Establishment of STO Cell Lines Expressing Green Fluorescent Protein and Mouse Leukemia Inhibitory Factor / 中国实验血液学杂志

OBJECTIVE@#To establish the STO cell lines expressing green fluorescent protein (GFP) and mouse leukemia inhibitory factor (LIF) , and try to culture the mouse embryonic stem cells (mESCs) by using the established STO-GFP-mLIF cells as the feeder layer.@*METHODS@#The lentiviral particles containing GFP and mLIF and puromycin-resistance gene were constructed and transduced into STO cell lines. The cell lines stably expressing GFP and mLIF genes were screened out. The expression level of the inserted exogenous LIF gene was tested by Western blot and ELISA. The STO-GFP-mLIF cells were treated with different concentrations of mitomycin C (5, 10, 15, 20 µg/ml) for different time (1.5, 2.5, 3, 3.5 hours) to prepare feeder layers and the cell proliferation level on feeder layer was observed. Mouse embryonic stem cells were cultured on mitomycin C-treated feeder layer and the growth of cell colonies was observed.@*RESULTS@#The expression level of LIF protein in STO-GFP-mLIF cells was up-regulated, as compared with STO cells (P＜0.05). It was confirmed that the optimal concentration and time for inhibiting the proliferetion of STO-GFP-mLIF cells by mitomycin C were 10 µg/ml and 3 hours respectively. The observation also found that the embryonic stem cells could develop into typic "birdnest" shaped stem cell colony on mitomycin C-treated feeder layer.@*CONCLUSION@#The stable STO cell lines effectively expressing green fluorescent protein and mouse leukemia inhibitory factor have been established successfully, which can maintain the undifferentiated state of mouse embryonic stem cells.

Role of mitochondrial permeability transition pore in mediating the effect of endomorphin-1 postconditioning against myocardial ischemia-reperfusion injury in rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of mitochondrial permeability transition pore (MPTP) opening in mediating the effect of endomorphine-1 postconditioning to alleviate myocardial ischemia-reperfusion (IR) injury in rats.</p><p><b>METHODS</b>Forty-five male SD rats were randomized equally for sham operation, myocardial IR injury, endomorphin-1 postconditioning, atractyloside (a MPTP opener) postconditioning, or endomorphin-1 + atractyloside postconditioning. The hemodynamic param-eters of the rats were monitored in real time via carotid artery cannulation to the left ventricle. After reperfusion, plasma samples were collected for biochemical analyses. The size of myocardial infarct area was detected using Evans blue and TTC double staining, and the myocardial expressions of apoptosis-related proteins Bax, Bcl-2 and cleaved caspase-3 were analyzed using Western blotting.</p><p><b>RESULTS</b>Myocardial IR injury resulted in significantly decreased heart rate and blood pressure in the rats (P<0.05). Compared with those in IR group, the rats with endomorphin-1 postconditioning showed significantly increased heart rate and blood pressure (P<0.05), lowered contents or activities of LDH, CK-MB, cTnI, IL-6, TNF-α, Cyt-C and MDA in the plasma (P<0.05), increased plasma SOD activity (P<0.05), reduced size of myocardial infarction, decreased myocardial expression of Bax and cleaved caspase-3 protein (P<0.05), and increased myocardial expression of Bcl-2 protein (P<0.05). All these changes induced by endomorphin-1 were obviously reversed by atractyloside postconditioning (P<0.05).</p><p><b>CONCLUSION</b>Endomorphin-1 postconditioning protects against myocardial IR injury in rats probably by inhibiting the opening of MPTP and reducing cardiac myocyte apoptosis via down-regulating cleaved caspase-3 expression.</p>

Effect of Endomorphin-1 on Maturation and Expression of TLR4 in Peripheral Blood Dendritic Cells Induced by High Glucose / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of endomorphin-1 (EM-1) on the maturation phenotype, cytokine secretion, T cell proliferation and TLR4 expression in human peripheral blood dendritic cells (PBDCs) stimulated and induced by high glucose, and to explore the regulatory mechanism of EM-1 on DC immune function.</p><p><b>METHODS</b>Peripheral blood mononuclear cells (PBMNCs) were induced into immature dendritic cells (imDCs). The high glucose was used as the stimulating factor, and the EM-1 was used as the interventional factor. Then, the experiments were divided into normal glucose group (NG group), high glucose group (HG group), high glucose plus EM-1 group (EM group) and high glucose plus EM-1 and naloxone group (Nal group), respectively. The PBDC's phenotype changes were detected by flow cytometry; ELISA was used to detect the changes of cytokines secreted by PBDCs co-cultured with autologous lymphocytes; CFSE was used to detect the proliferation of T lymphocytes. TLR4 expression on PBDC surface was detected by RT-PCR.</p><p><b>RESULTS</b>Compared with HG group, the expression of PBDC surface molecules CD86, CCR7 and CD36 was up-regulated in EM group (P<0.01), while the change of CD83 expression was not statistically significant. However, IL-12 and IL-10 secreted by PBDCs and the proliferation index of T-lymphocytes stimulated by PBDCs were both decreased in EM group. Compared with EM group, the expression of CD86, CCR7 and CD36 was decreased in Nal group (P<0.01), while the expression of CD83 was almost unchanged (P>0.05). T-lymphocyte proliferation index was increased very significantly in Nal group (P<0.01). The gray ratio of TLR4 in HG group was higher than that in NG group, while the gray ratio in EM group's was very significantly lower than that in HG group's (P<0.01). These results indicate that the high glucose can promote the expression of PBDC TLR4, while the EM-1 inhibits the expression of TLR4.</p><p><b>CONCLUSION</b>EM-1 up-regulates the expression of PBDC surface molecules CD86, CCR7 and CD36 stimulated and induced by high glucose, but inhibites the induction of PBDC to maturity by high glucose. And the secreted inflammatory cytokines IL-12 and IL-10 inhibites the proliferation of T lymphocytes derived from PBDCs, while naloxone inhibites the effect of EM-1. EM-1 inhibites the expression of TLR4 on PBDC surface induced by high glucose.</p>

Suppression of Mdr1 gene using RNA interference in cultured rat astrocytes induced by coriaria lactone / 中华神经科杂志

Objective To reverse P-glycoprotein-mediated muhiple drug resistance using RNA interference(RNAi)in cultured rat astrocytes.Methods Astroeytes overexpressing P-glyeoprotein induced by coriaria lactone were transfected with the short-hairpin RNA expression vector-p-SIREN shuttle designed to target Mdrl mRNA.The mRNA level of Mdrl gene was evaluated by real-time PCR;the P-glycoprotein was examined by immunocytochemistry and image analysis,meanwhile the rhodamine efflux was assessed by flow cytometry.Results The astrocyte model overexpressing P-glycoprotein were established and successfully transfected with the short-hairpin RNA expression vector-p-SIREN shuttle.The mRNA level of Mdrl gene was knocked down by 67.70%(P