自噬对成骨细胞在酸性微环境中的保护作用及其机制的初步研究

发布时间：2018-05-12 21:43

本文选题：骨折 + 酸性微环境　；参考：《山东大学》2017年硕士论文

【摘要】：现代外科学的发展为骨折的治疗带来了新的理念和技术,但术后骨不连的发病率仍旧很高,因此骨折愈合的机制及影响因素仍有待于深入研究。成骨细胞在骨折愈合中扮演重要的角色,其增殖分化与骨折周围的微环境息息相关,因此,研究成骨细胞在骨折局部微环境中的反应有着非常重要的意义。自噬可避免细胞受外界不利环境的影响,是用来维持细胞内环境平衡与稳定的重要保护机制。研究表明,缺血、低氧和氧化应激等可导致自噬的发生。然而,骨折局部同样存在酸性的微环境,但是酸性微环境是否诱导成骨细胞发生自噬目前尚不清楚。因此,对成骨细胞所在酸性微环境中的自噬反应以及相关机制进行研究,可能为促进骨愈合以及骨折术后骨不连的防治提供新的思路。目的:本研究通过构建小鼠骨折模型,分析骨折断端周围能否发生自噬反应。通过体外细胞实验模拟骨折周围的酸性微环境,分析酸性环境对成骨细胞活力和凋亡的影响,以及酸性微环境下成骨细胞是否能发生保护性自噬反应,从而提高成骨细胞的存活率,为促进骨折愈合提供新的策略。方法:1.动物实验分组及方法实验选取27只体重在(30±10)g,6周龄雄性KM大鼠,并随机分为3组:A组(6h),B组(24h),C组(36h),每组9只。小鼠右侧股骨建立骨折损伤,左侧正常侧作为对照。A,B,C三组大鼠分别在骨折6h,24h,36h后处死,取下骨折及正常侧骨组织作为样本,样本固定、骨组织脱钙、石蜡包埋、切片,进行组织免疫荧光染色,检测标志蛋白LC3,p62的表达水平。2.细胞实验的分组及方法实验按pH的不同将培养基随机分为三组分别为pH 6.4、6.8(实验组)及7.4组(对照组)。成骨细胞培养在96孔板内,经过不同pH的培养基处理12h,24h,48h时间后,采用MTT比色法检测酸性微环境下成骨细胞的细胞活力;细胞在不同pH的培养基内处理24h后,采用AnnexinV-PI染色法检测不同pH培养基对成骨细胞凋亡的影响;细胞免疫荧光用来检测,经不同pH的培养基处理6h后,LC3在细胞内的表达;透射电镜观察pH 6.4的培养基处理6h后自噬小体的数量及其形态的变化;免疫蛋白印记法检测自噬标志蛋白LC3及p62的表达以及转化以及监测酸性微环境下成骨细胞自噬流的产生。通过添加自噬抑制剂CQ抑制成骨细胞自噬反应,检测不同pH培养基培养24h后细胞凋亡情况,分析酸性微环境下自噬对凋亡的影响。采用SPSS16.0软件包进行分析,单因素方差分析对数据进行统计。结果:1.动物免疫荧光观察显示:免疫荧光用来检测LC3及p62的表达。骨折组,骨折断端LC3的表达相比对照组要强(p0.05),相反p62表达较对照组弱(p0.05)。即骨折断端发生了自噬。2.MTT比色法显示:实验组(pH6.4、6.8)与对照组(pH7.4)相比,在各个时间点(12h,24h,48h)细胞活力均小于对照组(p0.05),即酸性环境下成骨细胞的细胞活力受到抑制,与pH6.8组相比pH6.4组细胞活力更低,其差异具有显著性(p0.05)。即酸性pH微环境对成骨细胞的细胞活力是不利的且呈pH及时间依赖性。3.AnnexinV-PI染色法显示:24小时后,pH6.4和pH6.8组细胞凋亡数目较正常组pH(7.4)明显增多,且pH6.4组细胞凋亡数最多。即酸性微环境促使成骨细胞凋亡,其差异具有显著性(p0.05)。4.电镜观察显示:成骨细胞在酸性为pH6.4的培养液中培养6h后,自噬小体数目较正常组pH(7.4)明显增多,成骨细胞的形态结构发生了变化,呈双层膜包裹着细胞器或线粒体。即酸性环境可以诱导成骨细胞发生自噬反应。5.蛋白质免疫印记检测显示:pH6.4时,随着时间的延长(6h,12h,24h),LC3-Ⅱ/Ⅰ比值变低(p0.05),而p62逐渐升高(p0.05);即随着时间延长自噬减弱;6h时,随着pH的升高(pH6.4,6.8,7.4)LC3-Ⅱ/Ⅰ比值降低,相反p62逐渐升高(p0.05),即酸性pH越低,自噬越强。在相同条件加入自噬抑制剂氯喹后,因其阻碍了自噬小体与溶酶体的结合,导致LC3-Ⅱ的表达明显增加。即随着时间的延长自噬减弱。6.细胞免疫荧光检测显示:细胞在pH6.4的培养液处理6h后,采用免疫荧光检测LC3,红色荧光分布在细胞质内,且随pH的升高,荧光表达降低(p0.05)。说明酸性微环境可以促进自噬。7.加入自噬抑制剂后AnnexinV-PI染色法显:在相同的条件下,加入自噬抑制剂组比不加入抑制剂组细胞凋亡数明显增多(p0.05)。即在酸性微环境下,抑制自噬,促进了成骨细胞凋亡。结论:1.体外实验发现酸性pH微环境对成骨细胞的细胞活力是不利的,它可以促进成骨细胞凋亡,并且诱导成骨细胞发生自噬。2.体内实验发现成骨细胞在骨折部位可发生自噬反应。3.成骨细胞通过自噬抑制细胞的凋亡,从而提高成骨细胞在酸性微环境中的的存活率。
[Abstract]:The development of modern surgery has brought new ideas and techniques for the treatment of fracture, but the incidence of postoperative bone nonunion is still very high. Therefore, the mechanism and influencing factors of fracture healing still need to be studied. Osteoblasts play an important role in fracture healing, and their proliferation and differentiation are closely related to the microenvironment around fracture. It is of great significance to study the response of osteoblasts in the local microenvironment of fracture. Autophagy can avoid the effect of the adverse environment on the external environment, and it is an important protective mechanism for maintaining the balance and stability of the cell environment. In acidic microenvironment, it is not clear whether acid microenvironment induces autophagy in osteoblasts. Therefore, the study of autophagy and related mechanisms in the acidic microenvironment of osteoblasts may provide new ideas for the promotion of bone healing and the prevention and treatment of bone nonunion after fracture. A mouse model of fracture was used to analyze the autophagic reaction around the broken end of the fracture. The effects of acid environment on the activity and apoptosis of osteoblasts and the protective autophagy in the acidic microenvironment were analyzed by simulating the acid microenvironment around the fracture. To provide a new strategy to promote fracture healing. Methods: 1. group and method experiment of 1. animals were divided into 3 groups (30 + 10) and 6 weeks old male KM rats, and were randomly divided into 3 groups: A group (6h), B group (24h), C group (36h), 9 rats in each group. The left normal side was used as control.A, B, C three rats respectively in the fracture 6h, 3, 3. 3 After 6h, the fracture and the normal lateral bone tissue were taken as samples, the samples were fixed, bone tissue decalcified, paraffin embedded, sliced, tissue immunofluorescence staining, and tissue immunofluorescence staining, LC3, p62 expression level.2. cell experiments were divided into three groups randomly divided into pH 6.4,6.8 (experimental group) and 7.4 respectively according to the difference of pH. Group (control group). Osteoblasts were cultured in 96 orifice plates. After 12h, 24h and 48h were treated with different pH medium, MTT colorimetric assay was used to detect the cell viability of osteoblasts in acidic microenvironment. After the cells were treated with 24h in the culture base of different pH, the effect of different pH medium on the apoptosis of osteoblasts was detected by AnnexinV-PI staining. Cell immunofluorescence was used to detect the expression of LC3 in the cell after treating 6h with different pH medium. The number and morphological changes of autophagic corpuscles after 6h was treated with pH 6.4 medium; the expression of autophagic marker protein LC3 and p62 was detected by the immunoglobulin imprint method and the autophagy was transformed and the osteoblast under the acid microenvironment was monitored from the acid microenvironment. The effect of autophagy by adding autophagic inhibitor CQ to autophagy in osteoblasts was detected and apoptosis in different pH cultures was detected and the effect of autophagy on apoptosis in acid microenvironment was analyzed. SPSS16.0 software package was used to analyze the cell apoptosis. The data were analyzed by single factor analysis of variance. Results: 1. animal immunofluorescence observation showed that: 1. The expression of LC3 and p62 was detected by immunofluorescence. The expression of LC3 in fracture fracture group was stronger than that of the control group (P0.05), but the expression of p62 was weaker than that of the control group (P0.05). That is, the autophagic.2.MTT colorimetric method of the fracture end showed that the experimental group (pH6.4,6.8) was less than the control group (pH7.4) at all time points (12h, 24h, 48h) cells were less than those of the control group. The cell viability of osteoblasts in the acidic environment was inhibited, and the cell viability of the pH6.4 group was lower than that in the pH6.8 group, and the difference was significant (P0.05). That is, the acid pH microenvironment was negative to the cell viability of the osteoblasts and was shown by pH and time dependent.3.AnnexinV-PI staining: 24 hours later, pH6.4 and pH6.8 groups finely. The number of apoptotic cells increased significantly compared with the normal group pH (7.4), and the number of apoptotic cells in the pH6.4 group was the most. That is, the acid microenvironment resulted in the apoptosis of osteoblasts. The difference was significant (P0.05).4. electron microscope observation showed that the number of autophagic corpuscles in the acidic pH6.4 culture medium, the number of autophagic corpuscles increased significantly than the normal group pH (7.4), and osteoblasts The morphological structure changed, and the cell organelle or mitochondria were wrapped in the double layer membrane. That is, the acid environment can induce autophagy to induce autophagic reaction.5. protein immuno imprint detection show: pH6.4, with the prolongation of time (6h, 12h, 24h), LC3- II / I ratio decreases (P0.05), and p62 gradually increases (P0.05); that is, the autophagy decreases with time; At 6h, with the decrease of the ratio of pH (pH6.4,6.8,7.4) LC3- II / I, the opposite p62 gradually increased (P0.05), that is, the lower the acid pH, the stronger the autophagy. After adding the autophagic inhibitor chloroquine, the expression of the autophagosome and the lysosome was hindered by the combination of the autophagic inhibitor, which resulted in a significant increase in the expression of LC3- II. That is, the autophagy attenuated the.6. cell with the time of autophagy. Immunofluorescence detection showed that after the cells were treated with 6h in pH6.4 culture, LC3 was detected by immunofluorescence, the red fluorescence was distributed in the cytoplasm, and the fluorescence expression decreased with the increase of pH (P0.05). It indicated that the acid microenvironment could promote autophagic.7. to add autophagic inhibitor after AnnexinV-PI staining method: under the same condition, the autophagy inhibition was added. The number of apoptotic cells in the agent group was significantly increased (P0.05). That is, in the acidic microenvironment, inhibiting autophagy and promoting the apoptosis of osteoblasts. Conclusion: 1. in vitro, the acid pH microenvironment was found to be unfavorable to the cell viability of osteoblasts. It could promote osteoblast apoptosis and induce autophagy in.2. body. It was found that autophagy could induce autophagy.3. osteoblasts to inhibit the apoptosis of cells by autophagy, thus improving the survival rate of osteoblasts in the acid microenvironment.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R683

【参考文献】