ABSTRACT
Objective To construct a dual-luciferase reporter gene vector and validate the targeting relation ship between miR-299 and the COL4A3 gene,laying a foundation for the study on the effect of miR-299 in the chondrogenic differentiation of stem cells by regulating the COL4A3 gene.Methods This study was made from March,2018 to December,2018.Firstly,the potential binding sites between miR-299 and COL4A3-3'UTR were pre dicted using bioinformatics.Then,the wild and mutant COL4A3-3'UTR sequences were amplified by PCR and cloned into psiCHECK-2 plasmid to construct corresponding recombinant vectors.The vectors were validated by enzyme digestion and gene sequencing.Finally,the cells were resuscitated,amplified,transfected and divided into 4 groups:COL4A3-WT+miR-299/NC group,COL4A3-WT+miR-299-inhibitor/NC-inhibitor group,COL4A3-MUT+miR-299/NC group and COL4A3-MUT+miR-299-inhibitor/NC-inhibitor group.Each group contains 3 holes,respectively.Luciferase activity in each group was determined using a dual-luciferase assay kit.The statistical analysis was conducted and differences between groups were compared by t test.Probabilities lower than 5％(P＜0.05) were considered statistically significant.Results Enzyme digestion and DNA sequencing showed that the dual-luciferase reporter gene vector of psiCHECK-2-COL4A3 was constructed successfully.Luciferase assay demonstrated that in wild COL4A3 gene,luciferase activity reduced in the miR-299 transfection group (The average R/F value was 59.38％) compared with the NC group (The average R/F value was 100.00％),with a statistical significant difference (P＜0.05).In wild COL4A3 gene treated with inhibitor,luciferase activity increased in the miR-299-inhibitor group (The average R/F value was 153.98％) compared with the NC-inhibitor group (The average R/F value was 100.00％),with a statistical significant difference (P＜0.05).In mutant COL4A3 gene treated with inhibitor,no obvious statistical differences in luciferase activity were found between miR-299 transfection group (The average R/F value was 102.09％),miR-299-inhibitor group (The average R/F value was 108.51％) and NC group (The average R/F value was 104.70％),NC-inhibitor group (The average R/F value was 105.13％) and/9＞0.05.Conclusion The dual-luciferase reporter gene vector of the 3'UTR of the COL4A3 gene is constructed successfully.In addition,dual-luciferase assay further verifies the authenticity of miR-299 directly targeting the 3'UTR of the COL4A3 gene.
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Objective To construct MIA3 psicheck2 wild-type and mutant vectors targeting miR-374b,and provide the previous guarantee for the dual luciferase reporter assay.Methods The amplification primer was firstly designed according to mice MIA3-3'UTR sequence information,mice whole blood genomic DNA was taken as the template for PCR amplification of MIA3-3'UTR sequence,and the PCR product was cloned into psicheck2 dual luciferase reporter vector.Then,mutant primer was designed to mutate the MiR-374b seed sequence target TATTATA into AAATTAT so as to construct mutant vector.At last,the vector enzyme digestion evaluation and sequencing method was used to evaluate the constructed vectors.Results It could be seen from the analysis of agarose electrophoresis that the PCR amplification size of vector was consistent with the theoretical size.DNA sequencing evaluation showed that the MIA3-3'UTR-WT vector had been constructed successfully.The construction of mutant vector has successfully mutated the MiR-374b seed sequence target TATTATA into AAATTAT.Conclusion The successful construction of the vector will lay a foundation for the further evaluation on whether there is an actual binding site between the miR-374b and the chondrogenic differentiation-related target gene MIA3.
ABSTRACT
10.3969/j.issn.2095-4344.2013.26.004
ABSTRACT
BACKGROUND:The pathogenesis of avascular necrosis of the femoral head is still uncertain,so the treatment is not favorable.OBJECTIVE:To study the feasibility and mechanism of intramedullary injection with salvia miltiorrhiza for preventing of steroid-induced necrosis of the femoral head.DESIGN,TIME AND SETTING:Randomized controlled animal trial was performed at Zunyi Medical College between April 2005 and May 2007.MATERIALS:Thirty 6-month-old healthy rabbits,half amount of male and female,weighing(2.5?0.25) g,were randomly divided into three groups(n=10):control,model,and prevention groups.METHODS:Normal saline was injected into the control group.Prednisolone was injected into model group,7.5 mg/kg,two times per week for 8 weeks.Glucocorticoid-induced animal model in the prevention group was intramedullary injected with salvia miltiorrhiza,0.3-0.5 cm below the third trochanter of the femur,0.4 mL/kg,twice a week for 8 weeks.MAIN OUTCOME MEASURES:Blood lipids level,serum calcium and phosphorus were measured;X-ray,emission computerized tomographic(ECT) and histopathology were performed.RESULTS:The serum calcium and phosphorus in model group were remarkably reduced.X-ray showed inhomogeneous density of the femoral head,with irregular radiolucent area,obscure structures of bone-trabecula,but intact femoral head and normal joint space.The blood flow and blood pool showed decreasing radioactive distribution of the femoral head,and local concentration of nuclide in the delayed phase in the model group.Histopatholgical observation suggested that the cortical bone was thinned,with thin bone-trabecula and irregular arrangement,necrosed bone marrow and pimelosis and increased bone lacuna.The calcium-phosphorus product of prevent group was increased;X-ray showed normal femoral head appearance and normal bone density except unclear bone-trabecula.ECT showed the dynamic and static images of the prevention group were similar as normal imaging of rabbits.In addition,histopatholgical observation suggested the rate of empty lacuna was decreased,and bone trabecula minimally thinned with ordered arrangement.CONCLUSION:Salvia miltiorrhiza injection plays a good effective role in the prevention of steroid-induced avascular necrosis of femoral head through improving hemorheology and reducing rate of empty lacuna.