Objective To construct human brain-derived neurotrophic factor retroviral vector-pLXSN (hBDNFpLXSN), and to evaluate the bioactivity of hBDNF. Methods The genome mRNA was extracted from embryonic brain tissue of a 5-month-old infant, the hBDNF gene sequence was obtained with RT-PCR technology, and hBDNF-pLXSN constructed in vitro was used to infect the fibroblasts (NIH/3T3). The expression of hBDNF was identfied by the immunohistochemistry method, and the NIH/3T3 and BDNF biological activities were determined by culture of the PC12 cells and dorsal root gangl ia. Results The hBDNF-pLXSN was constructed successfully by sequencing analyses. The infected NIH/3T3 showed positive expression of hBDNF. The infected NIH/3T3 could product hBDNF. Bioactivity of the products could support the PC12cell survival and neurite growth in the primary cultures of dorsal root gangl ia neurons of mice. Conclusion hBDNF-pLXSNvirus has the abil ity to infect NIH/3T3 and make it expressed and secreted hBDNF with the biological activity. It can be used to treat facial paralysis as a gene therapy.
OBJECTIVE: To analysis the biological characteristics of human fibroblasts transfected by human telomerase reverse transcriptase (hTERT) eucaryotic expression plasmid pGRN145. METHODS: Fibroblasts from children’s foreskin were isolated and cultured in vitro, and the fibroblasts were transfected by pGRN145 with Lipofec-tAMINE PLUS Reagent. After strict screening of hygromycin B, the positive clones were subcultured. The telomerase activity was detected by RT-PCR and TRAP-PCR technique. The cell generation cycle and apoptosis rate were detected by flow cytometry to investigate the proliferative characteristics after transfection, and the chromosome karyotype of transformed cells was analyzed. The collagen secreted by transformed cells was detected by immunohistochemical staining. RESULTS: The morphological properties of fibroblasts did not change obviously after transfection. There were telomerase activity in transfected fibroblasts, while it could not be detected in pre-transfection fibroblasts. The cell generation cycle had no obvious changes between pre-transfection and post-transfection. However, the apoptosis rate of transfected fibroblasts were decreased compared with that of pre-transfection. The fibroblasts transfected by pGRN145 maintained the normal diploid karyotype, as well as the cells could normally secret type I and III collagen. CONCLUSION: The human fibroblasts transfected by pGRN145 has telomerase activity with prolonged life span of culture, which preliminarily proves the availability of establishing standard seeding cell lines of tissue engineering by hTERT plasmid transfection techniques.
Objective To observe the inhibitory effects of local co-transfection of tissuetype plasminogen activator(tPA) gene and proliferating cell nuclear antigen antisense oligodeoxynucleotides(PCNA-ASODN) on the intima proliferation and restenosis of autograft artery in rabbits. Methods One hundred and twenty male Zelanian rabbits were randomly divided into four groups(n=30, in each group): control group, PCNA-ASODN group, tPA group and tPA+PCNAASODN group. The left and right external iliac arteries (length 1.0 cm) were transplanted reciprocally. The transplanted arteries were respectively soaked in lipofection, PCNAASODN, pBudCE4.1/tPA and pBudCE4.1/tPA+PCNA-ASODN solution about 15 minutes. The transplanted arteries were sutured with 9-0 sutures soaked in PCNA-ASODN and pBudCE4.1/tPA solution. Each group were divided into five subgroups(n=6, in each subgroup) according to the sacrifice time (3 d, 7 d, 14 d, 28 d and 56 d after operation). On every sacrifice time point, the vascular specimens were harvested. The thrombocyte assembling and thrombus forming lining vessel wall were observed by scanning electron microscope. The pathological morphology of transplanted arteries were observed under microscope(HE). The intimal areas and stenosis ratio(%) of transplanted arteries were calculate and analyzed statistically among groups by computer system. The mRNA expression of tPA gene in transplanted ressel wall was detected with vevere transcriptionPCR(RT-PCR). The number of PCNA positive cells in transplanted vessel wall was counted by SP immunochemisty.Results The mRNA expression of tPA gene in the transplanted vessel wall in tPA and tPA+PCNA-ASODN groups was higher than that of the other two groups(P＜0.01).The number of PCNA positive cells in the transplanted arteries in PCNAASODN, tPA and tPA+PCNAASODN groups were significantly lower than that of control group(P＜0.05，P＜0.01). The intimal areas and degrees of luminal stenosis of PCNAASODN, tPA and tPA+PCNAASODN groups were lower than those of control group(P＜0.05，P＜0.01), and those of tPA＋ PCNA-ASODN group were lower than those of PCNA-ASODN and tPA groups(P＜0.05). Scanning electron microscopy showed that there were a few thrombocytes lining the vessel wall of tPA group and tPA+PCNAASODN group and no thrombus, whereas there were abundant thrombocytes and thrombi lining the vessel wall of the control group. Conclusion Co-transfection of tPA gene and PCNA-ASODN can effectively inhibit the proliferation of VSMC, hyperplasia of intima and restenosis of transplanted artery.
Objective
To investigate the ability of gene-loaded lipopolysaccharide-amine nanopolymersomes (LNPs) in inducing osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by in vitro gene transfection, where LNPs were used as a non-viral cationic carrier, and their properties were optimized during synthesis.
Methods
LNPs were synthesized by a graft-copolymerization method, and the effects of different pH environments during synthesis on physicochemical properties of LNPs and LNPs/plasmid of bone morphogenetic protein 2-green fluorescent protein (pBMP-2-GFP) complexes were explored. Then, optimized LNPs with maximum transfection efficiency and safe cytotoxicity in rat BMSCs were identified by cytotoxicity and transfection experiments in vitro. Thereafter, the optimized LNPs were used to mediate pBMP-2-GFP to transfect rat BMSCs, and the influences of LNPs/pBMP-2-GFP on osteogenic differentiation of BMSCs were evaluated by monitoring the cell morphology, concentration of BMP-2 protein, activity of alkaline phosphatase (ALP), and the formation of calcium nodules.
Results
The nitrogen content, particle size, and zeta potential of LNPs synthesized at pH 8.5 were lower than those of the other pH groups, with the lowest cytotoxicity (96.5%±1.4%) and the highest transfection efficiency (98.8%±0.1%). After transfection treatment, within the first 4 days, BMSCs treated by LNPs/pBMP-2-GFP expressed BMP-2 protein significantly higher than that treated by Lipofectamine2000 (Lipo)/pBMP-2-GFP, polyethylenimine 25K/pBMP-2-GFP, and the blank (non-treated). At 14 days after transfection, ALP activity in BMSCs treated by LNPs/pBMP-2-GFP was higher than that treated by Lipo/pBMP-2-GFP and the blank, comparable to that induced by osteogenic medium; with alizarin red staining, visible calcium nodules were found in BMSCs treated by LNPs/pBMP-2-GFP or osteogenic medium, but absent in BMSCs treated by Lipo/pBMP-2-GFP or the blank with apoptosis. At 21 days after transfection, transparent massive nodules were discovered in BMSCs treated by LNPs/pBMP-2-GFP, and BMSCs exhibited the morphologic features of osteoblasts.
Conclusion
LNPs synthesized at pH 8.5 has optimal transfection efficiency and cytotoxicity, they can efficiently mediate pBMP-2-GFP to transfect BMSCs, and successfully induce their directional osteogenic differentiation, whose inducing effect is comparable to the osteogenic medium. The results suggest that gene transfection mediated by LNPs may be a convenient and effective strategy in inducing directional differentiation of stem cells.
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 the transfection and expression of recombinant plasmid human vascular endothelial growth factor 165/pcDNA3. 1 （hVEGF165/pcDNA3. 1） in myocardial cells, and to build foundation for gene therapy and cell therapy of coronary artery disease （CAD）. Methods Myocardial cells were cultured in vitro and transfected by hVEGF165/pcDNA3.1 with liposome; then transient expressed protein was detected by reverse transcriptase-polymerase chain reaction （RT-PCR）, immunochemistry and Western blotting. Results A strap as hVEGF165 was obtained by RT-PCR, the protein of hVEGF165 was found in myocardial cells by immunochemistry and in supernatant by Western blotting. Conclusion The recombinant plasmid hVEGFI65/pcDNA3. 1 can be expressed in myocardial cells, and may be used in studying CAD by gene therapy and cell transplantation.
Objective
To investigate the effects of recombinant adenovirus-mediated co-transfection of carcinoembryonic antigen (CEA) gene and erythropoietin (EPO) gene on promoting hematopoietic stem cells directly producing erythrocyte vaccine against colon cancer.
Methods
The expression adenovirus vectors carrying CEA and EPO or green fluorescent protein (GFP) gene were constructed respectively, and recombinant adenovirus carrying CEA, EPO or GFP were packaged and produced respectively. The bone marrow-derived mesenchymal stem cells (MSCs) of mice were isolated and cultured in vitro by anti-CD117 magnetic bead separation, and were transfected with CEA (CEA group), EPO (EPO group) or GFP (blank vector group), co-transfected with CEA and EPO (CEA-EPO group). The expressionsof CEA and EPO gene and its protein after transfection in supernatant fluid of culture were detected by realtime-PCR and Western blot method in each group. We had checked and obtained the vaccine with co-transfection of CEA gene and EPO gene by cell red line marker antibody CD71 and GPA, then we carried on experiments with the vaccine in vitro and in vivo. There were 4 groups in our trail: blank vector group, CEA group, EPO group, and CEA-EPO group.
Results
We had successfully gathered the hematopoietic stem cells, flow cytometry analysis result showed that there were significant differences before and after purification for positive selected samples (P<0.05). The expressions of double genes (CEA-EPO gene) and protein showed CEA-EPO gene were successfully transfected into the hematopoietic stem cells. We had confirmed erythrocyte vaccine with co-transfection of CEA and EPO gene by antibody CD71 and GPA with flow cytometry. The monocytes cytotoxicity on colon cancer cell line CT26 showed that lysis of target cells of CEA-EPO group were higher than those of other 3 groups when in proportion of 40∶1 (P<0.05). In the experimentation of neoplasma format, the volume of tumor and mortality were smaller or lower, but survival time was longer of CEA-EPO group in2 weeks after treatment (P<0.05).
Conclusions
The erythrocyte vaccine with co-transfection of CEA gene and EPO gene has efficient anti-tumor effects on colon cancer. Not only can promote hematopoietic stem cell directly producing erythrocyte vaccine, but also can produce tumor antigen vaccine against colon cancer.
Objective To observe the expression of adenovirus vector coding for mouse endostatin gene（Ad-mES） in lung cancer cells and its antiangiogenic activity in human umbilical vein endothelial cells（ECV304） in vitro.Methods Lewis lung cancer（LLC） cells were transfected with Ad-mES at different multiplicity of infection（MOI）.The expression of mES in LLC cells and supernatant after 48 hours was detected by immunohistochemical staining and Western blot respectively.The inhibitory effect of supernatant at different MOI on ECV304 non-stamulated and stimulated by basic fibroblast growth factor（bFGF） was measured by methyl thiazolyl tetrazolium（MTT） assay.Results After transfected for 48 hours，endostatin was identified in the cell plasma of infected LLC and negative result was founded in non-infected LLC.Western blot revealed band of endostatin in 20 kDa in culture supernatant of infected LLC and negative results in non-infected LLC.The inhibitory effects on ECV304 cell proliferation were ber at higher MOI，and the difference was significant between stimulated and non-stamulated cells by bFGF（Plt;0.05）.Conclusion Ad-mES can transfect and express endostatin effectively in LLC with biological activity
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.
ObjectiveTo construct and identify the recombinant adenovirus vector expressing bone morphogenetic protein 2(BMP-2) and transforming growth factor β3(TGF-β3) genes,to observe the expressions of BMP-2 and TGF-β3 after transfected into bone marrow mesenchymal stem cells (BMSCs) of the Diannan small-ear pigs.
MethodsBMP-2 cDNA and TGF-β3 cDNA were amplified by PCR,and were subcloned into the pEC3.1(+) plasmid to obtain pEC-GIE 3.1-BMP-2 and pEC-GIE3.1-TGF-β3 plasmid respectively.They were subcloned into pGSadeno vector by homologous recombination reaction and HEK293 cells were transfected after linearization to obtain Ad-BMP-2 and Ad-TGF-β3.The BMSCs were isolated from the bone marrow of Diannan small-ear pig and cultured.The 3rd passage BMSCs were transfered with Ad-BMP-2(group A),Ad-TGF-β3(group B),Ad-BMP-2+Ad-TGF-β3(group C),and untransfected cells served as a control (group D).The expressions of BMP-2 and TGF-β3 genes and proteins were detected by PCR,immunofluorescence,and Western blot.The chondrogenic differentiation of BMSCs was evaluated by immunohistochemical of collagen type Ⅱ.
ResultsThe Ad-BMP-2 and Ad-TGF-β3 were constructed successfully and confirmed by PCR and sequencing.The expression clones of Ad-BMP-2 and Ad-TGF-β3 were packaged into maturated adenovirus successfully,the titer was 5.6×108 and 1.6×108 pfu/mL respectively.The PCR results showed a light band at 310 bp in group A and at 114 bp in group B,and both 310 bp and 114 bp bands in group C,but no band in group D.The image of immunofluorescence showed that there were red fluorescence and green fluorescence expressions in the cytoplasm of BMSCs at 72 hours after transfection in groups A and B,respectively;in group C,both red and green fluorescence expressions were detected,and no red or green fluorescence was detected in group D.The results of Western blot showed that there was a light band at 18×103 in group A and at 50×103 in group B;both 18×103 and 50×103 bands were detected in group C;but no band was detected in group D.The cells were positive for collagen type Ⅱ in groups A,B,and C;group C acquired strong collagen type Ⅱ staining when compared with group A and group B;in group D,the cells were negative for collagen type Ⅱ staining.
ConclusionThe recombinant adenovirus vector expressing BMP-2 and TGF-β3 are constructed successfully.The BMP-2 and TGF-β3 genes could be expressed effectively in BMSCs of Diannan small-ear pig after transfection,which could afford modified seeding cells for cartilage tissue engineering.
