OBJECTIVE To probe the possibility of direct transfer of exogenous gene into peripheral nerve and its following expression in vivo. METHODS The PCMV beta plasmid containing cytomegalovirus (CMV) promoter and Escherichia Coli (E. Coli), beta-Galactosidease (beta-Gal) structural gene (lacZ gene) was constructed and injected into the rabbit sciatic nerve. The control group was injected PBS solution. The injected nerves were sampled and tested by beta-Gal enzyme activity assay of the 5-bromo-4-chloro-3-indolyl-beta-D-galactoside and beta-Gal histochemical stain. RESULTS In the control group, no beta-Gal enzyme activity was detected in the different stages after operation, and beta-Gal histochemical stains showed positive. In the experimental group, enzyme activity could be detected from 2 days to 30 days after operation, and the histochemical stains showed negative. CONCLUSION The exogenous gene can be transferred into peripheral nerve and expressed with bioactivity, thus the gene therapy to accelerate the recovery of nerve is practical.
Objective To explore the human stromal cell-derived factor 1α (hSDF-1α) and human vascular endothel ial growth factor 165 (hVEGF165) mRNA expressions of the transfected cells after hSDF-1α gene and hVEGF165 gene were transfected into rat myoblasts in vitro so as to lay a foundation for further study on the synergistic effects of 2 genes on tissue engineered skeletal muscle vascularization. Methods The myoblasts of 1-day-old Sprague Dawley rats were cultured and purified by trypsin digestion assay in vitro and were identified by immunohistochemistry staining of Desmin. pproximately 70%-80% of confluent myoblasts were transfected with enhanced green fluorescent protein (EGFP)-hSDF-1α and EGFP-hVEGF165 genes in vitro (transfected group) and were not transfected (control group). The expressions of hSDF-1αand hVEGF165 mRNA and protein in the transfected cells were detected by RT-PCR, ELISA, and Western blot espectively.Results The cultured cells were identified as myoblasts by immunohistochemistry staining of Desmin. The expression ofgreen fluorescent protein was observed in transfected cells, indicating that hSDF-1α and hVEGF165 genes were transfected into myoblasts successfully. The mRNA and protein expressions of the 2 genes were positive in the transfected group by RT-PCR and Western bolt assay at 2, 4, 6, and 8 days after transfection, and were negative in the control group. The expressions of hSDF- 1α and hVEGF165 showed a stable low level in the control group, but the expressions of the proteins increased at 2 days and then showed gradual downtrend with time in the transfected group by ELISA assay. There were significant differences in the expressions of hSDF-1α and hVEGF165 proteins between different time points in the transfected group, and between 2 groups (P lt; 0.05). Conclusion hSDF-1α and hVEGF165 genes are successfully transfected into myoblasts in vitro, and mRNA and proteins of hSDF-1α and hVEGF165 can be expressed in the transfected myoblasts, which may provide the experimental evidence for the expressions of hSDF-1α and hVEGF165 mRNA and proteins in vivo successfully.
Objective To investigate the effect of transforming growth factor-β1 (TGF-β1) gene transfer on the biological characteristics of osteoblasts. Methods The expression of TGF-β1 in the transfected osteoblasts was detected by in situ hybridization and assay of TGF-β1 activity in the supernatant (minklung epithelium cell growth -inhibition test). The effects of gene transfer andsupernatant of the transfected osteoblasts on the proliferation and alkaline phosphatase(ALP) activity of osteoblasts were detected by 3 H-TdR and MTT. Results The results of in situ hybridization analysis suggested that the osteoblasts transfected by TGF-β1 gene could express TGF-β1 obviously. The complex medium, which was the mixture of serum-free DMEM and the activated supernatant according to 1∶1, 1∶2, 1∶4, could inhibit growth of Mv-1-Lu evidently and the ratios ofinhibition were 16.3%, 22.7%, 28.2% respectively. TGF-β1 gene transfer hadno effect on the biological characteristics of osteoblasts, but the activated supernatant of transfected osteoblasts stimulated proliferation and inhibited ALPactivity of osteoblasts. Conclusion TGF-β1 gene transfer promotes the expression of TGF-β1 and the biological characteristics of trasfected osteoblasts are stable, which is helpful for gene therapy of bone defects in vivo.
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.
Objective To determine whether fibroblasts can be used to promote endochondral bone formation in vivo by transfer of human bone morphogenetic protein-2(hBMP-2) into fibroblasts. Methods pcDNA3-hBMP-2 was constructed by use of gene clone and recombined technique.NIH3T3 fibroblasts were transfected with pcDNA3hBMP-2. The positive cell clones were selected with G418. In NIH3T3 fibroblaststransferred with pcDNA3-hBMP-2, the expression of hBMP-2 was determined by in situ hybridization and immunohistochemical analysis; alkaline phosphatase activity was measured. hBMP-2producing fibroblasts were implanted into nude mouse muscle to observe endochondral bone formation in vivo. Results pcDNA3-hBMP-2 was successfully constructed. In NIH3T3 fibroblasts transfected with -pcDNA3-hBMP-2,the BMP-2 expression was stable; alkaline phophatase activity was much higher than that in nontransfectedNIH3T3 cells. Endochondral bone formation invivo was observed at the site of implantation 4 weeks later.Conclusion Fibroblasts transfected by hBMP-2 gene can be used to promote endochondral bone formation in vivo.
Objective To explore the feasibility of high-pressure injection to transfer human thrombomodulin (hTM) gene into arterial wall of rabbits.Methods Eighty-four healthy New Zealand rabbits were randomly divided into three groups: pcDNA3.1/hTM plasmid group (n=28), pcDNA3.1(+)/neo plasmid group (n=28) and untransfected group (n=28). After gene transfection, the model of arterial injury-blocking was established. Then, the expressions of hTM mRNA and protein in arterial wall were examined by RT-PCR and immunohistochemistry at 3 d, 7 d, 14 d and 28 d after operation. Results Seventeen rabbits died accidentally from the day of operation to 3 d after operation. The expressions of hTM mRNA of different time points in pcDNA3.1/hTM plasmid group were significantly higher than that in pcDNA3.1(+)/neo plasmid group and untransfected group (Plt;0.01). For the expressions of hTM mRNA at different time points in pcDNA3.1(+)/neo plasmid group and untransfected group, the difference of inter-group and intra-group was not significant (Pgt;0.05). hTM protein was expressed in every group and mainly localized in the inner lining of arterial wall. The expressions of hTM protein at different time points in pcDNA3.1/hTM plasmid group were significantly higher than that in pcDNA3.1(+)/neo plasmid group and untransfected group (Plt;0.05). The expression of hTM protein at different time points in pcDNA3.1(+)/neo plasmid group and untransfected group kept relative constancy, the difference of inter-group and intra-group was also not significant (Pgt;0.05). Conclusion High-pressure injection is feasible to transfer pcDNA3.1/hTM plasmid into arterial wall of live animals.
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.
【Abstract】ObjectiveTo construct eukaryotic expression vector pSecTag2/HygroB-CD59 of human CD59 and transfect NIH3T3 cells after encapsulated by chitosan. MethodsThe human CD59 fragments were obtained by PCR form CD59-pGEM-T Easy Vector, cloned into the eukaryotic expression vector pSecTag2/HygroB, identified by restriction endonuclease’s digestion and DNA sequencing. After the particles of pSecTag2/HygroB-CD59 were encapsulated by chitosan, the NIH3T3 cells were transfected by chitosanCD59 nanoparticles and detected CD59 expression by immunohistochemistry stain. ResultsThe CD59 fragment was 312 bp. Its sequence was as same as CD59 cDNA in Genbank. After having been transfected by chitosan-CD59 nanoparticles in 24 hours, the 3T3 cells showed diffusely positive in the cytoplasms by anti-CD59 immunohistochemistry. ConclusionThe eukaryotic expression vector of human CD59 is constructed and transfected to NIH3T3 cells after encapsulated by chitosan. It will be very helpful for further study on transgenic livers.
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.
ObjectiveTo investigate the effect of overexpressing the Indianhedgehog (IHH) gene on the chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs) in a simulated microgravity environment. MethodsThe 2nd generation BMSCs from rabbit were divided into 2 groups: the rotary cell culture system (RCCS) group and conventional group. Each group was further divided into the IHH gene transfection group (RCCS 1 group and conventional 1 group), green fluorescent protein transfection group (RCCS 2 group and conventional 2 group), and blank control group (RCCS 3 group and conventional 3 group). RCCS group cells were induced to differentiate into chondrocytes under simulated microgravity environment; the conventional group cells were given routine culture and chondrogenic induction in 6 well plates. During differentiation induction, the ELISA method was used to detect IHH protein expression and alkaline phosphatase (ALP) activity, and quantitative real-time PCR to detect cartilage and cartilage hypertrophy related gene expressions, and Western blot to detect collagen typeⅡ, agreecan (ANCN) protein expression; and methylene blue staining and Annexin V-cy3 immunofluorescence staining were used to observe cell slide. ResultsAfter transfection, obvious green fluorescence was observed in BMSCs under fluorescence microscopy in RCCS groups 1 and 2, the transfection efficiency was about 95%. The IHH protein levels of RCCS 1 group and conventional 1 group were significantly higher than those of RCCS 2, 3 groups and conventional 2, 3 groups (P < 0.05); at each time point, ALP activity of conventional 1 group was significantly higher than that of conventional 2, 3 groups (P < 0.05); ALP activity of RCCS 1 group was significantly higher than that of RCCS 2 and 3 groups only at 3 and 7 days (P < 0.05). Conventional 1 group expressed high levels of cartilage-related genes, such as collagen typeⅡand ANCN at the early stage of differentiation induction, and expressed high levels of cartilage hypertrophy-related genes, such as collagen type X, ALP, and Annexin V at the late stage (P < 0.05). RCCS 1 group expressed high levels of cartilage-related genes and low levels of cartilage hypertrophy-related genes at all stages. The expression of collagen typeⅡprotein in conventional 1 group was significantly lower than that of conventional 2 and 3 groups at 21 days after induction (P < 0.05); RCCS 1 group expressed high levels of collagen typeⅡand ANCN proteins at all stages (P < 0.05). Methylene blue staining indicated conventional 1 group was stained lighter than conventional 2 and 3 groups at 21 days after induction; while at each time point RCCS 1 group was significantly deeper than RCCS 2 and 3 groups. Annexin V-cy3 immunofluorescence staining indicated the red fluorescence of conventional 1 group was stronger than that of conventional 2 and 3 groups at each time point. The expression of red fluorescence in each RCCS subgroup was weak and there was no significant difference between the subgroups. ConclusionUnder the simulated microgravity environment, transfection of IHH gene into BMSCs can effectively promote the generation of cartilage and inhibit cartilage aging and osteogenesis. Therefore, this technique is suitable for cartilage tissue engineering.