Objective To construct recombinant adenovirus vector co-expressing human interleukin (hIL)-10 and green fluorescent protein (GFP) for study of the expression of genes of interest in vascular smooth muscle cells （VSMCs）. Methods hIL-10 cDNA was amplified from pUCm-T/hIL-10 cDNA using polymerase chain reaction (PCR), and cloned into shuttle plasmid pShuttle-IRES-hrGFP-1. Kanamycin resistance screeninged for recombinant plasmids, which were linealized with PmeⅠand transformed into BJ5183-AD-1 containing pAdEasy-1 by electroporation after determining the insert’s sequence correct by NotⅠ and XholⅠdigestion， sequencing and basic local alignment search tool (BLAST). Prepared recombinant adenovirus plasmids were transformed into XL10-Gold cells. Amplified plasmids were transfected to AD-293 cells for packaging after being linearized with PacⅠ. PCR was used to determine target gene; The titer of the recombinant adenovirus was measured. VSMCs were transfected by recombinant adenovirus and viewed under fluorescence microscope. hIL-10 concentration in transfected VSMCs supernant was measured by enzyme linked immune sorbent assay (ELISA). Results Recombinant shuttle plasmids contained interest gene. Recombinant adenovirus had 30 kb and 3 kb fragments after digestion with PacⅠ. PCR indicated that the recombinant adenovirus contained interest gene. The titer of recombinant adenovirus was 3×10１0 efu/ml. Transfected VSMCs had GFP expression and hIL-10 concentration in supernatant was 25 ng/106 cells. Conclusion The recombinant adenovirus co-expressing hIL-10 and GFP is successfully constructed and could effectively express in VSMCs, this lays the foundation for the gene therapy of vascular intimal hyperplasia.
ObjectiveTo evaluate the effect of human heme oxygenase 1 augmentation in vein grafts by adenoviral mediated gene transfer of heme oxygenase 1 (Ad hHO 1) on intimal hyperplasia.MethodsTwenty one Japanese white rabbits were divided into three groups: control group, Ad null control group, and Ad hHO 1 group(each group 7 rabbits). During the operation of rabbits jugular vein into carotid artery interposition grafting, harvested rabbit jugular vein segments were exposed for 30min at room temperature to heparin saline, recombinant replication deficient adenovirus encoding hHO 1(Ad hHO 1, 1× 10 9pfu/ml), and nude recombinant replication deficient adenovirus (Ad null, 1×10 9pfu/ml). Quantitative histological studies of the vein segments were performed 28 days after operation. Protein of hHO 1 was detected with method of immunohistochemical staining(S P) in 14 days and 28 days after operation.ResultsThe average intimal thickness, medial thickness and intimal to medial(I/M) ratio were calculated for each group 28 days after bypass operation. Compared to intimal thickness, I/M ratio of control group veins and Ad null group veins,Ad hHO 1 group veins decreased significantly( P lt;0.01). There was no statistically difference in medial thickness ( P gt;0 05). Strong staining of hHO 1 was detected in vein grafts wall of Ad hHO 1 group.ConclusionAd hHO 1 gene therapy may inhibit intimal hyperplasia of vein grafts in rabbits.
Objective To investigate the effects of human insulin-like growth factor 1 (hIGF-1) gene transfected by recombinant adenovirus vector (Ad-hIGF-1) on the apoptosis of rabbit nucleus pulposus cells induced by tumor necrosis factor α (TNF-α). Methods The intervertebral disc nucleus pulposus were harvested from 8 healthy adult domestic rabbits (male or female, weighing 2.0-2.5 kg). The nucleus pulposus cells were isolated with collagenase II digestion and the passage 2 cells were cultured to logarithm growing period, and then they were divided into 3 groups according to culture condition: DMEM/F12 medium containing 10% PBS, DMEM/F12 medium containing 10% PBS and 100 ng/mL TNF-α, and DMEM/ F12 medium containing 10% PBS, 100 ng/ mL TNF-α, and Ad-hIGF-1 (multiplicity of infection of 50) were used in control group, TNF-α group, and Ad-hIGF-1 group, respectively. The results of transfection by adenovirus vector carrying hIGF-1 gene were observed by fluorescent microscopy; the expression of hIGF-1 protein was detected by Western blot, hIGF-1 mRNA expression by RT-PCR, and the cell apoptosis rate by TUNEL and flow cytometry. Results Green fluorescence was observed by fluorescent microscopy in Ad-hIGF-1 group, indicating that successful cell transfection. The expressions of hIGF-1 protein and mRNA were detected in Ad-hIGF-1 group by Western blot and RT-PCR, while the control group and TNF-α group had no expression. The cell apoptosis rates of TNF-α group, Ad-hIGF-1 group, and control group were 34.24% ± 4.60%, 6.59% ± 1.03%, and 0.40% ± 0.15%, respectively. The early apoptosis rates of TNF-α group, Ad-hIGF-1 group, and control group were 22.16% ± 2.69%, 5.03% ± 0.96%, and 0.49% ± 0.05%, respectively; the late cell apoptosis rates were 13.96% ± 4.86%, 10.68% ± 3.42%, and 0.29% ± 0.06%, respectively. Compared with TNF-α group, the cell apoptosis rates of Ad-hIGF-1 group and control group were significantly reduced (P lt; 0.05); the cell apoptosis rate of Ad-hIGF-1 group was significantly higher than that of control group (P lt; 0.05). Conclusion Ad-hIGF-1 could inhibit the apoptosis of nucleus pulposus cells induced by TNF-α.
Objective To investigate the feasibility of rabbit synovial-derived mesenchymal stem cells (SMSCs) differentiating into fibrocartilage cells by the recombinant adenovirus vector mediated by bone morphogenetic protein 2/7 (BMP-2/7) genes in vitro. Methods SMSCs were isolated and purified from 3-month-old New Zealand white rabbits [male or female, weighing (2.1 ± 0.3) kg]; the morphology was observed; the cells were identified with immunocytological fluorescent staining, flow cytometry, and cell cycles. The adipogenic, osteogenic, and chondrogenic differentiations were detected. The recombinant plasmid of pAdTrack-BMP-2-internal ribosome entry site (IRES)-BMP-7 was constructed and then was used to infect SMSCs. The cell DNA content and the oncogenicity were tested to determine the safety. Then infected SMSCs were cultured in incomplete chondrogenic medium in vitro. Chondrogenic differentiation of infected SMSCs was detected by RT-PCR, immunofluorescent staining, and toluidine blue staining. Results SMSCs expressed surface markers of stem cells, and had multi-directional potential. The transfection efficiency of SMSCs infected by recombinant plasmid of pAdTrack-BMP-2-IRES-BMP-7 was about 70%. The safety results showed that infected SMSCs had normal double time, normal chromosome number, and normal DNA content and had no oncogenicity. At 21 days after cultured in incomplete chondrocyte medium, RT-PCR results showed SMSCs had increased expressions of collegan type I and collegan type II, particularly collegan type II; the expressions of RhoA and Sox-9 increased obviously. Immunofluorescent staining and toluidine blue staining showed differentiation of SMSCs into fibrocartilage cells. Conclusion It is safe to use pAdTrack-BMP-2-IRES-BMP-7 for infecting SMSCs. SMSCs infected by pAdTrack-BMP-2-IRES-BMP-7 can differentiate into fibrocartilage cells spontaneously in vitro.
Objective To construct a recombinant adenovirus vector pAdxsi-GFP-NELL1 that co-expressing green fluorescent protein (GFP) and homo sapiens NEL-l ike 1 (NELL1) protein (a protein bly expressed in neural tissue encoding epidermal growth factor l ike domain), to observe its expression by transfecting the recombinant adenovirus into rat bone marrow mesenchymal stem cells (BMSCs) so as to lay a foundation for further study on osteogenesis of NELL1 protein. Methods From pcDNA3.1-NELL1, NELL1 gene sequence was obtained, then NELL1 gene was subcloned into pShuttle-GFP-CMV (-)TEMP vector which was subsequently digested with enzyme and insterted into pAdxsi vector to package the recombinant adenovirus vector (pAdxsi-GFP-NELL1). After verified by enzyme cutting and gel electrophoresis, pAdxsi-GFPNELL1 was ampl ified in HEK293 cells and purified by CsCl2 gradient purification, titrated using 50% tissue culture infective dose (TCID50) assay. The rat BMSCs were cultured and identified by flow cytometry and directional induction, then were infected with adenoviruses (pAdxsi-GFP-NELL1 and pAdxsi-GFP). NELL1 expression was verified by RT-PCR and immunofluorescence; GFP gene expression was verified by the intensity of green fluorescence under fluorescence microscope. Cell counting kit-8 (CCK-8) was used for investigate the influence of vectors on the prol iferation of rat BMSCs. Results Recombinant adenoviral vector pAdxsi-GFP-NELL1, which encodes a fusion protein of human NELL1, was successfully constructed and ampl ified with titer of 1 × 1011 pfu/mL. The primary BMSCs were cultured and identified by flow cytometric analysis, osteogenic and adipogenic induction, then were used for adenoviral transfection efficiency and cell toxicity tests. An multipl icity of infection of 200 pfu/cell produced optimal effects in transfer efficiency without excessive cell death in vitro. Three days after transfection with 200 pfu/cell pAdxsi-GFP-NELL1 or pAdxsi-GFP, over 60% BMSCs showed green fluorescent by fluorescence microscopy. Imunofluorescence with NELL1 antibody also revealed high level expression of human NELL1 protein in red fluorescent in these GFP expressing cells. RT-PCR analysis confirmed that the exogenous expression of NELL1 upon transfection with pAdxsi-GFPNELL1 at 200 pfu/cell, whereas NELL1 remained undetectable in Ad-GFP-transfected rat BMSCs. The prol iferative property of primary rat BMSCs after adenoviral NELL1 transfection was assayed by CCK-8 in growth medium. Growth curve demonstratedno significant difference among BMSCs transfected with pAdxsi-GFP-NELL1, pAdxsi-GFP, and no treatment control at 7 days (P gt; 0.05). Conclusion Recombinant adenovirus vector pAdxsi-GFP-NELL1 can steady expressing both GFP and NELL1 protein after being transfected into rat BMSCs. It provides a useful tool to trace the expression of NELL1 and investigate its function in vitro and in vivo.
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.
Objective To observe the tissue engineered bonefabricated with the cultured mesenchymal stem cells (MSCs) by the green fluorescent protein (GFP) gene transfer. Methods The recombinant Adeno-XTM-GFP expression vector was purified after being packed and proliferated by the HEK293 cells, and then it was used to infect the rabbit’s MSCs directly afer the virus titer was assayed. The cell morphological changes were observed under the inverted phase contrast microscope, and the expression of GFP was observed under the fluorescence microscope to confirm success of the labeling of MSCs.The GPFlabeled MSCs and the pure MSCs were cultured together in the conventional osteogenic supplements for 3 weeks, and then they were seeded onto the compound scaffold of the calcium phosphate cement (CPC) and the fibrin glue (FG) to form a new kind of the tissue engineered bone. It was implanted into the donator rabbit subcutaneously to be used as the experimental group; in contrast, the pure compound scaffold of the CPC-FG without any MSCs was implanted in the same rabbit as a control. The alkline phosphatase (ALP) activity assay was performed respectively at 1, 2 and 3 weeks after operation. GFP was observed under the laserconfocal microscope at 4 weeks after operation, and the formed new bone was harvested at 4 weeks and evaluated by the Masson staining, the immunohistochemistry staining of osteocalcin (OC) and collagen typeⅠ.Results The virus titer was 3×108pfu/ml after proliferation and purification. Expresstionof GFP was confirmed 96 h after MSCs were infected by the Adeno-XTM-GFP expression vector and the infection rate was proximally 50%-70%. In contrast to MSCs, division and proliferation of the GPF-labeled MSCs were not significantly different. The ALP activity in the experimental group (12.546±1.091, 16.567±0.659, 20.443±0.706) was significantly higher than that in the control group (0.453±0.113, 0.243±0.018, 0.308±0.056), respectively at 1, 2 and 3 weeks after operation (Plt;0.05). The tissue engineered bone formed at 4 weeks. There were newly-formed trabeculae around the pore of the compound scaffold, and theimmunohistochemistry staining of OC and collagen typeⅠ were positive. The laser confocal microscope revealed that the GFP-labeled cells existed in many newlyformed tissues，and the compound scaffold of CPC-FG was partly degraded. Conclusion The engineered bone is similar to the spongy bone and the composed cells originate from the cultured MSCs, all of which can be confirmed by the GFP gene transfer technique.