OBJECTIVE To investigate the role and mechanism of G protein-coupled receptor kinase 2(GRK2)involving in hepatocel ular carcinoma(HCC)progression.METHODS Cel Counting Kit 8 and tumor colony formation assay were designed to detect HCC cell proliferation,wound healing assay was to detect HCC migration.The correlation between GRK2 and early growth response-1(EGR1)were detected by RT-PCR and real-time PCR assays.Co-immunoprecipitation and Western blot assay were adopted to detect the relationship between GRK2and insulin-like growth factor 1 receptor(IGF-1R)signaling pathway.RESULTS In this study we find that GRK2plays an inhibition role in IGF1-induced HCC cell proliferation and migration.Overexpression of GRK2 causes a decrease in EGR1 expression,while knockdown of GRK2 leads to the dramatically increase in EGR1 expression in the treatment of IGF1.Through co-immunoprecipitation and Western blot assay,we confirm that GRK2can interact with IGF-1R and inhibiting IGF1-induced activation of IGF1R signaling pathway.Silencing EGR1attenuates GRK2 overexpression-caused inhibition of cell proliferation,tumor colony number and migrationactivity,while overexpressing of EGR1 restores the antiproliferative and migratory effect by GRK2 overexpression in HCCLM3 cells.CONCLUSION Taken together,these results suggest that GRK2 may inhibit IGF1-induced HCC cell growth and migration through down-regulation of EGR1 and indicate that enforced GRK2 may offer a potential therapeutic approach against HCC.
OBJECTIVE This study aimed to investigate the influence of IgD on T/B cell activation and construct h IgD-Fc-Igfusion protein to competitive inhibition IgD binding with IgDR.METHODS T/B cells were sorted by magnetic cell sorting.The differences of m IgD and IgD-R level between different T/B cell subtypes were detected by FCM.Serum IgD level was detected by ELISA.Human IgD-Fc-IgG1-Fc sequence was amplified by cross-PCR and then subcloned into PET28 a(+) empty vector.After prokaryotic expression through escherichia coli,we obtained the h IgD-Fc-Igfusion protein by affinity chromatograph.Western blot was used to identify the h IgD-Fc-Igfusion protein.Human peripheral blood monouclear cells(PBMC) and fibroblast like synoviocytes(FLS) proliferation were detected using a cell counting kit-8(CCK-8).RESULTS The percentage of CD3^+/CD4^+,CD3^+/IgD^+,CD3^+/CD4^+/IgD^+,CD3^+/IgD-R+and CD3^+/CD4^+/IgD-R+cells increased significantly in RA patients comparing to healthy people.IgD can stimulate PBMC proliferation.IgD(1,3,10,30 μg·mL^(-1)) stimulate PBMC proliferation significantly after 24 h.We obtained stable and active h IgD-Fc-Igfusion protein.The h IgD-Fc-Igfusion protein showed no effect on PBMC proliferation.But it could downregulate human IgD protein promoting proliferation effects in human PBMC.CONCLUSION This result suggests that IgD and IgDR play an important role on T/B cell activation in RA patients and the h IgD-Fc-Igfusion protein may competitively inhibit IgD′s function and may play an therapeutic role in autoimmune diseases.
Rheumatoid arthritis(RA)is an autoimmune disease,which is characterized by synovial inflammation.Hyperplasia sublining macrophages found in synovium is an early hallmark of RA and effective treatment results in their diminution.However,the origin of these sublining macrophages in synovium(including infiltrated macrophages and tissue-resident macrophages)are still unknown both in animal models of arthritis and RA patients,let alone the differences and feature of these macrophages.In rheumatic synovium,macrophages are submitted to a large variety of micro-environmental signals which influence the phenotypic polarization and activation of macrophages.Understanding the mechanisms and functional consequences of the heterogeneous macrophages will contribute to confirm their potential role in synovial inflammation development.Furthermore,research on macrophage plasticity to soft-control their phenotypic polarization could lead to novel therapeutic approaches.
