KLF4对小鼠成骨细胞成骨分化及基质分泌的影响

发布时间：2018-10-08 11:17

【摘要】：牙周炎(periodontal disease)是威胁人类口腔健康的常见病之一,是细菌及代谢产物介导的作用于牙周支持组织的具有慢性破坏性的疾病。牙周炎主要临床表现为牙槽骨的吸收破坏,病理学变化显示为骨新生的不足—骨生成与骨吸收失衡所致。由于牙周炎患病率高,与全身的健康密切相关,目前其临床治疗方式主要为对症处理,控制牙周炎的继续发展,对牙周炎所造成的骨质缺损尚无有效的治疗措施,因此深入探索骨形成的机制对于牙周炎的防治具有极其重要的作用。骨平衡是一个动态变化过程,成骨细胞所引起的骨生成与破骨细胞所导致的骨吸收之间的平衡统一是维持骨代谢稳态的关键。大量研究表明若骨形成少于骨吸收时,将导致如骨质疏松、牙周病、骨关节炎、风湿性关节炎等骨疾病;反之则导致骨硬化疾病。其中,成骨细胞为骨代谢平衡的主导因素之一,其活性可以被多种因素调节。因此骨生成的分子机制的研究对提高骨疾病所导致缺损的修复治疗效果具有临床意义。越来越多的研究都集中在成骨细胞的发生、增殖、分化中,这表明了在骨相关疾病中成骨细胞对骨再生的重要性。基于以上分析,本研究拟通过成骨细胞分离培养及鉴定培养出实验细胞,通过过表达核转录因子KLF4观察其对成骨细胞增殖及分化相关基因的影响,从而探讨KLF4对成骨细胞早期成骨分化的影响;最后本研究对成骨细胞分化过程中KLF4对骨平衡所涉及的基质分泌的影响,明确KLF4在成骨细胞成骨分化中发挥的作用,为牙周疾病临床诊治进一步提出理论依据。方法:1.利用分段酶消化法从新生小鼠颅骨片中获得成骨原代细胞,显微镜下观察细胞形态,经成骨诱导液分别培养9 d和21 d后采取ALP染色和茜素红染色对其进行鉴定。2.采用免疫荧光技术检测成骨细胞中KLF4的表达;成功构建携带KLF4的重组腺病毒载体(Ad-KLF4),成骨细胞经不同感染复数(multiplicity of infection,MOI)的Ad-KLF4转染后荧光显微镜下观察细胞绿色荧光表达情况并检测转染效率。将成骨细胞随机分为对照组(未转染组)、KLF4组(Ad-KLF4转染)和GFP组(Ad-GFP转染),经最佳MOI感染后MTT测定细胞增殖,Real-time PCR检测成骨相关基因碱性磷酸酶(alkaline phosphatase,ALP)、骨涎蛋白(bone sialoprotein,BSP)、核心蛋白结合因子2(runt-related transcription factor 2,RUNX2)的表达。3.采用western-blot检测成骨细胞成骨分化过程KLF4,MMP2及COL1的表达;siRNA KLF4或者Ad-KLF4处理成骨细胞后,在成骨细胞及成骨细胞早期分化过程(0d、2 d、4 d)通过wester-blot及Real-timePCR观察其对MMP2及COL1蛋白及基因表达的影响;结果:1.本实验利用酶消化法从新生小鼠颅骨片中获得细胞形态呈多边形、立方形的成骨原代细胞。ALP染色可见细胞浆内大量沙砾状蓝染阳性颗粒,茜素红染色可见呈细胞外成片红色矿化团块。通过细胞形态学的观察以及染色鉴定表明通过分段酶消化法可以分离培养出成骨原代细胞,为本实验的进一步顺利进行打下基础。2.经PCR扩增、基因测序成功构建出携带KLF4基因的重组腺病毒载体(Ad-KLF4)。成骨细胞经最适合MOI(100 PFU/ml)处理后,KLF4组可观察到KLF4蛋白及mRNA水平显著上升;成骨细胞增殖速率明显上升;成骨分化相关基因ALP、RUNX2、BSP的水平显著下降。3.随着成骨细胞成骨分化时间的延长,KLF4的表达不断上升,伴随着MMP2表达下降及COL1表达的升高。经40NM的KLF4 siRNA感染后,KLF4基因及蛋白表达明显下降,同时MMP2的表达上升,COL1的表达下降;经MOI=100 PFU/ml的Ad-KLF4感染后,KLF4表达水平显著上升,相反MMP2表达下调,COL1的基因及蛋白水平依然呈下降趋势。结论:1.本实验利用分段酶消化法成功获得成骨原代细胞,且第二代细胞生长状态好、活力好,能满足后续实验要求。2.Ad-KLF4转染成骨细胞后可使成骨细胞增殖速率增加,下调成骨分化基因ALP、BSP、RUNX2水平,最终抑制骨形成。3.在骨平衡中,KLF4可以负性调控成骨细胞中MMP2的表达,调控骨形成过程中基质的分泌,从而影响骨形成。
[Abstract]:Periodontitis is one of the common diseases threatening human oral health, and is a chronic and destructive disease mediated by bacteria and metabolites in periodontal support tissues. The main clinical manifestations of periodontitis are absorption and destruction of alveolar bone, and pathological changes show that the deficiency of bone regeneration is caused by imbalance of bone resorption and bone resorption. Because the prevalence of periodontitis is high, it is closely related to the health of the whole body, the current clinical treatment way is mainly symptomatic treatment, the continuous development of periodontitis is controlled, and no effective treatment measures are available for the bone defect caused by periodontitis, Therefore, deeply exploring the mechanism of bone formation plays an extremely important role in the prevention and treatment of periodontitis. Bone balance is a dynamic process, and the balance between bone resorption caused by osteoblasts and bone resorption caused by osteoclasts is the key to maintaining the homeostasis of bone metabolism. A large number of studies have shown that if bone formation is less than bone resorption, bone diseases such as osteoporosis, periodontal disease, osteoarthritis, rheumatic arthritis, and the like are caused; conversely, bone-hardening disease is caused. Among them, osteoblasts are one of the leading factors of bone metabolism balance, and their activity can be regulated by a variety of factors. Therefore, the study of molecular mechanism of bone formation is of clinical significance to improve the curative effect of repairing the defect caused by bone disease. More and more studies have focused on the occurrence, proliferation and differentiation of osteoblasts, indicating the importance of osteoblasts to bone regeneration in bone-related diseases. Based on the above analysis, this study intends to investigate the effects of KLF4 on osteoblast proliferation and differentiation related genes through the isolation and culture of osteoblasts and the identification and culture of experimental cells, and to investigate the effect of KLF4 on the early bone differentiation of osteoblasts by observing the effect of KLF4 on the proliferation and differentiation of osteoblasts. Finally, the effect of KLF4 on the bone balance in osteoblast differentiation was studied, and the role of KLF4 in osteoblast differentiation was defined, and further theoretical basis was put forward for clinical diagnosis and treatment of periodontal disease. Method: 1. Bone primary cells were obtained from the skull tablets of newborn mice by a segmented enzymatic digestion method. The morphology of the cells was observed under a microscope. ALP staining and scarlet staining were performed after 9 days and 21 days respectively after being cultured for 9 days and 21 days, respectively. The expression of KLF4 in osteoblasts was detected by immunofluorescence; the recombinant adenovirus vector (Ad-KLF4) carrying KLF4 was successfully constructed. The cell green fluorescence was observed under fluorescence microscope after Ad-KLF4 transfected with multiple infection (MOI), and transfection efficiency was detected. Osteoblasts were randomly divided into control group (non-transfected group), KLF4 group (Ad-KLF4 transfection) and GFP group (Ad-GFP transfection). After the best MOI infection, cell proliferation and real-time PCR were detected as bone-related gene alkaline phosphatase (ALP), bone sialoprotein (BSP). Expression of core protein binding factor 2 (RUNX2). The expression of KLF4, MMP2 and COL1 was detected by western-blot. After treatment of osteoblasts with siRNA KLF4 or Ad-KLF4, the effects of MMP2 and COL1 protein and gene expression were observed through wester-blot and Real-time PCR in the early differentiation of osteoblasts and osteoblasts (0d, 2d, 4d). In this experiment, the cell morphology was polygonal in the skull tablet of newborn mice by the enzyme digestion method. In the ALP staining, a large amount of sand-like blue-stained positive particles were observed in the cytoplasm of the cells, and the red-stained red-stained clusters of red cells in the cytoplasm of the cells were observed. Through the observation of cell morphology and the identification of staining, it is shown that the culture of primary cells can be separated by segmented enzymatic digestion, which will lay the foundation for the further progress of this experiment. The recombinant adenovirus vector (Ad-KLF4) carrying the KLF4 gene was successfully constructed by PCR amplification and gene sequencing. After treatment with MOI (100 PFU/ ml), the level of KLF4 protein and mRNA increased significantly in KLF4 group, and the rate of osteoblast proliferation increased significantly. With the prolongation of osteoblast differentiation time, the expression of KLF4 increased continuously with the decrease of MMP2 expression and the increase of COL1 expression. After the infection of KLF4 siRNA of 40NM, the expression of KLF4 gene and protein decreased obviously, while the expression of MMP2 increased and the expression of COL1 decreased. After the infection of Ad-KLF4 infected with MOI = 100 PFU/ ml, the expression level of KLF4 increased significantly, while the expression of MMP2 was down-regulated, and the gene and protein level of COL1 continued to decrease. Conclusion: 1. In this experiment, the bone primary cells were successfully obtained by the method of segmented enzyme digestion, and the second generation cells were good in growth state and good in activity, which could meet the follow-up experimental requirements. The proliferation rate of osteoblasts was increased after the transfection of osteoblasts with Ad-KLF4, and the ALP, BSP and RUNX2 levels of the bone-differentiated genes were down-regulated. Final inhibition of bone formation. 3. In bone balance, KLF4 can regulate the expression of MMP2 in osteoblasts and regulate the secretion of matrix during bone formation, thus affecting bone formation.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.4

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