活血通督汤对坐骨神经钳夹伤模型大鼠脊髓的保护作用及机制研究

发布时间：2018-06-05 10:30

本文选题：坐骨神经损伤 + 活血通督汤　；参考：《福建中医药大学》2017年硕士论文

【摘要】：目的通过观察活血通督汤对坐骨神经钳夹伤大鼠脊髓运动神经元的形态变化和相应节段脊髓组织iNOS、NGF、GDNF的表达情况,探讨HXTDD保护脊髓神经元的作用及机制。方法1、建立大鼠坐骨神经钳夹伤模型2月龄SD大鼠16只,随机分为假手术组(8只)和模型组(8只)。模型组:大鼠麻醉后制成坐骨神经钳夹伤模型;假手术组只暴露坐骨神经,但不致伤神经。两组术后常规饲料喂养4w后取材,分别通过HE染色、尼氏染色,观察光镜下相应节段脊髓前角运动神经元的形态变化和计数其神经元存活率,并以免疫组化分析iNOS蛋白表达情况,鉴定造模是否成功。2、活血通督汤对坐骨神经钳夹伤模型大鼠脊髓的作用观察2月龄SD大鼠48只,随机分为假手术组16只,模型组16只,活血通督汤组16只。根据上述实验造模操作,模型组和活血通督汤组建立大鼠坐骨神经钳夹伤模型,假手术组只暴露坐骨神经,但不致伤神经。各组术后待动物苏醒后立即开始给药干预,活血通督汤组灌服活血通督汤,假手术组、模型组灌服等剂量0.9%氯化钠溶液,每日2次,连续服用4w。给药后每周记录大鼠行为学变化情况。术后4周取材,相应节段脊髓组织行HE、尼氏染色法;免疫组化法检测脊髓组织iNOS表达量;以WB、qPCR检测各组大鼠脊髓iNOS、NGF、GDNF的蛋白及mRNA相对表达量。结果1、建立大鼠坐骨神经钳夹伤模型。2、大鼠行为学变化:术后每周进行行为学测定,活血通督汤组运动神经功能恢复快于模型组,差异有统计学意义(P0.01,P0.05)。3、大鼠相应节段脊髓组织HE染色:假手术组:脊髓神经细胞形态正常,细胞轮廓清晰,大而多角,核呈蓝紫色,基质着色均匀,有极性存在。模型组:脊髓神经元结构不清,形态多样,基质染色不均,细胞核缩小或不清,细胞周围空泡形成明显。活血通督汤组:其神经细胞外貌较模型组规整,有散在的尼氏体,核仁较清楚,空泡样改变极少,部分神经元有水肿。4、大鼠相应节段脊髓前角运动神经元存活率变化:术后4w,与假手术组相比,其余两组的脊髓前角运动神经元存活率显著下降(P0.01),但活血通督汤组存活的运动神经元较模型组多。5大鼠相应节段脊髓组织iNOS免疫组化法分析:模型组iNOS表达最高,且活血通督汤组明显高于假手术组(P0.01)。6大鼠相应节段脊髓组织iNOS、NGF、GDNF的蛋白表达:药物治疗4周后,活血通督汤组GDNF、NGF的的蛋白表达水平较模型组明显上升,iNOS表达较模型组有所下降,差异有统计学意义(P0.01,P0.05)。7大鼠相应节段脊髓组织iNOS、NGF、GDNFmRNA的表达:在受损脊髓组织中,iNOSmRNA的表达有所增强,模型组明显高于活血通督汤组跟假手术组,差异有统计学意义(P0.05);脊髓组织GDNFmRNA和NGFmRNA表达,活血通督汤组、模型组均较假手术组高,而活血通督汤组也高于模型组表达(P0.05)。结论1、活血通督汤能改善坐骨神经钳夹伤模型大鼠后肢功能障碍;2、活血通督汤能有效改善坐骨神经钳夹伤模型大鼠脊髓前角运动神经元的存活情况;3、活血通督汤能通过调控脊髓组织中iNOS、NGF、GDNF的表达,有效防治脊髓运动神经元的损伤,为进一步深入研究提供基础依据。
[Abstract]:Objective To observe the morphological changes of the spinal cord motoneurons and the expression of iNOS, NGF and GDNF in the corresponding segmental spinal cord tissue of the sciatic nerve clamp of rats with Huoxue Tong Du Decoction (Huoxue Tong Du Decoction), and to explore the effect and mechanism of HXTDD on the protection of spinal neurons. Method 1, 16 rats of the sciatic nerve clamp injury model of 2 month old SD rats were established and randomly divided into sham operation group (the group of 16 rats were randomly divided into sham operation group). 8 rats and model group (8). Model group: the rat model of sciatic nerve clamp injury was made after anesthesia. The sham operation group only exposed the sciatic nerve, but did not hurt the nerve. The two groups were fed after 4W and were fed with HE staining, Nissl staining, and observed the morphological changes of the motor neurons in the anterior horn of the spinal cord under the light microscope and the count of the nerve. The survival rate of the iNOS protein was analyzed by immunohistochemistry. The effect of.2 was identified and the effect of Huoxue Tong Du Decoction on the spinal cord of the sciatic nerve clamp was observed in 2 month old SD rats, and 16 rats were randomly divided into a sham operation group, 16 in the model group and 16 in the Huoxue Tong Du Decoction group. The rat model of the sciatic nerve clamp injury was established in the Tong Du Decoction group. The sham operation group only exposed the sciatic nerve, but did not hurt the nerve. After the animals were awakened, the rats were given the medicine intervention immediately after the animals were awakened. The group of Huoxue Tong Du Decoction group was filled with Huoxue Tong Du decoction, sham operation group, and the model group was given the dosage of 0.9% Sodium Chloride Solution, 2 times a day, every week after the continuous administration of 4w. administration. The changes in the behavior of the rats were recorded. 4 weeks after the operation, HE, Nissl staining was used in the corresponding segments of the spinal cord, and the expression of iNOS in the spinal cord tissue was detected by immunohistochemistry; the iNOS, NGF, GDNF protein and mRNA relative expression of iNOS, NGF, GDNF in the spinal cord of rats in each group were detected by WB and qPCR. Results 1, the rat sciatic nerve clamp injury model.2 and the behavior changes of rats were established. After a weekly behavioral test, the motor nerve function of the Huoxue Tong Du Decoction group was recovered faster than the model group. The difference was statistically significant (P0.01, P0.05).3, and the corresponding spinal cord tissue of the rat was HE staining: the sham operation group: the spinal nerve cell morphology was normal, the cell outline was clear, the large and multi angle, the nucleus was blue purple, the matrix coloring was even and polar existed. There was polarity existence. Type group: spinal cord neuron structure is not clear, morphological diversity, uneven matrix staining, cell nuclei narrowing or unclear, cell surrounding vacuoles formation obviously. Huoxue Tong Du group: the nerve cell appearance is more regular than the model group, there are scattered Nissl body, the nucleolus is clearer, the vacuoles change very few, some neurons have edema.4, the corresponding segment of the spinal cord anterior horn of the rat The survival rate of motor neuron: 4W after operation, compared with the sham group, the survival rate of the motor neuron in the other two groups decreased significantly (P0.01), but the surviving motoneurons in the Huoxue Tong Du Decoction group were compared with that of the model group in the corresponding segment spinal cord tissue with iNOS immunohistochemical method: the iNOS expression of the model group was the highest and the Huoxue Tong Du Decoction group was in the model group. The protein expression of iNOS, NGF, GDNF in the corresponding segment of the spinal cord of the sham group (P0.01).6 rats was significantly higher than that of the model group after 4 weeks of drug treatment, and the expression level of GDNF in the Huoxue Tong Du Decoction group was significantly higher than that in the model group, and the expression of iNOS was lower than that in the model group, and the difference was statistically significant (P0.01, P0.05).7 rat spinal cord tissue iNOS. The expression of GDNFmRNA: in the damaged spinal cord tissue, the expression of iNOSmRNA was enhanced. The model group was significantly higher than the Huoxue Tong Du Decoction group and the sham group (P0.05). The expression of GDNFmRNA and NGFmRNA in the spinal cord, the Huoxue Tong Du Decoction group, the model group were higher than the sham operation group, and the Huoxue Tong Du Decoction group was also higher than the model group (P0.05 Conclusion 1. 1, Huoxue Tong Du decoction can improve the hindlimb dysfunction of the sciatic nerve clamp injury model rats. 2, Huoxue Tong Du decoction can effectively improve the survival of the motor neurons of the anterior horn of the spinal cord of the sciatic nerve clamp injury model rats. 3, Huoxue Tong Du decoction can effectively control the spinal motor neurons by regulating the expression of iNOS, NGF, and GDNF in the spinal cord tissue. The damage will provide a basis for further study.
【学位授予单位】：福建中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5;R-332

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