本文選題：晚期糖基化終末產(chǎn)物 + 利拉魯肽　； 參考：《蘭州大學(xué)》2017年碩士論文

【摘要】：目的:本實驗通過檢測利拉魯肽(Liraglutide,Lira)對晚期糖基化終末產(chǎn)物(Advanced glycosylation end products,AGEs)誘導(dǎo)的人牙周膜細(xì)胞(periodontal ligament cells,PDLCs)增殖、凋亡、受體表達(dá)、成骨分化及炎癥因子表達(dá)的作用,探討h PDLCs在AGEs積累時,Lira對h PDLCs的保護(hù)及修復(fù)作用。方法:采用酶消化法聯(lián)合組織塊培養(yǎng)法分離培養(yǎng)原代h PDLCs,取3-6代細(xì)胞用于后續(xù)實驗,采用細(xì)胞化學(xué)染色及成骨誘導(dǎo)21天茜素紅染色法進(jìn)行h PDLCs鑒定。采用MTT法篩選Lira對AGEs誘導(dǎo)的h PDLCs的最佳作用濃度及作用時間。采用MTT法檢測Lira對AGEs誘導(dǎo)的h PDLCs增殖活性的影響。采用Hoechst33258熒光染色法及Annexin-V-FITC/PI雙染檢測凋亡水平。采用Realtime PCR及Western Blot檢測晚期糖基化終末產(chǎn)物受體RAGE及胰高血糖素樣肽受體GLP-1R的表達(dá)水平、成骨因子Runx2和ALP的表達(dá)水平、炎癥因子TNF-α和IL-6的表達(dá)水平。采用茜素紅染色法檢測礦化結(jié)節(jié)形成水平。結(jié)果:1.細(xì)胞化學(xué)染色:HE染色細(xì)胞呈纖維細(xì)胞樣長梭形外觀;抗波形絲蛋白染色陽性,抗角蛋白染色陰性,證明細(xì)胞來源為中胚層;茜素紅染色見礦化結(jié)節(jié)形成證明細(xì)胞具有成骨分化能力。綜上可鑒定細(xì)胞為人牙周膜細(xì)胞;2.MTT結(jié)果:AGEs對h PDLCs細(xì)胞增殖抑制作用的最佳作用濃度是100μg/m L,最佳作用時間是48h;Lira對AGEs誘導(dǎo)的h PDLCs的最佳作用濃度是100 nmol/m L,最佳作用時間是24h;與對照組相比,AGEs組的增殖抑制率顯著增高(P㩳0.01),Lira組的增殖抑制率顯著降低(P㩳0.01),AGEs+Lira組的增殖抑制率顯著高于Lira組(P㩳0.01),但低于對照組。3.Western Blot及Real-time PCR結(jié)果:(1)受體表達(dá):h PDLCs上RAGE蛋白及m RNA及GLP-1R蛋白及m RNA的表達(dá)。相比對照組,AGEs組顯著促進(jìn)RAGE的表達(dá)(P㩳0.01),抑制GLP-1R的表達(dá)(P㩳0.05);Lira組抑制RAGE的表達(dá)(P㩳0.05),促進(jìn)GLP-1R的表達(dá)(P㩳0.05),與AGEs組相比,AGEs+Lira組抑制RAGE的表達(dá)(P㩳0.05),促進(jìn)了GLP-1R的表達(dá)(P㩳0.05);(2)成骨因子:相比對照組,AGEs組抑制Runx2和ALP蛋白及m RNA的表達(dá)(P㩳0.01,P㩳0.05),Lira組促進(jìn)Runx2和ALP的表達(dá)(P㩳0.05,P㩳0.01),與AGEs組相比,AGEs+Lira組促進(jìn)Runx2和ALP的表達(dá)(P㩳0.05,P㩳0.01);(3)炎癥因子:相比對照組,AGEs組促進(jìn)IL-6及TNF-α蛋白及m RNA的表達(dá)(P㩳0.01),Lira組抑制IL-6及TNF-α的表達(dá)(P㩳0.05,P㩳0.01),與AGEs組相比,AGEs+Lira組抑制IL-6及TNF-α的表達(dá)(P㩳0.01);5.Hoechst33258染色Annexin-V-FITC/PI細(xì)胞流式術(shù)結(jié)果:對照組和Lira組的細(xì)胞核均未發(fā)生改變;AGEs組細(xì)胞核發(fā)生皺縮,染色質(zhì)濃縮或斷裂;AGEs+Lira組細(xì)胞發(fā)生核皺縮,染色質(zhì)濃縮或斷裂的細(xì)胞減少。相比對照組,AGEs組的凋亡率顯著增高(P㩳0.01);Lira組降低(P㩳0.01);相比AGEs組,AGEs+Lira組的凋亡率顯著降低(P㩳0.01),但高于對照組;7.茜素紅染色結(jié)果:AGEs組的紫紅色礦化結(jié)節(jié)明顯少于對照組,AGEs+Lira組的紫紅色礦化結(jié)節(jié)明顯多于AGEs組。經(jīng)Image J軟件對染色進(jìn)行分析,Lira組與對照組,對照組與AGEs組,AGEs+Lira組與AGEs組之間均表現(xiàn)出明顯差異(P㩳0.05,P㩳0.01,P㩳0.01)。結(jié)論:1.Lira對AGEs誘導(dǎo)的h PDLCs的最佳作用濃度是100 nmol/m L,最佳作用時間是24小時。2.h PDLCs上具有RAGE及GLP-1R受體的表達(dá)。Lira可促進(jìn)正常h PDLCs及AGEs誘導(dǎo)的h PDLCs上GLP-1R的表達(dá),抑制RAGE的表達(dá);3.Lira可促進(jìn)正常h PDLCs和AGEs誘導(dǎo)的h PDLCs增殖,抑制其凋亡,促進(jìn)成骨分化,抑制炎癥因子的表達(dá)。為糖尿病牙周病患者選擇Lira保護(hù)及修復(fù)牙周組織提供實驗依據(jù)。
[Abstract]:Objective: To investigate the proliferation, apoptosis, receptor expression, osteogenesis and expression of inflammatory factors in human periodontal ligament cells (periodontal ligament cells, PDLCs) induced by advanced glycation end products (Advanced glycosylation end products, AGEs) by Leila Lou (Liraglutide, Lira). Methods: the protection and repair effect of PDLCs. Methods: the original h PDLCs was isolated and cultured by enzyme digestion method combined with tissue mass culture. 3-6 generation of cells were used for follow-up experiments. Cytochemical staining and osteogenesis induced 21 days alizarin red staining for H PDLCs identification. The optimum concentration and effect of Lira on AGEs induced H PDLCs were selected by MTT method. Time. The effect of Lira on the proliferation of H PDLCs induced by AGEs was detected by MTT. Hoechst33258 fluorescence staining and Annexin-V-FITC/PI double staining were used to detect the apoptosis level. The expression level of receptor RAGE and glucagon like peptide in advanced glycosylated end products was detected by Realtime PCR and Western Blot. And expression level of ALP, expression level of inflammatory factor TNF- alpha and IL-6. Using alizarin red staining method to detect the formation level of mineralized nodules. Results: 1. cell chemical staining: HE stained cells showed fibrous cell like long spindle shape; anti wave silk protein staining positive, anti keratin staining negative, proved cell source was mesoderm; alizarin red staining found The formation of mineralized nodules proved that the cells had osteogenic differentiation ability. In addition, the identification cells were human periodontal cells; 2.MTT results: the optimum concentration of AGEs to h PDLCs cell proliferation inhibition was 100 mu g/m L, the best time was 48h, and the optimum concentration of Lira for AGEs induced H PDLCs was 100 nmol/m. Compared with the control group, the proliferation inhibition rate of the AGEs group was significantly increased (P 0.01), the proliferation inhibition rate in the Lira group was significantly lower (P? 0.01), and the proliferation inhibition rate in AGEs+Lira group was significantly higher than that in the Lira group (P? 0.01), but it was lower than the control group.3.Western Blot and Real-time PCR results: (1) receptor expression. Compared with the control group, AGEs group significantly promoted the expression of RAGE (P? 0.01), inhibited the expression of GLP-1R (P? 0.05), the Lira group inhibited the expression of RAGE (P? 0.05), and promoted the expression of GLP-1R (P? 0.05). The expression of ALP and m RNA (P? 0.01, P? 0.05), Lira group promoting the expression of Runx2 and ALP (P? 0.05, P? 0.01). 01), compared with the AGEs group, the AGEs+Lira group inhibited the expression of IL-6 and TNF- alpha (P? 0.01); 5.Hoechst33258 stained Annexin-V-FITC/PI cell flow cytometry results: the nuclei of the control group and the Lira group were not changed; the nucleus of the AGEs group crinkled, chromatin concentration or fracture; the AGEs+Lira group had nuclear crinkle, chromatin concentration or fracture Compared with the control group, the apoptosis rate in the AGEs group was significantly higher (P? 0.01) and the group Lira decreased (P? 0.01); compared with the AGEs group, the apoptosis rate of the AGEs+Lira group was significantly lower (P? 0.01), but higher than the control group; 7. alizarin red staining results: the purple red mineralized nodules in the AGEs group were significantly less than those in the control group, and the purple red mineralized nodules in the AGEs+Lira group were significantly more than those in the AGEs group. The staining was analyzed by Image J software, the Lira group and the control group, the control group and the AGEs group, the AGEs+Lira group and the AGEs group showed significant difference (P? 0.05, P? 0.01, P? 0.01). Conclusion: the best concentration of 1.Lira to AGEs induced H is 100. The best time is 24 hours. .Lira can promote the expression of GLP-1R on normal h PDLCs and AGEs induced H PDLCs and inhibit the expression of RAGE. 3.Lira can promote normal h PDLCs and AGEs induced proliferation, inhibit its apoptosis, promote osteogenic differentiation and inhibit the expression of inflammatory factors. It provides experimental basis for the selection of periodontitis patients to protect and repair periodontal tissues of diabetic periodontitis.

【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R587.2;R781.4

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