Objective To review the current concepts of gene therapy approachesmediated by adenovirus vectors for bone trauma and bone disease. Methods The recent literature concerned gene therapy mediated by adenovirus vectors was reviewed, which provides new insights into the treatments of bone trauma and bone disease. Results Adenovirus vectors was efficient, achieved high expression after transduction, and could transfer genes to both replicating and nonreplicating cells, such as osteoblasts, osteoclasts, fibroblasts, chondrocytes, bone marrow stromal cells, etc. Gene therapy mediated by adenovirus vectors achieved affirmative results in enhancing bone union and in curing bone diseases, such as osteoporosis and rheumatoid arthritis. Conclusion Gene therapy mediatedby adenovirus offers an exciting avenue for treatment of bone trauma and bone diseases.
ObjectiveTo investigate the proliferation and apoptosis effects of adenovirus-mediated interleukin-24 (Ad-IL-24) gene on Karpas299 cells in vitro. MethodsThe Karpas299 cells were divided into blank control group, Ad-IL-24 group, and the adenovirus which carrying green fluorescent protein gene group (Ad-GFP group). Karpas299 cells of Ad-IL-24 group were infected by adding 200.0 μL Ad-IL-24, Karpas299 cells of Ad-GFP group were infected by adding 200.0 μL Ad-GFP, but Karpas299 cells of blank control group were treated by adding 200.0 μL PBS. Cells' proliferation inhibition rates of 3 groups were detected by cell counting kit (CCK-8) method at 12, 24, and 48 hours after treatment, respectively, and the cells' apoptosis rates of 3 groups were detected by flow cytometry at 48 hours after treatment. ResultsAd-IL-24 can suppress the growth of Karpas299 cells, and the inhibition rate increased over time. Compared with Ad-GFP group at the same time, the cell' proliferation inhibition rate of Ad-IL-24 group was higher at 12, 24, and 48 hours after treatment (P<0.05). In addition, the cells' apoptosis rate of Ad-IL-24 group was higher than those of Ad-GFP group and blank control group at 48 hours after treatment (P<0.05). ConclusionAd-IL-24 can suppress the growth of Karpas299 cells and induce the apoptosis of it.
Objective To explore the effects of overexpression of human tissue inhibitors of metalloproteinase-1 (hTIMP-1) on proliferation of human liver cancer cell line HepG2 in vitro. Methods A recombinant adenoviral vector containing full-length cDNA of hTIMP-1 was generated and transfected into HepG2. The viral titer was checked by measuring GFP, and the expression of hTIMP-1 in vitro was detected by the techniques of Western blot and semi-quantitative RT-PCR. The ultrastructure was observed by transmission electron microscope and the effects of overexpression of hTIMP-1 on proliferation of HepG2 in vitro was analyzed by MTT assay and growth curve. Results The resultant AdhTIMP-1 was successfully constructed and the expression of hTIMP-1 was detected by Western blot and RT-PCR. The growth and proliferation of HepG2, which had been transfected with AdhTIMP-1, was significantly inhibited. Conclusion The proliferation of HepG2 was markedly inhibited by recombinant adenovirus-mediated overexpression of hTIMP-1, which may pave the way for further application in liver gene therapy.
Objective To transfect bone marrow mesenchymal stem cells (BMSCs) of rats by recombinant adenovirus Ad-human matrix metalloproteinase 1 (hMMP-1) in vitro so as to lay the experimental foundation for the treatment of liver fibrosis with a combination of BMSCs and hMMP-1 gene transplantation. Methods BMSCs were isolated from bone marrow of 2-3 weeks old Sprague Dawley rats by whole bone marrow adherence method and identified, then transfected by recombinant adenovirus Ad-hMMP-1 carrying enhanced green fluorescent protein (EGFP) marker in vitro. The green fluorescent expression was observed by fluorescence microscope and the transfection efficiency was detected by flow cytometry to determine the optimum multiplicity of infection (MOI). BMSCs at passage 3 were divided into 3 groups: untransfected BMSCs group (group A), Ad-EGFP transfected BMSCs group (group B), and Ad-hMMP-1-EGFP transfected BMSCs group (group C); the gene and intracellular protein of hMMP-1 were detected by RT-PCR and Western blot; the ELISA assay was used to detect the supernatant protein expression, and the hMMP-1 activity was measured by fluorescent quantification kit. Results The green fluorescent was observed in BMSCs transfected by recombinant adenovirus at 24 hours after transfection; the fluorescence intensity was highest at 72 hours; and the optimum MOI was 200. The cells of 3 groups entered the logarithmic growth phase on the 3rd day and reached plateau phase on the 6th day by MTT assay; no significant difference was found in the cell proliferation rate among 3 groups (P gt; 0.05). RT-PCR, Western blot, and ELISA assay showed high expressions of the hMMP-1 gene and protein in group C, but no expression in groups A and B. The hMMP-1 activity was 1.24 nmol/(mg · min) in group C, but hMMP-1 activity was not detectable in groups A and B. Conclusion The exogenous hMMP-1 gene is successfully transfected into BMSCs of rats via recombinant adenovirus and can highly express, which lays the experimental foundation for the treatment of liver fibrosis with a combination of BMSCs and hMMP-1 gene transplantation.
目的:寻找对腺病毒灭活的有效方法,来预防大规模生产腺病毒时所致的生产细胞的污染。方法:采用紫外线照射和不同浓度过氧乙酸的灭活方法灭活腺病毒,并将处理后的病毒加入到正常人胚肾293细胞中培养,观察细胞是否出现病变作为灭活效果判断标准。结果:我们发现一定强度紫外线照射至少2 h以上才能完全灭活腺病毒,过氧乙酸达到1 %的浓度和至少作用30 min,才能将完全病毒灭活。结论:紫外线照射2 h以上能有效灭活腺病毒,我们可以用于操作台的腺病毒灭活;1%浓度的过氧乙酸作用30 min以上能有效将腺病毒灭活,我们可以运用于实验室大空间的腺病毒灭活。
Objective To study the adenovirus-mediated human bone morphogenetic protein-2 gene （Ad-hBMP-2）transferred to the intervertebral disc cells of the New Zealand rabbit in vitro. Methods The cells of New Zealand white rabbitswere isolated from their lumbar discs. The cells were grown in the monolayer and treated with an adenovirus encoding the LacZ gene (Ad-LacZ) and Ad-hBMP-2 (50,100, 150 MOI,multiplicity of infection) in the Dulbecco’s Modified Eagle Medium and the Ham’s F-12 Medium in vitro. Three days after the Ad-hBMP-2 treatment,the expression of hBMP-2 in the cells that had been infected by different dosesof MOI was determined by immunofluorescence and the Western blot analysis, and the expression was determined in the cells with the Ad-LacZ treatment in a dose of 150 MOI. Six days after the Ad-hBMP-2 treatment, mRNA was extracted for the reverse transcription polymerase chain reaction (RT-PCR) and the difference was detected between the control group and the culture group that was treated withAd-hBMP-2 in doses of 50, 100 and 150 MOI so that the expressions of aggrecan and collagen ⅡmRNA could be observed. Results The expression of hBMP-2 in the cells was gradually increased after the transfection in an increasing dose, which was observed by immunofluorescence and the Western blot analysis. At 6 days the aggrecan and collagen type Ⅱ mRNA expressions were up-regulated by Ad-hBMP-2 after the transfection at an increasing viral concentration in the dosedependent manner. Conclusion The results show that Ad-hBMP-2 can transfect the rabbit intervertebral disc cells in vitro with a high efficiency rate and the expression of hBMP-2 after theinfection is dose-dependent in the manner. AdhBMP-2 after transfection can up-regulate the expression of aggrecan and collagen Ⅱ mRNA at an increasing viral concentration.
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.
ObjectiveTo evaluate the therapeutic effect of liver transplantation (LT) combined with adenovirus-mediated delivery of herpes simplex virus thymidine kinase / ganciclovir (ADV-TK/GCV) in treatment of patients with hepatocellular carcinoma (HCC), so as to benefit more patients with HCC beyond the Milan criteria. MethodsThe clinicopathologic data of patients with HCC underwent LT by the author team since 2007 were collected and analyzed. The patients were assigned into simple LT group and LT+ADV-TK/GCV group. The 5-year cumulative overall survival rate and relapse free survival rate of all LT patients and the patients with LT beyond the Milan criteria by simple LT and LT+ADV-TK/GCV therapy were compared. Meanwhile, Cox regression was used to analyze the risk factors affecting long-term overall survival rate and relapse free survival rate of all patients with HCC after LT. ResultsA total of 216 patients eligible for inclusion were collected in this study, including 134 patients in the simple LT group and 82 patients in the LT+ADV-TK/GCV group, 162 of whom beyond the Milan criteria, including 101 patients underwent the simple LT and 61 patients underwent the LT+ADV-TK/GCV. There were no statistical differences in the baseline data between the simple LT and LT+ADV-TK/GCV in all patients and patients beyond the Milan criteria (P>0.05). There were no statistical differences in 5-year overall survival rate and relapse free survival rate of all patients with HCC (P>0.05). The 5-year cumulative overall survival rate of the LT+ADV-TK/GCV group was better than that of the simple LT group in the patients beyond the Milan criteria (χ2=4.11, P=0.047), but it was not found that the 5-year cumulative relapse free survival rate had statistical difference (27-month survival time as the critical value, P=0.46, P=0.06). Cox regression multivariate analysis results showed that the larger cumulative tumor diameter, the preoperative elevated serum alpha fetoprotein (>400 μg/L), later TNM stage, and without combination of ADV-TK/GCV therapy increased the probability of shorter overall survival of patients after LT; and the patient’s older age, the larger cumulative tumor diameter, and later TNM stage increased the probability of shorter relapse free survival after LT, and it was not found that the combination of ADV-TK/GCV therapy had an impact on the relapse free survival. ConclusionLT combined with ADV-TK/GCV therapy can obviously improve overall survival among patients beyond the Milan criteria, more patients with advanced HCC will be candidates for LT combined with ADV-TK/GCV therapy.
ObjectiveTo construct a transgenic cell sheet of cartilage-derived morphogenetic protein 1 (CDMP-1) by adenovirus vector in vitro and to identify its biological activity. MethodsThe bone mesenchymal stem cells (BMSCs) were isolated from bone marrow of 1-month-old rabbit, and cultured in vitro. The 3rd-6th generation of BMSCs were used for experiment. The experiment was divided into 3 groups:BMSCs transfected by adenovirus (Ad)-cytomegalovirus (CMV)-human CDMP1 (hCDMP1)-internal ribosome entry site (IRES)-enhanced green fluorescent protein (EGFP) in group A, BMSCs transfected by Ad-CMV-EGFP in group B, and untransfected BMSCs in group C. The expression of green fluorescence was observed in 3 groups under fluorescent inverted microscope. MTT assay was used to detect the proliferation of the cells. The cell sheet was obtained by means of temperature-responsive culture dish for 14 days. The morphological and HE staining observations of the cell sheet were carried out. RT-PCR and Western blot were used to detect the expressions of hCDMP1 and collagen type II at gene and protein levels, while alcian blue staining was used to detect the expression of glycosaminoglycans (GAG). ResultsBright green fluorescence was observed in transfected cells at 72 hours under fluorescent inverted microscope, and the transfection efficiency was up to 90%. MTT assay showed approximate S-shaped growth curves in 3 groups, showing no significant difference in the absorbance (A) value among 3 groups within 9 days (P>0.05). The three-dimensional cell sheets were successfully harvested in vitro. The RT-PCR and Western blot showed that there were positive expressions of hCDMP1 and collagen type II in group A and negative expression in other 2 groups. HE staining and alcian blue staining showed that there were rich fibrous tissues, mass extracellular matrix, and dark blue metachromatic granules in group A, but there was less fibrous tissues and no specific blue metachromatic granules in other 2 groups; and the positive expression area was significantly lower and gray scale of GAG was significantly higher in group A than that in groups B and C (P<0.05). ConclusionA transgenic cell sheet of exogenous recombinant hCDMP1 by adenovirus vector can express collagen type II and GAG, so it has chondrogenic capacity. This technology that overcomes limitations in traditional tissue engineering, such as low cell-attachment efficiency and inflammatory reaction, may be a new tissue engineering approach for hard tissue reconstruction and is hopeful to build a large density of tissue engineered cartilage.
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.