神经生长因子基因修饰的许旺细胞治疗大鼠脊髓损伤的疗效观察及其机制研究
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摘要
第一部分许旺细胞的取材、纯化和鉴定
目的培养并纯化大鼠许旺细胞,为后续的实验提供细胞来源。
方法取新生3-4天SD大鼠的坐骨神经和臂丛神经,采用Ⅳ型胶原酶消化的半植块培养法获取原代大鼠许旺细胞；然后用免疫磁珠分选法获纯化许旺细胞。用S-100抗体进行免疫组织化学染色鉴定许旺细胞并计算细胞纯度。
结果取材后6-8小时可见到细胞贴壁,呈现出明显的梭形,遮光性强。免疫磁珠分选后可获得了高纯度的许旺细胞,免疫细胞化学染色见到大量的阳性细胞,阳性率达到95%以上。
结论肤酶和胶原酶联合消化法联合磁珠分选法可以获得高纯度的许旺细胞,为后续的实验提供了细胞来源。
神经生长因子基因载体的构建、转染及表达
目的构建绿色荧光蛋白标记的神经生长因子基因表达载体,并检测其表达效率。
方法采用双酶切法切取人神经生长因子基因序列,采用PCR法扩增,酶切后和载体EGFP-N1连接；感染感受态大肠杆菌后挑选单克隆菌株,提取质粒酶切鉴定证实为目的质粒后进行扩增。用Iipofectamine2000将质粒转染大鼠许旺细胞,用ELISA法检测神经生长因子表达量的变化。
结果PCR凝胶电泳结果显示切取的片段约为750bp左右,和目的基因片段大小相符合；提取质粒后双酶切显示切取片段和目的基因的大小相符合；转然后24小时在显微镜下可见到明显的绿色荧光,一周后上清液中NGF的含量较对照组明显升高。
结论成功构建出含有神经生长因子基因的EGFP-N1质粒,转染许旺细胞后能够促进NGF的表达。
高表达NGF的许旺细胞移植治疗脊髓损伤的疗效及其机制探讨
目的观察高表NG的许旺细胞移植治疗大鼠脊髓损伤的效果,并探讨其作用的可能机制。
方法用改良Allen法制备大鼠脊髓损伤模型,随机分为A、B、C、D四组,A组为转染含NGF基因的许旺细胞移植组,B组为许旺细胞移植组,C组为PBS对照组,D组为假手术组,造模一周后分别用108/ml含有转染NGF的许旺细胞的PBS液、108/m1许旺细胞的PBS液、PBS液注入A、B、C组大鼠脊髓损伤部位及其上下各5μ1。注射后一二、三、四周分别用BBB评分法观察大鼠双下肢运动功能,用肌电图检测大鼠的神经传导功能;处死后用免疫组织化学染色法检测脊髓损伤部位Bcl-2和6AP-43的表达变化。并取转染组的大鼠脊髓组织行纵行切片观察许旺细胞的迁移情况。
结果各时间点BBB评分结果显示D组>A组>B组>C组；肌电图检测结果显示中枢潜伏时传导速度A、B、C组间差别有统计学意义P<0.05,中枢波幅A、B、C组间未见明显差异(P>0.05),D组明显高于其它三组； Bc1-2和GAP-43免疫组化检测结果提示A>B>C组,D组仅有少量表达。大鼠脊髓组织纵行切片观察显示在脊髓损伤后一周损伤区有大量的绿色荧光蛋白表达,随后随着时间的延长,大鼠脊髓损伤区内的许旺细胞数量逐渐减少。
结论本研究证实NGF基因修饰的许旺细胞能够促进脊髓损伤的恢复,可能是通过抑制受损细胞的凋亡和促进神经轴突的生长有关。
Part1Purified and Identified of Schwann Cells from Rat
Object To culture and purify schwan cells of rat, which provides cells for following up experiments.
Method The sciatic and brachial plexus nerves were obtained from SD rats which were newborn3-4days. The primary schwann cells were acquried through these nerves digested by collagenase Ⅳ. The schwann cells were purified by immunomagnetic beads after cultured two generations. The S-100antibody were used to identify the schwann cells and calculate the purity by immunohistochemical staining.
Result The schwann cells were adhesioned after cultured6-8hours, showing a clear spindle. The purity of schwann cells were as high as95%indetified by immunohistochemical staining which were pueified by immunomagnetic beads.
Conclusion High purity of schwann cells were obtain through collagenase IV digestion method combined with magnetic bead sorting, providing a cell source for subsequent experiments.
Part2Construction and Expression of Retroviral Vector of pEGFP-N1/NGFβ
Object To construct retroviral vector carrying nerve growth factor (NGF) beta gene which were labelled by EGFP, and to observe its expression in rat Schwann cell (SC).
Method The NGF beta gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from pAAV-SP vector which was send by Dr Lynne Moor. By gene recombination technique, human NGF beta gene was inserted into vector pEGFP-N1. Then, we transfected the recombination plasmid into SC by lipofectamine2000, and collected the positive clones after G418selection and observed its expression under fluorescence microscope and detect the NGF concentration by ELISA.
Result The PCR production of NGF beta is about750bp. The recombinant plasmid was identified by restriction endonuclease analysis. The human NGF beta gene could be expressed successfully by SCs under fluorescence microscope and the concentration of NGF beta is higher than untransfection SCs.
Conclusion The recombination vector pEGFP-hNGFβ was construction successfully. The NGF beta gene could be efficient expressed by SC.
Part3Nerve Growth Factor Gene Modified Schwann Cells Inhabit Apoptosis and Promote Functional Recovery after Contusion Spinal Cord Injury in Sprague Dawley Rats
Object Schwann cells(SCs) which were modified by human nerve growth factor gene were injected into the injured spinal cord,to observe the functional recovery of hind limb and the expression of Bcl-2and GAP-43after spinal cord injury, and to explore the mechanism of SCs on the spinal cord injury.
Methods52SD rat were divided into four groups randomly:EGFP-N1-NGF transfected SCs transplantation group (group A)16rats,SCs transplantation group (group B)16rats, PBS control group (group C)12rats and sham group(group D)8rats. The modified Allen method was using to made spinal coed injury(SCI) model.BBB scores were observed to evaluate the behavior improvement and EMG detect the velocity of the central nerve at7d、14d、21d and28d, respectively. Then the animals were killed and the SCI areas were embedded by paraffin, tissue section and immunostaining of Bcl-2and GAP-43evaluate the Histology recovery after spinal cord injury. The migration of hNGF gene modified SCs in the group A were observed by fluorescence microscope.
Result The result of BBB scores showed that the animals in group A were higher than group B and group C, and animals in group B were higher than group C, the animals in group D were normal (P<0.05); There is significantly difference of central latency between group A、B and C, but were longer than group D; However, there were no significantly difference of central amplitude between group A、B and C. The immunostaining of Bcl-2and GAP-43showed that group A> group B> group C, the group D have little expression. The SCs in injured spinal cord were distributed in punctate, the SCs were granduly reduced as time follow.
Conclusion Our study confirmed that NGF gene modified SCs promote the recovery of spinal cord injury, probably by inhibiting the apoptosis of damaged cells and promoting the growth of the nerve axon.
目的培养并纯化大鼠许旺细胞,为后续的实验提供细胞来源。
方法取新生3-4天SD大鼠的坐骨神经和臂丛神经,采用Ⅳ型胶原酶消化的半植块培养法获取原代大鼠许旺细胞；然后用免疫磁珠分选法获纯化许旺细胞。用S-100抗体进行免疫组织化学染色鉴定许旺细胞并计算细胞纯度。
结果取材后6-8小时可见到细胞贴壁,呈现出明显的梭形,遮光性强。免疫磁珠分选后可获得了高纯度的许旺细胞,免疫细胞化学染色见到大量的阳性细胞,阳性率达到95%以上。
结论肤酶和胶原酶联合消化法联合磁珠分选法可以获得高纯度的许旺细胞,为后续的实验提供了细胞来源。
神经生长因子基因载体的构建、转染及表达
目的构建绿色荧光蛋白标记的神经生长因子基因表达载体,并检测其表达效率。
方法采用双酶切法切取人神经生长因子基因序列,采用PCR法扩增,酶切后和载体EGFP-N1连接；感染感受态大肠杆菌后挑选单克隆菌株,提取质粒酶切鉴定证实为目的质粒后进行扩增。用Iipofectamine2000将质粒转染大鼠许旺细胞,用ELISA法检测神经生长因子表达量的变化。
结果PCR凝胶电泳结果显示切取的片段约为750bp左右,和目的基因片段大小相符合；提取质粒后双酶切显示切取片段和目的基因的大小相符合；转然后24小时在显微镜下可见到明显的绿色荧光,一周后上清液中NGF的含量较对照组明显升高。
结论成功构建出含有神经生长因子基因的EGFP-N1质粒,转染许旺细胞后能够促进NGF的表达。
高表达NGF的许旺细胞移植治疗脊髓损伤的疗效及其机制探讨
目的观察高表NG的许旺细胞移植治疗大鼠脊髓损伤的效果,并探讨其作用的可能机制。
方法用改良Allen法制备大鼠脊髓损伤模型,随机分为A、B、C、D四组,A组为转染含NGF基因的许旺细胞移植组,B组为许旺细胞移植组,C组为PBS对照组,D组为假手术组,造模一周后分别用108/ml含有转染NGF的许旺细胞的PBS液、108/m1许旺细胞的PBS液、PBS液注入A、B、C组大鼠脊髓损伤部位及其上下各5μ1。注射后一二、三、四周分别用BBB评分法观察大鼠双下肢运动功能,用肌电图检测大鼠的神经传导功能;处死后用免疫组织化学染色法检测脊髓损伤部位Bcl-2和6AP-43的表达变化。并取转染组的大鼠脊髓组织行纵行切片观察许旺细胞的迁移情况。
结果各时间点BBB评分结果显示D组>A组>B组>C组；肌电图检测结果显示中枢潜伏时传导速度A、B、C组间差别有统计学意义P<0.05,中枢波幅A、B、C组间未见明显差异(P>0.05),D组明显高于其它三组； Bc1-2和GAP-43免疫组化检测结果提示A>B>C组,D组仅有少量表达。大鼠脊髓组织纵行切片观察显示在脊髓损伤后一周损伤区有大量的绿色荧光蛋白表达,随后随着时间的延长,大鼠脊髓损伤区内的许旺细胞数量逐渐减少。
结论本研究证实NGF基因修饰的许旺细胞能够促进脊髓损伤的恢复,可能是通过抑制受损细胞的凋亡和促进神经轴突的生长有关。
Part1Purified and Identified of Schwann Cells from Rat
Object To culture and purify schwan cells of rat, which provides cells for following up experiments.
Method The sciatic and brachial plexus nerves were obtained from SD rats which were newborn3-4days. The primary schwann cells were acquried through these nerves digested by collagenase Ⅳ. The schwann cells were purified by immunomagnetic beads after cultured two generations. The S-100antibody were used to identify the schwann cells and calculate the purity by immunohistochemical staining.
Result The schwann cells were adhesioned after cultured6-8hours, showing a clear spindle. The purity of schwann cells were as high as95%indetified by immunohistochemical staining which were pueified by immunomagnetic beads.
Conclusion High purity of schwann cells were obtain through collagenase IV digestion method combined with magnetic bead sorting, providing a cell source for subsequent experiments.
Part2Construction and Expression of Retroviral Vector of pEGFP-N1/NGFβ
Object To construct retroviral vector carrying nerve growth factor (NGF) beta gene which were labelled by EGFP, and to observe its expression in rat Schwann cell (SC).
Method The NGF beta gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from pAAV-SP vector which was send by Dr Lynne Moor. By gene recombination technique, human NGF beta gene was inserted into vector pEGFP-N1. Then, we transfected the recombination plasmid into SC by lipofectamine2000, and collected the positive clones after G418selection and observed its expression under fluorescence microscope and detect the NGF concentration by ELISA.
Result The PCR production of NGF beta is about750bp. The recombinant plasmid was identified by restriction endonuclease analysis. The human NGF beta gene could be expressed successfully by SCs under fluorescence microscope and the concentration of NGF beta is higher than untransfection SCs.
Conclusion The recombination vector pEGFP-hNGFβ was construction successfully. The NGF beta gene could be efficient expressed by SC.
Part3Nerve Growth Factor Gene Modified Schwann Cells Inhabit Apoptosis and Promote Functional Recovery after Contusion Spinal Cord Injury in Sprague Dawley Rats
Object Schwann cells(SCs) which were modified by human nerve growth factor gene were injected into the injured spinal cord,to observe the functional recovery of hind limb and the expression of Bcl-2and GAP-43after spinal cord injury, and to explore the mechanism of SCs on the spinal cord injury.
Methods52SD rat were divided into four groups randomly:EGFP-N1-NGF transfected SCs transplantation group (group A)16rats,SCs transplantation group (group B)16rats, PBS control group (group C)12rats and sham group(group D)8rats. The modified Allen method was using to made spinal coed injury(SCI) model.BBB scores were observed to evaluate the behavior improvement and EMG detect the velocity of the central nerve at7d、14d、21d and28d, respectively. Then the animals were killed and the SCI areas were embedded by paraffin, tissue section and immunostaining of Bcl-2and GAP-43evaluate the Histology recovery after spinal cord injury. The migration of hNGF gene modified SCs in the group A were observed by fluorescence microscope.
Result The result of BBB scores showed that the animals in group A were higher than group B and group C, and animals in group B were higher than group C, the animals in group D were normal (P<0.05); There is significantly difference of central latency between group A、B and C, but were longer than group D; However, there were no significantly difference of central amplitude between group A、B and C. The immunostaining of Bcl-2and GAP-43showed that group A> group B> group C, the group D have little expression. The SCs in injured spinal cord were distributed in punctate, the SCs were granduly reduced as time follow.
Conclusion Our study confirmed that NGF gene modified SCs promote the recovery of spinal cord injury, probably by inhibiting the apoptosis of damaged cells and promoting the growth of the nerve axon.
引文
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