【作者】 韦理；

【导师】 张俐；

【作者基本信息】 福建中医药大学 ， 中医骨伤科学， 2013， 博士

【摘要】 目的(1)观察丹参注射液对大鼠急性脊髓损伤后运动功能的作用。(2)探讨丹参注射液对大鼠急性脊髓损伤后运动功能的作用机制。方法1.动物造模、分组及干预方法(1)动物造模SD雄性大鼠144只,麻醉后俯卧位固定于手术台上,消毒后以T11为中心取后正中切口,咬除T10～T12椎板和棘突,用硬塑料片保护T10和T12脊髓,只暴露T11脊髓。采用改良Alleni去致伤脊髓：以10g不锈钢杆自三维立体定向仪上经套筒自由坠落4cm,撞击Tll脊髓,然后移去不锈钢杆,打击区脊髓硬脊膜下可见充血,观察到鼠尾痉挛性摆动,双下肢及躯体痉挛收缩,示造模成功。另选取SD雄性大鼠48只作为假手术组,采用上述手术方法,不撞击脊髓。(2)分组144造模大鼠,随机分为观察组、对照组和SCI组,每组各48只,3组又各分为造模后1、3、7及14天组,每组12只。(3)干预方法观察组：于造模后1h给予丹参注射液1.78mL/kg/天,腹腔内注射,每天1次,按存活时间分别注射1、3、7、14天。对照组：采用大剂量冲击疗法,于造模后1h腹腔注射甲基强的松龙30mg/kg,45min后按5.4mg/kg/h计算23h总量,分4次腹腔注射。假手术组和SCI组：不进行任何干预。分别于手术后1天、3天、7天、14天,分组分批取材。2.实验指标检测(1)各组大鼠一般状态比较(2)行为学评分采用改良Rivlin斜板法评分来评价大鼠的脊髓功能。(3)组织病理学观察光学显微镜下观察脊髓造模后1天、3天、7天、14天的细胞结构变化。(4)原位杂交检测包括胶质细胞源性神经营养因子(GDNF)、乙酰胆碱转化酶(ChAT)、突触体素(synaptophysin).突触素(synapsin)、突触结合蛋白I(sytI)。3.统计学处理将所得数据用SPSS11.0版统计软件进行统计。结果1.各组大鼠一般状态比较损伤1天后,各组大鼠毛发散乱,后肢无法运动,呼吸较浅,饮食减少,尿失禁或尿潴留。损伤3天后观察组与对照组毛色散乱,后肢能轻微活动,但不能支撑爬行,SCI组后肢毛色干枯、散乱,无任何活动,均呼吸较浅、饮食减少、尿失禁或尿潴留。损伤7天后,观察组和对照组动物毛色变润泽、整齐,后肢活动增强,无法支撑爬行,呼吸正常,饮食有所增加,尿失禁或尿潴留有所改善,SCI组无明显变化。损伤14天后观察组和对照组动物毛色润泽、整齐,后肢活动增强,稍可支撑爬行,呼吸正常,饮食接近正常,排尿趋于正常,SCI组毛发散乱,后肢轻微活动,呼吸接近正常,饮食有所增加,尿失禁或尿潴留有所改善。2.行为学评分各组损伤前斜板试验临界角比较,差异无统计学意义(P>0.05)。造模后1天,观察组斜板试验临界角低于治疗前及对照组同期(P<0.01)；造模后3天,观察组斜板试验临界角与造模后1天比较,差异无统计学意义(P>0.05);造模后7天,观察组斜板试验临界角高于造模后3天及SCI组同期(P<0.01),低于对照组(P<0.05)；造模后14天,观察组斜板试验临界角高于造模后7天及SCI组同期(P<0.01,P<0.05),与对照组无显著差异(P>0.05)。3.组织病理学改变脊髓造模后1天,SCI组脊髓损伤灰质区明显出血、水肿,部分神经元变性坏死；观察组和对照组部分神经元坏死,但出血、水肿较轻。造模后3天,SCI组脊髓损伤灰质区仍明显出血、水肿,神经元坏死增多；观察组出血、水肿减轻,神经元坏死稍增多；对照组出血、水肿减轻,神经元坏死增多。造模后7天、14天,SCI组脊髓损伤灰质区出血、水肿减轻：观察组和对照组出血、水肿均明显减轻。4.原位杂交检测结果(1)胶质细胞源性神经营养因子(GDNF)表达造模后1天,观察组GDNF阳性产物吸光度值低于对照组同期(P<0.05),高于SCI组同期(P<0.01)；造模后3天,高于造模后1天及SCI组同期(P<0.01,P<0.05),低于对照组同期(P<0.05)；造模后7天,与造模后3天及对照组同期无显著差异(P>0.05,P>0.05),高于SCI组同期(P<0.01)；造模后14天,与造模后7天及对照组同期无显著差异(P>0.05,P>0.05),高于SCI组同期(P<0.01)。(2)乙酰胆碱转化酶(ChAT)表达造模后1天,观察组的ChAT阳性产物吸光度值低于对照组(P<0.01),与SCI组无显著差异(P>0.05)；在造模后3天、7天的乙酰胆碱转化酶阳性产物吸光度值低于对照组(P<0.05),高于SCI组(P<0.01)；在造模后14天与对照组无显著差异(P>0.05),高于SCI组(P<0.01)。(3)突触体素(synaptophysin)表达造模后1天、3天,观察组的synaptophysin阳性产物吸光度值与对照组和SCI组无显著差异(P>0.05),在造模后7天、14天高于SCI组(P<0.01),与对照组无显著差异(P>0.05)。(4)突触素I(synapsinI)表达造模后1天、3天,观察组的synaptophysin吸光度值与对照组和SCI组无显著差异(P>0.05),在造模后7天、14天高于SCI组(P<0.01),与对照组无显著差异(P>0.05)。(5)突触结合蛋白I(synaptotagmin, SytI)表达观察组在造模后1天sytI阳性产物吸光度值与对照组和SCI组无显著差异(P>0.05)；造模后3天低于对照组(P<0.05),高于SCI组(P<0.01)；在造模后7天、14天高于SCI组(P<0.01),与对照组无显著差异(P<0.05)。结论(1)丹参注射液对急性脊髓损伤运动功能具有保护作用(2)丹参注射液对急性脊髓损伤灰质的保护机制：可能通过促进脊髓灰质突触体素、突触素Ⅰ、突触结合蛋白Ⅰ、胶质细胞源性神经营养因子、乙酰胆碱转化酶合成,加快脊髓灰质突触的功能恢复。更多还原

【Abstract】 ObjectiveTo observe effect of Danshen injections on Acetylcholine transferase, Glial cell line-derived neurotrophic factor, synaptophysin, synapsinI and Synaptic adhesion proteins I in the gray matter of rat after acute spinal cord injury. To deduce the mechanism about the effect of Salvia miltiorrhiza on acute spinal cord injury.Methods1.Grouping and making rat models with acute spinal cord injury:144Male SD rats were randomly divided into observation group, control group and SCI group (48in each group) Spinal cords of rats in these groups were injured with Modified Allen method. Other spinal cords of48rats in the sham group were not injured in addition to the same surgical procedure as that in SCI group.2. Delivery method(1) Observation group:Salvia miltiorrhiza injection was injected into rats’abdominal cavity in one hour (Once a day, and respectively injected for one time, three times, seven times, fourteen times according to different groups)(2) Control group:Used large dose of shock therapy, methylprednisolone was injected into rats’ abdominal cavity with30mg/kg first, and then respectively injected into rats’ abdominal cavity in0.5hour, lhour,2hour and4hour with4.5*23/4mg/kg.3.Indexes detection(1)Behavioral score:Assessing the spinal cord function with inclined plate method improved by Rivlin (2) Histopathology:With optical microscope, observating the change in cell structure in1days、3days、7days and14days after acute spinal injury.(3)hybridization in situ:Including acetylcholine transferase, glial cell line-derived neurotrophic factor, synaptophysin, synapsinI and synaptic adhesion proteinsl.4. Statistical analysis:The data will be calculated with SPSS11.0.Results1. Behavioral score:The critical angle of inclined plane test was significantly reduced in observation group and control group on the first day, the third day after modeling. There was not significant difference between observation group and control group(P>0.05). There was a slight rebound in critical angle on the seventh day and the fourteenth day after modeling. The critical angle in control group was greater than that of the observation group on the seventh day(P<0.05). There was not significant difference between observation group and control group in the critical angle of inclined plane test on the fourteenth day(P>0.05).2. Histopathology:Bleeding, edema, thin nissl bodies in cytoplasm were found in damage zone of gray matter of observation group and control group on the first day after modeling, with part of denatured and necrotic neurons. Bleeding and edema were reduced in observation group on the third day, with slightly increased neuronal necrosis. Bleeding and edema were reduced in control group on the third day, with increased neuronal necrosis. Bleeding and edema were significantly reduced in damage zone on the seventh day and the fourteenth day, with thick nissl bodies in cytoplasm.4. Glial cell line-derived neurotrophic factor(GDNF):The absorbance value of GDNF positive products in observation group was less than that of control group(P<0.05), but more than that of SCI group(P<0.01)on the first day after modeling. It was less than that of control group(P<0.05), but more than that of SCI group(P<0.05)on the third day. It was not significant difference from control group(P>0.05), but more than that of SCI group(P<0.01)on the seventh day and the fourteenth day(P>0.05).3. Acetylcholine-converting enzyme(ChAT):The absorbance value of ChAT positive products in observation group was less than that of control group(P<0.01), but not significant difference from SCI group(P>0.05)on the first day after modeling. It was less than that of control group(P<0.05), but more than that of SCI group(P<0.01)on the third day and the seventh day. It was more than that of SCI group(P<0.01), but was not significant difference from control group(P>0.05)on the fourteenth day.5. synaptophysin:The absorbance value of synaptophysin positive products in observation group was not significant difference compared with control group and SCI group on the first day and the third day after modeling(P>0.05). It was more than that of SCI group(P<0.01), but was not significant difference compared with control group(P>0.05)on the seventh day and the fourteenth day.6. synapsin I:The absorbance value of synapsin I positive products in observation group was not significant difference compared with control group and SCI group on the first day and the third day after modeling(P>0.05). It was more than that of SCI group(P<0.01),but was not significant difference compared with control group(P>0.05)on the seventh day and the fourteenth day.7. Synaptic adhesion proteins I (syt I):The absorbance value of syt I positive products in observation group was not significant difference compared with control group and SCI group on the first day after modeling(P>0.05). It was less than that of control group(P<0.05), but more than that of SCI group(P<0.01)on the third day. It was more than that of SCI group(P<0.01), but was not significant difference compared with control group(P>0.05)on the seventh day and the fourteenth day.Conclusion1. Animal model in this experimentation was repeatable, reliable and controllable.2. Protection of Salvia for spinal cord gray matter after acute spinal cord injurySalvia could increase ChAT, GDNF, synaptophysin, synapsinI and sytI in spinal cord gray matter after acute spinal cord injury. The mechanisms maybe that Salvia increases the active of ChAT to restore the motor function of spinal cord gray matter, and increases the active of associated protein in synaptic to promote the transfer of nerve impulses.更多还原