Human Endostatin Gene Transfered Into CD34+ Hematopoietic Stem Cells From Human Umbilical Cord Blood

Objective: To discuss the feasibility of Transfer Human endostatin gene into CD34+ hematopoietic stem cells from human umbilical cord. Methods :A 550 base human endostatin cDNA with 5' and 3' untranslated sequences reconstructed in retrovirus vector was cloned in Ecoli.Then prolification of recombinant plasmids digested with restriction enzymes (Hind Ⅲ and ClaⅠ).After that,the recombinant plasmid plncx/endo was transferred into virus-packing cell PA317 by electroporation method. The clones of the cells transferred with human endostatin were selected by G418.Targeted NIH3T3 cells were infected with the virus granules secreted from PA317 and also selected by G418.The insertion and expression of endostatin gene in NIH3T3 and PA317 cells were analysed with PCR.Mini-MACS was used to isolated CD34+ hematopoietic stem cells from human umbilical blood.And then the retrovirus containing human endostatin cDNA generated by these PA317 cells were used to transfect human umbilical cord CD34+ hematopoietic stem cells to build endostatin-transferred cells.RT-PCR and Western blot analysis were applied to examine the transfection ,expression and excretion of endostatin 72 hours later. Results:Positive clones which strongly resisted G418 were selected and endostatin gene retrovirus granules with high biological activity were prepared.PCR results confirmed the stable transfer and high expression of human endostatin in PA317 and NIH3T3 cells.FCM (flowcytometry)identified that CD34+ hematopoietic stem cells from cord blood were 6～20×105,occupied more than 90%.RT-PCR proved that genome of endostatin-transferred CD34+ hematopoietic stem cells contained a 550bp fragment of endostatin .The expression and excretion of human endostatin from endoatatin-transferred hematopoietic stem cells were confirmed by Westernblot analysis. Conclusion:Electroporation is a useful method to transduct recombinational gene into cells. Human endostatin gene can be transferred into CD34+ hematopoietic stem cells which can provide us a new biotherapy for personal control of solid tumor growth.