蜂毒肽抑制终板软骨细胞中基质金属蛋白酶表达的相关性研究

发布时间：2018-05-15 05:32

本文选题：蜂毒 + 抑制　；参考：《蚌埠医学院》2017年硕士论文

【摘要】：背景:椎间盘退行性病变(Intervertebral Disc Degeneration,IVDD)已成为临床上常见的脊柱退变性疾病,主要表现为下腰痛及神经根病变,严重影响人们的生活和工作。但目前的主要治疗手段均属对症治疗,尚无有效的延缓或逆转椎间盘退变的治疗方案。软骨终板细胞中基质金属蛋白酶(matrix metalloproteinase,MMP)的表达及炎症的发生是终板退变的原因,也被认为是导致椎间盘退变的主要原因。蜂毒肽(Melittin)是从蜂蜜(西方蜂蜜)中提取的一种多肽,由于其具有延缓软骨退变的功效,从而被广泛地应用于中国、韩国及日本的传统医学中。研究表明,Melittin可以通过抑制NF-κB信号通路转导从而下调MMP的表达。因此,如果能实验证明Melittin能够通过抑制终板软骨细胞(Endplate Chondrocytes,EPCs)中MMP表达延缓其退变,进而抑制椎间盘退变,这就为Melittin在预防椎间盘退变方面提供了可能性。目的:建立大鼠EPCs细胞的体外自然传代的退变模型;观察大鼠EPCs的细胞学特性及生物学特性;对比大鼠退变EPCs与正常EPCs的形态学及Ⅱ型胶原蛋白表达差异;研究Melittin对大鼠EPCs中MMP表达的抑制情况。方法:1.无菌条件下取大鼠腰椎的终板软骨组织,用Ⅱ型胶原酶消化分离椎间盘终板软骨组织后培养,胰蛋白酶消化后传代培养;2.选取P1代及P5代使用倒置显微镜观察细胞形态,并行甲苯胺蓝染色及免疫荧光染色来对其进行鉴定;3.使用MTT法确定Melittin对于大鼠EPCs的最适浓度;4.随机选取P1代及P5代细胞进行实验分组:A.P1代空白对照;B.P1代+最适浓度的Melittin;C.P5代空白对照;D.P5代+最适浓度的Melittin;5.对干预后的四组细胞使用蛋白印迹分析法(western blot)检测MMP-3蛋白的表达情况。结果:1.培养出的EPCs传代后逐渐由多角形变为梭型;2.甲苯胺蓝染色细胞质中的酸性粘液物质(如蛋白多糖)则被染成深蓝色,免疫荧光染色标记Col-2,可见细胞骨架部分明显阳性表现(呈绿色荧光),P5代较P1代Col-2表达明显减弱,以上所见可证明所培养细胞为软骨细胞;3.MTT法测定Melittin的最适浓度为1μg/ml;4.蜂毒肽对于退变EPCs中的MMP蛋白的表达有抑制作用,并能够延缓终板软骨退变,表现为B、D较A、C组MMP蛋白表达明显减弱,B组较D组MMP蛋白表达明显减弱,其差别具有统计学意义(p0.05)。结论:Melittin能够抑制EPCs中MMP的表达,从而具有一定的终板软骨保护作用,为防治脊柱退变性疾病提供一定的实验依据。
[Abstract]:Background: Intervertebral Disc Degeneration (IVDD) has become a common degenerative disease of the spine clinically, mainly characterized by lower back pain and nerve root lesions, which seriously affect people's life and work. But the main treatment means are symptomatic treatment, and there is no effective treatment to delay or reverse the treatment of intervertebral disc degeneration. The expression of matrix metalloproteinase (matrix metalloproteinase, MMP) in cartilage endplate cells and the occurrence of inflammation is the cause of endplate degeneration, and is also considered to be the main cause of degeneration of the intervertebral disc. Melittin is a polypeptide derived from honey (Western honey), because it has the effect of retarding cartilage degeneration. It is widely used in traditional medicine in China, South Korea and Japan. Studies have shown that Melittin can down regulate the expression of MMP by inhibiting the NF- kappa B signaling pathway. Therefore, if it can prove that Melittin can retard the degeneration by inhibiting the MMP expression in the terminate cartilage cells (Endplate Chondrocytes, EPCs), and then inhibit the vertebra. Intervertebral disc degeneration, which provides the possibility of Melittin in preventing the degeneration of intervertebral disc. Objective: to establish a natural generation of degeneration model of rat EPCs cells in vitro; to observe the cytological and biological characteristics of EPCs in rats; to compare the morphology of the degenerative EPCs with the normal EPCs and the difference in the expression of type II collagen; and to study Melittin to the large. The inhibition of expression of MMP in rat EPCs. Methods: 1. under aseptic conditions, the end plate cartilage tissue of the lumbar vertebrae was taken and cultured with type II collagenase to isolate the intervertebral disc cartilage tissue. Trypsin was digested and cultured. 2. the cell morphology was observed by P1 and P5 by inverted microscope, and toluidine blue staining and immunofluorescence were used. Staining to identify it; 3. MTT was used to determine the optimal concentration of Melittin for EPCs in rats; 4. randomly selected P1 and P5 cells for experimental grouping: A.P1 generation blank control; B.P1 + + optimum concentration Melittin; C.P5 generation blank control; D.P5 generation + optimum concentration Melittin; 5. cells using Western blot analysis for the prognosis of four groups. The expression of MMP-3 protein was detected by method (Western blot). Results: 1. the cultured EPCs gradually changed from polygon to shuttle type, and the acid mucus in the cytoplasm of 2. toluidine blue (such as proteoglycan) was dyed dark blue and immunofluorescent staining labeled Col-2, so the cytoskeleton part was obviously positive (green fluorescence), P5 The expression of P1 generation Col-2 was obviously weakened. The above results showed that the cultured cells were chondrocytes, and the optimum concentration of Melittin was 1 u g/ml, and 4. melittin could inhibit the expression of MMP protein in the degenerative EPCs, and could delay the degeneration of the endplate cartilage, which showed B, D compared to A, and C group, the expression of MMP protein decreased obviously. The expression of protein was obviously weakened, and the difference was statistically significant (P0.05). Conclusion: Melittin can inhibit the expression of MMP in EPCs, thus it has a certain end plate cartilage protection, and provides a certain experimental basis for the prevention and treatment of spinal degenerative disease.

【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R681.5

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