The Effect Of Lingo-1Gene Silencing By RNA Interference On Myeline Repairment In Experimental Autoimmune Encephalomyelitis

BACKGROUNDMultiple sclerosis is a kind of demyelinated disease of central nervous system(CNS) and is a common disabled neurological disease disease in young people. The failure of remyelination and axonal injure are the pathological basis of perpetual neurological damage. Repeated demyelination could result in axonal injure. Therefore, it is the key to restore myelin sheaths as soon as possible. Leucine rich repeat and Ig domain containing1(Lingo-1) negatively regulates myelin formation in CNS.EAE as an animal model of MS is widely used in studies.We inhibit Lingo-1expression by RNA interference,observe the effects of Lingo-1inhibition on oligodendrocytes and EAE and explore the possible mechanisms, which makes a try for Lingo-1blockade treatment. Our study is divided into three parts.Part OneConstruction and screening of lentiviral vectors encoding Lingo-1shRNA on oligodendrocytesObjective:to screen out lentiviral vectors encoding Lingo-shRNA with higher silencing efficiency in oligodendrocytes of mice.Methods:1.Oligodendrocytes were obtained from the cerebral cortex of newborn within48h C57BL/6mice for primary culture. Cell survival rate and purity were compared after different separated and purified methods, which was a basis of exploring a better means for cell culture. The cells were identified by Lingo-1,MBP immunofluorescence.2.After LV/Lingo-lshRNA transduced oligodendrocytes, MTT tests were performed to investigate the effect of transduction on cell survival.3.Lingo-1mRNA level was detected by Real-Time fluorescent quantitative PCR(RT-qPCR) and Lingo-1protein level was detected by Western blot at96h after lentivirus transduction. The Lingo-1staining in oligodendrocytes after transduction was observed and Lingo-1positive rates were counted by immunofluorescence. Through the above tests, the lentiviral vectors with the best inhibited effect were screened out.Results:1.The cell purities of primarily-cultured oligodendrocytes after mild trypsinization+short-time succession method and vibration-separation method were similar (P>0.05). The cell survival rate of mild trypsinization+short-time succession method was higher(96.46+2.26%vs80.25+1.55%, P<0.05). The cultured cells were identified Lingo-1and MBP positive.2.MTT tests showed that there was no difference in OD value between RNA interference group and normal control (P>0.05)3.The silencing efficiency of LV/Lingo-1shRNA(1) and LV/Lingo-1shRNA(2) on Lingo-1mRNA was36%and37%,obviously higher than the other groups(P<0.05, P(0.05). The silencing efficiency of LV/Lingo-1shRNA(1) and LV/Lingo-1shRNA(2) on Lingo-1mRNA was27.6%and28.3%,obviously higher than the other groups (P<0.05, P <0.05). The immunofluorescence results confirmed that the Lingo-1protein contents of LV/Lingo-1shRNA(1) and LV/Lingo-1shRNA(2) group were distinctly reduced. Accordingly, LV/Lingo-1shRNA(1) and LV/Lingo-1shRNA(2) was the lentiviral vectors with higher silencing efficiency.Conclusions:LV/Lingo-1shRNA(1) and LV/Lingo-1shRNA(2) could effectively silence the Lingo-1expression in oligodendrocytes with no distinct effect on cell survival. LV/Lingo-1shRNA(2) was used for the following study. Part TwoEffect of lentiviral vectors encoding Lingo-1shRNA on oligodendrocytesObjective:to explore the effect of lentiviral vectors encoding Lingo-1shRNA on oligodendrocytes in mice.Methods:1.The Lingo-1levels in oligodendrocytes were detected by RT-qPCR and Western blot on day1,3,7,14after transduction by the lentiviral vectors with the best inhibited effect respectively. Silencing efficiency was evaluated at different times.2.MBP positive rates of oligodendrocytes were counted at different times after transduction via immunofluorescence to know about the effect of LV/Lingo-1shRNA on cell differentiation and maturation.3.P-RhoA protein was detected by Western blot to explore the possible channel which Lingo-1shRNA acted on.Results:1.The silencing efficiency on Lingo-1mRNA of RNA interference(RNAi) group was13.2%,45.1%and57.0%on day3,7,14after LV/Lingo-1shRNA(2) transduction respectively,which was higher than control (P (0.05, P<0.05, P<0.05). The silencing efficiency on Lingo-1protein of RNAi group was27%、47%and53%on day3,7,14after LV/Lingo-1shRNA(2) transduction respectively,which was higher than control (P<0.05, P<0.01, P<0.01).2.The MBP positive rate was (45.4±5.5)%on day1after LV/Lingo-1shRNA(2) transduction,which was not significantly different from control. On day3and5, the MBP positive rate of RNAi group was (89.7±7.9)%and (99.6±4.6)%, which was obviously higher than control (P<0.01, P<0.05).3.There were no markedly difference on p-RhoA protein expression between RNAi group and control group at any time (P>0.05) Conclusions:The silencing efficiency increasingly enhances with time during a certain period. Lingo-1silencing promotes faster and earlier maturation of oligodendrocytes in vitro. Part ThreeEffect of Lingo-1gene silencing on experimental autoimmune encephalomyelitisObjective:To explore Lingo-1gene silencing on locomotor function and myelin repairment of experimental autoimmune encephalomyelitis(EAE) mice.Methods:1.Lingo-1expression in brain tissue of EAE mice was detected by RT-PCR and Western blot on day1,3,7,14,21,30after onset.2.EAE mice with the locomotor score between2and3were divided into5groups:5×109Tu/mlLV/Lingo-l-shRNA group,5×108Tu/mlLV/Lingo-l-shRNA group,5×107Tu/ml LV/Lingo-1-shRNA group, LVCON053group and untreated group.Lingo-1level was detected by RT-PCR and Western-Blot at different times after transduction.3.Locomotor function of different groups was assessed at different times. Luxol fast staining was performed to know about the situations of myelin repairment.Results:1.Attack occurred7-10days after inoculation. In the present study, the overall incidence of EAE in mice was81.3%, and the mean motor function score at onset was2.45±0.94. Lingo-1mRNA and protein expression levels were significantly higher in the untreated EAE mice group than normal mice at any time(P<0.01). The levels first increased and then decreased with time. On day30, expression levels of Lingo-1in untreated EAE mice were still higher than normal mice. 2.The expression levels of Lingo-1mRNA expression were significantly decreased in the5×108TU/ml LV/Lingo-1-shRNA and5×109TU/ml LV/Lingo-1-shRNA groups at day3after LV/Lingo-1shRNA injection (2.45±0.15,2.06±0.18,2.14±0.08vs2.80±0.10,P<0.05). On day14, the expression of Lingo-1mRNA in the5×108TU/ml LV/Lingo-1-shRNA and5×109TU/ml LV/Lingo-1-shRNA groups were close to that of the normal group (1.19±0.11,1.46±0.10vs.1.03±0.09, P>0.05, P<0.05). The expression of Lingo-1mRNA in the5×107TU/ml LV/Lingo-1-shRNA did not verge on that of the normal group until day30. The Lingo-1protein expression also decreased significantly in the LV/Lingo-1-shRNA treated groups on day7(1.99±0.07,2.08±0.06vs.3.14±0.04,3.08±0.05, P<0.001), but reached close to the normal group on day21. The Lingo-1expression was also reduced in the5×107TU/ml LV/Lingo-1-shRNA group, but the reduction rate was significantly lower than the5×108TU/ml LV/Lingo-1-shRNA and5×109TU/ml LV/Lingo-1-shRNA groups (2.53±0.10vs.1.99±0.13,2.08±0.10, P<0.001, P<0.01).3.From day7on, motor function of mice treated with different concentrations of LV/Lingo-1-shRNA (5×107,5×108, and5×109TU/ml) was significantly improved, compared to the untreated or LVCON053groups (3.11±0.13,2.42±0.13,2.96±0.10vs3.56±0.15,3.87±0.12,P<0.01,P<0.01, P<0.05). Motor function improved most significantly in the5×108TU/ml LV/Lingo-1-shRNA group (0.87±0.11vs.3.05±0.13, P<0.001)by day30.4.Among different LV/Lingo-1-shRNA treated groups, myelination in5×10’TU/ml LV/Lingo-1-shRNA group or the5×109TU/ml LV/Lingo-1-shRNA group was better than untreated group. Most significant repairment appeared in the5×108TU/ml LV/Lingo-1-shRNA group (P<0.05),but the degree of myelination was still lower than normal group (P<0.05)Conclusions:LV/Lingo-1shRNA by ICV injection is an effective method to silence Lingo-1expression.Lingo-1silencing could ameliorate motor function and promote formation of myelin sheaths.But the effects do not enhance with the increase of LV/Lingo-1-shRNA dose.