【作者】 史册；

【导师】 孙宏晨；

【作者基本信息】 吉林大学 ， 口腔医学， 2010， 硕士

【摘要】 破骨细胞是参与骨吸收的主要细胞,其分化调控是个极其复杂的过程。通过对破骨细胞分化和功能调控的研究,不仅对正确认识疾病的发生机制具有重要的理论意义,而且对进一步防治相关疾病具有潜在的实际意义。TWEAK作为一种细胞因子,在生理和病理状态下与破骨细胞的分化有关。本研究通过建立体外破骨细胞培养方法,用所构建的同一启动子的腺病毒载体携带EGFP和TWEAK基因转染体外破骨细胞培养体系中的细胞;通过TRAP阳性细胞计数及骨吸收陷窝面积的测量,结果表明TWEAK在体外能延长破骨细胞的生存时间,并能显著促进破骨细胞的骨吸收功能。口颌系统巨细胞病变是一种非肿瘤性病损,被分为中心性巨细胞病变和外周性巨细胞病变。通过对二者临床病理学的分析,研究二者在发病性别和年龄上的异同。病变中多核巨细胞的组织学发生及其病理生理学机制是研究的热点。研究表明,这种多核巨细胞是破骨细胞样细胞。但是这种破骨细胞样细胞的来源及其分化调控是否与真正破骨细胞相同,还有待研究。本研究采用免疫组织化学方法,从破骨细胞的分化途径研究巨细胞病变中多核巨细胞的分化,结果表明其分化是由RANKL-RANK介导的,RANKL通过自分泌和旁分泌机制发挥其生物学效应。因此,破骨细胞培养体系的建立为体外研究破骨细胞的分化调控提供了可行的方法;研究破骨细胞转基因的可行性,为进一步利用转基因技术治疗骨吸收性疾病奠定理论基础;通过对破骨细胞样细胞分化途径的研究,对正确认识口颌系统巨细胞病变的发生、发展和转归具有重要的理论意义。更多还原

【Abstract】 Bone is a dynamic, active tissue. The process of bone remodeling is composed of osteoblast mediated bone formation and osteoclast mediated bone resorption, the persistence of which can make the balance of mineralization and the integrity of structure. The resorption, atrophy and defect of the jaw bone, caused by inflammation, damage, tumor or tumor-like lesion, congenital abnormality, aging, etc, is one of the most common clinical manifestation in stomatology. It not only affects beautiful outlook, function such as mastication, pronunciation, foodintake, etc, and the further restoration, but also seriously affects physical and mental health of the patient, and the quality of life. What we meet is how to find an effective way to prevent from and inhibit bone resorption, and promote bone formation. So the research of the origin and differentiation regulation of osteoclast not only has great theoretical significance in recognizing the process of bone resorption in both physiological and pathological condition, but also has great practical significance in the treatment of bone resorption diseases by inhibition of osteoclast differentiation and function.TWEAK, as a cytokine, is found to be related with the differentiation of the cells in bone remodeling. But there’s no literature on the effect of TWEAK on matured osteoclast. We researched the feasibility of transfecting exogenous gene into osteoclast mediated by adenovirus and at the same time we researched adenovirus mediated TWEAK on osteoclast. It settles theoretic bases for further study on treatment of bone resorption diseases by transgene therapy.Giant cell granuloma of stomatognathic system is a kind of non-tumor lesion. Pathologically, it is characterized by proliferation of fibroblasts and appearance of multinucleated giant cells, which shows a proliferative reaction to local damage or inflammation. There are 2 kinds of GCG, central giant cell granuloma (CGCG) and peripheral giant cell granuloma(PGCG). They occur in different tissues and there’s no accepted argument on whether the nature of the lesion is the same. The origin of the histology and the mechanism of pathophysiology of the MGCs in the lesion is hot spots of scientific research. Researchers found that MGCs in GCG showed some phenotypes of osteoclast and they had the function of bone resorption when cultured in vitro. So it is a kind of osteoclast-like cell. But it needs further study about the origin of the osteoclast-like cells and whether the differentiation of which is the same as genuine osteoclast.Thus we try to culture osteoclast in vitro, construct adenovirus carrying EGFP and TWEAK gene, and transfect cultured cells; we study the feasibility of transfecting osteoclast and the effect of TWEAK on the survival and function of osteoclast; By clinical pathological study of CGCG and PGCG, we try to show their similarities and differences; By immunohistochemistry, we try to study the mechanism of differentiation of MGCs in GCG. It has great theoretical significance in recognizing the onset, development and turnover of GCG.At first, we isolated and cultured matured osteoclast by mechanical method. By TRAP staining and bone resorption assay, we established a system of culturing osteoclast. Then we transfected the cultured cells using adenovirus carrying TWEAK gene. The result showed there were more TRAP positive cells in TWEAK group than in control group (P<0.05). And the area of bone resorption lacuna was much larger in TWEAK group than that in control group (P<0.01). By statistically analyzing gender, ages and clinical characteristics, we found female patients are 2 times as many as male patients and ages of PGCG was older than CGCG (P<0.05); CGCG occurs in hard tissue, while PGCG occurs in soft tissue, and they show different clinical characteristics and imaging manifestation. By immunohistochemistry method, the result showed immunoreactivity for CD68 was found in the cytoplasm of MGCs and parts of ovoid mononucleated stromal cells. Immunoreactivity for S-100 was negative in MGCs, spindle shaped and ovoid mononucleated stromal cells. Immunoreactivity for RANKL was found in the cytoplasm of MGCs, parts of ovoid mononucleated cells and vascular endothelial cells (VECs). Immunoreactivity for RANK was found in the cytoplasm of MGCs and parts of mononucleated stromal cells. There’s no difference of expression pattern of these protein in CGCG and PGCG.However, it still needs further research on the signal pathways of the effect of TWEAK on osteoclast, the transgene of osteoclast, the differentiation and signal pathways of MGCs in GCG of stomatognathic system. Conclusion:1. We have successfully isolated and cultured matured osteoclast, which is TRAP positive and have the function of bone resorption. It provides a feasible method for studying differentiation and function of osteoclast in vitro, and settle the basis for studying the mechanism of bone resorption;2. TWEAK could prolong the survival time of osteoclast to some extent, and TWEAK could significantly promote the function of osteoclast;3. In giant cell granuloma of stomatognathic system, there are 2 times of female patients than male ones. The age of PGCG is older than that of CGCG;4. The differentiation of osteoclast-like cells in giant cell granuloma of stomatognathic system is mediated by RANKL-RANK pathway, and RANKL exerts its biological effects by autocrine and paracrine.更多还原