ABSTRACT
Spinal nerve injury causes mechanical allodynia and structural imbalance of neurotransmission, which were typically associated with calcium overload. Storeoperated calcium entry (SOCE) is considered crucial elements-mediating intracellular calcium homeostasis, ion channel activity, and synaptic plasticity. However, the underlying mechanism of SOCE in mediating neuronal transmitter release and synaptic transmission remains ambiguous in neuropathic pain. Neuropathic rats were operated by spinal nerve ligations. Neurotransmissions were assessed by whole-cell recording in substantia gelatinosa. Immunofluorescence staining of STIM1 with neuronal and glial biomarkers in the spinal dorsal horn. The endoplasmic reticulum stress level was estimated from qRT-PCR. Intrathecal injection of SOCE antagonist SKF96365 dose-dependently alleviated mechanical allodynia in ipsilateral hind paws of neuropathic rats with ED 50 of 18 μg. Immunofluorescence staining demonstrated that STIM1 was specifically and significantly expressed in neurons but not astrocytes and microglia in the spinal dorsal horn. Bath application of SKF96365 inhibited enhanced miniature excitatory postsynaptic currents in a dosage-dependent manner without affecting miniature inhibitory postsynaptic currents. Mal-adaption of SOCE was commonly related to endoplasmic reticulum (ER) stress in the central nervous system. SKF96365 markedly suppressed ER stress levels by alleviating mRNA expression of C/ EBP homologous protein and heat shock protein 70 in neuropathic rats. Our findings suggested that nerve injury might promote SOCE-mediated calcium levels, resulting in long-term imbalance of spinal synaptic transmission and behavioral sensitization, SKF96365 produces antinociception by alleviating glutamatergic transmission and ER stress. This work demonstrated the involvement of SOCE in neuropathic pain, implying that SOCE might be a potential target for pain management.
ABSTRACT
Objective To derive hematopoietic stem cells with functional properties of hematopoietic reconstitution from murine embryonic stem (ES) cells. Methods ES-D3 cells by formation of the day-4 embryoid bodies (4dEBs) were induced into hematopoietic stem cells by co-culture with murine bone marrow endothelial cell-conditional medium (mBMEC-CM) and the fetal liver stromal cell-conditional medium (FLSC-CM). This experiment was designed to 4 groups (mBMEC-CM + FLSC-CM group, mBMEC-CM group, FLSC-CM group, and the control group). Results The total cell numbers, CD34 + cell numbers, and colony numbers formed in the mBMEC-CM + FLSC-CM group were the highest among the 4 groups. The cells in the mBMEC-CM + FLSC-CM group resumed the hematopoietic system of the mice after being transplanted with the inducing cells. Conclusion The culture condition combing mBMEC-CM with FLSC-CM can promote murine ES cells differentiating into hematopoietic stem cells with functional properties of hematopoietic reconstitution.
ABSTRACT
The present study investigated the effects of the serum-free conditioned media of the bone marrow endothelial cells on CFU-F for potential mechanisms upon which hematopoiesis may be regulated by them within the bone marrow microenvironment. After obtaining the serum-free conditioned media of human and murine purified bone marrow endothelial cells (hBMEC-CM and mBMEC-CM) in vitro, MW > 10 kD, 3 - 10 kD and < 3 kD components were sifted out from these media by means of serial ultrafiltration. Assays of CFU-F were performed to test the effects of BMEC-CM and their ultrafiltrated components. The results showed that every one of hBMEC-CM, mBMEC-CM and their MW < 3 kD components exerted a suppressive effect on the proliferation of corresponding CFU-F but MW > 10 kD and 3 - 10 kD components. The BMEC-CM and MW < 3 kD components decreased the number as well as the size of CFU-F. There were the markedly negative concentration-dependent relations between the concentrations of MW < 3 kD component and the numbers of CFU-F. These findings suggested that bone marrow endothelial cells in culture could secrete at least a humoral factor (molecular weight less than 3 kD) which has an inhibitory effect on the growth of CFU-F.
ABSTRACT
AIM: To purify murine yolk sac endothelial cells (mYS-EC) and investigate the cytokines mRNA expression in mYS-EC. METHODS: The murine yolk sacs were digested with 0.1% collagenase, resuspended in DMEM and counted after digestion and centrifugation. The yolk sac adherent cells were cultured in DMEM containing 15% FBS with 10% mBMEC-CM or 5?g/L VEGF, ECGF and bFGF. The phagocytose function and expression of vWF were evaluated via particle phagocytosis and immunohistochemistry method. Atlas cDNA expression array was used for analysis of cytokine expression in mYS-EC. RESULTS: Colonies consisting of pure yolk sac endothelial cells were obtained in liquid culture system containing 15% FBS and 10% mBMEC-CM or 5?g/L VEGF, ECGF and bFGF. For complete purification of the endothelial cells, subsequent passage was also necessary. Cellular cord formed during passage culture. The endothelial cells were round or oval sharp in morphology, positive in phagocytosis and factor VIII related antigen (von Willebrand's Factor,vWF). The mRNA expressions of cytokines, such as TGF-?2, TNF-?, IFN-?, FL, BMP-4, MIP-1?, BMP-2A, FLT2, endothelin 2, thymosin ?10, IL-6, IL-13, IL-9, SCYA5 and ACBP were detected in mYS-ECs. CONCLUSION: mYS-EC was purified and expanded in vitro . The mRNA expression of 15 kinds of cytokines was detected in mYS-ECs by Atlas arrays.