自噬相关因子Beclin1、LC3和p62在慢性氟中毒大鼠软骨中的表达意义

发布时间：2018-05-13 15:47

本文选题：慢性氟中毒 + 自噬　；参考：《贵州医科大学》2017年硕士论文

【摘要】：目的:通过检测染氟大鼠软骨组织中自噬相关因子Beclin1、LC3、p62及凋亡通路中Bcl-2的表达水平变化及氟致大鼠原代软骨细胞中自噬相关因子中Beclin1、LC3蛋白及mRNA的表达情况,探讨自噬在慢性氟中毒大鼠软骨损伤中的作用。方法:1、选用36只SD大鼠,自由饮用自来水,随机分成3组:对照组(水氟浓度1 mg/L)、低氟组(水氟浓度为5 mg/L)、高氟组(水氟浓度为50 mg/L)每组各12只,实验6个月后,成功建立氟中毒的模型,观察大鼠氟斑牙情况,氟离子电极法测定尿氟含量及灰化-氟离子选择电极法测骨氟含量;光镜下观察软骨组织病理变化,免疫组织化学法(IHC)和蛋白印迹(Western blot)法检测软骨组织中Beclin1、LC3、p62、Bcl-2蛋白表达情况。2、采用甲苯胺蓝鉴定SD大鼠原代软骨细胞后,分为对照组(氟浓度0mg/L)及不同浓度氟处理组(氟浓度分别为5、10、20、40mg/L)培养48h后噻唑蓝检测细胞增殖情况,Wb及逆转录PCR法检测各组细胞中Beclin1、LC3因子的蛋白及其mRNA表达情况,激光共聚焦显微镜下观察Beclin1、LC3在内质网上的表达水平。结果:1、与对照组相比较,染氟组大鼠均出现不同程度的氟斑牙;低氟组及高氟组尿氟(2.72±0.11、4.43±0.14)、低氟组及高氟组骨氟含量(237.67±8.33、270.25±9.83)与对照组尿氟(1.83±0.10)、骨氟(182.58±12.02)相比逐渐升高,差异有统计学意义(P0.05);2、HE结果显示:与对照组比较,低氟组、高氟组软骨细胞柱明显变细且排列紊乱。3、免疫组化结果显示:低氟组、高氟组大鼠软骨组织中Beclin1、LC3、p62、Bcl-2蛋白表达分别是:Beclin1蛋白(67.75±8.67,85.75±12.29)、LC3蛋白(68.17±12.18,86.58±11.07)与对照组Beclin1蛋白(52.92±8.63)、LC3蛋白(50.92±9.36)相比表达升高;与对照组p62蛋白(71.42±8.72)、Bcl-2蛋白(101.58±6.73)相比,低氟组、高氟组的p62蛋白(55.92±8.22,39.58±7.07)和Bcl-2蛋白(78.00±6.22,59.17±5.25)表达降低(P0.05)。4、Wb结果显示,与对照组Beclin1蛋白(0.40±0.08)和LC3蛋白(0.42±0.06)相比,低氟组Beclin1蛋白(0.50±0.06)和高氟组Beclin1蛋白(0.63±0.06)以及低氟组LC3蛋白(0.56±0.08)和高氟组LC3蛋白(0.74±0.08)的表达明显升高;与对照组p62蛋白(0.79±0.09)和Bcl-2蛋白(1.15±0.10)相比,p62蛋白在低氟组、高氟组分别为(0.59±0.06,0.27±0.03),Bcl-2蛋白在低氟组、高氟组分别(0.61±0.08,0.37±0.07)(P0.05)。5、提取并鉴定原代软骨细胞后,MTT结果显示:在24h、48h、72h各时间段,软骨细胞增殖率在5 mg/L染氟组细胞为(1.15±0.07、1.19±0.04、1.07±0.22)、10mg/L染氟组细胞为(1.16±0.13、1.22±0.06、1.07±0.18),与对照组(1.02±0.04、1.01±0.11、1.00±0.05)比较,以48h和72h升高为明显,差异有统计学意义(P0.05)。而5 mg/L、10mg/L染氟组细胞两两相比,细胞增殖率在各时间段增殖无明显变化,差异无统计学意义(P0.05)。6、培养48 h后在染氟组(10mg/L、20mg/L、40mg/L)中,Beclin1蛋白分别为(0.64±0.10、0.77±0.14、1.88±0.21)、LC3蛋白分别为(0.72±0.11、1.36±0.19、2.02±0.26)及Beclin1mRNA分别为(0.52±0.05、0.67±0.12、0.99±0.13);LC3mRNA分别为(0.72±0.12、0.94±0.07、1.19±0.13),表达均高于对照组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、激光共聚焦显微镜观察发现,培养48 h后,染氟组(10mg/L、20mg/L、40mg/L)软骨细胞内质网上Beclin1蛋白表达分别为(238.33±16.01、350.67±27.39、455.67±29.54)和LC3蛋白表达分别为(440.67±36.07、439.33±35.02、499.67±24.17),与对照组Beclin1蛋白(185.67±20.82)和LC3蛋白(217.67±25.01)相比,差异有统计学意义(P0.05)。10、20、40mg/L的染氟组细胞内质网的积分光密度分别为(267.93±24.10;275.13±26.66;293.17±25.43),与对照组细胞内质网的积分光密度(273.40±26.42)比较,差异无统计学意义(P0.05)。结论:过量氟引起大鼠软骨组织中自噬和凋亡相关因子Beclin1、LC3、p62、Bcl-2蛋白表达异常;在不同浓度氟促进大鼠软骨细胞增殖及凋亡,同时可能通过内质网应激,激活自噬相关因子,引起氟致大鼠软骨细胞中内质网功能异常,可能是造成慢性氟中毒大鼠关节软骨损害的发生机制之一。
[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.

【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R599.1

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