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 To construct recombinant lentiviral expression vectors of porcine transforming growth factor β1 (TGF-β1) gene and transfect bone marrow mesenchymal stem cells (BMSCs) so as to provide TGF-β1 gene-modified BMSCs for bone and cartilage tissue engineering. Methods The TGF-β1 cDNA was extracted and packed into lentiviral vector, and positive clones were identified by PCR and gene sequencing, then the virus titer was determined. BMSCs were isolated frombone marrow of the 2-month-old Bama miniature pigs (weighing 15 kg), and the 2nd and 3rd generations of BMSCs wereharvested for experiments. BMSCs were then transfected by TGF-β1 recombinant lentiviral vectors (TGF-β1 vector group)respectively at multi pl icity of infection (MOI) of 10, 50, 70, 100, and 150; then the effects of transfection were detected bylaser confocal microscope and Western blot was used to determine the optimal value of MOI. BMSCs transfected by empty vector (empty vector group) and non-transfected BMSCs (non-transfection group) were used as control group. RT-PCR, immunocytochemistry, and ELISA were performed to detect the expressions of TGF-β1 mRNA, TGF-β1 protein, and collagen type II. Results Successful construction of recombinant lentiviral vectors of porcine TGF-β1 gene was identified by PCR and gene sequencing, and BMSCs were successfully transfected by TGF-β1 recombinant lentiviral vectors. Green fluorescence was observed by laser confocal microscope. Western blot showed the optimal value of MOI was 70. The expression of TGF-β1 mRNA was significantly higher in TGF-β1 vector group than in empty vector group and non-transfection group (P lt; 0.05). Immunocytochemistry results revealed positive expression of TGF-β1 protein and collagen type II in BMSCs of TGF-β1 vector group, but negative expression in empty vector group and non-transfection group. At 21 days after transfection, high expression of TGF-β1 protein still could be detected by ELISA in TGF-β1 vector group. Conclusion TGF-β1 gene can be successfully transfected into BMSCs via lentiviral vectors, and long-term stable expression of TGF-β1 protein can be observed, prompting BMSCs differentiation into chondrocytes.
Objective To introduce the characteristics of tetrahedral framework nucleic acids (tFNA), focusing on its application in the treatment of osteoarthritis (OA) and relationship with microRNA (miRNA), and prospect the application of tFNA in the treatment of OA and the new idea of constructing miR-tFNA functional complex to treat OA. Methods Recent studies were extensively reviewed to analyze the mechanism of tFNA and its relationship with OA and miRNA. Results tFNA, a new type of new carrier, can not only play an indirect role in the treatment of OA as a small molecular carrier with therapeutic effect, but also play a direct role through the regulation of chondrocytes. It can bind with the miRNA that can regulate OA. The therapeutic effect of constructing tFNA functional complex loaded with miRNA has been verified in various diseases, and tFNA has advantages compared with other vectors. Conclusion tFNA, a novel framework nucleic acid structure, plays an important role in the treatment of OA. Constructing miR-tFNA functional complex may be an innovative idea in the treatment of OA.
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.
Objective To investigate the ability of gene-loaded lipopolysaccharide-amine nanopolymersomes (LNPs) in inducing osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by in vitro gene transfection, where LNPs were used as a non-viral cationic carrier, and their properties were optimized during synthesis. Methods LNPs were synthesized by a graft-copolymerization method, and the effects of different pH environments during synthesis on physicochemical properties of LNPs and LNPs/plasmid of bone morphogenetic protein 2-green fluorescent protein (pBMP-2-GFP) complexes were explored. Then, optimized LNPs with maximum transfection efficiency and safe cytotoxicity in rat BMSCs were identified by cytotoxicity and transfection experiments in vitro. Thereafter, the optimized LNPs were used to mediate pBMP-2-GFP to transfect rat BMSCs, and the influences of LNPs/pBMP-2-GFP on osteogenic differentiation of BMSCs were evaluated by monitoring the cell morphology, concentration of BMP-2 protein, activity of alkaline phosphatase (ALP), and the formation of calcium nodules. Results The nitrogen content, particle size, and zeta potential of LNPs synthesized at pH 8.5 were lower than those of the other pH groups, with the lowest cytotoxicity (96.5%±1.4%) and the highest transfection efficiency (98.8%±0.1%). After transfection treatment, within the first 4 days, BMSCs treated by LNPs/pBMP-2-GFP expressed BMP-2 protein significantly higher than that treated by Lipofectamine2000 (Lipo)/pBMP-2-GFP, polyethylenimine 25K/pBMP-2-GFP, and the blank (non-treated). At 14 days after transfection, ALP activity in BMSCs treated by LNPs/pBMP-2-GFP was higher than that treated by Lipo/pBMP-2-GFP and the blank, comparable to that induced by osteogenic medium; with alizarin red staining, visible calcium nodules were found in BMSCs treated by LNPs/pBMP-2-GFP or osteogenic medium, but absent in BMSCs treated by Lipo/pBMP-2-GFP or the blank with apoptosis. At 21 days after transfection, transparent massive nodules were discovered in BMSCs treated by LNPs/pBMP-2-GFP, and BMSCs exhibited the morphologic features of osteoblasts. Conclusion LNPs synthesized at pH 8.5 has optimal transfection efficiency and cytotoxicity, they can efficiently mediate pBMP-2-GFP to transfect BMSCs, and successfully induce their directional osteogenic differentiation, whose inducing effect is comparable to the osteogenic medium. The results suggest that gene transfection mediated by LNPs may be a convenient and effective strategy in inducing directional differentiation of stem cells.
Objective To evaluate the characterization, biocompatibil ity in vitro and in vivo, and antimicrobial activity of an injectable vancomycin-loaded borate glass/chitosan composite (VBC) so as to lay the foundation for its further cl inical application. Methods The sol id phase of VBC was constituted by borate glass and vancomycin, liquid phase was a mixture of chitosan, citric acid, and glucose with the proportion of 1 ∶ 10 ∶ 20. Solid phase and liquid phase was mixed withthe ratio of 2 ∶ 1. Vancomycin-loaded calcium sulfate (VCS) was produced by the same method using calcium sulfate instead of borate glass and sal ine instead of chitosan, as control. High performance liquid chromatography was applied to detect the release rate of antibiotics from VBC and VCS, and minimum inhibitory concentration (MIC) was tested by using an antibiotic tube dilution method. The structure of the VBC and VCS specimens before and 2, 4, 8, 16, and 40 days after immersion in D-Hank’s was examined by scanning electron microscopy, and the phase composition of VBC was analysed by X-ray diffraction after soaked for 40 days. Thirty-three healthy adult New Zealand white rabbits (weighing, 2.25-3.10 kg; male or female) were used to establ ish the osteomyel itis models according to Norden method. After 4 weeks, the models of osteomyel itis were successfully established in 28 rabbits, and they were randomly divided into 4 groups (groups A, B, C, and D). In group A (n=8), simple debridement was performed; in groups B and C (n=8), defect was treated by injecting VCS or VBC after debridement; and in group D (n=4), no treatment was given. The effectiveness of treatment was assessed using radiological and histological techniques after 2 months. Results The releases of vancomycin from VBC lasted for 30 days; the release rate of vancomycin reached 75% at the first 8 days, then could reached more than 90%. The releases of vancomycin from VCS lasted only for 16 days. The MIC of VBC and VCS were both 2 μg/mL. The VCS had a smooth glass crystal surface before immersion, however, it was almost degradated after 4 days. The fairly smooth surface of the VBC pellet became more porous and rougher with time, X-ray diffraction analysis of VBC soaked for 40 days indicated that the borate glass had gradually converted to hydroxyapatite. After 2 months, the best result of treatment was observed in group C, osteomyelitis symptoms disappeared. The X-ray scores of groups A, B, C, and D were 3.50 ± 0.63, 2.29 ± 0.39, 2.00 ± 0.41, and 4.25 ± 0.64, respectively; Smeltzer scores were 6.00 ± 0.89, 4.00 ± 0.82, 3.57 ± 0.98, and 7.25 ± 0.50, respectively. The scores were significantly higher in group D than in groups A, B, and C (P lt; 0.05), and in group A than in groups B and C (P lt; 0.05). The scores were higher in group B than in group C, but no significant difference was found (P gt; 0.05). Conclusion The VBC is effective in treating chronic osteomyelitis of rabbit by providing a sustained release of vancomycin, in addition to stimulating bone regeneration, so it may be a promising biomaterial for treating chronic osteomyelitis.
ObjectiveTo summarize the possible roles and relevant mechanisms of solute carrier family 3 member A2 (SLC3A2) gene in hepatocellular carcinoma (HCC), and explore its clinical application prospects and value in the diagnosis, treatment, and prognosis of patients with HCC. MethodThe literature on reseaches of the SLC3A2 gene and its association with HCC both domestically and internationally in recent years was reviewed and summarized. ResultsNotably, the SLC3A2 exhibited obviously elevated expression in the HCC tissue as compared with the normal liver tissue. It mainly affected the disease progression of HCC by regulating the intracellular and extracellular amino acids transport, inhibiting the ferroptosis of cells, activating the mechanistic target of rapamycin complex signaling pathways and integrin signaling pathway, and played an important role in the diagnosis, treatment, and prognosis of patients with HCC. ConclusionFrom the results of literature review collected, SLC3A2 might be closely associated with the migration, invasion, proliferation, and apoptosis of HCC cell, and it is expected to serve as an indicator for evaluating survival and prognosis of patients with HCC, and become one of the effective treatment targets for HCC in future.
目的 构建含小鼠血管内皮生长因子（mVEGF）的重组慢病毒表达载体，包装成病毒颗粒后感染NS-1小鼠骨髓瘤细胞株，以便进一步探索VEGF在骨髓瘤病理生理机制中的作用。 方法 聚合酶链反应法扩增mVEGF基因，克隆入含嘌呤霉素抗性的pCDH慢病毒表达载体，构建出表达mVEGF的慢病毒表达载体pCDH-mVEGF；采用磷酸钙法将慢病毒系统三质粒pCDH-mVEGF、psPAX2、pMD2.G共转染293FT细胞包装病毒，分别收集转染后48 h和72 h病毒上清并感染靶细胞NS-1，初次感染72 h后开始采用嘌呤霉素筛选稳定株，筛选2周后采用ELISA法检测稳定株细胞培养上清中mVEGF的表达，建立出稳定高表达mVEGF的NS-1小鼠骨髓瘤细胞株。 结果 成功构建重组慢病毒表达质粒pCDH-mVEGF，并包装成慢病毒颗粒，感染NS-1细胞株后获得靶基因的稳定高表达。 结论 成功构建出含mVEGF的慢病毒表达载体pCDH-mVEGF，慢病毒系统能有效介导目的基因在NS-1小鼠骨髓瘤细胞株中稳定表达，病毒包装成功并能有效感染NS-1细胞，为进一步探索VEGF在骨髓瘤病理生理机制中的作用奠定了基础。
Objective To investigate the stable, high effective and low toxicity gene transfer vectors’ basics in research and clinical application.Methods Current status of hepatocytedirected gene transfer vectors were reviewed by history document and contemporary experimental advances analysis.Results Viral and nonviral vector systems could both transduce target genes to liver effectively with various transduction rates based on their corresponding biological characteristics.Conclusion Hepatocyte is the effective target for gene therapy of liverrelated genetic, neoplastic and infectious diseases, although the security needs to be further evaluated.
Objective To clone human bone morphogenetic protein 2 ( BMP-2) gene and construct the gene’s eukaryotic expression vector. Methods The total RNA was extracted from human osteosarcoma cells, the human BMP-2 cDNA was amplified by RT-PCR and inserted into pGEM-T vector. The positive clones were screened out, and the n the recombinant plasmid was confirmed by restriction enzyme digestion, PCR and the analysis of nucleotide sequence. The BMP-2 cDNA in the pGEM-T cloning vec tor was inserted into the pcDNA3.1(+) eukaryotic expression vector. Results The agarose electrophoresis showed that the fragments of BMP-2, pGEMT and pcDNA3.1(+) were 1.2 kbp, 4.0 kbp and 5.0 kbp, respectively. The result of nucleotide sequence confirmed that the cDNA sequence, which was inserted into pGEM-T and pcDNA3.1(+) plasmid was human BMP-2. Conclusion The pcDNA3.1(+)-hBMP-2 eukaryotic vector can be successfully constructed.