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BACKGROUND:Adrenomedulin gene transfection can strength the anti-apoptotic ability of bone marrow mesenchymal stem cels under ischemia and hypoxia, but its mechanism is not yet clear. OBJECTIVE:To investigate the effect of adrenomedulin on the expression of apoptosis-related proteins, Bax, Bcl-2 and Caspase-3, in bone marrow mesenchymal stem cels under hypoxia and ischemia. METHODS:Bone marrow mesenchymal stem cels of Sprague-Dawley rats were isolated, cultured and purified, and then cultured in serum-free medium under hypoxic condition for 0, 3, 6, 9, 12 hours. Then, western blot assay was employed to detect the expression of Bax, Bcl-2 and Caspase-3 so as to determine the optimal hypoxia time that was determined at 6 hours of hypoxia. Depending on whether adrenomedulin pretreatment was done, the cels were divided into control group (with no adrenomedulin pretreatment before hypoxia and ischemia) and adrenomedulin groups with different concentrations (1, 10, 100 μg/L). Afterwards, the expression of Bax, Bcl-2 and Caspase-3 was detected by using western blot assay. RESULTS AND CONCLUSION:(1) After cultured in serum-free medium under hypoxia for 0, 3, 6, 9, 12 hours, the expression of Bax, Bcl-2 and Caspase-3 in bone marrow mesenchymal stem cels were increased (P < 0.05);at 6 hours of hypoxia, the Bax/Bcl-2 ratio and Caspase-3 expression reached the minimum value (P < 0.05). (2) At 6 hours of hypoxia, the expression of Bax and Caspase-3 protein as wel as Bax/Bcl-2 ratio became the lowest in the 100 μg/L group compared with the 1 and 10 μg/L groups, but the expression of Bcl-2 protein reached the peak (P < 0.05). These findings indicate that adrenomedulin can reduce the expression of Bax/Bcl-2 ratio and Caspase-3 protein in bone marrow mesenchymal stem cels cultured in serum-free medium under hypoxic conditions, which is in a dose-dependent manner.
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BACKGROUND:Previous studies have demonstrated that hepatocyte growth factor (HGF) gene transfection can improve the effectiveness of bone marrow mesenchymal stem cel transplantation, but the mechanism is stil unclear. OBJECTIVE:To observe the effects of HGF gene transfection on c-MET, Bax, Bcl-2, Caspase-3 of bone marrow mesenchymal stem cel s cultured under hypoxia and serum-free conditions. METHODS:(1) Bone marrow mesenchymal stem cel s were isolated and amplified in vitro by differential adhesion method. The infection efficiency of recombinant adenovirus Ad-HGF in bone marrow mesechymal stem cel s was tested by x-gal staining. (2) Bone marrow mesenchymal stem cel s were cultured under hypoxia and serum-free conditions for 0, 3, 6, 9, 12 hours. RT-PCR and western blot assays were used to evaluate the expression of Bax, Bcl-2, Caspase-3. (3) Bone marrow mesenchymal stem cel s were cultured under hypoxia and serum-free conditions for 6 hours, and RT-PCR and western blot assays were adopted to detect HGF, c-Met, Bax, Bcl-2 and Caspase-3. (4) Cel scratch test was used to detect the effect of HGF transfection on the migration of bone marrow mesenchymal stem cel s cultured under hypoxia and serum-free conditions for 6 hours. RESULTS AND CONCLUSION:(1) Transfection efficiency of bone marrow mesenchymal stem cel s was increased with multiplicity of infection in a dose-dependent manner. When the multiplicity of infection was 150, the transfection efficiency was 96.4%. (2) Expressions of Bax and Bcl-2 were gradual y increased with hypoxia time (P<0.05). The Bax/Bcl-2 ratio and Caspase-3 expression reach the minimum at 6 hours of hypoxia (P<0.05). (3) Compared with the control and Ad-LacZ groups, the expressions of HGF, c-Met, Bcl-2 increased, and the expressions of Bax and Caspase-3 decreased in the Ad-HGF group after 6 hours of culture under hypoxia and serum-free conditions (P<0.05). There was no significant difference between the control and Ad-LacZ groups. (4) The mobility of bone marrow mesenchymal stem cel s was higher in the Ad-HGF group than the control group and Ad-LacZ groups after 6 hours of culture under hypoxia (P<0.05). These findings indicate that transfection of HGF in bone marrow mesenchymal stem cel s can increase the expression of c-Met, Bcl-2 and decrease the expression of Bax, Caspase-3 under hypoxia and serum-free conditions, which also enhance the mobility of bone marrow mesenchymal stem cel s under hypoxia and serum-free conditions.
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Objective To investigate effects of transplantation of mesenchymal stem cells combining with two different gene transfection on the treatment of myocardial infarction in rats. Methods MSCs were isolated and expanded using the preplating method. Ad-ADM or Ad-HGF expression in MSCs and its secretion in culture medium were measured by ELISA. The left anterior descending branch of rats was ligated to establish a myocardial infarction model. Then the pretreated MSCs were labeled by DAPI, through injection directly into the infarcted myocardium at open thorax surgery. Four weeks later, cardiac function was evaluated using echocardiography. Hearts were harvested. Immunohistochemistry staining of factor Ⅷ was used to evaluate the density of capillary vessels in infarction area. Immunohistochemistry staining of troponin I ( TNI) and connexin 43 were used to evaluate the differentiation and expression of implanted MSCs. Fluorescence microscopy was used to identify the DAPI-labeled cells. Results The expression of ADM or HGF was traced in culture medium and has a time-effect relationship. DAPI-labeled transplantion MSCs were founded in the hearts of the recipients and expressed TNI in all MSCs transplantation groups. Immunohistochemical studies demonstrated that intense immunostaining for connexin 43 was higher in Ad-ADM plus MSCs group and Ad-HGF plus MSCs group, compared with MSCs group. A combination of Ad-ADM or Ad-HGF trensfection and MSCs transplantation demonstrated a further increase in capillary density and improved LVEF as compared with MSCs alone. There is no statistical significance between Ad-ADM plus MSCs group or Ad-HGF plus MSCs group in all index. Conclusion Ad-ADM or Ad-HGF transfection enhanced the angiogenic potency of MSCs transplantation and improved cardiac function. The treatment of myocardial infarction in rats in both Ad-ADM plus MSCs group and Ad-HGF plus MSCs group was not able to statistics significance.
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AIM:To observe the effect of micronised fenofibrate(lipanthyl)on lipotoxicity and insulin sensitivity(IS)in spontaneously hypertensive rats(SHR)with high-fat diet.METHODS:Twenty-seven SHR were randomly divided into three groups:normal chow group(n=9),high-fat diet group(n=9)and micronised fenofibrate treatment group(n=9).Micronised fenofibrate 100 mg?kg-1?d-1 was given orally to SHR,which diet on high-fat diet for three months.Intramuscular lipids were observed and lipids accumulation index(LAI)was calculated.Nonesterified fatty acid,glucose and insulin were determined in all rats.RESULTS:(1)Compared to SHR in normal chow diet group,body weight and the level of serum TG and TC increased significantly and the level of HDL-C decreased significantly in SHR fed with high-fat diet(P