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pIRES2-BDNF-VEGF165真核表达载体的构建与鉴定
栗炳南，李卫东，林俊堂，丰慧根
- 新乡医学院生命科学技术学院，河南省新乡市 453003
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修回日期:2013-09-06出版日期:2013-12-10发布日期:2013-12-10 -
通讯作者:栗炳南，新乡医学院生命科学技术学院，河南省新乡市 453003 -
作者简介:栗炳南☆，男，1983年生，河南省新乡市人，汉族，2011年中南大学湘雅医学院毕业，博士，讲师，目前主要从事神经系统疾病的发病机制与治疗方面的研究。 并列第一作者：李卫东★，新乡医学院生命科学技术学院在读硕士。 -
基金资助:实验由新乡医学院重点领域招标课题(ZD2011-16)*；河南省教育厅科学技术研究重点项目(13A180850)*共同资助
Construction and Identification of pIRES2-BDNF-VEGF165 bicistronic eukaryotic expression vector
Li Bing-nan, Li Wei-dong, Lin Jun-tang, Feng Hui-gen
- Department of Life Sciences and Technology, Xinxiang Medical University, Xinxiang 453003, Henan Province, China
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Revised:2013-09-06Online:2013-12-10Published:2013-12-10 -
Contact:Li Bing-nan, Department of Life Sciences and Technology, Xinxiang Medical University, Xinxiang 453003, Henan Province, China bingnanli120@yeah.net -
About author:Li Bing-nan☆, Ph.D., Lecturer, Department of Life Sciences and Technology, Xinxiang Medical University, Xinxiang 453003, Henan Province, China bingnanli120@yeah.net Li Wei-dong, Studying for master’s degree, Department of Life Sciences and Technology, Xinxiang Medical University, Xinxiang 453003, Henan Province, China -
Supported by:the Tender Subject of Key Research Areas of Xinxiang Medical Uuniversity in 2011, No. ZD2011-16*; Key Projects in Scientific Research of Henan Provincial Education Department, No. 13A180850*
摘要：
背景：人脑源性神经营养因子(Brain-derived neurotrophic factor，BDNF)和血管内皮生长因子165(vascular endothelial growth factor 165，VEGF165)在细胞分化过程中有重要作用。病毒载体临床应用存在安全隐患，利用真核表达载体表达蛋白为解决安全性问题提供了一种方法。 目的：构建双基因共表达载体pIRES2-BDNF-VEGF165并对其进行鉴定。 方法：采用PCR的方法从人外周血单个核细胞的基因组DNA中获取人脑源性神经营养因子基因，然后将人脑源性神经营养因子的cDNA片段插入到pIRES2-EGFP多克隆位点构建为pIRES2-BDNF-EGFP。人血管内皮生长因子165 cDNA片段是通过双PCR的方法从pIRES2-VEGF165-EGFP质粒中获取，接着将血管内皮生长因子165 cDNA片段以替换EGFP的方式插入pIRES2-BDNF-EGFP中，最后构建成为含有即内部核糖体进入位点的pIRES2-BDNF-VEGF165双基因共表达载体。通过双酶切和 DNA测序方法对其鉴定，将重组的双基因共表达载体感染 HEK293细胞，利用RT-PCR与 Western-blot 方法检测双基因的表达。 结果与结论：DNA测序显示，提取的人脑源性神经营养因子和血管内皮生长因子165均与基因库报道序列一致，片段长度分别为744 bp和576 bp。构建的pIRES2-BDNF-VEGF165双基因共表达载体经Eco RⅠ/Bam HⅠ切出BDNF条带，经BDNF/NotⅠ双酶切后可见 IRES- VEGF165基因片段，经 Eco RⅠ/NotⅠ双酶切后可见BDNF-IRES-VEGF165基因片段。RT-PCR 与Western-blot 方法检测显示，此载体转染后，HEK293 细胞均能表达人脑源性神经营养因子和血管内皮生长因子165 mRNA和蛋白。结果证实，实验成功构建了人脑源性神经营养因子和血管内皮生长因子165 双基因真核表达载体。
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引用本文
栗炳南，李卫东，林俊堂，丰慧根. pIRES2-BDNF-VEGF165真核表达载体的构建与鉴定[J]. 中国组织工程研究, doi: 10.3969/j.issn.2095-4344.2013.50.016.
Li Bing-nan, Li Wei-dong, Lin Jun-tang, Feng Hui-gen. Construction and Identification of pIRES2-BDNF-VEGF165 bicistronic eukaryotic expression vector[J]. Chinese Journal of Tissue Engineering Research, doi: 10.3969/j.issn.2095-4344.2013.50.016.
Amplification of BDNF and VEGF165 genes
BDNF gene was obtained from the genomic DNA of human peripheral blood mononuclear cells by PCR, and the size of BDNF gene was 744 bp. The VEGF165 gene was obtained from pIRES2-VEGF165-EGFP plasmid by PCR, and the size of VEGF165 gene was 576 bp (Figure 1).
BDNF gene was obtained from the genomic DNA of human peripheral blood mononuclear cells by PCR, and the size of BDNF gene was 744 bp. The VEGF165 gene was obtained from pIRES2-VEGF165-EGFP plasmid by PCR, and the size of VEGF165 gene was 576 bp (Figure 1).
Identification of plasmid pIRES2-BDNF-EGFP
The plasmid pIRES2-BDNF-EGFP was cut by Eco RⅠand Bam HⅠdouble enzyme. A gene fragment with 744 bp was obtained, which was in full agreement with BDNF gene (Figure 2).
The plasmid pIRES2-BDNF-EGFP was cut by Eco RⅠand Bam HⅠdouble enzyme. A gene fragment with 744 bp was obtained, which was in full agreement with BDNF gene (Figure 2).
Identification of plasmid pIRES2-BDNF-VEGF165
The plasmid pIRES2-BDNF-VEGF165 was cut by Eco RⅠand Bam HⅠ double enzyme. And a fragment about 744 bp would be obtained. This indicated BDNF gene inserted into the plasmid pIRES2-BDNF-VEGF165. The
plasmid pIRES2-BDNF-VEGF165 was cut by Bam HⅠand NotⅠdouble enzyme, and a fragment about 1 164 bp would be obtained, indicating IRES-VEGF165 gene could be inserted into the plasmid pIRES2-BDNF-VEGF165. The plasmid pIRES2-BDNF-VEGF165 was cut by Eco RⅠand NotI double enzyme, and a fragment about 1 914 bp would be obtained, indicating BDNF-IRES-VEGF165 gene inserted into the plasmid pIRES2-BDNF-VEGF165. The sequence of the plasmid pIRES2-BDNF-VEGF165 was in accordance with gene sequence in Gene Bank (Figure 3).
The plasmid pIRES2-BDNF-VEGF165 was cut by Eco RⅠand Bam HⅠ double enzyme. And a fragment about 744 bp would be obtained. This indicated BDNF gene inserted into the plasmid pIRES2-BDNF-VEGF165. The
plasmid pIRES2-BDNF-VEGF165 was cut by Bam HⅠand NotⅠdouble enzyme, and a fragment about 1 164 bp would be obtained, indicating IRES-VEGF165 gene could be inserted into the plasmid pIRES2-BDNF-VEGF165. The plasmid pIRES2-BDNF-VEGF165 was cut by Eco RⅠand NotI double enzyme, and a fragment about 1 914 bp would be obtained, indicating BDNF-IRES-VEGF165 gene inserted into the plasmid pIRES2-BDNF-VEGF165. The sequence of the plasmid pIRES2-BDNF-VEGF165 was in accordance with gene sequence in Gene Bank (Figure 3).
To illustrate mRNA expression by pIRES2-EGFP, pIRES2-BDNF/EGFP and pIRES2-BDNF/VEGF165 transfected HEK293 cells, we evaluated the expression of BDNF and VEGF165 by RT-PCR analysis. RT-PCR was performed using BDNF-specific primers and the β-actin sequence served as an internal standard. GFP expression was monitored in pIRES2-EGFP and pIRES2-BDNF/EGFP transfected HEK293 cells by inverted fluorescence microscopy. Expression of BDNF mRNA was higher in either pIRES2-BDNF/EGFP or pIRES2-BDNF/VEGF165 transfected HEK293 cells than that pIRES2-EGFP transfected HEK293 cells or negative controls (P < 0.01).
As shown above, expression of VEGF165 mRNA was higher in pIRES2-BDNF/VEGF165 transfected HEK293 cells than the other three (P < 0.01, Figure 5). These results demonstrated that the BDNF and VEGF165 had been introduced successfully into HEK293 cells by pIRES2-BDNF/EGFP and pIRES2-BDNF/VEGF165.
Western blot analysis of the expression of BDNF and VEGF165 in HEK293 cells
The results suggested that exogenous BDNF protein was strongly expressed in pIRES2-BDNF/EGFP and pIRES2-BDNF/VEGF165 transfected HEK293 cells, but the pIRES2-EGFP transfected HEK293 cells and non-transfected expression of endogenous BDNF was very low (Figure 6). As shown above, western blot analysis also revealed that exogenous VEGF165 protein was strongly expressed in pIRES2-BDNF/VEGF165 transfected HEK293 cells, but endogenous VEGF165 very low in the pIRES2-EGFP, pIRES2-BDNF/EGFP transfected HEK293 cells (Figure 7). These results also demonstrated that BDNF and VEGF165 had been introduced successfully into HEK293 cells by pIRES2-BDNF/VEGF165.
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Spinal cord injury is caused by ischemia, hypoxia, or caused by the lack of a variety of cytokines[1]. In recent years, vascular endothelial growth factor (VEGF) family has been confirmed to play an important role in the regulation of angiogenesis, tissue repair and neuron protective[2-3]. To know VEGF and its biological effect and the regulation after spinal cord injury during the process of spinal cord substantial capillary regeneration and the process of nerve protection will contribute to the further understanding and research of VEGF. Although the major function of VEGF in the nervous system is associated with angiogenesis, in recent studies, VEGF in the nervous system have been shown to have other important functions[4]. As VEGF can maintain the growth and survival of brain and spinal cord capillary, VEGF has a direct neurotrophic effect and can promote stem cell proliferation and neuritis outgrowth in the central nervous system[5-6]. Although hypoxia, inflammation and injury can lead to a boost expression of endogenous VEGF in the spinal cord
In this study, we attempted to build a co-expression plasmid based on double genes, brain-derived neurotrophic factor and vascular endothelial growth factor, to transfect HEK293 cells and to detect the its expression, thereby providing the experimental basis for the next experiment of this subject on co-transfected mesenchymal stem cells, on the improvement of the internal environment at spinal cord injured sites, on the regulation of differentiation direction of mesenchymal stem cells, on the increasing of the number of newborn neurons, and on the promotion of functional recovery of the spinal cord.
parenchyma, the VEGF secreted by cells themselves is not sufficient to fight against neuronal apoptosis induced by hypoxia-ischemia. Therefore, exogenous VEGF is necessary, which can protect against apoptosis of endothelial cells and promote angiogenesis, and in the other hand it can play a direct role in neural protection and promote axonal regeneration[7-8]. All those studies provide a theoretical guidance and new ideas for the use of exogenous VEGF in the treatment of spinal cord injuries, so that VEGF is expected to become one of the effective ways to treat spinal cord injuries.
Brain-derived neurotrophic factor (BDNF) is the most abundant nerve growth factor in the central nervous system[9]. Studies have found that BDNF and its receptor express and transport actively in multiple areas of the brain tissue, and can play an important role in survival, growth and development of neurons, in the maintenance and regulation of neurons, as well as in the damage repair of neurons[10-11]. The diversified expression of BDNF and its activity determines its potential role in the pathogenesis and treatment of nervous system diseases.
Animal experiments have confirmed that BDNF can produce beneficial effects on the repair of neuronal function and different cells of central nervous system[12].Therefore, increasing the endogenous BDNF or adding exogenous BDNF may become an effective treatment way to treat spinal cord injuries or other nervous system diseases. BDNF have a direct impact on downstream cellular signals related to the activity and function of nerve cells, can effectively treat neurological diseases caused by different factors, and even can treat a part of diseases from pathophysiology aspect[13-14]. However, how to achieve effective therapeutic dose and secure method in the BDNF treatment and how to transport BDNF insistently into brain tissue is still a challenge in current studies and in human clinical trials.
Design
An eukaryotic expression vector construction experiment.
Time and setting
The experiment was completed in the Life Science College of Xinxiang Medical University, China from November 2011 to August 2013.
Materials
Escherichia coli (E. coli) DH5α was kept in the Stem Cell and Biological Treatment Center at Xinxiang Medical University. Fresh anticoagulant blood samples were donated from a volunteer in this project group who is a male, 30 years old, with no disease and good general situation, and there was no disease found after physical examination. We had informed the purpose, significance and possible discomfort of this experiment to the donor and gotten the permission of this volunteer who signed the informed consent.
Vectors
pIRES2-EGFP plasmid was purchased from Beijing TianDz Inc, pIRES2-VEGF165-EGFP plasmid was preserved in the Stem Cell and Biological Treatment Center at Xinxiang Medical University.
Cells
HEK293 cells were kept in the Stem Cell and Biological Treatment Center at Xinxiang Medical University.
Related reagents and instruments on cconstruction of eukaryotic expression vector on double gene consisting of BDNF and VEGF165
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Methods
Design of the primers
BDNF (NM-001102654.1) and VEGF165 gene sequence (NM-001025368.2) in Gene Bank as the template were used to design the primers. The coding sequence of human BDNF gene fragment was totally on the exon2 of the genomic DNA. Two primers were designed to amplify BDNF. Eco RⅠ restriction enzyme was used to cut sites and protective base was added in the upstream prime; and Bam HⅠ restriction enzyme was used to cut sites and protective base was added in the downstream primer. The length of amplified fragment was 744 bp.
Two primer pairs were designed to amplify the human VEGF165 from the pIRES2-VEGF165-EGFP (Table 1). Forward-long primer was four bases longer than forward-short at the 5’ end, so did primer reverse-long than reverse-short. The underlined bases in forward primers were introduced parts of sequence of Bst XⅠ site, and in reverse primers of NotI site, which could be used to assemble the Bst XⅠ and Not Ⅰ sticky ends. The length of amplified fragment was 576 bp.
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Table 1 The primers of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor 165 (VEGF165)
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Construction of pIRES2-BDNF-EGFP
The genomic DNA of human peripheral blood mononuclear cells severed as the template and the primer of BDNF was used to amplify BDNF gene. The coding sequence of human BDNF gene fragment was totally on the exon2 of the genomic DNA. A total of 20 μL PCR reaction system was added, including the genomic DNA 0.5 μL (10 ng), 2×Prime STAR Max DNAPolymerase 10 μL, RNase-Free Water 7.5 μL, upstream primer and downstream primer 2 μL. The reaction conditions were: 95 ℃ for 5 minutes, 98 ℃ for 10 seconds, 55 ℃ for 5 seconds, and 72 ℃ for 5 seconds, totally 30 cycles, and finally, 72 ℃ for 5 minutes. PCR product was purified by the PCR Purification Kit, then the PCR product and plasmid pIRES2-EGFP was cut by Eco RⅠand Bam HⅠ. After digested, PCR product was purified by the PCR Purification Kit. The DNA fragment was inserted into the plasmid by T4 DNA ligase, and 20 μL system was added, including pIRES2-EGFP 2 μL, cDNA (BDNF) 8 μL, T4 DNA ligase 0.2 μL at 22 ℃ for 30 minutes. The DNA fragment was translated into E. coli DH5α and placed onto LB plate (kanr) at 37 ℃ in an incubator for 16 hours. Monoclonal colony was picked up and shaken for 12-16 hours at 37 ℃ at a speed of 225 r/min. The plasmid was extracted and identified by using Eco RⅠ and Bam HⅠ double enzyme digestion. The recombinant plasmid pIRES2-BDNF-EGFP was obtained.
PCR ampli?cation of VEGF165
Two parallel PCRs were set up using forward-long/forward-short or reverse-long/reverse-short primer pairs, with the pIRES2-VEGF165-EGFP plasmid by as templates. Both ampli?cations were subject to 30 cycles of 94 ℃ for 30 seconds, 54 ℃ for 30 seconds and 72 ℃ for 1 minute, followed by a 5- minute extension at 72 ℃ using Prime STAR Max DNA Polymerase. Another two parallel PCRs were performed using EXTaqDNA polymerase. The expected DNA fragments in the four PCR products were purified separately using DNA gel extraction kit and quantified by Nano Drop 2000UV–Vis Spectrophotometers.
Construction of pIRES2-BDNF/VEGF165
PIRES2-BDNF-EGFP was digested with Bst XⅠand Not Ⅰ, followed by phenol: chloroform extraction and ethanol precipitation. Meanwhile, each pooled VEGF165 PCR products amplified with Prime STAR Max DNA Polymerase were mixed, denatured at 94 ℃ for 4 minutes and re-annealed at 65 ℃ for 2 minutes. After a ligation reaction, the annealed VEGF165 and linearized pIRES2-BDNF-EGFP (molar ratio equal to 4:1) were incubated with T4 DNA ligase at 16 ℃ for 3 hours. The two ligation products were used to transform E. coli DH5α CaCl2 competent cells following standard method. Screening of the transformants was performed by Bst XⅠplus Not Ⅰ digestion following alkalinelysis plasmid preparation, and the recombinants were confirmed by DNA sequencing.
In vitro transfection of HEK293 cells
HEK293 cells were maintained in Dulbecco’s modified Essential Medium (DMEM) containing 10% fetal bovine serum and100 mg/L penicillin/streptomycin. Cells were maintained in a humidified environment at 37 ℃ and 5% CO2. Cell viability was monitored with trypan blue exclusion method. The viability was over 95% in all experiments. Cells were seeded at a density of 5×105 cells/well in a 6-well tissue culture plate and cultured for 24 hours up to 60%-80% confluence. HEK293 Cells were either mock infected or infected with the pIRES2-EGFP, pIRES2-BDNF/EGFP and pIRES2-BDNF/VEGF165 three vectors using Lipofectamine™ 2000 respectively for 2 hours at 37 ℃ at 5 μg per well. Two hours later the transfection medium was removed, and fresh complete growth medium was added. Twenty-four hours post-transfection, then they were observed under the inverted fluorescent microscope. Expression of the BDNF and VEGF165 gene by RT-PCR and western blot were analyzed after 3 days.
Semi-quantitative RT-PCR detection of VEGF and BDNF mRNA expression
To detect the BDNF and VEGF165 mRNA expression levels in HEK293 cells which either mock infected or infected with the pIRES2-EGFP, pIRES2-BDNF/EGFP, and pIRES2-BDNF/VEGF165 three vectors respectively 3 days later, the BDNF and VEGF165 expression was primarily assessed by means of RT-PCR (Table 2). The methods is that each collected cells were added per 1 mL TRIZOL reagent and pipetting repeatedly, extract total cellular RNA, in accordance with the instruction manual steps, transcribe the extracted total RNA reverse into cDNA. Then amplified the Reverse transcripted cDNA with the PCR, Reverse transcription of the cDNA was amplified with the PCR, and the reaction conditions were pre-denaturation under 94 ℃ for 5 minutes; 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 30 seconds, make 35 cycles; when under 72 ℃, 10 minutes will be extended. 5 μL PCR amplification product was taken and added into 1 μL loading buffer to run on 20 g/L agarose gel for electrophoresis under 100 V voltage. Next shot by gel imaging system, and the gray value of each band was analyzed.
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Table 2 The semi-quantitative RT-PCR primers of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor 165 (VEGF165)
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Western blot detection of VEGF and BDNF protein expression
A standard western blotting protocol was used to detect BDNF and VEGF165 protein expression in HEK293 cells which either mock infected or infected with the pIRES2-EGFP, pIRES2-BDNF/EGFP and pIRES2-BDNF/VEGF165 three vectors respectively three days later. We collected the culture supernatants of infected HEK293 cells without serums which were processed for western blot using an anti-BDNF and anti-VEGF165 antibody. The 20 μg protein extracted from transfected HEK293 cells was separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and denaturing conditions and transferred to polyvinylidene difluoride (PVDF) membrane. The membranes were incubated in a 1:500 dilution of polyclonal rabbit anti-BDNF antibody (Santa Cruz Biotechnology, CA, USA) or 1:500 dilution of polyclonal rabbit Anti-VEGF165 antibody(Santa Cruz Biotechnology), polyclonal rabbit Anti-β-Actin(1:500, Santa-Cruz)was used as the loading control. After washing with TBS for three times(10 minutes once), the samples were incubated with horseradish peroxidase conjugated goat anti-rabbit lgG (1:5 000) at room temperature for 2 hours. TBS washing (3×15 minutes per time), and X-ray film was performed to observe the final results. The amount of signals was quantified with Gel-Pro analyzer software.
Main outcome measures
mRNA and protein expressions of VEGF and BDNF were detected by RT-PCR and western blot assay.
Statistical analysis
SPSS 13.0 statistical software was used on experimental data for statistical processing, the data were described as mean±SD. Intergroup comparisons were tested by one-way analysis of variance, and a value of P < 0.05 was considered statistically significant.
To improve the cloning efficiency and simplify the cloning procedure, we developed twin PCR to create insert DNA with single-stranded overhangs at the ends which were complimentary to restriction enzyme digested vector[15]. As shown in Figure 1, PCR products were reassembled via complimentary pairing after heat-denaturing and re-annealing. Four types of molecules with equal proportions were created, two of which were blunt-ended and the other two were sticky-ended. Theoretically, half of the sticky-ended products are suitable for a certain ligation. Using twin PCR strategy, hybridized PCR products flanked with all kinds of sticky ends that are compatible with almost all restriction enzymes can be generated solely based on primer designing and PCR amplification. Since no restriction enzyme digestion is required for the PCR products, twin PCR overcomes the problems caused by end sensitivity of restriction enzymes, and the internal restriction sites within the amplified sequences will not limit the selection of cloning sites. This is particularly useful for cloning long PCR products, which have more opportunities to contain kinds of restriction enzymes sites.
In this study, BDNF and VEGF165 were successfully inserted into a bicistronic eukaryotic expression vector and a plasmid pIRES2-BDNF-VEGF165 was constructed successfully. We plan to transfect this plasmid expressed by double genes into mesenchymal stem cells in the next experiment, and to achieve the stable transfected cell lines. We surmised that in promoting neuronal regeneration, mesenchymal stem cells modified by the double genes will be more significant compared with the single gene modified cells.
In this study, VEGF165 was directly inserted into pIRES2-BDNF-EGFP at the BstXI and NotI sites. Those more efficient and economical enzymes, such as BstXI and NotI, could always be chosen for vector digestion. It is not any longer necessary to buy so many kinds of restriction enzymes for cloning. Meanwhile, our cloning method has other advantages. It is so simple that the whole procedure, including PCR, enzyme digestion, ligation and transformation, could be completed within 8 hours, leading to the substantial saving in time, cost and labor. In addition, interested inserts are amplified using proof-reading DNA polymerase, which provides a faithful guarantee for the PCR products. These advantages make it a universal technique for the directional cloning of PCR products.
VEGF is a specific growth factor; it can promote the division, proliferation and the migration of vascular endothelial growth factor, thereby to promote angiogenesis[16-18]. It has been confirmed that the VEGF can promote the proliferation of vascular endothelial cells, thereby enhancing the capillary density[19]. So VEGF has been widely studied and used in the treatment of various ischemic diseases such as coronary atherosclerotic heart disease, cerebral ischemia, peripheral neuritis[20-21]. Some scholars also use it for skin flap to promote its vascularisation, then to improve skin flap survival rate[22]. However, there are now few studies about gene transfection of VEGF in the protection and treatment of spinal cord injuries.
BDNF is a kind of protein which can promote the growth activity of nervus, it was firstly isolated and purified from porcine brain in 1982 by Barde et al. A substantial portion of the amino acid sequence of BDNF are the same with nerve growth factor, so there are called by a joint name as neurotrophins[23]. Scholars have made a lot of research about the reconstruction and repair role of BDNF after spinal cord injury and found that BDNF can improve the survival of sensory neuron and the activity of damaged spinal cord, moreover it can promote repair and regeneration of neurons[24]. In addition, BDNF can also stimulate sprouting of adult axons and dendrites, enhancing the release of synaptic neurotransmitter and strengthening signal transduction among synaptic[25]. BDNF is the most important neurotrophic factor in the nervous system, a large number of studies have shown that the content of BDNF changes in a certain regularity with the time extension of the spinal cord injury[26], which indicates that the complexity pathological process that BDNF involved in spinal cord injury. With the continuous development of transgenic technology and the deepening study of the pathology on spinal cord injury, BDNF transgene therapy applied to the repair of movement spinal cord injury is inevitable.
In this group of experiments, we constructed the eukaryotic expression vector co-expressing BDNF and VEGF165, then to transfect HEK293 cells. The results showed that HEK293 cells highly expressed the BDNF and VEGF165, providing the basis for the usage of cells as vector to transmit BDNF and VEGF165 to spinal cord injury site. In addition, this experimental group also has the following characteristics: (1) Two genes were encoded in one eukaryotic expression vector, and an internal nucleic acid was used into a locus sequence (IRES), connecting the BDNF and VEGF165. Controlled by a CMV promoter, both two genes could express effectively. Although the IRES is a commonly used constituent element of the double gene expressed vector[27-28], when under the different systems, the expressing level of the two genes connected with IRES are different. The gene close to the promoter often expresses higher, and usually the gene of downstream the IRES expresses lower and even cannot express effectively[29]. However in this group of experiments we demonstrated both BDNF and VEGF165 connected with IRES can obtain a high efficiency expression. (2) The BDNF, VEGF165, and human mesenchymal stem cells as well as eukaryotic expression vector as the research subjects, may be more conducive to neural regeneration and reconstruction after spinal cord injury, and may be more helpful for transforming research findings to clinical application. The way infecting cells as vectors in vitro at first place avoids the potential safety hazard that caused by injecting eukaryotic expression vectors directly in vivo, and prevents the ethics problem of leading in heterologous gene directly.
In the study on repair of spinal cord injury, cells are playing two roles: the target cells and carrier cells. Target cells play its role by replacing the regenerated tissue with cells, then rebuilding neural pathways. Carrier cells are used as a media of gene expression or the medium on therapeutic cytokine releasing, so as to promote the recovery of neural engineering. By summarizing the present researches, mesenchymal stem cells have the characteristics as the target cells and the carrier cells, which is ideally suitable for gene therapy research on spinal cord injury[30]. Currently there are two categories of vectors used for gene therapy that is viral vectors and non-viral vectors[31]. Although viral vectors can be highly transfected and expressed stably, it is easy to be integrated into the chromosomes of target cells. Therefore, the security is low, and we should process it with caution. Most of non-viral vectors are various kinds of plasmids, appearing as an episome form after entering the target cells, with the advantages of high security and large capacity. In this experiment, the utilized pIRES2-EGFP eukaryotic expression vector can express high levels of the target genes. However, because the BDNF and VEGF are small molecule proteins, they are easily degraded in the body, with a short half-life, and meanwhile it is not easy to make multiple dosing[31-34].
By gene recombination, we successfully constructed the eukaryotic expression vector of BDNF and VEGF165, and these two genes can be co-expressed in HEK293 cells. By checking and analyzing the national and international relative literatures, there is no study about gene therapy by BDNF and VEGF165 to promote differentiation of mesenchymal stem cells for spinal cord injury, so this experiment will provide new methods for in vivo animal experiments in the next step.
文章构思特点
在基因治疗中，联合应用多个具有协同作用的治疗基因通常可产生较单基因更为理想的效果。但是与国内外同类研究水平的比较以往进行多基因联合治疗的方法存在不同缺陷，利用携带不同基因的独立载体系统同时转染靶细胞的方法，虽然可自由调节各表达载体的比例，但各基因的总表达效率低下；而用传统方法构建的多启动子表达载体，因为每个治疗基因都需要一个包括启动子、终止子等表达元件在内的完整基因表达盒，使得载体过于庞大，操作困难；而将2个基因融合表达的策略可能因蛋白结构相互影响而导致功能丧失。利用IRES构建多基因共表达载体，则可大大提高转移及表达效率。
近年来研究者常常采用几种神经营养因子的单基因重组载体直接注射或转染相应细胞后对脊髓损伤动物模型进行治疗，结果均取得了令人欣喜的疗效，为临床的应用带来了曙光。但是大量不同单基因重组载体的应用，增加了基因治疗载体的用量，不可避免的增加了非治疗相关的外源基因序列的导入（如载体中抗性筛选序列等），从而为临床的应用增加了不安全因素。实验采用内部核糖体进入位点序列构建了能够分别表达的脑源性神经营养因子)与血管内皮细胞生长因子165双基因真核表达载体，即增加了外源基因的表达，同时可以减少基因治疗载体的用量，减少了不安全因素，为临床的进一步应用带来了希望。
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