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Transfection and Expression of RI Gene on Human Umbilical Blood Stem Cells

Author: ShangZuoYing
Tutor: DengYanJie
School: Dalian Medical University
Course: Obstetrics and Gynaecology
Keywords: Human umbilical cord blood CD34 cells RI gene Transfection Stem cell transplantation Tumor
CLC: R73-3
Type: Master's thesis
Year: 2003
Downloads: 32
Quote: 0
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Abstract


The tumor is one of the major diseases that endanger human health, current treatment methods have their own lack of specific anti-tumor is still a lack of an efficient, with fewer side effects. A large number of experiments confirmed that angiogenesis is necessary for the growth of tumors sustained vascular tumor cells provide oxygen and nutrients to exclude metabolites. If the tumor nodule diameter of more than 1-2mm, and also the lack of new blood capillaries and the growth of small blood vessels in the tumor tissue degradation. Therefore, if the inhibition of tumor tissue angiogenesis, can inhibit the growth of solid tumors, and limit its transfer. Ang (Angiogenin / vascular endothelial growth factor) is an important tumor tissue pro-angiogenic factors, RI (Ribonuclease inhibitor / ribonuclease inhibitor) is its efficient inhibitor, is a promising anti-angiogenic drugs. The umbilical cord blood stem cells is now recognized as a good carrier of the gene into, it has a high rate of successful transplantation, strong proliferative potential, graft-versus-host disease light and anti-tumor immune cells and cord blood derived convenient, easy collection. Since 1989, the success of a cord blood stem cell gene transfer has increasingly become a focus for researchers since the world's first umbilical cord blood transplantation for the treatment of Fanconi anemia. At present, the umbilical cord blood stem cells is its incomparable advantages are widely used in gene therapy of cancer, genetic diseases and other diseases. In this experiment, the retroviral vector, the RI gene transfection has been isolated and purified human umbilical cord blood stem cells, and is highly expressed on which lay the foundation for the next stem cell transplantation in the treatment of tumors (ie: will carry the RI gene umbilical cord blood stem cell screening and in vitro cultured and amplified, transported back to the cancer patients in vivo, by inhibiting tumor angiogenesis, inhibit the growth of solid tumors, and restricting their transfer.) This new anti-tumor therapy, at home and abroad There were no reports. Objective: To investigate the RI gene transfection and expression in human umbilical cord blood stem cells. Method: ① by alkaline lysis, extraction pLNCX-ri; (2) with a liposome transfection method, the the RI gene into the packaging cell line PA317 and detect whether there is a positive cell clones by G418 selection; (3) NIH3T3 cells to target cells measuring viral titers, screening cell lines with high drug production; ④ the immunomagnetic separation human umbilical cord blood stem cells, enriched CD34 ~ cells and CD34 ~ cell content measured by flow cytometry; The ⑤ retroviral vector , pLNCX-ri transfected with CD34 ~ cells, and then detected by G418 selection gene transfection efficiency and elected positive cells cloned; ⑥ PCR method identification the whether the pLNCX-ri is integrated into the chromosome; ⑦ with the westem seven lot detected after transfection Ban the expression of the gene. Results: (1) by agarose gel electrophoresis, extracted pLNCX ri purity higher: 2 G418 in 500 pg/m1 concentration RI gene transfected PA317 cells positive cell clones; (3) detection of NIH3T3 cells a virus titer of of 2.5 x IOOCFU / ml; ④ using flow cytometry CD34 cell content% .15%; ⑤ the G418 at 500 RA g/m1 the concentration, CD34 transfected cells efficiency reached 17.8 %, and the positive cell clones; ⑥ by the PCR method identification, pLNCx ri has been integrated into the chromosome; (7) via the Westem a lot detected after transfection with positive expression of genes of classes. Conclusion: Ban gene able to import human cord blood CD34 cells and highly expressed on it.

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