六味地黄丸对兔椎间盘体外退变模型间盘细胞MAPK信号级联的影响
发布时间：2019-06-10 17:28
【摘要】：目的:通过观察六味地黄丸对兔椎间盘体外退变模型椎间盘细胞MAPK信号级联的影响,探讨六味地黄丸防治椎间盘退变的疗效。方法:含药血清制备:6月龄清洁级新西兰白兔5只,体重2.0-3.2kg之间,湖南中医药大学动物中心提供,以六味地黄丸悬液灌胃,按人和动物体表面积比计算动物等效给药量给药,空白对照组灌以等量生理盐水。每天两次,共7天,自然喂养,标准饲养环境。在最后一次灌胃4小时后,经心脏穿刺采血,离心、过滤、灭活后备用。选取6月龄清洁级新西兰兔20只,参照Haschtmann方法建立整体椎间盘体外培养体系,空气栓塞法处死动物,无菌条件下采用后正中入路或经腹腔前入路取出带终板的L1-L6椎间盘100只,尽量去除终板上附着的骨组织及韧带,保留终板软骨的完整性,连续以0.9%Na Cl溶液,聚烯吡酮磺溶液以及含55 mmol/L柠檬酸钠、50μg/m L庆大霉素的Hanks液漂洗。椎间盘样本置于六孔板中,放在转鼓上(转鼓的转速为10r?h-1),以含5%胎牛血清、50μg/m L庆大霉素、20mmol/L柠檬酸钠的DMEM/F12培养,置于37℃、5%CO2、100%相对湿度温箱中过夜。随机分为空白对照组,TNF-α组、p38-JNK/SAPK阻断组,六味地黄丸血清组、TNF-α+含六味地黄丸血清组,将过夜后标本接种于六孔板,加入8ml含10%胎牛血清、50μg/m L庆大霉素、25μg/m L维生素C的DMEM/F12,置于37℃、5%CO2、100%相对湿度温箱中培养,每两天换液一次。TNF-α组培养液中另含10ng/μl TNF-α2μl;p38-JNK阻断组另含p38MAPK特异性阻断剂SB203580,JNK特异性阻断剂SP600125各20μmol/L各10μl,10ng/μl TNF-α2μl,六味地黄丸血清组培养液中另含六味地黄丸血清10%;TNF-α+含六味地黄丸血清组含10ng/μl TNF-α2μl,六味地黄丸血清10%。分别于培养的第2、4、8、14天收集标本待测。结果:同一时间点的组间比较,1.TNF-α组与空白组比较,有显著差异P0.05,TNF-α组的JNK及P38通路的m RNA含量、蛋白表达量均明显高于空白组,在光镜下纤维环结构的裂隙明显增加,髓核细胞的形态变化明显。说明椎间盘已进入退变过程,造模成功。2.P38.JNK阻断剂组与TNF-α+六味地黄丸血清组比较,P0.05,P38.JNK阻断剂组的JNK及P38通路的m RNA含量、蛋白表达量均明显低于TNF-α+六味地黄丸血清组。TNF-α+六味地黄丸血清组与TNF-α组比较,P0.05,有明显差异。TNF-α+六味地黄丸血清组的JNK及P38通路的m RNA含量、蛋白表达量均明显低于TNF-α组,可说明六味地黄丸可一定程度阻滞JNK及P38通路激活,延缓椎间盘退变。不同时间点的组内比较,1.TNF-α组的JNK及P38通路的m RNA含量、蛋白表达量在2d与4d、4d与8d、8d与14d相比有明显差异P0.05,含量一直呈上升趋势,在光镜下随着时间的推移,纤维环结构的裂隙明显增加,髓核细胞的形态变化明显,可说明造模成功。2.TNF-α+六味地黄丸血清组JNK及P38通路的m RNA含量、蛋白表达量在2d与4d、4d与8d、8d与14d相比有明显差异P0.05,表达含量呈上升趋势,但是上升趋势明显低于TNF-α组,也说明了六味地黄丸可一定程度阻滞JNK及P38通路激活,延缓椎间盘退变。结论:六味地黄丸可通过一定程度的抑制兔椎间盘细胞JNK、P38通路的基因和蛋白表达,减轻椎间盘细胞的炎症反应,延缓椎间盘退变进程,起到保护椎间盘细胞的作用。
[Abstract]:Objective: To study the effect of Liuwei Dihuang pill on the control of disc degeneration in the disc of rabbit intervertebral disc in vitro. Methods: The preparation of drug-containing serum:5 rabbits with 6-month-old clean-grade New Zealand white rabbits and 2.0-3.2kg of body weight, were supplied by the animal center of the Chinese University of Traditional Chinese Medicine in Hunan Province. The body surface area of the six-part-six-month-old New Zealand white rabbits was given by gavage, and the equivalent dose of the animals was calculated according to the body surface area of the human and the animals. The blank control group was given the same amount of normal saline. Twice daily for 7 days, natural feeding, standard feeding environment. After the last gastric administration for 4 hours, the blood was collected by the heart, centrifuged, filtered and inactivated for backup. 20 of the six-month-old clean-grade New Zealand rabbits were selected, the whole-disc in-vitro culture system was established with reference to the Haschtmann method, the animals were sacrificed by the air embolism method, The bone tissue and ligament attached to the endplates were removed as much as possible, the integrity of the end plate cartilage was preserved, and the Hanks solution containing 55 mmol/ L sodium citrate and 50. m u.g/ m L of gentamicin was continuously rinsed with a 0.9% Na Cl solution, a polyalkenylone sulfonate solution, and a 55 mmol/ L sodium citrate, 50.mu. g/ m L Gentamicin. The disc sample was placed in a six-well plate, placed on a drum (the rotational speed of the drum was 10 r? h-1), cultured in DMEM/ F12 containing 5% fetal bovine serum,50 & mu; g/ m L of gentamicin, and 20 mmol/ L sodium citrate, and was placed overnight at 37.degree. C.,5% CO2, and 100% relative humidity. and randomly divided into a blank control group, a TNF-antigen group, a p38-JNK/ SAPK blocking group, a six-flavor dihuang pill serum group, a TNF-1 + containing Liuwei Dihuang pill serum group, an overnight sample is inoculated on a six-well plate,8 ml of DMEM/ F12 containing 10% fetal bovine serum,50 & mu; g/ m L of gentamicin and 25 & mu; g/ m L of vitamin C is added, and is placed at 37 & deg; C; Culture in 5% CO2 and 100% relative humidity incubator, and once every two days. The other contains 10 ng/. mu.l of TNF-HC2.mu. l in the culture solution of TNF-JNK, and the other p38MAPK-specific blocking agent SB203580 and JNK-specific blocking agent SP600125 in the p38-JNK blocking group contains 10. mu. l,10 ng/ mu. l of TNF-2. mu. l, and the other six-wei Dihuang pill serum is 10% in the serum group culture solution of the Liuwei Dihuang Pills. The serum group of TNF-1 + containing Liuwei Dihuang pill contains 10 ng/. mu.l of TNF-2. mu. l, and the serum of Liuwei Dihuang pill is 10%. The specimens were collected on day 2,4,8 and 14 of culture, respectively. Results: Compared with the blank group, the amount of mRNA and protein expression of the JNK and P38 in the TNF-linked group were significantly higher than that of the blank group, and the fracture of the fiber ring structure was significantly increased under the light microscope. The morphological changes of the nucleus pulposus cells were significant. The results showed that the expression of mRNA and protein of JNK and P38 in the group of JNK and P38 in the group of P38. JNK blocker group and TNF-1 + Liuwei Dihuang pill group were significantly lower than that in the serum group of TNF-1 + Liuwei Dihuang pill. The serum group of TNF-1 + Liuwei Dihuang pill group was compared with that of TNF-1 group, P 0.05, and there was a significant difference. The expression of mRNA and protein of JNK and P38 in the serum of TNF-1 + Liuwei Dihuang Pills was significantly lower than that of the TNF-1 group, and the activation of JNK and P38 pathway could be blocked to a certain extent, and the degeneration of the disc was delayed. The expression of m-RNA of JNK and P38 in TNF-1 group was significantly different between 2d and 4d, 4d, 8d, 8d and 14d. 2. The expression of m-RNA of JNK and P38 in the serum group of TNF-1 + 6-wei Dihuang Pills was significantly different from that of the 4-d,4-d, and 8d, 8d, and 14d, and the expression level was on the rise. However, that increase trend is obviously lower than that of the TNF-1 group, and the six-wei-dihuang pill can block the activation of JNK and P38 via a certain degree, and delay the degeneration of the disc. Conclusion: Liuwei Dihuang Wan can inhibit the expression of the gene and protein of the JNK and P38 pathway of the rabbit intervertebral disc cells to a certain extent, reduce the inflammatory reaction of the intervertebral disc cells, delay the disc degeneration, and play a role in protecting the intervertebral disc cells.
【学位授予单位】：湖南中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R285.5;R-332
本文编号：2496611
[Abstract]:Objective: To study the effect of Liuwei Dihuang pill on the control of disc degeneration in the disc of rabbit intervertebral disc in vitro. Methods: The preparation of drug-containing serum:5 rabbits with 6-month-old clean-grade New Zealand white rabbits and 2.0-3.2kg of body weight, were supplied by the animal center of the Chinese University of Traditional Chinese Medicine in Hunan Province. The body surface area of the six-part-six-month-old New Zealand white rabbits was given by gavage, and the equivalent dose of the animals was calculated according to the body surface area of the human and the animals. The blank control group was given the same amount of normal saline. Twice daily for 7 days, natural feeding, standard feeding environment. After the last gastric administration for 4 hours, the blood was collected by the heart, centrifuged, filtered and inactivated for backup. 20 of the six-month-old clean-grade New Zealand rabbits were selected, the whole-disc in-vitro culture system was established with reference to the Haschtmann method, the animals were sacrificed by the air embolism method, The bone tissue and ligament attached to the endplates were removed as much as possible, the integrity of the end plate cartilage was preserved, and the Hanks solution containing 55 mmol/ L sodium citrate and 50. m u.g/ m L of gentamicin was continuously rinsed with a 0.9% Na Cl solution, a polyalkenylone sulfonate solution, and a 55 mmol/ L sodium citrate, 50.mu. g/ m L Gentamicin. The disc sample was placed in a six-well plate, placed on a drum (the rotational speed of the drum was 10 r? h-1), cultured in DMEM/ F12 containing 5% fetal bovine serum,50 & mu; g/ m L of gentamicin, and 20 mmol/ L sodium citrate, and was placed overnight at 37.degree. C.,5% CO2, and 100% relative humidity. and randomly divided into a blank control group, a TNF-antigen group, a p38-JNK/ SAPK blocking group, a six-flavor dihuang pill serum group, a TNF-1 + containing Liuwei Dihuang pill serum group, an overnight sample is inoculated on a six-well plate,8 ml of DMEM/ F12 containing 10% fetal bovine serum,50 & mu; g/ m L of gentamicin and 25 & mu; g/ m L of vitamin C is added, and is placed at 37 & deg; C; Culture in 5% CO2 and 100% relative humidity incubator, and once every two days. The other contains 10 ng/. mu.l of TNF-HC2.mu. l in the culture solution of TNF-JNK, and the other p38MAPK-specific blocking agent SB203580 and JNK-specific blocking agent SP600125 in the p38-JNK blocking group contains 10. mu. l,10 ng/ mu. l of TNF-2. mu. l, and the other six-wei Dihuang pill serum is 10% in the serum group culture solution of the Liuwei Dihuang Pills. The serum group of TNF-1 + containing Liuwei Dihuang pill contains 10 ng/. mu.l of TNF-2. mu. l, and the serum of Liuwei Dihuang pill is 10%. The specimens were collected on day 2,4,8 and 14 of culture, respectively. Results: Compared with the blank group, the amount of mRNA and protein expression of the JNK and P38 in the TNF-linked group were significantly higher than that of the blank group, and the fracture of the fiber ring structure was significantly increased under the light microscope. The morphological changes of the nucleus pulposus cells were significant. The results showed that the expression of mRNA and protein of JNK and P38 in the group of JNK and P38 in the group of P38. JNK blocker group and TNF-1 + Liuwei Dihuang pill group were significantly lower than that in the serum group of TNF-1 + Liuwei Dihuang pill. The serum group of TNF-1 + Liuwei Dihuang pill group was compared with that of TNF-1 group, P 0.05, and there was a significant difference. The expression of mRNA and protein of JNK and P38 in the serum of TNF-1 + Liuwei Dihuang Pills was significantly lower than that of the TNF-1 group, and the activation of JNK and P38 pathway could be blocked to a certain extent, and the degeneration of the disc was delayed. The expression of m-RNA of JNK and P38 in TNF-1 group was significantly different between 2d and 4d, 4d, 8d, 8d and 14d. 2. The expression of m-RNA of JNK and P38 in the serum group of TNF-1 + 6-wei Dihuang Pills was significantly different from that of the 4-d,4-d, and 8d, 8d, and 14d, and the expression level was on the rise. However, that increase trend is obviously lower than that of the TNF-1 group, and the six-wei-dihuang pill can block the activation of JNK and P38 via a certain degree, and delay the degeneration of the disc. Conclusion: Liuwei Dihuang Wan can inhibit the expression of the gene and protein of the JNK and P38 pathway of the rabbit intervertebral disc cells to a certain extent, reduce the inflammatory reaction of the intervertebral disc cells, delay the disc degeneration, and play a role in protecting the intervertebral disc cells.
【学位授予单位】：湖南中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R285.5;R-332
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