Objective To investigate the effect of TIMP-2 gene that was transfected by adenovirus on extracellular matrix of abdominal aortic through assessing the changes of morphology and histopathology of the rat models with abdominal aortic aneurysm. Methods The rat models with abdominal aortic aneurysm were constructed by intraluminally perfusing porcine pancreatic elastase. Twenty-four SD rats with aneurysm were then randomly divided into 3 groups: AdTIMP-2 group (perfused locally with solution of TIMP-2 gene transfected by adenovirus vector to abdominal aorta), AdCMV group (transfected by non-viral vector), and PBS group. After 14 days, the concentrations of elastin and collagen that were collected from the samples of aortic wall were measured by image analysis system and the fixed aortic tissues were examined by light microscopy and some other specific staining methods. Results None of abdominal aortic aneurysm developed in TIMP-2 gene transfected group, with significantly higher rates of developed aneurysm in the other groups (P＜0.01). The diameters of arteries on day 14 in the AdTIMP-2 group were (2.33±0.06) mm, which were significantly smaller than those in the AdCMV group 〔（3.52±0.11） mm〕 and PBS group 〔（3.43±0.09） mm〕. The elastic fibers and collagenous fibers were preserved with more integrity in AdTIMP-2 group and inflammation cells that were observed in adventitia of artery were also less than those of the other groups. Conclusion TIMP-2 gene transfected by adenovirus vector could restore the degradation of extracellular matrix that was aroused by elastase and could block the formation of abdominal aortic aneurysm, which may provide a new strategy for the treatment of abdominal aortic aneurysm.
Objective To construct the recombinant of hepatocellular carcinoma-targeting adenovirus containing r-Caspase-3 gene and provide the gene therapic strategy for hepatocellular carcinoma. Methods The pAdTrack-EAFP-PALB was constructed and the r-Caspase-3 gene was subcloned into the vector. The linearized shuttle plasmid was homogenously recombined with AdEasy-1 in BJ5183 cells. The candidate clone was analyzed by restriction endonuclease digestion and sequencing, and then pAdEasy-EAFP-PALB/r-Caspase-3 vector was digested with PacⅠand transfected into AD293 cells for packaging and amplifying, recombinant virus was constructed successfully. Infection titer and efficiency of recombinant virus were monitored by green fluorescent protein (GFP) expression. The expression of r-Caspase-3 in infected HepG2 cells was detected by RT-PCR and Western blot. The apoptosis of HepG2 cells was detected by SRB dyeing method. Results Shuttle vector pAdTrack-EAFP-PALB/r-Caspase-3 was correct after identification by restriction endonuclease analysis and sequencing. By PCR and PacⅠ restriction endonuclease analysis， the homologous recombinant of pAdEasy-EAFP-PALB/r-Caspase-3 was successful. The expression of GFP was observed when linearized pAdEasy-EAFP-PALB/r-Caspase-3 was transfected into AD293 cells. AD293 cells could be infected repeatedly by recombinant adenovirus. The expression of r-Caspase-3 gene on HepG2 cells was detected by RT-PCR and Western-blot methods respectively, which confirmed that the Ad-EAFP-PALB/r-Caspase-3 was constructed successfully. The specificity of Ad-EAFP-PALB/r-caspase-3 which targeting induced hepatocellular carcinoma cells was founded by SRB dyeing test. Conclusion The Recombinant of hepatocellular carcinoma-targeting adenovirus containing r-Caspase-3 gene was constructed successfully and which established the foundation of r-Caspase-3 gene therapy in future research to hepatocellular carcinoma.
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 construct recombinant adenovirus expressing nerve growth factor (NGF) and myelin associated glycoprotein (MAG) (Ad-NGF-MAG) and to investigate its effect on repair and regeneration of sciatic nerve injury in rats. MethodsNGF and MAG gene sequences were cloned into shuttle plasmid pCA13 of adenovirus type 5. After packed in HEK293 cells, the recombinant Ad-NGF-MAG underwent sequence and identification. Thirty-two male Sprague Dawley rats were randomly divided into 4 groups (n=8): control group (normal control), adenovirus vector group (Ad group), Ad-NGF group, and Ad-NGF-MAG group. The sciatic nerve injury model was established by transection of the right sciatic nerve; then, the empty adenovirus vector, Ad-NGF, and Ad-NGF-MAG were injected into the gastrocnemius muscle of the affected limb at a dose of 1×108 PFU every other day for 3 times in Ad group, AdNGF group, and Ad-NGF-MAG group, respectively. The right sciatic nerve was exposed only, and then the incision was closed in the control group. The sciatic nerve function index (SFI) was measured, and neuro-electrophysiology was observed; mRNA and protein expressions of NGF and MAG were detected by RT-PCR and Western blot; and histological examination was performed at 31 days after operation. ResultsRecombinant adenovirus vectors of Ad-NGF and Ad-NGF-MAG were constructed successfully. All rats survived and incision healed by first intension. The SFI, nerve conduction velocity, evoked potential amplitude, and latent period of Ad-NGF-MAG group were significantly better than those of Ad group and Ad-NGF group (P < 0.05). MAG mRNA and protein expressions of Ad-NGF-MAG group were the highest in all the groups (P < 0.05). The expressions of NGF mRNA and protein increased in Ad-NGF group and AdNGF-MAG group when compared with control group and Ad group (P < 0.05). Histological examination showed that the nerve had good continuity in control group; nerve fibers disarranged in Ad group; neurons connections formed in some nerve fibers of Ad-NGF group, but nerve fibers arrange disorderly; and the growth of the nerve were ordered and wellstructured in Ad-NGF-MAG group. ConclusionAd-NGF-MAG can effectively promote the growth of the nerve and inhibit the form of abnormal branches, facilitating the repair of sciatic nerve injury in rats.
Objective To explore the effects of bone marrow mesenchymal stem cells (BMSCs) transfected with adenovirus hepatocyte growth factor (Ad-HGF) on wound repair in diabetic rats. Methods BMSCs from male Wistar rats were isolated by density gradient centrifugation, cultured, and transfected with Ad-HGF. The multi pl icity of infection was 100. Diabetic models were establ ished in 20 female Wistar rats by diets in high fat and sugar plus intraperitoneal injection ofstreptozotocin (30 mg/kg). Then 2 full-thickness skin wounds (approximately 1.5 cm in diameter) were made on the dorsum. The rats were randomly divided into 4 groups (n=5 rats). After wounding, the 0.3 mL suspensions of BMSCs (group A), Ad- HGF (group B), BMSCs transfected with Ad-HGF (group C), and PBS (group D) were injected directly into the derma of wounds. The transverse diameter and longitudinal diameter of wound were measured at 21 days after treatment. At 7 days and 28 days after treatment, HE staining was performed to evaluate wound heal ing. The contents of hydroxyprol ine and advanced glycosylation end products (AGEs) in the wounds were measured by enzyme l inked immunosorbent assay and fluorospectrophotometer, respectively, at 3, 7, 14, and 28 days after treatment. Results At 21 days after treatment, the wounds almost healed in group C, and the transverse diameter and longitudinal diameter were 0 and (0.110 ± 0.024) cm, respectively. But the wounds healed partially in groups A, B, and D, and the transverse diameter and longitudinal diameter were (0.470 ± 0.051) cm and (0.590 ± 0.041) cm, (0.390 ± 0.042) cm and (0.480 ± 0.032) cm, and (0.700 ± 0.068) cm and (0.820 ± 0.068) cm, respectively. There were significant differences in wound heal ing between group C and groups A, B, and D (P lt; 0.05). The wound heal ing time of group C [(20.5 ± 1.9) days] was significantly shorter (P lt; 0.05) than those of groups A, B, and D [(28.3 ± 1.9), (25.9 ± 2.3), and (36.6 ± 5.1) days]. At 7 days, the HE staining showed that evident epidermis transportation, collagen formation, and leukocytes infiltration were observed in group C. At 28 days, the HE staining showed that the epidermis in group C was significantly thinner and more regular than those in other groups, and the decreased collagen and many small vessels were observed in group C. The content of hydroxyprol ine in group C was higher than those in groups A, B, and D at 7 days and 14 days (P lt; 0.05). The contents of AGEs in group C was lower than those in groups A, B, and D at 14 days and 28 days (P lt; 0.05). Conclusion Transplantation of BMSCs transfected with Ad-HGF can accelerate the wounds repair in diabetic rats.
ObjectiveTo detect the expression of indoleamine2, 3-dioxygenase (IDO) and HBV preS mRNA in HepG2 cells and its inhibitory effect on the proliferation of human peripheral blood lymphocyte. MethodsThe AdIDOEGFPpreS was transfected to human hepatocarcinoma cell line HepG2 and the specific fragment of IDO mRNA and HBV preS mRNA in these HepG2 cells were detected by RT-PCR. Then the transfected HepG2 cells were cocultured with human peripheral blood lymphocyte and the inhibitory effect of IDO on the proliferation of human peripheral blood lymphocyte was observed. ResultsThe IDO and HBV preS mRNA were successfully transferred to HepG2 cells, so the specific fragment of IDO and HBV preS mRNA could be found in the transfected HepG2 cells but not in the control group HepG2 cells by RT-PCR. Furthermore, the relative expression intensity of IDO and HBV preS were 1.27 and 1.18, respectively. When co-cultured with human peripheral blood lymphocyte, the counts per minute in the transfected HepG2 cells 〔(7 471±1 375) beats/min〕 was significantly less than that in the control group 〔(13 821±1 997) beats/min〕, Plt;0.001. ConclusionThe target gene IDO and HBV preS can be transferred and expressed in HepG2 cells successfully, which can obviously suppress the proliferation of human peripheral blood lymphocyte.
ObjectiveTo compare the osteogenic effect of bone marrow mesenchymal stem cells (BMSCs) transfected by adenovirus-bone morphogenetic protein 2-internal ribosome entry site-hypoxia inducible factor 1αmu (Ad-BMP-2-IRES-HIF-1αmu) and by Ad-cytomegalovirus (CMV)-BMP-2-IRES-human renilla reniformis green fluorescent protein 1 (hrGFP-1) single gene so as to optimize the source of osteoblasts. MethodsBMSCs were separated and cultured from 1-month-old New Zealand white rabbit. The BMSCs at passage 3 were transfected by virus. The experiment was divided into 4 groups (groups A, B, C, and D) according to different virus: BMSCs were transfected by Ad-BMP-2-IRES-HIF-1αmu in group A, by Ad-CMV-BMP-2-IRES-hrGFP-1 in group B, by Ad-CMV-IRES-hrGFP-1 in group C, and BMSCs were not transfected in group D. The optimum multiplicity of infection (MOI) (50, 100, 150, and 200) was calculated and then the cells were transfected by the optimum MOI, respectively. The expression of BMP-2 gene was detected by immunohistochemistry staining after transfected, the expressions of BMP-2 protein and HIF-1α protein were detected by Western blot method. The osteogenic differentiation potential was detected by alkaline phosphatase (ALP) activity and Alizarin red staining. ResultsThe optimum MOI of groups A, B, and C was 200, 150, and 100, respectively. The expression of BMP-2 was positive in groups A and B, and was negative in groups C and D by immunohistochemistry staining; the number of positive cells in group A was more than that in group B (P ﹤ 0.05). The expression of BMP-2 protein in groups A and B was significantly higher than that in groups C and D (P ﹤ 0.05), group A was higher than group B (P ﹤ 0.05). The expression of HIF-1α protein in group A was significantly higher than those in the other 3 groups (P ﹤ 0.05), no significant difference was found among the other 3 groups (P ﹥ 0.05). ALP activity in groups A and B was significantly higher than that in groups C and D (P ﹤ 0.05), group A was higher than group B (P ﹤ 0.05). Calcium nodules could be seen in groups A and B, but not in groups C and D; the number of calcium nodules in group A was higher than that in group B (P ﹤ 0.05). ConclusionThe expression of BMP-2 and osteogenic effect of BMSCs transfected by Ad-BMP-2-IRES-HIF-1αmu (double genes in single carrier) are higher than those of BMSCs transfected by Ad-CMV-BMP-2-IRES-hrGFP-1 (one gene in single carrier).
Objective Targeted adenoviral gene delivery from peripheral nerves was used to integrally analyse the characterization and time course of LacZ gene (AdLacZ) retrograde transfer to spinal cord and transgene product anterograde labeling ofperipheral nerve. Methods Recombinant replication-defective adenovirus containing AdLacZ was administrated to the cut proximal stumps of median and tibial nerves in Wister rats. Then the transected nerve was repaired with 10-0 nylon sutures. At different time point postinfection the spinal cords of C5 to T1 attached with DRGs and brachial plexuses, or L2 to L6 attached with DRGs and lumbosacralplexuses were removed. The removed spinal cord and DRGs were cut into 50 μm serialcoronal sections and processed for X-gal staining and immunohistochemical staining. The whole specimens of brachial or lumbosacral plexuses attaching with theirperipheral nerves were processed for X-gal staining. The number of X-gal stained neurons was counted and the initial detected time of retrograde labeling, peaktime and persisting period of gene expression in DRG sensory neurons, spinal cord motor neurons and peripheral nerves were studied. Results The gene transfer was specifically targeted to the particular segments of spinal cord andDRGs, and transgene expression was strictly unilaterally corresponding to the infected nerves. Within the same nerve models, the initial detected time of gene expression was earliest in DRG neurons, then in the motor neurons and latest in peripheral nerves. The persisting duration of β-gal staining was shortest in motor neurons, then in sensory neurons and longest in peripheral nerves. The initial detected time of β-gal staining in median nerve models was earlier in mediannerve models compared with that in the tibial nerve models. Although the initial detected time and the beginning of peak duration of β-gal staining were not same, the decreasing time of β-gal staining in motor and sensory neurons of thetwo nerve models were started at about the same day 8 post-infection. The labeled neurons were more in tibial nerve-models than that in median nerve models. Within the same models, the labeled sensory neurons of DRGs were morethan labeled motor neurons of ventral horn. The β-gal staining was tenser in median nerves than that in tibial nerves. However the persisting time of β-gal staining was longer in tibial nerve models. Conclusion The b gene expression in neurons and PNS renders this system particularly attractive for neuroanatomical tracing studies. Furthermore this gene delivery method allowing specific targeting of motor and sensory neurons without damaging the spinal cord might offer potentialities for the gene therapy of peripheral nerve injury.
Objective To observe effects of the direct impaction onthe cell survival and the bone formation of the tissue engineered bone modified by the adenovirus mediated human bone morphogenetic protein 2 (Adv-hBMP2) gene and to verify the feasibility of the impacted grafting with it. Methods The marrow stromal cells (MSCs) were separated from the canine bone marrow and were cultured. MSCs were transfected with the Adv-hBMP2 gene and combined with the freeze-dried cancellous bone (FDB) to form the tissue engineered bone. Four days after the combination, the tissue engineered bone was impacted in a simulated impactor in vitro and implanted in the mouse. The cell survivals were evaluated with SEM 1 and 4 days after the combination, immediately after the impaction, and 1 and 4 days after the impaction, respectively. The bone formation and the allograft absorption were histologically evaluated respectively. Results There were multiple layers of the cells and much collagen on FDB before the impaction. Immediately after the impaction, most of the cells on the direct contact area disappearedand there was much debris on the section. Some of the cells died and separatedfrom the surface of FDB at 1 day, the number of the cells decreased but the collagen increased on the surface at 4 days. Histologically, only the fibrous tissue was found in FDB without the cells, the bone formation on FDB was even in distribution and mass in appearance before the impaction, but declined and was mainly on the periphery after the impaction in the AdvhBMP2 modified tissue-engineered bone. Conclusion The simulated impaction can decrease the cells survival and the bone formation of the AdvhBMP-2 modified tissue-engineered bone. The survival cells still function well.It is feasible to use the tissue engineered bone in the impaction graft.
Objective To construct replication-defective adenovirus containing tk gene (ADV-tk). Methods Recombinant adenovirus of ADV-tk was constructed using homologous recombination in cells. After the interested tk gene fragment in the recombinant plasmid obtained was confirmed by PCR, the titre of purified recombinant adenovirus was detected. In vitro study, tk gene in SMMC7721 cells transfected by ADV-tk was investigated by RT-PCR. In vivo study, ADV-tk was injected intraperitoneally into BALB/c nude mice with liver cancer and apoptosis cells in tumor were observed. Results Recombinant adenovirus containing ADV-tk was proved successfully. The titre of purified recombinant adenovirus was 1.4×1010 pfu/ml. In vitro study, tk was integrated and expressed by SMMC-7721 cells. In vivo study, with the injection of ADV-tk, apoptosis cells in tumor increased. Conclusion A replication-defective adenovirus containing tk gene is successfully constructed, which may useful for further research on tumor suicide gene therapy with ADV-tk.