RNA干扰小鼠NgR基因表达促进脊髓损伤修复的实验研究
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摘要
中枢神经系统(CNS)损伤后轴突难以再生的原因之一是轴突再生抑制性因子的存在, Nogo–A, MAG和Omgp通过一个共同的受体(NgR)发挥神经抑制作用, NgR是轴突再生抑制因子作用的集中点。
本实验采用RNA干涉技术使NgR基因沉默,阻断抑制因子的传导通路,促进中枢神经轴突再生。应用RT-PCR﹑Western-Blot和免疫组织化学检测海马细胞内源性NgR基因沉默效果;体内实验表明,通过载体介导的特异性NgR干涉小发夹能够抑制NgR在脊髓神经元中的表达,同时脊髓损伤轴突再生特异性蛋白GAP-43的表达上调。
实验证明,质粒介导的RNA干涉小发夹能够有效地沉默NgR基因,抑制NgR在脊髓神经元中的表达可以促进脊髓损伤的轴突再生。
Background
Spinal cord injury (SCI) is a major problem in orthopaedics and neurodurgery. Until now, no breakthrough has been made. However, with the rapid development of modern science and technology and preclinical medicine especially the molecular biology, a series of experiments have been conducted concerning spinal cord regeneration, and invigorating results have been obtained. Currently, many methods are applied in the study of the SCI regeneration, including neurotrophic factors, antagonists for growth inhibitory factors, electrostimulation, neural transplantation involved peripheral nerve, embryonic spinal cord, neural stem cell, olfactory ensheathing cell and gene therapy, which could make recoveries of SCI of adult mammal to some extent.
The regeneration of central nervous system (CNS) injury is always a hot spot as well as a difficulty of neuroscience. More and more researchers are focusing their eyes on the studies of SCI. At present, the inability of CNS neurons to regenerate and the growth-inhibitory molecules present in local environment are regarded as the main reasons for the failure of axon regeneration. And myelin and keloid-related inhibitory molecules may take a major role. So far, three major inhibitors from central nervous myelin sheath have been identified as Nogo-A, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp). These inhibitors are generated from oligodendrocytes and some neuron and could be found in the central and peripheral myelin sheath and on the surface of oligodendrocytes. These myelin proteins could induce growth cone collapse and inhibit axon outgrowth. Recent findings indicate that they inhibit axon growth by binding a common receptor, the Nogo-66 receptor (NgR).
Mouse NgR is a protein of 473 amino acids, containing a transposition signal sequence and eight adjacent leucine-rich repeats (LRR) at its N terminal and a specific C-terminal unit. LRR domain shares a moderate amino acid homology (35%) with other proteins containing the same domain. The results from enzyme digest of NgR on the membrance have revealed that its C-terminal is an incidental component for signal of glycosylphosphatidy- linositol (GPI), which is similar to the GPI-anchored receptors. In Genebank, no obvious comparability has been observed between the total cDNA and NgR, except that only few expressed sequence tags (EST) could match some fragments of NgR sequence accurately. Rat NgRcDNA shares an amino acid homology of 89% with human NgRcDNA. In situ hybridization has proved the wide distribution of NgRcDNA in various kinds of SCI neurons, including cerebral cortex neurons, hippocampi neurons, cerebellum Purkinje cells and pons neurons. The distribution of NgR is consistent with that of its mRNA. NgR is expressed in CNS neurons but not in oligodendrocytes. However, Nogo could be expressed in oligodendrocytes, which illustrates that Nogo-A exerts inhibitory effect on central nerve just by interacting with the receptor NgR.
RNA interference (RNAi) is a process of posttransc- riptional gene silencing by sequence-specific mRNA degradation, which is triggered by short double-stranded RNA. The main mechanism is that the dsRNAs get processed into small fragments of 21-23 nt by an RNase, which could be used as templates to cleave and destroy the cognate RNA at specific sites and with specific distance.
Small interfering RNA (siRNA) is an extremely effective tool for reducing gene expression of target genes in a variety of organisms and cell types (e.g., worms, fruit flies, plants, and mammalian). The ability of transfected siRNAs to suppress the transcription of specific genes has proved a useful technique to probe gene function, gene knockout, antiviral research, and gene therapy in mammalian cells. This simple, effective and specific method for down-regulating gene expression holds enormous scientific, commercial, and therapeutic potential.
Herein, we used small vector-mediated hairpin RNA (shRNA) to mediate NgR gene silencing in rat neurons. The shRNA could down-regulate NgR gene expression, blocking its interactions with myelin-related inhibitors (like Nogo-A, MAG, and OMgp), and therefore, promoting the axonal growth and the recovery of injured spinal cord. The results may provide a new strategy for systematic investigations on the therapy of SCI.
Objective:
1. 1.Design and synthesize small hairpin RNAs homologous to NgR and construct siNgR expression vector.
2. 2.Transfect the constructed siNgR expression vector into mice hippocampal cells cultured in vitro to interfere with NgR expression in hippocampus, and screen out the shRNA with the highest inhibitory effect.
3. Discuss the influences of interference of NgR gene on axon regeneration after SCI.
Methods:
1. Two NgR-specific siRNAs were chemically synthesized and the vector pMU6shRNA-NgR was constructed. The construction was verified for further experiments.
2. Mice hippocampal cells were cultured in vitro. Then, the specific vector-mediated siRNA was transfected singly into hippocampal cells with liposomal transfection reagent. RT-PCR, Western-Blot, and immunohistochemical analysis were carried out to detect the silencing of endogenous NgR gene. The pMU6shRNA-NgR with the highest inhibition effect was screened out for the following experiments.
3. Established a contusion model of SCI, which resulted in mice with incomplete paraplegia.
4. Vectors incuding NgR-specific siRNAs were locally injected into zones of SCI. The expression of NgR mRNA was detected using RT-PCR and the presence of NgR protein was tested using Western-Blot.
5. Detect the expression of GAP-43 protein using immunocytochemistry and predict the axon regeneration after NgR gene silencing.
Results:
1. The expression vector for NgR siRNA pMU6sh-NgR has been successfully constructed.
2. NgR siRNAs have been proved to inhibit the expression of NgR in mice hippocampal cells. The expression of endogenous NgR mRNA has been down-regulated 57%and NgR protein has been down-regulated 52%with statistical significance (P<0.05).
3. .In the mice model of SCI, the expression of CAP-43, a specific protein related to the neurite outgrowth after SCI, was found to increase after the injection of NgR siRNAs with statistical significance (P<0.05).
Conclusions:
We have demonstrated that chemically-synthesized NgR-specific siRNAs can effectively inhibit NgR expression in cultured mice hippocampal cells. NgR gene silencing is efficient and specific, and the effect is the most effective at 3 days post transfection. In mice SCI model, the level of NgR mRNA and protein was down-regulated significantly by the two siNgR. The increasing expression of GAP-43 mRNA could promote axon regeneration of mice after SCI.
To sum up, our future work is to investigate NgR signal conduction pathway and other associated genes by multiple gene combination RNAi strategy. We hope to develop gene therapy medicine that could inhibit NgR, proliferation specifically and effectively.
本实验采用RNA干涉技术使NgR基因沉默,阻断抑制因子的传导通路,促进中枢神经轴突再生。应用RT-PCR﹑Western-Blot和免疫组织化学检测海马细胞内源性NgR基因沉默效果;体内实验表明,通过载体介导的特异性NgR干涉小发夹能够抑制NgR在脊髓神经元中的表达,同时脊髓损伤轴突再生特异性蛋白GAP-43的表达上调。
实验证明,质粒介导的RNA干涉小发夹能够有效地沉默NgR基因,抑制NgR在脊髓神经元中的表达可以促进脊髓损伤的轴突再生。
Background
Spinal cord injury (SCI) is a major problem in orthopaedics and neurodurgery. Until now, no breakthrough has been made. However, with the rapid development of modern science and technology and preclinical medicine especially the molecular biology, a series of experiments have been conducted concerning spinal cord regeneration, and invigorating results have been obtained. Currently, many methods are applied in the study of the SCI regeneration, including neurotrophic factors, antagonists for growth inhibitory factors, electrostimulation, neural transplantation involved peripheral nerve, embryonic spinal cord, neural stem cell, olfactory ensheathing cell and gene therapy, which could make recoveries of SCI of adult mammal to some extent.
The regeneration of central nervous system (CNS) injury is always a hot spot as well as a difficulty of neuroscience. More and more researchers are focusing their eyes on the studies of SCI. At present, the inability of CNS neurons to regenerate and the growth-inhibitory molecules present in local environment are regarded as the main reasons for the failure of axon regeneration. And myelin and keloid-related inhibitory molecules may take a major role. So far, three major inhibitors from central nervous myelin sheath have been identified as Nogo-A, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp). These inhibitors are generated from oligodendrocytes and some neuron and could be found in the central and peripheral myelin sheath and on the surface of oligodendrocytes. These myelin proteins could induce growth cone collapse and inhibit axon outgrowth. Recent findings indicate that they inhibit axon growth by binding a common receptor, the Nogo-66 receptor (NgR).
Mouse NgR is a protein of 473 amino acids, containing a transposition signal sequence and eight adjacent leucine-rich repeats (LRR) at its N terminal and a specific C-terminal unit. LRR domain shares a moderate amino acid homology (35%) with other proteins containing the same domain. The results from enzyme digest of NgR on the membrance have revealed that its C-terminal is an incidental component for signal of glycosylphosphatidy- linositol (GPI), which is similar to the GPI-anchored receptors. In Genebank, no obvious comparability has been observed between the total cDNA and NgR, except that only few expressed sequence tags (EST) could match some fragments of NgR sequence accurately. Rat NgRcDNA shares an amino acid homology of 89% with human NgRcDNA. In situ hybridization has proved the wide distribution of NgRcDNA in various kinds of SCI neurons, including cerebral cortex neurons, hippocampi neurons, cerebellum Purkinje cells and pons neurons. The distribution of NgR is consistent with that of its mRNA. NgR is expressed in CNS neurons but not in oligodendrocytes. However, Nogo could be expressed in oligodendrocytes, which illustrates that Nogo-A exerts inhibitory effect on central nerve just by interacting with the receptor NgR.
RNA interference (RNAi) is a process of posttransc- riptional gene silencing by sequence-specific mRNA degradation, which is triggered by short double-stranded RNA. The main mechanism is that the dsRNAs get processed into small fragments of 21-23 nt by an RNase, which could be used as templates to cleave and destroy the cognate RNA at specific sites and with specific distance.
Small interfering RNA (siRNA) is an extremely effective tool for reducing gene expression of target genes in a variety of organisms and cell types (e.g., worms, fruit flies, plants, and mammalian). The ability of transfected siRNAs to suppress the transcription of specific genes has proved a useful technique to probe gene function, gene knockout, antiviral research, and gene therapy in mammalian cells. This simple, effective and specific method for down-regulating gene expression holds enormous scientific, commercial, and therapeutic potential.
Herein, we used small vector-mediated hairpin RNA (shRNA) to mediate NgR gene silencing in rat neurons. The shRNA could down-regulate NgR gene expression, blocking its interactions with myelin-related inhibitors (like Nogo-A, MAG, and OMgp), and therefore, promoting the axonal growth and the recovery of injured spinal cord. The results may provide a new strategy for systematic investigations on the therapy of SCI.
Objective:
1. 1.Design and synthesize small hairpin RNAs homologous to NgR and construct siNgR expression vector.
2. 2.Transfect the constructed siNgR expression vector into mice hippocampal cells cultured in vitro to interfere with NgR expression in hippocampus, and screen out the shRNA with the highest inhibitory effect.
3. Discuss the influences of interference of NgR gene on axon regeneration after SCI.
Methods:
1. Two NgR-specific siRNAs were chemically synthesized and the vector pMU6shRNA-NgR was constructed. The construction was verified for further experiments.
2. Mice hippocampal cells were cultured in vitro. Then, the specific vector-mediated siRNA was transfected singly into hippocampal cells with liposomal transfection reagent. RT-PCR, Western-Blot, and immunohistochemical analysis were carried out to detect the silencing of endogenous NgR gene. The pMU6shRNA-NgR with the highest inhibition effect was screened out for the following experiments.
3. Established a contusion model of SCI, which resulted in mice with incomplete paraplegia.
4. Vectors incuding NgR-specific siRNAs were locally injected into zones of SCI. The expression of NgR mRNA was detected using RT-PCR and the presence of NgR protein was tested using Western-Blot.
5. Detect the expression of GAP-43 protein using immunocytochemistry and predict the axon regeneration after NgR gene silencing.
Results:
1. The expression vector for NgR siRNA pMU6sh-NgR has been successfully constructed.
2. NgR siRNAs have been proved to inhibit the expression of NgR in mice hippocampal cells. The expression of endogenous NgR mRNA has been down-regulated 57%and NgR protein has been down-regulated 52%with statistical significance (P<0.05).
3. .In the mice model of SCI, the expression of CAP-43, a specific protein related to the neurite outgrowth after SCI, was found to increase after the injection of NgR siRNAs with statistical significance (P<0.05).
Conclusions:
We have demonstrated that chemically-synthesized NgR-specific siRNAs can effectively inhibit NgR expression in cultured mice hippocampal cells. NgR gene silencing is efficient and specific, and the effect is the most effective at 3 days post transfection. In mice SCI model, the level of NgR mRNA and protein was down-regulated significantly by the two siNgR. The increasing expression of GAP-43 mRNA could promote axon regeneration of mice after SCI.
To sum up, our future work is to investigate NgR signal conduction pathway and other associated genes by multiple gene combination RNAi strategy. We hope to develop gene therapy medicine that could inhibit NgR, proliferation specifically and effectively.
引文
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