人胶质细胞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的构建与鉴定

doi:10.3969/j.issn.2095-4344.2014.29.015

中国组织工程研究 ›› 2014, Vol. 18 ›› Issue (29): 4675-4682.doi: 10.3969/j.issn.2095-4344.2014.29.015

• 组织构建与生物活性因子 tissue construction and bioactive factors • 上一篇下一篇

人胶质细胞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

修回日期:2014-05-01 出版日期:2014-07-09 发布日期:2014-07-09
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，河南省新乡市 453003
基金资助:
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)

Construction and identification of pIRES₂-GDNF-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

Revised:2014-05-01 Online:2014-07-09 Published:2014-07-09
Contact: Li Bing-nan, Department of Life Sciences and Technology, Xinxiang Medical University, Xinxiang 453003, Henan Province, China
About author:Li Bing-nan, Ph.D., Lecturer, Department of Life Sciences and Technology, Xinxiang Medical University, Xinxiang 453003, Henan Province, China Li Wei-dong, Department of Life Sciences and Technology, Xinxiang Medical University, Xinxiang 453003, Henan Province, China Li Bing-nan and Li Wei-dong contributed equally to this work.
Supported by:
the Tender Subject of Key Research Areas of Xinxiang Medical University in 2011, No. ZD2011-16; Key Projects in Scientific Research of Henan Provincial Education Department, No. 13A180850.

摘要/Abstract

摘要：

背景：人胶质细胞源性神经营养因子(glial cell line - derived neurotrophic factor，GDNF)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。

目的：构建双基因共表达载体pIRES₂-GDNF-VEGF₁₆₅并对其进行鉴定。

方法：采用PCR法从人外周血单个核细胞的基因组DNA中获取人胶质细胞源性神经营养因子基因，然后将人胶质细胞源性神经营养因子的cDNA片段插入到pIRES₂-EGFP多克隆位点构建成为pIRES₂-GDNF-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，最后构建成为含有内部核糖体进入位点(IRES)的pIRES₂-GDNF-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，提取的人胶质细胞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，相对分子质量分别为636 bp和576 bp。构建的pIRES₂-GDNF-VEGF₁₆₅双基因共表达载体经Bgl II/Bam HI切出GDNF条带，经Bam HI/Not I双酶切后切出 IRES-VEGF₁₆₅片段，经Bgl II/Not I双酶切后切出GDNF-IRES-VEGF₁₆₅片段。RT-PCR与Western-blot方法检测显示，此载体转染后，HEK293细胞均能表达人胶质细胞源性神经营养因子和血管内皮生长因子165 mRNA和蛋白。说明实验成功构建了能表达人胶质细胞源性神经营养因子和血管内皮生长因子165的双基因真核表达载体。

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

关键词: 组织构建, 组织工程, 胶质细胞源性神经营养因子, 血管内皮生长因子165, 真核双表达载体, 内部核糖体进入位点, 转染, 双PCR

Abstract:

BACKGROUND: Human glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor 165 (VEGF₁₆₅) are essential genes for cell differentiation.

OBJECTIVE: To construct and identify pIRES₂-GDNF-VEGF₁₆₅ bicistronic eukaryotic expression vector.

METHODS: Human GDNF genes were obtained from the genomic DNA of human peripheral blood mononuclear cells by PCR. Then the GDNF cDNA fragment was inserted into the multiple cloning sites of pIRES₂-EGFP, to generate the bicistronic eukaryotic expression plasmid pIRES₂-GDNF-EGFP. The VEGF₁₆₅gene was obtained from pIRES₂-VEGF₁₆₅-EGFP plasmid by twin PCR. Then VEGF₁₆₅cDNA fragment was cloned into the pIRES₂-GDNF-EGFP, instead of EGFP, to create a double gene co-expressing vector plasmid pIRES₂-GDNF-VEGF₁₆₅ containing internal ribosome entry sites. Then pIRES₂-GDNF-VEGF₁₆₅was used to transfect HEK293 cells. RT-PCR and western blot analysis were performed to test the co-expression of double genes.

RESULTS AND CONCLUSION: DNA sequencing analysis demonstrated that the GDNF and VEGF₁₆₅ were exactly consistent with the sequence recorded in the GenBank. The size of GDNF gene was 636 bp and the size of VEGF₁₆₅ gene was 576 bp. Enzyme digestion analysis indicated that, pIRES₂-GDNF-VEGF₁₆₅ bicistronic eukaryotic expression vector inserted GDNF band by Bgl II/Bam HI, inserted IRES-VEGF₁₆₅ fragment by Bam HI/Not I, and inserted GDNF-IRES-VEGF₁₆₅ fragment by Bgl II/Not I. RT-PCR and western blot analysis showed that, after HEK293 cells were transfected with pIRES₂-GDNF-VEGF₁₆₅, double genes were expressed at the mRNA and protein levels. The pIRES₂-GDNF-VEGF₁₆₅ bicistronic eukaryotic expression vector is successfully constructed.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: glial cell line-derived neurotrophic factor, vascular endothelial growth factor, carrier proteins, tissue engineering

中图分类号:

R318

栗炳南，李卫东，林俊堂，丰慧根. 人胶质细胞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的构建与鉴定[J]. 中国组织工程研究, 2014, 18(29): 4675-4682.

Li Bing-nan, Li Wei-dong, Lin Jun-tang, Feng Hui-gen. Construction and identification of pIRES₂-GDNF-VEGF₁₆₅ bicistronic eukaryotic expression vector[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(29): 4675-4682.

图/表（结果） 2

Amplification of GDNF and VEGF₁₆₅ genes

GDNF gene was obtained from the genomic DNA of human peripheral blood mononuclear cells by overlap PCR. The size of GDNF gene was 636 bp. The VEGF₁₆₅gene was obtained from pIRES₂-VEGF₁₆₅-EGFP plasmid by twin PCR, and the size of VEGF₁₆₅ gene was 576 bp (Figure 1).

Identification of plasmid pIRES₂-GDNF-EGFP

The plasmid pIRES₂-GDNF-EGFP was cut by Bgl II and Bam HI double enzyme. A gene fragment with 636 bp was obtained, which was in full agreed with GDNF gene (Figure 2A).

Identification of plasmid pIRES₂-GDNF-VEGF₁₆₅

The plasmid pIRES₂-GDNF-VEGF₁₆₅ was cut by Bgl II and Bam HI double enzyme. And a fragment about 636 bp would be obtained. This indicated GDNF gene inserted into the plasmid pIRES₂-GDNF-VEGF₁₆₅. The sequence of the plasmid pIRES₂-GDNF-VEGF₁₆₅ was in accordance with gene sequence in Gene Bank. The plasmid pIRES₂-GDNF-VEGF₁₆₅was cut by Bam HI and Not I double enzyme. And a fragment about 1 183 bp would be obtained. This indicated IRES-VEGF₁₆₅ gene inserted into the plasmid pIRES₂-GDNF-VEGF₁₆₅. The plasmid pIRES₂-GDNF- VEGF₁₆₅ was cut by Bgl II and Not I double enzyme. And a fragment about 1 825 bp would be obtained. This indicated BDNF-IRES-VEGF₁₆₅ gene inserted into the plasmid pIRES₂-GDNF-VEGF₁₆₅. The sequence of the plasmid pIRES₂-BDNF-VEGF₁₆₅ was in accordance with gene sequence in Gene Bank (Figure 2B).

RT-PCR analysis of the expression of GDNF and VEGF₁₆₅ in HEK293 cells

To illustrate mRNA expression by pIRES₂-EGFP, pIRES₂-GDNF/EGFP, and pIRES₂-GDNF/VEGF₁₆₅transduced HEK293 cells, we evaluated the expression of GDNF and VEGF₁₆₅ by RT-PCR analysis. RT-PCR was performed using GDNF-specific primers and the β-actin sequence as an internal standard. GFP expression was monitored in pIRES₂-EGFP and pIRES₂-GDNF/EGFP transduced HEK293 cells by inverted fluorescence microscopy. Expression of GDNF mRNA was higher in either pIRES₂-GDNF/EGFP or pIRES₂-GDNF/VEGF₁₆₅ transduced HEK293 cells than that pIRES₂-EGFP transduced HEK293 cells or negative control (Figure 3A, B). As shown above, expression of VEGF₁₆₅ mRNA was higher in pIRES₂-GDNF/VEGF₁₆₅-transduced HEK293 cells than the other three (Figure 3C, D). These results demonstrated that the GDNF and VEGF₁₆₅ had been introduced successfully into HEK293 cells by pIRES₂-GDNF/EGFP and pIRES₂-GDNF/VEGF₁₆₅. After the HEK293 cells were transduced by pIRES₂-GDNF/EGFP, we passaged them continually and then monitored the mean percentage of expression of GFP under fluorescence microscopy. There was no decrease in GFP fluorescence, illustrating the maintenance of transgenic expression in the transduced cells.

Western blot analysis of the expression of GDNF and VEGF₁₆₅ in HEK293 cells

To illustrate protein expression by pIRES₂-EGFP, pIRES₂-GDNF/EGFP, and pIRES₂-GDNF/VEGF₁₆₅transduced HEK293 cells, we evaluated the expression of GDNF and VEGF₁₆₅ by western blot assay. After 72 hours of transfection, we collected the culture supernatants of infected HEK293 cells without serums which were processed for western blot analysis using an anti-GDNF and anti-VEGF₁₆₅ antibody. β-actin served as an internal standard. The results suggested that exogenous GDNF protein was strongly expressed in pIRES₂-GDNF/EGFP and pIRES₂-GDNF/VEGF₁₆₅ transduced HEK293 cells, but the pIRES₂-EGFP transduced HEK293 cells and no transduced cells, the expression of endogenous GDNF was very low (Figures 4A, B). As shown above, western blot analysis also revealed that exogenous VEGF₁₆₅ protein was strongly expressed in pIRES₂-GDNF/VEGF₁₆₅ transduced HEK293 cells, and expression of endogenous VEGF₁₆₅ was very low in the pIRES₂-EGFP, pIRES₂-GDNF/EGFP transduced HEK293 cells (Figures 4C, D). These results also demonstrated that GDNF and VEGF₁₆₅ had been introduced successfully into HEK293 cells by pIRES₂-GDNF/VEGF₁₆₅.

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引言

Spinal cord injury can cause severe spinal nerve dysfunction^[1]. With the development of molecular therapy, gene therapy is considered to be a new choice for treatment of spinal cord injury^[2]. Vascular endothelial growth factor (VEGF) has the effect of promoting angiogenesis^[3]. Increasing evidence of recent studies showed that, VEGF also can promote the growth of nerve cells^[4]. On the one hand, VEGF can stimulate the regeneration nerve and its nutrient vessels to repair spinal cord injury^[5]; on the other hand, VEGF can increase the activity of peripheral nervous system nerve cells through vegetative nerve and mitogen activation^[6].

Glial cell line derived neurotrophic factor (GDNF) is a novel neurotrophic factor, belongs to TGF-p superfamily, which was isolated and purified from the supernatant from rat B49 cells in 1993 by Lin et al^[7-10]. GDNF is one of the most effective neurotrophic factors for supporting the growth of motor neurons in vitro, it can not only save the programmed death of motor neurons during the development process, but also promote the survival of cortical neurons after spinal cord injury^[11-15]. In addition to nutritional and protective effect on the midbrain dopaminergic neurons, GDNF also has nutritional and protective effect to different degree on the sensory and motor neurons^[16-18].

In this study, we constructed the co-expression plasmid of GDNF and VEGF, and then transfected HEK293 cells and detect the expression. This provides the experimental

basis for the following experiment of co-transfecting mesenchymal stem cells, improving the internal environment of spinal cord injury, regulating the differentiation of mesenchymal stem cells, increasing the number of newborn neurons, and promoting functional recovery of spinal cord.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

材料方法

Design

A gene 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 DH5α are kept in Stem Cells and Biological Treatment Center at Xinxiang Medical University, China. Fresh anticoagulant blood samples are donated from a volunteer in this project group. This volunteer is a 30-year-old male, with no disease previously. There was no disease detected by physical examination. We have informed the donor of the purpose, significance and possible discomfort of this experiment, and have got the permission of this volunteer, who signed the informed consent.

Vector

pIRES₂-EGFP plasmid was purchased from Beijing TianDz Inc. pIRES₂-VEGF₁₆₅-EGFP plasmid are preserved in Stem Cells and Biological Treatment Center at Xinxiang Medical University in China.

Cells

HEK293 cells are kept in Stem Cells and Biological Treatment Center at Xinxiang Medical University in China.

Methods

Design of the primers

GDNF (NM_000514.3) and VEGF₁₆₅ gene sequence (NM-001025368.2) in Gene Bank served as the template and was used to design the primers. Overlap-PCR, an efficient and rapid method, was used to clone human GDNF gene CDS (coding sequence) from genomic DNA. The procedure included four primers and three-step PCRs. The Coding Sequence of human GDNF gene fragment includes two exons (exon1 + exon2). Two primer pairs (forward-primer and reverse-primer) were designed to amplify GDNF-1 (exon1), Bgl II restriction enzyme cut site was added in forward-primer which is the outermost primers in the upstream. Other two primer pairs (forward-primer and reverse-primer) were designed to amplify the exon2 (GDNF-2). SmalI restriction enzyme cut site was added in reverse-primer which is the outermost primers in the downstream. The length of amplified fragment was 636 bp.

Two primer pairs were designed to amplify the human VEGF₁₆₅ from the pIRES₂-VEGF₁₆₅-EGFP. Forward-long primer was 4 bases longer than forward-short at the 5’ end, so did primer reverse-long than reverse-short. The forward primers were introduced parts of sequence of Bst XI site, and in reverse primers of Not I site, which will be used to assemble the Bst XI and Not I sticky ends. The length of amplified fragment was 576 bp.

Construction of pIRES₂-GDNF-EGFP

The genomic DNA of human peripheral blood mononuclear cells severed as the template and the primer of GDNF was used to amplify GDNF gene. In this study, Overlap-PCR, an efficient and rapid method, was used to clone human GDNF gene CDS (coding sequence) from genomic DNA. The procedure included four primers and three-step PCRs. Human GDNF gene consists of two exons and the CDS contains 636 bp. In the first step three PCRs were performed to generate extended exon1 (GDNF-1), exon2 (GDNF-2) that contained overlapped nucleotides and were used as the templates for second ligation PCR. Two primer pairs were designed to amplify exon1 (GDNF-1). Other two primer pairs were designed to amplify the exon2 (GDNF-2). Secondly, exon1 (GDNF-1) and exon2 (GDNF-2) were spliced together. Lastly, the two exons (GDNF-1 and GDNF-2) were linked together with outermost primers and the templates from the second step. As an efficient and rapid method, overlap-PCR is feasible and acceptable for gene cloning from genomic DNA.

The genomic DNA of human peripheral blood mononuclear cells severed as the template and the primer of GDNF was used to amplify GDNF gene. A total of 20 μL PCR reaction system was added, including genomic DNA 0.5 μL (10 ng), 2 × Prime STAR Max DNA Polymerase

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 55 seconds, 30 cycles; finally, maintained at 72 ℃ for 5 minutes. PCR product was purified by the PCR Purification Kit, then the PCR product and plasmid pIRES₂-EGFP was cut by Bgl II and Sma I. After digestion, PCR product was purified by the PCR Purification Kit. The DNA fragment was inserted into the plasmid by T4 DNA ligase, 20 μL system was added, including pIRES₂-EGFP 2 μL, cDNA (GDNF) 8 μL, T4 DNA ligase 0.2 μL, at 22 ℃ for 30 minutes. The DNA fragment was translated into Escherichia coli DH5α and placed on LB plate (kanr) at 37 ℃ incubator for 16 hours. Monoclonal colony was picked up and shaken for 12-16 hours at 37 ℃ with a speed of 225 r/minute. The plasmid was extracted and identified by using Bgl II and Bam HI double enzyme digestion. The recombinant plasmid pIRES₂-GDNF-EGFP was obtained.

PCR amplification 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 amplifications were subjected 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 EXTaq DNA polymerase. The expected DNA fragments in the four PCR products were puri?ed separately using DNA gel extraction kit and quanti?ed by Nano Drop 2000 UV-Vis Spectrophotometers.

Construction of pIRES₂-GDNF/VEGF₁₆₅

PIRES₂-GDNF-EGFP was digested with Bst XI and Not I, followed by phenol: chloroform extraction and ethanol precipitation. Meanwhile, each pooled VEGF1₆₅ PCR products ampli?ed with Prime STAR Max DNA Polymerase were mixed, denatured at 94 ℃ for

4 minutes and re-annealed at 65 ℃ for 2 minutes. For a ligation reaction, the annealed VEGF₁₆₅ and linearized pIRES₂-GDNF-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 Escherichia coli DH5a CaCl₂ competent cells following standard method. Screening of the transformants was performed by Bst XI plus Not I digestion following alkalinelysis plasmid preparation, and the recombinants were con?rmed by DNA sequencing.

Two parallel PCRs were set up using either forward-long/reverse-long (PCR products A) or forward-short/reverse-short primer pairs (PCR products B), with the pIRES₂-VEGF₁₆₅-EGFP plasmid (double ring means this plasmid in this figure) by as templates. Equimolar of the two PCR products A and B were mixed, heat denatured and re-annealed to generate hybridized DNA fragments. Four types of double-stranded DNA molecules with equal proportions are generated via random complementary pairing. They are molecule I, II, III and IV. Two of them, I and II, are blunt-ended and another two are sticky-ended. The molecule III possess a 5’ Bst XI sticky end and a 3’ Not I sticky end, which allow the fragment to be cloned directionally into pIRES₂-BDNF-EGFP digested with Bst XI and Not I.

In vitro transduction of HEK293 cells

HEK293 cells were maintained in Dulbecco’s modified Essential Medium (DMEM) containing 10% newborn bovine serum and 100 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 × 10⁵cells/well in a six-well tissue culture plate and cultured for 24 hours to 60%-80% confluence. HEK293 cells were either mock infected or infected with the pIRES₂-EGFP, pIRES₂-GDNF/EGFP and pIRES₂-GDNF/VEGF₁₆₅ three vectors using Lipofectamine^TM 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, they were observed under the inverted fluorescent microscope. The expression of the BDNF and NT-3 gene was detected by RT-PCR and western blot analysis 3 days later.

RT-PCR detection of the expression of GDNF and VEGF₁₆₅ in HEK293 cells

To detect the GDNF and VEGF₁₆₅ mRNA expression levels in HEK293 cells which were either mock infected or infected with the pIRES₂-EGFP, pIRES₂-GDNF/EGFP and pIRES₂-GDNF/VEGF₁₆₅respectively, the GDNF and VEGF₁₆₅ expression was primarily assessed by means of RT-PCR. The collected cells were added per 1 mL TRIZOL reagent and pipetting repeatedly, total cellular RNA was extracted in accordance with the instruction of manual steps. Then the extracted total RNA was transcribed and reversed into cDNA. Then the reverse transcription of the cDNA was amplified with the PCR, and the reaction conditions were pre-denaturation under 94 ℃ for 5 minutes; 94 ℃ for 30 seconds, 55 ℃ for

30 seconds, 72 ℃ for 30 seconds, 35 cycles, followed by

72 ℃ extension for 10 minutes. 5 μL of PCR amplification product was added with 1 μL loading buffer, placed in

20 g/L agarose gel electrophoresis at 100 V. Subsequently the cells were observed with gel imaging system, and the absorbance value of each band was analyzed.

Western blot analysis of the expression of GDNF and VEGF₁₆₅ in HEK293 cells

A standard western blot protocol was used to detect BDNF and NT-3 protein expression in HEK293 cells which were either mock infected or infected with the pIRES₂-EGFP, pIRES₂-GDNF/EGFP and pIRES₂-GDNF/VEGF₁₆₅vectors respectively. We collected the culture supernatants of infected HEK293 cells without serums for western blot analysis, which was assayed using an anti-GDNF and anti-NT-3 antibody. The 20 μg protein extracted from the culture supernatants of transduced HEK293 cells was separated on SDS-PAGE under reducing and denaturing conditions, and transferred to polyvinylidene difluo-ride (PVDF) membrane. The membranes were incubated in a 1:500 dilution of polyclonal rabbit anti-GDNF antibody (Santa Cruz Biotechnology, USA) or 1:250 dilution of polyclonal rabbit anti-VEGF₁₆₅ antibody (Santa Cruz Biotechnology), polyclonal rabbit anti-β-actin (1:500, Santa-Cruz Biotechnology) was used as the loading control. After washing with TBS for three times (10 minutes per time), cells were incubated with horseradish peroxidase conjugated goat anti-rabbit lgG (1:5 000) at room temperature for 2 hours. After TBS washing (3 times, for 15 minutes each), X-ray film was performed to observe the final results. The amount of signal was quantified with Gel-Pro analyzer software.

Main outcome measures

Expressions of GDNF and VEGF₁₆₅ mRNA and protein were detected by RT-PCR and western blot assay.

Statistical analysis

We using SPSS 13.0 statistical software on experimental data for statistical processing, the data are described in mean±SD, data comparisons between groups were tested by one-way analysis of variance. A P < 0.05 was considered statistically significant difference.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

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In this article, VEGF₁₆₅ was directly inserted into pIRES₂-GDNF-EGFP at the Bst XI and Not I sites. Those more efficient and economical enzymes, such as Bst XI and Not I, could always be chosen for vector digestion. It is not any longer necessary to buy so many kinds of restriction enzymes for cloning. 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.

GDNF is a protein found in recent years and has been cloned its gene, originally isolated from mouse glial cell line B49 cells, in vitro can activate the uptake of lateral dopamine neurons in ventral embryonic rats, and can promote neuronal survival^[20]. This factor is composed of two monomers by glycosylation two sulfur bond into two body, monomer is composed of 134 amino acids^[21]. The seven cysteine residues in the molecule conformation and transforming growth factor superfamily, and its sequence homology near 20%, belongs to the TGF superfamily distant from the structure^[22]. The experimental study indicated that GDNF is a neurotrophic factor with multi effect, which function on the sensory neurons of nutrition, exercise and dopamine, and its effect is the change in the different period of individual development^[23]. Because GDNF is a protein, not through the blood brain barrier and spinal cord barrier, also cannot be administered through the digestive tract, it must be through the effective methods to make it into the spinal cord or the surrounding, thus playing the best biological effect^[24]. The current administration methods are: (1) direct or indirect perfusion: concrete ways is intraventricular, cerebellomedullary cistern and a margin of administration, because this method in the operation is complicated and can increase the infection, aggravate the injury of spinal cord, so its application is limited^[25]. (2) The method of gene therapy for spinal cord injury is to cell transplantation of genetically modified GDNF such as glial cells, fibroblasts, thus providing micro-environment to promote neuronal growth, the method is obviously better than that of perfusion method^[26].

In recent years, VEGF has a direct effect on the growth of nerve cells, it can effectively reduce the spinal cord blood flow occlusion occurred after spinal cord injury^[27]. Neural stem cells can effectively enhance the angiogenesis in vivo and blood-brain barrier density, improve the neurological function score and spinal cord tissue necrosis in vitro through the transfer of VEGF gene^[28]. VEGF also can influence the apoptosis of neurons in spinal cord after injury^[29]. Application of VEGF in treating diabetes, ischemic neuropathy, nerve regeneration, Parkinson’s disease, Alzheimer’s disease and multiple sclerosis, also show its neuroprotective effect^[30].

In this study, we used the eukaryotic expression vector co-expressing GDNF and VEGF₁₆₅, and then transfect HEK293 cells with it. The results showed that HEK293 cells can highly express GDNF and VEGF₁₆₅, which provided the basis for the use of cells as a vector to transfer GDNF and VEGF₁₆₅ to spinal cord injury site.

In addition, GDNF and VEGF₁₆₅ genes were successfully inserted into a bicistronic eukaryotic expression vector and a plasmid pIRES₂-GDNF-VEGF₁₆₅ was constructed successfully. We plan to transfect this plasmid expressed by double genes into mesenchymal stem cells in the next experiment, in order to achieve the stable transfected cell lines. We surmised that double genes modified mesenchymal stem cells are better than single gene modified cells in promoting neuronal regeneration.

By gene recombination we successfully constructed eukaryotic expression vector of GDNF and VEGF₁₆₅, and these two genes can be co-expressed in HEK293 cells. Checking and analyzing the relative literatures, there is no study about gene therapy by using GDNF and VEGF₁₆₅ to promote differentiation of mesenchymal stem cells for spinal cord injury, so this experiment will provide new methods for the following animal experiments.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

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人胶质细胞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的构建与鉴定

Construction and identification of pIRES₂-GDNF-VEGF₁₆₅ bicistronic eukaryotic expression vector

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人胶质细胞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的构建与鉴定

Construction and identification of pIRES2-GDNF-VEGF165 bicistronic eukaryotic expression vector

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Construction and identification of pIRES₂-GDNF-VEGF₁₆₅ bicistronic eukaryotic expression vector