本文選題：蜂毒 + 抑制��； 參考：《蚌埠醫(yī)學(xué)院》2017年碩士論文

【摘要】：背景:椎間盤(pán)退行性病變(Intervertebral Disc Degeneration,IVDD)已成為臨床上常見(jiàn)的脊柱退變性疾病,主要表現(xiàn)為下腰痛及神經(jīng)根病變,嚴(yán)重影響人們的生活和工作。但目前的主要治療手段均屬對(duì)癥治療,尚無(wú)有效的延緩或逆轉(zhuǎn)椎間盤(pán)退變的治療方案。軟骨終板細(xì)胞中基質(zhì)金屬蛋白酶(matrix metalloproteinase,MMP)的表達(dá)及炎癥的發(fā)生是終板退變的原因,也被認(rèn)為是導(dǎo)致椎間盤(pán)退變的主要原因。蜂毒肽(Melittin)是從蜂蜜(西方蜂蜜)中提取的一種多肽,由于其具有延緩軟骨退變的功效,從而被廣泛地應(yīng)用于中國(guó)、韓國(guó)及日本的傳統(tǒng)醫(yī)學(xué)中。研究表明,Melittin可以通過(guò)抑制NF-κB信號(hào)通路轉(zhuǎn)導(dǎo)從而下調(diào)MMP的表達(dá)。因此,如果能實(shí)驗(yàn)證明Melittin能夠通過(guò)抑制終板軟骨細(xì)胞(Endplate Chondrocytes,EPCs)中MMP表達(dá)延緩其退變,進(jìn)而抑制椎間盤(pán)退變,這就為Melittin在預(yù)防椎間盤(pán)退變方面提供了可能性。目的:建立大鼠EPCs細(xì)胞的體外自然傳代的退變模型;觀察大鼠EPCs的細(xì)胞學(xué)特性及生物學(xué)特性;對(duì)比大鼠退變EPCs與正常EPCs的形態(tài)學(xué)及Ⅱ型膠原蛋白表達(dá)差異;研究Melittin對(duì)大鼠EPCs中MMP表達(dá)的抑制情況。方法:1.無(wú)菌條件下取大鼠腰椎的終板軟骨組織,用Ⅱ型膠原酶消化分離椎間盤(pán)終板軟骨組織后培養(yǎng),胰蛋白酶消化后傳代培養(yǎng);2.選取P1代及P5代使用倒置顯微鏡觀察細(xì)胞形態(tài),并行甲苯胺藍(lán)染色及免疫熒光染色來(lái)對(duì)其進(jìn)行鑒定;3.使用MTT法確定Melittin對(duì)于大鼠EPCs的最適濃度;4.隨機(jī)選取P1代及P5代細(xì)胞進(jìn)行實(shí)驗(yàn)分組:A.P1代空白對(duì)照;B.P1代+最適濃度的Melittin;C.P5代空白對(duì)照;D.P5代+最適濃度的Melittin;5.對(duì)干預(yù)后的四組細(xì)胞使用蛋白印跡分析法(western blot)檢測(cè)MMP-3蛋白的表達(dá)情況。結(jié)果:1.培養(yǎng)出的EPCs傳代后逐漸由多角形變?yōu)樗笮?2.甲苯胺藍(lán)染色細(xì)胞質(zhì)中的酸性粘液物質(zhì)(如蛋白多糖)則被染成深藍(lán)色,免疫熒光染色標(biāo)記Col-2,可見(jiàn)細(xì)胞骨架部分明顯陽(yáng)性表現(xiàn)(呈綠色熒光),P5代較P1代Col-2表達(dá)明顯減弱,以上所見(jiàn)可證明所培養(yǎng)細(xì)胞為軟骨細(xì)胞;3.MTT法測(cè)定Melittin的最適濃度為1μg/ml;4.蜂毒肽對(duì)于退變EPCs中的MMP蛋白的表達(dá)有抑制作用,并能夠延緩終板軟骨退變,表現(xiàn)為B、D較A、C組MMP蛋白表達(dá)明顯減弱,B組較D組MMP蛋白表達(dá)明顯減弱,其差別具有統(tǒng)計(jì)學(xué)意義(p0.05)。結(jié)論:Melittin能夠抑制EPCs中MMP的表達(dá),從而具有一定的終板軟骨保護(hù)作用,為防治脊柱退變性疾病提供一定的實(shí)驗(yàn)依據(jù)。
[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.

【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R681.5

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