Objective To construct the recombinant adeno-associated virus vector with human bone morphogenetic protein 4 gene（AAV-hBMP4）. Methods The hBMP-4 gene primer was designed basing on the corresponding gene sequence in GenBank. EcoR I site was introduced into the upstream of the primer and Sal Ⅰ site into downstream. The hBMP-4 gene was amplifiedwith the template of EX-A0242-M01-hBMP-4, then was cloned into pUC18 vectorto construct recombinant plasmid pUC18-hBMP-4. The plasmids pUC18-hBMP-4 and plasmid pSNAV cut by EcoR Ⅰ and Sal Ⅰenzyme, the fragments were collected and linked with T4 DNA ligase at 16℃ over night, recombinant plasmid pSNAVhBMP-4 was obtained. The recombinant plasmid was then transfected into BHK21 cells using Lipofectamine TM2000. The G418 resistant cells were obtained consequently. Thesecells were infected with HSV1-rc/△UL2 which has the function of packaging andcopying the recombinant AAV. After purification, the construction of recombinant AAV-hBMP-4 was completed. Results The construction of the recombinant pSNAV-hBMP-4 was confirmed by PCR electrophoresis and digestion with restriction enzyme. The gene sequence in the recombinant pSNAV-hBMP-4 wascorrect. The virus titer was about 1.5×1012 μg/ml.The purity of the virus was more than 95% using the SDSPAGE method. Conclusion With this method, high virus titers and purity of AAV-hBMP-4 can be acquired successfully and it is useful to bone tissue engineering.
Objective To use an improved technique to construct the recombinant adeno-associated virus 2 (rAAV2) mediated gene which can transfer human tissue inhibitor of metalloproteinase-1 (TIMP1). Methods Human TIMP1 gene was amplified from pDNR-LIB plasmid by PCR and cloned into the rAAV2 vector pSNAV to recombinant pSNAV-TIMP1, then was transferred into BHK-21 cells by means of lipofectamine. Using G418 selection, a mixed cell named BHK-21/rAAV2-TIMP1 was isolated, which was capable to express TIMP1. The cell was subsequently infected with recombinant herpes simplex virus 1 (rHSV1-rc/△UL2) that was able to package the rAAV2-TIMP1. After purification, rAAV2-TIMP1 was obtained. Results The rAAV2 carrying human TIMP1 gene was constructed successfully. The viral titer of the rAAV2-TIMP1 was 1×1012 v.g./ml. Conclusion rAAV2-TIMP1 was constructed successfully, which would provide experimental basis for carrying the TIMP1 into hepatocellular carcinoma effectively and inhibiting the invasiveness and migratory capacity of hepatocellular carcinoma in vitro and in vivo models.
Objective To study the time effect of the gene expression 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 so as to lay a theoretical foundation for gene therapy of osteonecrosis. Methods The best multipl icity of infection (MOI) of BMSCs transfected with rAAV was detected by fluorescent cell counting. The 3rd generation rabbit bone mesenchymal stem cells (BMSCs) were transfected with rAAV-hVEGF165-internal ribosome entry site (IRES)-hBMP-7 (experimental group) and green fluorescent protein (GFP) labeled rAAV-IRES-GFP (control group), respectively. The expression of GFP was observed by inverted fluorescent microscope. The expressions of hVEGF165 and hBMP-7 were assessed by RT-PCR assay and Western blot assay in vitro. The transfected cells in 2 groups were prepared into suspension with 5 × 106 cells/mL, and injected into the rabbit thigh muscles of experimental group 1 (n=9) and control group 1 (n=9), respectively. The muscle injected with rAAV-IRES-GFP was sl iced by frozen section method and the expression of GFP protein was observed by inverted fluorescent microscope. The expressions of hVEGF165 and hBMP-7 were assessed by Western blot assay and ELISA assay in vivo. Results The best MOI of BMSCs transfected with rAAV was 5 × 104 v.g/cell. In vitro, the expressions of GFP, hVEGF165, and hBMP-7 genes started at 1 day after transfection, the expressions obviously increased at 14 days after transfection, and the expression maintained the b level at 28 days after transfection. In vivo, the expressions of GFP, hVEGF165, and hBMP-7 genes could be detected at 2 weeks after injection, and b expressions were shown at 6 to 8 weeks after injection. The values of hVEGF165 and hBMP-7 were (248.67 ± 75.58) pg/mL and (4.80 ± 0.61) ng/mL respectively in experimental group 1, and were (32.28 ± 8.42) pg/mL and (0.64 ± 0.42) ng/mL respectively in control group 1; showing significant differences between 2 groups (P lt; 0.05). Conclusion The rAAV-hVEGF165-IRES-hBMP-7 has efficient gene expression ability.
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.
【Abstract】ObjectiveTo construct a recombinant adeno-associated virus(rAAV2) vectors carrying the combined transcriptional regulatory sequences of α-fetoprotein enhancer and albumin promoter for the purpose of targeted gene therapy for hepatocellular carcinoma (HCC). MethodsThe fragment of combined transcriptional regulatory sequences of α-fetoprotein enhancer and albumin promoter was amplified through polymerase chain reaction (PCR) and cloned into the promoter site of pAAV-IRES-hrGFP instead of the CMV promotor in AAV Helper-Free System to construct the rAAV2 expression plasmid pAAV-IRES-hrGFP-EP. Then the packaging cell lines (HEK 293 cell) was co-transfected with the pAAV-IRES-hrGFP-EP together with the control plasmid pAAV-RC and pHelper in AAV Helper-Free System by means of lipofectamine.The recombinant adenoassociated virus vector(rAAV2-EP) carrying the combined transcriptional regulatory sequences of α-fetoprotein enhancer and albumin promoter was packaged and amplified in the HEK 293 cell. Then the viral titer was checked by GFP. ResultsThe recombinant adeno-associated virus vector(rAAV2-EP) carrying the combined transcriptional regulatory sequences of α-fetoprotein enhancer and albumin promoter was constructed successfully, the b green fluorescence was observed in HEK 293 cells under fluorescence microscope. The viral titer was 1.2×105. ConclusionConstruction of the recombinant adeno-associated virus vector rAAV2-EP driven by the combined transcriptional regulatory sequences of α-fetoprotein enhancer and albumin promoter would provide a sound basis and improved vector for targeted gene therapy for HCC.
Objective To verify the potential of the recombinant adeno-associated virus 2 (rAAV2) vector as a strategy for human transforming growth factor β1 (hTGF-β1) gene transfer in degenerative intervertebral discs of rabbit, to investigate the gene transduction efficacy and to quantify the biologic effects on the proteoglycan level after gene transferring. Methods Rabbit models of disc degeneration were established by injecting the 25 μL fibronectin fragment (Fn-f, 1 mmol/ L), 4 weeks later,saline with or without virus was injected directly into 96 lumbar discs of 24 mature New Zealand white rabbits (male or female and weighing 1.7-2.2 kg) which were divided into 3 groups (n=8). Group A received the 25 μL rAAV2-hTGF-β1 (1 × 1012 vg/mL); group B received rAAV2-enhanced green fluorescent protein (rAAV2-EGFP); and group C received PBS. Two rabbits of groups A, C were killed 1 week after injection, the immunohistochemical staining for hTGF-β1 was performed on the sl ices of nucleus pulposus (NP) tissues. At 4, 8, and 12 weeks after gene transferring, NP tissues were harvested and cultured to quantify the changes of the proteoglycan level using 35S-sulfate incorporation assay. The expression of EGFP in group B was observed 12 weeks after injection. Results Immunohistochemical staining showed that extensive and intense positive immunohisochemical staining for hTGF-β1 were seen in group A when compared with group C 1 week after gene transferring. The nucleus pulposus tissues from the group A exhibited an increased synthesis of proteoglycan, which was significantly more than that from groups B and C (P lt; 0.05), and no significant difference was observed between group B and group C. The expression of EGFP in group B was high at 12 weeks. Conclusion The discs injected with rAAV2-hTGF-β1 can highly expressed the therapeutic proteins for more than 12 weeks, it is suggested that rAAV2 should be an valid vector for transferring exogenous genes in the degenerative disc. The therapeutic factors hTGF-β1 can efficiently increase the proteoglycan synthesis of the degenerative NP cells.
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 The human amniotic epithel ial cells (hAECs) are a recently identified new type of stem cells.It has previously been shown that hAECs express hepatocyte-related gene and possess intracellular features and functional properties of hepatocytes. The hAECs may be a candidate seed cell for l iver regeneration. To research the survival and migrationin vivo of hAECs via adeno-associated virus-mediated the green fluorescent protein gene (AAV-GFP) transfection, and toexplore the expression of hepatocyte-l ike function. Methods Thirty nude mice (aging 6-8 weeks, half males and females, and weighing 20-22 g) were randomly divided into 3 groups (groups A, B, and C, n=10). The mice of groups A and C were made the 2/3 partial hepatectomy model, and the mice of group B underwent open abdominal operation without hepatectomy. The hAECs transfected by AAV-GFP were transplanted into the inferior end of the spleen in groups A and B with a cell density of 5 × 106/mL and a volume of 0.2 mL; the same volume of normal sal ine was injected in group C. At 4 hours, the nude mice were sacrificed and the samples of l iver, spleen, heart, lung, brain, and kidney were harvested and the general observation, histological observation, and immunofluorescence detection were performed for the hAECs survival, migration, and the functional properties of hepatocytes. Results No tumor tissue was found in l iver and spleen of 3 groups, and HE staining showed no tumor cells. There were a lot of roundl ike and deeply-stained cells with less cytoplasm and large nucleus in the spleen and the l iver of group A; no abnormal cells were found in l iver and spleen of groups B and C and in kidney, heart, bung, and brain of groups A, B, and C. The GFP+ cells were detected in the spleen and l iver of group A with expressing human albumin, but no GFP+ cells was found in l iver and spleen of groups B and C and in heart, kidney, lung, and brain of groups A, B, and C. Conclusion AAV-GFP infected hAECs transplanted into SCID nude mice with hepatectomy can keep the hepatocyte-l ike function. It will be beneficial to further identify their biological characteristics.
In the present study, packaging system composed of pAAV-CMV-GFP, pAAV-RC and pHelper were transfected into human embryonic kidney 293 cells (HEK293 cells) mediated by polyethyleneimine (PEI) to explore an optimal transfection condition. Different total plasmid DNA dosages (1, 2, 3, 4, 5, 6μg) and different PEI/Plasmid ratios (1:1, 3:1, 5:1, 7:1) were tested with detection of green fluorescence protein (GFP) with ImagePro Plus6.0 Software. Then transfection efficiency of the optimized transfection system was further observed for different time periods(12, 24, 36, 48, 60, 72 h). The results showed that total plasmid dosage of 4μg/well with PEI/plasmid ratio of 3:1~5:1 was an efficient transfection condition. Transfection efficiency-time curve was an S-shaped curve. Transfection efficiency reached a plateau at 60 h after transfection. The optimized conditions for PEI-mediated transfection at the optimal time result in enhanced transfection efficiency of triple plasmid into HEK293 cells.