.50% ± 0.33% vs 1.82% ± 0.19%, P = 0.0001). Increased mRNA expression of IL-17 and ROR-γt was also observed in ECA patients compared with HC (P = 0.0041 and P = 0.0004, respectively), while the mRNA expression of IL-6 and tumor necrosis factor-α (TNF-α) showed significant decreases (P = 0.0049 and P < 0.0001, respectively). No obvious correlations were found between the frequencies of MDSCs and Th17 cells in the peripheral blood from ECA patients(r = -0.1725, P = 0.3534). Arg1 mRNA levels were positively correlated with levels of IL-6 (r = 0.6404, P = 0.0031) and TNF-α (r = 0.7646, P = 0.0001). Similarly, iNOS mRNA levels were also positively correlated with levels of IL-6 (r = 0.6782, P = 0.0007) and TNF-α (r = 0.7633, P < 0.0001). CONCLUSION: This study reveals the relationship between circulating MDSCs and Th17 cells, which may lead to new immunotherapy approaches for ECA based on the associated metabolites and cytokines.

[A preliminary study on the effect of lincomycin on the immune function of dendritic cell line DC2.4].

AIM: To explore the effect of lincomycin (lin) on the immune function of dendritic cell (DCs) line DC2.4. METHODS: Three experimental groups, namely, DC2.4 cells group, DC2.4 Cells+LPS group and DC2.4 cells +LPS +lin group were established (LPS and lin were 500 ng/mL). The morphological changes in each group were observed under inverted microscope. The MHC class II , CD86 and CD80 on the DC2.4 cells were detected by flow cytometer. The effects of lipopolysaccharide (LPS) and LPS +lin on the DC2.4 cells immuno-stimulatory capacity were evaluated by allogeneic mixed leukocytes reaction (MLR) between DC2.4 cells and T cells. ELISA was adopted to quantitate the level of IFN-γin the supernatant of cultured DC2.4 cells and T cells in each group. RESULTS: DC2.4 cells showed typical morphology of immature DCs. When stimulated with LPS or LPS combined with lin, DC2.4 cells exhibited typical maturate DCs modality. LPS of 500 ng/mL could significantly up-regulate the expression of MHC class II , CD86 and CD80 on DC2.4 cells and also augment stimulatory action of DC2.4 cells on T cells proliferation and secretion of IFN-γ. However, compared with LPS alone, the treatment of lin (500 ng/mL) combined with LPS down-regulated the immmuno-regulation function of DC2.4 cells. CONCLUSION: Lin can partly inhibit the immuno-regulation function of maturate DC2.4 cells.

[Construction and identification of recombinant adenovirus vector pAdEasy-GFP-GITRL].

AIM: To construct recombinant adenovirus vector pAdEasy-GFP-GITRL and detect the viral titer. METHODS: GITRL gene was obtained by double digestion using Bgl II and Sal I, and cloned into the baculovirus transfer vector(pAdtrack-CMV), then the recombinant adenovirus vector (pAdtrack-CMV-GITRL) was digested by restrictive endoenzyme Pme I. The linear recombinant adenorirus vector and pAdEasy-1 were cotransfected into HEK293 cells by co-precipitate of calcium phosphate. Recombinant adenovirus was packaged and purified in HEK293A cells. RESULTS: Recombinant adenovirus vector pAdEasy-GFP-GITRL was constructed successfully and high titer of recombinant adenovirus was obtained (2.0 x 109 pfu/mL). Western blotting analysis also revealed the expression of GITRL by recombinant adenovirus vector. CONCLUSION: The construction of recombinant adenovirus vector pAdEasy-GFP-GITRL and recombinant adenovirus will facilitate the potential GITRL gene therapy.