摘要 Objective: We aimed to provide an alternative cell source for cell therapy in leukoaraiosis (LA). Methods: Olfactory ensheathing cells (OECs) from the olfactory bulb were isolated,cultured,and purified.Next,the lentivirus carrying human VEGF165 gene was constructed and transfected into OECs. Results: The proliferative capacity of primary OECs was strong.OECs were infected with different multiplicity of infection.The expression level of VEGF was confirmed by real-time PCR with specific primers for GAPDH and VEGF,indicating that the genetically engineered OECs-VEGF produced VEGF with functional activity. Conclusions: Our data showed that these engineered OECs-VEGF highly express functional VEGF and retain the characteristics of astrocytes and Schwann cells,providing an alternative cell source for cell therapy in LA.
Abstract: Objective: We aimed to provide an alternative cell source for cell therapy in leukoaraiosis (LA). Methods: Olfactory ensheathing cells (OECs) from the olfactory bulb were isolated,cultured,and purified.Next,the lentivirus carrying human VEGF165 gene was constructed and transfected into OECs. Results: The proliferative capacity of primary OECs was strong.OECs were infected with different multiplicity of infection.The expression level of VEGF was confirmed by real-time PCR with specific primers for GAPDH and VEGF,indicating that the genetically engineered OECs-VEGF produced VEGF with functional activity. Conclusions: Our data showed that these engineered OECs-VEGF highly express functional VEGF and retain the characteristics of astrocytes and Schwann cells,providing an alternative cell source for cell therapy in LA.
20170712150516 Figure 1 Morphology and identification of OECs isolated from rat olfactory bulbs. a. The primary OECs showed a typical spindlelike morphology with two or three processes or appearance (left picture: 40× magnification and right picture: 100× magnification). b. OECs formed colonies in the DF12 culture medium supplemented with 15% FBS, 2 μM forskolin, and 10 ng/mL bFGF (left picture: 40× magnification and right picture: 100× magnification). c. OECs were subjected for the immunofluorescence staining of p75 (Green) with Hoechst for nuclei labeling (left picture with 40× magnification and right picture with 100× magnification).
20170712150533 Figure 2 Construction of VEGF lentivirus. a–b. Human VEGF gene was cloned into the lentivirus vector with the marker of EGFP. The representative picture of transfected 293T cells with the VEGF lentivirus vector. c. The total protein of transfected 293T cells was subjected to western blotting for the VEGF specific antibody detection.
20170712150542 Figure 3 The infection efficiency with serial MOIs of control lentivirus. The OECs were infected with serial MOIs of control lentivirus: 1, 10, 50, and 100. The infection efficiency was monitored through fluorescence microscope with Hoechst for nuclei labeling.
20170712150607 Figure 4 Quantification of infection efficiency for control lentivirus and the detection of secreted VEGF by OECs-VEGF. a. The percentage of GFP+ cells with different MOIs of control lentivirus: 1, 10, 50, and 100. b. The VEGF standards and their absorbance at 450 nm were plotted. c. Quantification of secreted VEGF by control OECs and OECs-VEGF. D. The log/log curve for standards with R2 = 0.99.
20170712150627 Figure 5 The functional activity of secreted VEGF by OECs-VEGF. The supernatant of cultivated OECs-VEGF was collected and the HUVECs were treated with the supernatant for up to 72 h. The OD450-OD460 values of HUVECs were compared between OECs, EGFP-OECs, and VEGF-OECs groups (* demonstrates P < 0.05).
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2017, Vol. 3 
