| It has been proved that VEGF (Vascular endothelial growth factor) could be secreted by most kinds of tumor cells. VEGF could promote tumor growth by enhancing mesenchymal cells'angiogenesis. Therefore, many studies focused on inhibiting tumor growth by blocking the effect of VEGF in recent years.Present studies showed that VEGF was expressed not only in solid tumor cells, but also in leukemia cells. Except for high expression of VEGF, leukemia cells and cell lines express Flt-1 and KDR (VEGF receptors) too. VEGF induces angiogenesis and leukemia cells'proliferation by autocrine or paracrine pathway so that forming a bad cycle in leukemia cells growth.methods of decreasing production of VEGF by anti-VEGF drugs and blocking functional receptors of VEGF are adopted to inhibit leukemia cells'proliferation. It is known that VEGF take effect through binding receptors among the mesenchymal cell membrane. The therapy strategy of employing soluble receptor of VEGF to compete with VEGF and to inhibit its biological functions has been proved to be rather effective.As the extracellular part of protein VEGFR1(Flt1), sFlt-1 is coded by montage of Flt-1mRNA. Because protein sFlt-1 is composed of 6 Ig-like regions lack of transmembrane and intracellular amino acid sequence, even if binding with VEGF, it would not transduct biological signals to downstream bio-molecules. Comparing with KDR, sFlt-1's affinity with VEGF is 10 fold stronger. We can find the nature sFlt-1 in human vmbilical vein endothelial cells cultured in vitro.Without the help of heparin, sFlt-1 can restrain almost all (82%) cell proliferation and metastasis induced by VEGF. At one theme, lacking transmembrane region and TPK domain, sFlt-1 don't transduct biological signals while capturing VEGF. So sFlt-1 is used to compete to combine with VEGF secreted by tumor cell in circulation to inhibit its biological activity. At another theme, because sFlt-1 can compete to bind with membrane receptor to form heterogenous dimer, sFlt-1 can inhibit VEGF's biological functions by blocking phosphorylation of RTK and signal transduction. So, researchers are paying more attentions to molecule bridge technology, which use gene transfection assay to transfect sFlt-1cDNA sequence to target site.In this study, adopting reverse transcription virus as expression carrier, we transfected sFlt-1 cDNA gene sequence to the umbilical cord blood monocyte cell, and transplanted the monocyte recombined with sFlt-1 cDNA gene sequence to nude mice radiated .Then, in different time points, the varying of sFlt-1 gene's expression and secretion was observed and recorded in protein and functional level in vitro. This study is necessary for further animal experiment. The concrete methods and results are as follows.1. In this study, sFlt-1cDNA was amplified from plasmid DNA including sFlt-1cDNA sequence by PCR. According with previous datas, a single band was present in 2230bp after electrophoresis. The sequencing plasmid was constructed at the same time. The sequencing showed that there was no gene mutation in 1st, 2nd, 3rd and 4th Ig-like loop of sFlt-1 gene.2. The reverse transcription virus expressing carrier sFlt-1 cDNA was constructed and was testified by BamHI,ClaI incision and PCR. The carrier sequence was transfected into packaging cell and virus particles were harvested .3. The total RNA of umbilical cord blood monocyte cell infected by virus carrier was raised and the recombination of sFlt-1 gene was identified by RT-PCR. The result of electrophoresis showed that the target gene band was present in transfected group but not in control group. It proved that there existed sFlt-1 gene expression in transcriptional level. The transfection ratio detected by Flow Cytometry was 13.5%.4. The umbilical cord blood monocyte cell recombined with sFlt-1 cDNA sequence were transplanted to radiated nude mice by bones injection into medullary cavity .The nude mice were killed in 48 hours, 7 days. 14 days and 21days after transplantation. Bone marrow cells in bilateral femur and tibia were washed out by medium of IMDM in serious asepsis condition. The positive expression of protein sFlt-1 has been proved by ELISA in transplanted nude mice's bone marrow of 7,14 and 21days. It unraveled that recombined umbilical cord blood monocyte cell could express protein sFlt-1 and secrete it into bone marrow. The result showed that the secretion of sFlt-1 could not be detected in 48 hours after transplantation, rise to the peak in 7 days and maintain stable expression during 14 to 21days though declined faintly.5. The K562 cells were cocultured with bone marrow of 7 ,14 and 21days'transplanted mice. The result of MTT showed that proliferation extent of cocultured K562 cells decreased,the VEGF in supernatant descended of K562 cells treated by supernatant,it elucidated that sFlt-1 suppressed the autocrine pathway.6,human- originated gene emerged in bone marrow cells when 2days after transplantation ,in the fresh peripherial blood,bone marrow and spleen cell suspension of nude mice 7days dectected by PCR assay,such results certified that the stem cells differentiated planted in bone marrow can arrive the non-hematopoietic organ ,it explained that stem cells have excellent homing after transplanting into bone marrow.The study showed that, 7,14 and 21days after transplanting recombined umbilical cord blood monocyte cell to nude mice by bone marrow injection, secretion of protein sFlt-1 was detected, The concentration of VEGF in culture medium of K562 was inhibited by secreted sFlt-1. Therefore, tumor growth was inhibited also. Our study provides a foundation for further animal experiment of leukemia's anti-VEGF therapy.
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