慢病毒介导RNA干扰NgR基因对新生大鼠缺氧缺血性脑白质损伤的修复作用
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摘要
目的:
构建NgR特异性siRNA慢病毒重组体,在新生大鼠缺氧缺血性脑白质损伤模型水平,探讨NgR特异性siRNA慢病毒重组体干扰大鼠NgR mRNA表达及其对新生大鼠缺氧缺血性脑白质损伤的修复作用。
方法:
1、建立P3新生SD大鼠缺氧缺血性脑白质损伤模型,从生物行为学、组织形态学进行验证。
2、设计、构建NgR特异性siRNA慢病毒重组体并鉴定。
3、Western Blot方法外源筛靶、内源筛靶,选择最有效的靶点,并包装成慢病毒。
4、40只SD大鼠随机分为假手术组、模型组、空病毒组、慢病毒组(每组10只),新生大鼠缺氧缺血性脑白质损伤3天后(P6)经侧脑室注射给药,观察大鼠的行为学改变。
5、80只SD大鼠随机分为假手术组、模型组、空病毒组、慢病毒组(每组20只),新生大鼠缺氧缺血性脑白质损伤3天后(P6)经侧脑室注射给药,RT-PCR检测P6、P7、P9、P13 SD大鼠NgR mRNA表达情况。
结果:
1、结扎P3新生SD大鼠双侧颈总动脉建立缺氧缺血性脑白质损伤模型,从生物行为学、组织形态学进行验证,模型组都出现了有意义的改变。
2、设计、构建NgR特异性siRNA慢病毒重组体经PCR鉴定、基因测序仪测序,结果与设计序列相符,目的基因序列准确无误,慢病毒重组体构建成功。
3、筛选最有效的靶点,包装成慢病毒,慢病毒重组体浓度为:E+9TU/ml。
4、模型组、空病毒组和慢病毒组大鼠的行为学相对于假手术组出现明显改变,慢病毒组大鼠恢复情况明显好于模型组和空慢病毒组。
5、模型组和空病毒组大鼠的NgR mRNA表达量相对于假手术组明显增高,慢病毒组NgR mRNA表达比假手术组增加,但明显少于模型组和空慢病毒组。
结论:
本研究采用结扎P3新生SD大鼠双侧颈总动脉建立缺氧缺血性脑白质损伤模型,为后续的实验提供了可靠的动物模型;设计、构建NgR特异性siRNA慢病毒重组体并将其应用到动物模型,在活体动物水平探讨NgR特异性siRNA慢病毒重组体干扰大鼠NgR mRNA表达及其对新生大鼠缺氧缺血性脑白质损伤的修复作用。实验结果说明在活体内经侧脑室注射慢病毒重组体转染后可以实现NgR基因的沉默。本研究认为慢病毒介导的RNA干扰有可能成为治疗缺氧缺血性脑白质损伤的有效手段之一,为进一步应用临床试验提供理论依据。
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.
构建NgR特异性siRNA慢病毒重组体,在新生大鼠缺氧缺血性脑白质损伤模型水平,探讨NgR特异性siRNA慢病毒重组体干扰大鼠NgR mRNA表达及其对新生大鼠缺氧缺血性脑白质损伤的修复作用。
方法:
1、建立P3新生SD大鼠缺氧缺血性脑白质损伤模型,从生物行为学、组织形态学进行验证。
2、设计、构建NgR特异性siRNA慢病毒重组体并鉴定。
3、Western Blot方法外源筛靶、内源筛靶,选择最有效的靶点,并包装成慢病毒。
4、40只SD大鼠随机分为假手术组、模型组、空病毒组、慢病毒组(每组10只),新生大鼠缺氧缺血性脑白质损伤3天后(P6)经侧脑室注射给药,观察大鼠的行为学改变。
5、80只SD大鼠随机分为假手术组、模型组、空病毒组、慢病毒组(每组20只),新生大鼠缺氧缺血性脑白质损伤3天后(P6)经侧脑室注射给药,RT-PCR检测P6、P7、P9、P13 SD大鼠NgR mRNA表达情况。
结果:
1、结扎P3新生SD大鼠双侧颈总动脉建立缺氧缺血性脑白质损伤模型,从生物行为学、组织形态学进行验证,模型组都出现了有意义的改变。
2、设计、构建NgR特异性siRNA慢病毒重组体经PCR鉴定、基因测序仪测序,结果与设计序列相符,目的基因序列准确无误,慢病毒重组体构建成功。
3、筛选最有效的靶点,包装成慢病毒,慢病毒重组体浓度为:E+9TU/ml。
4、模型组、空病毒组和慢病毒组大鼠的行为学相对于假手术组出现明显改变,慢病毒组大鼠恢复情况明显好于模型组和空慢病毒组。
5、模型组和空病毒组大鼠的NgR mRNA表达量相对于假手术组明显增高,慢病毒组NgR mRNA表达比假手术组增加,但明显少于模型组和空慢病毒组。
结论:
本研究采用结扎P3新生SD大鼠双侧颈总动脉建立缺氧缺血性脑白质损伤模型,为后续的实验提供了可靠的动物模型;设计、构建NgR特异性siRNA慢病毒重组体并将其应用到动物模型,在活体动物水平探讨NgR特异性siRNA慢病毒重组体干扰大鼠NgR mRNA表达及其对新生大鼠缺氧缺血性脑白质损伤的修复作用。实验结果说明在活体内经侧脑室注射慢病毒重组体转染后可以实现NgR基因的沉默。本研究认为慢病毒介导的RNA干扰有可能成为治疗缺氧缺血性脑白质损伤的有效手段之一,为进一步应用临床试验提供理论依据。
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.
引文
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2. Litt J,Taylor H,Klein N,Hack M. Learning disabilities in children with very low birthweight:Prevalenee,neuropsychological correlates and edueational interventions.J Learn Disabil,2005,8:130-141
3. Miller SP,Ferriero DM,Leonard C,PiecuehR,Glidden D,Partridge JC,Perez M,Mukherjee P,Vigneron D,Barkovieh AJ.Early brain injury in Premature newborns detected with magnetic resonance imaging is associated with adverse neurodeveloPmenial outcome.J Pediatr,2005,147:609-616
4.Blumenthal1.Periventrieular leucomalacia:areview.Eur J Pediatr,2004, 63:435-442
5.Feeriero DM.Neontal brain injury.N Engl J Med,2004,351:1985-1995
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