携带CTLA4Ig基因腺病毒载体对共同导入致敏大鼠脊髓的LacZ基因表达的影响
|
摘要
目的:随着社会的发展,各种创伤发生越来越多,伴随的神经系统损伤的数量也迅速的增加。但由于神经系统修复的复杂性和特殊,神经系统损伤的修复至今仍是一个非常棘手的难题。随着分子生物学的发展,对于神经系统损伤的修复采用基因治疗的方法,将外源性的神经营养因子的基因通过载体定向的导入神经断端,利用受体细胞长期的高效的分泌神经营养因子,促进神经再生轴突的生长,从而促进神经损伤的修复,以期获得良好的效果。虽然利用转基因技术为神经的损伤修复提供了一个非常好的方向,但由于受体对外源性基因及载体的免疫排斥作用,使得外源性基因的长期高效的表达不能实现,这也就限制了神经修复的效果。针对这一瓶颈,本实验采用导入带有细胞毒T淋巴细胞相关抗原4免疫球蛋白(Cytotoxic T lymphocyte-associated antigen 4 immunoglobulin, CTLA4Ig)基因腺病毒AdV(Ad CTLA4Ig)和携带LacZ基因的AdV(AdLacZ)微量注射器共同导入大鼠脊髓腰膨大处作为实验组,将未携带外源性基因的Ad0和携带LacZ基因的AdV(AdLacZ)通过微量注射器共同导入大鼠脊髓腰膨大处作为对照组,两组通过比较β-gal在脊髓表达的变化,并通过多聚酶链反应(PCR)监测腺病毒注入后在脊髓量的变化及消失时间和用逆转录多聚酶链反应(PT-PCR)法检测CTLA4Ig基因和LacZ基因在大鼠脊髓的表达,以探讨CTLA4Ig诱导机体对AdLacZ的局部免疫耐受的作用及其机理。
方法:选用7周龄健康雌性Wister大鼠54只,体重200～250g。将大鼠随机分成四组,A,B每组21只,C,D组每组6只。在往大鼠脊髓注射病毒之前7天,在胸背侧皮下注射AdLacZ 1μl进行预处理。对照组(A组、C组)导入AdlacZ+Ad0,实验组(B组、D组)导入AdlacZ+AdCTLA4Ig。将大鼠经腹腔注射氯胺酮/塞拉嗪麻醉(75-100mg/kg+5mg/kg),麻醉成功后固定于脑立体定位仪上。取长约3cm后正中切口,去除T13椎板暴露脊髓腰膨大部位。利用微量注射器及ST-III型手动推进器,于脊髓后正中动脉右侧1mm处注射AdlacZ(1×109pfu/ml)和Ad0(5×109pfu/ml)各1μl(A组、C组)或AdLacZ(1×109 pfu/ml)和AdCTLA4Ig(5×109 pfu/ml)各1μl(B组、D组)。针尖向头侧呈45度斜行进针,斜行刺入深度为2.5mm。注射速度为1μl/min,注射完毕后滞针5min,缓慢拔针。充分止血、冲洗切口,局部喷洒抗生素预防感染,关闭切口。逐层关闭切口,结束手术。自然条件下恢复清醒,相同条件下喂养进行观察。C组和D组于距第一次手术30天后于脊髓后正中动脉左侧1mm处注射AdlacZ(1×109pfu/ml)和Ad0(5×109pfu/ml)各1μl(C组)或AdLacZ(1×109 pfu/ml)和AdCTLA4Ig(5×109 pfu/ml)各1μl(D组)。其余手术方法相同。实验组和对照组在转染腺病毒后的9时间点取大鼠脊髓注射点远近端5mm的腰膨大节段,注射点头侧5mm的脊髓节段进行厚50μm的连续冰冻横切片,对实验组和对照组中X-gal染色阳性的切片进行计数,并通过多聚酶链反应(PCR)监测腺病毒注入后在脊髓量的变化及消失时间,用逆转录多聚酶链反应(PT-PCR)法检测CTLA4Ig基因和LacZ基因在大鼠脊髓的表达情况。
结果:
1 X-gal, CTLA4Ig转基因表达:LacZ基因和CTLA4Ig基因转染脊髓双侧的前角运动细胞和周围的胶质细胞。转基因表达范围局限于注射点上下各0.5cm区段,表达高峰时有转基因表达的阳性冰冻横切片数≤120片。切片观察可见实验对照组的X-gal染色阳性表达时间约为15天;而实验组的X-gal染色阳性表达时间约为60天。实验组脊髓阳性切片计数显示β-gal在脊髓腰膨大表达的高峰期均在4天和9天,15天后β-gal在脊髓腰膨大X-gal染色阳性表达量逐步减少,在60天仍能见到低表达量。统计学分析表明两组之间在高峰期间阳性切片数有显著差异(P<0.05)。并且AdLacZ基因在脊髓表达的时间明显延长。
2腺病毒液PCR检测:腺病毒的表达量随着浓度的降低而逐渐减小。采用病毒液10倍系列稀释提取后DNA经PCR反应可检测出腺病毒的特异性条带的最低稀释度为104。AdlacZ(1×109pfu/ml)与Ad0(5×109 pfu/ml)特异性条带的最低稀释度一致。
3脊髓标本腺病毒PCR检测:用PCR法从DNA水平分别检测腺病毒在实验对照组、实验组的表达情况,腺病毒的DNA量是随时间延长逐渐下降的,实验对照组至39天检测不到腺病毒的表达,实验组60天时仍能检测到腺病毒的表达。
4 RT-PCR实验的结果
4.1β-gal RNA在大鼠脊髓腰膨大处局部表达
用RT-PCR法从mRNA水平分别检测β-gal在实验对照组、实验组的表达情况:转染2天后两组均可检测出β-gal mRNA在大鼠脊髓腰膨大组织的表达,对照组在30天处未检测β-gal的目标条带,实验组在60天处仍可见到β-gal表达的目标条带,但亮度以明显变暗,可见在初次导入腺病毒及第二次导入腺病毒30天β-gal mRNA仍有低量的表达。
4.2 CTLA4Ig mRNA在大鼠脊髓腰膨大组织局部的表达,用RT-PCR法从mRNA水平分别检测CTLA4Ig mRNA在实验组的表达情况。转染2天后实验组可检测出CTLA4Ig mRNA在大鼠脊髓腰膨大组织的表达,在初次导入腺病毒的60天和第二次导入腺病毒的30天处可见到CTLA4Ig mRNA的目标条带,可见最亮处为初次导入的4天和9天处,第二次导入腺病毒的第9天和初次导入腺病毒的第9天出相比较暗。
结论:在预先致敏的大鼠体内,将AdV介导的CTLA4Ig基因注射入脊髓腰膨大处能有效地抑制局部T淋巴细胞的浸润,从而减轻由AdV介导的局部炎症反应,抑制机体对病毒载体的排斥反应,显著延长LacZ基因在脊髓的表达时间,并且能明显增强与其共同导入的LacZ基因表达强度。而且AdCTLA4Ig对体液免疫应答有明显的抑制作用,能明显延长腺病毒在体内的存留时间。
Objective: With the development of society, all kinds of trauma has occurred more and more nervous system damage associated with the number rapidly increased. but because of the complexity of the nervous system and special repair nervous system damage repair is still a very difficult problem. With the development of molecular biology, for the repair of injured nervous system using gene therapy approach, exogenous neurotrophic factor gene by vector-oriented nerve stump into the use of receptor cells and efficient secretion of the long-term neurotrophic factor, promoting the growth of nerve axons, thus promoting nerve injury repair, in order to obtain good results. Although the use of transgenic technology for nerve repair provides a very good direction, but because of exogenous receptor gene and vector role of immune rejection, making a long-term efficiency of exogenous gene expression can not be achieved, which also limits the the effect of nerve repairing. In response to this bottleneck, the present study, to import with a cytotoxic T lymphocyte-associated antigen-4 Ig (Cytotoxic T lymphocyte- associated antigen 4 immunoglobulin, CTLA4Ig) gene adenovirus AdV (Ad CTLA4Ig) and carrying LacZ gene AdV (AdLacZ ) micro-syringe into the common rat spinal cord lumbar enlargement division, as the experimental group, will not carry the exogenous gene Ad0 and carrying LacZ gene AdV (AdLacZ) through the micro-syringe into the common rat spinal cord lumbar enlargement division, as the control group, two groups of by comparing theβ-gal expression changes in the spinal cord and by polymerase chain reaction (PCR) to monitor the spinal cord after injection of adenovirus amount of change and disappearance time and the use of reverse transcription-polymerase chain reaction (PT-PCR) to detect CTLA4Ig gene and LacZ gene expression in the rat spinal cord to explore the CTLA4Ig induce local immune tolerance to the AdLacZ the role of its mechanism.
Methods: Rats were primed by subcutaneous injection of AdLacZ into the dorsal thorax 7 days before injection of virus into the Spinal Cord. 54 Wister female rats aged 7 weeks were randomly divided into 4 groups: group A, C, the AdlacZ+Ad0 transfer group and group B,D, the AdlacZ+AdCTLA4Ig transfer group. After anesthetized,the animal were fixed on the stereotaxic instrument. About 3cm length of posterior median incision was made, T13 vertebral plate was removed and the spinal cord was exposed. At the site 1.0mm right to the posterior median artery of spinal cord, 1.0μl (1×109pfu/ml )AdlacZ and 1.0μl (5×109pfu/ml) Ad0 in group A,C, or 1.0μl (1×109pfu/ml) AdlacZ and 1.0μl (5×109pfu/ml) AdCTLA4Ig in group B,D was injected with micro-injector under a propeller by the same person. The needle tip is 45℃headward and downward at the depth of 2.5mm. The injecting speed is 1μl/min. Needles were stuck 5min and withdrew slowly after the injection. The wound was rinsed, stopped bleeding thoroughly, sprinked antibiotics to and then closed. group C and group D after 30 days from the first operation after the median artery in the left side of the spinal cord 1mm injections AdlacZ (1×109pfu/ml) and Ad0 (5×109pfu/ml) of 1μl (group C) or AdLacZ (1×109 pfu / ml), and AdCTLA4Ig (5×109 pfu / ml) of 1μl (group D). The remaining operations the same way. The experimental group and control group transfected with adenovirus to take after the 9 time points near and far side of rat spinal cord injection sites 5mm segment of the lumbar enlargement, injection nodding side of the spinal segment to 5mm thick frozen cross-sections 50μm continuum, the experimental group and the control group, X-gal staining of the sections counted, and by polymerase chain reaction (PCR) to monitor the spinal cord after injection of adenovirus amount of change and disappearance time, using reverse transcription-polymerase chain reaction (PT-PCR) Detection of CTLA4Ig gene and LacZ gene expression in rat spinal cord.
Results:
1 X-gal, CTLA4Ig transgene expression: LacZ gene and CTLA4Ig gene transfection of the spinal cord anterior horn motor cells of bilateral and surrounding glial cells. Transgenic expression of the upper and lower limit the scope of the 0.5cm section of the injection site and expressed from time to time the peak of transgenic expression of the number of positive frozen cross-sections≤120 Pian. Slice the control group were observed under experimental X-gal staining positive expression of time is about 15 days; while the experimental group of X-gal staining positive expression of time is about 60 days. Count of the experimental group of spinal cord sections showed positiveβ-gal expression in the spinal cord lumbar enlargement of the peak are in four days and 9 days, 15 days after theβ-gal in the spinal cord lumbar enlargement of X-gal staining positive for expression of the gradual decrease in the 60 Days can still see the low-expression. Statistical analysis showed that between the two groups during the height of the number of positive biopsy were significantly different (P <0.05). And AdLacZ gene expression in the spinal cord significantly longer time.
2 Ad. PCR tests: the expression of adenovirus with the decrease of the concentration gradually decreased. Solution using 10-fold serial dilutions of the virus extracted DNA by PCR reaction can detect adenovirus-specific bands of the lowest dilution of 104. AdlacZ (1×109pfu/ml) and Ad0 (5×109 pfu / ml) specific bands of the lowest dilution line.
3 PCR detection of adenovirus samples of spinal cord: The PCR method from the adenovirus DNA levels were detected in the experimental control group, the expression of the experimental group, adenovirus of the DNA volume is decreasing with time, the experimental control group to 39 days detected the expression of adenovirus in the experimental group 60 days can still detect the expression of adenovirus .
4 RT-PCR results of the experiment
4.1 The expression ofβ-gal RNA in the rat lumbar spinal cord enlargement at local
Using RT-PCR method from the mRNA levels were detectedβ-gal in the experimental control group, the expression of the experimental group: 2 days after transfection of the two groups can be detected inβ-galmRNA lumbar enlargement in the rat spinal cord tissue of the control group Service was not detected in the 30-day goal ofβ-galm bands, the experimental group at 60 days, one can still see the expression ofβ-galm target band, but in order to significantly darken the brightness can be seen in the initial and secondary adenovirus-Import Import Adenovirus 30-dayβ-galmRNA is still a low level of expression.
4.2 CTLA4Ig mRNA in the rat spinal cord lumbar enlargement of local expression of the organization (see figure 20) using RT-PCR method to detect the CTLA4IgmRNA from the mRNA level of expression in the experimental group. Transfected 2 days after the experimental group could be detected CTLA4IgmRNA lumbar enlargement in the rat spinal cord tissues, in the first 60 days of adenovirus into the second import of adenovirus at 30 days of the objectives can be seen CTLA4IgmRNA bands can be seen most light and for the first time in four days and 9 days into office, the second into the first nine days of adenovirus initial import of adenovirus compared to the first 9 days out of dark.
Conclusion: In the pre-sensitized rats, the AdV-mediated CTLA4Ig gene was injected into the spinal cord lumbar enlargement Department can effectively inhibit local T lymphocyte infiltration, thereby reducing by AdV-mediated local inflammatory response, inhibit the body of the virus vector rejection, significantly increased the expression of LacZ gene in the spinal cord the time, and can significantly enhance its co-import intensity of LacZ gene expression. And AdCTLA4Ig on humoral immune response significantly inhibited, could significantly prolong adenovirus in vivo lifetime.
方法:选用7周龄健康雌性Wister大鼠54只,体重200～250g。将大鼠随机分成四组,A,B每组21只,C,D组每组6只。在往大鼠脊髓注射病毒之前7天,在胸背侧皮下注射AdLacZ 1μl进行预处理。对照组(A组、C组)导入AdlacZ+Ad0,实验组(B组、D组)导入AdlacZ+AdCTLA4Ig。将大鼠经腹腔注射氯胺酮/塞拉嗪麻醉(75-100mg/kg+5mg/kg),麻醉成功后固定于脑立体定位仪上。取长约3cm后正中切口,去除T13椎板暴露脊髓腰膨大部位。利用微量注射器及ST-III型手动推进器,于脊髓后正中动脉右侧1mm处注射AdlacZ(1×109pfu/ml)和Ad0(5×109pfu/ml)各1μl(A组、C组)或AdLacZ(1×109 pfu/ml)和AdCTLA4Ig(5×109 pfu/ml)各1μl(B组、D组)。针尖向头侧呈45度斜行进针,斜行刺入深度为2.5mm。注射速度为1μl/min,注射完毕后滞针5min,缓慢拔针。充分止血、冲洗切口,局部喷洒抗生素预防感染,关闭切口。逐层关闭切口,结束手术。自然条件下恢复清醒,相同条件下喂养进行观察。C组和D组于距第一次手术30天后于脊髓后正中动脉左侧1mm处注射AdlacZ(1×109pfu/ml)和Ad0(5×109pfu/ml)各1μl(C组)或AdLacZ(1×109 pfu/ml)和AdCTLA4Ig(5×109 pfu/ml)各1μl(D组)。其余手术方法相同。实验组和对照组在转染腺病毒后的9时间点取大鼠脊髓注射点远近端5mm的腰膨大节段,注射点头侧5mm的脊髓节段进行厚50μm的连续冰冻横切片,对实验组和对照组中X-gal染色阳性的切片进行计数,并通过多聚酶链反应(PCR)监测腺病毒注入后在脊髓量的变化及消失时间,用逆转录多聚酶链反应(PT-PCR)法检测CTLA4Ig基因和LacZ基因在大鼠脊髓的表达情况。
结果:
1 X-gal, CTLA4Ig转基因表达:LacZ基因和CTLA4Ig基因转染脊髓双侧的前角运动细胞和周围的胶质细胞。转基因表达范围局限于注射点上下各0.5cm区段,表达高峰时有转基因表达的阳性冰冻横切片数≤120片。切片观察可见实验对照组的X-gal染色阳性表达时间约为15天;而实验组的X-gal染色阳性表达时间约为60天。实验组脊髓阳性切片计数显示β-gal在脊髓腰膨大表达的高峰期均在4天和9天,15天后β-gal在脊髓腰膨大X-gal染色阳性表达量逐步减少,在60天仍能见到低表达量。统计学分析表明两组之间在高峰期间阳性切片数有显著差异(P<0.05)。并且AdLacZ基因在脊髓表达的时间明显延长。
2腺病毒液PCR检测:腺病毒的表达量随着浓度的降低而逐渐减小。采用病毒液10倍系列稀释提取后DNA经PCR反应可检测出腺病毒的特异性条带的最低稀释度为104。AdlacZ(1×109pfu/ml)与Ad0(5×109 pfu/ml)特异性条带的最低稀释度一致。
3脊髓标本腺病毒PCR检测:用PCR法从DNA水平分别检测腺病毒在实验对照组、实验组的表达情况,腺病毒的DNA量是随时间延长逐渐下降的,实验对照组至39天检测不到腺病毒的表达,实验组60天时仍能检测到腺病毒的表达。
4 RT-PCR实验的结果
4.1β-gal RNA在大鼠脊髓腰膨大处局部表达
用RT-PCR法从mRNA水平分别检测β-gal在实验对照组、实验组的表达情况:转染2天后两组均可检测出β-gal mRNA在大鼠脊髓腰膨大组织的表达,对照组在30天处未检测β-gal的目标条带,实验组在60天处仍可见到β-gal表达的目标条带,但亮度以明显变暗,可见在初次导入腺病毒及第二次导入腺病毒30天β-gal mRNA仍有低量的表达。
4.2 CTLA4Ig mRNA在大鼠脊髓腰膨大组织局部的表达,用RT-PCR法从mRNA水平分别检测CTLA4Ig mRNA在实验组的表达情况。转染2天后实验组可检测出CTLA4Ig mRNA在大鼠脊髓腰膨大组织的表达,在初次导入腺病毒的60天和第二次导入腺病毒的30天处可见到CTLA4Ig mRNA的目标条带,可见最亮处为初次导入的4天和9天处,第二次导入腺病毒的第9天和初次导入腺病毒的第9天出相比较暗。
结论:在预先致敏的大鼠体内,将AdV介导的CTLA4Ig基因注射入脊髓腰膨大处能有效地抑制局部T淋巴细胞的浸润,从而减轻由AdV介导的局部炎症反应,抑制机体对病毒载体的排斥反应,显著延长LacZ基因在脊髓的表达时间,并且能明显增强与其共同导入的LacZ基因表达强度。而且AdCTLA4Ig对体液免疫应答有明显的抑制作用,能明显延长腺病毒在体内的存留时间。
Objective: With the development of society, all kinds of trauma has occurred more and more nervous system damage associated with the number rapidly increased. but because of the complexity of the nervous system and special repair nervous system damage repair is still a very difficult problem. With the development of molecular biology, for the repair of injured nervous system using gene therapy approach, exogenous neurotrophic factor gene by vector-oriented nerve stump into the use of receptor cells and efficient secretion of the long-term neurotrophic factor, promoting the growth of nerve axons, thus promoting nerve injury repair, in order to obtain good results. Although the use of transgenic technology for nerve repair provides a very good direction, but because of exogenous receptor gene and vector role of immune rejection, making a long-term efficiency of exogenous gene expression can not be achieved, which also limits the the effect of nerve repairing. In response to this bottleneck, the present study, to import with a cytotoxic T lymphocyte-associated antigen-4 Ig (Cytotoxic T lymphocyte- associated antigen 4 immunoglobulin, CTLA4Ig) gene adenovirus AdV (Ad CTLA4Ig) and carrying LacZ gene AdV (AdLacZ ) micro-syringe into the common rat spinal cord lumbar enlargement division, as the experimental group, will not carry the exogenous gene Ad0 and carrying LacZ gene AdV (AdLacZ) through the micro-syringe into the common rat spinal cord lumbar enlargement division, as the control group, two groups of by comparing theβ-gal expression changes in the spinal cord and by polymerase chain reaction (PCR) to monitor the spinal cord after injection of adenovirus amount of change and disappearance time and the use of reverse transcription-polymerase chain reaction (PT-PCR) to detect CTLA4Ig gene and LacZ gene expression in the rat spinal cord to explore the CTLA4Ig induce local immune tolerance to the AdLacZ the role of its mechanism.
Methods: Rats were primed by subcutaneous injection of AdLacZ into the dorsal thorax 7 days before injection of virus into the Spinal Cord. 54 Wister female rats aged 7 weeks were randomly divided into 4 groups: group A, C, the AdlacZ+Ad0 transfer group and group B,D, the AdlacZ+AdCTLA4Ig transfer group. After anesthetized,the animal were fixed on the stereotaxic instrument. About 3cm length of posterior median incision was made, T13 vertebral plate was removed and the spinal cord was exposed. At the site 1.0mm right to the posterior median artery of spinal cord, 1.0μl (1×109pfu/ml )AdlacZ and 1.0μl (5×109pfu/ml) Ad0 in group A,C, or 1.0μl (1×109pfu/ml) AdlacZ and 1.0μl (5×109pfu/ml) AdCTLA4Ig in group B,D was injected with micro-injector under a propeller by the same person. The needle tip is 45℃headward and downward at the depth of 2.5mm. The injecting speed is 1μl/min. Needles were stuck 5min and withdrew slowly after the injection. The wound was rinsed, stopped bleeding thoroughly, sprinked antibiotics to and then closed. group C and group D after 30 days from the first operation after the median artery in the left side of the spinal cord 1mm injections AdlacZ (1×109pfu/ml) and Ad0 (5×109pfu/ml) of 1μl (group C) or AdLacZ (1×109 pfu / ml), and AdCTLA4Ig (5×109 pfu / ml) of 1μl (group D). The remaining operations the same way. The experimental group and control group transfected with adenovirus to take after the 9 time points near and far side of rat spinal cord injection sites 5mm segment of the lumbar enlargement, injection nodding side of the spinal segment to 5mm thick frozen cross-sections 50μm continuum, the experimental group and the control group, X-gal staining of the sections counted, and by polymerase chain reaction (PCR) to monitor the spinal cord after injection of adenovirus amount of change and disappearance time, using reverse transcription-polymerase chain reaction (PT-PCR) Detection of CTLA4Ig gene and LacZ gene expression in rat spinal cord.
Results:
1 X-gal, CTLA4Ig transgene expression: LacZ gene and CTLA4Ig gene transfection of the spinal cord anterior horn motor cells of bilateral and surrounding glial cells. Transgenic expression of the upper and lower limit the scope of the 0.5cm section of the injection site and expressed from time to time the peak of transgenic expression of the number of positive frozen cross-sections≤120 Pian. Slice the control group were observed under experimental X-gal staining positive expression of time is about 15 days; while the experimental group of X-gal staining positive expression of time is about 60 days. Count of the experimental group of spinal cord sections showed positiveβ-gal expression in the spinal cord lumbar enlargement of the peak are in four days and 9 days, 15 days after theβ-gal in the spinal cord lumbar enlargement of X-gal staining positive for expression of the gradual decrease in the 60 Days can still see the low-expression. Statistical analysis showed that between the two groups during the height of the number of positive biopsy were significantly different (P <0.05). And AdLacZ gene expression in the spinal cord significantly longer time.
2 Ad. PCR tests: the expression of adenovirus with the decrease of the concentration gradually decreased. Solution using 10-fold serial dilutions of the virus extracted DNA by PCR reaction can detect adenovirus-specific bands of the lowest dilution of 104. AdlacZ (1×109pfu/ml) and Ad0 (5×109 pfu / ml) specific bands of the lowest dilution line.
3 PCR detection of adenovirus samples of spinal cord: The PCR method from the adenovirus DNA levels were detected in the experimental control group, the expression of the experimental group, adenovirus of the DNA volume is decreasing with time, the experimental control group to 39 days detected the expression of adenovirus in the experimental group 60 days can still detect the expression of adenovirus .
4 RT-PCR results of the experiment
4.1 The expression ofβ-gal RNA in the rat lumbar spinal cord enlargement at local
Using RT-PCR method from the mRNA levels were detectedβ-gal in the experimental control group, the expression of the experimental group: 2 days after transfection of the two groups can be detected inβ-galmRNA lumbar enlargement in the rat spinal cord tissue of the control group Service was not detected in the 30-day goal ofβ-galm bands, the experimental group at 60 days, one can still see the expression ofβ-galm target band, but in order to significantly darken the brightness can be seen in the initial and secondary adenovirus-Import Import Adenovirus 30-dayβ-galmRNA is still a low level of expression.
4.2 CTLA4Ig mRNA in the rat spinal cord lumbar enlargement of local expression of the organization (see figure 20) using RT-PCR method to detect the CTLA4IgmRNA from the mRNA level of expression in the experimental group. Transfected 2 days after the experimental group could be detected CTLA4IgmRNA lumbar enlargement in the rat spinal cord tissues, in the first 60 days of adenovirus into the second import of adenovirus at 30 days of the objectives can be seen CTLA4IgmRNA bands can be seen most light and for the first time in four days and 9 days into office, the second into the first nine days of adenovirus initial import of adenovirus compared to the first 9 days out of dark.
Conclusion: In the pre-sensitized rats, the AdV-mediated CTLA4Ig gene was injected into the spinal cord lumbar enlargement Department can effectively inhibit local T lymphocyte infiltration, thereby reducing by AdV-mediated local inflammatory response, inhibit the body of the virus vector rejection, significantly increased the expression of LacZ gene in the spinal cord the time, and can significantly enhance its co-import intensity of LacZ gene expression. And AdCTLA4Ig on humoral immune response significantly inhibited, could significantly prolong adenovirus in vivo lifetime.
引文
1 Lundborg G.A 25-year perspective of peripheral nerve surgery: evolving neuroscientific concepts and clinical significance. J Hand Surg[Am]. 2000,25(3):391-414
2 Lundborg G, Longo FM, Varon S . Nerve regenerat ion model and trophic factors in vivo. Brain Res, 1982; 232:157
3 Bessis N,Garcia Cozar F J, Boissier M.C. Immune response to gene therapy vectors: influence on vector function and effector mechanisms. Gene Therapy, 2004,11 Suppll:S10-S17
4 Wang GM, Ma JB, Jin YZ, et al. Long-term survival of cardiac allografts induced by cyclophosphamide combined with CTLA4Ig-gene transfer mediated by adenoviral vector. Sheng Li Xue Bao. 2007 ,25 (1):71-78
5 Nakagawa I, Musaaki M, Ijima K, et al. Persistent and secondary adenovirus-mediated hepatic gene expression using adenovirus vector containing CTLA4IgG. Hum Gene Ther, 1998, 9(12):1739-1745
6 Saito I, Oya Y, Yamamoto K, et al. Construction of ondefective adenovirus type 5 bearing 2.8 kilobase hepatitis B virus DNA near the right end of its genome. J Virol,1985,54(3):711-719
7 Niwa H, Yamamura K, Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene, 1991,108(2):193-200
8 Roelvink PW, Mi Lee G, Einfeld DA, et al. Identification of a conserved receptor-binding site on the fiber proteins of CAR-recognizing enoviridae. Science1999,286(5444):1568-1571
9 Wickham TJ. Targeting adenovirus. Gene her,2000,7(2):110-114
10 Du L Yang, P Hou S, et al. No association of CTLA-4 polymorphisms with susceptibility to Beh?et disease. British Journal of Ophthalmology. 2009, 93(10): 1378-1381
11 Ellis J H, Burden MN, Vinogradov DV, et al. Interactions of CD80 andCD86 with CD28 and CTLA4.J Immunol,1996,156(8):2700-2709
12 McAdam, A J, Farkash, et al. B7 costimulation is critical for antibody class switching and CD8(+) cytotoxic T-lymphocyte generation in the host response to vesicular stomatitis virus. J. Virol. 2000. 74:203-208
13 Chitnis Tanuja, Najafian Nader, Abdallah, Kald A. CD28-independent induction of experimental autoimmune encephalomyelitis. The Journal of Clinical Investigation,2001, 107(5): 575-583
14 Kitajima T, Caceres-Dittmar G, Tapia FJ, et al. T cell-mediated terminal maturation of dendritic cells:loss of adhesive and phagocytotic capacities. I Immunol,1996,157(6):2340-2347
15 Kremer, Joel M, Westhovens, Rene, Leon, Marc, et al. Treatment of Rheumatoid Arthritis by Selective Inhibition of T-Cell Activation with Fusion Protein CTLA4Ig. The New England Journal of Medicin. 2003, 349(20): 1907-1915
16 Nakagawa I, Musaaki M, Ijima K, et al. Persistent and secondary adenovirus-mediated hepatic gene expression using adenovirus vector containing CTLA4IgG.Hum Gene Ther,1998,9(12):1739-1745
17王德利.腺病毒载体的研究进展.海军总医院学报,2004,12(4)227-230
18蒋国平,谢海洋,郑树森.重组CTLA4Ig腺病毒对巨噬细胞活性及移植后排斥反应的抑制作用.中国药学杂志,2005,9( 17):1352-1356
19 Nakagawa I, Musaaki M, Ijima K, et al. Persistent and secondary adenovirus-mediated hepatic gene expression using adenovirus vector containing CTLA4IgG. Hum Gene Ther, 1998, 9(12):1739-1745
20韩久卉,张英泽,陈炜.腺病毒介导的LacZ基因靶向性导入脊髓及示踪周围神经的动态过程.中国修复重建外科杂志, 2005, 19(3):215-220
1 Lundborg G. A 25-year perspective of peripheral nerve surgery: evolving neuroscientific concepts and clinical significance. J Hand Surg. 2000,25(3):391~414
2 Lundborg G. Nerve regeneration and repair. Acta Orthop Scand. 1987, 58(2):145~169
3 Lundborg G, Longo FM, Varon S. Nerve regenerat ion model and trophic factors in vivo. Brain Res. 1982, 232:157
4 Lundborg G, Longo FM, Varon S. Nerve regenerat ion model and trophic factors in vivo. Brain Res. 1982, 232:157
5李坚,郭梦和等.神经再生室桥接断离面神经的实验研究.中国临床康复. 2002, 6:1748~1749.
6 HALL S. anoxal regenernation though acellular muscle graft. J Anat. 1997,190:57~71
7崔凤国,薛玉柏.静脉桥接配合神经生长因子治疗周围混合神经损伤.中国矫形外科杂志.2000,7:1221~1222
8 Weiss P. The technology of nerve regenerat ion: sutureless tubulation and related methods of repair. Neuro surg. 1944, 1:400
9 Lundborg G, Longo FM, Varon S. Nerve regeneration model and trophic factors in vivo. Brain Res. 1982, 232:157
10 Rice DH, Burstein FD, Newman A. Use of polytetrafluor inated ethylene compound in peripheral nerve grafting. Arch Otolaryn-gol, 1985, 111:259
11 Lundborg G, Longo FM, Varon S. Nerve regeneration model and trophic factors in vivo. Brain Res.1982,232:157
12 Jeng CB, Coggeshall RE. Permeable tubes increase the length of the gap that regenerating axons can span. Brain Res.1987,408:239
13 A ebischer P, Winn SR, Valentini RF, et al. Blind ended sem- ipermeable guidance channels support peripheral nerve regeneration in the absence of a distal nerve stump. Brain Res.1988,454:179
14 MerleM, Dellon AL, Campbell JN, et al. Complication fromsilicone polymer entubulization of nerves. Microsurg. 1989,10:130
15 Tong XJ, Shimada H, M izutani Y, et al. Sciatic nerve regeneration navigated by lam in infibronectin double coated biodegradable collagen grafts in rats. Brain Res. 1994, 663:155
16 Evans DR, Brandt K, W idmerM S, et al. In vivo evaluation of poly (L -lactide acid) porous conduits for peripheral nerve regeneration. Biomaterials.1999,20 (12):1109
17 Kiyotani T, TeramachiM, Takimoto Y, et al. Nerve regeneration across a 25mm gap bridged by a polyglycolic acid-collagen tube: ahistological and electrophysiological evaluation of regenerated nerves. Brain Res.1996,740:66
18 Mack innon SE, Dellon AL. Clinical nerve reconst ruction with a bioabsorbable polyglycolic acid tube. Plast Reconstr Surg. 1990, 85 (3):419
19 Hadlock T, Elisseeff J, L anger R, et al. A tissue-engineered conduit for peripheral nerve repair. Arch Otolaryngol Head Neck Surg.1998, 124:1081
20 Meek M F, Den DunnenW F, Schakenraad JM, et al. Peripheralnerve regeneration and functional recovery after reconstruction with a thin walled biodegradable poly (DL–lactide-Εcaprolactone) nerve guide. CellsMater.1997,7(1):53
21 Rosen JM, Padilla JA, Siedman J, et al. Artificial nerve graft using glycolide trimethylene carbonate as a nerve conduit filled withcollagen compared to sutured autografte in a rat model. J RehabilRes Dev. 1992, 29 (2):1
22 Hoppen HJ, Leenslag JW, Pennings AJ. Two-ply biodegradeable nerve guide: basic aspectes of design, construction and biological performance. Biomaterials. 1990, 11:286
23 Hadlock T, Elisseeff J, Langer R, et al. A tissue-engineered conduit for peripheral nerve repair. Arch Otolaryngol Head Neck Surg, 1998, 124:1081
24 Kiyotani T, TeramachiM, Takimoto Y, et al. Nerve regeneration across a 25mm gap bridged by a polyglycolic acid-collagen tube: a histological and electrophysiological evaluation of regenerated nerves.Brain Res. 1996,740:66
25 Varon S Williams LR. Peripheral nerve regeneration in a silicone model chamber:cellcular and molecular aspects. Peripheral Never Rep Regen .1986,1:9
26 Hoppen HJ, L eenslag JW, Pennings AJ. Two-ply biodegradable nerve guide: basic aspectes of design, construction and biological performance. Biomaterials. 1990, 11:286
27 Chang AS, Yannas IV, Krarup C, et al. Conduction properties in peripheral nerve fibers regenerated by biodegradable polymermatrix. Mat Res Soc Symp Proc.1989,110:3
28 Chamberlain LJ, Yannas IV, Hsu HP, et al. Collagen-GA Gsubstrate enhances the quality of nerve regeneration through collagen tubes up to level of auto graft. Exp Neuro. 1998,154:315
29 Labrador RO, ButiM, Navarro X. Influence of collagen and lam in gels concent ration on nerve regeneration after resection and tube repair. Exp Neuro, 1998,149:243
30 Ceballos D, Navarro X, Dubey N, et al. Magnetically aligned collagen gel filling a collagen nerve guide improves peripheral nerve regeneration. Exp Neuro. 1999,158:290
31 Barde YA. Trophic factor andneuron survival. Neuron, 1989, 2(6): 1525~1534
32 Lundborg G, Longo FM, Varon S. Nerve regenerat ion model and trophic factors in vivo. Brain Res.1982, 232:157
33 Seckel BR. Enhancement of peripheral nerve regeneration. Muscle Nerve. 1990,13:785
34 Lingo FM.et al. Neuronontrphic activities accumulate in vivo within silicone nerve regeneration chambers. Brain Res. 1993,261:109
35 Varon S Williams LR. Peripheral nerve regeneration in a siliconemodel chamber: cellcular and molecular aspects. Peripheral NeverRep Regen. 1986,1:9
36 Lew in SL, Utley DS, Terris DJ, et al. Simultaneous treatment with BDNT and CNTF after peripheral nerve transaction and repair enhances rate of functional recovery compared with BDNF treatment alone. Laryngo scope. 1997,107:992
37 Varon S Williams LR. Peripheral nerve regeneration in a silicone model chamber: cellcular and molecular aspects. Peripheral Never Rep Regen.1986,1:9
38 Seckel BR. Enchancement of peripheral nerve regeneration. Muscle Nerve. 1990,13:785
39 Williams LR. spatial-temporal progress of pertheral nerve vegeneration within a silicone chamber parameters for a bioassay. J Comp Neurlo. 1983,218:460
40 Williams LR, Varon S. Modification offibrin matrix formation in situenhances nerve vegeneration in silicone chamber. J Comp Neurlo. 1985,231:209
41 Williams LR. Competences nerve tissue as distal insert prompting nerve regeneration in silicone chamber. Brain Res.1984,293:201
42宋修竹,顾玉东.神经再生室中神经再生过程和微环境的研究.中华手外科杂志.200,17(1):39-40
43 Myers RR, Heckman HM, Powell HC. Endoneurial fluid is hypertonic. Results of microanalysis and its significance in neuropathy. J Neuropath Exp Neurol. 1983,42:217-224
44 Lundborg G, Longo FM, Varon S. Nerve regeneration model and trophic factors in vivo. Brain Res. 1982, 232:157
45 Varon S. Neuronotrophic and neurite promoting factors and their clinical potentials. Dev Neurosci. 1984,6:73
46 Weiss P, Taylor AC. Further experimental evidence against neueotroposm in nerve regeneration. J Exp Zool.1944,95:233
47 Politis MJ. Tropism in nerve regeneration in vivo. Attraction of regeneration axons by diffusible factor derive from cells in distal nerve stumps of transected peripheral nerve. Brain Res. 1982,253:1
48 Lundborg G. Tissue specificity in nerve regeneration. Scand J plast Reconstr Surg.1986,20:279
49 Scaravilli F. The influence of distal envixonment on peripheral n-erve regeneration across a gap. J Neurocytol. 1984,13:1027
50 Mackinnon SE. A study of neurophisn in a primate model. J Hand surg(Am ). 1986,11:888
51 Seckel BR. Target specific nerve regeneration through a nerve guid in the rat. Plast Reconstr Surg. 1986,78:793
52 Politis MJ. Tropism in nerve regeneration in vivo. Attraction of regeneration axons by diffusible factor derive from cells in distal nerve stumps of transected peripheral nerve. Brain Res.1982,253:1
53 Brushart TM. Preferential reinnervation of motor nerves by regeneration motor axons. J Neurosci. 1988,8:1026
2 Lundborg G, Longo FM, Varon S . Nerve regenerat ion model and trophic factors in vivo. Brain Res, 1982; 232:157
3 Bessis N,Garcia Cozar F J, Boissier M.C. Immune response to gene therapy vectors: influence on vector function and effector mechanisms. Gene Therapy, 2004,11 Suppll:S10-S17
4 Wang GM, Ma JB, Jin YZ, et al. Long-term survival of cardiac allografts induced by cyclophosphamide combined with CTLA4Ig-gene transfer mediated by adenoviral vector. Sheng Li Xue Bao. 2007 ,25 (1):71-78
5 Nakagawa I, Musaaki M, Ijima K, et al. Persistent and secondary adenovirus-mediated hepatic gene expression using adenovirus vector containing CTLA4IgG. Hum Gene Ther, 1998, 9(12):1739-1745
6 Saito I, Oya Y, Yamamoto K, et al. Construction of ondefective adenovirus type 5 bearing 2.8 kilobase hepatitis B virus DNA near the right end of its genome. J Virol,1985,54(3):711-719
7 Niwa H, Yamamura K, Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene, 1991,108(2):193-200
8 Roelvink PW, Mi Lee G, Einfeld DA, et al. Identification of a conserved receptor-binding site on the fiber proteins of CAR-recognizing enoviridae. Science1999,286(5444):1568-1571
9 Wickham TJ. Targeting adenovirus. Gene her,2000,7(2):110-114
10 Du L Yang, P Hou S, et al. No association of CTLA-4 polymorphisms with susceptibility to Beh?et disease. British Journal of Ophthalmology. 2009, 93(10): 1378-1381
11 Ellis J H, Burden MN, Vinogradov DV, et al. Interactions of CD80 andCD86 with CD28 and CTLA4.J Immunol,1996,156(8):2700-2709
12 McAdam, A J, Farkash, et al. B7 costimulation is critical for antibody class switching and CD8(+) cytotoxic T-lymphocyte generation in the host response to vesicular stomatitis virus. J. Virol. 2000. 74:203-208
13 Chitnis Tanuja, Najafian Nader, Abdallah, Kald A. CD28-independent induction of experimental autoimmune encephalomyelitis. The Journal of Clinical Investigation,2001, 107(5): 575-583
14 Kitajima T, Caceres-Dittmar G, Tapia FJ, et al. T cell-mediated terminal maturation of dendritic cells:loss of adhesive and phagocytotic capacities. I Immunol,1996,157(6):2340-2347
15 Kremer, Joel M, Westhovens, Rene, Leon, Marc, et al. Treatment of Rheumatoid Arthritis by Selective Inhibition of T-Cell Activation with Fusion Protein CTLA4Ig. The New England Journal of Medicin. 2003, 349(20): 1907-1915
16 Nakagawa I, Musaaki M, Ijima K, et al. Persistent and secondary adenovirus-mediated hepatic gene expression using adenovirus vector containing CTLA4IgG.Hum Gene Ther,1998,9(12):1739-1745
17王德利.腺病毒载体的研究进展.海军总医院学报,2004,12(4)227-230
18蒋国平,谢海洋,郑树森.重组CTLA4Ig腺病毒对巨噬细胞活性及移植后排斥反应的抑制作用.中国药学杂志,2005,9( 17):1352-1356
19 Nakagawa I, Musaaki M, Ijima K, et al. Persistent and secondary adenovirus-mediated hepatic gene expression using adenovirus vector containing CTLA4IgG. Hum Gene Ther, 1998, 9(12):1739-1745
20韩久卉,张英泽,陈炜.腺病毒介导的LacZ基因靶向性导入脊髓及示踪周围神经的动态过程.中国修复重建外科杂志, 2005, 19(3):215-220
1 Lundborg G. A 25-year perspective of peripheral nerve surgery: evolving neuroscientific concepts and clinical significance. J Hand Surg. 2000,25(3):391~414
2 Lundborg G. Nerve regeneration and repair. Acta Orthop Scand. 1987, 58(2):145~169
3 Lundborg G, Longo FM, Varon S. Nerve regenerat ion model and trophic factors in vivo. Brain Res. 1982, 232:157
4 Lundborg G, Longo FM, Varon S. Nerve regenerat ion model and trophic factors in vivo. Brain Res. 1982, 232:157
5李坚,郭梦和等.神经再生室桥接断离面神经的实验研究.中国临床康复. 2002, 6:1748~1749.
6 HALL S. anoxal regenernation though acellular muscle graft. J Anat. 1997,190:57~71
7崔凤国,薛玉柏.静脉桥接配合神经生长因子治疗周围混合神经损伤.中国矫形外科杂志.2000,7:1221~1222
8 Weiss P. The technology of nerve regenerat ion: sutureless tubulation and related methods of repair. Neuro surg. 1944, 1:400
9 Lundborg G, Longo FM, Varon S. Nerve regeneration model and trophic factors in vivo. Brain Res. 1982, 232:157
10 Rice DH, Burstein FD, Newman A. Use of polytetrafluor inated ethylene compound in peripheral nerve grafting. Arch Otolaryn-gol, 1985, 111:259
11 Lundborg G, Longo FM, Varon S. Nerve regeneration model and trophic factors in vivo. Brain Res.1982,232:157
12 Jeng CB, Coggeshall RE. Permeable tubes increase the length of the gap that regenerating axons can span. Brain Res.1987,408:239
13 A ebischer P, Winn SR, Valentini RF, et al. Blind ended sem- ipermeable guidance channels support peripheral nerve regeneration in the absence of a distal nerve stump. Brain Res.1988,454:179
14 MerleM, Dellon AL, Campbell JN, et al. Complication fromsilicone polymer entubulization of nerves. Microsurg. 1989,10:130
15 Tong XJ, Shimada H, M izutani Y, et al. Sciatic nerve regeneration navigated by lam in infibronectin double coated biodegradable collagen grafts in rats. Brain Res. 1994, 663:155
16 Evans DR, Brandt K, W idmerM S, et al. In vivo evaluation of poly (L -lactide acid) porous conduits for peripheral nerve regeneration. Biomaterials.1999,20 (12):1109
17 Kiyotani T, TeramachiM, Takimoto Y, et al. Nerve regeneration across a 25mm gap bridged by a polyglycolic acid-collagen tube: ahistological and electrophysiological evaluation of regenerated nerves. Brain Res.1996,740:66
18 Mack innon SE, Dellon AL. Clinical nerve reconst ruction with a bioabsorbable polyglycolic acid tube. Plast Reconstr Surg. 1990, 85 (3):419
19 Hadlock T, Elisseeff J, L anger R, et al. A tissue-engineered conduit for peripheral nerve repair. Arch Otolaryngol Head Neck Surg.1998, 124:1081
20 Meek M F, Den DunnenW F, Schakenraad JM, et al. Peripheralnerve regeneration and functional recovery after reconstruction with a thin walled biodegradable poly (DL–lactide-Εcaprolactone) nerve guide. CellsMater.1997,7(1):53
21 Rosen JM, Padilla JA, Siedman J, et al. Artificial nerve graft using glycolide trimethylene carbonate as a nerve conduit filled withcollagen compared to sutured autografte in a rat model. J RehabilRes Dev. 1992, 29 (2):1
22 Hoppen HJ, Leenslag JW, Pennings AJ. Two-ply biodegradeable nerve guide: basic aspectes of design, construction and biological performance. Biomaterials. 1990, 11:286
23 Hadlock T, Elisseeff J, Langer R, et al. A tissue-engineered conduit for peripheral nerve repair. Arch Otolaryngol Head Neck Surg, 1998, 124:1081
24 Kiyotani T, TeramachiM, Takimoto Y, et al. Nerve regeneration across a 25mm gap bridged by a polyglycolic acid-collagen tube: a histological and electrophysiological evaluation of regenerated nerves.Brain Res. 1996,740:66
25 Varon S Williams LR. Peripheral nerve regeneration in a silicone model chamber:cellcular and molecular aspects. Peripheral Never Rep Regen .1986,1:9
26 Hoppen HJ, L eenslag JW, Pennings AJ. Two-ply biodegradable nerve guide: basic aspectes of design, construction and biological performance. Biomaterials. 1990, 11:286
27 Chang AS, Yannas IV, Krarup C, et al. Conduction properties in peripheral nerve fibers regenerated by biodegradable polymermatrix. Mat Res Soc Symp Proc.1989,110:3
28 Chamberlain LJ, Yannas IV, Hsu HP, et al. Collagen-GA Gsubstrate enhances the quality of nerve regeneration through collagen tubes up to level of auto graft. Exp Neuro. 1998,154:315
29 Labrador RO, ButiM, Navarro X. Influence of collagen and lam in gels concent ration on nerve regeneration after resection and tube repair. Exp Neuro, 1998,149:243
30 Ceballos D, Navarro X, Dubey N, et al. Magnetically aligned collagen gel filling a collagen nerve guide improves peripheral nerve regeneration. Exp Neuro. 1999,158:290
31 Barde YA. Trophic factor andneuron survival. Neuron, 1989, 2(6): 1525~1534
32 Lundborg G, Longo FM, Varon S. Nerve regenerat ion model and trophic factors in vivo. Brain Res.1982, 232:157
33 Seckel BR. Enhancement of peripheral nerve regeneration. Muscle Nerve. 1990,13:785
34 Lingo FM.et al. Neuronontrphic activities accumulate in vivo within silicone nerve regeneration chambers. Brain Res. 1993,261:109
35 Varon S Williams LR. Peripheral nerve regeneration in a siliconemodel chamber: cellcular and molecular aspects. Peripheral NeverRep Regen. 1986,1:9
36 Lew in SL, Utley DS, Terris DJ, et al. Simultaneous treatment with BDNT and CNTF after peripheral nerve transaction and repair enhances rate of functional recovery compared with BDNF treatment alone. Laryngo scope. 1997,107:992
37 Varon S Williams LR. Peripheral nerve regeneration in a silicone model chamber: cellcular and molecular aspects. Peripheral Never Rep Regen.1986,1:9
38 Seckel BR. Enchancement of peripheral nerve regeneration. Muscle Nerve. 1990,13:785
39 Williams LR. spatial-temporal progress of pertheral nerve vegeneration within a silicone chamber parameters for a bioassay. J Comp Neurlo. 1983,218:460
40 Williams LR, Varon S. Modification offibrin matrix formation in situenhances nerve vegeneration in silicone chamber. J Comp Neurlo. 1985,231:209
41 Williams LR. Competences nerve tissue as distal insert prompting nerve regeneration in silicone chamber. Brain Res.1984,293:201
42宋修竹,顾玉东.神经再生室中神经再生过程和微环境的研究.中华手外科杂志.200,17(1):39-40
43 Myers RR, Heckman HM, Powell HC. Endoneurial fluid is hypertonic. Results of microanalysis and its significance in neuropathy. J Neuropath Exp Neurol. 1983,42:217-224
44 Lundborg G, Longo FM, Varon S. Nerve regeneration model and trophic factors in vivo. Brain Res. 1982, 232:157
45 Varon S. Neuronotrophic and neurite promoting factors and their clinical potentials. Dev Neurosci. 1984,6:73
46 Weiss P, Taylor AC. Further experimental evidence against neueotroposm in nerve regeneration. J Exp Zool.1944,95:233
47 Politis MJ. Tropism in nerve regeneration in vivo. Attraction of regeneration axons by diffusible factor derive from cells in distal nerve stumps of transected peripheral nerve. Brain Res. 1982,253:1
48 Lundborg G. Tissue specificity in nerve regeneration. Scand J plast Reconstr Surg.1986,20:279
49 Scaravilli F. The influence of distal envixonment on peripheral n-erve regeneration across a gap. J Neurocytol. 1984,13:1027
50 Mackinnon SE. A study of neurophisn in a primate model. J Hand surg(Am ). 1986,11:888
51 Seckel BR. Target specific nerve regeneration through a nerve guid in the rat. Plast Reconstr Surg. 1986,78:793
52 Politis MJ. Tropism in nerve regeneration in vivo. Attraction of regeneration axons by diffusible factor derive from cells in distal nerve stumps of transected peripheral nerve. Brain Res.1982,253:1
53 Brushart TM. Preferential reinnervation of motor nerves by regeneration motor axons. J Neurosci. 1988,8:1026