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Experimental Studies On The Construction And Biological Characteristics Of Human Bone Marrow Mesenchymal Stem Cell Line Genetically Modified By Human Proenkephalin Gene

Posted on:2011-09-14Degree:MasterType:Thesis
Country:ChinaCandidate:S Y CaiFull Text:PDF
GTID:2154360308984951Subject:Anesthesia
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Backgroud and ObjectivesThe treatment of chronic pain that seriously affected the quality of human life is one of the problems that have not yet settled. The current drugs treatment may inevitably cause various defects, for this reason, they can not get satisfactory curative effect. Bio-analgesia that simulates the characteristion of human physiological secret may be a new direction of the future pain treatment. Transplantation of transgeneic cells for analgesia is considered as a kind of efficient treatment method. Bone marrow mesenchymal stem cells are rich in sourse and posse satisfactory histocompatibility, lower immunogenicity and multi-differentiation potential. They are ideal choice of cells for gene therapy. Enkephalin is an endogenous opioid peptide and widely distributed in peripheral and CNS. It is an important analgesic media in CNS. This project will construct a recombinant retroviral vector with human proenkephalin gene by the RT-PCR method, and establish a human mesenchymal stem cell line that expresses PENK gene and secrets enkaphalin and identify its biological characteristics. It lays the foundation on the further experimental study of gene therapy for pain.Methods1 Construction of recombinant retroviral vector with human proenkephalin geneTotal RNA was extracted from human pheochromocytoma tissue by Trizol DAB method. The full-length of proenkephalin cDNA was amplified by RT-PCR method. The amplification products were cut by the endonuclease of EcoR I/Sal I and cloned to the pBABE-puro vector after right sequenced. Recombinant plasmid was identified by endonuclease digestion method and sequence analysis.2 Construction and biological characteristics of human bone marrow mesenchymal stem cell line genetically modified by human proenkephalin geneThe primary culture hMSCs were isolated from the human bone marrow. The packaging cell line, Phenix-293T cell line was transfected by the recombinant pBABE-PENK plasmid to acquire virus,then the recombinant virus was collectted and used to infect hMSCs. The continuous passages of human bone marrow mesenchymal stem cell line genetically modified by human proenkephalin gene and untransformed hMSCs were adopted. Morphology and growth features after subculture, freezing and recovering were observed and compared. The cell surface markers CD44/CD29/CD34/CD45 that hMSCs express were detected by flow cytometry. Then their differentiation potentiality to lipoblasts and osteoblasts was tested respectively. Cells differentiated into adipocytes that accumulate lipid vacuoles were stained by oil red.Cells differentiated into osteoblasts that produce mineralized matrices were stained by alizarin red. Stable expression of PENK gene and enkephalin protein were detected by RT-PCR, immunofluorescence and enzyme linked immunosorbent assay (ELISA) method respectively.Results1 Recombinant plasmid pBABE-PENK was digested with EcoR I/Sal I, and was confirmed by sequence analysis. The full coding region of the PENK gene was included in the recombinant plasmid pBABE-PENK.2 The cell line was maintained for more than 20 passages in vitro. Subculture, freezing and recovering had no influence on cellular shape and proliferation(P=0.386). Compared with the 4th generation of hBMSCs, its proliferation capability has no significant differences(P=0.873).The cell surface markers: CD29(P=0.136),CD44(P=0.352),CD34(P=0.466),CD45(P=0.474)detected by flow cytometry have no significant differences. Moreover, the cells before and after transfection can differentiate into adipocytes and osteoblasts. Compared with hBMSCs, the expression of proenkephalin gene and enkephalin protein markly increase according to the result of RT-PCR and immunofluorescence method respectively. The concentration of enkephalin in the culture medium rose apparently according to the ELISA method in the hMSC-PENK cells(P<0.001).Conclusions1 Recombinant retroviral vector with human PENK gene was constructed successfully.2 The human mesenchymal stem cell line that expresses PENK gene and secrets enkaphalin was established successfully. It could be cultured long-termly in vitro and keep the property of fission and proliferate, and it still remains the same differentiated phenotypes and multilineage differentiation potential as its original cells. The human mesenchymal stem cell line that expresses PENK gene could be used as vehicle in transplantation of transgeneic cells for analgesia.
Keywords/Search Tags:Analgesic, Enkephalin, pBABE puro, human mesenchy stem cells
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