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Objective To study dynamic changes of gene expressions and protein synthesis of Runx2 (runt-related transcription factor-2), Osterix, osteocalcin and AJ18 inside the femoral head with steroid-induced osteonecrosis after mechanical stress stimulation in rats. Methods A total of 50 Wistar rats (half male in sex) weighing 250-270 g (mean 260 g) were involved in this study and randomly divided into experimental group (40 rats) and normal group (10 rats). The rats in experimental group were injected with dexamethasone (20 mg/kg) via bilateral gluteus maximus alternatively once a week and then trained on laboratory animal treadmill twice weekly to make rat model of femoral head necrosis. After identifying the successfully induced model by Hematoxylin and eosin stain, glucocorticoid injection was ceased and the experimental group was randomly divided into model control group, 4 weeks, 6 weeks, 8 weeks groups after hormone training stopped. Then, total RNA and total protein were extracted from femoral head for detecting dynamic changes of genes expressions and proteins synthesis of Runx2, Osterix, osteocalcin and A J18 after mechanical stress stimulation inside the femoral head with steroid-induced osteonecrosis by means of real-time quantitative PCR and Western blot assay. Results In 4 weeks, 6 weeks, 8 weeks groups after hormone training stopped, the gene expressions and proteins synthesis of Runx2, Osterix and osteocalein were reduced more significantly compared with model control group, mBNA expression values of Runx2, Osterix and osteoealcin were 0. 1809, 0. 1639, 0. 1374 and 0. 4219, 0. 3026, 0.2652 and 0. 2857, 0.2027, 0. 1583 times of those in model control group. The expressions of Runx2, Osterix and osteocalcin showed a downward trend with time. The mBNA expression and protein synthesis of AJ18 at 4th, 6th and 8th weeks after hormone training stopped were 2.6391,4. 2718 and 5. 3165 times of model control group. Conclusions In addition to hormonal factors, inappropriate mechanical stress inhibits expressions and proteins synthesis of Runx2, Osterix and osteocalcin, while the expression and protein synthesis of AJ18 are upgraded in early steroid-induced femoral head necrosis in rats.
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BACKGROUND:Research has been proved that Fasudil,a Rho kinase inhibitor,can effectively inhibit the onset of secondary spinal cord injury.OBJECTIVE:To investigate the synergistic effect of bone marrow masenchymal stem cell (BMSC) transplantion and Fasudil on motor functional recovery following spinal cord injury.DESIGN,TIME AND SETTING:A randomized controlled animal experimant was performed at institute of Endocrinology,Tianjin Medical University from November 2008 to March 2009.MATERIALS:A 1-month-old SD rat was obtained to extract BMSCs.Another 30 healthy female SD rats were used to establish spinal cord injury models,and they were then randomly divided into single injury group,cell transplantation group,and cell transplantation + Fasudil group,with 10 rats for each group.Fasudil was provided by Tianjin Hongri Pharmaceutical Co.,Ltd.METHODS:One week after modeling,spinal cord injury was exposed in the cell transplantation group and cell transplantation+Fasudil group,and 10 μL BMSC suspension was inserted into the injured region.Otherwise,6 hours later rats in the cell transplantation +Fasudil group were treated with an intraperitoneal injection of 10 mg/kg Fasudil,twice a day for one successive week.MAIN OUTCOME MEASURES:Hindlimb motor function was detected using inclined plane test.The Phosphorylated-ERM protein expression was detected by hernatoxylin-eosin staining,pathology and horseradish peroxidase (HRP) nerve trace,and Western blot.RESULTS:Eight weeks after modeling,degree of inclined plane was significantly increased in cell transplantation group and cell transplantation + Fasudil group compared with single injury group (P ＜ 0.05,P ＜ 0.01);while the increased value in the cell transplantation group was significantly greater than cell transplantation + Fasudil group (P ＜ 0.05).Broken myeloid tissue and cavitation were observed in the single injury group;a few of neuraxis-like structures were observed in the cell transplantation group and cell transplantation + Fasudil group,but the cavity in the cell transplantation group was larger than cell transplantation + Fasudil group.HRP-pesitive nerve fibers were detected at T_8 segment or even above in the single injury group and increased in cell transplantation group,in particular in cell transplantation + Fasudil group.Phospho-ERM protein expression in the single injury group and cell transplantation group was significantly greater than cell transplantation + Fasudil group (P ＜ 0.05).CONCLUSION:BMSC transplantation can promote hindlimb motor functional recovery following spinal cord injury,while the combined application of cell transplantation and Fasudil may cause a synergistic effect.
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<p><b>OBJECTIVE</b>To explore the strategies which reduce the amount of xenoantigen Galalpha1,3Gal.</p><p><b>METHODS</b>Human alpha-galactosidase gene and alpha1,2-fucosyltransferase gene were transferred into cultured porcine vascular endothelial cells PEDSV.15 and human alpha-galactosidase transgenic mice were produced. The Galalpha1,3Gal on the cell surface and susceptibility of cells to human antibody-mediated lysis were analyzed.</p><p><b>RESULTS</b>Human alpha-galactosidase gene alone reduced 78% of Galalpha1,3Gal on PEDSV.15 cell surface while human alpha-galactosidase combined with alpha1,2-fucosyltransferase genes removed Galalpha1,3Gal completely. Decrease of Galalpha1,3Gal could reduce susceptibility of cells to human antibody-mediated lysis, especially during co-expression of alpha-galactosidase gene and alpha1,2-fucosyltransferase gene. RT-PCR indicated positive human alpha-galactosidase gene expression in all organs of positive human alpha-galactosidase transgenic F1 mice including heart, liver, kidney, lung, and spleen, the amount of Galalpha1,3Gal antigens on which was reduced largely. 58% of spleen cells from F1 mice were destroyed by complement-mediated lysis compared with 24% of those from normal mice.</p><p><b>CONCLUSIONS</b>Human alpha-galactosidase gene and alpha1,2-fucosyltransferase gene effectively reduce the expression of Galalpha1,3Gal antigens on endothelial cell surface and confers resistance to human serum-mediated cytolysis. The expression of human alpha-galactosidase in mice can also eliminate the Galalpha1,3Gal antigens in most tissues and decrease the susceptibility of spleen cells to human serum-mediated cytolysis.</p>
Subject(s)
Animals , Humans , Mice , Antigens, Heterophile , Metabolism , Cell Death , Cells, Cultured , Disaccharides , Metabolism , Endothelial Cells , Metabolism , Fucosyltransferases , Genetics , Metabolism , Graft Rejection , Genetics , Mice, Transgenic , Spleen , Cell Biology , Swine , Transfection , alpha-Galactosidase , Genetics , MetabolismABSTRACT
Objectives:To establish hybridomas secreting monoclonal antibodies(McAbs) against O6-methylguanine-DNA methyltransferase(MGMT) and to observe the relationship between MGMT expression and clinical responses to ACNU and BCNU in human brain tumors.Methods:The hybridomas were established by cell fusion.MGMT expression in 60 glioma specimens was detected by means of immunohistochemical assay.Results: Seven hybridomas secreting McAbs against MGMT were obtained.Thirty tumor specimens had no detectable or low level of MGMT expression(Mer-), while 30 specimens had high level of MGMT expression(Mer+). The Mer- patients showed more sensitive to ACNU and BCNU than the Mer+ patients.Conclusions: The high specific hybridomas secreting monoclonal antibodies(McAbs) against MGMT were established.The preliminary study indicated that MGMT negative tumors were sensitive to ACNU and BCNU, whereas MGMT positive ones were more resistant to nitrosourea drugs.