口腔扁平苔藓与NF-κB相关细胞因子和幽门螺杆菌关系的研究及药物对牙龈成纤维细胞增殖作用的影响
发布时间：2019-05-15 20:39
【摘要】：第一部分口腔扁平苔藓与NF κB相关细胞因子和幽门螺杆菌关系的研究目的:口腔扁平苔藓(Oral lichen planus OLP)是一种与自身免疫相关常见口腔疾病,目前为止OLP病因机制尚未完全明确。近年来核转录因子-κB(nuclearfactor-KB NF-κB)引导的炎症通路及其介导的炎症介质的紊乱一直成为研究的热点。NF-κB可能通过引起细胞因子网络紊乱,进一步介导炎症反应参与口腔扁平苔藓疾病的发生。杨凤英等采用基因芯片技术筛选出与OLP关系最密切的是幽门螺杆菌感染上皮细胞信号传导通路ECS-HP(Epithelial cell signaling in Helicobacter pylori infection)[1],口腔中幽门螺杆菌(Helicobacter pylori HP)感染与口腔扁平苔藓疾病的发生存在密切关系,HP也与慢性胃炎、消化性溃疡、胃黏膜相关淋巴组织淋巴瘤、胃癌的发病密切相关。HP感染胃壁粘膜后可以激活NF-κB通路并介导炎症反应。口腔粘膜与胃壁粘膜均来自于外胚层,口腔黏膜感染HP后引起的炎症机制可能与胃壁粘膜一致。口腔中HP感染激活NF-κB炎症通路在OLP发病中的作用有待进一步研究。本研究采用酶联免疫吸附实验(ELISA),检测口腔扁平苔藓患者与健康者血清、唾液中IL-8、RANTES的蛋白水平变化,通过观察血清和唾液中IL-8、RANTES等炎症因子的变化水平以及感染幽门螺杆菌OLP患者血清和唾液中细胞因子变化水平,初步探讨NF-κB炎症通路在口腔扁平苔藓发病中的作用机制以及OLP与HP的关系。方法:1采用ELISA方法对口腔扁平苔藓患者和健康者血清、唾液中IL-8、RANTES的含量进行测定,分析IL-8、RANTES在血清、唾液中表达。病例纳入标准:口腔扁平苔藓患者30例(糜烂型14例,普通型16例)和健康志愿者30例,分别收集口腔扁平苔藓患者和健康志愿者的唾液、血清样本。2采用ELISA方法检测HP感染阳性及HP阴性的口腔扁平苔藓患者血清、唾液中IL-8、RANTES的表达,分析HP感染与NF-κB通路在口腔扁平苔藓发病中的关系及其关键因子的表达。3幽门螺杆菌感染检测:快速尿素酶试验法检测口腔扁平苔藓患者牙菌斑中的幽门螺杆菌,探讨OLP患者和HP感染的相关性。结果:1口腔扁平苔藓患者与对照组相比血清和唾液中IL-8、RANTES显著增高,P0.05,差别有统计学意义;2口腔扁平苔藓血清中糜烂型与普通型IL-8、RANTES细胞因子表达水平差别无统计学意义,P0.05,口腔扁平苔藓唾液中糜烂型IL-8含量较普通型扁平苔藓显著升高,P0.05;3将口腔扁平苔藓分为HP感染阳性和HP感染阴性,血清中HP感染阳性较HP感染阴性者IL-8、RANTES表达水平显著增高,P0.05。唾液中HP感染阳性较HP感染阴性者IL-8、RANTES表达水平差别无统计学意义,P0.05。结论:1口腔扁平苔藓患者血清和唾液中IL-8、RANTES细胞因子水平与对照组相比均有不同程度的升高,提示口腔扁平苔藓的发病可能与NF κB通路介导的炎症反应相关,阻断NF κB炎症通路的激活可能成为治疗口腔扁平苔藓新的靶点,为口腔扁平苔藓的治疗提供新思路与方法。2血清中HP感染阳性较HP感染阴性者IL-8、RANTES表达水平显著增高,提示口腔扁平苔藓的发病可能与口腔中HP感染有一定相关性,并进一步介导免疫炎症反应。第二部分药物对牙龈成纤维细胞增殖作用的影响目的:目前药物性牙龈增生的机制尚未完全明确。一些研究表明牙龈炎、牙周炎菌斑的刺激可能有助于牙龈增生发展。本课题利用健康人牙龈组织原代培养牙龈成纤维细胞,分别应用硝苯地平、IL-1以及两种药物共同干预牙龈成纤维细胞,观察不同药物对牙龈成纤维细胞的增殖活性的影响,进一步探讨药物性牙龈增生机制以及药物性牙龈增生与牙龈炎的关系,为临床上药物性牙龈增生治疗提供新的思路。方法:1牙龈成纤维细胞原代培养:按照原代细胞组织块培养方法,将牙龈组织块接种于25m L细胞培养瓶中,观察原代细胞游出状态。细胞爬满瓶底约80%时进行传代。使用0.25%胰酶消化传代培养,首次传代按1:1的比例,再次传代以1:3的比例进行。取第5代牙龈成纤维细胞用于实验。其余细胞冻存备用。2牙龈成纤维细胞鉴定:倒置显微镜下观察原代细胞形态学变化;光镜下观察原代细胞HE染色及免疫组化结果(抗波形蛋白、抗角蛋白)。3细胞增殖实验:采用甲基噻唑基四唑(methyl thiazolyltetrazolium,MTT)法测定不同条件培养液下细胞数量变化(A组为空白对照组,不加任何药物;B组为硝苯地平组,又分为3个浓度亚组,B1组加入硝苯地平1200μg/L,B2组加入硝苯地平360μg/L,B3组加入硝苯地平108μg/L;C组为IL-1组,加入IL-1β10ng/ml;D组为硝苯地平+IL-1组,分为3个亚组,D1组=B1组+C组,D2组=B2组+C组,D3组=B3组+C组),在酶联免疫仪上测定490 nm处各孔吸光度值(OD值)。本实验重复3次。利用SPSS21.0软件分析数据。结果:1原代培养细胞从组织块游离出的时间约为10天,倒置显微镜镜下观察可见散在的贴壁伸展细胞,细胞形态呈长梭形,第11天组织块周围细胞明显增多,组织块周围形成细胞生长晕形态。原代第12天,可见细胞呈长梭形,胞核圆形或卵圆形,细胞数量明显增多。约12-14天细胞逐渐融合;2牙龈成纤维细胞鉴定:经传代培养后牙龈成纤维细胞形态逐渐向典型的成纤维样细胞转化,细胞生长呈漩祸状或放射走行,有极性且排列紧密,突起变短,呈长梭形、纺缍形、不规则三角形态(见Fig.1)。HE染色示细胞呈梭形或不规则三角形态,核圆或卵圆,胞核内可见分裂象,胞浆粉红,胞核蓝染(见Fig.2)。光镜下观察细胞免疫组织化学染色,波形蛋白表达为强阳性,呈棕黄着色,定位于细胞的胞质中,阳性颗粒在胞质内分布均匀,细胞核内染色阴性,细胞外未见阳性表达,细胞核经苏木精复染为蓝色,说明细胞来源于外胚间充质(见Fig.3),对照组细胞染色阴性。角蛋白表达为阴性(见Fig.4);3细胞增殖实验:不同浓度培养液作用后细胞增殖结果:培养0h、24h、48h、72h、96h各组OD值变化见Table 1。结论:1原代培养的细胞抗波形蛋白表达阳性、抗角蛋白表达阴性以及形态学上观察结果符合牙龈成纤维细胞,保留此细胞株,以便用于今后实验。2三种不同浓度硝苯地平都在一定程度上促进牙龈成纤维细胞细胞增殖,当硝苯地平浓度为1200μg/L时细胞增殖作用最明显,说明硝苯地平局部药物浓度可能在牙龈增生方面有着重要作用;3 IL-1浓度为10ng/ml对牙龈成纤维细胞的增殖活性产生抑制。硝苯地平与IL-1共同作用于牙龈成纤维细胞时,对牙龈成纤维细胞增殖作用不明显,有可能是牙龈炎症对硝苯地平引起的牙龈增生效果不明显。
[Abstract]:The relationship between the first part of oral lichen planus and NF-B-associated cytokines and Helicobacter pylori: Oral lichen planus OLP is a common oral disease associated with autoimmune diseases, so far the cause mechanism of OLP has not been completely clear. In recent years, the inflammatory pathway and the disorder of the mediated inflammatory mediators of the nuclear transcription factor-BNF-B have been the focus of the study. NF-B may further mediate inflammatory response to the occurrence of oral lichen planus by causing a disorder of the cytokine network. The infection of Helicobacter pylori (HP) in the oral cavity is closely related to the occurrence of oral lichen planus disease. HP is also closely related to chronic gastritis, digestive tract ulceration, gastric mucosa-associated lymphoid tissue lymphoma, and gastric cancer. The NF-B-B pathway can be activated and the inflammatory response can be mediated by the HP infection of the gastric wall mucosa. The mucosa of the oral mucosa and the stomach wall all come from the ectodermal layer, and the inflammatory mechanism caused by the infection of HP in the oral mucosa may be consistent with the mucosa of the stomach wall. The role of HP infection in the oral cavity in the pathogenesis of OLP is to be further studied. In this study, an enzyme-linked immunosorbent assay (ELISA) was used to detect the changes of the levels of IL-8 and RANTES in the serum and saliva of oral lichen planus, and by observing the levels of IL-8 in serum and saliva, The changes of the inflammatory factors such as RANTES and the level of cytokines in the serum and saliva of the patients with Helicobacter pylori OLP were studied. The mechanism of the inflammatory pathway in the oral lichen planus and the relationship between OLP and HP were discussed. Methods:1 The levels of IL-8 and RANTES in the serum and saliva of oral lichen planus and healthy persons were determined by ELISA, and the expression of IL-8 and RANTES in serum and saliva was analyzed. The cases were included in 30 cases of oral lichen planus (14 cases of erosion type,16 cases of common type) and 30 healthy volunteers. The saliva of oral lichen planus and healthy volunteers were collected separately. The expression of IL-8 and RANTES in the serum and saliva of the oral lichen planus with positive and negative HP infection was detected by ELISA. The relationship between HP infection and NF-B pathway in the pathogenesis of oral lichen planus was analyzed and the expression of the key factors was analyzed. The relationship between OLP and HP infection was discussed by the rapid urease test in the detection of Helicobacter pylori in the dental plaque of the oral lichen planus. Results: The levels of IL-8 and RANTES in the serum and saliva of 1 oral lichen planus were significantly higher than those in the control group. The level of IL-8 in the saliva of the oral lichen planus was higher than that of the normal lichen planus, P 0.05;3. The oral lichen planus was divided into HP infection positive and HP infection negative. There was no significant difference in the expression of IL-8 and RANTES in the patients with positive HP infection in saliva, P0.05. Conclusion: The level of IL-8 and RANTES in the serum and saliva of the oral lichen planus is higher than that of the control group, suggesting that the incidence of the oral lichen planus may be related to the inflammatory response mediated by the NF-B pathway. Blocking the activation of the NF-B inflammatory pathway may be a new target for the treatment of oral lichen planus, providing a new thought and method for the treatment of oral lichen planus. It is suggested that the incidence of oral lichen planus may be associated with HP infection in the oral cavity and further mediate the immune inflammatory response. The effect of the second part of the drug on the proliferation of gingival fibroblasts is that the mechanism of drug-induced gingival hyperplasia is not yet completely clear. Some studies have shown that the stimulation of plaque in gingivitis and periodontitis may contribute to the development of gingival hyperplasia. In this paper, the gingival fibroblasts were cultured in the gingival tissues of healthy people, and nifedipine, IL-1 and the two drugs were applied to the gingival fibroblasts, and the effects of different drugs on the proliferation of gingival fibroblasts were observed. To further study the relationship between drug-induced gingival hyperplasia and drug-induced gingival hyperplasia and gingivitis, and provide a new way for the treatment of drug-induced gingival hyperplasia. Methods:1. Primary culture of gingival fibroblasts: the gingival tissue mass was inoculated into a 25 m L cell culture flask according to the primary cell culture method, and the primary cell was observed. The cells were passaged at about 80% of the bottom of the flask. The subculture was digested with 0.25% trypsin, and the first passage was carried out at a ratio of 1:1, and the passage was carried out at a ratio of 1:3. The 5th generation of gingival fibroblasts was used for the experiment. The rest of the cells were cryopreserved.2 Gingival fibroblast identification: the morphological changes of primary cells were observed under an inverted microscope; the HE staining and the immunohistochemical results of primary cells (anti-keratin and anti-keratin) were observed under light microscope. The changes of the number of cells under different conditions were determined by MTT method. The group A was the blank control group without any drug. The group B was the nifedipine group, and it was divided into 3 concentration sub-groups, and the B1 group was added nifedipine 1200. m u.g/ L and the B2 group was added with nifedipine 360. m In group B3, Nifedipine 108. mu.g/ L was added; group C was IL-1, IL-1 and 10 ng/ ml were added; the D group was nifedipine + IL-1, divided into 3 subgroups, D1 group = B1 group + C group, D2 group = B2 group + C group, D3 group = B3 group + C group), The absorbance value (OD value) of each hole at 490 nm was measured on an enzyme-linked immunoassay. This experiment was repeated three times. The software was used to analyze the data. Results: The free time of the primary cultured cells from the tissue mass was about 10 days, and the adherent stretching cells of the primary cultured cells were observed under the inverted microscope. The cell morphology of the primary cultured cells was long and the number of cells around the tissue mass in the 11th day was significantly increased, and the cell growth halo was formed around the tissue mass. On the 12th day of primary culture, the cells were found to be long, round or oval, and the number of cells increased significantly. from about 12 to 14 days, the cells are gradually fused; the gingival fibroblasts are characterized in that the morphology of the gingival fibroblasts is gradually transformed to a typical fibroblast-like cell after subculture, Irregular triangular shape (see Fig.1). HE staining showed that the cells were in the form of a shuttle or an irregular triangle, a nucleus or an egg circle, a split image in the nucleus of the nucleus, pink of the cytoplasm, and blue staining of the nucleus (see Fig.2). The immunohistochemical staining of the cells was observed under the light microscope. The expression of the fluorescent protein was strongly positive. It was found to be brown and yellow, which was localized in the cytoplasm of the cell. The positive particles were distributed in the cytoplasm. The positive expression was not found in the cell. The nuclei were stained with hematoxylin to blue. The cells were derived from the human mesenchymal stem cells (see Fig.3), and the control group cells were stained with negative staining. The expression of keratin was negative (see Fig.4);3-cell proliferation experiment: the proliferation of cells after different concentration of culture medium: the changes of OD values in each group after incubation for 0 h,24 h,48 h,72 h, and 96 h are shown in Table 1. Conclusion:1. The expression of anti-keratin in primary cultured cells is positive, the anti-keratin expression is negative and the morphological observation is in line with the gingival fibroblasts, and the cell line is preserved. so as to be used for future experiments. The three different concentrations of nifedipine promote the proliferation of the gingival fibroblast cells to a certain extent, and the cell proliferation effect is most obvious when the concentration of nifedipine is 1200 & mu; g/ L, and the concentration of the local drug of nifedipine can play an important role in the gingival hyperplasia; The proliferation of gingival fibroblasts was inhibited by the concentration of 3IL-1 at 10 ng/ ml. The effect of nifedipine and IL-1 on the proliferation of gingival fibroblasts is not obvious, and the effect of gingival inflammation on the gingival hyperplasia caused by nifedipine is not obvious.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.5
本文编号：2477740
[Abstract]:The relationship between the first part of oral lichen planus and NF-B-associated cytokines and Helicobacter pylori: Oral lichen planus OLP is a common oral disease associated with autoimmune diseases, so far the cause mechanism of OLP has not been completely clear. In recent years, the inflammatory pathway and the disorder of the mediated inflammatory mediators of the nuclear transcription factor-BNF-B have been the focus of the study. NF-B may further mediate inflammatory response to the occurrence of oral lichen planus by causing a disorder of the cytokine network. The infection of Helicobacter pylori (HP) in the oral cavity is closely related to the occurrence of oral lichen planus disease. HP is also closely related to chronic gastritis, digestive tract ulceration, gastric mucosa-associated lymphoid tissue lymphoma, and gastric cancer. The NF-B-B pathway can be activated and the inflammatory response can be mediated by the HP infection of the gastric wall mucosa. The mucosa of the oral mucosa and the stomach wall all come from the ectodermal layer, and the inflammatory mechanism caused by the infection of HP in the oral mucosa may be consistent with the mucosa of the stomach wall. The role of HP infection in the oral cavity in the pathogenesis of OLP is to be further studied. In this study, an enzyme-linked immunosorbent assay (ELISA) was used to detect the changes of the levels of IL-8 and RANTES in the serum and saliva of oral lichen planus, and by observing the levels of IL-8 in serum and saliva, The changes of the inflammatory factors such as RANTES and the level of cytokines in the serum and saliva of the patients with Helicobacter pylori OLP were studied. The mechanism of the inflammatory pathway in the oral lichen planus and the relationship between OLP and HP were discussed. Methods:1 The levels of IL-8 and RANTES in the serum and saliva of oral lichen planus and healthy persons were determined by ELISA, and the expression of IL-8 and RANTES in serum and saliva was analyzed. The cases were included in 30 cases of oral lichen planus (14 cases of erosion type,16 cases of common type) and 30 healthy volunteers. The saliva of oral lichen planus and healthy volunteers were collected separately. The expression of IL-8 and RANTES in the serum and saliva of the oral lichen planus with positive and negative HP infection was detected by ELISA. The relationship between HP infection and NF-B pathway in the pathogenesis of oral lichen planus was analyzed and the expression of the key factors was analyzed. The relationship between OLP and HP infection was discussed by the rapid urease test in the detection of Helicobacter pylori in the dental plaque of the oral lichen planus. Results: The levels of IL-8 and RANTES in the serum and saliva of 1 oral lichen planus were significantly higher than those in the control group. The level of IL-8 in the saliva of the oral lichen planus was higher than that of the normal lichen planus, P 0.05;3. The oral lichen planus was divided into HP infection positive and HP infection negative. There was no significant difference in the expression of IL-8 and RANTES in the patients with positive HP infection in saliva, P0.05. Conclusion: The level of IL-8 and RANTES in the serum and saliva of the oral lichen planus is higher than that of the control group, suggesting that the incidence of the oral lichen planus may be related to the inflammatory response mediated by the NF-B pathway. Blocking the activation of the NF-B inflammatory pathway may be a new target for the treatment of oral lichen planus, providing a new thought and method for the treatment of oral lichen planus. It is suggested that the incidence of oral lichen planus may be associated with HP infection in the oral cavity and further mediate the immune inflammatory response. The effect of the second part of the drug on the proliferation of gingival fibroblasts is that the mechanism of drug-induced gingival hyperplasia is not yet completely clear. Some studies have shown that the stimulation of plaque in gingivitis and periodontitis may contribute to the development of gingival hyperplasia. In this paper, the gingival fibroblasts were cultured in the gingival tissues of healthy people, and nifedipine, IL-1 and the two drugs were applied to the gingival fibroblasts, and the effects of different drugs on the proliferation of gingival fibroblasts were observed. To further study the relationship between drug-induced gingival hyperplasia and drug-induced gingival hyperplasia and gingivitis, and provide a new way for the treatment of drug-induced gingival hyperplasia. Methods:1. Primary culture of gingival fibroblasts: the gingival tissue mass was inoculated into a 25 m L cell culture flask according to the primary cell culture method, and the primary cell was observed. The cells were passaged at about 80% of the bottom of the flask. The subculture was digested with 0.25% trypsin, and the first passage was carried out at a ratio of 1:1, and the passage was carried out at a ratio of 1:3. The 5th generation of gingival fibroblasts was used for the experiment. The rest of the cells were cryopreserved.2 Gingival fibroblast identification: the morphological changes of primary cells were observed under an inverted microscope; the HE staining and the immunohistochemical results of primary cells (anti-keratin and anti-keratin) were observed under light microscope. The changes of the number of cells under different conditions were determined by MTT method. The group A was the blank control group without any drug. The group B was the nifedipine group, and it was divided into 3 concentration sub-groups, and the B1 group was added nifedipine 1200. m u.g/ L and the B2 group was added with nifedipine 360. m In group B3, Nifedipine 108. mu.g/ L was added; group C was IL-1, IL-1 and 10 ng/ ml were added; the D group was nifedipine + IL-1, divided into 3 subgroups, D1 group = B1 group + C group, D2 group = B2 group + C group, D3 group = B3 group + C group), The absorbance value (OD value) of each hole at 490 nm was measured on an enzyme-linked immunoassay. This experiment was repeated three times. The software was used to analyze the data. Results: The free time of the primary cultured cells from the tissue mass was about 10 days, and the adherent stretching cells of the primary cultured cells were observed under the inverted microscope. The cell morphology of the primary cultured cells was long and the number of cells around the tissue mass in the 11th day was significantly increased, and the cell growth halo was formed around the tissue mass. On the 12th day of primary culture, the cells were found to be long, round or oval, and the number of cells increased significantly. from about 12 to 14 days, the cells are gradually fused; the gingival fibroblasts are characterized in that the morphology of the gingival fibroblasts is gradually transformed to a typical fibroblast-like cell after subculture, Irregular triangular shape (see Fig.1). HE staining showed that the cells were in the form of a shuttle or an irregular triangle, a nucleus or an egg circle, a split image in the nucleus of the nucleus, pink of the cytoplasm, and blue staining of the nucleus (see Fig.2). The immunohistochemical staining of the cells was observed under the light microscope. The expression of the fluorescent protein was strongly positive. It was found to be brown and yellow, which was localized in the cytoplasm of the cell. The positive particles were distributed in the cytoplasm. The positive expression was not found in the cell. The nuclei were stained with hematoxylin to blue. The cells were derived from the human mesenchymal stem cells (see Fig.3), and the control group cells were stained with negative staining. The expression of keratin was negative (see Fig.4);3-cell proliferation experiment: the proliferation of cells after different concentration of culture medium: the changes of OD values in each group after incubation for 0 h,24 h,48 h,72 h, and 96 h are shown in Table 1. Conclusion:1. The expression of anti-keratin in primary cultured cells is positive, the anti-keratin expression is negative and the morphological observation is in line with the gingival fibroblasts, and the cell line is preserved. so as to be used for future experiments. The three different concentrations of nifedipine promote the proliferation of the gingival fibroblast cells to a certain extent, and the cell proliferation effect is most obvious when the concentration of nifedipine is 1200 & mu; g/ L, and the concentration of the local drug of nifedipine can play an important role in the gingival hyperplasia; The proliferation of gingival fibroblasts was inhibited by the concentration of 3IL-1 at 10 ng/ ml. The effect of nifedipine and IL-1 on the proliferation of gingival fibroblasts is not obvious, and the effect of gingival inflammation on the gingival hyperplasia caused by nifedipine is not obvious.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.5
【参考文献】
相关期刊论文 前10条
1 姚小武;林敏校;陈仕生;卢子正;;幽门螺杆菌感染与口腔扁平苔藓和鳞状细胞癌相关性的初步探讨[J];中华口腔医学研究杂志(电子版);2014年05期
2 倪靖;束蓉;;服用硝苯地平后牙龈增生以及未增生患者牙龈成纤维细胞生物特性比较[J];口腔医学研究;2012年11期
3 浦光瑞;张虹;;口腔扁平苔藓中MMP-2和MMP-9的表达及意义[J];大连医科大学学报;2012年05期
4 王翔宇;孙克勤;李霞;崔娟;任娟;李欣欣;霍俊锋;;IL-1β诱导人牙龈成纤维细胞TRAF6表达[J];中国公共卫生;2011年06期
5 金早蓉;宋斌;李曙霞;方红辉;;口腔粘膜扁平苔藓患者口腔多部位幽门螺杆菌的检测及其意义[J];当代医学;2010年19期
6 郭万伟;柏树令;王军;范军;田晓红;;NF-κB及调节活化正常T细胞表达和趋化因子在升主动脉瘤形成中的表达[J];解剖学报;2010年01期
7 倪靖;束蓉;;硝苯地平对药物性牙龈增生患者牙龈成纤维细胞增殖的影响[J];上海交通大学学报(医学版);2010年01期
8 许彦枝;杨凤英;;口腔黏膜扁平苔藓差异基因筛选及其通路分析[J];中华临床医师杂志(电子版);2010年01期
9 宋铁芳;杨婷;李佩玲;;热休克蛋白27与恶性肿瘤关系的研究进展[J];实用肿瘤学杂志;2008年05期
10 张敏;冉玉平;;趋化因子RANTES与皮肤病[J];中国皮肤性病学杂志;2007年05期
，本文编号：2477740
本文链接：https://www.wllwen.com/yixuelunwen/kouq/2477740.html
最近更新
教材专著
