【作者】 张云；

【导师】 郑筱祥；

【作者基本信息】 浙江大学 ， 生物医学工程， 2007， 博士

【摘要】 成骨细胞和破骨细胞为骨组织的主要功能细胞，成骨细胞增殖和分化可形成新骨；破骨细胞活动则诱导骨吸收。有研究表明牙槽骨丢失是骨吸收增加或骨形成降低或两者共同作用的结果，因此作为牙周组织靶细胞的成骨细胞和破骨细胞前体细胞-骨髓单核／巨噬细胞在牙周炎的发生、发展中发挥重要的作用。同时牙龈卟啉菌(Porphyromonas gingival，Pg)和伴放线杆菌(Actinobacillus actinomycetemcomitan，Aa)等革兰氏阴性菌细胞壁的主要成分脂多糖(Lipopolysaccharide，LPS)为一种非常重要的细胞因子，可作用于牙周组织介导牙周炎的发生和牙周组织损伤。但大肠杆菌(Escherichia coli)LPS对牙周组织靶细胞-成骨细胞和骨髓单核细胞分化和功能的影响目前还不清楚。本研究着眼于以上两个靶细胞在牙槽骨吸收中的作用，探讨了Escherichia coli LPS对成骨细胞和骨髓单核细胞的影响及其可能的作用机理和药物干预机制。本论文综合应用激光共聚焦显微术、流式细胞术、细胞免疫化学和免疫印迹等现代研究技术，分别从细胞、分子水平研究了LPS对成骨细胞分化的影响及其诱导破骨细胞骨吸收作用，并探讨了PI3K／Akt信号通路在以上模型中的作用。此外，还研究了中药单体葛根素对成骨细胞骨形成和E．coli-LPS诱导的骨吸收干预及其可能的作用机理。本研究利用LPS诱导成骨细胞凋亡研究了LPS对成骨细胞生长和分化的影响。结果表明LPS能明显抑制成骨细胞骨形成，并诱导成骨细胞凋亡、骨架F-actin重排、线粒体膜电位丢失和Akt的去磷酸化。阻断PI3K活性可明显促进LPS抑制成骨细胞生长和Akt失活，而且应用PI3K激活剂IGF-Ⅰ(insulin-like growth factorⅠ，IGF-Ⅰ)可减弱PI3K抑制剂LY24002诱导的成骨细胞生长抑制和Akt去磷酸化作用，但是IGF-Ⅰ对LPS所刺激的上述反应没有影响。提示LPS可抑制成骨细胞增殖分化，其机制可能是通过诱导细胞凋亡和Akt失活而进行，但与PI3K活性无关。同时，本研究还建立LPS诱导骨吸收模型，探讨了LPS诱导破骨细胞形成和骨吸收作用。结果显示LPS能诱导破骨细胞形成、骨吸收并激活Akt而导致p-AktSer473和p-AktThr308水平明显增加。而且PI3K的另一抑制剂Wortmanin可显著抑制破骨细胞形成和Akt的活化，并诱导成熟破骨细胞凋亡，表明LPS诱导破骨细胞分化、吸收和存活可能与PI3K／Akt信号通路激活有关。此外，本研究还探讨了葛根提取物-葛根素对骨形成和骨吸收的干预作用。结果发现葛根素可明显促进骨形成并诱导Akt活化和核转位，且这种活化作用能被LY294002所阻断。同时，葛根素还可显著抑制LPS诱导的骨吸收和Akt活化，并促进成熟破骨细胞及其前体细胞-骨髓单核细胞凋亡。由此可知，葛根素可促进骨形成和抑制LPS诱导骨吸收，其机理可能与PI3K／Akt通路激活或灭活相关。综上所述，本研究主要创新体现在：1．本研究通过建立LPS诱导成骨细胞凋亡模型，论证了E．coli-LPS可抑制牙周靶细胞-成骨细胞增殖与分化而影响牙槽骨形成。2．利用LPS诱导破骨细胞形成和骨吸收证实了E．coli-LPS可刺激牙周另一靶细胞-骨髓单核细胞分化为破骨细胞，并促进骨吸收，提示刺激骨髓单核细胞的分化、融合可诱导或加重牙槽骨吸收。3．本研究通过对PI3K／Akt通路在E．coli-LPS诱导牙周靶细胞凋亡和骨吸收模型中的作用研究，为深入探讨LPS诱导的牙槽骨吸收和牙周炎发病机制提供一个新的信号通路，并为研制开发治疗该病药物提供新的作用靶点和一定的理论基础。4．本研究还证实了葛根素可促进骨形成和抑制E．coli-LPS诱导的破骨细胞形成和骨吸收，因此葛根素可用于预防和辅助治疗LPS感染的牙周病，并为该药在牙周疾病上的应用提供了一定的理论依据。更多还原

【Abstract】 Bone tissue is composed of two distinct coll populations, namely the bone-forming ostcoblasts (OBs) derived from the mesenchymal stromal lineage, and the bone-resorbing osteoclasts (OCs), which arc of haematopoietic stem cell origin. Previous studies indicated that alveotar bone destruction results from the increase of bone resorption by OCs, or the reduction of bone formation by OBs, and or an imbalance between the two aspects. Then, the gingival giant cells such as osteoblasts and bone marrow monocytes (BMMs) play important roles in periodontitis. In addition, lipopolysaccharide (LPS) from gram-negative bacteria such as Porphyromonas gingival (Pg), Actinobacillus actinomycctemcomitan (Aa) has been considered a key factor in the development of chronic inflammation leading to the destruction of periodontal tissue. However, the effect of LPS from Escherichia coli on the above cells remains unknown. This study was perfomed to observe the role of osteoblasts and BMMs in gingival resorption, and periodontal disease, and to understand its possible mechanism. And anti-resorptive drug was also investigated in this study.In the present study, confocal laser scanning microscopy, flow cytometry, and immunofluroscence and western Blots were used to observe the effect of E.coli-LPS on ostcoblasts differentiation and ostcoclasts resorption in vitro, and further to understand the role of phosphatidylinositol 3-kinasc/Akt (PI3K/Akt) pathway in the process. In addition, we examined the effects of puerarin on bone formation and LPS-induced osteolysis and studied their possible molecullar mechanisms.Calvaria ostcoblasts isolated from newborn SD rats were used to observe the effect of LPS on osteoblastic growth and differentiation. Data showed that LPS significantly inhibited osteoblastic bone formation, and resulted in osteoblastic apoptosis and F-actin rearrangment. This dysfuction was accompanied by loss of mitochondrial membrane potential (ΔΨm) and dephosphorylation of Akt. Inhibition of PI3K with LY294002 enhanced LPS-induced inhibition of ostcoblasts growth and dephosphorylation of Akt.Furthmore, the PI3K stimulator IGF-Ⅰwas found to significantly weaken LY294002-triggercd dephosphorylation of Akt and growth inhibition. But LPS effect on Akt was not affected by IGF-Ⅰ. This meant that LPS inhibited osteoblasts differentiation and induced cell apoptosis might be through inactivation of Akt, but independently of PI3K.To study whether LPS induced bone resorption, osteoclasts were obtained from bone marrow monocytes (BMMs) of 6-9 weeks SD rats, and found evidence of PI3K/Akt pathway in the process. Results indicated that LPS significantly stimulated osteoclasts formation, bone resorption and activation of Akt at Ser473 and Thr308, which was obviously blocked by another PI3K specific inhibitor Wortmanin. Furthermore, Wortmanin triggered rapid apoptosis of mature osteoclasts and Akt inavtivation. These findings suggested that LPS stimulated osteoclasts differentiation, resorption and survival, which might be mediated by activation of the PI3K/Akt signaling pathway.In addition, this study examined the effects of puerarin from Chinese Pueraria lobata (Wild.) Ohwi on bone formation and on LPS-induced osteolysis. Data suggested that puerarin caused a significant increase in cell viability, alkaline phosphatase (ALP) activity and mineral nodules formation in osteoblasts, suggesting that puerarin had a stimulatory effect on osteoblastic bone formation. The functional improvement by puerarin was accompanied by activation and translocation of Akt. Furthermore, the activation of Akt and its redistribution were blocked by LY294002. These results indicated that puerarin stimulated activation of Akt in a PI3K-dependent manner. Then, puerarin can promote bone formation in cultured rat osteoblasts, which might be mediated by activation of the PI3K/Akt pathway. On the other hand, puerarin significantly inhibited LPS-induced osteolysis, and triggered rapid apoptosis of mature osteoclasts and their precursor cells. And puerarin blocked LPS-stimulated Akt activation, and caused an obvious decrease in Akt phosphorylation at Ser473 and Thr308, implying an important role for Akt pathway in the anti-osteolysis action. These resuts indicated puerarin could protect against LPS-stimulated alveolar bone loss by triggering cell apoptosis and inactivation of Akt. Taken together, puerarin from Chinese Pueraria lobata (Wild.) Ohwi might stimulate bone formation and inhibit bone resorption by activation or inactivation of PI3K/Akt pathwy. Based on the above results, some innovative points have been drawn as below:1. LPS from Escherichia coli inhibited osteoblastic growth and differentiation, suggesting that E. coli-LPS may prevent new bone formation in gingival bone tissue.2. E. coli-LPS induced osteoclasts formation and bone resorption, indicating that E.coli-LPS can regulate periodontal BMMs, and result in the reduction of alveolar bone resorption.3. We studied the roe of PI3K/Akt pathway in the E.coli LPS-triggered osteoblasts apoptosis and osteolysis, and offerd a new signaling pathway for understanding periodontal bone resorption, and some academic data for developing anti-resorption drugs.4. Puerarin from Pueraria lobata (Wild.) Ohwi might stimulate bone formation and inhibit LPS-inducd bone resorption, which have therapeutic value in effective in treating alveolar bone destruction and periodontitis, and offer some data for its application in these diseases.更多还原