In Vitro Study Of Olfactory Ensheathing Glia Activated By Light-Sensitive Gene

Objective: The researches of cell transplantation to repair spinal cord injury havetwo shortcomings. The first, lack of antagonism to inhibitory factors in spinal cord injuryregenerative microenvironment; the second, lack of control and regulation of the cellstransplanted into injured spinal cord. Even if cells were transplanted into injured spinalcord, there were still a large number of axonal regeneration inhibitors in spinal cordmicroenvironment. Recent studies had found that the Nogo receptor(NgR/p75NTR/Lingo-1) complex on axonal membrane play a role of the commonchannel in suppression of axonal regeneration and myelination. The hydrophilic Lingo-1fragment (Lingo-1-Fc) may competitive bind sites on NgR to hinder the combination ofthe Lingo-1and NgR. Then, the inhibitory effects of Lingo-1were antagonized. In thesame time, the inhibitory factors in the microenvironment also were decreased. Inaddition, the activation and regulation of function of cells implanted into the injuredspinal cord were still insufficient. OEG cells have been widely used in basic and clinicalstudies of spinal cord injury, and showed effects of neuroprotection, crosssing the gliascar, remyelination, promotion of axon regeneration. The light-sensitive genes, ChR2,can be transducted and expressed in neurons. ChR2encodes cell membrane cationicchannel protein which could be activated by photostimulation of blue light. ActivatedChR2cationic channel protein may open and result in cations internal flow that couldlead to cell action potential and excitation. According to the results of astrocyte, will theOEG cells be activated by blue light with light-sensitive gene technology and secretLingo-1-Fc fragment to antagonize the inhibitory factors, and enhance its effects in repairof spinal cord injury? This study is going to attempt to study the issue with the method oflight-sensitive gene technology in vitro study of OEG. The study may provide scientificand theoretical basis for OEG transplantation with light activation in vivo. This mayresults in the coordination of promoting agent and inhibtory factors in spinal cord injury microenvironment and may obtain better restoration effect.Methods: In this study, lentiviral vector of light-sensitive gene ChR2andLingo-1-Fc gene were built and were transducted into rat OEG cells in vitro. The proteinand gene exprssion of ChR2and Lingo-1-Fc were analized by RT-PCR, ELISA, cellprotein positioning technology mthods. When valid transfection was certified, thefabricated LED blue illumination device were designed and made to photostimulate theOEG cells according to the LED flash circuit. With blue light photostimuation andregulation, the biological responses of OEG cells were observed in this vitro study,especially for changes of OEG cell survival, proliferation, migration, secretion function.The neuroprotective effects were observe by the method of human neuroblastoma cellscultured in supernatant of light stimulated OEG cells.Results: The OEG cells can be effectively, safely, efficiently transfected byconstucted lentiviral vectors. The best transfection MOI was10. The expression andsecretion of target gene tranducted into OEG cells can be detected with method of gene andprotein. The wavelength of blue LED light470nm, the radiant illumination of this bluelight was enough for activation of light-sensitive protein ChR2. Compared with the simplepurified OEG, the proliferation activity, secretion of neurotrophic factors, migrationcapability, and neuroprotective effects were enhanced among OEG cells stimulated by bluelight in vitro study.Conclusions: OEG cells can be controled and regulated by light-sensitive genetechnology. Light stimulation in vitro enhanced the physiological functions of the OEGcells. The photostimulation promoted the neurotrophic factor secretory function,enhanced the capability migration and proliferation, reinforced neuroprotective effects oflight-activated OEG. This study may provide the scientific and theoretical basis forfurther research of OEG transplantation regulated with light-sensitive gene tchnology invivo.