川芎嗪对顺铂诱导大鼠耳蜗毛细胞凋亡的干预机制研究

发布时间：2018-06-09 03:41

本文选题：药物性耳聋 + 顺铂　；参考：《辽宁中医药大学》2015年博士论文

【摘要】：第一部分顺铂腹腔注射诱导大鼠耳聋模型的实验研究目的:探索顺铂腹腔注射诱导大鼠耳聋模型的最佳剂量及实验条件。材料与方法:健康Wister大白鼠50只,雌雄各半,体重200±20g,6-8周龄。购于中国医科大学实验动物中心。将50只大鼠按照随机数字法平均分为5组:空白对照组;模型1组;模型2组;模型3组;模型4组,每组10只。除空白对照组外,其余四组大鼠均经腹腔给予不同剂量的顺铂注射液注射,方法如下:空白对照组:每只大鼠给予生理盐水注射液腹腔注射,剂量为1ml/100g·d-1,连续给药5天。模型1组:每只大鼠给予0.4mg/ml浓度的顺铂注射液腹腔注射,剂量为0.5ml/100g·d-1,连续给药5天(相当于2mg/Kg)。模型2组:每只大鼠给予0.8mg/ml浓度的顺铂注射液腹腔注射,剂量为0.5ml/100g·d-1,连续给药5天(相当于4mg/Kg)。模型3组:每只大鼠给予1.6mg/ml浓度的顺铂注射液腹腔注射,剂量为0.5ml/100g·d-1,连续给药5天(相当于8mg/Kg)。模型4组:每只大鼠给予3.2mg/ml浓度的顺铂注射液腹腔注射,剂量为0.5ml/100g·d-1,连续给药5天(相当于16mg/Kg)。实验结束后,对各组大鼠听阈ABR阈值进行检测,同时观察耳蜗毛细胞的病理改变。结果:1.各组大鼠的一般状态及死亡率空白对照组大鼠精神状态良好,活动度正常,食水量正常,给予击掌噪音刺激,耳廓反应灵敏。模型1组大鼠精神状态较好,活动度略有减少,食水量正常,给予击掌噪音刺激,耳廓反应较空白组反应略显迟钝,至实验结束,未出现死亡大鼠。模型2、3、4组大鼠精神状态较差,尤其3、4组,蜷缩于笼子角落,很少活动,食水量也明显减少,模型4组大鼠在实验的第4-5天几乎没有进食,给予击掌噪音刺激,耳廓反应非常迟钝,模型4组大鼠耳廓反应阴性。模型2组大鼠未见死亡情况出现,而模型3、4组大鼠出现死亡。模型3组于实验第3天死亡1只、第5天死亡2只,总死亡率30%;模型4组于实验第2天死亡1只、第3天死亡1只、第4天死亡1只,第5天死亡3只,总死亡率50%。2.各组大鼠ABR检测结果各组大鼠ABR听阈检测结果显示:与空白对照组大鼠比较,模型1、2、3、4组大鼠ABR阈值显著升高(p0.01),有统计学差异。由模型1、2、3、4组组间比较结果可知,随着顺铂注射剂量的加大,ABR阈值明显升高。3.耳蜗基底膜毛细胞形态学观察(1)耳蜗基底膜铺片各组大鼠耳蜗基底膜铺片结果显示:空白对照组大鼠耳蜗基底膜铺片清晰可见完整的三排毛细胞,排列均匀、整齐,无缺失及坏死细胞出现。模型1组大鼠基底膜铺片上,明显可见毛细胞缺失情况,但大部分排列仍然较为均匀整齐,仅偶见缺失。模型2组大鼠基底膜铺片可见大面积形态模糊的毛细胞排列,大体形态排列仍较为整齐,模型3组和4组大鼠基底膜毛细胞形态丧失,无法辨认毛细胞形态,偶见3-5个毛细胞轮廓排列在一起。(2)耳蜗病理各组大鼠耳蜗病理切片HE染色结果显示:通过病理切片HE染色,可清晰的分辨耳蜗毛细胞形态及排列情况。空白对照组大鼠耳蜗毛细胞排列整齐均匀,可见毛状结构附着于基底膜上。模型1组大鼠耳蜗基底膜毛细胞排列均匀整齐,偶见缺失,但毛状结构稀疏。模型2组大鼠耳蜗基底膜毛细胞缺失明显,毛状结构溶解。模型3组和4组大鼠耳蜗基底膜毛细胞缺失明显,排列紊乱,几乎见不到毛状结构附着。结论:1.顺铂给予大鼠腹腔注射的方法,可导致大鼠ABR阈值的升高及耳蜗毛细胞的损伤。2.顺铂2mg/kg,日一次,连续5日给药的剂量可导致大鼠耳蜗损伤,但不足以作为耳聋动物模型,用于科研实验。3.顺铂8mg/kg和16mg/kg,日一次,连续5日给药的剂量可导致大鼠耳蜗毛细胞过度损伤,不适合作为耳聋动物模型,用于科研实验。4.顺铂4mg/kg,日一次,连续5日给药的剂量可导致大鼠耳蜗损伤,损伤程度适中,模型稳定,可作为耳聋动物模型,用于科研实验。第二部分川芎嗪对顺铂诱导耳聋模型大鼠耳蜗毛细胞凋亡内部途径干预机制的实验研究目的:探索川芎嗪对顺铂诱导的耳聋模型大鼠耳蜗毛细胞凋亡早期内部途径的干预作用和机制。材料与方法:将62只大鼠采用随机数字法平均分为两组,分别为:正常组(N组,11只),模型复制组(M组,51只)。正常组大鼠不做处理,正常饲养;模型复制组大鼠采用顺铂腹腔注射的方法诱导药物性耳聋模型。模型复制成功后,再次采用随机数字法,将正常组大鼠随机分成两组:正常对照组(N1组)、川芎嗪对照组(N2组);将模型复制组大鼠随机分为两组:模型对照组(M1)、川芎嗪治疗组(M2)。实验第1-5天:正常组给予生理盐水腹腔注射,5ml/kg体重,连续注射5天。模型复制组给予腹腔注射顺铂注射液,4mg/kg体重,连续注射5天。实验第6-12天:正常对照组给予生理盐水腹腔注射,5ml/kg体重,连续注射7天。川芎嗪对照组给予川芎嗪注射液腹腔注射,140mg/kg体重,连续注射7天。模型对照组给予生理盐水腹腔注射,5ml/kg体重,连续注射7天。川芎嗪治疗组给予川芎嗪注射液腹腔注射,140mg/kg体重,连续注射7天。末次给药后1h,处死大鼠,剥离耳蜗组织,动存于-80℃冰箱中,用于p53、bax、bcl-2、caspase-9、caspase-3 m RNA及蛋白表达水平检测。结果:1.模型评价结果模型复制后,N组和M组大鼠ABR检测结果显示:与正常组大鼠比较,模型复制组大鼠ABR阈值显著升高(p0.01),有统计学意义。N组大鼠耳蜗基底膜毛细胞排列整齐,分布均匀,无明显的细胞缺失情况;M组大鼠耳蜗基底膜毛细胞大部分变形,排列紊乱,可见大量溶解破坏的毛细胞,细胞间连接残缺不全。2.各组大鼠耳蜗组织中p53、bax、bcl-2、caspase-9、caspase-3蛋白表达水平实验结果显示:与空白对照组比较,模型对照组及模型加川芎嗪组大鼠耳蜗组织中bax、p53、caspase-3、9蛋白表达水平显著升高,而bcl-2蛋白表达水平显著下降(p0.01);与模型对照组比较,模型加川芎嗪组大鼠耳蜗组织中bax、p53蛋白表达水平显著降低,而bcl-2蛋白表达水平显著升高(p0.01);空白对照组及空白加川芎嗪组组间比较,各指标均无显著性差异(p0.05)。3.各组大鼠耳蜗组织中p53、bax、bcl-2、caspase-9、caspase-3 m RNA表达水平实验结果显示:与空白对照组比较,模型对照组及模型加川芎嗪组大鼠耳蜗组织中bax、p53、caspase-3、9 m RNA表达水平显著升高,而bcl-2 m RNA表达水平显著下降(p0.01);与模型对照组比较,模型加川芎嗪组大鼠耳蜗组织中bax、p53、caspase-3、9 m RNA表达水平显著降低,而bcl-2 m RNA表达水平显著升高(p0.01);空白对照组及空白加川芎嗪组组间比较,各指标均无显著性差异(p0.05)。结论:1.给予大鼠顺铂腹腔注射(剂量4mg/kg),每日一次,连续5天,可成功诱导药物性耳聋大鼠模型。2.川芎嗪可显著下调模型大鼠耳蜗组织中的p53、bax、caspase-9、caspase-3 m RNA及蛋白表达水平,上调bcl-2 m RNA及蛋白表达水平。3.川芎嗪对药物性耳聋模型大鼠耳蜗毛细胞的抗凋亡作用可能是通过阻断或抑制细胞凋亡起始阶段的内部途径而实现的。第三部分川芎嗪对顺铂诱导耳聋模型大鼠耳蜗毛细胞凋亡外部途径干预机制的实验研究目的:探索川芎嗪对顺铂诱导的耳聋模型大鼠耳蜗毛细胞凋亡早期外部途径的干预作用和机制。材料与方法:将62只大鼠采用随机数字法平均分为两组,分别为:正常组(N组,11只),模型复制组(M组,51只)。正常组大鼠不做处理,正常饲养;模型复制组大鼠采用顺铂腹腔注射的方法诱导药物性耳聋模型。模型复制成功后,再次采用随机数字法,将正常组大鼠随机分成两组:正常对照组(N1组)、川芎嗪对照组(N2组);将模型复制组大鼠随机分为两组:模型对照组(M1)、川芎嗪治疗组(M2)。实验第1-5天:正常组给予生理盐水腹腔注射,5ml/kg体重,连续注射5天。模型复制组给予腹腔注射顺铂注射液,4mg/kg体重,连续注射5天。实验第6-12天:正常对照组给予生理盐水腹腔注射,5ml/kg体重,连续注射7天。川芎嗪对照组给予川芎嗪注射液腹腔注射,140mg/kg体重,连续注射7天。模型对照组给予生理盐水腹腔注射,5ml/kg体重,连续注射7天。川芎嗪治疗组给予川芎嗪注射液腹腔注射,140mg/kg体重,连续注射7天。末次给药后1h,处死大鼠,剥离耳蜗组织,动存于-80℃冰箱中,用于fas/fas L、caspase-8m RNA及蛋白表达水平检测。结果:1.各组大鼠耳蜗组织中fas/fas L、caspase-8蛋白表达水平实验结果显示:与空白对照组比较,模型对照组及模型加川芎嗪组大鼠耳蜗组织中fas/fas L、caspase-8蛋白表达水平显著升高(p0.01);与模型对照组比较,模型加川芎嗪组大鼠耳蜗组织中fas/fas L、caspase-8蛋白表达水平显著降低(p0.01);空白对照组及空白加川芎嗪组组间比较,各指标均无显著性差异(p0.05)。2.各组大鼠耳蜗组织中fas/fas L、caspase-8 m RNA表达水平实验结果显示:与空白对照组比较,模型对照组及模型加川芎嗪组大鼠耳蜗组织中fas/fas L、caspase-8 m RNA表达水平显著升高(p0.01);与模型对照组比较,模型加川芎嗪组大鼠耳蜗组织中fas/fas L、caspase-8 m RNA表达水平显著降低(p0.01);空白对照组及空白加川芎嗪组组间比较,各指标均无显著性差异(p0.05)。结论:1.川芎嗪可显著下调模型大鼠耳蜗组织中的fas/fas L、caspase-8 m RNA及蛋白表达水平。2.川芎嗪对药物性耳聋模型大鼠耳蜗毛细胞的抗凋亡作用可能是通过阻断或抑制细胞凋亡起始阶段的外部途径而实现的。
[Abstract]:The first part of the experimental study on induced deafness model of rats by intraperitoneal injection of cisplatin in order to explore the optimal dosage and experimental conditions of induced deafness model induced by intraperitoneal injection of cisplatin. Materials and methods: 50 healthy Wister rats, male and female, weight 200 + 20g, 6-8 weeks old, were bought at the experimental animal center of China Medical University. 50 rats were used in accordance with the experimental animal center. The random number method was divided into 5 groups: blank control group, model 1, model 2, model 3, model 4, 10 in each group. Except for blank control group, the other four groups of rats were injected with different doses of Cisplatin Injection intraperitoneally, the method was as follows: each rat was given intraperitoneal injection of saline injection, the dose of 1ml/100g. D-1, 5 days of continuous administration. Model 1: each rat was given an intraperitoneal injection of 0.4mg/ml concentration of Cisplatin Injection with a dose of 0.5ml/100g. D-1 for 5 days (equivalent to 2mg/Kg). Model 2: each rat was given an intraperitoneal injection of 0.8mg/ml concentration, the dose was 0.5ml/100g. D-1, and a continuous dose of 5 days (equivalent to 4mg/Kg). Model 3 groups: Each rat was given an intraperitoneal injection of 1.6mg/ml concentration of Cisplatin Injection, with a dose of 0.5ml/100g D-1, and a continuous dose of 5 days (equivalent to 8mg/Kg). Model 4: each rat was given a 3.2mg/ml concentration of Cisplatin Injection intraperitoneal, a dose of 0.5ml/100g D-1, and a continuous dose of 5 days (equivalent to 16mg/Kg). At the end of the experiment, the threshold ABR threshold of rats in each group was completed. The values were detected and the pathological changes of the cochlear hair cells were observed at the same time. Results: 1. the general state and death rate of the rats in each group were in good mental state, normal activity, normal water intake, irritation of the noise of the palm, and the sensitive auricle reaction. The 1 groups of rats in the model were better, the activity degree was slightly reduced and the water content was normal. With the stimulation of the noise, the response of the auricle was slightly slower than that in the blank group. To the end of the experiment, there was no death in the rat. The model 2,3,4 group had a poor mental state, especially in the 3,4 group, curled up in the corner of the cage, with little activity and a significant decrease in the amount of water. The 4 rats in the model group had almost no feeding on the 4-5 day of actual test, giving the ear noise stimulation, ear irritation. The auricle reaction was very slow in the model 4 rats. There was no death in the model group of rats. There was no death in the model 2 rats, but the model 3,4 group died. The model 3 groups died 1 in the third day experiment and 2 in fifth days, the total mortality was 30%. The model 4 group died second days, 1, third days death, and total death The result of ABR hearing threshold detection in each group of rats in each group was 50%.2.. The results of ABR hearing threshold of rats in each group showed that compared with the blank control group, the ABR threshold of the model 1,2,3,4 group increased significantly (P0.01), and there was a statistical difference. The results of the model 1,2,3,4 group showed that with the increase of the injection dose of cisplatin, the threshold of ABR increased significantly in the.3. cochlear base. Morphological observation of membrane hair cell (1) the result of the laying of the cochlear basement membrane in the cochlear basement membrane sheets showed that the complete three rows of hair cells were clearly visible in the basal membrane of the rats in the blank control group, and the arrangement was uniform and tidy, and the absence of the missing and necrotic cells appeared. The loss of hair cells in the basal membrane of the 1 groups of rats was obviously visible, but the loss of hair cells was obvious. Most of the permutations were still even and neatly, only missing. The 2 groups of rats in the base membrane of the model group showed a large area of fuzzy hair cells arranged in large area. The morphology of the basal membrane of the model 3 and the 4 groups of rats lost the form of hair cells in the basement membrane, and the outline of 3-5 hair cells could not be identified. (2) ears (2). The HE staining results of the cochlear pathological sections of the rats in each group showed that the morphology and arrangement of the cochlear hair cells could be clearly identified by pathological section HE staining. The hair cells of the cochlear hair cells in the blank control group were arranged neatly and evenly, and the hair like structure attached to the basement membrane. The 1 groups of rat cochlear basal membrane hair cells were arranged evenly and neatly. The loss of hair like structure was sparse. The loss of hair cells in the basal membrane of the cochlea of model 2 rats was obvious, and the hair like structure dissolved. The 3 groups and 4 groups of rat cochlea basal membrane hairy cells were lost obviously, the arrangement was disorderly, and almost no hairy structure attached. Conclusion: 1. the method of intraperitoneal injection of cisplatin in rats can lead to the increase of ABR threshold and ear of rats. The damage of the cochlear cells is.2. cisplatin 2mg/kg. Once a day, the dose of 5 days of continuous administration can cause cochlear injury in rats, but it is not enough to be used as an animal model of deafness. It is used for research and experiment.3. cisplatin 8mg/kg and 16mg/kg. On the other day, the dose of 5 days of continuous administration can lead to excessive damage of the rat cochlear capillary cell, which is not suitable to be used as a deafness animal model. Research experiment.4. cisplatin 4mg/kg, once a day, the dose of 5 days continuous administration can lead to cochlear injury in rats, the degree of injury is moderate, the model is stable, and it can be used as a deafness animal model and used in scientific research. The second part of the experimental study on the intervention mechanism of Ligustrazine on the internal pathway of cochlear hair cell withering in the deafness model rats induced by cisplatin The effect and mechanism of Ligustrazine on the early internal pathway of cochlear hair cell apoptosis induced by cisplatin in the deafness model of cisplatin. Materials and methods: 62 rats were divided into two groups by random number method: normal group (group N, 11), model replicating group (group M, 51). Normal rats were not treated, normal rearing, model replicating group was large. The rat model of deafness was induced by intraperitoneal injection of cisplatin. After the model replication was successful, the rats were randomly divided into two groups: normal control group (group N1) and ligustrazine control group (group N2). The model replicas group was randomly divided into two groups: model control group (M1), ligustrazine treatment group (M2). Experiment 1-5 Day: normal group was given intraperitoneal injection of saline, 5ml/kg weight, continuous injection for 5 days. Model replicating group was given intraperitoneal injection of Cisplatin Injection, 4mg/kg weight, continuous injection for 5 days. On day 6-12: normal control group was given intraperitoneal injection of saline, 5ml/kg weight, continuous injection for 7 days. Ligustrazine control group was given intraperitoneal injection of ligustrazine injection. Injection, 140mg/kg weight, continuous injection for 7 days. The model control group was given intraperitoneal injection of saline, 5ml/kg weight, continuous injection for 7 days. Ligustrazine treatment group was given intraperitoneal injection of ligustrazine injection, 140mg/kg weight, continuous injection for 7 days. After the last dose of 1H, the rats were killed and cochlear tissue was stripped at -80 centigrade refrigerator and used for p53, Bax, Bcl-2, CASPA. Se-9, caspase-3 m RNA and protein expression level detection. Results: after the 1. model evaluation model was replicated, the results of ABR detection in N and M group rats showed that the ABR threshold of the model replicas rats increased significantly (P0.01), and there was statistical significance in the.N group of the rat cochlea basal membrane hair cells arranged neatly and evenly distributed, without obvious finer. In the M group, most of the rat cochlear basal membrane hair cells were deformed and arranged in disorder. A large number of dissolving and damaged hair cells were seen, and the intercellular connection was incomplete in the cochlear tissues of.2. rats. The experimental results of p53, Bax, Bcl-2, caspase-9, and caspase-3 protein expression in the rat cochlear tissues were shown: compared with the blank control group, the model control group and the model plus Sichuan were compared with the blank control group. The expression level of Bax, p53, caspase-3,9 protein in the cochlear tissue of the Ligustrazine group increased significantly, while the expression level of Bcl-2 protein decreased significantly (P0.01). Compared with the model control group, the expression level of Bax, p53 protein in the rat cochlear tissue of Ligustrazine group was significantly decreased, and the level of Bcl-2 protein expression was significantly increased (P0.01), and the blank control group and the empty space were empty. The results of p53, Bax, Bcl-2, caspase-9, caspase-3 m RNA expression in the cochlear tissues of rats in each group of.3. groups showed no significant difference (P0.05) in the cochlear tissue of each group. The results showed that the level of Bax, p53, caspase-3,9, in the model control group and the model plus Ligustrazine group were significantly higher than that in the control group. The expression level of Bcl-2 m RNA decreased significantly (P0.01). Compared with the model control group, the RNA expression level of Bax, p53, caspase-3,9 m in the rat cochlear tissue of the model tetramethylpyrazine group decreased significantly, while the Bcl-2 m RNA expression level increased significantly, and there was no significant difference in the indexes between the blank control group and the blank and ligustrazine group. 05). Conclusion: 1. intraperitoneal injection of cisplatin (dose 4mg/kg), once a day for 5 days, can successfully induce.2. tetramethylpyrazine in the rat model of drug-induced deafness, which can significantly reduce the level of p53, Bax, caspase-9, caspase-3 m RNA and protein expression in the rat cochlear tissue of model rats, up Bcl-2 m RNA and protein expression level.3. tetramethylpyrazine on drug sex The anti apoptosis effect of the cochlear hair cells in the deafness model rats may be realized by blocking or inhibiting the internal pathway of the initial stage of apoptosis. Third experimental study on the mechanism of the intervention mechanism of the external pathway of the apoptosis of the cochlear hair cells induced by cisplatin in the deafness model of the rat: explore the deafness model induced by TMP The intervention and mechanism of the early external pathway of the rat cochlear hair cell apoptosis. Materials and methods: 62 rats were divided into two groups by random number method, the normal group (group N, 11), model replicating group (group M, 51). The normal group rats were not treated, and the rats in the model replicating group were induced by cisplatin intraperitoneal injection. After the model replication was successful, the normal group rats were randomly divided into two groups: normal control group (group N1) and ligustrazine control group (group N2). The model replicating group was randomly divided into two groups: model control group (M1), tmw treatment group (M2). The normal group was given intraperitoneal injection of saline. Injection, 5ml/kg weight, continuous injection for 5 days. The model replicating group was given intraperitoneal injection of Cisplatin Injection and 4mg/kg body weight for 5 days. On day 6-12: normal control group was given intraperitoneal injection of saline, 5ml/kg weight, continuous injection for 7 days. Ligustrazine control group was given intraperitoneal injection of ligustrazine injection, 140mg/kg weight, continuous injection for 7 days. The control group was given intraperitoneal injection of saline, 5ml/kg weight, continuous injection for 7 days. Ligustrazine treatment group was given intraperitoneal injection of ligustrazine injection, 140mg/kg weight, continuous injection for 7 days. After the last administration, 1H, rats were killed and cochlear tissue was stripped at -80 C refrigerator and used for the detection of fas/fas L, caspase-8m RNA and protein expression level. Results: 1. The experimental results of fas/fas L and caspase-8 protein expression in the cochlear tissues of rats in each group showed that: compared with the blank control group, the level of fas/fas L and caspase-8 protein expression in the rat cochlear tissues of the model control group and the model Ligustrazine group increased significantly (P0.01). Compared with the model control group, the model plus Ligustrazine group had fas/fas in the cochlear tissue of the Ligustrazine group. L, Caspase-8 protein expression level was significantly decreased (P0.01), and there was no significant difference between the blank control group and the blank Kagawa group (P0.05) the fas/fas L in the cochlear tissue of each group of.2. rats, the experimental results of the caspase-8 m RNA expression showed that the model control group and the model plus Ligustrazine group were compared with the blank group. The expression level of fas/fas L and caspase-8 m RNA in the worm tissues was significantly higher (P0.01). Compared with the model control group, the expression level of fas/fas L and caspase-8 m RNA in the rat cochlear tissue of the model plus Ligustrazine group was significantly lower (P0.01), and there was no significant difference between the blank control group and the blank Kagawa group. Conclusion: 1. Ligusticum chuanxiong. The anti apoptosis effect of fas/fas L, Caspase-8 m RNA and protein expression level.2. tetramethylpyrazine on the cochlear hair cells of drug-induced deafness model rats may be achieved by blocking or inhibiting the external pathway of the initial stage of apoptosis.
【学位授予单位】：辽宁中医药大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R965

【参考文献】