探讨糖尿病大鼠椎间盘中TGF-β和IGF-1的含量变化及其对糖尿病大鼠椎间盘的作用意义

发布时间：2018-04-04 14:20

本文选题：椎间盘　切入点：糖尿病　出处：《河北医科大学》2017年硕士论文

【摘要】：目的:测定链脲佐菌素处理后造成糖尿病状态的大鼠和正常大鼠椎间盘组织中转化生长因子β(TGF-β)和胰岛素样生长因子-1(IGF-1)在三个不同时期的含量变化,并探讨含量之间差异的意义。方法:1 3月龄成熟雄性SD大鼠30只,随机分为实验组以及对照组,每组SD大鼠各15只,实验组SD大鼠采用腹腔单次注射链脲佐菌素溶液(strep-tozotoein,STZ),通过链脲佐菌素对胰腺胰岛β-细胞的制毒作用,致使胰岛素生成障碍,从而造成糖尿病状态,建立糖尿病大鼠模型,对照组SD大鼠不做特殊处理,仅给予腹腔单次注射柠檬酸盐缓冲液。2分别在第2、4、6周时,从实验组和对照组各取5只大鼠,用1%戊巴比妥钠进行腹腔注射处死大鼠后,取其各组SD大鼠新鲜腰椎间盘组织,用4%多聚甲醛进行固定,梯度乙醇脱水,包埋成石蜡组织块。分别行苏木素-伊红(HE)染色法,二甲苯透明后,中性树脂封片显微镜下观察两组大鼠椎间盘组织病理变化情况。免疫组化法,脱蜡加入一抗二抗后,辣根酶标记,DAB显色显微镜下观察两组大鼠椎间盘组织中TGF-β和IGF-1的含量。选取其中最有意义的,阳性表达最强的5个视野进行计算,以细胞质着色和阳性细胞率为判断标准,统计两组大鼠椎间盘中TGF-β和IGF-1的表达情况。结果:1经链脲佐菌素注射液处理后的糖尿病组模型大鼠,血糖浓度明显升高,与正常组对比,差异有统计学意义(P0.05)。糖尿病组血糖浓度分别在第2、4、6周时相对于正常组,差异有统计学意义(P0.05)。2在3个不同时期,实验组大鼠的椎间盘均出现不同程度的退行性改变,HE染色显示可见脊索细胞减少,增生出较多胶原纤维,终板呈不同程度的钙化,出现类软骨细胞,纤维环呈蜿蜒状排列,髓核基质排列紊乱、减少,呈不同程度的皱缩,髓核及纤维环向外膨出;部分髓核细胞排列成簇,这是椎间盘退变的标志。而对照组正常大鼠,椎间盘结构正常,终板由一层透明软骨组成,纤维环具有规则的层状结构,胶原束排列整齐平行,以成纤维细胞为主,髓核与纤维环分界清楚,其中成条索状分布的脊索细胞较为丰富,基质中细胞充实。3免疫组化法测定,对照组大鼠中TGF-β染色阳性率在3个不同时期无差异(P=0.368),对照组大鼠中IGF-1染色阳性率在3个不同时期无差异(P=0.368);实验组大鼠中TGF-β的阳性染色率在3个不同时期无差异(P=0.311),实验组大鼠中IGF-1的阳性染色率在3个不同时期无差异(P=0.727)。实验组大鼠椎间盘组织中TGF-β的含量在3个不同时期均明显低于对照组(P0.05)。实验组大鼠椎间盘组织中IGF-1的含量在3个不同时期均明显低于对照组(P0.05)。结论:本研究结果显示实验组中STZ诱导的糖尿病大鼠椎间盘中TGF-β和IGF-1含量较对照组正常大鼠明显减少,在椎间盘HE染色中可以观察到,实验组糖尿病大鼠椎间盘的形态发生了退变性改变,这种TGF-β和IGF-1含量的降低,引起了椎间盘内基质的降解,抑制软骨基质的合成,使其未分化间充质细胞的分化减退,加速了椎间盘内的胶原水解,降低椎间盘内II型胶原和蛋白多糖等软骨细胞表型的能力,最终使椎间盘因为缺少足够的基质水分而发生弹性下降、结构紊乱、稳定性及代谢生理功能异常等改变,导致椎间盘退变。本研究结果证明了糖尿病使椎间盘内TGF-β和IGF-1的含量发生降低,这可能是导致糖尿病患者椎间盘发生退行性改变的原因之一。这为今后糖尿病患者预防、治疗椎间盘发生的退行性改变提供了新的治疗思路。
[Abstract]:Objective: to determine the streptozotocin treated by diabetes status and normal rat intervertebral disc tissue transforming growth factor beta (TGF- beta) and insulin-like growth factor -1 (IGF-1) in three different periods of the content changes, and to explore the significance of the difference between the content. Method: 13 month old adult male 30 SD rats were randomly divided into experimental group and control group, each group of SD rats, 15 rats in each experimental group, SD rats by a single intraperitoneal injection of streptozotocin solution (strep-tozotoein, STZ), on pancreatic islet beta cell precursor by STZ, resulting in insulin production obstacles, resulting in the diabetic state, establish the model of diabetic rats, the control group of SD rats did not do special treatment, only received a single intraperitoneal injection of citrate buffer.2 respectively in 2,4,6 weeks, from each experimental group and control group 5 rats, abdominal with 1% pentobarbital sodium The rats were sacrificed after injection, the group of SD rats fresh lumbar intervertebral disc tissue was fixed with 4% paraformaldehyde, dehydrated in graded ethanol and embedded into paraffin blocks respectively. Hematoxylin and eosin (HE) staining, xylene transparent, observe two groups of rat intervertebral disc tissue the change of neutral resin mounting microscope. Immunohistochemical method, adding a two anti anti dewaxing, HRP, DAB color microscope TGF- P and IGF-1 two groups of rat intervertebral disc tissues. The most significant, the 5 strongest vision positive expression was calculated by cytoplasmic staining and positive cell rate of the expression of statistical criteria, two groups of rats intervertebral discs TGF- beta and IGF-1. Results: 1 the diabetic group rats by streptozotocin injection after treatment, blood glucose concentration increased significantly, compared with the normal group, the difference was statistically significant (P0.05). The blood glucose concentration in the diabetic group were 2,4,6 weeks when compared with normal group, the difference was statistically significant (P0.05.2) in 3 different periods, the intervertebral disc of the experimental rats showed varying degrees of degenerative change, HE staining showed that the notochord cells decreased, more collagen fiber hyperplasia, endplate was different degrees of calcification, like cartilage cells, the fiber ring is arranged in the form of winding, nucleus pulposus disorder, reduce, showed different degrees of shrinkage, nucleus pulposus and annulus bulge outward; part of the nucleus pulposus cells arranged in clusters, which is a sign of intervertebral disc degeneration. While the control group of normal rats, intervertebral disc structure normally, a layer of transparent cartilage endplate, layered annulus has rules, collagen bundles arranged parallel to fibroblasts, nucleus pulposus and annulus clear boundaries, which become a notochord cell cord distribution is abundant, medium In full.3 cells were determined by immunohistochemical method, the rats in the control group TGF- beta staining was no difference in 3 different periods (P=0.368), IGF-1 control group rats in the positive rate had no difference in 3 different periods (P=0.368); the positive staining of experimental rats in TGF- beta rate the differences in the 3 different periods (P=0.311), the positive staining of IGF-1 in experimental group rats was no difference in the 3 different periods (P=0.727). The content of the experimental group of rat intervertebral disc tissue of TGF- beta in 3 different periods were significantly lower than the control group (P0.05). The experimental group of rat intervertebral disc the fabric in the content of IGF-1 in 3 different periods were significantly lower than the control group (P0.05). Conclusion: the results of this study showed that the experimental group in STZ induced diabetic rats intervertebral discs TGF- beta and IGF-1 content compared with the control group of normal rats was significantly reduced, the intervertebral disc HE staining can be observed in the experimental group The morphogenesis of the intervertebral disc degenerative change, reduce the TGF- beta and IGF-1 content, caused the degradation of intervertebral disc matrix, inhibit the synthesis of cartilage matrix, the differentiation of undifferentiated mesenchymal cells decreased, accelerates the hydrolysis of collagen in the intervertebral disc, reduce the ability of phenotype intradiscal and collagen II protein sugar of cartilage cells, the intervertebral disc because of the lack of sufficient and elastic matrix moisture decreased, structure disorder, metabolic stability and physiological function abnormal, intervertebral disc degeneration. The results of this study proved that the content of diabetes intradiscal TGF- beta and IGF-1 incidence decreased, which may lead to intervertebral in patients with diabetes mellitus one of the causes of disc degeneration. This is the future of diabetes prevention, provide a new treatment strategy for degenerative intervertebral disc changes.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R681.5

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