[Therapeutic Effect of SPK1 Gene Transfected Adipose Derived Mesenchymal Stem Cells on Experimental Autoimmune Encephalomyelitis Mice and Its Effect on T Helper Cell 17/Regulatory T Cells Balance].

Objective To investigate the therapeutic effect of SPK1 gene transfected adipose derived mesenchymal stem cells(ADMSC)on experimental autoimmune encephalomyelitis mice and the effect on T helper cell 17(Th17)/regulatory T(Treg) cells balance. Methods EAE was induced by myelin oligodendrocyte glycoprotein 35-55 in mice.Totally 44 mice were randomly divided into four groups:normal control group(NC group),model group(EAE group),ADMSC group,and ADMSC-SPK1 group.Forty days after injection,the pathological changes of brain and spinal cord,Th17/Treg-related inflammatory markers in brain tissue,expressions of interleukin-17A(IL-17A)and forkhead box protein p3(Foxp3)in brain and spinal cord tissue,and flow cytometric results of spleen immune cells were detected. Results Forty days after the injection,serious inflammatory cell infiltration and demyelination occurred in the brain and spinal cord of EAE group,whereas demyelination and axonal injury were improved in ADMSC group and ADMSC-SPK1 group.Compared with EAE group,the ADMSC group and ADMSC-SPK1 group had significantly improved levels of IL-17A(Z=1.021,P=0.017;Z=1.193, P=0.009)and tumor necrosis factor-α(TNF-α)(Z=2.540,P=0.004; Z=3.005, P=0.006).The expression level of IL-17A in mouse brain and spinal cord tissues was significantly reduced in the ADMSC group(t=10.323,P=0.013;t=7.422,P=0.008),and it was significantly lower in the ADMSC-SPK1 group than in the ADMSC group (t=14.244, P=0.017; t=16.865,P=0.006).The expression level of Foxp3 in the ADMSC group was significantly increased(t=14.544, P=0.008;t=9.420,P=0.002),and it was significantly higher in the ADMSC-SPK1 group than ADMSC group(t=9.654,P=0.005; t=11.535, P=0.009).The proportion of IL-17+IFN-γ+T cells in spleen decreased in ADMSC-SPK1 group,while that of CD25+Foxp3+Treg cells increased. Conclusions ADMSC transfected with SPK1 has an ideal therapeutic effect on EAE mice,and the effect is superior to ADMSC without SPK1 transfection.The mechanism may be that ADMSC-SPK1 can markedly reduce the Th17/Treg cell ratio and decrease the release of related inflammatory cytokines in EAE mice.

[Effect of baicalein on proliferation and migration in multiple myeloma cell lines RPMI 8226 and U266 cells].

OBJECTIVE: To investigate the effect of baicalein on proliferation and migration of multiple myeloma (MM) cell lines and its molecular mechanism. METHODS: The MM cell line RPMI-8226 and U266 cells were used as the model, and treated with different concentration and time of baicalein the effect of baicalein on the MM cells proliferation was assessed by MTT assay. With or without baicalein or Interleukin-6 (IL-6) treatment, the ß-catenin protein level was analyzed by immunofluorescence assay and western blot assay and mRNA levels of ß-catenin, c-myc, cyclin D1 and integrin 7 gene by RT-PCR. Transwell chamber migration assay was used to detect the cells migration ability with different concentration of baicalein cultured. RESULTS: Baicalein inhibited the MM cell line RPMI 8226 and U266 cell proliferation in a dose- and time-dependent manner. It simultaneously inhibited ß-catenin protein level to resist the effect of IL-6 on inducing MM cell proliferation, and resulted in decrease of ß-catenin, c-myc, cyclinD1 and integrin ß7 mRNA levels. Baicalein also decreased migration ability of MM cells in a dose-dependent manner by SDF-1. CONCLUSION: Baicalein can inhibit MM cells proliferation and migration, and its molecular mechanisms are associated with inhibition of proliferation related genes ß-catenin, c-myc, cyclin D1 and integrin ß7 expression.

[Relationship between CD45 molecule expression and apoptotic sensitivity of myeloma cells].

OBJECTIVE: To investigate the effects of CD45 expression on induction of apoptosis in multiple myeloma cells. METHODS: Melphalan was used to induce myeloma cell line U266 apoptosis. Serum-free culture was used to induce CD45RB gene or empty plasmid transfected U266 apoptosis. The glucose-free culture was used to induce high CD45 (CD45(hi)) or low CD45 (CD45(low)) expression AMO1 apoptosis. Intraperitoneal inoculation was used to compare the survival of CD45(-) or CD45(+) U266 cells in mice. The number of apoptotic cells and mitochondrial membrane potential (MMP) was detected by flow cytometry. Western blotting was used to detect the cytochrome C release from mitochondrial and caspase-9 activation. RESULTS: Melphalan treatment induced 45% of CD45(+) and 30% of CD45(-) U266 cells apoptosis. Compared with the CD45(low) AMO1 cells, CD45(hi) cells were more susceptible to apoptosis. In serum-free culture for five days, 60% of CD45RB transferred U266 cells underwent apoptosis, while in the empty plasmid transfected ones, apoptotic cell number was not significantly increased. The survival time of CD45(-) U266 cells in the SCID-hIL-6 mice was 5 times that of CD45(+) cells. After melphalan treatment, 60% of the CD45(+) U266 cells lost MMP, while only 30% of CD45(-) U266 cells, and 10% of control cells did so. After UV irradiation, CD45(+) U266 cells mitochondria released more cytochrome C, leading to more caspase-9 activation. CONCLUSION: CD45 expression is involved in mitochondria-mediated apoptotic process and increases apoptotic sensitivity of myeloma cells under a variety of stimulation.