过表达EGF、CNTF诱导反应性星形胶质细胞逆分化促进脊髓损伤修复的实验研究
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摘要
神经干细胞（neural stem cells, NSCs）目前被认为是治疗中枢神经系统损伤最有临床应用前景的细胞，但NSCs的来源、致瘤性以及移植后的免疫排斥反应等诸多方面的问题又面临着巨大的挑战。研究发现，在神经系统受到机械性、化学性或病理性损伤后，损伤区周围的星形胶质细胞能够表达NSCs的特异性标记物nestin，呈现神经前体细胞或NSCs的特性，并且经过体外培养可以获得NSCs，这一现象的发生机制尚未完全明确，据推测可能与星形胶质细胞周围的微环境变化引起星形胶质细胞逆分化有关。因此星形胶质细胞逆分化的特性为神经系统损伤修复提供了一条新的思路。本实验的目的是构建表皮生长因子（Epidermal GrowthFactor, EGF）、睫状神经营养因子（Ciliary Neurotrophic Factor, CNTF）的真核表达载体后，将重组载体在体外细胞模型进行共转染，观察重组载体对反应性星形胶质细胞（Reactive astrocytes, RAS）逆分化的影响，继而将重组载体共转染脊髓损伤大鼠，观察重组载体在体内环境中对RAS逆分化的影响及促进脊髓损伤修复的效果。为下一步从蛋白和基因水平研究EGF、CNTF调节RAS逆分化的分子机制打下基础。
本研究共分为三部分。在实验一中，我们将3周龄的SD大鼠脱颈处死后，在无菌、无RNA酶污染、冰冷的条件下利用Trizol试剂提取出大鼠颌下腺及坐骨神经组织总RNA。利用逆转录聚合酶链反应（RT-PCR）分别扩增出EGF及CNTF基因功能片段，电泳分析结束后回收纯化PCR产物，回收产物分别与空白质粒pSecTag2/Hygro B同时进行BamHⅠ和HindⅢ双酶切，两组酶切产物经T4DNA连接酶作用后，分别转化入感受态大肠杆菌JM109，LB琼脂平板培养过夜。经PCR初筛，两组阳性克隆分别行BamHⅠ和HindⅢ双酶切鉴定后送测序，测序结果使用BLAST软件与GeneBank数据库进行同源性分析。正确构建成功的载体命名为pSecTag2/Hygro B-EGF、pSecTag2/Hygro B-CNTF。随后将重组载体在脂质体试剂Lipofectamine2000的介导下分别单独及共转染cos-7细胞检测重组载体的表达活性。转染48h后分别收集三组细胞培养上清液，Western blot鉴定重组EGF、CNTF蛋白在cos-7细胞各组中的表达情况。结果发现：单独转染pSecTag2/HygroB-EGF、pSecTag2/hygro B-CNTF的cos-7细胞培养上清分别在6kD、22kD左右处出现阳性条带，共转染组在6kD、22kD均出现阳性条带，对照组呈阴性结果。证实重组EGF、CNTF蛋白在cos-7细胞中共表达成功。
在实验二中，我们首先在体外分离培养出大鼠脊髓组织的星形胶质细胞，并在体外用细胞划痕的方法建立RAS细胞模型，经免疫荧光染色发现细胞呈GFAP和nestin表达双阳性，说明星形胶质细胞在受到刺激后发生了逆分化，表达神经干细胞的特异性蛋白，这与前期其他学者的研究结果相一致。接着，我们将构建成功的真核表达载体pSecTag2/Hygro B-EGF、pSecTag2/Hygro B-CNTF共转染RAS，在RAS中过表达重组EGF、CNTF蛋白，发现随着共转染时间的推移，RAS表达nestin的强度不断增强，培养到第6d，视野中基本都是GFAP和nestin双阳性细胞，nestin的表达强度也有显著提高。而共转染真核表达载体的正常星形胶质细胞未出现nestin阳性细胞。转染空白质粒的RAS仅有少量的GFAP阳性细胞表达nestin，且表达强度相对较弱。并且对nestin的表达进行半定量分析发现，未损伤的正常星形胶质细胞没有明显的表达nestin。转染空白质粒的RAS的nestin表达强度相对较弱。而共转染第2~6d，nestin的表达强度出现逐渐上升的趋势，在第6d时，nestin的表达量达到最高。以转染空白质粒的RAS相对光密度值为1，转染第2d的相对光密度值为1.03±0.13，第4d为1.14±0.16，第6d为1.23±0.27。这些结果提示将重组EGF、CNTF真核表达载体在体外共转染RAS的细胞模型，能够上调RAS的nestin表达，促进RAS的逆分化过程，但是其具体分子机制有待进一步研究。
在实验三中，我们将4月龄SPF级SD大鼠随机分为EGF转染组（A组），CNTF转染组（B组），CNTF与EGF共转染组（C组）和对照组（D组）4组，每组24只。采用改良Allen’s法制备大鼠脊髓损伤模型后，应用Alzet微渗透压泵分别向A、B、C组和D组持续转染重组EGF与CNTF质粒和空白质粒各5μg（A组单独EGF转染，B组单独CNTF转染，C组EGF与CNTF共转染，D组为空白质粒）。在术后1、2、4、6周采用BBB运动功能评分系统，斜板运动试验检测大鼠运动功能恢复情况；采用RT-PCR和Western blot方法检测EGF与CNTF基因的表达；采用免疫荧光双标染色和组织学观察EGF与CNTF对反应性星形胶质细胞增殖分化的影响以及脊髓组织病理变化情况。结果发现，术后2、4、6周运动功能评分C组BBB评分明显高于其余三组，差异有统计学意义（P<0.05）。RT-PCR和Western blot方法检测显示A组大鼠脊髓组织可见EGF，B组可见CNTF mRNA与蛋白的表达，C组可见共同表达，而D组则未见明显表达。组织学观察示C组脊髓结构以及神经元形态恢复较好且逐渐趋于正常，且免疫荧光双标染色示C组脊髓组织中Nestin阳性的星形胶质细胞数较对照组明显增多，差异有统计学意义（P<0.05）。这些结果提示重组EGF、CNTF能够在体内环境中通过促进RAS的逆分化从而促进脊髓损伤大鼠神经功能的恢复。
综上所述，在体外RAS细胞模型中过表达EGF和CNTF蛋白能够促进RAS的逆分化过程。在脊髓损伤大鼠中过表达EGF和CNTF蛋白能够促进RAS逆分化过程并且促进脊髓损伤大鼠神经功能的恢复。
Neural stem cells (NSCs) are regards as a perspective candidate for neural treatment inclinical therapies for central nerve system injury and neurological discorders.Unfortunately, NSCs sources supply, tumor formation and persistence of pronouncedimmue response make this technique face great challenge for its therapeutic applications.Astrocytes within injured central nerve system region resulting from mechanical,chemical and pathological unjury may undergo a process of de-differentiation, thesede-differentiated astrocytes express nestin, the marker of neural progenitor cells, orsometimes possess neural precursor/stem cells characteristics. How the astrocytes makede-differentiation following injury is largely unclear. It presumption that astrocytesde-differentiation may correlate to intimate microenviroment around them. The data invitro shows astrocytes from injured spinal cord may acquire NSCs in culture conditions,the approved viewpoint is of astrocytes de-differentiation profiles. The property ofastrocytes de-differentiation offers a potential useful method for treatment of centralnerve system injury. The purpose of this experiment is to bulid the eukaryoticexpression vector of epidermal growth factor (EGF) and ciliary neurotrophic factor(CNTF). The recombinant vectors were co-transfected into reactive astrocytes (RAS) toobserve its effect on RAS de-differentiation in vitro. Then the recombinant vectors wereco-transfected with a spinal cord injury in rats, to observed its effect on RASde-differentiation in vivo and promote the repair of spinal cord injury.
The study is divided in three parts. In the first experiment, a SD rat aged3weeks was selected and killed by dislocation of neck, the submandibular gland and sciatic nervewere quickly taken out under cold condition, then dissected the submandibular glandand sciatic nerve, after that, the total RNA of the submandibular gland and sciatic nervewas extracted under the condition without RNA enzyme contamination with Trizolreagents. Using revers transcription PCR, the cDNA fragments of EGF and CNTF geneswere amplified from total RNAs respectively. After the end of electrophoretic analysis,the purified PCR products were collected. Then the amplified fragments wererespectively inserted into eukaryotic expression vector pSecTag2/Hygro B to constructthe recombined plasmid that encoded EGF and CNTF cDNA. The plasmids carryingEGF and CNTF genes were transfected alone respectively into competence JM109E.coli. After preliminary screening by PCR, the two positive clone were digested bydouble restriction enzyms BamH Ⅰ and Hind Ⅰ. The vectors which build correctly werenamed pSecTag2/Hygro B-EGF and pSecTag2/Hygro B-CNTF. The purified plasmidscarrying EGF and CNTF genes were transfected alone respectively or cotransfected intocos-7cells by liposome method. After transfecting48hours, collecting cell culturemedium and then the expression proteins were detected by Western blot. The resultsshowed that, a specific positive band was detected in6kD in signal transfectionpSecTag2/Hygro B-EGF group, a specific positive band was detected in22kD in signaltransfection pSecTag2/Hygro B-CNTF group, co-transfected group in both6kD and22kD, the control group showed a negative result. These results confirmed that therecombinanted EGF and CNTF protein were correctly expression in cos-7cells.
In the second experiment, we first isolated and cultured rat spinal cord astrocytes invitro, then established the RAS cell model by scratch method. The immunofluorescencestaining showed that the RAS GFAP and nestin expression in double positive, thisconfirmed that astrocytes ungo de-differentiation by stimulation, and expression ofneural stem cell specific protein, which in consistent with the preliminary findings of other reserachs. Then, we on-transfected the successful constracted eukaryoticexpression vector pSecTag2/Hygro B-EGF and pSecTag2/Hygro B-CNTF into RAS tooverexpression recombinant EGF and CNTF proteins. As time goes on, RAS expressnestin constantly enhance the strength, at6d, the field of view are basically GFAP andnestin double positive cells, the expression of nestin also significantly enhanced. But thenormal astrocytes do not appear nestin positive cells with co-transfection eukaryoticexpression vectors. The RAS which transfected with blank plasmid only a small numberof GFAP-positive cells express nestin, and the expression intensity is relatively weak.And the semiquantitative analysis found that no significant expression of nestin innoramal astrocytes. The RAS transfected with blank plasmind expression nestin isrelatively weak. From2to6day, the nestin expression intensity gradually rising inco-transfection group, at the6th day, the nestin expression reach the highest. Set therelative optical density value of1in blank plasmid transfection group, at2day therelative optical density value is1.03±0.13, the4d is1.14±0.16, the6d is1.23±0.27.These results suggest that the recombinant EGF and CNTF eukaryotic expression vectorwere co-transfected the RAS cell model in vitro, that can up-regulated nestin expressionin RAS, and promote RAS de-differentiation, but the specific molecular mechanismneeds further research.
In the third experiment, Ninety-six rats were randomly divided into four groups, andeach group contained24rats. The rats in EGF uni-transfection group (group A), CNTFuni-transfection group (group B), EGF and CNTF co-transfection group (group C) andcontrol group (group D). The rats received a spinal cord contusive injury at T10levelusing modified Allen’s method. EGF and CNTF recombinant plasmid and blankplasmid were transfected into the damaged areas of exprimental group and controlgroup respectively by Alzet pumps. At1,2,4,6weeks, Basso-Beattle-Bresnahan (BBB)Rating Scale was used to observe the recovery of motor function. The expressions of EGF and CNTF DNA and protein in injured spinal cord were detected by RT-PCR andWestern blot techniques. And double immunofluorescence and histopathologicexaminations were performed to study the proliferation and differentiation of thereactive astrocyte and pathological change after SCI. The results showed that, At2,4,6weeks after SCI, the BBB scores in the group C was significantly higher than that inother three groups (P<0.05). RT-PCR and Western blot showed that the DNA andprotein expressions of EGF and CNTF were observed in group A, B, C and noexpression was seen in group D. Histologic observation showed that the morphology ofspinal cord and neurons in group C was better than that in the other three groups andwas close to the normal tissue. The number of Nestin+astrocytes in group C wassignificantly more than that in other three groups (P<0.05). These results suggested thatthe recombinant EGF and CNTF in vivo environment by promoting the RASde-differentiation neurological function recovery in rats with spinal cord injury.
In summary, overexpression of EGF and CNTF protein in vitro model of RAS cells cancontribute to the process of de-differentiation of the RAS. Overexpression of EGF andCNTF protein can promote RAS de-differentiation and promotion of spinal cord injuryin rats recovery of neurological function in spinal cord injury in rats.
本研究共分为三部分。在实验一中，我们将3周龄的SD大鼠脱颈处死后，在无菌、无RNA酶污染、冰冷的条件下利用Trizol试剂提取出大鼠颌下腺及坐骨神经组织总RNA。利用逆转录聚合酶链反应（RT-PCR）分别扩增出EGF及CNTF基因功能片段，电泳分析结束后回收纯化PCR产物，回收产物分别与空白质粒pSecTag2/Hygro B同时进行BamHⅠ和HindⅢ双酶切，两组酶切产物经T4DNA连接酶作用后，分别转化入感受态大肠杆菌JM109，LB琼脂平板培养过夜。经PCR初筛，两组阳性克隆分别行BamHⅠ和HindⅢ双酶切鉴定后送测序，测序结果使用BLAST软件与GeneBank数据库进行同源性分析。正确构建成功的载体命名为pSecTag2/Hygro B-EGF、pSecTag2/Hygro B-CNTF。随后将重组载体在脂质体试剂Lipofectamine2000的介导下分别单独及共转染cos-7细胞检测重组载体的表达活性。转染48h后分别收集三组细胞培养上清液，Western blot鉴定重组EGF、CNTF蛋白在cos-7细胞各组中的表达情况。结果发现：单独转染pSecTag2/HygroB-EGF、pSecTag2/hygro B-CNTF的cos-7细胞培养上清分别在6kD、22kD左右处出现阳性条带，共转染组在6kD、22kD均出现阳性条带，对照组呈阴性结果。证实重组EGF、CNTF蛋白在cos-7细胞中共表达成功。
在实验二中，我们首先在体外分离培养出大鼠脊髓组织的星形胶质细胞，并在体外用细胞划痕的方法建立RAS细胞模型，经免疫荧光染色发现细胞呈GFAP和nestin表达双阳性，说明星形胶质细胞在受到刺激后发生了逆分化，表达神经干细胞的特异性蛋白，这与前期其他学者的研究结果相一致。接着，我们将构建成功的真核表达载体pSecTag2/Hygro B-EGF、pSecTag2/Hygro B-CNTF共转染RAS，在RAS中过表达重组EGF、CNTF蛋白，发现随着共转染时间的推移，RAS表达nestin的强度不断增强，培养到第6d，视野中基本都是GFAP和nestin双阳性细胞，nestin的表达强度也有显著提高。而共转染真核表达载体的正常星形胶质细胞未出现nestin阳性细胞。转染空白质粒的RAS仅有少量的GFAP阳性细胞表达nestin，且表达强度相对较弱。并且对nestin的表达进行半定量分析发现，未损伤的正常星形胶质细胞没有明显的表达nestin。转染空白质粒的RAS的nestin表达强度相对较弱。而共转染第2~6d，nestin的表达强度出现逐渐上升的趋势，在第6d时，nestin的表达量达到最高。以转染空白质粒的RAS相对光密度值为1，转染第2d的相对光密度值为1.03±0.13，第4d为1.14±0.16，第6d为1.23±0.27。这些结果提示将重组EGF、CNTF真核表达载体在体外共转染RAS的细胞模型，能够上调RAS的nestin表达，促进RAS的逆分化过程，但是其具体分子机制有待进一步研究。
在实验三中，我们将4月龄SPF级SD大鼠随机分为EGF转染组（A组），CNTF转染组（B组），CNTF与EGF共转染组（C组）和对照组（D组）4组，每组24只。采用改良Allen’s法制备大鼠脊髓损伤模型后，应用Alzet微渗透压泵分别向A、B、C组和D组持续转染重组EGF与CNTF质粒和空白质粒各5μg（A组单独EGF转染，B组单独CNTF转染，C组EGF与CNTF共转染，D组为空白质粒）。在术后1、2、4、6周采用BBB运动功能评分系统，斜板运动试验检测大鼠运动功能恢复情况；采用RT-PCR和Western blot方法检测EGF与CNTF基因的表达；采用免疫荧光双标染色和组织学观察EGF与CNTF对反应性星形胶质细胞增殖分化的影响以及脊髓组织病理变化情况。结果发现，术后2、4、6周运动功能评分C组BBB评分明显高于其余三组，差异有统计学意义（P<0.05）。RT-PCR和Western blot方法检测显示A组大鼠脊髓组织可见EGF，B组可见CNTF mRNA与蛋白的表达，C组可见共同表达，而D组则未见明显表达。组织学观察示C组脊髓结构以及神经元形态恢复较好且逐渐趋于正常，且免疫荧光双标染色示C组脊髓组织中Nestin阳性的星形胶质细胞数较对照组明显增多，差异有统计学意义（P<0.05）。这些结果提示重组EGF、CNTF能够在体内环境中通过促进RAS的逆分化从而促进脊髓损伤大鼠神经功能的恢复。
综上所述，在体外RAS细胞模型中过表达EGF和CNTF蛋白能够促进RAS的逆分化过程。在脊髓损伤大鼠中过表达EGF和CNTF蛋白能够促进RAS逆分化过程并且促进脊髓损伤大鼠神经功能的恢复。
Neural stem cells (NSCs) are regards as a perspective candidate for neural treatment inclinical therapies for central nerve system injury and neurological discorders.Unfortunately, NSCs sources supply, tumor formation and persistence of pronouncedimmue response make this technique face great challenge for its therapeutic applications.Astrocytes within injured central nerve system region resulting from mechanical,chemical and pathological unjury may undergo a process of de-differentiation, thesede-differentiated astrocytes express nestin, the marker of neural progenitor cells, orsometimes possess neural precursor/stem cells characteristics. How the astrocytes makede-differentiation following injury is largely unclear. It presumption that astrocytesde-differentiation may correlate to intimate microenviroment around them. The data invitro shows astrocytes from injured spinal cord may acquire NSCs in culture conditions,the approved viewpoint is of astrocytes de-differentiation profiles. The property ofastrocytes de-differentiation offers a potential useful method for treatment of centralnerve system injury. The purpose of this experiment is to bulid the eukaryoticexpression vector of epidermal growth factor (EGF) and ciliary neurotrophic factor(CNTF). The recombinant vectors were co-transfected into reactive astrocytes (RAS) toobserve its effect on RAS de-differentiation in vitro. Then the recombinant vectors wereco-transfected with a spinal cord injury in rats, to observed its effect on RASde-differentiation in vivo and promote the repair of spinal cord injury.
The study is divided in three parts. In the first experiment, a SD rat aged3weeks was selected and killed by dislocation of neck, the submandibular gland and sciatic nervewere quickly taken out under cold condition, then dissected the submandibular glandand sciatic nerve, after that, the total RNA of the submandibular gland and sciatic nervewas extracted under the condition without RNA enzyme contamination with Trizolreagents. Using revers transcription PCR, the cDNA fragments of EGF and CNTF geneswere amplified from total RNAs respectively. After the end of electrophoretic analysis,the purified PCR products were collected. Then the amplified fragments wererespectively inserted into eukaryotic expression vector pSecTag2/Hygro B to constructthe recombined plasmid that encoded EGF and CNTF cDNA. The plasmids carryingEGF and CNTF genes were transfected alone respectively into competence JM109E.coli. After preliminary screening by PCR, the two positive clone were digested bydouble restriction enzyms BamH Ⅰ and Hind Ⅰ. The vectors which build correctly werenamed pSecTag2/Hygro B-EGF and pSecTag2/Hygro B-CNTF. The purified plasmidscarrying EGF and CNTF genes were transfected alone respectively or cotransfected intocos-7cells by liposome method. After transfecting48hours, collecting cell culturemedium and then the expression proteins were detected by Western blot. The resultsshowed that, a specific positive band was detected in6kD in signal transfectionpSecTag2/Hygro B-EGF group, a specific positive band was detected in22kD in signaltransfection pSecTag2/Hygro B-CNTF group, co-transfected group in both6kD and22kD, the control group showed a negative result. These results confirmed that therecombinanted EGF and CNTF protein were correctly expression in cos-7cells.
In the second experiment, we first isolated and cultured rat spinal cord astrocytes invitro, then established the RAS cell model by scratch method. The immunofluorescencestaining showed that the RAS GFAP and nestin expression in double positive, thisconfirmed that astrocytes ungo de-differentiation by stimulation, and expression ofneural stem cell specific protein, which in consistent with the preliminary findings of other reserachs. Then, we on-transfected the successful constracted eukaryoticexpression vector pSecTag2/Hygro B-EGF and pSecTag2/Hygro B-CNTF into RAS tooverexpression recombinant EGF and CNTF proteins. As time goes on, RAS expressnestin constantly enhance the strength, at6d, the field of view are basically GFAP andnestin double positive cells, the expression of nestin also significantly enhanced. But thenormal astrocytes do not appear nestin positive cells with co-transfection eukaryoticexpression vectors. The RAS which transfected with blank plasmid only a small numberof GFAP-positive cells express nestin, and the expression intensity is relatively weak.And the semiquantitative analysis found that no significant expression of nestin innoramal astrocytes. The RAS transfected with blank plasmind expression nestin isrelatively weak. From2to6day, the nestin expression intensity gradually rising inco-transfection group, at the6th day, the nestin expression reach the highest. Set therelative optical density value of1in blank plasmid transfection group, at2day therelative optical density value is1.03±0.13, the4d is1.14±0.16, the6d is1.23±0.27.These results suggest that the recombinant EGF and CNTF eukaryotic expression vectorwere co-transfected the RAS cell model in vitro, that can up-regulated nestin expressionin RAS, and promote RAS de-differentiation, but the specific molecular mechanismneeds further research.
In the third experiment, Ninety-six rats were randomly divided into four groups, andeach group contained24rats. The rats in EGF uni-transfection group (group A), CNTFuni-transfection group (group B), EGF and CNTF co-transfection group (group C) andcontrol group (group D). The rats received a spinal cord contusive injury at T10levelusing modified Allen’s method. EGF and CNTF recombinant plasmid and blankplasmid were transfected into the damaged areas of exprimental group and controlgroup respectively by Alzet pumps. At1,2,4,6weeks, Basso-Beattle-Bresnahan (BBB)Rating Scale was used to observe the recovery of motor function. The expressions of EGF and CNTF DNA and protein in injured spinal cord were detected by RT-PCR andWestern blot techniques. And double immunofluorescence and histopathologicexaminations were performed to study the proliferation and differentiation of thereactive astrocyte and pathological change after SCI. The results showed that, At2,4,6weeks after SCI, the BBB scores in the group C was significantly higher than that inother three groups (P<0.05). RT-PCR and Western blot showed that the DNA andprotein expressions of EGF and CNTF were observed in group A, B, C and noexpression was seen in group D. Histologic observation showed that the morphology ofspinal cord and neurons in group C was better than that in the other three groups andwas close to the normal tissue. The number of Nestin+astrocytes in group C wassignificantly more than that in other three groups (P<0.05). These results suggested thatthe recombinant EGF and CNTF in vivo environment by promoting the RASde-differentiation neurological function recovery in rats with spinal cord injury.
In summary, overexpression of EGF and CNTF protein in vitro model of RAS cells cancontribute to the process of de-differentiation of the RAS. Overexpression of EGF andCNTF protein can promote RAS de-differentiation and promotion of spinal cord injuryin rats recovery of neurological function in spinal cord injury in rats.
引文
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