RNA干扰ROCK-Ⅱ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(central nervoussystem,CNS)损伤后,髓鞘相关性轴突生长抑制因子Nogo-A、髓磷脂相关糖蛋白(myelin-associated glycoprotein,MAG)、少突胶质细胞-髓磷脂糖蛋白(oligodendrocyte myelinglycoprotein,Omgp)共同作用于高亲和受体NgR,在p75NTR的参与下,激活内源性的Rho后通过活化下游的效应分子ROCK,使其作用底物肌球蛋白磷酸酶磷酸化,从而影响细胞微结构的骨架肌动蛋白系统,最终抑制轴突生长。
为了研究特异性shRNA-ROCK-Ⅱ对ROCK-Ⅱ基因表达的抑制和对轴突再生修复的作用,在体外和体内中分别进行了实验研究。体外试验中,将构建的小发夹片段RNA (small hairpin RNA,shRNA)与质粒结合后,通过脂质体介导转染自行培养并纯化、鉴定正确的少突胶质细胞后,应用RT-PCR、免疫组化方法检测ROCK-Ⅱ基因沉默效果,并对转染后的细胞在特定的时间点进行流式细胞术检测细胞生长周期情况。结果表明,体外构建的特异性shRNA-ROCK-Ⅱ有效地沉默了ROCK-Ⅱ基因表达。体内实验中通过制作的大鼠脊髓损伤模型进行分组研究,根据体外实验中观察到的转染后基因抑制的有效时间点,在mRNA水平和蛋白表达水平对ROCK-Ⅱ基因的表达进行评价,特异性shRNA-ROCK-Ⅱ抑制了损伤脊髓组织中的ROCK-Ⅱ基因的表达,同时脊髓损伤后轴突再生特异性生长标志物GAP-43蛋白的表达上调,进一步为说明神经细胞轴突具有再生的倾向提供了依据。实验证明,ROCK-Ⅱ基因的抑制与损伤后脊髓的轴突再生有正相关性,特异性shRNA-ROCK-Ⅱ抑制ROCK-Ⅱ基因的表达可以促进脊髓损伤的轴突再生。
本实验利用小干扰RNA(small or short inferenceRNA,siRNA)的有效性和特异性,有效抑制了ROCK-Ⅱ基因的表达,阻断了Rho-ROCK传导通路,抑制了NogoA、MAG、Omgp等轴突生长抑制因子的作用,为脊髓损伤的治疗提供了新的方法。
Spinal cord injury is a severe disabling central nervous system diseases, in recent years, the incidence of it have a clear upward trend. The pathological basis can be divided into primary injury and secondary injury. Among them, primary injury is irreversible. Therefore, to prevent the occurrence of secondary injury, as far as possible to retain residual neurological function and promote nerve regeneration has become the focus of SCI research and treatment. To date, regeneration and functional reconstruction after spinal cord injury have been plagued problems to medical workers.
In recent years, experimental and theoretical studies have shown that the key to repairing of spinal cord injury are improve the microenvironment of axon growth and reduce apoptosis of nerve cells and promote axon growth. We have confirmed that Nogo-A, MAG, OMgP are major factors in inhibiting axonal regeneration until now. They are united by a common receptor (NgR), in the P75NTR participation, to enable it bonding with theαhelix structural domain of ROCK by activating the Rho, thereby activating ROCK. When ROCK is activated, ROCK will make myosin phosphatase (MP) which is it’s substrate to phosphorylation, then the substrate will be inactivation, thus affecting the actin system. The final result is the axon growth cone will be retraction, degradation, collapse, inhibit axon growth.
ROCK is a Rho-associated coiled-coil forming protein kinase, also known as Rho-kinase. ROCK belongs to serine / threonine protein kinase, the molecular weight of it is about 160 kDa. ROCK mainly exists in the cytoplasm. ROCK, there are two subtypes ROCKⅠand ROCKⅡ, ROCKⅡmainly expressed in the brain, while the ROCKⅠexpressed mainly in non-neural tissues, such as the lung, kidney and skeletal muscles. Two kinds of subtypes in structure has 65% homology, ROCK is the most important downstream effector molecule of Rho.
ROCK involved in regulation of MLC phosphorylation is a classic way of Rho-kinase system signaling pathway. ROCK increase the level of MLC phosphorylation by direct phosphorylation of MLC and indirectly inhibit MLCP activity, thereby increasing myosin interaction with actin to regulate actin cytoskeleton reorganization, leading to growth cone collapse and neurite retraction. At present, the studies which Rho / ROCK signaling pathway by regulating the gene expression involved in the development of a variety of diseases are in progress. Studies have found that Rho / ROCK system have inhibitory effect in spinal cord injury repair. The inhibitory molecule which comes from myelin is one of the chief hindrances to central nervous system axonal regeneration after injury. Founding Rho / ROCK signal transduction pathway not only further clarifies the mechanism of its role, but also provides a new research ideas and therapeutic targets for regeneration after CNS injury, which has potential application value.
RNA interference is a post-transcriptional gene silencing. Small interfering RNA (small or short RNA) can trigger some kind of post-transcriptional control procedures to identify homologous sequence mRNA, specifically cutting them, thus blocking its translation.
In recent years, Rho-ROCK pathway in the nervous system mainly focused on specificity inhibitors for the Rho and ROCK. The experimental study which using RNA interference to treat acute spinal cord injury in vitro and in vivo has not been reported. So we designed experiments using RNA interference ROCK-Ⅱin adult rat model of SCI to promote cell axon regeneration for the study of the therapeutic effect of ROCK-Ⅱin cell apoptosis in the acute phase of spinal cord injury.
In this study, using RNA interference method to silence ROCK-Ⅱgene specifically, inhibiting the mRNA, blocking Rho-ROCK pathway, so that the activity of myosin phosphatase downstream is inhibited, thereby promoting axon regeneration of injured spinal cord cells, providing a new strategy and method to research and treatment of post-SCI.
1. Methods
In vitro experiments, we designed four shRNA sequences for the ROCK-Ⅱgene in rat, and connected with the cloning vector pGPU6/GFP/Neo, then syntheticed recombinant plasmid, enzyme digestion and sequencing at last. At the same time, making spinal cord oligodendrocyte separation, purification, identification, and putting the recombinant plasmid transfect into oligodendrocytes. After that, detecting the expression level of ROCK-Ⅱby using RT-PCR and immunohistochemistry, and testing the transfected cells by flow cytometry to observe the cell apoptosis, thus evaluate the shRNA interference effects which we designed.
In vivo experiment, we chose the utility frag in vitro experiment first, then recombinated it with lipoplast. Preparation of spinal cord injury model in rats accordance with the modified Allen's, and randomized three groups, each group respectively 1,3,7,10,15 day randomly selected three were killed, and put spinal cord injury samples for RT-PCR and Western Blot analysis. Meanwhile, to detected GAP-43 in the treatment group and untreated group by using Western Blot in spinal cord injury areas.
2. Results
In vitro experiments, I constructed four disruption plasmids for the ROCK-Ⅱgene of rats successfully, and identified as positive plasmids after restriction enzyme digestion, sequencing is correct, then transfected the recombinant plasmids into oligodendrocytes. The RT-PCR results showed that ROCK-Ⅱgene in the relative expression level had a downward trend, which showed that four interfering vector pairs ROCK-Ⅱgene are inhibitory, but ROCK-Ⅱ-2951 fragment inhibited the best results. The data obtained are with statistical significance.
In vivo experiments, in accordance with modified Allen's method I prepared rat model of spinal cord injury successfully. I put the recombinant plasmid into the spinal cord injury zone through the method of liposome-mediated. Western Blot and RT-PCR analysis showed that the treatment group ROCK-Ⅱgene in the damage zone relative level of expression of a downward trend, but the untreated group ROCK-Ⅱgene expression continued to rise. The expression of ROCK gene in treatment group and untreated group and normal group had statistically significant difference, P <0.05. At the same time, as compared with the untreated group, the expression of GAP-43 was significantly upward in treatment group. Two sets of data had statistically significant difference, P <0.05.
3.Conclusion
In this experiment, ROCK as a target goal, I constructed a small hairpin structure in inhibiting its expression successfully, this hairpin structure can inhibited endogenous ROCK-Ⅱgene expression in oligodendrocyte cells specifically, which gene silencing effect of a high efficiency, specificity, and in effect five days after transfection, the most obvious. In the rat animal model of SCI , the experimental results confirmed that specific transfected shRNA were able to successfully interfere with the expression levels of mRNA of ROCK-Ⅱgene, and it was a reliable evidence which the expression of GAP-43 increase is a tendency to axonal regeneration.
To sum up, our future work is to investigate Rho-ROCK signal conduction pathway and other associated genes by multiple gene combination RNAi strategy. We hope to develop gene therapy medicine that inhibits ROCK-Ⅱproliferation specifically and effectively.
为了研究特异性shRNA-ROCK-Ⅱ对ROCK-Ⅱ基因表达的抑制和对轴突再生修复的作用,在体外和体内中分别进行了实验研究。体外试验中,将构建的小发夹片段RNA (small hairpin RNA,shRNA)与质粒结合后,通过脂质体介导转染自行培养并纯化、鉴定正确的少突胶质细胞后,应用RT-PCR、免疫组化方法检测ROCK-Ⅱ基因沉默效果,并对转染后的细胞在特定的时间点进行流式细胞术检测细胞生长周期情况。结果表明,体外构建的特异性shRNA-ROCK-Ⅱ有效地沉默了ROCK-Ⅱ基因表达。体内实验中通过制作的大鼠脊髓损伤模型进行分组研究,根据体外实验中观察到的转染后基因抑制的有效时间点,在mRNA水平和蛋白表达水平对ROCK-Ⅱ基因的表达进行评价,特异性shRNA-ROCK-Ⅱ抑制了损伤脊髓组织中的ROCK-Ⅱ基因的表达,同时脊髓损伤后轴突再生特异性生长标志物GAP-43蛋白的表达上调,进一步为说明神经细胞轴突具有再生的倾向提供了依据。实验证明,ROCK-Ⅱ基因的抑制与损伤后脊髓的轴突再生有正相关性,特异性shRNA-ROCK-Ⅱ抑制ROCK-Ⅱ基因的表达可以促进脊髓损伤的轴突再生。
本实验利用小干扰RNA(small or short inferenceRNA,siRNA)的有效性和特异性,有效抑制了ROCK-Ⅱ基因的表达,阻断了Rho-ROCK传导通路,抑制了NogoA、MAG、Omgp等轴突生长抑制因子的作用,为脊髓损伤的治疗提供了新的方法。
Spinal cord injury is a severe disabling central nervous system diseases, in recent years, the incidence of it have a clear upward trend. The pathological basis can be divided into primary injury and secondary injury. Among them, primary injury is irreversible. Therefore, to prevent the occurrence of secondary injury, as far as possible to retain residual neurological function and promote nerve regeneration has become the focus of SCI research and treatment. To date, regeneration and functional reconstruction after spinal cord injury have been plagued problems to medical workers.
In recent years, experimental and theoretical studies have shown that the key to repairing of spinal cord injury are improve the microenvironment of axon growth and reduce apoptosis of nerve cells and promote axon growth. We have confirmed that Nogo-A, MAG, OMgP are major factors in inhibiting axonal regeneration until now. They are united by a common receptor (NgR), in the P75NTR participation, to enable it bonding with theαhelix structural domain of ROCK by activating the Rho, thereby activating ROCK. When ROCK is activated, ROCK will make myosin phosphatase (MP) which is it’s substrate to phosphorylation, then the substrate will be inactivation, thus affecting the actin system. The final result is the axon growth cone will be retraction, degradation, collapse, inhibit axon growth.
ROCK is a Rho-associated coiled-coil forming protein kinase, also known as Rho-kinase. ROCK belongs to serine / threonine protein kinase, the molecular weight of it is about 160 kDa. ROCK mainly exists in the cytoplasm. ROCK, there are two subtypes ROCKⅠand ROCKⅡ, ROCKⅡmainly expressed in the brain, while the ROCKⅠexpressed mainly in non-neural tissues, such as the lung, kidney and skeletal muscles. Two kinds of subtypes in structure has 65% homology, ROCK is the most important downstream effector molecule of Rho.
ROCK involved in regulation of MLC phosphorylation is a classic way of Rho-kinase system signaling pathway. ROCK increase the level of MLC phosphorylation by direct phosphorylation of MLC and indirectly inhibit MLCP activity, thereby increasing myosin interaction with actin to regulate actin cytoskeleton reorganization, leading to growth cone collapse and neurite retraction. At present, the studies which Rho / ROCK signaling pathway by regulating the gene expression involved in the development of a variety of diseases are in progress. Studies have found that Rho / ROCK system have inhibitory effect in spinal cord injury repair. The inhibitory molecule which comes from myelin is one of the chief hindrances to central nervous system axonal regeneration after injury. Founding Rho / ROCK signal transduction pathway not only further clarifies the mechanism of its role, but also provides a new research ideas and therapeutic targets for regeneration after CNS injury, which has potential application value.
RNA interference is a post-transcriptional gene silencing. Small interfering RNA (small or short RNA) can trigger some kind of post-transcriptional control procedures to identify homologous sequence mRNA, specifically cutting them, thus blocking its translation.
In recent years, Rho-ROCK pathway in the nervous system mainly focused on specificity inhibitors for the Rho and ROCK. The experimental study which using RNA interference to treat acute spinal cord injury in vitro and in vivo has not been reported. So we designed experiments using RNA interference ROCK-Ⅱin adult rat model of SCI to promote cell axon regeneration for the study of the therapeutic effect of ROCK-Ⅱin cell apoptosis in the acute phase of spinal cord injury.
In this study, using RNA interference method to silence ROCK-Ⅱgene specifically, inhibiting the mRNA, blocking Rho-ROCK pathway, so that the activity of myosin phosphatase downstream is inhibited, thereby promoting axon regeneration of injured spinal cord cells, providing a new strategy and method to research and treatment of post-SCI.
1. Methods
In vitro experiments, we designed four shRNA sequences for the ROCK-Ⅱgene in rat, and connected with the cloning vector pGPU6/GFP/Neo, then syntheticed recombinant plasmid, enzyme digestion and sequencing at last. At the same time, making spinal cord oligodendrocyte separation, purification, identification, and putting the recombinant plasmid transfect into oligodendrocytes. After that, detecting the expression level of ROCK-Ⅱby using RT-PCR and immunohistochemistry, and testing the transfected cells by flow cytometry to observe the cell apoptosis, thus evaluate the shRNA interference effects which we designed.
In vivo experiment, we chose the utility frag in vitro experiment first, then recombinated it with lipoplast. Preparation of spinal cord injury model in rats accordance with the modified Allen's, and randomized three groups, each group respectively 1,3,7,10,15 day randomly selected three were killed, and put spinal cord injury samples for RT-PCR and Western Blot analysis. Meanwhile, to detected GAP-43 in the treatment group and untreated group by using Western Blot in spinal cord injury areas.
2. Results
In vitro experiments, I constructed four disruption plasmids for the ROCK-Ⅱgene of rats successfully, and identified as positive plasmids after restriction enzyme digestion, sequencing is correct, then transfected the recombinant plasmids into oligodendrocytes. The RT-PCR results showed that ROCK-Ⅱgene in the relative expression level had a downward trend, which showed that four interfering vector pairs ROCK-Ⅱgene are inhibitory, but ROCK-Ⅱ-2951 fragment inhibited the best results. The data obtained are with statistical significance.
In vivo experiments, in accordance with modified Allen's method I prepared rat model of spinal cord injury successfully. I put the recombinant plasmid into the spinal cord injury zone through the method of liposome-mediated. Western Blot and RT-PCR analysis showed that the treatment group ROCK-Ⅱgene in the damage zone relative level of expression of a downward trend, but the untreated group ROCK-Ⅱgene expression continued to rise. The expression of ROCK gene in treatment group and untreated group and normal group had statistically significant difference, P <0.05. At the same time, as compared with the untreated group, the expression of GAP-43 was significantly upward in treatment group. Two sets of data had statistically significant difference, P <0.05.
3.Conclusion
In this experiment, ROCK as a target goal, I constructed a small hairpin structure in inhibiting its expression successfully, this hairpin structure can inhibited endogenous ROCK-Ⅱgene expression in oligodendrocyte cells specifically, which gene silencing effect of a high efficiency, specificity, and in effect five days after transfection, the most obvious. In the rat animal model of SCI , the experimental results confirmed that specific transfected shRNA were able to successfully interfere with the expression levels of mRNA of ROCK-Ⅱgene, and it was a reliable evidence which the expression of GAP-43 increase is a tendency to axonal regeneration.
To sum up, our future work is to investigate Rho-ROCK signal conduction pathway and other associated genes by multiple gene combination RNAi strategy. We hope to develop gene therapy medicine that inhibits ROCK-Ⅱproliferation specifically and effectively.
引文
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