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 construct small interfering RNA（siRNA） eukaryotic expression vector specific for human hnRNP K gene，and to observe its silencing effects on hnRNP K gene in A549 cells.Methods The expression vectors of pSUPER/hnRNP K siRNAa，pSUPER/hnRNP K siRNAc and pSUPER/siRNAn were constructed by gene recombination and then transfected into the A549 lung carcinoma cell line by using Lipofectamine2000（a and c respectively represented A and C fragments in hnRNP K coding sequence contained 19 nts，n represented nonsense fragment as control）.The mRNA and protein were harvested after 24 h and analyzed for the expression of hnRNP K by RT-PCR and Western blotting respectively.Results The siRNA vector targeted to hnRNP K successfully decreased hnRNP K mRNA and protein levels 24 h after transfection in A549 cells.Relative expressed doses of hnRNP K mRNA in lung cancer cells transfected by hnRNP K siRNAa and hnRNP K siRNAc respectively were 0.24±0.53 and 0.28±0.57 after 24 h，which were significantly lower than that in the control group（both Plt;0.01）.The gray scale values of hnRNP K protein were 0.23±0.11 and 0.28±0.09 respectively，which were also significantly lower than those in the control group（both Plt;0.05）.And pSUPER/hnRNP K siRNAa was the most effective one.Conclusion Eukaryotic expression vector of siRNA specific for hnRNP K is successfully constructed，which lays the basis for the function study of hnRNP K gene and its application in the treatment of lung carcinoma.
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 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.
ObjectiveTo investigate the effect of expressions of nucleoside transporters subtype (hENT1 and hENT2) on 5-fluorouracil(5-FU) cytotoxicity in breast cancer cell lines(MDA-MB-231, MDA-MB-468, SK-BR-3, MCF-7). MethodsFour breast cancer cell lines were chosen to detect the mRNA expressions of hENT1 and hENT2 by RT-PCR. Cells were incubated in the medium with a serial concentrations of 5-FU from 1.28×104 ng/L to 2.00×108 ng/L for 48 h. Then the cell proliferation in each cell line was measured by MTT assay and the IC50 was evaluated. Results①The mRNA expressions of hENT1 and hENT2 in the MDA-MB-231, MDA-MB-468, or SK-BR-3 cells were significantly higher than thoes in the MCF-7 cells(P < 0.05). The mRNA expression of hENT2 was detected in the MDA-MB-231, MDA-MB-468, or SK-BR-3 cells, not detected in the MCF-7 cells. 2MTT showed that IC50 of 5-FU in the MDAMB-231, MDA-MB-468, or SK-BR-3 cells was significantly lower than that in the MCF-7 cells(P < 0.05). There was no statistical difference of IC50 among the three lines(MDA-MB-231, MDA-MB-468, and SK-BR-3)(P > 0.05).③The three lines(MDA-MB-231, MDA-MB-468, and SK-BR-3) with lower IC50 of 5-FU highly expressed hENTs, and MCF-7 cell with the higher IC50 of 5-FU expressed less hENTs. ConclusionsThe expressions of hENTs in breast cancer cell lines can significantly influence 5-FU cytotoxic effect. It is implicated that the hENTs expressions might be the clue to the choice of nucleoside anticancer drugs in clinic.
Objective To study efficiency and security of the recombinant adenoviralmediated gene transfer to the donor heart during the heart transplantation. Methods A total of 140 healthy male Wistar rats,aged 10 weeks, weighing 200250 g, were equally divided into the donor group and the recipient group, and then 70 rats in the recipient group were randomly andequally divided into 2 subgroups: the gene transfer group and the control group. The rat model of heterotopic heart transplantation(Abdomen)was developed, the donor hearts were removed and their coronary arteries were perfused with 800 μlof the recombinant adenoviral vectors encoding the β-galactosidase gene（Ad-LacZ）. The grafts were stored in the 4℃ cold saline solution for 30 minutes, and then the syngeneic transplant was performed. In the control group, saline of tales doses was perfused. The donor hearts were harvested at 3, 5, 7, 14, and 28days (n=7)after transplantation, and the β-galactosidase activity was assessed by the X-gal staining. At 28 days the major organs of the recipients were tested by the histopathological analysis and the polymerase chain reaction of the adenoviral E1A sequences. Results The successful gene transfer of the βgalactosidase gene was demonstrated in the adenovirus-perfused hearts, with no staining in the control group. The gene expression reached a peak level at 3, 5 and 7 days, and the averaged numbers of the total βgalactosidase positive staining cells per slice were 66.4±23.1, 91.3±32.4 and 68.7±22.7, respectively, with no significant difference between the groups (Pgt;0.05). At 14 days the gene expression gradually declined (32.1±13.9), and the significant difference was found when compared with that at 3, 5 and 7 days (Plt;0.05). At 28 days the cells positive for β-galactosidase were sparse (3.9±3.4), and the gene transfer was significantly less efficient compared with that at 3, 5, 7 and 14 days (Plt;0.05). The major organs of the recipients were not affected seriously at 28 days. No virus spread to other organs in this experimental protocol. Conclusion The ex vivo adenoviralmediated gene transfer intracoronarily to the donor heart during the heart transplantation is feasible and safe.
To construct the recombinant adeno-associated virus (rAAV) vector co-expressinghVEGF165 and hBMP-7 depending on internal ribosome entry site (IRES) sequence, to measure the virus titer and to ver ify the correct recombination. Methods The AAV helper-free system was used to generate the rAAV co-expressing hVEGF165 and hBMP-7 genes. The IRES sequence from the bicistronic eukaryotic expression plasmid pIRES was cut down and subcloned into the ITR/MCS containing vector pAAV-MCS to get pAAV-MCS A-IRES-MCS B, in which upstream MCS A and downstream MCS B was constructed. The hVEGF165 and hBMP-7 genes were ampl ified by PCR and inserted into MCS A and MCS B respectively. The recombinant expression plasmid pAAV-hVEGF165-IRES-hBMP-7 was co-transfected into AAV-293 cells with pHelper and pAAV-RC for packaging of recombinant AAV. The green fluorescent protein (GFP) labeled rAAVIRES GFP was simultaneously packaged by using the parallel plasmid pAAV-IRES-GFP. The efficiency of rAAV packagingwas monitored under fluorescent microscope and recombinant viral particles were harvested from infected AAV-293 cells.The virus titer was measured through infecting AAV-HT1080 cells, and the recombinant rAAV-hVEGF165-IRES-hBMP-7was verified by PCR of the exogenous interest genes of hVEGF165 and hBMP-7. Results The recombinant plasmid pAAVhVEGF165- IRES-hBMP-7 was verified by double digestion. Using the AAV helper-free system, GFP expression could be observed under fluorescent microscope 72 hours after triple plasmid co-transfection and the system provided a high packing ratio of 95%-100%. The rAAV has a high purity and high titer of 5.5 × 1011vp/mL, and AAV-HT1080 cell could be infected at a ratio of 90%. The recombinant virus was confirmed by PCR of exogenous hBMP-7 and hVEGF165 genes. Conclusion Re combinant rAAV-hVEGF165-IRES-hBMP-7 was successfully constructed with a high virus titer, which may offer the basement
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.
This research aims to construct a lentiviral expression vector carrying the extracelluar domain (ED) of human hepatocyte growth factor receptor (C-Met), and to express it in transfected 293T cells. The extracellular domain of C-Met was amplified by RT-PCR, ligated with lentiviral expression vector p RRL-CMV-ED, and then expressed in 293T cell line. The expressed protein was purified and identified by RT-PCR and Western blot. The enzyme digestion and sequence analysis showed that the lentiviral expression vector p RRL-CMV-ED was constructed correctly. The size of amplified genes was about 2 700 bp. The purified protein with Ni-affinity column was about 105 kD analyzed by SDS-PAGE. The Western blot and ELISA results showed that the expressed protein which could bind to HGF specifically was the extracelluar domain of human hepatocyte growth factor receptor. This research may lay a foundation for further study of anti-C-MET monoclonal antibody and neutralizing antibody.