Abstract


BBACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) are easily isolated and amplified, and facilitate the exogenous gene transfer and expression. In the human medicine, it is believed that BMSCs are ideal therapeutic cells and target cells in the gene therapy.
OBJECTIVE: To investigate liposome-mediated cytosine deaminase (CD) gene transfecting rabbit BMSCs and its gene expression.
DESIGN: A single sample observation.
SETTING: Dalian Research and Development Center for Stem Cell and Tissue Engineering; Department of Biochemistry, College of Basic Medical Science, Dalian Medical University.
MATERIALS: This study was performed at in the Dalian Research and Development Center for Stem Cell and Tissue Engineering; Department of Biochemistry, College of Basic Medical Science, Dalian Medical University from March 2006 to June 2007. New Zealand big-ear white rabbits of either gender, weighing 2.0-2.5 kg, with the age of 5 months old, were included in this study.
METHODS: The CD gene was obtained from E.coli JM109 DNA by polymerase chain reaction (PCR). The fragment was cloned into pMD19-T vector. After restriction enzyme BamHI/XhoI digestion analysis and DNA sequence analysis, pIRES2-AcGFP1-CD eukaryotic expression plasmid was constructed. Meanwhile, BMSCs were harvested, cultured and identified. After enzyme digestion of eukaryotic expression plasmid, the rabbit BMSCs were transfected by Lipofectamine 2000-mediated method. Twenty-four hours after transfection, expression of green fluorescent protein was observed under an inverted fluorescent microscope.
MAIN OUTCOME MEASURES: Construction of eukaryotic expression plasmid and identification of CD gene-transferred BMSCs.
RESULTS: CD gene was cloned and connected to eukaryotic expression plasmid with green fluorescence. Twenty-four hours after transfecting rabbit BMSCs, it was found under an inverted microscope that under the excitation of 488 nm blue light, green fluorescence appeared in the pIRES2-AcGFP1-CD and pIRES2-AcGFP1 empty-plasmid transfected BMSCs, but not in the non-transfected ones. It indicates that CD gene successfully transferred BMSCs.
CONCLUSION: BMSCs are ideal vectors in the CD gene therapy.

INTRODUCTION

Bone marrow mesenchymal stem cells (BMSCs), existing in the bone marrow, are another kind of stem cells with infinite proliferation and multi-directional differentiation potential besides hematopoietic stem cells. Under some inducing conditions, such cells can directionally differentiate into the tissue cells from various germinal layers, especially the tissue cells from mesoderm and neural ectoderm, such as osteoblast, chondroblast, adipocyte, tendon cell, muscle cell, nerve cell and so on. BMSCs can grow in adherence, be easily isolated and amplified in vitro, and also facilitate exogenous gene transfer and expression. In the human medicine, it is believed that BMSCs are a kind of ideal therapeutic cells and of target cells in the gene therapy.
Cytosine deaminase (CD) is expressed by an E. coli. It only exists in the bacterium and fungi, but not in the mammals. It metabolizes nontoxic 5- flurocytosine into the commonly used. Meanwhile, it produces powerful bystander effects, which kill transgenic and some non-transgenic cells. In this study, we directionally cloned CD gene into pIRES2-AcGFP1 expression vector, transferred BMSCs with pIRES2-AcGFP1-CD recombinant plasmid, in order to provide scientific experiment and theoretical basis for the study on further expression of CD gene and future gene therapy of tumor.

MATERIALS AND METHODS

Materials
This study was performed at the Dalian Research and Development Center for Stem Cell and Tissue Engineering; Department of Biochemistry, College of Basic Medical Science, Dalian Medical University from March 2006 to June 2007. New Zealand big-ear white rabbits of either gender, weighing 2.0-2.5 kg, with the age of 5 months old, were provided by the Laboratory Animal Center, Dalian Medical University. Reagents: E. coli JM109, DH5α, restriction enzyme BamHI/XhoI, Wild Range DNA Marker (500- 12 000), DNA Ligation Kit Ver.2.0, Agarose Gel DNA Purification Kit Ver.2.0, pMD19-T Vector, LA Taq [Takara Biotechnology Co.,Ltd, Dalian, China], plasmid pIRES2-AcGFP1(Clotech), LipofectamineTM 2000 liposome (Invitrogen), plasmid purification kit (Qiagen Company) and low-glucose DMEM medium( Hyclone Company) were used in this study.


Methods
CD gene cloning
Design and synthesis of polymerase chain reaction (PCR) primer: Based on the 5'end and 3'end of open reading frame of CD gene, a pair of CD gene specific primer was designed according to the nucleotide sequence of CD gene provided by GeneBank database. Distribution of restriction enzyme sites were analyzed and restriction enzyme with non-enzyme digested CD sequence was selected. One XhoⅠsite was designed at 5'end, one BamHI site was designed at 3'end, and protective bases were added. Upstream primer: Cd1: 5'-CTG ACT CGA GAT GTC GAA TAA CGC TTT AC-3'; downstream primer: Cd2: 5'-CAG TGG ATC CTC AAC GTT TGT AAT CGA TGG-3'. Primer was synthesized Takara Biotechnology Co.,Ltd, Dalian, China.
Extraction of E. coli JM109 genome DNA: E.coli JM109 genome DNA was extracted by routine molecule cloning operation [1].
Synthesis of target gene fragment CD gene: The total volume of PCR system was 50 μL supplemented with 10×PCR buffer(Mg2+ plus), 10 mmol/L dNTP, 300 ng genome DNA（template）, 1.25 U LA Taqenzyme and 1.0 μmol/L primer. The conditions for PCR amplification were as follows: After initial denaturation at 94 ℃ for 5 minutes, 30 cycles at 94 ℃ for 45 s, 55 ℃ for 45 s, and 72 ℃ for 45 s were performed, followed by a final elongation at 72 ℃ for 7 minutes. 10 μL PCR product was taken. After 2 μL buffer solution was added, PCR product was identified by 1% agarose gel electrophoresis (1×TAE, room temperature, constant voltage 110 V, ethidium bromide staining, final concentration 0.5 mg/L). Compared with DNA Marker, amplified fragment was 1 284 bp, which was in accordance with the data announced by GeneBank database. PCR product was retrieved according to the instruction of Agarose Gel DNA Purification Kit Ver.2.0.
Construction of pMD19-T-CD cloning vector: According to the operation of DNA Ligation Kit Ver.2.0, by TA cloning, CD gene was connected to pMD19-T vector. Recombinant plasmid pMD19-T-CD was transformed into E.coli DH5α and plasmid was extracted. Restriction enzyme EcoRΙ,ＢamHΙ digestion analysis and 1% agarose gel electrophoresis analysis were performed.
CD gene sequence analysis: After restriction enzyme digestion, purified plasmid pMD19-CD was sequenced with reagent (BigDyeR Terminator v3.1 Cycle Sequencing kit) on an automatic sequencer (ABI PRISMTM3730). Sequence analysis was carried out twice, including forward and reverse reactions. Consensus primer was used in the sequencing.

Construction of pIRES2-AcGFP1-CD eukaryotic expression plasmids
1% agarose gel electrophoresis analysis confirmed that XhoⅠand BamHI digested pIRES2-AcGFP1 plasmid was linear after cut. According to DNA Ligation Kit Ver.2.0 operation, purified CD gene fragments were connected to linear plasmids. Amplification and identification of pIRES2-AcGFP1-CD: The connected product was transformed into E.coli DH5α． Positive clones were screened from kanamycin (50 mg/L)-resistant plat plate. Ten transformed colonies were randomly selected and inoculated in the LB medium supplemented with kanamycin (50 mg/L), overnight at 37 ℃, and plasmids were extracted. XhoⅠand BamHI were used to digest pIRES2-AcGFP1-CD. Enzymolysis products were also identified by 1% agarose gel electrophoresis analysis. Purification of pIRES2-AcGFP1- CD recombinant plasmid: Positive clones with recombinant plasmid screened in the last step were inoculated in LB medium supplemented with kanamycin (50 mg/L) at 1:100 for amplification culture, overnight at 37 ℃. Plasmids were extracted with a plasmid purification kit (QIAGEN). The following procedures were the same as those in the above step.

Sampling and culture of rabbit BMSCs
The big-ear white rabbits were anesthetized by marginal intravenous perfusion of 30 g/L pentobarbitone (1 mL/kg body mass). Then, a No. 18 myeloid puncture needle was connected to a 10 mL syringe, which contained about 0.2 mL diluted heparin sodium (3 000 U). About 3-4 mL bone marrow was taken and then slowly perfused into a centrifuge tube, in which 4 mL Ficoll separating medium was pre-added. Thereafter, density gradient centrifugation was performed at 2 500 r/min for 25 minutes. Cloudlike monocytes were taken, rinsed twice with phosphate buffer solution (PBS), re-suspended with DMEM medium supplemented with fetal bovine serum (0.15 volume faction), regulated at 2×108 L-1 and inoculated in a culture flask. Subsequently, the culture flask was cultured at 37 ℃ in a CO2 (0.05 volume faction) incubator. Forty-eight hours later, half of the medium was renewed, and then it was renewed every two or three days. About on day 10, cells completely covered the bottom of culture flask. Passage began when 80%-90% of the cells reached the confluence.

Identification of rabbit BMSCs
The suspension (5×107 L-1 ) of well-grown BMSCs of the 5th generation was inoculated to 24-well plate with lysine-pretreated cover slips in each well, and in which 250 μL suspension was added. When 80%-90% cells reached the confluence (about 48 hours after culture), culture medium was renewed with osteogenetic inductor in 12 wells, serving as inducing group, and DMEM supplemented with fetal bovine serum (0.15 volume fraction) was still used in the other 12 wells, serving as control group. Corresponding medium was renewed in each well two or three times a week. Three weeks later, cells were stained with alkaline phosphatase. The supernatant fluid in the 24-well plate was carefully sucked out. Cover slides were slightly rinsed two or three times with PBS, fixed for 15- 30 minutes with cold acetone (or ethanol, 0.95 volume faction) at room temperature, rinsed several times with distilled water and dried. The 24-well plate with osteogenetic incubating fluid was incubated for 2-4 hours in the incubator, and then it was rinsed with distilled water after osteogenetic incubating fluid was sucked out. Subsequently, calcium nitrate (20 g/L) and cobaltous nitrate (20 g/L) were separately spread on the 24-well plate for 2 minutes in order, and then washed away with distilled water. Thereafter, ammonium sulfide (10 g/L) was spread for 2 minutes and washed away with distilled water. Cell nucleuses were re-stained with methyl green (2%), then naturally dried, fixed with gum, and finally observed under an inverted microscope and photographed. BMSCs of the 5th generation were cultured for 3 days. When 80 % of them reached the confluence, they were induced for 14 days with adipogenic inductor. After culture solution was carefully discarded, cells were rinsed once with PBS, fixed in formaldehyde (0.04 volume fraction) for 1 hour at 4 ℃, rinsed with ethanol (0.7 volume fraction), stained with old red, balanced for 5-10 minutes at room temperature, rinsed with ethanol (0.7 volume fraction) and deionized water, stained with haematoxylin for 2 minutes, observed under a microscope and photographed in order.

Transfecting BMSCs with CD gene by liposome-mediated method
Before transfection, BMSCs of the 5th generation were inoculated into the 6-well plate without antibiotic medium at 2×105/ well. 4 μg recombinant plasmid)pIRES2-AcGFP1-CD) and empty vector （pIRES2-AcGFP1）were separately dissolved in 250 μL serum-free medium and balanced for 5 minutes. 6 μL Lipofectamine2000 was dissolved in 250 μL serum-free medium and balanced for 5 minutes. The above two were mixed （total volume 500 μL）and incubated at room temperature for 30 minutes. The 500 μL mixture was added to each well. Afterward, the 6-well plate was cultured for 6 hours in a CO2 (0.05 volume fraction) incubator at 37 ℃, and fresh serum medium was used. Twenty-four and twenty-eight hours later, green fluorescence could be found under a fluorescent microscope.

RESULTS

PCR amplification of CD gene
E.coli JM109 genome DNA was used as template for PCR amplification. Agarose gel electrophoresis analysis of PCR product showed that a specific band appeared between 1 000 bp and 1 500 bp, which was in accordance with prospective amplified length (Figure 1).

CD gene cloning
The constructed pMD19-T-CD plasmid was digested by restriction enzyme XhoⅠ/BamHI, and then 1 300 bp fragment and pMD19-T vector were obtained (Figure 2).

CD gene sequence analysis
Sequencing result of CD gene is shown in Figure 3. Sequence analysis demonstrated that the whole length of CD gene nucleotide sequence was 1 284 bp coded with 427 amino acids. Its 99% was in accordance with nucleotide sequence provided by GeneBank database, and 1% difference may be induced by mutation. CD gene-coded 427 amino acids were completely consistent with those provided by GeneBank database.

 Construction of pIRES2-AcGFP1-CD eukaryotic expression plasmid
The sequenced pMD19-T-CD plasmid was digested with XhoⅠand BamHI, and CD gene product was retrieved. Under the effect of DNA rapid joinase, XhoⅠand BamHI digested pIRES2-AcGFP1 plasmids were connected in orientation, and pIRES2-AcGFP1-CD recombinant expression vector was obtained. Agarose gel electrophoresis analysis of XhoⅠ/BamHI-digested product showed that a band appeared between 1 000 bp and 1 500 bp, and its length was the same as CD gene length (Figure 4). It confirms that pIRES2-AcGFP1-CD eukaryotic expression vector is successfully constructed.

Primary and passage culture of rabbit BMSCs
BMSCs were primarily cultured. Three days later, medium was initially renewed. Under the contrast phase microscope, spindle-shaped mononuclear cells with typical fibroblast-like appearance adhered to the wall in coefficient distribution. On day 7 of primary culture, diffusedly distributed adherent cells increased, and they became from spindle-shaped into long spindle shaped. On day 10, original single cells or cell colonies formed many cell clones. On day 14, each cell clone was expanded until the culture flask bottom was completely covered. After digested with 1.25 g/L trypsin for 4 days, the cells could fuse. After 3 to 5 passages, they were basically purified (Figure 5).

Identification of rabbit BMSCs
Alkaline phosphatase is the representative enzyme for osteoblasts and osteoblastic differentiation. As one of phenotype characteristics of osteoblasts, it plays a key role in the in vitro calcification. In this study, after staining the cells cultured for 3 weeks with alkaline phosphatase, we found a great number of black particles or massive sediments, presenting alkaline phosphatase staining positive, but we did not found any obvious changes in the control group (Figure 6).

Six days after adipogenic induction of BMSCs, small lipid droplets appeared in the cytoplasm, and gradually fused into small round sparkling and lucent lipid droplets with high refraction, which mainly gathered around the nucleus. With the induction time going, lipid droplets appeared in more and more cells, and occupied the space from cell membrane to nucleus. After further induction, small round lipid droplets gradually fused into big ones. Cells gradually become from long spindle shaped into oval shape or polygon. In each cell, nucleus was jostled to one side by large lipid droplets. The cells were stained with oil red, and nuclei were re-stained by haematoxylin. Then under the inverted microscope, we found that lipid droplets presented salmon pink and nuclei presented blue, cytoplasm contained lipid droplets of different size, and nuclei were jostled to one side by lipid droplets (Figure 7).

Observation of gene transfection and green fluorescent protein expression
Transfecting rabbit BMSCs for 24 hours, by an inverted fluorescence microscope, we found green fluorescence in pIRES2-AcGFP1-CD and pIRES2-AcGFP1 empty vector-transfected cells, but did not found green fluorescence in the non-transfected cells (Figure 8).

DISCUSSION

Recently, with the progression of study, people have tried to use the stem cells with the capacity of troposm to tumor and carrying exogenous gene. However, they found that neural stem cells on which expectations are initially centered presented good tropism to glioma and gene modification [2-5], but their tissue sampling, laboratory application generalization and ethical problems limited their application. In order to solve such problems, investigators paid their attention to BMSCs which were easily obtained and operated, and were not limited by ethics. Under the effect of in vivo microenvironment, BMSCs transplanted into brain tissue can spontaneously differentiate into neural precursor cells and nerve cells. BMSCs have distinct advantages in the gene therapy of nervous system diseases: they can migrate into central nervous system in vivo [6-7]; In addition, as gene therapy vector of glioma, BMSCs, the same as neural stem cells, have the same tumor tracing and good gene modification functions [8-11]. Based on above-mentioned advantages, studies on BMSCs as the vectors for gene therapy of glioma were successively developed.
CD is a kind of enzyme in the metabolic bypass of E.coli. It only exists in the bacterium and fungi. In 1992, Austin et al [12] and Anderson et al [13] firstly cloned a 3 kb DNA fragment with CD gene activity and Mr 52000 from E.coli. It is a kind of enzyme, and can catalyze 5-fluorocytosine deaminase to transform into 5- fluorocytosine. 5-fluorocytosine in the cells transforms into 5 fluorouracil -deoxyuridine monophosphate, then inhibits deoxythymidylate synthetase, prevents deoxyuridine monophosphate from methylating deoxythymidine monophosphate, and influences DNA synthesis, thus, tumor cells can be killed [14]. Because 5- fluorocytosine has a series of adverse effects, so it is limited in the clinical application. But mammals do not have cytosine deaminase[15], so 5- fluorocytosine can not be transformed into 5-fluorouracil. By gene transfection technique, CD gene is transferred into mammalian cells, thus mammalian cells can transform 5- fluorocytosine without cytotoxicity into 5-fluorouracil with cytotoxicity. Lü et al[16] transduced CD gene into human glioma U251 cell strain and incubated them with different concentrations of 5-fluorocytosine. They found that CD gene could enhance the sensitivity of U251 cells to 5-fluorocytosine and transform 5-fluorocytosine into 5- fluorouracil, inhibiting tumor growth. Barresi et al [17] perfused C6 glioma cells mixed with ST14A cells with and without E.coli CD gene separately into rat brains, and treated them with 5-fluorocytosine, and then they found that under the condition of absence of CD gene, 5-fluorocytosine had no effects on tumor.
In this study, we successfully cloned CD gene, and found that gene sequence result was in accordance with the sequence from GeneBank. We subcloned CD gene onto pIRES2-AcGFP1 plasmid in order to construct pIRES2-AcGFP1-CD eukaryotic expression vector. We used CD gene to transfect BMSCs by liposome-mediated method, and 24 hours later, we observed the expression of green fluorescent protein under an inverted fluorescence microscope. The above results demonstrated that pIRES2-AcGFP1-CD eukaryotic expression vector had been successfully transferred into BMSCs. It will provide theoretical evidence and experimental basis for the treatment of tumor in central nervous system with CD gene combined with 5- fluorocytosine. After transfection, Western blotting is necessary for further verifying the expression of CD protein. Some problems need to be solved, such as whether the characteristics of BMSCs will change after transfection, how to shorten screening time in clinical application, and how to enhance transfection rate.

REFERENCES

1 Sambrook J, Russell D W. Molecular Cloning: A Laboratory Manual. Beijing: Science Press 2002:728
2 Aboody KS, Brown A, Rainov NG，et al. Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial gliomas. Proc Natl Acad Sci U S A 2000;97(23): 12846-12851
3 Benedetti S, Pirola B, Pollo B, et al. Gene therapy of experimental brain tumors using neural progenitor cells. Nat Med 2000;6(4); 447-450
4 Yang SY, Liu H，Zhang JN. Gene therapy of rat malignant gliomas using neural stem cells expressing IL-12. DNA Cell Biol 2004;23(6): 381-389
5 Ehtesham M, Kabos P, Gutierrez MA, et al. Induction of glioblastoma apoptosis using neural stem cell-mediated delivery of tumor necrosis factor-related apoptosis-inducing ligand. Cancer Res 2002;62(24): 7170-7174
6 Li Y, Chopp M, Chen J, et al. Intrastriatal transplantation of bone marrow nonhematopoietic cells improves functional recovery after stroke in adult mice. J Cereb Blood Flow Metab 2000;20(9): 1311-1319
7 Chen J, Li Y, Wang L, et al. Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats. Stroke 2001;32(4):1005-1011
8 Honma T, Honmou O, lihoshi S, et al. Intravenous infusion of immortalized human mesenchymal stem cells protects against injury in a cerebral ischemia model in adult rat. Exp Neurol 2006;199(1):56-66
9 Liu H, Honmou O, Harada K, et al. Neuroprotection by PlGF gene-modified human mesenchymal stem cells after cerebral ischaemia. Brain 2006;129(Pt 10):2734-2745
10 Lu L，Zhao C, Liu Y, et al. Therapeutic benefit of TH-engineered mesenchymal stem cells for Parkinson's disease. Brain Res Brain Res Protoc 2005;15(1):46-51
11 Nakamizo A, Marini F, Amano T, et al. Human bone marrow-derived mesenchymal stem cells in the treatment of gliomas. Cancer Res 2005; 65(8):3307-3318
12 Austin EA, Huber BE. A first step in the development of gene therapy for colorectal carcinoma: cloning, sequencing, and expression of Escherichia coli cytosine deaminase. Mol Pharmacol 1993;43(3): 380-387
13 Anderson L, Kilstrup M, Neuhard J. Pyrimidine, purine and nitrogen control of cytosine deaminase synthesis in Escherichia coli K 12. Involvement of the glnLG and purR genes in the regulation of codA expression. Arch Microbiol 1989;152(2):115-118
14 Wiebke EA, Grieshop NA, Loehrer PJ, et al. Antitumor effects of 5-fluorouracil on human colon cancer cell lines：antagonism by levamisole. J Surg Res 2003;111(1): 63-69
15 Smythe WR. Prodrug/drug sensitivity gene therapy: current status. Curr Oncol Rep 2000;2(1):17-22
16 Lv SQ, Yang H, HE JQ, et al. Effects of CD/5-FC suicide gene therapy system on human malignant glioma cells in vitro. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2003;35(5):430-434
17 Barresi V, Belluardo N, Sipione S, et al. Transplantation of prodrug-converting neural progenitor cells for brain tumor therapy. Cancer Gene Ther 2003;10(5):396-402

pIRES2-AcGFP1-CD真核表达载体构建
及其在骨髓间充质干细胞中的表达☆○

宋 飞1,2，陈一曲1，马学虎1，葛 丹1，刘天庆1，马郁芳3，崔占峰○1,4
1大连市干细胞与组织工程研发中心(大连理工大学)，辽宁省大连市 116023；大连医科大学，2第二附属医院神经外科，3基础医学院生化教研室，辽宁省大连市 116027；4牛津大学化学工程系，英国牛津
宋 飞☆，男，1968年生，辽宁省沈阳市人，汉族，大连医科大学在读博士，副主任医师，主要从事骨髓间充质干细胞及神经干细胞的研究。
摘要
背景：骨髓间充质干细胞在体外易分离和扩增，还易于外源基因的转入及表达，在人类医学上被认为是一种理想的治疗性细胞和基因治疗中的靶细胞。
目的：探讨脂质体介导胞嘧啶脱氨酶基因转染兔骨髓间充质干细胞及其基因的表达。
设计：单一样本观察。
单位：大连市干细胞与组织工程研发中心，大连医科大学基础医学院生化室。
材料：实验于2006-03/2007-06在大连市干细胞与组织工程研发中心及大连医科大学基础医学院生化室完成。新西兰大白耳兔，5月龄，雌雄不拘，体质量2.0~2.5 kg。
方法：以大肠杆菌JM109基因组DNA为模板，用PCR方法获得目的基因片段，定向克隆至载体pMD19-T，限制性内切酶消化鉴定、基因测序后，构建pIRES2-AcGFP1-CD真核表达质粒。同时对兔骨髓间充质干细胞取材、培养、鉴定。真核表达质粒经酶切鉴定后，采用Lipofectamine 2000介导法转染经过鉴定的兔骨髓间充质干细胞。并于转染后24 h 在倒置荧光显微镜下观察绿色荧光蛋白的表达。
主要观察指标：表达载体的构建及基因转染骨髓间充质干细胞鉴定。
结果：实验克隆出胞嘧啶脱氨酶基因，并将其与带荧光的真核表达载体pIRES2-AcGFP1连接。经转染兔骨髓间充质干细胞24 h 后，在倒置荧光显微镜488 nm 蓝光激发下观察，pIRES2-AcGFP1-CD组和pIRES2-AcGFP1空载体组均可见细胞发出绿色荧光，未经转染的细胞未见发出绿色荧光，说明胞嘧啶脱氨酶基因成功转染了骨髓间充质干细胞。
结论：骨髓间充质干细胞有望成为胞嘧啶脱氨酶基因治疗中的理想载体。
关键词：胞嘧啶脱氨酶；基因转染；骨髓间充质干细胞
中图分类号: R394.2 文献标识码: A 文章编号: 1673-8225(2008)08-01568-05
宋飞，陈一曲，马学虎，葛丹，刘天庆，马郁芳，崔占峰. pIRES2-
AcGFP1-CD真核表达载体构建及其在骨髓间充质干细胞中的表达[J].中国组织工程研究与临床康复,2008,12(8):1568-1572
[www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1568(ps).pdf]
(Edited by Zhou SL/Song LP/Wang L)