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Egr-1启动子基因调控FLT3配基基因表达的实验研究
免疫学杂志 2000年第3期第16卷 基础免疫学
作者：杜楠 裴雪涛 罗成基 李梁 邹仲敏 冯凯 白慈贤
单位：杜楠（第三军医大学复合伤研究所，重庆 400038）；罗成基（第三军医大学复合伤研究所，重庆 400038）；邹仲敏（第三军医大学复合伤研究所，重庆 400038）；裴雪涛（军事医学科学院放射医学研究所实验血液研究室，北京 100850）；李梁（军事医学科学院放射医学研究所实验血液研究室，北京 100850）；冯凯（军事医学科学院放射医学研究所实验血液研究室，北京 100850）；白慈贤（军事医学科学院放射医学研究所实验血液研究室，北京 100850）
关键词：辐射诱导基因；造血生长因子；基因治疗；辐射；骨髓基质细胞
［摘 要］目的 探索辐射诱导基因调控序列启动造血生长因子基因表达及观察其对造血恢复的作用。方法 本实验将带有Egr-1调控序列启动的FLT3配基（FL）和EGFP双顺反子基因表达载体（Egr-EF）转染骨髓基质细胞系HFCL（称HFCL／EF）；用RT—PCR鉴定细胞内目的基因的mRNA表达；采用FACS观察EGFP绿色荧光表达的阳性细胞；用ELISA方法检测HFCL／EF细胞培养上清FL的含量；观察HFCL／EF培养上清液对CD34＋细胞的增殖作用。结果 在HFCL／EF细胞中证实有外源性基因EGFP和FL的整合和表达，在辐照16h后的HFCL／EF细胞培养上清液中表明FL含量较照射前明显增高（P＜0.01）；同时证实辐射10d后HFCL／EF培养上清液对CD34＋造血祖细胞的作用较辐射前具有明显的扩增作用（P＜0.01）。结论 Egr-1调控序列启动的造血生长因子基因在辐射后表达明显增高并促进造血祖细胞增殖作用。
［中图分类号］R818.52＋1 ［文献标识码］A
In v itro studies on the expression of FLT3 Ligand regulated by Egr-1 regulated sequence
DU Na，LUO Cheng-ji，ZOU Zhong-min
（Institute of Combined Injury，the Third Military Medical University，Chongqing 400038，China）
PEI Xue-tao，LI Liang，FENG Kai，BAI Ci-xian
（Department of Experimental Hematology，Institute of Radiation Medicine，Beijing 100850，China）
［Abstract］Objective To explore the regulating effects of radiation inducible gene on the expression of hematopoiedtic growth factor genes.Methods The human FLT3 Ligand （FL）cDNA and EGFP cDNA were linked together with IRES and then inserted into the eukaryotic expression vector pCI-Egr，which was constructed by substituting CMV promoter in pCIneo with the Egr-1 promoter（Egr-EF）.The expression of FL in HFCL／EF cells were confirmed with RT-PCR、ELISA、FACS and cell culture.Results The activity of EGFP in transfected cells increased after exposure to 2.5Gy.The amounts of secreted FL in serum-free supernatants of HFCL／EF were significantly higher than the control group.FL cDNA was successfully expressed in the cells by ELISA and RT-PCR analysis.At day 10 of culture the number of CD34＋ cells in HFCL／EF culture supernatants was significantly higher than that of non-radiation group.Conclusion These results showed that radiation can enhance the ability of the supernatants containing FL of HFCL／EF to expand early hematopoietic progenitor cells and protect hematopoietic cells from radiation-injury effects.
［Key words］hematopoietic growth factor； radiation inducible gene；gene therapy； radiation； bone marrow stromal cell
［Article ID］1000-8861（2000）03-0166-06
Recent studies have shown that ionizing radiation exposure can induce the expression of certain immediate-early genes that code for transcription factors［1,2］.These including members of early growth response（EGR），the jun／fos and nuclear factor κB（NF-κB） gene families. Previous studies have demonstrated that induction of Egr-1 gene transcription is mediated by activation of CC（A＋T）6GG in the Egr-1 mofits promoter and gene therapy using viral vectors containing the radiation-inducible Egr-1 promoter provides a potential approach for controlling gene transcription by ionizing radiation［3］.
FLT3 Ligand（FL） is a novel hematopoietic cytokine，involved in regulation of early hematopoiesis［4］.It stimulates alone，or in combination with other growth factors，the proliferation of highly enriched human and murine hematopoie-tic stem cells in vitro. However，daily subcutaneous administration of FL can be associated with dose-limiting systemic side effects，and as an alternative approach，we demonstrate that local tissue-specific high-level expression of cytokines can be largely confirmed to within the marrow space.This is achieved by cytokine gene transfer to marrow stromal cells.Thought the cytokine that stimulate hematopoiesis have often provd to expert radioprotective effects as well，no absolute correlation has been demonstrated between the expression of the cytokine regulated by radio-inducible gene and potential radioprotection.The present study shows that ionizing radiation can activate bicistronic eukaryotic expression vectors containing enhanced green fluorescent protein（EGFP） and human FL cDNA by the Egr-1 promoter after transduction of bone marrow stromal cells in vitro.These studies suggest that gene therapy using above expression vector may be a useful strategy for the treatment of radiation-injury.
1 MATERIALS AND METHODS
1.1 The construction of recombinant expression vector The human FL cDNA （pUC18／FL provi-ded by Dr Wang LS，Indianana University，USA） and EGFP cDNA （pEGFP-N1 Clontech）were linked together with internal ribome entry site（IRES）of 5’nontranslation region from poliavirus and then inserted into the eukaryotic expression vector pCI-Egr-1［5］，which was constructed by substituting CMV promoter in pCI neo with the Egr-1 promoter（Egr-EF）.
1.2 Transfection Human bone marrow stromal cell line（human fibroblast cell line，HFCL）［6］were maintained in Dulbecco’s modified Eagle’s medium （DMEM，Gibco）meium／10％ fetal calf serum at 37°C and 5％ CO2.On the day of transfection，the vector was transferred into HFCL by lipofectinTM.The transfected cell clones （HFCL／EF） have been selected by the addition G418（1000μg／ml）.Clone no.4 have been used in the studies.Umbilical cord blood （CB） samples were obtained from umbilical tissues according to approved procedures.Low-density CB cells were collected after separation on Ficoll-Hypaque，and CD34＋ enriched cells were collected with a MACS laboratory separation system（miltenti bioteo germany） as previously described［7］.CD34＋ culture medium was prepared by supplementing Iscove’s Modified Dulbecco’s medium（IMDM） with 12.5％ horse serum，12.5％ fetal calf serum （FCS），50ng／ml SCF，20ng／ml IL-3 and 20ng／ml IL-6.
1.3 FACS analysis for EGFP Recombinant expression vector containing EGFP reporter gene was transferred into HFCL／EF to characterize the re-gulatory function of the CC（A＋T）6GG-rich sequence after exposure to γ-radiation by 60Co source at 0.5～20.0 Gy.These results were confirmed by fluorescence microscope and fluorescence activated cell sorter （FACS）.
1.4 RT-PCR for FL mRNA Total RNA was isolated by using the RNeasy method（TRIZOL Reagent，Gibco），estimated spectrophotometrically，from irradiated transfected stromal cells.RT-PCR technique was used to determine the expression of FL mRNA transcripts in transfected stromal cells as the manufacturer’s instructions（Boehringer Mannheim）.The following primer sets for human FL were obtained from Takara（Dalian，China）：FL primer： upstream primer 5’-TGC TGC TGA GCT CGG GAC TC-3’，downstream primer 5’-AGT TCT GCA GAG TGA TCC AG-3’，amplifying a 500-bp fragment.The human β-actin primers are from TaKaRa as an internal control for RNA integrity and relative quantitation： upstream primer 5’-GTG GGC CGC TCT AGG CAC CA-3’；downstream primer，5’-CGG TTG GCC TTA GGG TTC AGG GGG G-3’，amplifying a 245-bp fragment.Conditions used were as follows： reverse transcription at 50°C for 30min followed by denaturation at 94°C for 2min.The DNA amplification consisted of 35 cycles of 94°C for 30sec，60°C for 30sec，and 70°C for 1 min.A final elongation step of 7min at 70°C followed the 35 amplification cycles，after which the samples were cooled rapidly and stored at 4°C.The mRNA for human β-actin was amplified for 35 cycles in the same PCR tube by using primers from TaKaRa as an internal control for RNA integrity and relative quantitation.The amplified products were resolved on 1.5％ agarose gel［8］.
1.5 ELISA for FL Human FL levels in cell culture supernatant of HFCL／EF before and after radiation were measured by enzyme linked immunosorbent assay（ELISA） kits （Santa Cruz） according to the manufacturers’ recommendations.Time course of Egr-1 promoter activation by ioni-zing radiation was observed at 0，8，16，24，32，40，48 h.
1.6 The effects of supernatants from irradiated cultures of HFCL／EF on CD34＋ cells Supernatants were collected from confluent plates of FL-producing cell lines cultures 16h after irradiation by 60Co source at 2.5Gy.CD34＋ cells （1×104 cells／well，repeatly 6 wells per sample） were cultured in 24 well places containing 30％ cultured supernatants of transfected cells，10－4 mol／L 2-mercaptoethanol，10－6mol／L hydrocortisone，and other cytkines（SCF 50ng／ml，IL-3 20ng／ml and IL-6 20ng／ml） in 1 ml serum-free medium （CellGro SCGM，Boehringer Ingelheim）.The positive control was medium containing recombinant FL（Immunex）100ng／ml，negative controls were mediums containing non-irradiated cell cultured supernatants or no recombinant cytokines.Cultures were incubated at 37°C with 5％ CO2 in air for 7d.
Flow cytometry.Two-color flow cytometry was performmed on a FacScan（Becton Dickinson，Mountain View，CA） as previous described.Briefly，CB cells were incubated on ice for 30 min in presence of satureating amounts of monoclonal anti-CD34-fluorescein isothiocyanate （FITC；HPCA-1；Becton Dickinson） and anti-CD38-phycoerythrin（PE；Leu-17；Becton Dicknson） antibodies.IgG1 isotype controls conjugated to FITC and PE were also included.Analysis of the CD34＋ content of all samples before and after expansion was performed with FACS and fluorescence microscope.
1.7 Inhibition of proliferation of CD34＋ cells by anti-FL antibodies The effect of neutralizing antibodies against FL on stroma-conditioned medium-induced induced proliferation of CD34＋ cells was examined by adding the antibodies to fluid cultures at final concentratons of 1μg／ml anti-FL，or 5μg／ml normal goat IgG（Santa Cruz） on day 0.Cultures were supplemented with additional antibodies at the same concentration on day 3，6，9.Cell were scored on day 10.
2 RESULTS
2.1 Construction of Egr-EF and transfec-tion Fig1 shows that bicistronic eukaryotic expression vector PCIneo containing human FL and EGFP cDNA directed by Egr-1 gene promoter was constructed.The recombinant plasmid and PCIneo were transferred into human bone marrow stromal cell lines HFCL with Lipofectin.After selection of the transduced HFCL in G418 and re-expansiion to confluency，supernatant of the positive clone no.4 was harvested for EILSA or bioassay of human FL，and green fluorescent of EGFP was shown in transduced cells by FACS.
Fig 1 The construction of recombinant bicistronic eukaryotic expression vector
2.2 The expression of EGFP regulated of the Egr-1 promoter in HFCL The radiation-inducible Egr-1 promoter placed upstream of EGFP-IRES-FL is transcriptionally induced in HFCL cells following irradiation.We found the activity of EGFP in transfected cells increased by 3.1 fold at 16h after exposure to 2.5 Gy as compared to non-transfected cells （see Fig 2）.
Fig 2 Induced expression of EGFP in HFCL／EF cells transfected
with Egr-EF after exposure to irradiation
2.3 ELISA for FL The amounts of secreted FL in serum-free supernatants of HFCL／EF increased by 795.64±107.21pg／ml，which was significantly higher than the preirradiated group （123.04±25.61pg／ml），The two-tailed p value was below 0.05.The addition of neutralizing antibodies against FL（FL group was 0.32×104 cells／well ） completely abrogated the proliferation of CD34＋ cells by stroma conditioned medium（IgG group was 4.83×104 cells ／well）.
2.4 The effects of cultured supernatants from irradiated HFCL／EF on expansion of CD34＋ cells The effects of FL in HFCL／EF serum-free supernatants on expansion of CD34＋ cells derived from cord blood in the presence of SCF，IL-3 and IL-6 were studied.These results indicated that at day 7 of culture the number of CD34＋ cells increased by 173.09±11.58×103 cells／ml，which was significantly higher than that of non-irradiation group （68.0±13.73×103 cells／ml）. Incubation of CD34＋ cells for 1week on performed supernatants of HFCL／EF did not drastically alter their cell surface phenotype（see Fig 3），though increased production of mature cells and decreased in the percentage of CD34＋ cells were observed，the total amount of CD34＋ were increased.It showed that radiation can enhance the ability of the supernatants containing FL of HFCL／EF to expand early hematopoietic progenitor cells.
Fig 3The phenotype of hematopoietic progenitor cells following culture
with supernatant of transduced cells after 10d
a：The phenotype of CD34＋ cells isolated from cord blood cells using MACS and
analyzed using a FACSTM flow cytometer.b：The phenotype of CD34＋ cells
in culture medium containing supernatant of HFCL／EF following radiation at day 10.
c：The phenotype of CD34＋ cells in culture medium containing supernatant of
HFCL／EF following radiation at day 10.d：The phenotype of CD34＋ cells
in culture medium containing supernatant of HFCL cells at day 10.
2.5 RT-PCR for FL mRNA expressed in the transfected cells Fig4 shows that the RT-PCR-amplified products resolved on 1.5％ agarose gel in 1×TAE buffer pH 8.0 and visualized with 0.5μg／ml ethidium bromide.The negative controls were RT-PCR reactions performed in the absence of any template or in the absence of the enzyme.The RT-PCR-amplified products resolved on 1.5％ agarose gel showed a single band of the expected size for the FL product amplified by the TaKaRa primers，in comparison to a positive control，which was the 500bp amplified product of the specific FL cDNA fragment supplied by PUC 18／FL with the PCRAmplimer set.The amplified product for hu-man β-actin mRNA in the same tube was detected as a single band of the expected size （245bp）.These results indicated that FL mRNA transcripts were detected and highly expressed in HFCL／EF cells after irradiation by RT-PCR analysis.
Fig 4Expression of FL mRNA in transduced and non-transduced cells by RT-PCR analysis
Lain 1：marker：DL 15000＋2000
Lain 2： the expression of β-actin mRNA in HFCL cells
Lain 3： the expression of FL and β-actin mRNA in HFCL／EF cells
3 DISCUSSION
There have been high expectation that stimulatory hematopoietic factors would have been an important impact on irradiatherapy by reducing irradiation-induced neutropenia.Radiation induced death has been attributed to damage of the hematopoietic system followed by infection largely with enteric bacteria.Several cytokines with hematopoietic or immune stimulatory activities are known to protect hematopoietic tissues from radiation-induced death including FL，SCF，GM-CSF，G-CSF and so on.The majority of the cytokines protect the host from infection by reducing radiation damage of primitive hematopoietic cells.FLT3 Ligand（FL） was recently cloned from T cells，and shown to enhance the in vitro growth of hematopoietic stem cells，primitive myeloid progenitors，and lymphoid progenitors［3］.Like many hematopoietic factors，the dose of recombinant FL（give as a daily subcutaneous injection） is limited by potential system toxicities，including pericarditic，fluid retention，fever and rash.As an alternative to daily systemic subcutaneous injection，localized expression restricted to stromal cells within the marrow may facilitate similar hematopoietic effects without systemic toxicity by gene transfer technique［9］.Promoters of expressive vectors are generally constitutive promoters，a potential disadvantage is the lack of tissue-specific or developmental stage-specific expression.Inducibi-lity of the transgene expression is，therefore，not dependent on hematopoietic regulation when need after irradiation.However，an alternate approach to improve the specifity of gene therapy involves using radiosensitive promoter to activate gene expression selectively in the radiation field［4］.Pre-vious studies have demonstrated that irradiation increases transcription of the Egr-1 gene［1］.The Egr-1 promoter sequence responsible for this effect has been localized to six CC（A＋T）6GG（CarG） elements［2］.the potentiality of the Egr-1 promoter in allowing a discrete expression of the transgene at the specific site，and only when triggered［10］.The temporal control of the transgene expression is made possible because the Egr-1 promoter is inducible by radiation； The spatial control is made possible by the fact that ionizing radiation can be delivered precisely within a specific volume of tissue.The present studies provided an experimental basis that the expression vectors containing the radiation-inducible Egr-1 promoter may be a useful strategy for cytokine gene therapy to protect hematopoiesis from irradiation-induce damage.We demonstrate the utility of the enhance green fluorescent protein as a simple reporter and rapid selectable marker.The coordinate expression of the reporter gene and therapeutic genes in a single vector is markedly advantageous.IRES from poio viruses were used to construct a bicitronic expression vector PCI-Egr，where EGFP cDNA was used as a reporter gene FL cDNA as gene of interest，so that the Egr-1 promoter derived the expression of a bicistronic mRNA.We demonstrated that trans-cripts of the Egr-1 gene were significantly increased in stromal cells after exposure to 2.0～10 Gy of irradiation by inducing the expression of the EGFP cDNA in stromal cells with the Egr-EF expression vector in vitro.RT-PCR and ELISA ana-lyses showed that the expression of FL was increased in the transfected stromal cells after irradiation.The results suggested that the bicitronic eukaryotic expression vector containing EGFP and FL cDNA directed with Egr-1 gene promoter was successfully expressed in stromal cells.
Our studies show that not only the levels of FL were increased in culture supernatant，but the supernatant containing FL had a significantly effect on expansion of CD34＋ cells.It shows that radiation can enhance the ability of supernatants containing FL of HFCL／EF to expand early hematopoietic progenitor cells by radiation-inducible gene and the vector was used in potentiality of the cytokine gene therapy.These studies provide the foundation for improved clinical applications of gene therapy protocols employing non-virally transduced marrow stromal cells.
［Foundation item］This work is supported by Natural Science Foundation of China（39900040） and Natural Science Outstan-ding Youth Foundation of China（39825111）.
［Biography］Du Nan（1962-），male，borned in Shijiazhuang，Hebei province，doctorate，vice-professor，have mainly been engaged in studies in hematology and oncology.
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［Receive］1999-09-29；［Revised］2000-03-15