OBJECTIVE: To construct eukaryotic expression vector of rat myogenin gene for further study on its functions in skeletal muscle denervated atrophy and repair. METHODS: The cloning vectors (containing full length of myogenin cDNA and two restriction sites: Hind III and Xho I) were first cut by two restriction endonuclease: Hind III and Xho I, and the same as the eukaryotic expression vector; then, the myogenin cDNA and the digested vector were ligated by T4 DNA ligase, and recombinant eukaryotic expression vector was formed. Its length was certificated by agarose gel electrophoresis analysis, digestion with Hind III and Xho I, PCR; and the rightness of the myogenin cDNA sequence was confirmed by sequencing. RESULTS: The results of agarose gel electrophoresis analysis, digestion, and PCR confirmed the right length of inserted DNA, which was the same as the myogenin cDNA, and the sequencing result of pcDNA3-myogenin was identical with the reported. CONCLUSION: pcDNA3-myogenin a eukaryotic expression vector, is successfully constructed.
Objective To study the interferencing and anti-tumor effects of lentiviral vector of siRNA targeting IGF1R and EGFR gene of the liver cancer cell. Methods The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and connected to the pLVTHM vector, named pLVTHM-IGF1R, into whom the EGFR-siRNA expression frame containing H1 promotor synthesized by RT-PCR was cloned to generate pLVTHM-IGF1R-EGFR-siRNA. The 293T cells were cotransfected by 3 plasmids of pLVTHM-IGF1R-EGFR-siRNA, psPAX2 and pMD2G to enclose LVTHM-IGF1R-EGFR-siRNA, which was amplified in large amount and purified by caesium chloride density gradient centrifugation for measurement of virus titer. SMMC7721 cells infected by LVTHM-IGF1R-EGFR-siRNA were infection group, the untreated SMMC7721 cells and blank vector plasmid LVTHM were two control groups (SMMC7721 cell group and blank vector group). The effect of LVTHM-IGF1R-EGFR-siRNA on IGF1R and EGFR expressions of SMMC7721 cells were detected by RT-PCR and Western blot. The antitumor potential of LVTHM-IGF1R-EGFR-siRNA to SMMC7721 cells was evaluated by Cell Counting Kit-8 assay for cell growth and TUNEL for apoptosis respectively. Results LVTHM-IGF1R-EGFR-siRNA was constructed successfully. Functional pfu titers of LVTHM-IGF1R-EGFR-siRNA was 4.58×109 pfu/ml. Protein and mRNA expression of IGF1R and EGFR of infection group were less than those of blank vector group and SMMC7721 cell group （P＜0.05）, LVTHM-IGF1R-EGFR-siRNA was more effective to inhibit the proliferation and promote apoptosis of SMMC7721 cells （P＜0.05）. Conclusion LVTHM-IGF1R-EGFR-siRNA expressing IGF1R-EGFR-siRNA can inhibit the expression of IGF1R and EGFR, and may be used for further investigation of gene therapy of liver cancer.
ObjectiveTo clone full-length cDNA of rat galectin-9 and construct recombinant adenovirus granule containing rat galectin-9 gene. MethodsThe galectin-9 gene was amplified by RT-PCR from rat liver tissue and inserted orientationally into plasmid pDC316-GFP digested by restriction endonucleases NotⅠ and HindⅢ. The recombinant pDC316-GFP-galectin-9 shuttle plasmid was identified by PCR, restriction endonuclease digestion and sequencing, and then co-transfected with rescue plasmid pBHGlox△E1.3Cre into HEK-293 cells by liposome reagent. Recombinant adenovirus vector containing rat galectin-9 gene (Ad5-galectin-9) was generated by sitespecific recombination and confirmed by PCR, and then Ad5-galectin-9 was propagated in HEK-293 cells and purified. The infectious titer of viral stock was determined by TCID50 assay. ResultsConstruction of pDC316-GFP-galectin-9 shuttle plasmid was confirmed to be correct by PCR, restriction endonuclease digestion and sequencing. Construction of recombinant adenovirus Ad5-galectin-9 was confirmed to be correct by PCR. The infective titer of Ad5-galectin-9 was 1.4×109 U/ml. ConclusionRecombinant adenovirus vector containing rat galectin-9 gene (Ad5-galectin-9) is successfully constructed, which provides the foundation of further research on the function of galectin-9 gene.
Objective To study the effect of recombinant adeno-associated virus (rAAV) vector co-expressing human vascular endothel ial growth factor 165 (hVEGF165) and human bone morphogenetic protein 7 (hBMP-7) genes on bone regeneration and angiopoiesis in vivo so as to provide a theoretical basis for the gene therapy of avascular necrosis of thefemoral head (ANFH). Methods Twenty-four male adult New Zealand rabbits were made the ischemic hind l imb model and divided into 4 groups (n=6). The 3rd generation rabbit bone marrow mesenchymal stem cells (BMSCs) were transfected with the following 4 virus and were administered intramuscularly into the ischemic thigh muscle of 4 groups, respectively: rAAVhVEGF165- internal ribosome entry site (IRES)-hBMP-7 (group A), rAAV-hVEGF165-green fluorescent protein (GFP) (group B), rAAV-hBMP-7-GFP (group C), and rAAV-IRES-GFP (group D). At 8 weeks after injection, the blood flow of anterior tibial artery in the rabbit hind l imb was detected by ultrasonographic image. Immunohistochemical staining for CD34 was performed to identify the prol iferation of capillary. Another 24 male adult New Zealand rabbits were made the femur muscle pouch model and divided into 4 groups (n=6). The above 4 BMSCs transfected with rAAV were administered intramuscularly into the muscle pouch. At 8 weeks after injection, X-ray radiography was used to assess orthotopic bone formation, and von Kossa staining to show mineral ization. Results No symptoms of local or systemic toxicity were observed after rAAV injection. At 8 weeks after injection, the ratio of ischemic to normal blood flow and the number of capillaries in group A were the highest among 4 groups (P lt; 0.05). The ratio of ischemic to normal blood flow and the number of capillaries in group B were significantly higher than those in group C and group D (P lt; 0.05). However, there was no significant difference between group C and group D (P gt; 0.05). At 8 weeks after injection, orthotopic ossification and mineral ization were evidently detected in group A and group C, and group A was ber than group C. No obvious evidence of orthotopic ossification and mineral ization were observed in group B and group D. Conclusion rAAV-hVEGF165-IRES-hBMP-7 vector has the biological activities of inductive bone regeneration and angiopoiesis in vivo.
Objective To validate the advantage of repairing bone defect by staphylococcus aureus injection carried in collagen membrane. Methods Twentyfour adult New Zealand rabbits were divided into two groups randomly. After the experimental model of standard bone defect had been made by operation, collagen membrane/staphylococcus aureus injection and staphylococcus aureus injection with the same quantity were transplanted in bone defect areas of the two groups respectively. The reconstructed tissues were observed by general method, X-ray, histology, and immunohistochemistry at 2nd、4th、6th、8th week respectively. Results The experimental group showed that new bone proliferated distinctly in bone defect areaand the proliferation lasted long, and no excessive connective tissue in defectarea. X-ray observation showed that there was continual callus growth in transplantation area in early stage and the distribution of new bones was even in the group. Histological observation showed that there were many new bone growth centers in bone defect area, trabecular bones were sequentially distributed, and mature bone replacement was complete. Immunohistochemical examination showed that bone morphogenetic protein (BMP) could be seen for a long time and BMP took up a large part in the new bone tissues. Conclusion Collagen membrane could prevent parenchyma from penetrating into bone defect area and provide room for new bone growth. As the carrier of staphylococcus, collagen membrane could reduce the overflow of staphylococcus and improve its curative effect as well.
Objective To investigate the neuroprotective effects of recombinant adeno-associated virus (rAAV) expressing vascular endothel ial growth factor (VEGF) on traumatic spinal cord injury (SCI) of rat and its mechanisms. Methods The 144 male Sprague Dawley rats were randomly divided into 4 groups, and each group contained 36 rats. The rats in sham group (group A) received dorsal laminectomy without SCI and microinjection, the rats in model control group (group B), rAAV-green fluorescent protein (GFP) group (group C), and rAAV-hVEGF165-GFP group (group D) received dorsallaminectomy with SCI and injection of 20 μL sal ine, rAAV-GFP viruses, or rAAV-hVEGF165-GFP viruses, respectively. At 3 and 7 days after operation, Basso-Beattie-Bresnahan (BBB) score was used to evaluate the neurologic function. At 7 days after operation, Nissl’s body staining was used to evaluate the histopathological changes; apoptosis was confirmed by transmission electron microscope examination and TUNEL staining; the expression of aquaporin 4 (AQP-4) was detected by Western blot assay. At 1, 3, 5, and 7 days, ELISA assay was used to detect the VEGF165 protein expression. Results According to BBB scores, the neurologic function in group D was significantly better than those in groups B and C at 3 and 7 days after operation (P lt; 0.05). Nissl’s body staining showed that tissue damage in group D was significantly milder than those in groups B and C at 7 days after operation (P lt; 0.05). ELISA results showed that VEGF165 protein expression was slowly-released in low dose in group D, and the expression in group D was significantly higher than that in groups A, B, and C at 3, 5, and 7 days after operation (P lt; 0.05). The results of transmission electron microscope and TUNEL staining showed that apoptosis rate of spinal cord neurons in group D was significantly lower than that in groups B and C at 7 days after operation (P lt; 0.05). The results of Western blot showed that AQP-4 expression in group D was significantly decreased when compared with that in groups B and C at 7 days after operation (P lt; 0.05). Conclusion TherAAV expressing VEGF has neuroprotective effects by inhibiting apoptosis of spinal cord neurons and relieving spinal cord edema.
Objective To investigate the physicochemicalproperties of the calcium phosphate cement (CPC) containing Danshen composite injection and its drug release rate. Methods This experiment included 4 groups and each group contained 6 specimens. CPC (2 g) was mixed with the setting solution that served as thecontrol group; 0.1,0.5 and 1.0 ml of Danshen composites injection (concentration, 1 000 mg/ml; pH, 7.35) were respectively added to CPC (2 g), which were used as the experimental groups 1, 2 and 3. The resulting specimens were investigated by the X-ray diffraction (XRD), the fourier transformed infrared spectroscopy(FTIR), and the scanning electron microscope (SEM).ResultsThe XRD analysis showed that the control group had a typical diffraction pattern of the hydroxypatite (HAP), which was consistent with the standard patternof HAP. When more Danshen was added in the experimental groups, the diffractionpeaks of HAP gradually decreased; when the diffraction angle 2θ was about 25.92°, the HAP peaks disappeared. Based on the FTIR analysis, with an increase of the drug concentration, the absorption peak of the hydroxy groups decreased. The SEM showed that the size of the CPC particle was related to the drug concentration; with an increase of the drug concentration, the CPC particle increased in number, resulting in an increasing trend of coacervation. The elution test showed that the drugrelease rate and capacity varied with the different concentrationsof Danshen. The initial release rate was relatively great, but after 96 hours the rate slowed down, lasting for a long time. Conclusion The physicochemical properties of CPC do not change when a proper dose (0.1 ml/2 g) of Danshen isadded to CPC. The Danshen composite can be effectively released from CPC, and so CPCcan be used as an ideal drugdelivery carrier for Danshen composite.
Bone morphogenetic protein (BMPs) has been so far regarded as one of the highly potent osteoinductive growth factors. Recombinant human bone morphogenetic proteins have been utilized extensively in the disciplines of orthopedics, stomatology, etc. For clinical application, BMPs are usually loaded in carriers with a controlled-release system, to maintain concentration to induce de novo bone formation at the desired site. In this article, the research advancements of the carriers and release systems of BMP are reviewed.
Objective Bioactive borate glass (BG) has good biocompatibil ity and biodegradation. To investigate the feasibilty of bioactive borate glass as a carrier of the antibiotic controlled-releasing by implanting vancomycin-loaded BG (VBG)into the focus of tibia chronic osteomyel itis after debridement. Methods VBG and vancomycin-loaded calcium sulfate (VCS) were prepared with a vancomycin content of 80 mg/g. Sixty-five New Zealand white rabbits, weighing 2.12-3.91 kg (mean, 2.65 kg), were used. The tibia chronic osteomyel itis rabbit models were establ ished by injecting methicill in-resistant Staphylococcus aureus (MRSA, 0.1 mL, 1 × 109 cfu/mL) into the right tibia of 65 rabbits. After 3 weeks of injection, 54 rabbits of successful models were randomly divided into groups A (n=11), B (n=11), C (n=16), and D (n=16). Simple debridement was performed in group A; BG, VCS, and VBG were implanted into the infection sites of groups B, C, and D respectively after thorough debridement. A sample of the debrided tissues was harvested for bacterial examination. The vancomycin serum levels were determined in groups C and D at 1, 2, 4, 10, 24, and 48 hours after operation. The boron serum levels were determined in groups B and D at 10, 24, 48, 72, and 120 hours after operation. After 8 weeks, the effectiveness was assessed radiographically, bacteriologically, and histopathol ogically. Results Ten rabbits died after operation. No vancomycin was detected in group C; the vancomycin level increased gradually, reached the highest level at 4 hours after operation, and then decreased rapidly in group D. No boron was detected in group B; the boron reached the highest serum level at 10 hours after operation, and then decreased gradually in group D. At 8 weeks, calcium sulfate degraded in group C; BG degraded partially in group D; and no obvious degradation was observedin group B. The repair effect was better in group D than in group C. There was no significant difference in radiograph scoring between groups A, B, C and D (P gt; 0.05) before operation, but there was significant difference between group D and groups A, B, C (P lt; 0.05) at 8 weeks after operation. The bacterial culture showed that all the MRSA results were positive in 4 groups. At 8 weeks, the negative rates of MRSA examination were 36.36%, 18.18%, 73.33%, and 81.25% respectively in groups A, B, C, and D, showing significant differences between group D and groups A, B (P lt; 0.05). The histopathological observation showed that a large number of new bones formed and no foreign body reaction occurred in group D. The histopathologic scores of groups A, B, C, and D were 6.45 ± 3.62, 7.55 ± 3.36, 4.27 ± 2.91, and 3.81 ± 3.04 respectively, showing significant differences between group D and groups A, B, and between group C and group B (P lt; 0.05). Conclusion VBG can improve the repair of bone defect in the treatment of chronic osteomyel itis.
Objective To summarize the research progress of microRNA (miRNA) and its non-viral vector in intervertebral disc degeneration (IDD) and to investigate the potential of non-viral vector delivery of miRNA in clinical application. Methods The related literature about the role of miRNA in IDD and its non-viral delivery system was reviewed and analyzed. Results MiRNA can regulate the related gene expression level and further participate in the pathophysiologic process in degenerated intervertebral disc, miRNA delivered by various non-viral vectors has obtained an ideal effect in some diseases. Conclusion MiRNA plays a great role in the cellular and molecular mechanisms of IDD, as a safe and effective strategy for gene therapy, non-viral vector provides new possibilities for IDD treated with miRNA.