神经肽CGRP、SP、NPY对人OA软骨细胞的影响研究
发布时间：2019-02-18 10:26
【摘要】：目的:利用神经肽CGRP、SP、NPY干预体外培养人OA软骨细胞,同时检测MMP-l、MMP-3、MMP-13的分泌。探索神经肽CGRP、SP、NPY对人OA软骨细胞增殖的同时是否抑制MMP-l、MMP-3、MMP-13的分泌。为探讨神经肽增殖软骨细胞,寻找OA的药物治疗提供理论基础。方法:1、采用II型胶原酶消化法处理人膝关节软骨标本从而取得大量高纯度的OA软骨细胞,采用II型胶原免疫组化法对所取得的OA软骨细胞进行鉴定,甲苯胺蓝染色法检测OA软骨细胞的存活率。2、分为实验组和对照组,分别用神经肽CGRP、SP、NPY对OA软骨细胞进行干预,对照组用等量PBS处理。采用MTT法检测各组细胞的增殖并绘制曲线。3、Ki67免疫法检测细胞增殖率。4、采用Westen Blot检测细胞MMP-l、MMP-3、MMP-13的表达。结果:1、OA软骨细胞体外培养后呈三角或多角等不规则形状,向四周爬行生长,并且前三代细胞基本上能较好的保持原有的细胞表型、形态及功能。2、在所测定的时间点内(3d、5d、7d)所测得的增殖神经肽CGRP、SP、NPY对OA软骨细胞均有增殖作用,其中20μm CGRP在第5天增殖达最高峰(1.048±0.181);20μm SP在第5天增殖达最高峰(1.132±.0537);50μm NPY第5天增殖达最高峰(0.806±0.115)。各组在所测定的时间点内(3d、5d、7d)均较空白组(P0.001),差异显著。3、ki67免疫组化示软骨细胞呈椭圆形、梭形、多角形或者不规则形;棕色阳性颗粒包绕细胞核,空白对照组可见少量细胞内多核,处于分裂增殖状态。神经肽干扰组可见大部分细胞多核,处于分裂增殖状态。Ki67阳性率为空白组(0.265±0.031)x100%,CGRP组为(0.562±0.042)x100%、SP组为(0.558±0.036)x100%、NPY组为(0.649±0.055)x100%,较空白组差异显著(P0.001)。4、west blot示神经肽处理细胞后与空白组对照比较,经过Quantity one图像分析。MMP-1、MMP-3、MMP-13蛋白相对表达量显著下调。其中MMP1蛋白相对表达量NPY组为1.744±0.238和CGRP组为1.562±0.346较空白组4.951±2.445下调显著,MMP3蛋白相对表达量CGRP组为1.432±0.823较空白组4.389±1.572下调显著,MMP13蛋白相对表达量NPY组为0.629±0.266和CGRP组为0.732±0.089较空白组3.182±3.129下调显著,神经肽各组较空白组差异显著(P0.05)。结论:1、II型胶原酶一步消化法对于OA软骨细胞培养,可以获得稳定的OA软骨细胞。2、神经肽CGRP、SP、NPY对OA软骨细胞均有增殖作用,并且在第5天是可以达到最高峰,随着时间延长逐渐趋于稳定。3、神经肽CGRP、SP、NPY可以促进抑制MMP-1、MMP-3、MMP-13蛋白的产生,从而能阻止OA软骨细胞的凋亡,为治疗OA提供理论基础。
[Abstract]:Aim: to investigate the effect of neuropeptide CGRP,SP,NPY on human OA chondrocytes cultured in vitro and to detect the secretion of MMP-l,MMP-3,MMP-13. To explore whether neuropeptide CGRP,SP,NPY inhibits the secretion of MMP-l,MMP-3,MMP-13 while inhibiting the proliferation of human OA chondrocytes. To explore the proliferation of neuropeptide chondrocytes and search for the drug treatment of OA provides a theoretical basis. Methods: 1. A large number of OA chondrocytes were obtained by II collagenase digestion, and the OA chondrocytes were identified by II collagen immunohistochemical method. The survival rate of OA chondrocytes was detected by toluidine blue staining. 2 the chondrocytes were divided into experimental group and control group. OA chondrocytes were treated with neuropeptide CGRP,SP,NPY and the control group was treated with the same amount of PBS. The proliferation of cells in each group was detected by MTT method and the curve was drawn. 3The proliferative rate of cells was detected by immunocytochemistry. 4. The expression of MMP-l,MMP-3,MMP-13 was detected by Westen Blot. Results: 1 OA chondrocytes were cultured in vitro with irregular shape such as triangle or polygonal, crawling around, and the first three generations of cells could maintain the original phenotypic cells, morphology and function. 2. The proliferative neuropeptide CGRP,SP,NPY obtained at the measured time point (3 d ~ 5 d ~ 7 d) could proliferate OA chondrocytes, and 20 渭 m CGRP reached a peak of (1.048 卤0.181) on the 5th day. The proliferation of 20 渭 m SP reached the highest peak on the 5th day (1.132 卤0537) and 50 渭 m NPY on the 5th day (0.806 卤0.115). Compared with the control group (P0.001), the difference was significant in each group (3 days and 5 days after 7 days). The immunohistochemical staining showed that the chondrocytes were elliptical, fusiform, polygonal or irregular in shape. The brown positive granules wrapped around the nucleus, while in the blank control group, a small number of polynuclei were found in the cells, which were in the state of mitosis and proliferation. The positive rate of Ki67 was (0.265 卤0.031) x 100 and (0.562 卤0.042) x 100 in the control group and (0.558 卤0.036) x 100 in the SP group. The NPY group was (0.649 卤0.055) x 100g, which was significantly different from the blank group (P0.001). 4The blot showed that the cells were treated with neuropeptide and compared with the control group. The results of Quantity one image analysis. MMP-1,MMP-3,. The relative expression of MMP-13 protein was significantly down-regulated. The relative expression of MMP1 protein in NPY group (1.744 卤0.238) and CGRP group (1.562 卤0.346) was significantly lower than that in blank group (4.951 卤2.445). The relative expression of MMP3 protein in CGRP group (1.432 卤0.823) was significantly lower than that in blank group (4.389 卤1.572). The relative expression of MMP13 protein in NPY group (0.629 卤0.266) and CGRP group (0.732 卤0.089) was significantly lower than that in blank group (3.182 卤3.129). Conclusion: 1 stable OA chondrocytes can be obtained by one-step digestion of type II collagenase on OA chondrocytes. 2. Neuropeptide CGRP,SP,NPY can proliferate OA chondrocytes and reach the peak on the 5th day. The neuropeptide CGRP,SP,NPY can promote the inhibition of the production of MMP-1,MMP-3,MMP-13 protein, thus prevent the apoptosis of OA chondrocytes, and provide a theoretical basis for the treatment of OA.
【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R684.3
本文编号：2425779
[Abstract]:Aim: to investigate the effect of neuropeptide CGRP,SP,NPY on human OA chondrocytes cultured in vitro and to detect the secretion of MMP-l,MMP-3,MMP-13. To explore whether neuropeptide CGRP,SP,NPY inhibits the secretion of MMP-l,MMP-3,MMP-13 while inhibiting the proliferation of human OA chondrocytes. To explore the proliferation of neuropeptide chondrocytes and search for the drug treatment of OA provides a theoretical basis. Methods: 1. A large number of OA chondrocytes were obtained by II collagenase digestion, and the OA chondrocytes were identified by II collagen immunohistochemical method. The survival rate of OA chondrocytes was detected by toluidine blue staining. 2 the chondrocytes were divided into experimental group and control group. OA chondrocytes were treated with neuropeptide CGRP,SP,NPY and the control group was treated with the same amount of PBS. The proliferation of cells in each group was detected by MTT method and the curve was drawn. 3The proliferative rate of cells was detected by immunocytochemistry. 4. The expression of MMP-l,MMP-3,MMP-13 was detected by Westen Blot. Results: 1 OA chondrocytes were cultured in vitro with irregular shape such as triangle or polygonal, crawling around, and the first three generations of cells could maintain the original phenotypic cells, morphology and function. 2. The proliferative neuropeptide CGRP,SP,NPY obtained at the measured time point (3 d ~ 5 d ~ 7 d) could proliferate OA chondrocytes, and 20 渭 m CGRP reached a peak of (1.048 卤0.181) on the 5th day. The proliferation of 20 渭 m SP reached the highest peak on the 5th day (1.132 卤0537) and 50 渭 m NPY on the 5th day (0.806 卤0.115). Compared with the control group (P0.001), the difference was significant in each group (3 days and 5 days after 7 days). The immunohistochemical staining showed that the chondrocytes were elliptical, fusiform, polygonal or irregular in shape. The brown positive granules wrapped around the nucleus, while in the blank control group, a small number of polynuclei were found in the cells, which were in the state of mitosis and proliferation. The positive rate of Ki67 was (0.265 卤0.031) x 100 and (0.562 卤0.042) x 100 in the control group and (0.558 卤0.036) x 100 in the SP group. The NPY group was (0.649 卤0.055) x 100g, which was significantly different from the blank group (P0.001). 4The blot showed that the cells were treated with neuropeptide and compared with the control group. The results of Quantity one image analysis. MMP-1,MMP-3,. The relative expression of MMP-13 protein was significantly down-regulated. The relative expression of MMP1 protein in NPY group (1.744 卤0.238) and CGRP group (1.562 卤0.346) was significantly lower than that in blank group (4.951 卤2.445). The relative expression of MMP3 protein in CGRP group (1.432 卤0.823) was significantly lower than that in blank group (4.389 卤1.572). The relative expression of MMP13 protein in NPY group (0.629 卤0.266) and CGRP group (0.732 卤0.089) was significantly lower than that in blank group (3.182 卤3.129). Conclusion: 1 stable OA chondrocytes can be obtained by one-step digestion of type II collagenase on OA chondrocytes. 2. Neuropeptide CGRP,SP,NPY can proliferate OA chondrocytes and reach the peak on the 5th day. The neuropeptide CGRP,SP,NPY can promote the inhibition of the production of MMP-1,MMP-3,MMP-13 protein, thus prevent the apoptosis of OA chondrocytes, and provide a theoretical basis for the treatment of OA.
【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R684.3
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