Lentiviral Vector Mediated RNA Interfere Gene NgR To Repair Hypoxic-ischemic Neonatal White Matter Damage In Newborn Rat

Objective:Construct the effective NgR (Nogo receptor) specific siRNA Lentivirus vector and explore the effects of NgR specific siRNA Lentivirus recombination in NgR mRNA expression on newborn rat hypoxic-ischemia neonatal white matter damage and its repair function on newborn rat white matter damage from animal model level.Methods:1. The model of newborn (P3) SD rat white matter damage was established and validated based on biological behavior and histology.2. Design, construct and identification of the effective NgR specific siRNA Lentivirus vector.3. Western Blot to exogenous target sieve, endogenous target sieve to select the most effective target and to package into lentivirus.4. 40 SD rats were divided into control, model, control Lentivirus and recombinant Lentivirus groups randomly (10 rats each).Drugs were intracerebroventricular microinjected three days after newborn rat white matter damage.Behavioral and histological changes were observed.5. 80 SD rats were divided into control, model, control Lentivirus and recombinant Lentivirus groups randomly (20 rats each). Drugs were intracerebroventricular microinjected three days after newborn rat (P6) white matter damage and RT-PCR on P6 P7 P9 P13 SD rats to observe the expression of NgR mRNA.Results:1. Newborn (P3) SD rat was be established the model of white matter damage, significant biological behavioral and histological changes were observed in model.2. The constructed effective NgR specific siRNA Lentivirus vector was identified by PCR and DNA sequencing and was proven to be identical to the designed sequence.3. Select the most effective target and package into lentivirus, The lentivirus titer reached E+9TU/ml.4. Obvious biological behavioral was observed in model group, control lentivirus vector group and recombination group compare with control group, however, healing in recombination group was better than model group and control lentivirus vector group.5. Obvious enhance was observed in the NgR mRNA expression in model and control lentivirus vector groups compare with control group, the NgR mRNA expression in recombination group was observed enhance than control group, while significant decrease of NgR mRNA expression was observed in recombination lentivirus vector group.Conclusion:In this study , bilateral common carotid artery of newborn (P3) SD rat was set up to established the model of white matter damage, which provide reliable rat model for success experiments.The effective NgR specific siRNA lentivirus vector was designed and constructed and its effects on rat NgR mRNA expression level and repair function on newborn (P3) SD rat white matter damage were explored on live animal level.The result indicating the NgR gene silencing occurred after intracerebroventricular microinjection of recombinant lentivirus vectors.It demonstrated the promising role of lentivirus conducted RNAi in healing the white matter damage and provided the theoretical basis for further clinic trial.