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 evaluate the transfection efficiency and expression level of hepatocyte growth factor (HGF) by transfecting a recombinant adenovirus carrying HGF gene (Ad-HGF) into bone marrow mesenchymal stem cells (BMSCs) and to explore the effect of the expression supernatant on BMSCs in vitro so as to lay a foundation for the manufacture of gene medicine which expresses efficient cell factors. Methods Rat BMSCs were isolated using Percoll density gradient method and cultured according to the adherent property of BMSCs. The expression of c-Met was detected by immunohistochemical examination. BMSCs were infected with a recombinant adenovirus carrying green fluorescent protein gene (Ad-GFP) at multipl icity of infection (MOI, 0, 25, 50, 100, and 200 pfu/cell). To select an optimal MOI, the transfection efficiency and the degree of cell damage were assayed by flow cytometry and MTT, respectively, at 48 hours after transfecting. The expression of HGF in BMSCs transfected with optimal MOI Ad-HGF was measured with ELISA assay. MTT method was used to evaluate the prol iferation effect of HGF expression supernatant on BMSCs. Results Immunohistochemical staining showed that BMSCs expressed c-Met receptor for HGF. At 48 hours after transfecting with different MOI Ad-GFP (0, 25, 50, 100, and 200 pfu/cell), the transfection efficiencies were 0.34% ± 0.04%, 40.72% ± 0.81%, 61.72% ± 1.04%, 85.33% ± 0.83%, and 17.91% ± 0.63%, respectively; and the highest transfection efficiency was observed at 100 pfu/cell MOI. The cell damage was obviously observed when MOI was 200 pfu/cell. The expression of HGF in BMSCs reached the highest level after being transfected with 100 pfu/cell MOI Ad-HGF for 48 hours. The expression product could stimulate the prol iferation of BMSCs. The prol iferation of BMSCs gradually rose with the increase of HGF protein, and reached the highest level at 10% (320 pg). Conclusion BMSCs can be transfected efficiently with Ad-HGF and express HGF protein, which stimulates the prol iferation of BMSCs. It suggests that BMSCs is an ideal repair cells with gene vector.
Objective To investigate a change in the differentiation and biological function of the cultured rat fibroblast (FB) transfected by the myoblast determining gene (MyoD) and the connexin 43 (Cx43) gene and to explore the possible mechanism of the MyoD and Cx43 genes on treatment of ischemic heart disease (IHD). Methods The gene cloning technology was used to construct the eukaryotic expressed plasmid vector pLenti6/V5-DEST-MyoD and pLenti6/V5DEST-Cx43 in which MyoD cDNA or Cx43 cDNA was inserted. The RFL-6 FB cells were transfected with exogenetic MyoD cDNA or Cx43 cDNA via lipofectamine, followed by the Blasticidin (50 μg/ml) selection, according to the lentiviral expression system (ViraPower) protocol. The expression and the biological functions of MyoD and Cx43 in the transfectants were testified by RT-PCR, Western blot, and molecular and immunocytochemical methods. The mophological structure changes of the cells were observed under microscope before and after the transfection. Results The expression of MyoD and Cx43 was detected in the MyoD and Cx43 genes transfected FB with RT-PCR and Western blot. The immunocytochemical methods indicated the expressionsof the MyoD and Cx43 genes, while desmin and αactin were found in these cells. The myotubes were found from the cultures incubated a week in the differentiation medium, in which the transfected cells had a characteristic of the filamentsin their cytoplasm and showed a myoblast morphology. Conclusion MyoD cDNA can induce the cultured FB to differentiate into the myoblasts and Cx43 cDNA can enhance the gap junctional intercellular communication between the cell and the cell. Thus, a further experimental foundation for the therapy of IHD can be provided.
Objective To construct a bioengineered dermis containing microencapsulated nerve growth factor (NGF) expressing -NIH3T3 cells and to study the effect of the microencapsule on the bioengineered dermis and acute wound healing. Methods A recombinant NGF (PcDNA3.1+/NGF) was constructed and transfected intoNIH-3T3 cells using FuFENETM6 transfection reagent. Positive cell strain was cultured and enclosed in alginate-polylysine-alginate(APA) microcapsules in vitro. Bioengineered dermis was incorporated with NGF-expressing micorencapsules and human fibroblast cells as seed cells using tissue engineering method. The characteristics of the dermis were described by the content of Hydroxyproline(Hyp), HE staining. The content of NGF in the dermis culturing supernatant was measured by ELISA method. These bioengineered dermis were transplanted onto the acute circular full thickness excisional wounds on the dorsum of each swine to observe the rate of reepithelization and wound healing: NGFNIH3T3 microencapsulations(group A), NIH3T3 microencapsulations( group B), empty microencapsulations (group C), NGF incorporated with collagenⅠ( group D) and blank (group E as control group). Results NGF can be tested stably about 124.32 pg/ml in the dermis culturing supernatant after 6 weeks, and the content of Hyp in group A was 69.68±6.20(mg/g wet weight) and increased about 2 times when compared with control groups after 1 week. The tissue engineering skin grafts which can secrete NGF were used to ure the acute wounds and the rate of reepithelization was promoted. The periods of wound healing were 25±2 days in group A, 34±3 days in group B, 34±2 days in group C, 33±2 days in group D and 40±3 days in group E.The period of wound healing was decreased about 10 days at least. Conclusion NGF-expressing NIH3T3 microencapsulates can promote the quality of bioengineered dermis and alsopromote acute wound healing.
【Abstract】 Objective To review the research progress of possible mechanism of indoleamine 2, 3-dioxygenase(IDO) in immunological regulation and function of transplantation immunity. Methods The advances in the IDO location, immunological regulatory mechanism and function of transplantation immunity were introduced based on the recent related l iterature. Results IDO played an immunoregulatory role by locally depleting tryptophan in tissue microenvironment which resulted in immunosuppression of allogeneic T-cell prol iferation. IDO cDNA was del ivered to chromosome in interesting cells by gene transfection and stimulated to express, which was associated with a prolongation in allograft survival in vivo . Conc lu sion IDO offers a new way in transplantation immunity, and this provid novel method for elevating allograft survival rate.
ObjectiveTo observe transforming growth factor β3 (TGF-β3) gene expression and the chondrogenesis of bone marrow mesenchymal stem cells (BMSCs) after TGF-β3 gene is transfected into BMSCs of Diannan small-ear pig.
MethodsRecombinant adenovirus 5 (rAd5) was extracted as gene vector and packed into recombinant adenovirus rAd5-TGF-β3, double enzyme digestion and PCR identification were performed. BMSCs were isolated and cultured from bone marrow of 2-month-old Diannan small-ear pigs (weighing, 12-15 kg), and the 2nd generation of BMSCs were harvested for experiments. The experiments were divided into 3 groups. BMSCs were transfected with rAd5-TGF-β3 as experimental group and with empty vector as control group, and non-transfected BMSCs were used as blank control group. The transfection efficiency of exogenous gene was identified by flow cytometry, TGF-β3 protein expression by immunofluorescence and Western blot. The cell morphology of experimental group was observed by inverted phase contrast microscope, and the expression of collagen type II in each group was detected by Western blot.
ResultsThe rAd5-TGF-β3 recombinant adenovirus was successfully constructed and transfected into BMSCs. Green fluorescence was observed by immunofluorescence microscope. Flow cytometry test showed the best transfection at 72 hours (transfection efficiency of 84.86%). Immunofluorescence staining showed that the expression of TGF-β3 protein was obvious at 72 hours; Western blot showed that there was a TGF-β3 positive band with a relative molecular mass of 30×103, while the control group and blank control group had no positive band. Obvious chondrogenic differentiation was observed in the experimental group after transfection in vitro, while the control group and blank control group had no obvious chondrogenic differentiation. Western blot showed that there was collagen type II positive band with a relative molecular mass of 130×103 at 21 days after culture, while the control group and blank control group had no positive band.
ConclusionrAd5-TGF-β3 gene can be successfully transfected into BMSCs via adenovirus vectors, and stable expression of TGF-β3 protein can be observed, enhancing BMSCs differentiation into chondrocytes, which may provide an experimental basis for gene therapy of joint cartilage defects.
Objective To investigate the possibility of constructing eukaryoticexpression vector for human angiopoietin 1（hAng-1），transfecting it to bonemarrow mesenchymal stem cells (MSCs) so as to repair bone defect. Methods The eukaryotic expression vector pcDNA3-hAng-1 was constructed by recombinant DNA technique, transfected into MSCs by liposome DOTAP, and selected with G418. The hAng-1 expression of mRNA and protein was detected by reverse transcript-PCR and Western Blot. Results After the recombinant eukaryotic expressionvector for hAng-1 was digested with Xho-I and BamH-I, electrophoresis revealed 1.4 kb fragment for hAng-1 gene and 5.4 kb fragment for pcDNA3 vector. In the transfected MSCs, the mRNA and protein expression of hAng-1 gene were detected with reverse transcriptPCR and Western Blot. Conclusion The constructed eukaryotic expression vector hAng-1 could be expressed in the transfected MSCs, thus to provide the basis for bone repair with tissue engineering.
Objective To investigate the possibility of constructing eukaryotic expression vector for human glial derived neurotrophic factor （hGDNF）, transfecting it to spinal cord tissue of rats so as to treat acute spinal cord injury. Methods The eukaryotic expression vector pcDNA3-hGDNF was constructed by recombinant DNA technique, transfected into glial cell and neuron of spinal cord by liposome DOTAP as experimental group. In control group, mixture of empty vector and liposome was injected. The mRNA and protein expressions of hGNDF were detected by RT-PCR and Western blot. Results After the recombinant eukaryotic expression vector for hGDNF was digested with Hind III and XbaⅠ, electrophoresis revealed 400 bp fragment for hGDNF gene and 5 400 bp fragment for pcDNA3 vector. In the transfected spinal cord tissue, the mRNA and protein expressions of hGDNF gene were detected with RT-PCR and Western blot. Conclusion The constructed eukaryotic expression vector pcDNA3hGDNF could be expressed in the transfected spinal cord tissue of rat, so it provide basis for gene therapy of acute spinal cord injury.
Objective To explore the effects of the basic fibroblast growth factor（bFGF） gene transfection on the meniscal fibrochondrocytes with the reconstructed lentivirus and to observe the response of the meniscal fibrochondrocytes to the bFGF gene transfection. Methods The cultured meniscal fibrochondrocytes were isolated from the same 3-monthold New Zealand rabbit. The cultured first-generation meniscal fibrochondrocytes were divided into 3 groups:Group A (experimental group), Group B (control group), and Group C (blank group). Each group comprised the cells in a 24hole flask in which each hole contained 2×104 cells. At the confluence of 60%, the fibrochondrocytes in Group A were cultured with the reconstructed lentivirus carrying the bFGF gene. The fibrochondrocytes in Group B were cultured with the lentivirus carrying no bFGF gene. The fibrochondrocytes in Group C were cultured without any intervention. After 48 h, the cell cycle, the collagen synthesis ability, the expression of bFGF, and the cell proliferation ability in each group were investigated. Results In Group A, the bFGF expression of 870±60 pg/ml was detected in the cells 48 h afterthe co-culture; however, in Group B and Group C, no expression of bFGF was found. After the co-culture for 6 days, the results of the MTT colorimetry revealed that the cells in Group A had an absorbtance of 0.427±0.037, which had a significant difference when compared with that in Group B and Group C (0.320±0.042,0.308±0.034,Plt;0.01). The cell cycle was significantly shorter in GroupA than in Group B and Group C (Plt;0.05); The durations of G1, S and G2M of the cells in Group A were 16.28, 12.60 and 11.04 h, but those in Group B and Group C were 23.61, 16.90, 21.33 h and 21.56, 19.80, 21.41 h, respectively. The disintegration per minute of the cells was significantly greater in Group A than in Group B and Group C (7281.69±805.50 vs 5916.40±698.11 and 5883.57±922.63，Plt;0.05). Conclusion The lentivirus vector can transfer the bFGF gene into the meniscal fibrochondrocytes, resulting in an increase of the cell proliferation and the collagen synthesis.
Objective To construct lentiviral vector carrying the human hepatocyte growth factor (hHGF) gene, and then to get hHGF gene/modified bone marrow mesenchymal stem cells (BMSCs) by infecting the BMSCs. Methods The hHGF gene was obtained with PCR from pcDNA-hHGF plasmid. The recombination lentiviral vector plasmid hHGF was constructed with Age I digestion and gene recombinant, then was identified with PCR and sequencing. Mediated by Lipofectamine2000, the three plasmids system of lentiviral vector including pGC-E1-hHGF, pHelper 1.0, and pHelper 2.0 was co-transfected to 293T cells to produce hHGF gene. The supernatant was collected and concentrated by ultracentrifugation and the titer of lentivirus was measured by real-time quantitative PCR. The BMSCs were infected by the constructed lentivirus and the multipl icities of infection (MOI) was identified with fluorescent microscope, the efficiency of infection with flow cytometry (FCM) analysis, the hHGF level with ELISA analysis, and the expression of hHGF gene with RT-PCR. Results Lentiviral vector carrying hHGF gene was constructed successfully. The titer of lentivirus was 1 × 108 TU/mL. The infection efficiency of BMSCs by hHGF lentiviral was high and reached 98% by FCM, and the best MOI was 10. A great mount of green fluorescence was observed with the fluorescent microscope at 28 days after infection. Peak concentration of hHGF secreted by BMSCs/hHGF reached 40.5 ng/mL at 5 days. The concentration could maintain a high level until 28 days after infection. RT-PCR showed that BMSCs/hHGF could express hHGF gene. Conclusion By lentiviral vector, hHGF gene was integrated into BMSCs genome, and it can express stably.
