Objective To investigate the role of calcium- and integrin-binding protein-1（CIB1） in oxidized lowdensity lipoprotein（OX-LDL） inhibiting migration of mouse macrophages. Methods To silence CIB1 express of mouse macrophages by RNA interference, then incubating mouse macrophages with OX-LDL, cell migration and cell spreading of mouse macrophages were analyzed. Results At 24-72h after macrophages transfected CIB1 siRNA, the express of CIB1 protein was restrained obviously. To silence CIB1 express could increase migration and spreading of mouse macrophages significantly. Conclusions CIB1 plays the important role in intracellular modulating mechanism of OX-LDL inhibiting mouse macrophages migration.
Objective To study the effect of signal-selective parathyroid hormone (PTH) analogue peptide on Wnt signal ing factors in osteoblasts isolated from neonatal mouse, and provide theoretical basis for the mechanism of PTH’s function in bone metabolism. Methods Osteoblasts were isolated from calvaria of 2-3-day-old C57BL neonatal mouse and identified by alkal ine phosphatase (ALP) staining, and Alizarin red staining. The cells at passage 1 were divided into 4 groups: control group, PTH (1-34) group, G1R19 (1-34) group, and G1R19 (1-28) group. Then the medium was changed to α-MEM supplemented with 1%FBS. After 12 hours, trifluoroacetic acid or three peptides [(10 nmol/L PTH (1-34), 10 nmol/L G1R19 (1-34), and 100 nmol/L G1R19 (1-28)] were added into the culture medium. After 4 hours, the cells were washed gently ithcold PBS 3 times before total RNA was isolated. The expressions of Wnt related genes were measured by quantitative eal-time PCR. Results Most of the cells were polygonal and triangular; the cells were positive for ALP staining with blue cytoplasm at 14 days and the Al izarin red staining showed the formation of red mineral ized nodules in the special mineral ization induction medium at 28 days. The expressions of osteocalcin mRNA and Wnt5b mRNA in PTH (1-34) group, G1R19 (1-34) group, and G1R19 (1-28) group were significantly higher than those in control group (P lt; 0.05); the expression of Wnt2 mRNA was significantly lower than that in control group (P lt; 0.05); the expression of β-catenin mRNA in PTH (1-34) group was significantly higher than that in control group (P lt; 0.05); the expression of Wnt7b mRNA in PTH (1-34) group and G1R19 (1- 34) group was higher than that in control group, and the G1R19 (1-34) group was higher than PTH (1-34) group and G1R19 (1-28) group (P lt; 0.05). Conclusion In the Wnt-related factors, PTH (1-34) and G1R19 (1-34) affect mainly canonical Wnt signal factors, but the G1R19 (1-28) chiefly acts on non-canonical Wnt signal factors.
ObjectiveTo establish a mouse model of pressure overload-induced heart failure via suprasternal notch approach. MethodsMale mice were separated into a sham group and an experiment group. Through suprasternal notch approach, the aortic arch port between the origin of the right innominate and left common carotid arteries was partially clipped with tantalum clip, which had a remaining opening of 0.35 mm or 0.25 mm in diameter to cause progres-sively increased afterload. Echocardiography was performed 10 weeks after aortic arch clipped or sham surgery to deter-mine left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular posterior wall end-diastolic thickness (LVPWD), ejection fraction (EF) and fractional shortening (FS). After hemodynamic recordings were completed, mouse body weight (BW) and heart weight (HW) were measured for obtaining HW/BW ratio (mg/g). After heart function examination, mice blood sample was collected for evaluation of serum N-terminal pro-brain natriuretic peptide (NT-proBNP). At the end, part of left ventricular free wall was excised, and hematoxylin and eosin stain was made for histopathological examination. ResultsThe HW/BW, LVEDD and serum NT-proBNP significantly increased in the experiment group compared with those in the sham group (P < 0.01, respectively). The LVPWD, EF and FS significantly decreased compared with the sham group (P < 0.01, respectively). Histopathological examination showed malalignment and rupture of cardiac muscle fibers, hypertrophy and degeneration of myocardial cells, part of which had local or patchy necrosis in left ventricule postoperatively 10 weeks. ConclusionThe model of pressure overload-induced heart failure in mice established through suprasternal approach is simple, minimally invasive and reliable.
Objective To investigate the expression of micro-dystrophin gene in myoblast cultured in vitro, to explore the possibil ity of combining myoblast transplantation with gene transfer for Duchenne muscular dystrophy therapy. Methods Competent Escherichia coli JM109 was prepared, which transformed with plasmid pSL139, and positive clones were picked to cultivate. Plasmid was extracted with Alkal ine lysis method and cutted with both Pvu I and Cla I enzyme. Agarose gel electrophoresis was employed to take pictures. Ten healthy 5-7 days old male C57/BL10 mice were selected, weighing4-5 g, the primary and subcultured myoblasts were cultured with multi-step enzymatic digestion and differential adhesionmethod, and Desmin immunofluorescent method was used to identfy. The 3rd generation myoblasts that were transfected with plasmid pSL139 mediated by l iposome served as the experimental group, untransfected cells served as the control group. After 48 hours of transfection, the expressions of micro-dystrophin mRNA and protein in myoblasts were detected with RTPCR and cell immunofluorescent methods, and the transfection efficiency was caculated. Results After pSL139 plasmids being digested and for 40 minutes agarose gel of electrophoresis, 3.75 kb fragment of target gene and vector were observed. The cells were almost uniform, and triangular or diamond shape after 24-48 hours of culture; the cells turned to fusion manner and could be passaged after 4-6 days. Desmin immunofluorescent result showed that green fluorescence was seen in cytoplasm of most 2nd myoblasts, and the purity of the myoblasts was above 90%. At 48 hours after transfection of myoblasts with plasmid pSL139, RT- PCR results showed that about 300 bp fragment was seen in the experimental group and the control group, and the brightness was higher in experimental group. Immunofluorescent staining displayed that green fluorescence was seen in the cytoplasm of the myoblasts in the experimental group and no green fluorescence in the control group; the expression efficiency of positive cells for micro-dystrophin was 45%-55% in experimental group. Conclusion Micro-dystrophin gene can highly express at the levels of mRNA and protein respectively in myoblasts transfected with plasmid pSL139 mediated by l iposome.
Objective To study the effect of transforming growth factor β1(TGF-β1) and insulin-like growth factor 1(IGF-1) during the induction course from marrow mesenchymal stem cells (MSCs) to chondrocytes and to observe the effect of cell density on cell induction. Methods Differential time adherent methods were used to purify MSCs obtained from the bone marrow of Kunming mice. MSCs were cultured under special conditionsto induce themto differentiate into chondrocytes. Toluidine blue staining and immunofluoresence were used to identify those induced chondrocytes.TGF-β1 and IGF-1 were used individually or in combination under two different culture patterns: pellet culture and monolayer culture. According to different growth factors, experiment included 3 experimental groups(TGF-β1+IGF-1 group,10 ng/mland 50 ng/ml respectively;TGF-β1 group, 10 ng/ml; and IGF-1 group, 50 ng/ml) and control group(without growth factor). In TGF-β1+IGF-1 group, toluidine blue staining and immunofluoresence staining were carried out at 14 days and 21 days. The effect ofTGF-β1 and IGF-1 on the expression of collagen Ⅱgene was detected by RT-PCR at 7, 14 and 21 days of induction; the expressionsof collagen Ⅱ were compared between two culture patterns. Results In TGF-β1+IGF-1 group, the histological examination and immunofluoresence showed that those inducted chondyocytes could express collagen Ⅱ at 14 days. The gel electrophoresis results showed that the fragment of collagen Ⅱ gene was seen in TGF-β1+IGF-1 group andTGF-β1 group and that no fragment ofcollagen Ⅱ gene was seen in IGF-1 group and control group. The expression of collagen Ⅱ gene was ber in TGF-β1+ IGF-1 group than inTGF-β1 group, showing significant difference(Plt;0.05). Cells expressed more collagen Ⅱ under pellet culture than under monolayer culture. Conclusion IGF-1 could enhance the effect ofTGF-β1 during the induction course from MSCs to chondrocytes. A certain extent of high cell density is more effective for MSCs to differentiate into chondrocytes.
Allogeneic mouse model of peritoneal heart transplant is a microscopic surgery on small animal with complex techniques. For a beginner, a learning curve of this surgical technique has to be experienced. The learning curve contains three stages:(1) to be familiar with the local anatomy of either donor or recipient mouse; (2) to be capable of collecting donor heart and well preparing the major peritoneal vessels of recipient; (3) to be skillful in the anastomosis of major vessels. The bottleneck of the learning curve is the valid skill of vascular anastomosis. The stepwise essentials are to "understand, be familiar, be accurate, and be quick" in the learning curve.
【Abstract】 Objective To investigate the role of myosin l ight chain (Myl) in myogenesis in vitro. Methods The extraocular muscle, diaphragm and gastrocnemius muscle myoblasts (eMb, dMb and gMb) were isolated and purified from 12 3-week-old C57BL/6 mice by using the enzyme digestion and Preplate technique, and then were subcultivated. The Myl expression in Mb was detected by RT-PCR and Western blot analysis; the Mb prol iferation activity was tested by methylene blue assay, and the myotube formation was observed. After anti-Myl antibody (1, 2, 3, 8, 16 ng/mL) was induced in the Mb culture (experimental group), the abil ity of prol iferation of myoblasts and the myotube formation were identified. Meanwhile, the Mb which was cultured without anti-Myl antibody was indentified as the control group. Results The results of RT-PCR and Western blot analysis showed that Myl1 and Myl4 mRNA and Myl protein were expressed in eMb, dMb and gMb at 24 hours after seeding, and their expression level were lower in eMb than in dMb and gMb (P lt; 0.01), and the latter two did not show any significant difference (P gt; 0.05). Myl2 and Myl3 mRNA was not detected in these three myoblasts. The prol iferation assay showed that the eMb prol iferated faster as compared with dMb and gMb (P lt; 0.01). eMb began to yield myotubes at 40 hours after seeding and dMb and gMb at 16 hours after seeding. At 6 days, the number of myotubes derived from eMb was (137.2 ± 24.5)/ field, which was significantly larger than that of myotubes from dMb [(47.6 ± 15.5) / field ] and gMb [(39.8 ± 5.1) field ] (P lt; 0.01). There was not statistically significant difference between the latter two groups (P gt; 0.05). After the antibody treatment, the absorbency values of the eMb, dMb and gMb in the experimental groups at each antibody concentration point were significantly higher than those in the corresponding control groups (P lt; 0.05), and the dose-dependent way was performed.The numbers of myotubes from dMb at 16 hours were (48.2 ± 7.1)/ well in the experimental group and (23.4 ± 4.9)/ well in the control group, and at 6 days were (40.6 ± 10.2)/ field in the experimental group and (63.1 ± 6.1)/ field in the control group.There was statistically significant difference between the experimental and control groups (P lt; 0.01). Conclusion Myl may play a role in myogenesis through the negative effect on the myoblast prol iferation.
Objective To investigate the effects of hypertonic saline (HTS) treatment on the function and susceptibility to sepsis of reticuloendothelial system (RES) in mice with hemorrhagic shock. Methods Forty percent of total blood volume of male Balb/c mice was withdrawn by cardiac puncture. Two hours later, the mice were treated with blood infusion and normal saline (10 ml/kg) or 7.5% NaCl (10 ml/kg).The survival rate of the mice was observed after cecal ligation and puncture (CLP). The phagocytosis function of the RES was measured by carbon clearance rate(α) and carbon amount ingested by the macrophages of liver and spleen. In vitro, the peritoneal phagocyte function in solutions of different osmotic pressor was measured by assaying neutral red amount taken in. Results The survival rate after CLP in HTS treated group was 70%, whereas all the mice in the normal saline group died. At the third hour after hemorrhagic shock, the RES carbon clearance rate(α) and carbon amount ingested by the macrophages of liver in the HTS treated mice were 5.61±0.42 and 0.59±0.19 respectively, significantly higher than those in the normal saline treated mice (4.15±0.62， 0.42±0.16). In vitro, hyperosmolarity below 40 mmol/L had no significant effects on the phagocytosis activity of peritoneal macrophages in mice. Conclusion Treating hemorrhagic shock with HTS can decrease the susceptibility to sepsis and improve the RES phagocytosis function indirectly.
Objective To establish a modified mouse abdominal heterotopic heart transplantation model in order to increase the graft survival rate and reduce operative complications. Methods The heart was transplanted into the abdomen by anastomosing the donor ascending aorta and pulmonary artery to the recipient abdominal aorta and infrahepatic vena cava respectively. Hilar tissue was not alone ligated, meanwhile recipient lumbar vein was not ligated. Recipient abdominal aorta and infrahepatic vena cava were not isolated, but were liberated and obstructed simultaneously. Results Two hundred and twenty-nine formal transplantations were performed with the successful rate of 97.82% (224/229). The syngeneic graft survival time was more than 6 months. Complications: Aorta thrombus was found in 2 mice (0.87%), inferior vena cava thrombus in 1 mouse (0.44%), heart torsion in 4 mice (1.75%), hemorrhage in 4 mice (1.75%), crural paralysis in 2 mice (0.87%), intestinal obstruction in 1 mouse (0.44%), and no anesthetic accident happened. Conclusions The meliorated mouse abdominal heterotopic heart transplantation model is simple and reliable, which can reduce the operation time. Thus, the meliorated method provides a useful technique for immunologic transplantation research.
Objective To observe the expressions of CXC chemokine receptor 4 (CXCR4) in muscle satell ite cells in situ of normal and cardiotoxin-intoxicated muscle tissues so as to further investigate the molecular mechanism involving inmuscle regeneration such as progressing muscular dystrophy (PMD) for seeking the way to cure muscle retrogression. Methods The muscle injured model of 12 C57 male mice was made by injecting cardiotoxin (5 μg per mouse) in left quadriceps femoris, their right quadriceps femoris was used as control without any injection. The histological, immunohistochemical analysis and RT-PCR were done to investigate the expression of CXCR4 in the quadriceps femoris in situ after 1 day, 4 days, 1 week, 2 weeks, 4 weeks and 6 weeks. Results HE staining results demonstrated that the muscle tissues experienced the process from muscle injury, repair to regeneration. The result of immunohistochemistry showed that the expressions of CXCR4 in injured muscle tissue were 1 955.6 ± 150.3, 2 223.2 ± 264.3, 2 317.6 ± 178.7, 3 066.5 ± 269.6, 1 770.9 ± 98.7 and 1 505.7 ± 107.1 at 1 day, 4 days, 1 week, 2 weeks, 4 weeks and 6 weeks after injection of cardiotoxin, there was significant difference when compared with normal muscle (640.3 ± 124.0, P lt; 0.001). The RT-PCR showed that the expressions of CXCR4 mRNA in injured muscle tissue were0.822 ± 0.013, 0.882 ± 0.025, 1.025 ± 0.028, 1.065 ± 0.041, 0.837 ± 0.011 and 0.777 ± 0.015 at 1 day, 4 days, 1 week, 2 weeks, 4 weeks and 6 weeks after injection of cardiotoxin, there was significant difference when compared with normal muscle (0.349 ± 0.006, P lt; 0.001). Conclusion CXCR4 may be the critical protein in the process of muscle impairment and reparation.