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Objective:To evaluate the role of phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway in reduction of acute lung injury (ALI) by bone marrow mesenchymal stem cells (MSCs)-conditioned medium in rats.Methods:Thirty-two healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 200-250 g, were divided into 4 groups ( n=8 each) according to the random number table method: control group (group C), group ALI, ALI + bone marrow MSCs-conditioned medium group (group M), and ALI + bone marrow MSCs-conditioned medium + LY294002 group (group L). A rat model of ALI was developed by endotracheal inhalation of aerosolized lipopolysaccharide (LPS) 5 mg/kg in anesthetized animals in group ALI, group M and group L. At 1 h after LPS administration, serum-free DMEM medium 1 ml was intravenously injected in group C and group ALI, bone marrow MSCs-conditioned medium 1 ml was intravenously injected in group M, PI3K inhibitor LY294002 30 mg/kg was intraperitoneally injected at 2 h before the model was developed, and bone marrow MSCs-conditioned medium 1 ml was intravenously injected at 1 h after the model was developed in group L. The blood samples were taken from the heart at 24 h after LPS administration, and serum was isolated for determination of the serum levels of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) (by enzyme-linked immunosorbent assay). Then the rats were sacrificed and lung tissues were obtained for determination of the lung wet/dry weight ratio (W/D ratio), expression of Akt and phosphorylated Akt (p-Akt) (by Western blot) and for microscopic examination of the pathological changes which were scored.The p-Akt/Akt ratio was calculated. Results:Compared with group C, the W/D ratio, lung injury score and serum levels of TNF-α and IL-6 were significantly increased, the expression of p-Akt in lung tissues was down-regulated, the ratio of p-Akt/Akt was decreased ( P<0.05), and pathological changes were found in lung tissues in group ALI.Compared with group ALI, the W/D ratio, lung injury score and serum levels of TNF-α and IL-6 were significantly decreased, the expression of p-Akt in lung tissues was up-regulated, the ratio of p-Akt/Akt was increased ( P<0.05), and pathological changes were significantly attenuated in group M, and no significant change was found in the parameters mentioned above in group L ( P>0.05). Compared with group M, the W/D ratio, lung injury score and serum levels of TNF-α and IL-6 were significantly increased, the expression of p-Akt in lung tissues was down-regulated, the ratio of p-Akt/Akt was decreased ( P<0.05), and pathological changes were accentuated in group L. There were no significant differences in the expression of Akt in lung tissues between the four groups ( P>0.05). Conclusions:PI3K/Akt signaling pathway is involved in reduction of ALI by bone marrow MSCs-conditioned medium in rats.
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Objective To evaluate the role of nuclear factor erythroid 2-related factor 2 ( Nrf2)∕heme oxygenase-1 ( HO-1) signaling pathway in bone marrow mesenchymal stem cells ( MSCs)-induced re-duction of endotoxin-induced acute lung injury ( ALI) in rats. Methods Thirty-two clean-grade healthy male Sprague-Dawley rats, weighing 180-250 g, were divided into 4 groups ( n=8 each) using a random number table method:control group (group C), ALI group, MSCs group and brusatol plus MSCs group ( group B+MSCs) . Lipopolysaccharide 20 mg∕kg was intravenously infused to establish the model of acute lung injury. Phosphate buffered saline ( PBS) 1 ml was intravenously infused at 1 h after establishing the model in group ALI. The equal volume of sterile saline and PBS was given instead in group C. PBS (1 ml) containing MSCs 1×106 cells was intravenously infused at 1 h after establishing the model in group MSCs. Nrf2 inhibitor brusatol 0. 4 mg∕kg was intraperitoneally injected every other day during 10 days before estab-lishing the model, and MSCs were given at 1 h after establishing the model in group B+MSCs. Bronchoalve-olar lavage fluid ( BALF) was collected and lung tissues were removed at 6 h after establishing the model. The protein concentration and neutrophil count in BALF were determined, and the wet∕dry weight ratio ( W∕D ratio) was calculated. The pathological changes of lung tissues were observed by hematoxylin-eosin staining. The expression of Nrf2 and HO-1 (by Western blot), activities of myeloperoxidase (MPO) and superoxide dismutase ( SOD) and content of malondialdehyde ( MDA) were determined. Results Com-pared with group C, the W∕D ratio and total cell count and protein concentration in BALF were significantly increased, the MPO activity was enhanced, the MDA content was increased, the SOD activity was weak-ened, and the expression of Nrf2 was up-regulated (P<0. 05), and the pathological changes were accentu-ated in group ALI. Compared with group ALI, the W∕D ratio and total cell count and protein concentration in BALF were significantly decreased, the MPO activity was weakened, the MDA content was decreased, the SOD activity was enhanced, the expression of Nrf2 and HO-1 was up-regulated (P<0. 05), and the pathological changes were significantly attenuated in group MSCs. Compared with group MSCs, the total cell count was significantly increased, the MPO activity was enhanced, the MDA content was increased, the expression of Nrf2 and HO-1 was down-regulated ( P<0. 05) , and the pathological changes were accentua-ted in group B+MSCs. Conclusion Nrf2∕HO-1 signaling pathway is involved in bone marrow MSCs-in-duced reduction of endotoxin-induced acute lung injury in rats.
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Objective To construct F344 rat bone marrow mesenchymal stem cell line (MSC) modified with human hepatocyte growth factor (hHGF) gene.Methods Recombinant virus containing hHGF was obtained by transfecting the packaging cell line 293 FT with lentiviral vector pLV/EF1α-hHGF-IRES-eGFP.MSCs derived from F344 rat bone marrow were then tranfected with packed lentiviral vector.Purified MSCs expressing hHGF was obtained by screening culture with G418.MSCs and MSCs transfected with empty vector were used as control.The expression of hHGF protein was detected by Western blot (eGFP-MSCs).The hHGF-transfected MSCs were cultured in osteoblast-inducing culture medium and osteoblast phenotype was assayed by alizarin Red staining.The cells were also cultured in adipogenesis medium and stained with Oil Red O for identification.Results The expression of hHGF protein was significantly up-regulated in the hHGF-MSCs as compared with MSCs and eGFP-MSCs.hHGF-MSCs readily differentiated into mineralizing cells or adipocytes when incubated in differentiation medium.Conclusion A F344 rat MSC line that stably expresses HGF is successfully established.
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Objective To investigate the effect of human hepatocyte growth factor (hHGF) genetic modification on the ameliorating effects of mesenchymal stem cells (MSCs) implantation on pulmonary microvascular rarefaction in a rat model of pulmonary hypertension (PH).Methods MSCs were obtained from F344 rats and transduced with lentiviral vector modified with human HGF (hHGF-MSCs) or empty vector (EGFP-MSCs).Sixty-six 7 week old male F344 rats weighing 180-250 g were used in this study.PH was induced by left pneumonectomy and subcutaneous monocrotaline (MCT) 60 mg/kg injected at 2 weeks after operation.The animals with PH were randomly divided into 3 groups:control group (group C),EGFP-MSCs group (group E) and HGF-MSCs group (group H).Groups H and E received hHGF-MSCs or EGFP-MSCs 5 × 105 in DMEM 1 ml iv at 3 weeks after subcutaneous MCT injection,while group C received plain DMEM 1 ml.Mean pulmonary arterial pressure (mPAP) was measured and right ventricular hypertrophy and angiogenesis in the lung were assessed and the content of rat HGF (rHGF) and hHGF protein in lung tissue and pulmonary capillary density (by immuno-histochemistry) was measured at 2 weeks after MSCs implantation.The survival rates within 45 days after MCT administration were compared among the 3 groups.Results No hHGF was detected in groups C and E.Both hHGF-MSCs and EGFP-MSCs significantly reduced MPAP and right ventricular hypertrophy and increased pulmonary capillary density and survival rates in groups H and E as compared with group C and the efficacy of hHGF-MSCs was significantly greater than that of EGFP-MSCs.Barium angiography revealed that distal pulmonary vasculature was significantly increased in group H as compared with groups E and C.The survival of the rats receiving hHGF-MSCs was significantly longer in group H than that in groups E and C.Conclusion hHGF genetic modification can improve the ameliorating effects of MSCs implantation on PH-related microvascular rarefaction.
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Objective To investigate the effect of transplantation of bone marrow mesenchymal stem cells (MSCs) genetically modified with human hepatocyte growth factor gene (hHGF) on angiogenesis in the rat lung.Methods Twenty F344 rats,aged 2 months,weighing 200-250 g,were randomly divided into 2 groups ( n =10 each):HGF group and control group (group C).MSCs genetically modified with hHGF was injected through the external jugular vein in group HGF.While the equal volume of DMEM culture medium (1 ml) was given instead in group C.The mean pulmonary artery pressure was detected at 28 days after transplantation.Then the rats were sacrificed and the lungs were removed for determination of the content of hHGF,expression of proliferating cell nuclear antigen (to reflect the degree of endothelial cell proliferation showed by the small pulmonary vessels) and Ⅷ factor (to reflect the density of the small pulmonary vessels),and microscopic examination.Results Compared with group C,no significant change was found in mean pulmonary artery pressure ( P ＞ 0.05),while the content of hHGF,degree of endothelial cell proliferation,and density of the small pulmonary vessels were significantly increased in group HGF ( P ＜ 0.01).No change was found in the structure of the small pulmonary vessels in group HGF.Conclusion Transplantation of MSCs genetically modified with hHGF can promote angiogenesis in the rat lung.
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Objective To investgate the changes in the expression of hepatocyte growth factor (HGF)and c-met in the lungs in a rat model of pulmonary hypertension.Methods Eighty 7 week old male SD rats weighing 180-250 g were randomly divided into 2 groups ( n =40 each ):control group (group C) and pulmonary hypertension group (group PH).Pulmonary hypertension was induced by left pneumonectomy and subcutaneous monocrotaline (MCT) 60 mg/kg 2 weeks later.Pulmonary artery pressure and the ratio between the weight of right ventricle and left ventricle + interventricular septum ( RV/LV + S) were measured at 7,14,21 and 28 d after MCT administration.HGF and c-met protein and mRNA expression and TGF-β content in the lung tissue were determined.Results Pulmonary hypertension and right ventricular hypertrophy associated with hypertrophy of pulmonary artery tunica media and muscularization of small pulmonary arteries developed after MCT administration in PH group.In PH group HGF protein and mRNA expression in the lungs was significantly down-regulated as compared with group C.There were no significant differences in c-met protein and mRNA expression in the lungs between the 2 groups.The TGF-β content in the lungs was significantly increased in group PH as compared with group C.Conclusion Decrease in HGF production in the lungs plays an important role in the pulmonary hypertension.Increasing of pulmonary TGF-β may play an important role in the down-regulation of pulmonary HGF expression during pulmonary hypertension.
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Objective To know the influence of wanning traditional Chinese medicine dipping for newborns on their sleeping quality and appetite. Methods Divided 600 newborns into the traditional Chinese medicine dipping group, swimming group and the single bath group according to their sequence of birth, there were 200 cases in each group. Different nursing cares were used in the different group sepa-rately. Compared the condition of sleep quality and appetite among the three groups. Results The condi-tion of sleep quality and appetite in the traditional Chinese medicine dipping group was significant better than those in the other groups with few cry and scream. Conclusions Wanning traditional Chinese medicine dipping for newborns can ameliorate their quality of sleep and improve their aapetite.
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Objective To investigate the sinusoidal endothelia) cell (SEC) apoptosis induced by hepatic ischemia-reperfusion (I/R) and the effect of propofol on the hepatic cell apoptosis in vivo. Methods For hepatic I/R study we utilized a rat model of 70% liver ischemia according to Kohli, et al . Twenty-four male SD rats weighing 250-350 g were randomly assigned to 3 equal groups of eight animals : group A was subjected to 30 min of liver ischemia followed by 6 h of reperfusion and normal saline was infused iv at the onset of reperfusion, at a rate 10 ml ? kg-1 ? h -1 for 60 min; group B was subjected to the same J/ R as in group A but instead of NS propofol 20 mg " kg was given iv followed by continuous infusion of 0.5% propofol at 50 mg? kg-1 ? h-1 for 60 min; group C underwent sham operation followed by intravenous NS infusion at 10 ml kg h for 60 min. Blood samples and liver biopsies were obtained at the end of 6h of reperfusion, for determination of plasma alanine aminotransferase (ALT) activity and hepatic malondialdehyde (MDA) content, apoptosis and microscopic examination (light and electron microscopy) . Apoptosis was determined both qualitatively and quantitatively by DNA laddering and TUNEL methods. Results In group A there was a significant increase in apoptotic hepatocytes and SEC after I/R as compared with group C ( P
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Objective To determine the feasibility of achieving local transgenic expression in the rat lung using bone marrow mesenchymal stem cells ( MSCs) transfected with Lac-Z gene. Methods Primary cultures of bone marrow MSCs from Lewis rats were transfected with the pSV-?-galactosidase control vector and labelled with a fluorescent, membrane impermeable dye DAPI. The transfected and labelled MSCs (5?105 cells/animal) were injected into the jugular vein of syngenetic recipient rats. The animals were killed at 48 h and 8 wk after injection respectively. The lungs, spleens, livers, kidneys and skeletal muscle were then excised and examined under fluorescene microscope. The transgenic expression of Lac-Z gene was detected by incubating with the X-gal chromogen.Results Only a few DAPI labelled MSCs could be identified in the spleen, liver, kidney and skeletal muscle, whereas a large amount of DAPI labelled MSCs could be identified in the lung and most of them lodged in the lung parenchyma and air sac at 48h and 8wk after intravenous injection of transfected MSCs. After incubation with the X-gal chromogen, microscopic examination showed that a large number of MSCs with multiple intense blue staining were scattered throughout the lung. On the contrary only a few cells with blue staining could be identified in the spleen and kidney and no MSCs with blue staining could be seen in the liver pancreas and skeletal muscle. Conclusion Genetically modified MSCs injected into the jugular vein can target the lung effectively and achieve local transgenic expression for a long time.