低氧特异性重组腺相关病毒VEGF165的构建与表达

目的构建VEGF165低氧启动rAAV载体,用于缺血性脑血管病的治疗.方法通过分子生物学的方法,构建VEGF165低氧启动rAAV载体,采用磷酸钙和氯仿-PEG8000/NaCl-氯仿法构建低氧启动rAAV病毒.通过体内外低氧诱导,验证VEGF165低氧启动rAAV对低氧的特异性.结果体内外实验证实,VEGF165低氧启动rAAV仅在低氧诱导后表达具有生物活性的VEGF蛋白,对低氧具有特异性.结论VEGF165低氧启动rAAV构建成功,可望用于缺血性疾病的基因治疗.     

降纤酶对脊髓损伤后VEGF的表达及细胞凋亡的作用

目的 观察降纤酶对大鼠脊髓损伤后血管内皮生长因子(VEGF)的表达及细胞凋亡的影响.方法 SD大鼠随机分为假手术组、对照组、干预组,每组各30只.各组分别于伤后各个时间点进行运动功能(BBB)评分,并随机处死5只,损伤部位取材进行HE染色和免疫组化染色,在光镜下观察、计算VEGF阳性细胞数及凋亡细胞数.结果 假手术组的BBB评分为21分,对照组、干预组在各时间点评分明显降低,干预组在5d及之后的各时间点BBB评分明显高于对照组(P＜0.05),尤其7d、14 d、28 d时间点两组有显著性差异(P＜0.01).脊髓损伤后对照组VEGF在脊髓损伤及损伤周边区高表达,5d达高峰,7d、14d表达仍较明显,28 d见少量表达.干预组各时间点VEGF的表达和对照组相比明显增加(P＜0.05),其中3d、5d、7d、14 d时间点差异显著(P＜0.01).凋亡细胞在脊髓损伤后逐渐增多,1d达高峰,此后逐渐减少;干预组各个时间点凋亡细胞数较对照组明显减少(P＜0.05),其中3d、5d、7d时间点差异显著(P＜0.01).相关性分析发现对照组1～5d时间点VEGF阳性细胞数和细胞凋亡数呈负相关(r=-0.90052,P＜0.05).结论 降纤酶可能通过促进VEGF的表达减少细胞凋亡,从而对急性脊髓损伤起治疗作用.     

腺相关病毒介导人血管内皮生长因子165基因转染后人骨髓基质细胞的成骨分化

背景：现阶段诱导骨髓基质细胞分化成骨的方法大致分为在化学药物作用、细胞因子作用以及转基因作用下的分化等。 目的：观察腺相关病毒介导人血管内皮生长因子165基因转染对人骨髓基质细胞成骨能力的影响。 设计、时间及地点：细胞学体外观察，于2008-03/12在郧阳医学院附属人民医院临床研究所和骨关节科完成。 材料：人骨髓基质细胞取自健康成年男性自愿者。人血管内皮生长因子165基因重组腺相关病毒、β半乳糖酐酶基因重组复制缺陷性腺相关病毒由课题组前期构建。 方法：抽取健康志愿者髂前上棘骨髓，采用全骨髓法分离培养人骨髓基质细胞。体外扩增纯化后取50%融合的P2代细胞，随机分为4组：腺相关病毒介导人血管内皮生长因子组向细胞培养液中加入人血管内皮生长因子165基因重组腺相关病毒1×1010 OPU/mL，孵育24 h后换为普通完全培养液继续培养。β半乳糖酐酶基因重组腺相关病毒组加入半乳糖酐酶基因重组复制缺陷性腺相关病毒，其余处理与前组相同。阳性对照组向培养液中添加地塞米松、抗坏血酸和β-甘油磷酸钠。空白对照组仅加入普通完全培养液，不进行特殊处理。 主要观察指标：全自动生化分析仪检测培养上清碱性磷酸酶活性，免疫组化染色观察血管内皮生长因子的表达，Von Kossa染色检测人骨髓基质细胞中骨胶原结节形成。 结果：处理后2周，腺相关病毒介导人血管内皮生长因子组与阳性对照组的培养上清碱性磷酸酶活性基本相似(P > 0.05)，而β半乳糖酐酶基因重组腺相关病毒组、空白对照组的培养上清碱性磷酸酶活性均明显降低(P < 0.01)。血管内皮生长因子主要在骨髓基质细胞的胞浆内表达，腺相关病毒介导人血管内皮生长因子组阳性细胞数明显多于其他3组。腺相关病毒介导人血管内皮生长因子组和阳性对照组可见大量红色骨胶原结节形成，明显多于β半乳糖酐酶基因重组腺相关病毒组、空白对照组。 结论：基因重组腺相关病毒人血管内皮生长因子165转染对人骨髓基质细胞成骨能力具有促进作用。     

外源性血管内皮生长因子对大鼠损伤脊髓神经元的作用

目的研究外源性血管内皮生长因子(VEGF)局部应用对大鼠脊髓损伤(SCI)后脊髓灰质神经元的作用。方法采用改良Allen氏重量打击法建立大鼠急性SCI模型,将72只模型大鼠随机分为VEGF治疗组〔人工基底膜(Matrigel蛋白胶)10μL+重组人VEGF1650.2μg〕、基底膜对照组(Matrigel蛋白胶10μL)和空白对照组(生理盐水10μL)3组,分别经蛛网膜下腔给药。于SCI后1、2、3、4周分别观察模型鼠脊髓横切面灰质神经元数量、神经元截面积和尼氏体密度恢复情况。结果 SCI后1~4周VEGF治疗组脊髓横切面灰质神经元数量(分别为76.34±6.25、108.25±11.74、144.33±13.22、165.96±18.11)和神经元截面积〔分别为(177.87±14.42)(、229.63±21.74)(、340.55±32.66)(、390.30±46.19)μm2〕均高于同期基底膜对照组〔神经元数量分别为40.89±7.20、50.61±13.11、59.78±9.46、80.13±10.84,神经元截面积分别为(108.98±15.03)(、148.53±18.38)、(163.22±16.77)(、208.87±29.58)μm2〕和空白对照组〔神经元数量分别为37.66±6.69、52.36±5.17、60.58±9.13、78.56±13.56,神经元截面积分别为(114.55±14.22)(、139.24±15.17)、(157.11±20.26)(、185.23±21.65)μm2〕(P<0.01,P<0.05)。SCI后1周时VEGF治疗组神经元尼氏体密度(0.2089±0.0210)与基底膜对照组(0.2012±0.0145)、空白对照组(0.1982±0.0106)比较差异均无统计学意义(P>0.05),SCI后2、3、4周,VEGF治疗组尼氏体密度(分别为0.2489±0.0217、0.2908±0.0327、0.3508±0.0362)亦大于基底膜对照组(分别为0.2198±0.0214、0.2406±0.0198、0.2605±0.0322)和空白对照组(分别为0.2044±0.0231、0.2356±0.0311、0.2582±0.0245)(均P<0.05)。两对照组各指标均无统计学差异。结论局部应用外源性VEGF可能对SCI后大鼠脊髓灰质神经元具有保护作用。     

高压氧对大鼠脊髓损伤后血管内皮生长因子表达的影响

目的通过观察高压氧对成年大鼠脊髓损伤后不同时期血管内皮生长因子（VEGF）表达变化的影响,探讨高压氧对脊髓损伤的治疗作用机制。方法雌性SD大鼠55只,体质量250-300g。随机分为脊髓损伤组（对照组）、脊髓损伤＋高压氧治疗组各25只（n=5）,假手术组5只。采用改良A llen＇s法制作T8脊髓打击伤动物模型,各组分别于伤后1 d、1 w、2 w、4 w和8 w进行行为学观察,运动功能评分方法（BBB）评分后损伤部位取材,免疫组化染色及图像分析检测组织中VEGF的表达。结果假手术组的评分21分,治疗组和对照组与假手术组比较在各时间点评分明显降低,治疗组在各时间段BBB评分明显高于对照组（P〈0.05）。假手术组显示除脊髓中央管周围可见到极少量VEGF表达外,其它部位几乎不表达;对照组脊髓损伤1 d VEGF在大鼠脊髓损伤区及损伤周边区高表达,主要出现在软脊膜和脊髓灰、白质血管壁上的血管内皮细胞胞浆内,脊髓灰、白质内的神经胶质细胞和巨噬细胞胞浆内也出现表达,一般脊髓白质中更为多见,1 w后下调,而治疗组1 d至2 wVEGF的表达较对照组明显增加,差异有显著性（P〈0.05）,4 w、8 w时各组比较无统计学差异。结论急性脊髓损伤可上调VEGF在脊髓血管内皮细胞、胶质细胞和巨噬细胞内的表达,高压氧可能通过促进VEGF表达,促进血管内皮细胞生长从而对脊髓损伤起到治疗作用。     

细胞移植治疗脊髓损伤的最新进展

一般认为脊髓损伤(spinal cord injury,SCI)造成患者局部甚至全身的瘫痪.大小便失禁和损伤平面以下的感觉消失等功能缺失是永久性的.     

重组腺相关病毒对中枢神经系统特异性转染的基础研究

中枢神经系统(CNS)疾病的基因治疗研究中，基因转染载体的选择非常重要。在现有的病毒载体中，重组腺相关病毒(recombined adeno—associated virus，rAAV)安全性好，无致突变性和免疫原性，可以重复多次使用；同腺病毒相比，rAAV的弥散性更强；同单纯疱疹病毒相比，rAAV无神经毒性；rAAV     

重组腺相关病毒载体在中枢神经系统转基因研究中的应用

重组腺相关病毒（recombinant adeno-associated virus,rAAV）作为转基因载体具有无神经毒性和低免疫原性等优点,因此在基因治疗的基础研究和临床应用领域倍受重视.在中枢神经系统（central nervous system,CNS）,以rAAV为载体已经成功转导了脑和脊髓的多个部位.随着国内外对rAAV载体研究的日趋深入,其优势已为学术界所公认,现已应用于数项CNS疾病临床前期的基因治疗,其有可能成为转基因治疗CNS疾病的最佳载体.     

携带cdc2-siRNA重组腺相关病毒的包装

背景：细胞分裂周期2(cell division cycle 2, cdc2)在神经变性疾病如阿尔茨海默病的神经元变性过程中扮演了重要角色。以腺相关病毒为载体对cdc2基因进行沉默,可能对神经变性疾病的神经元起保护作用。 目的：包装携带cdc2-siRNA的重组腺相关病毒(recombinant adeno-associated virus, rAAV)。 设计、时间及地点：空白对照实验,于2007-10/2008-08在武汉同济医院神经科实验室完成。 材料：Helper Free 腺相关病毒系统(pAAV-MCS-EGFP、p-RC、p-Helper)及AAV-293细胞为Stratagene公司产品。 方法：应用分子生物学方法构建生成pAAV-MCS-U6-cdc2-siRNA-EGFP表达质粒;用磷酸钙法将该质粒和p-RC、p-Helper质粒共转染AAV-293细胞,包装生成携带cdc2-siRNA的重组腺相关病毒 (rAAV-U6-cdc2-siRNA-EGFP)。病毒感染AAV-293细胞后行Western Blot 检测cdc2-siRNA对细胞内cdc2基因的沉默效果,并用斑点杂交方法测定该病毒的滴度。 主要观察指标：pAAV-MCS-U6-cdc2-siRNA-EGFP质粒中插入片段U6-cdc2-siRNA的测序;3质粒pAAV- MCS-U6-cdc2-siRNA-EGFP、p-RC、p-Helper共转染AAV-293细胞; rAAV-U6-cdc2-siRNA-EGFP感染AAV-293细胞后cdc2的表达。 结果：①DNA测序证明U6-cdc2-siRNA已成功构建到表达质粒pAAV-MCS-EGFP中。②AAV-293细胞表达绿色荧光蛋白,共转染成功。③包装得到的重组腺相关病毒 (rAAV-U6-cdc2-siRNA-EGFP)感染AAV-293细胞后能显著下调 AAV-293细胞cdc2基因的表达量。④rAAV-U6-cdc2-siRNA-EGFP的滴度为1x1012 v.g/mL。 结论：携带cdc2-siRNA重组腺相关病毒的包装获得成功,它具有沉默cdc2基因表达的功能。     

重组真核表达质粒pEGFP/hVEGF165转染兔成骨细胞条件的优化*

背景：血管内皮生长因子能够提高骨细胞的增殖能力,促进骨折局部血管发生,在骨缺损修复过程中发挥着至关重要的作用。 目的：优化重组真核质粒pEGFP/hVEGF165转染兔成骨细胞的条件,为构建新型组织工程骨种子细胞提供实验基础。 方法：体外分离、培养并鉴定兔成骨细胞。分别应用0.8,1.0,1.2 μg的质粒和1.5,2.0,2.5,3.0 μL脂质体两两组合,通过脂质体法将重组真核表达质粒pEGFP/hVEGF165转染入兔成骨细胞,转染48 h后倒置荧光显微镜下观察并测定转染率。 结果与结论：随着脂质体剂量的增加,成骨细胞死亡增多。转染48 h后,倒置荧光显微镜下可见细胞质内发绿色荧光的小颗粒成弥散分布,或在核周浓集成块状。其中,脂质体2.5 μL和质粒1.2 μg条件下的转染率最高,可达56%。说明质粒与脂质体之间的交互作用能够显著影响转染率,脂质体2.5 μL和质粒1.2 μg是转染兔成骨细胞的最佳条件。     

Establishment of a recombinant adeno-associated virus expressing hVEGF165*☆

BACKGROUND： Because certain gene vectors could have deleterious effects in the central nervous system, the choice of a safe and effective vector system has become more important for gene therapy of nerve regeneration. OBJECTIVE： To construct a non-pathogenic, recombinant adeno-associated virus （AAV） simultaneously expressing human vascular endothelial growth factor 165 （hVEGF165） and green fluorescent protein （GFP）. DESIGN, TIME AND SETTING： A randomized controlled experiment was performed at the Virology Laboratory of Shaanxi Provincial Center for Disease Control and Prevention between March and September 2007. MATERIALS： AAV helper-free system, AAV-293 packaging cell line, and AAV HT-1080 cells were purchased from Stratagene, USA. E. coli DH5α was a stocked strain from Centers for Disease Control and Prevention of Shaanxi, China. Plasmid pUC18-hHVEGF165 was a gift from Zhibin Shi. METHODS： The hVEGF165 gene was amplified by PCR from pUC18-hHVEGF165 and inserted into plasmid pAAV-IRES-hrGFP to construct recombinant plasmid pAAV-hVEGF165-IRES-hrGFP. Subsequently pAAV-hVEGF165-IRES-hrGFP, pAAV-RC （the rep/cap-gene containing plasmid）, and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hrGFP packaging through homologous recombination. The efficiency of AAV packaging was monitored under a fluorescent microscope, and the recombinant viral particles were harvested from infected AAV-293 cells, and further concentrated and purified. AAV HT-1080 cells were infected with the recombinant virus AAV-hVEGF165-IRES-hrGFP. MAIN OUTCOME MEASURES： Recombinant virus titer was measured by fluorescent cell counting, and infection efficiency was detected by Fluorescence Activated Cell Sorter （FACS） upon infecting AAV-HT1080 cells. The recombination with the exogenous gene was verified by PCR. RESULTS： The PCR amplified products were verified as hVEGF165 gene by DNA sequencing, and the recombinant pAAV-hVEGF165-IRES-GFP was confirmed by double digestion. The system prov     

Establishment of a recombinant adeno-associated virus expressing hVEGF_(165)

BACKGROUND: Because certain gene vectors could have deleterious effects in the central nervous system, the choice of a safe and effective vector system has become more important for gene therapy of nerve regeneration. OBJECTIVE: To construct a non-pathogenic, recombinant adeno-associated virus (AAV) simultaneously expressing human vascular endothelial growth factor 165 (hVEGF165) and green fluorescent protein (GFP). DESIGN, TIME AND SETTING: A randomized controlled experiment was performed at the Virology Laboratory of Shaanxi Provincial Center for Disease Control and Prevention between March and September 2007. MATERIALS: AAV helper-free system, AAV-293 packaging cell line, and AAV HT-1080 cells were purchased from Stratagene, USA. E. Coli DH5α was a stocked strain from Centers for Disease Control and Prevention of Shaanxi, China. Plasmid pUC18-hHVEGF165 was a gift from Zhibin Shi. METHODS: The hVEGF165 gene was amplified by PCR from pUC18-hHVEGF165 and inserted into plasmid pAAV-IRES-hrGFP to construct recombinant plasmid pAAV-hVEGF165-IRES-hrGFP. Subsequently pAAV-hVEGF165-IRES-hrGFP, pAAV-RC (the rep/cap-gene containing plasmid), and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hrGFP packaging through homologous recombination. The efficiency of AAV packaging was monitored under a fluorescent microscope, and the recombinant viral particles were harvested from infected AAV-293 cells, and further concentrated and purified. AAV HT-1080 cells were infected with the recombinant virus AAV-hVEGF165-IRES-hrGFP. MAIN OUTCOME MEASURES: Recombinant virus titer was measured by fluorescent cell counting, and infection efficiency was detected by Fluorescence Activated Cell Sorter (FACS) upon infecting AAV-HT1080 cells. The recombination with the exogenous gene was verified by PCR. RESULTS: The PCR amplified products were verified as hVEGF165 gene by DNA sequencing, and the recombinant pAAV-hVEGF165-IRES-GFP was confirmed by double digestion. The system provided a high packaging ratio of 95%, and the purified recombinant virus had a high titer of 5.5×1011 virus particles/mL. The AAV-HT1080 cells were infected at a ratio of 90.4%. The recombinant virus was confirmed by PCR to contain the exogenous hVEGF165 gene. CONCLUSION: The non-pathogenic rAAV-hVEGF165-GFP vector, carrying hVEGF165 and GFP reporter gene, was successfully constructed with a high titer and infection efficiency.     

Regulatory effect of vascular endothelial growth factor on blood spinal cord barrier in presyrinx state of experimental syringomyelia☆

BACKGROUND： Vascular endothelial growth factor （VEGF） is able to regulate blood spinal cord barrier function as well as influence neovascularization and cause edema. OBJECTIVE： Through establishment of a rabbit model of syringomyelia, to explore the correlation between VEGF protein and mRNA expressions and function of blood spinal cord barrier and edema degree of spinal cord in presyrinx state. DESIGN, TIME AND SETTING： Randomized controlled animal study was performed in the Tumor Institute of the Fourth Hospital, Hebei Medical University from January to June 2007. MATERIALS： Atotal of 0.6 mL Kaolin solution （250 g/L, 37℃） was injected into the cisterna magna of 40 rabbits in the kaolin group to establish syringomyelia models. Goat anti-rabbit VEGF monoclonal antibody was provided by DIACLONE Company, USA; RT-PCR related reagents were provided by Huamei Bioengineering Co., Ltd., Beijing. METHODS： Sixty Chinese white rabbits were divided randomly into two groups： Kaolin group （n = 40） and control group （n = 20）. Physiological saline （0.6 mL at 37℃） was injected in rabbits of control group. On days 1,3, 7, 14 and 21 after kaolin injection, cervical cords samples were harvested after sacrifice of animal. MAIN OUTCOME MEASURES： VEGF protein and mRNA expressions were detected by immunohistochemistry and RT-PCR on days 1, 3, 7, 14, and 21 after kaolin injection. A quantitative analysis of blood spinal cord barrier function was performed by Evans blue technique. Water content of the spinal cord was measured by dry-wet weight technique. The correlation between the expression of VEGF protein and mRNA and the function of blood spinal cord barrier in the upper cervical cord of the presyrinx state was analyzed by linear correlation analysis. RESULTS： The water content and Evans blue content increased in the kaolin group on days 1 and 3 postoperatively compared with the control group （F = 7.387, 61.35, P 〈 0.05, 0.01）, and reached a peak on day 7 （F = 135.94, 528.35, P 〈 0.01?     

Regulatory effect of vascular endothelial growth factor on blood spinal cord barrier in presyrinx state of experimental syringomyelia

BACKGROUND:Vascular endothelial growth factor (VEGF) is able to regulate blood spinal cord barrier function as well as influence neovascularization and cause edema. OBJECTIVE: Through establishment of a rabbit model of syringomyelia,to explore the correlation between VEGF protein and mRNA expressions and function of blood spinal cord barrier and edema degree of spinal cord in presyrinx state. DESIGN,TIME AND SETTING: Randomized controlled animal study was performed in the Tumor Institute of the Fourth Hospi...     

Electrical fields and the promotion of endogenous angiogenesis in a rat spinal cord injury model

BACKGROUND:Previous studies have shown that direct current electrical fields affect development and growth of human microvascular endothelial cells,but the role of electrical fields on promoting angiogenesis in tissues following spinal cord injury remains poorly understood.OBJECTIVE:To determine the effects of electrical fields on angiogenesis and spinal cord repair following traumatic spinal cord injury in rats.DESIGN,TIME AND SETTING:A randomized,controlled,animal experiment was performed at the Chongqing Key Laboratory of Neurology,Affiliated Hospital of Chongqing Medical University,China from September 2007 to August 2008.MATERIALS:Hydrogen blood flow detector(Soochow University Medical Instrument,China),Power Lab System(AD Instruments,Colorado Springs,CO,USA)and mouse anti-vascular endothelial growth factor(VEGF)monoclonal antibody(Sigma-Aldrich,St.Louis,MO,USA)were used in this study.METHODS:A total of 60 healthy,adult,Sprague Dawley rats were equally and randomly assigned to sham-surgery,model,and electrical field groups.The Allen's weight-drop method was used to induce complete spinal cord injury in the model and electrical field groups.Rats in the electrical field group were implanted with silver needles and electrical fields(350 V/m)were applied following traumatic injury.MAIN OUTCOME MEASURES:Latency of somatosensory-evoked potential was detected and spinal cord blood flow was measured by hydrogen blood flow detector.Microvascular density was determined by histological analysis.VEGF expression in the spinal cord was observed by immunohistochemical staining.RESULTS:Recovery of spinal cord blood flow was significantly increased in the electrical field group(at 1,2,4,8,and 24 days after injury)compared with the model group(P < 0.05 or P <0.01).Latency of P1 waves in somatosensory-evoked potential of electrical field group(at 1,2,4,8,and 24 days after injury)was significantly shorter than the model group(P < 0.05 or P < 0.01).Microvascular density and VEGF expression were greater in the electrical field group compared with the model group at 24 days after injury(P< 0.01).CONCLUSION:Electrical fields(350 V/m)promoted angiogenesis within injured rat tissue following spinal cord injury and improved spinal cord function.Electrical fields could help to ameliorate spinal cord injury.The mechanisms of action could be related to increased VEGF expression.     

Effects of Jisuikang on hemorheology and inflammatory factors in rats following spinal cord injury

BACKGROUND: Trauma can damage the spinal cord or cauda equina to different degrees. Previous studies have verified that traditional Chinese medicine has effects on spinal cord injury via a variety of pathways. OBJECTIVE: To observe changes in hemorheology and inflammatory factors in spinal cord injury rats following treatment with the Chinese medicine Jisuikang, to verify the dose-dependent effect of Jisuikang, and to compare its effects with the effects of prednisone. DESIGN, TIME AND SETTING: A randomized study was performed at the Research Institute of Orthopedics, and Experimental Center of First Clinical Medical College, Nanjing University of Traditional Chinese Medicine, China from September 2007 to March 2008. MATERIALS: Jisuikang powdered extract, composed of milkvetch root (30 g), Chinese angelica (12 g), red peony root (12 g), earthworm (10 g), szechwan lovage rhizome (10 g), peach seed (10 g) and safflower (10 g), was provided by the Experimental Center, First Clinical Medical College, Nanjing University of Traditional Chinese medicine. Each gram of powdered extract was equivalent to 6.47 g crude drug. METHODS: A total of 72 Sprague Dawley rats were randomly assigned into 6 groups (n = 12). Rat models of spinal cord injury were established using the occlusion method. Rats in the model group were treated with distilled water. Rats in the 25 g/kg, 12.5 g/kg, and 6.25 g/kg Jisuikang groups were given 25 g/kg, 12.5 g/kg, or 6.25 g/kg Jisuikang by gavage, for 14 days. Rats in the prednisone group received 0.06 g/kg prednisone by gavage, for 7 days. Rats in the normal group were given the same volume of distilled water. The volume of administration was 15 mL/kg.MAIN OUTCOME MEASURES: Rat serum interleukin-10, tumor necrosis factor-α (TNF-α), nitric oxide, nitric oxide synthase levels, malondialdehyde content, superoxide dismutase activity and whole blood viscosity were measured in each group. Spinal cord around the site of the model was collected. Half the spinal cord was used for histopathologic examination. The other half was used for measurement of nitric oxide and NOS levels, malondialdehyde contents, and superoxide dismutase activity. RESULTS: Superoxide dismutase activity was higher in the 25 g/kg Jisuikang group than in the model group. Malondialdehyde contents, nitric oxide and NOS levels were lower in the 25 g/kg and 12.5 g/kg Jisuikang groups compared with the model group. Whole blood viscosity was lower in the 25 g/kg and 12.5 g/kg Jisuikang groups compared with the model group (P < 0.05-0.01). Serum TNF-α content was lower in each Jisuikang group compared with the model group (P < 0.05-0.01). Serum interleukin-10 levels were greater in the prednisone group and each Jisuikang group compared with the model group (P < 0.01). Mild hemorrhage and necrosis in the rat spinal cord, and unclear neural cell swelling were seen in the 25 g/kg Jisuikang group. Severe hemorrhage and necrosis in the rat spinal cord, and distinct neural cell swelling were seen in the 12.5 g/kg Jisuikang group. Edema in the white matter was found in the 6.25 g/kg Jisuikang group. Pathological changes in the prednisone group were identical to the 25 g/kg and 12.5 g/kg Jisuikang groups. CONCLUSION: Jisuikang inhibits nitric oxide synthase expression, reduces nitric oxide and TNF-α levels, decreases malondialdehyde content, increases interleukin-10 levels and superoxide dismutase activity, improves indices of hemorheology, and prevents secondary changes in spinal cord injury, resulting in relieving pathological changes in spinal cord tissue. The outcome was significant in the 25 g/kg Jisuikang group compared with the 12.5 g/kg Jisuikang group.     

Effects of Jisuikang on hemorheology and inflammatory factors in rats following spinal cord injury*☆

BACKGROUND： Trauma can damage the spinal cord or cauda equina to different degrees. Previous studies have verified that traditional Chinese medicine has effects on spinal cord injury via a variety of pathways. OBJECTIVE： To observe changes in hemorheology and inflammatory factors in spinal cord injury rats following treatment with the Chinese medicine Jisuikang, to verify the dose-dependent effect of Jisuikang, and to compare its effects with the effects of prednisone. DESIGN, TIME AND SETTING： A randomized study was performed at the Research Institute of Orthopedics, and Experimental Center of First Clinical Medical College, Nanjing University of Traditional Chinese Medicine, China from September 2007 to March 2008. MATERIALS： Jisuikang powdered extract, composed of milkvetch root （30 g）, Chinese angelica （12 g）, red peony root （12 g）, earthworm （10 g）, szechwan lovage rhizome （10 g）, peach seed （10 g） and safflower （10 g） was provided by the Experimental Center, First Clinical Medical College, Nanjing University of Traditional Chinese medicine. Each gram of powdered extract was equivalent to 6.47 g crude drug. METHODS： A total of 72 Sprague Dawley rats were randomly assigned into 6 groups （n = 12）. Rat models of spinal cord injury were established using the occlusion method. Rats in the model group were treated with distilled water. Rats in the 25 g/kg, 12.5 g/kg, and 6.25 g/kg Jisuikang groups were given 25 g＆g, 12.5 g/kg, or 6.25 g/kg Jisuikang by gavage, for 14 days. Rats in the prednisone group received 0.06 g/kg prednisone by gavage, for 7 days. Rats in the normal group were given the same volume of distilled water. The volume of administration was 15 mL/kg. MAIN OUTCOME MEASURES： Rat serum interleukin-10, tumor necrosis factor- α （TNF-α ）, nitric oxide, nitric oxide synthase levels, malondialdehyde content, superoxide dismutase activity and whole blood viscosity were measured in each group. Spinal cord around the site of the model was collected. Half the     

Hepatocyte growth factor promotes endogenous repair and functional recovery after spinal cord injury

Many therapeutic interventions using neurotrophic factors or pharmacological agents have focused on secondary degeneration after spinal cord injury (SCI) to reduce damaged areas and promote axonal regeneration and functional recovery. Hepatocyte growth factor (HGF), which was identified as a potent mitogen for mature hepatocytes and a mediator of inflammatory responses to tissue injury, has recently been highlighted as a potent neurotrophic and angiogenic factor in the central nervous system (CNS). In the present study, we revealed that the extent of endogenous HGF up-regulation was less than that of c-Met, an HGF receptor, during the acute phase of SCI and administered exogenous HGF into injured spinal cord using a replication-incompetent herpes simplex virous-1 (HSV-1) vector to determine whether HGF exerts beneficial effects and promotes functional recovery after SCI. This treatment resulted in the significant promotion of neuron and oligodendrocyte survival, angiogenesis, axonal regrowth, and functional recovery after SCI. These results suggest that HGF gene delivery to the injured spinal cord exerts multiple beneficial effects and enhances endogenous repair after SCI. This is the first study to demonstrate the efficacy of HGF for SCI.