神經(jīng)肽CGRP、SP、NPY對(duì)人OA軟骨細(xì)胞的影響研究
發(fā)布時(shí)間：2019-02-18 10:26
【摘要】：目的:利用神經(jīng)肽CGRP、SP、NPY干預(yù)體外培養(yǎng)人OA軟骨細(xì)胞,同時(shí)檢測(cè)MMP-l、MMP-3、MMP-13的分泌。探索神經(jīng)肽CGRP、SP、NPY對(duì)人OA軟骨細(xì)胞增殖的同時(shí)是否抑制MMP-l、MMP-3、MMP-13的分泌。為探討神經(jīng)肽增殖軟骨細(xì)胞,尋找OA的藥物治療提供理論基礎(chǔ)。方法:1、采用II型膠原酶消化法處理人膝關(guān)節(jié)軟骨標(biāo)本從而取得大量高純度的OA軟骨細(xì)胞,采用II型膠原免疫組化法對(duì)所取得的OA軟骨細(xì)胞進(jìn)行鑒定,甲苯胺藍(lán)染色法檢測(cè)OA軟骨細(xì)胞的存活率。2、分為實(shí)驗(yàn)組和對(duì)照組,分別用神經(jīng)肽CGRP、SP、NPY對(duì)OA軟骨細(xì)胞進(jìn)行干預(yù),對(duì)照組用等量PBS處理。采用MTT法檢測(cè)各組細(xì)胞的增殖并繪制曲線。3、Ki67免疫法檢測(cè)細(xì)胞增殖率。4、采用Westen Blot檢測(cè)細(xì)胞MMP-l、MMP-3、MMP-13的表達(dá)。結(jié)果:1、OA軟骨細(xì)胞體外培養(yǎng)后呈三角或多角等不規(guī)則形狀,向四周爬行生長(zhǎng),并且前三代細(xì)胞基本上能較好的保持原有的細(xì)胞表型、形態(tài)及功能。2、在所測(cè)定的時(shí)間點(diǎn)內(nèi)(3d、5d、7d)所測(cè)得的增殖神經(jīng)肽CGRP、SP、NPY對(duì)OA軟骨細(xì)胞均有增殖作用,其中20μm CGRP在第5天增殖達(dá)最高峰(1.048±0.181);20μm SP在第5天增殖達(dá)最高峰(1.132±.0537);50μm NPY第5天增殖達(dá)最高峰(0.806±0.115)。各組在所測(cè)定的時(shí)間點(diǎn)內(nèi)(3d、5d、7d)均較空白組(P0.001),差異顯著。3、ki67免疫組化示軟骨細(xì)胞呈橢圓形、梭形、多角形或者不規(guī)則形;棕色陽(yáng)性顆粒包繞細(xì)胞核,空白對(duì)照組可見(jiàn)少量細(xì)胞內(nèi)多核,處于分裂增殖狀態(tài)。神經(jīng)肽干擾組可見(jiàn)大部分細(xì)胞多核,處于分裂增殖狀態(tài)。Ki67陽(yáng)性率為空白組(0.265±0.031)x100%,CGRP組為(0.562±0.042)x100%、SP組為(0.558±0.036)x100%、NPY組為(0.649±0.055)x100%,較空白組差異顯著(P0.001)。4、west blot示神經(jīng)肽處理細(xì)胞后與空白組對(duì)照比較,經(jīng)過(guò)Quantity one圖像分析。MMP-1、MMP-3、MMP-13蛋白相對(duì)表達(dá)量顯著下調(diào)。其中MMP1蛋白相對(duì)表達(dá)量NPY組為1.744±0.238和CGRP組為1.562±0.346較空白組4.951±2.445下調(diào)顯著,MMP3蛋白相對(duì)表達(dá)量CGRP組為1.432±0.823較空白組4.389±1.572下調(diào)顯著,MMP13蛋白相對(duì)表達(dá)量NPY組為0.629±0.266和CGRP組為0.732±0.089較空白組3.182±3.129下調(diào)顯著,神經(jīng)肽各組較空白組差異顯著(P0.05)。結(jié)論:1、II型膠原酶一步消化法對(duì)于OA軟骨細(xì)胞培養(yǎng),可以獲得穩(wěn)定的OA軟骨細(xì)胞。2、神經(jīng)肽CGRP、SP、NPY對(duì)OA軟骨細(xì)胞均有增殖作用,并且在第5天是可以達(dá)到最高峰,隨著時(shí)間延長(zhǎng)逐漸趨于穩(wěn)定。3、神經(jīng)肽CGRP、SP、NPY可以促進(jìn)抑制MMP-1、MMP-3、MMP-13蛋白的產(chǎn)生,從而能阻止OA軟骨細(xì)胞的凋亡,為治療OA提供理論基礎(chǔ)。
[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 鹵0.181) on the 5th day. The proliferation of 20 渭 m SP reached the highest peak on the 5th day (1.132 鹵0537) and 50 渭 m NPY on the 5th day (0.806 鹵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 鹵0.031) x 100 and (0.562 鹵0.042) x 100 in the control group and (0.558 鹵0.036) x 100 in the SP group. The NPY group was (0.649 鹵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 鹵0.238) and CGRP group (1.562 鹵0.346) was significantly lower than that in blank group (4.951 鹵2.445). The relative expression of MMP3 protein in CGRP group (1.432 鹵0.823) was significantly lower than that in blank group (4.389 鹵1.572). The relative expression of MMP13 protein in NPY group (0.629 鹵0.266) and CGRP group (0.732 鹵0.089) was significantly lower than that in blank group (3.182 鹵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.
【學(xué)位授予單位】：川北醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R684.3
本文編號(hào)：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 鹵0.181) on the 5th day. The proliferation of 20 渭 m SP reached the highest peak on the 5th day (1.132 鹵0537) and 50 渭 m NPY on the 5th day (0.806 鹵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 鹵0.031) x 100 and (0.562 鹵0.042) x 100 in the control group and (0.558 鹵0.036) x 100 in the SP group. The NPY group was (0.649 鹵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 鹵0.238) and CGRP group (1.562 鹵0.346) was significantly lower than that in blank group (4.951 鹵2.445). The relative expression of MMP3 protein in CGRP group (1.432 鹵0.823) was significantly lower than that in blank group (4.389 鹵1.572). The relative expression of MMP13 protein in NPY group (0.629 鹵0.266) and CGRP group (0.732 鹵0.089) was significantly lower than that in blank group (3.182 鹵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.
【學(xué)位授予單位】：川北醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R684.3
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