ABSTRACT
Human nerve growth factor (NGF) is a nerve cell growth regulation factor, which can provide nutrition for the neurons and promote the neurites outgrowth. In order to produce large-scale recombinant human nerve growth factor (rh-beta-NGF), we constructed a plasmid vector, which can stably express the rh-beta-NGF in the HEK293 cell lines. First, the plasmid of pCMV-beta-NGF-IRES-dhfr was constructed and transformed into HEK293 cells. Then MTX pressurized filter and limiting dilution methods were used to obtain monoclonal HEK293 cell lines. After stepwise reducing serum in culture media, the cells eventually adapted to serum-free medium and secreted rh-beta-NGF. SDS-PAGE analysis revealed that the expression product owned a molecular weight of about 13 kDa and a purity of more than 50%. The peptide mapping sequencing analysis demonstrated the sequences of rh-beta-NGF matched with the theoretical ones. Later we purified this protein by ion exchange and molecular sieve chromatograph. Finally, our experimental results exhibited that the recombinant cell lines can stably express rh-beta-NGF with a high efficiency of more than 20 pg/cell x day. In addition, this protein could successfully induce differentiation of PC12 cells. In summary, our recombinant HEK293 cells can express bio-active rh-beta-NGF with great efficiency and stability, which supply a valid basis to large-scale production of rh-beta-NGF.
Subject(s)
Humans , Cell Differentiation , Genetic Vectors , HEK293 Cells , Nerve Growth Factor , Plasmids , Recombinant ProteinsABSTRACT
AIM: To prepare Nano-Liposome encapsulated MAGE3/HSP70(NL M3H) and study its character and antitumor immunity in mouse. METHODS: NL M3H was prepared by the thin film-dispersion ultrasonic. The shape and size of NL M3H were detected by electron microscope. The encapsulation rate, drug-carrying capacity, stability and the releasing character were tested by Sephedex-G100 gel filtration. The mouse was immunized by NL M3H, and the antitumor immunity was detected by ELISPOT and LDH release assay. RESULTS: The mean size of NL M3H was lower than 100 nm. The encapsulation rate was 38％.The drug content was 0.038 g/L. NL M3H has good stability after stored in 4℃ for 6 months. The releasing profile showed that 74 percent of proteins was released during the first 24 hours in saline. The results of ELISPOT and LDH release assay showed that NL M3H generated tumor specific cytotoxic T lymphocyte(CTL)to damage tumor cel1. CONCLUSION: NL M3H has novel characters, it can generate specific CTL to kill tumor cell, and can be used as new kind of vaccine agsinst tumor.
ABSTRACT
Objective To investigate the mechanism of recombinant human bone morphogenetic protein-2(rhBMP-2)in repairing hematopoietic injury in mice irradiated with γ-ray.To prepare SRY gene probe and study the effect of rhBMP-2 in repairing hematopoietic injury in mice by in situ hybridization.Methods Twenty-two BALB/c female mice were randomly divided into the irradiated group and BMP treated group,respectively.Bone marrow cells of normal male mice were transplanted into 22 female mice post-irradiation to 8.5 Gy of 60 Co γ rays.The left femurs of the survived female mice were re-irradiated with 9 Gy 14 days later.Mice in BMP treated group were given rhBMP-2 20 mg/kg while those in control group were treated with 0.9% saline by intraperitoneal injection every day for 6 days.These mice were killed 14 days later and paraffin sections of femurs were made.The SRY gene was detected with in situ hybridization.Results There were more positive blots in the left femurs of the mice in irradiated group than those in BMP treated group(T=155.0,P＜0.05).The number of positive blots between the left femurs of the mice in irradiated group and the right femurs of the mice in two groups was not significantly different(T=92.0,78.5,P＞0.05).The number of positive blots in the left femurs of the mice in BMPtreated group was significantly less than those in the right femurs of the mice in two groups(T=155.0,55.0,P＜0.05).Conclusions No donor cell of male mice was detected in the left femurs of BMP treated group,suggesting that rhBMP-2 promoted the restoration of residuary bone marrow cells.Thus,rhBMP-2 promotes the proliferation or differentiation of residuary mesenchymal stem cells,improves hematopoietic microenvironment and accelerates the hematopoietic restoration.
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Objective To investigate the influence of gene transfection antiangiogenesis on microvessel and relative cytokine of hypertrophic scar of rabbits' ear.Methods The hypertrophic scar of rabbtis' ear was reproduced.On the 10th day after epithelization,Ad-METH-1 was injected into tissue of scar.30 days later,the microvessel of scar-tissue was detected by microcirculation microscope.Meanwhile.H&E and immunohistochemical stains were performed.Then the results were analyzed.Results 30 days after Ad-METH-1 injection.in experimental groups,the microvascular count of scar tissue was 12.38±2.56,the percentage of VEGF positive cells was 17.64%,and the percentage of bFGF positive cell was 18.24%:while in the control groups,the microvascular count of scar tissue was 48.12±6.46.the percentage of VEGF positive cell was 31.34%.and the percentage of bFGF positive eell was 28.26%.Results revealed that the count of microvessel of scar tissue in the experimental groups was lower than that in the control groups,between which there was the difference in statistics(P＜0.01).and that the percentage of VEGF and bFGF positive cells of scar tissue in the expenmental groups were lower than that in the control groups.between which there was the difference in statistics(P＜0.05).Conclusion Ad-METH-1 has marked inhibitory effects on scar tissue hyperplasia of rabbits' ear,angiogenesis and expression of VEGF and bFGF.Using antiangiogenesis therapy at the early phase could inhibit the formation of hypertrophic scar.Gene transfection antiangiogenesis therapy could bid fair to become an effective method to prevent and treat hypertrophic scar.
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Objective To determine whether cultured neural stem cell expresses morphogen molecule sonic hedgehog′s functional receptor patched. Methods Cultured neural stem cell clones were subjected to RT\|PCR after passaged several times in vitro ,the amplification production was sequenced and labeled by digoxingemin and in situ hybridization technique was carried out to detect the cryosection of the neural stem cell clones. Results Most cells in the stem cell clones were positive for sonic hedgehog functional receptor patched,no significant difference was found among the positive cells and the center and peripheral of the stem cell clones.Conclusion\ The sonic hedgehog signal transduction may have important role in the proliferation and differentiation process of neural stem cell.\;[
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Objective To investigate the expression of anti-apoptosis gene bag-1 and its relation to the differentiation of intrahepatic cholangiocarcinoma (ICC). Methods Immunohistochemical techniques were adopted to study the expression of bag-1 in ICC tissue ( n=48) and para-hepatocarcinoma bile duct ( control group, n=25). Results Expression of bag-1 in the ICC group was significantly higher than that in the control group. In the ICC group (P