【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.
【Abstract】 Objective To investigate the secretion of target gene and differentiation of BMSCs transfected by TGF-β1 and IGF-1 gene alone and together into chondrocytes and to provide a new method for culturing seed cells in cartilage tissue engineering. Methods The plasmids pcDNA3.1-IGF-1 and pcDNA3.1-TGF-β1 were ampl ified and extracted, then cut by enzymes, electrophoresed and analyzed its sequence. BMSCs of Wistar rats were separated and purificated by the density gradient centrifugation and adherent separation. The morphologic changes of primary and passaged cells were observed by inverted phase contrast microscope and cell surface markers were detected by immunofluorescence method. According to the transfect situation, the BMSCs were divided into 5 groups, the non-transfected group (Group A), the group transfected by empty vector (Group B), the group transfected by TGF-β1 (Group C), the group transfected by IGF-1 (Group D) and the group transfected both by TGF-β1 and IGF-1 (Group E). After being transfected, the cells were selected, then the prol iferation activity was tested by MTT and expression levels were tested by RT-PCR and Western blot. Results The result of electrophoresis showedthat sequence of two bands of the target genes, IGF-1 and TGF-β1, was identical with the sequence of GeneBank cDNA. A few adherent cells appeared after 24 hours culture, typical cluster formed on the forth or fifth days, and 80%-90% of the cells fused with each other on the ninth or tenth days. The morphology of the cells became similar after passaging. The immunofluorescence method showed that BMSCs were positive for CD29 and CD44, but negative for CD34 and CD45. A few cells died after 24 hoursof transfection, cell clone formed at 3 weeks after selection, and the cells could be passaged at the forth week, most cells became polygonal. The boundary of some cells was obscure. The cells were round and their nucleus were asymmetry with the particles which were around the nucleus obviously. The absorbency values of the cells tested by MTT at the wavelength of 490 nm were0.432 ± 0.038 in group A, 0.428 ± 0.041 in group B, 0.664 ± 0.086 in group C, 0.655 ± 0.045 in group D and 0.833 ± 0.103 in group E. The differences between groups A, B and groups C, D, E were significant (P lt; 0.01). The differences between groups A and B or between C, D and E were not significant (P gt; 0.05)。RT-PCR and Western blot was served to detect the expression of the target gene and protein. TGF-β1 was the highest in group C, 0.925 0 ± 0.022 0, 124.341 7 ± 2.982 0, followed by group E, 0.771 7 ± 0.012 0, 101.766 7 ± 1.241 0（P lt; 0.01）; The expression of IGF-1 was the highest in group E, 1.020 0 ± 0.026 0, 128.171 7 ± 9.152 0, followed by group D, 0.465 0 ± 0.042 0, 111.045 0 ± 6.248 0 （P lt; 0.01）. And the expression of collagen II was the hignest in group E, 0.980 0 ± 0.034 0, 120.355 0 ± 12.550 0, followed by group C, 0.720 0 ± 0.026 0, 72.246 7 ± 7.364 0（P lt; 0.01）. Conclusion The repairment of cartilage defects by BMSCs transfected with TGF-β1 and IGF-1 gene together hasa good prospect and important significance of cl inic appl ication in cartilage tissue engineering.
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.
目的:探讨丙型肝炎病毒非结构蛋白NS4B在原发性肝癌发生中的作用,及其发生机制。方法:设置对照组、空白载体PCXN2组、转染NS4B组。使用脂质体介导法,转染丙型肝炎病毒非结构蛋白重组质粒NS4B进入Chang肝细胞内,并用G418筛选。绘制生长曲线,分别用流式细胞仪检测瞬时表达及稳定表达时肝细胞凋亡率,用均数±标准差表示,统计学分析采用Dunnett t检验及q检验。结果:瞬时表达各组(PCXN2,NS4B)凋亡率分别为:(918±060)%,(445±053)%。稳定表达各组(PCXN2,NS4B)凋亡率分别为:(1575±209)%,(366±034)%。与PCXN2组比较Plt;001。结论:NS4B抑制肝细胞凋亡率,可能导致肝细胞异常增殖,诱导肝癌发生。
Objective To compare the effect of mosaicplasty, mosaicplasty with gene enhanced tissue engineering and mosaicplasty with the gels of non-gene transduced BMSCs in alginate on the treatment of acute osteochondral defects. Methods Western blot test was conducted to detect the expression of hTGF-β1, Col II and Aggrecan in 3 groups, namely hTGF-β1 transduction group, Adv-βgal transduction group and blank control group without transduction. Eighteen 6-month-old Shanghai mascul ine goats weighing 22-25 kg were randomized into groups A, B and C (n=6). BMSCs were isolatedfrom the autologous bone marrow of groups B and C, and were subcultured to get the cells at passage 3. In group B, the BMSCs were transduced with hTGF-β1. For the animals of 3 groups, acute cyl indrical defects 5 mm in diameter and 3 mm in depth were created in the weight bearing area of the medial femoral condyle of hind l imbs. In group A, the autologous osteochondral mosaicplasty was performed to repair the defect; in group B, besides the mosaicplasty, the dead space between the cyl indrical grafts and the host cartilage were injected with the suspension of hTGF-β1 gene transduced autogenous BMSCs in sodium alginate, and CaCl2 was dropped into it to form calcium alginate gels; in group C, the method was the same as the group B, but the BMSCs were not transduced. General condition of the goats after operation was observed, the goats were killed 12 and 24 weeks after operation to receive gross and histology observation, which was evaluated by the histological grading scale of O’Driscoll, Keeley and Salter. Immunohistochemistry and TEM observation were performed 24 weeks after operation. Results Western blot test showed the expression of the hTGF-β1, Col II and the Aggrecan in the hTGF-β1 transduction group were significantly higher than that of the Adv-βgal transduction and the blank control groups. All the goats survived until the end of experiment and all the wounds healed by first intention. Gross observation revealed the boundaries of the reparative tissue in group B were indistinct, with smooth and continuous surfaces of the whole repaired area; while there were gaps in the cartilage spaces of groups A and C. Histology observation showed the dead space between the cyl indrical grafts in group A had fibrocartilage-l ike repair tissue, fill ing of fibrous tissue or overgrowth of the adjacent cartilage; the chondrocytes in group B had regular arrangements, with favorable integrations; while the dead space between the cyl indrical grafts in group C had fibrocartilage-l ike repair tissue, with the existence of gaps. The histology scores of group B at different time points were significantly higher than that of groups A and C, and group C was better than group A (P lt; 0.05); for group B, significant difference was detected between 12 weeks and 24 weeks in the histology score (P lt; 0.05）. Immunohistochemistry staining for Col II 24 weeks after operation showed the chondrocytes and lacuna of the reparative tissue in group B was obviously stained, while groups A and C presented l ight staining. TEM observation showed there were typical chondrocytes in the reparative tissue in group B, while parallel or interlaced arrangement collagen fiber existed in groups A and C. Conclusion Combining mosaicplasty with tissue engineering methods can solve theproblem caused by single use of mosaicplasty, including the poor concrescence of the remnant defect and poor integration with host cartilages.
Objective To construct the rhesus monkey Schwann cells (SCs) modified with human glial cell derived neurotrophic factor (hGDNF) gene. Methods The coding sequence of hGDNF amplified by PCR from pUC19-hGDNF was inserted into eukaryotic expression vector pBABE-puro. The recombinant eukaryotic expression vector pBABE-puro-hGDNF was identified with restriction enzyme digestion and DNA sequencing. The SCs were isolated from rhesus monkeys, cultured and purified. The SCs were transfected with the recombinant retrovirus vector containing hGDNF gene. The mRNA and protein expressions of hGDNF were analyzed by real-time fluorescent quantitative PCR and Western blot. Results The PCR product of hGDNF coding sequence was a 596 bp specific segment. The recombinant eukaryotic expression vector was digested into a 596 bp specific segment by specific restriction enzyme and another segment. The 596 bp segment confirmed by DNA sequencing was consistent with hGDNF sequence on GenBank. Restriction enzyme digestion and sequencing results showed that the coding sequence of hGDNF was successfully inserted into the recombinant retrovirus vector and the mRNA and protein expressions of hGDNF were significantly higher in transfected SCs than non-transfected SCs (P lt; 0.05). Conclusion The rhesus monkey SCs modified with hGDNF gene are successfully constructed and hGDNF can be released continuously and stably, which will provide a foundation for the further research about cell therapy of hGDNF-SCs in the repair of injured nerve.
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 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 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.