【作者】 张姗姗；

【导师】 凌天牖；

【作者基本信息】 中南大学 ， 内科学， 2012， 博士

【摘要】 背景与目的口腔黏膜下纤维性变(oral submucous fibrosis, OSF)是发生于口腔黏膜的一种慢性、隐匿性疾病,其发病与咀嚼槟榔习惯关系密切,已经被世界卫生组织定义为癌前状态。与其他纤维化疾病一样,OSF目前尚无特效的治疗方法。同时合适动物模型的缺乏已经制约了对其具体病因、发病机制及治疗方法的进一步研究。姜黄素是中药姜黄的主要活性成分,已有报道称其具有较好的抗纤维化的作用。本研究拟通过局部注射博来霉素(Bleomycin, BLM)制备出重复性较好的OSF动物模型,然后在此基础上结合体外实验探讨姜黄素用于治疗OSF的可行性及作用机理。方法首先,进行动物模型的制备。SD大鼠100只,随机分为正常组、对照组和实验组。实验组大鼠在异氟烷的麻醉状态下,于双侧颊黏膜下注射BLM稀释液,每天一次,共持续八周。对照组采用磷酸盐缓冲液(Phosphate Buffered Saline, PBS)代替BLM稀释液。正常组不给任何干预。在规定时间处死大鼠后,采用HE染色、masson染色、羟脯氨酸碱水解法、免疫组化、western blotting及透射电镜分别检测各组大鼠颊黏膜组织病理、胶原水平、肌成纤维细胞(myofibroblast, MFB)、TGF-β1、IFN-γ及超微结构的变化情况。然后,SD大鼠35只,随机分为正常组、对照组、低剂量姜黄素组和高剂量姜黄素组。正常组大鼠不给予任何干预。其余大鼠在颊黏膜局部注射BLM4周后,采用不同剂量的姜黄素模型大鼠进行灌胃4周。大鼠处死后,采用HE染色、免疫组化和western blotting观察其颊黏膜组织病理、MFB、胶原水平、TGF-β1、 IFN-y和IL-13的表达水平。最后,采用体外细胞原代培养的方法,培养人正常颊黏膜成纤维细胞(fibroblast, FB),并利用TGF-β1将其诱导成为MFB表型。不同浓度姜黄素干预后,利用MTT、流式细胞术、western blotting及ELISA分别观察细胞增殖、细胞周期、凋亡、相关凋亡蛋白及上清液中Ⅰ、Ⅲ型胶原的改变。结果1、SD大鼠颊黏膜局部注射1mg/ml的BLM8周可以诱导出临床和病理上与人OSF相似的改变；2、较高剂量姜黄素可以降低BLM局部注射4周(病理改变接近人OSF早、中期)的SD大鼠颊黏膜组织中TGF-β1和IL-13的表达,提高IFN-y的表达,减少MFB；但对Ⅰ、Ⅲ型胶原的改变不明显；3、2.5-40μM的姜黄素可以抑制体外培养的FB和MFB增殖,且对MFB的抑制作用更加明显,并且将MFB阻滞在G0/G1期。10-40μM的姜黄素可以诱导MFB凋亡,提高Bax蛋白的表达,降低Bcl-2蛋白的表达,降低其上清液中Ⅰ、Ⅲ型胶原的表达水平。结论1、SD大鼠颊黏膜局部注射1mg/ml的BLM8周可以制备出类似人OSF病变的实验性OSF大鼠模型；该模型制作快捷,重复性好,但仍然存在一定的局限性。2、姜黄素可以改善lmg/ml BLM颊黏膜局部注射4周的(病理改变接近人OSF早、中期)SD大鼠的张口度,降低主要致纤维化细胞因子TGF-β1的表达,阻止纤维化的进一步发展；3、在体外姜黄素可以通过抑制MFB增殖,促进其凋亡,减少Ⅰ、Ⅲ型胶原生成,发挥抗纤维化作用。更多还原

【Abstract】 Background and ObjectiveOral submucous fibrosis (OSF) is a chronic and concealed disease of oral mucosa. It is closely related to betel quid (BQ) chewing habit and has been defined by WHO as one of the precancerous conditions. The same as other fibrotic diseases, there is still no definitive and effective treatment for OSF in the moment. The shortage of satisfied animal model of OSF has become a bottleneck slowing down the researches on the pathogeny, pathogenesis and clinical treatment of OSF.Curcumin is the primary active substance isolated from Curcuma Longa L. rhizome, and has been reported as an anti-fibrotic drug. Regard this as the starting point, we expected to create a stable and reliable animal model of OSF by locally injecting bleomycin (BLM), and then explored the feasibility of treatment with curcumin for OSF combined with in vivo and vitro experiment.MethodsFirstly,100Sprague-Dawley (SD) rats were randomly divided into3groups. Rats in the experimental group were injected BLM diluent to the bilateral buccal mucous membrane, once a day for8weeks, when they were under Isoflurane (inhalation) anesthesia. Rats in the control group were injected PBS instead of BLM diluents. Rats in the normal group did not receive any intervention. After the rats were executed, HE staining, masson staining, the soda hydrolysis in the measurement of hydroxyproline, immunohistochemistry, western blotting and transmission electron microscope were used to detect the changes of the histopathological features, levels of collagens, myofibroblast (MFB), TGF-β1、IFN-γ and ultrastructure.Secondly,35SD rats were randomly divided into4groups. Rats in the normal group did not receive any intervention. Rats in the control group, high curcumin group and low curcumin group were injected BLM diluent to the bilateral buccal mucous membrane, once a day for4weeks. Then curcumin solution of different dosages was given to the30rats by gavages, once a day for4weeks. After all the rats were executed, HE staining, immunohistochemistry and western blotting were used to detect the changes of the histopathological features, levels of collagens, MFB, TGF-β1、IFN-γ and IL-13.Thirdly, MFB were generated by incubating fibroblasts, obtained from human oral mucosa, with TGF-β1. MTT, PI staining, and FACS assays were used to investigate curcumin’s effect on proliferation and cell cycle of fibroblast (FB) and MFB. Annexin V/PI binding and FACS assays were used to examine apoptosis of MFB, western blotting to determine the levels of Bcl-2and Bax, and enzyme-linked immunosorbant assay was employed to examine the levels of collagen type Ⅰ and Ⅲ in the supernatants of myofibroblasts.Results1、Local injection of1mg/ml BLM diluent to the bilateral buccal mucous membrane of SD rats, once a day for8weeks, can create similar changes with human OSF in many ways of clinical symptoms and histopathological changes.2、The higher doses of curcumin decreased the expression of TGF-β1and IL-13, increased the expression of IFN-y and reduced MFB in the bucal mucous of the rats which were injected BLM diluents for4weeks. The levels of type Ⅰ,Ⅲ collagens had no significant change.3、Curcumin of2.5-40μmol/L inhibited proliferation of FB and MFB; MFB are more sensitive to curcumin than FB. Curcumin of10-40μmol/L also disturbed the cell cycle, induced apoptosis and decreases the generation of collagen type I and III in MFB. Curcumin induces apoptosis in MFB by down-regulating Bcl-2/Bax ratio.Conclusions1、Local injection of lmg/ml BLM diluent to the bilateral buccal mucous membrane of SD rats, once a day for8weeks, can create an experimental rat model of OSF. This model was of good repeatability and can be conveniently made. But there were still some defects in it.2、Curcumin can improve the mouth opening degree of the model rats, decreases the expression of TGF-β1, and prevent the further development of fibrosis.3. Curcumin play an anti-fibrosis role in vitro by inhibitting MFB proliferation, inducing apoptosis and decreasing the generation of collagen type Ⅰ and Ⅲ.更多还原