［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.

1MATERIALS AND METHODS

1.1 The construction of recombinant expression vectorThe 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 TransfectionHuman 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 lipofectin^TM.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.

1.3FACS analysis for EGFPRecombinant 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）.

1.4RT-PCR for FL mRNATotal 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