Expression of lentivirus-mediated rat prodynorphin gene in bone marrow mesenchymal stem cells / 医学研究生学报

ABSTRACT

Objective Dynorphins have advantages in powerful analgesic effect, high safety, no respiratory depression and no addiction, which is the emphasis of analgesic research at present. The aim of the article was to explore the expression of lentivirus-mediated rat prodynorphin gene in bone marrow mesenchymal stem cells( BM-MSCs) and contribute to the subsequent studies on bio-logical analgesia in cancer pain of rat model. Methods BM-MSCs were isolated and proliferated using the adherence screening meth-od, and further identified by flow cytometry, adipogenic and osteogenic differentiation experiments. The PDYN lentiviral vectors in rats were transfected into BM-MSCs after construction. The expression of green fluorescent protein (GFP) was detected under inversion fluo-rescence microscope and the best multiplicity of infection ( MOI ) of virus was screened by western blot. There are three groups in the ex-periment blank group, experimental group ( PCDH-CMV-PDYN-EF1-copGFP ) and empty vector group ( PCDH-CMV-MCS-EF1-copGFP). PYDN gene was determined by qPCR and western blot, while DYN protein was detected by immunochemical method.Results BM-SMCs were in longspindle-shape and fibrocyte-like adherent growth, most in expression of CD29, CD44 and CD90, and a few in CD45. The oil red-O staining of the induced cells by adipogenic differentiation was positive. The mineralized nodules formed in the induced cells by osteogenic differentiation were orange after alizarin red staining. Flow cytometry detection showed the positive rates of CD29, CD90, CD44 and CD45 were respectively (99.80±0.19)%, (99.62±0.24)%, (96.86±1.27)%, (0.82±0.06)%, while after transfection the positive rates were (99.59±0.34)%, (98.06±1.27)%, (95.23±0.71)%, (10.23±0.59)%, representing no sig-nificant difference before and after PDYN transfection. Lentiviral vector of PCDH-CMV-PDYN-EF1-copGFP was successfully construc-ted after the identification of PCR amplification, cloning and sequencing. The titer of recombined lentiviruses was 5×106IU/mL. The best MOI of lentiviruses was 100 according to the results of GFP and western blot. Western blot and qPCR suggested PDYN gene signif-icantly increased in BM-MSCs after lentiviral transfection ( P<0.05) , and immunohistochemical staining indicated DYN protein also in-creased greatly. Conclusion BM-MSCs are successfully cultured and the overexpressed rat PDYN gene lentivirus vector is also suc-cessfully constructed;PDYN gene is highly and stably expressed and DYN protein is secreted in BM-MSCs.