本文選題：慢性氟中毒 + 自噬�。� 參考：《貴州醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:通過檢測染氟大鼠軟骨組織中自噬相關(guān)因子Beclin1、LC3、p62及凋亡通路中Bcl-2的表達水平變化及氟致大鼠原代軟骨細(xì)胞中自噬相關(guān)因子中Beclin1、LC3蛋白及mRNA的表達情況,探討自噬在慢性氟中毒大鼠軟骨損傷中的作用。方法:1、選用36只SD大鼠,自由飲用自來水,隨機分成3組:對照組(水氟濃度1 mg/L)、低氟組(水氟濃度為5 mg/L)、高氟組(水氟濃度為50 mg/L)每組各12只,實驗6個月后,成功建立氟中毒的模型,觀察大鼠氟斑牙情況,氟離子電極法測定尿氟含量及灰化-氟離子選擇電極法測骨氟含量;光鏡下觀察軟骨組織病理變化,免疫組織化學(xué)法(IHC)和蛋白印跡(Western blot)法檢測軟骨組織中Beclin1、LC3、p62、Bcl-2蛋白表達情況。2、采用甲苯胺藍(lán)鑒定SD大鼠原代軟骨細(xì)胞后,分為對照組(氟濃度0mg/L)及不同濃度氟處理組(氟濃度分別為5、10、20、40mg/L)培養(yǎng)48h后噻唑藍(lán)檢測細(xì)胞增殖情況,Wb及逆轉(zhuǎn)錄PCR法檢測各組細(xì)胞中Beclin1、LC3因子的蛋白及其mRNA表達情況,激光共聚焦顯微鏡下觀察Beclin1、LC3在內(nèi)質(zhì)網(wǎng)上的表達水平。結(jié)果:1、與對照組相比較,染氟組大鼠均出現(xiàn)不同程度的氟斑牙;低氟組及高氟組尿氟(2.72±0.11、4.43±0.14)、低氟組及高氟組骨氟含量(237.67±8.33、270.25±9.83)與對照組尿氟(1.83±0.10)、骨氟(182.58±12.02)相比逐漸升高,差異有統(tǒng)計學(xué)意義(P0.05);2、HE結(jié)果顯示:與對照組比較,低氟組、高氟組軟骨細(xì)胞柱明顯變細(xì)且排列紊亂。3、免疫組化結(jié)果顯示:低氟組、高氟組大鼠軟骨組織中Beclin1、LC3、p62、Bcl-2蛋白表達分別是:Beclin1蛋白(67.75±8.67,85.75±12.29)、LC3蛋白(68.17±12.18,86.58±11.07)與對照組Beclin1蛋白(52.92±8.63)、LC3蛋白(50.92±9.36)相比表達升高;與對照組p62蛋白(71.42±8.72)、Bcl-2蛋白(101.58±6.73)相比,低氟組、高氟組的p62蛋白(55.92±8.22,39.58±7.07)和Bcl-2蛋白(78.00±6.22,59.17±5.25)表達降低(P0.05)。4、Wb結(jié)果顯示,與對照組Beclin1蛋白(0.40±0.08)和LC3蛋白(0.42±0.06)相比,低氟組Beclin1蛋白(0.50±0.06)和高氟組Beclin1蛋白(0.63±0.06)以及低氟組LC3蛋白(0.56±0.08)和高氟組LC3蛋白(0.74±0.08)的表達明顯升高;與對照組p62蛋白(0.79±0.09)和Bcl-2蛋白(1.15±0.10)相比,p62蛋白在低氟組、高氟組分別為(0.59±0.06,0.27±0.03),Bcl-2蛋白在低氟組、高氟組分別(0.61±0.08,0.37±0.07)(P0.05)。5、提取并鑒定原代軟骨細(xì)胞后,MTT結(jié)果顯示:在24h、48h、72h各時間段,軟骨細(xì)胞增殖率在5 mg/L染氟組細(xì)胞為(1.15±0.07、1.19±0.04、1.07±0.22)、10mg/L染氟組細(xì)胞為(1.16±0.13、1.22±0.06、1.07±0.18),與對照組(1.02±0.04、1.01±0.11、1.00±0.05)比較,以48h和72h升高為明顯,差異有統(tǒng)計學(xué)意義(P0.05)。而5 mg/L、10mg/L染氟組細(xì)胞兩兩相比,細(xì)胞增殖率在各時間段增殖無明顯變化,差異無統(tǒng)計學(xué)意義(P0.05)。6、培養(yǎng)48 h后在染氟組(10mg/L、20mg/L、40mg/L)中,Beclin1蛋白分別為(0.64±0.10、0.77±0.14、1.88±0.21)、LC3蛋白分別為(0.72±0.11、1.36±0.19、2.02±0.26)及Beclin1mRNA分別為(0.52±0.05、0.67±0.12、0.99±0.13);LC3mRNA分別為(0.72±0.12、0.94±0.07、1.19±0.13),表達均高于對照組Beclin1蛋白(0.32±0.10)、LC3蛋白(0.43±0.07)及Beclin1、LC3mRNA[(0.22±0.03);(0.33±0.06)](P0.05)。7、激光共聚焦顯微鏡觀察發(fā)現(xiàn),培養(yǎng)48 h后,染氟組(10mg/L、20mg/L、40mg/L)軟骨細(xì)胞內(nèi)質(zhì)網(wǎng)上Beclin1蛋白表達分別為(238.33±16.01、350.67±27.39、455.67±29.54)和LC3蛋白表達分別為(440.67±36.07、439.33±35.02、499.67±24.17),與對照組Beclin1蛋白(185.67±20.82)和LC3蛋白(217.67±25.01)相比,差異有統(tǒng)計學(xué)意義(P0.05)。10、20、40mg/L的染氟組細(xì)胞內(nèi)質(zhì)網(wǎng)的積分光密度分別為(267.93±24.10;275.13±26.66;293.17±25.43),與對照組細(xì)胞內(nèi)質(zhì)網(wǎng)的積分光密度(273.40±26.42)比較,差異無統(tǒng)計學(xué)意義(P0.05)。結(jié)論:過量氟引起大鼠軟骨組織中自噬和凋亡相關(guān)因子Beclin1、LC3、p62、Bcl-2蛋白表達異常;在不同濃度氟促進大鼠軟骨細(xì)胞增殖及凋亡,同時可能通過內(nèi)質(zhì)網(wǎng)應(yīng)激,激活自噬相關(guān)因子,引起氟致大鼠軟骨細(xì)胞中內(nèi)質(zhì)網(wǎng)功能異常,可能是造成慢性氟中毒大鼠關(guān)節(jié)軟骨損害的發(fā)生機制之一。
[Abstract]:Objective: To investigate the changes of the expression of autophagy related factors Beclin1, LC3, p62 and apoptosis pathway in the cartilage tissue of rats with fluorine, and the expression of Beclin1, LC3 protein and mRNA in the autophagy related factors in the primary chondrocytes of the rats, and to explore the role of autophagy in the cartilage injury of chronic fluorosis rats. Methods: 1, 36 Only SD rats were free to drink tap water and were randomly divided into 3 groups: the control group (water fluoride concentration 1 mg/L), low fluorine group (water fluoride concentration 5 mg/L), high fluorine group (water fluoride concentration 50 mg/L) 12 each. After 6 months, the model of fluorosis was successfully established, the fluorine plaque in rats was observed, fluorine content of urine and the selection of ash fluorine ion selection by fluorine ion electrode method. The fluoride content of bone was measured by the electrode method. The pathological changes of cartilage tissue were observed under light microscope. The expression of Beclin1, LC3, p62 and Bcl-2 protein in cartilage tissue was detected by immunohistochemistry (IHC) and Western blot (Western blot). The primary chondrocytes were identified by toluidine blue, and they were divided into control group (fluorine concentration 0mg/L) and different concentration fluorine treatment group. The concentration of fluorine was 5,10,20,40mg/L) and the cell proliferation was detected by thiazolium after 48h culture. The expression of Beclin1, LC3 factor and mRNA in each cell was detected by Wb and reverse transcriptase PCR. The expression level of Beclin1 and LC3 on the endoplasmic reticulum was observed under confocal laser scanning microscope. Results: 1, compared with the control group, the rats in the fluorine dyed group all appeared. The Urine Fluorine in the low fluorine group and the high fluorine group was (2.72 + 0.11,4.43 + 0.14). The bone fluoride content in the low fluorine group and the high fluorine group (237.67 + 8.33270.25 + 9.83) was higher than the control group (1.83 + 0.10), and the bone fluorine (182.58 + 12.02). The difference was statistically significant (P0.05). 2, HE results showed that compared with the control group, the low fluorine group and the high fluorine group were soft. The osteocyte column was obviously thinner and arranged in disorder.3. The immunohistochemical results showed that the expression of Beclin1, LC3, p62, Bcl-2 protein in the cartilage tissue of the high fluorine group was Beclin1 protein (67.75 + 8.67,85.75 + 12.29), LC3 protein (68.17 + 12.18,86.58 + 11.07) was compared with the control group Beclin1 protein (52.92 + 8.63), LC3 protein (50.92 + 9.36). As compared with the control group p62 protein (71.42 + 8.72), Bcl-2 protein (101.58 + 6.73), low fluorine group, p62 protein (55.92 + 8.22,39.58 + 7.07) and Bcl-2 protein (78 + 6.22,59.17 + 5.25) in the high fluorine group decreased (P0.05).4, Wb results showed that the Beclin1 protein (0.) in the low fluorine group was compared with the control group Beclin1 protein (0.40 + 0.08) and LC3 protein (0.42 + 0.06). 50 + 0.06) and high fluorine group Beclin1 protein (0.63 + 0.06) and low fluorine group LC3 protein (0.56 + 0.08) and high fluorine group LC3 protein (0.74 + 0.08), compared with the control group p62 protein (0.79 + 0.09) and Bcl-2 protein (1.15 + 0.10), p62 protein in low fluorine group and high fluorine group (0.59 + 0.06,0.27 +%), Bcl-2 protein in low fluorine group, high fluorine Group (0.61 + 0.08,0.37 + 0.07) (P0.05).5 respectively, after the extraction and identification of primary chondrocytes, the results of MTT showed that the proliferation rate of chondrocytes in 24h, 48h, 72h was (1.15 + 0.07,1.19 + 0.04,1.07 + 0.22) in 5 mg/L, and that of 10mg/L fluoride group was (1.16 + 0.13,1.22 + 0.18), and the control group was 1.02 + 1.16 + 0.11, 1 + 0.05) compared with 48h and 72h, the difference was statistically significant (P0.05). But 5 mg/L, 10mg/L stained fluorine group 22 compared with the cell proliferation rate, there was no significant difference in proliferation rate at each time period, the difference was not statistically significant (P0.05).6, after culture 48 h (10mg/L, 20mg/L, 40mg/L), Beclin1 protein was 0.64 +. 0.14,1.88 + 0.21), LC3 proteins were (0.72 + 0.11,1.36 + 0.19,2.02 + 0.26) and Beclin1mRNA respectively (0.52 + 0.05,0.67 + 0.12,0.99 + 0.13), LC3mRNA respectively (0.72 + 0.12,0.94 + 0.07,1.19 + 0.13), respectively higher than the control group Beclin1 protein (0.32 + 0.10), LC3 protein (0.43 + 0.07) and 0.22 + 0.03; 5).7, the laser confocal microscope observed that after 48 h, the expression of Beclin1 protein in the endoplasmic reticulum of the fluoride group (10mg/L, 20mg/L, 40mg/L) were (238.33 + 16.01350.67 + 27.39455.67 + 29.54) and LC3 protein respectively (440.67 + 36.07439.33 + 35.02499.67 + 24.17), respectively, and that of the control group (185.67 + 20.82). Compared with the protein (217.67 + 25.01), the integral light density of the cell endoplasmic reticulum in P0.05.10,20,40mg/L was (267.93 + 24.10; 275.13 + 26.66; 293.17 + 25.43), and compared with the integral light density of the cell endoplasmic reticulum (273.40 + 26.42) in the control group, the difference was not statistically significant (P0.05). Conclusion: excessive fluoride caused rats The expression of autophagy and apoptosis related factors Beclin1, LC3, p62, Bcl-2 protein expression in cartilage tissue is abnormal. At different concentrations of fluorine, it promotes the proliferation and apoptosis of chondrocytes in rats, and may activate autophagy related factors through endoplasmic reticulum stress, causing abnormal endoplasmic reticulum function in the chondrocytes of rats induced by fluorine, which may cause chronic fluorosis rats' joint soft joints. One of the mechanisms of bone damage.

【學(xué)位授予單位】：貴州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R599.1

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