【作者】 刘海凤；

【导师】 李万胜；

【作者基本信息】 河北医科大学 ， 妇产科学， 2010， 硕士

【摘要】 目的:宫颈癌是全球发病率居第二位的妇女恶性肿瘤,在我国的发病率居首位[1],其高发区连接成片,且发病人群有年轻化的趋势[2],成为严重威胁妇女健康的疾病之一。宫颈癌的发生、发展是一个由量变到质变,由渐变到突变的过程。局部浸润和淋巴结转移不仅影响手术效果,而且直接关系患者预后。淋巴结转移是宫颈癌的主要转移途径,在早期即可发生,有文献报道,无淋巴结转移者五年生存率为82.2%,有淋巴结转移者五年生存率仅为50.8%[3]。血管生成是从已有的微血管系统形成新生血管的过程,是一种对生理和病理过程都十分关键的生物学过程。血管生成的调节有赖于血管生成因子和血管静止因子之间的平衡,它们分别促进和抑制肿瘤血管的生成。如果没有血管,氧气和营养物质的扩散就会受到抑制,那么肿瘤也不会生长。血管形成过程可有许多生长因子触发,这些生长因子可由肿瘤细胞自身和(或)其周围的基质细胞分泌的[4]。随着最近几年对肿瘤血管形成的研究,人们发现许多生长因子都具有使血管生成的功能,血管生成相关性趋化因子和细胞因子在肿瘤血管生成过程中有重要作用,其中最初被描述为嗜中性化学诱导物的CXC趋化因子IL-8正越来越多的引起人们的注意。IL-8是1987年Yoshimur等[5]发现的第一个趋化因子,当时被命名为单核细胞衍生的中性粒细胞趋化因子,是与炎症相关的CXC趋化因子。随着对IL-8及其受体生物学活性和作用机制研究的深入, Strieter等[6]发现IL-8的NH2末端有一谷氨酸一亮氨酸一精氨酸( Glu-Leu-Arg )组成的三联体氨基酸基序一ELR基序,该基序在IL-8促血管生成中起重要作用。Murphy等[7]也发现IL-8参与肿瘤血管形成,与肿瘤的侵润、转移密切相关。随后研究者在小细胞肺癌[8]和胃癌[9]及卵巢癌[10]等也得出了类似的结果,提示IL-8参与肿瘤的发生和发展。IL-8可以促进肿瘤细胞的增生,使肿瘤细胞向内皮细胞迁移[11];调节肿瘤细胞及周围基质细胞分泌MMP-2 , MMP-9并增强其活性,从而促进胶原酶活性,提高肿瘤细胞的体[12]。IL-8也可以促进血管内皮细胞的增生、迁移、抑制其自发性凋亡及增加血管的通透性[13],有助于肿瘤细胞转移。另外,有研究显示淋巴管内皮细胞可通过分泌趋化因子吸引肿瘤细胞,进而主动促进肿瘤细胞的淋巴结转移[13]。但对IL-8在宫颈癌中的作用研究甚少,Tjiong等人[14]通过比较宫颈分泌物和阴道冲洗液中IL-8的水平认为宫颈癌和CIN病例阴道局部可能产生高水平的IL-8和IL-6,这些细胞因子有可能对局部炎症反应和肿瘤生长有一定作用。日本的Fujimot等人[15]的研究表明IL-8与宫颈癌的血管生成及预后相关,但并未阐明其分子作用机制。吴素慧等[16]对早期宫颈鳞癌进行了cDNA基因芯片筛查,结果表明IL-8在有淋巴结转移宫颈癌组织中的表达较无淋巴结转移者高3.4倍,推测其可能在早期宫颈癌的浸润、转移中起重要作用。尽管宫颈癌的治疗研究已近百年,成绩显著,但其疗效还未能达到普遍接近“早期宫颈癌5年生存率90%-100%”的水平。为进一步探讨早期宫颈癌发生、发展及淋巴结转移机制,以尽早有效地诊断、治疗宫颈癌,近些年来,随着细胞工程和基因工程技术的发展,肿瘤免疫治疗亦得以迅猛发展,细胞因子基因治疗成为目前研究的热点。本实验通过动物实验,应用免疫组化法比较CD34及CD57在宫颈癌移植瘤组织中的表达情况,观察IL-8对宫颈癌侵袭和转移能力的影响,并探索其作用机制,以期为宫颈癌的诊治和预后判断提供新的指标。现多采用免疫组织化学技术对肿瘤微血管进行定量计算,即微血管密度计数,目前反映微血管密度的标记物有CD34, CD31, CD105和vs因子等,其中CD34是较敏感和较特异的肿瘤新生血管内皮细胞表面标志, CD34又称为人定向造血干细胞抗原,在造血干细胞、血管内皮细胞等都有表达。在肿瘤组织中,其表达与肿瘤血管内皮细胞的增殖、移行有关,并被认为是判断肿瘤患者预后的一个独立因素。NK细胞是机体免疫中一类对多种靶细胞有自发细胞毒活性的效应细胞,在抗肿瘤中主要发挥非特异性免疫作用,NK细胞在启动杀瘤作用时不需要复杂的抗原呈递作用,也不受主要组织相容复合体(MHC)的限制,能直接释放特异性杀伤蛋白-穿孔素(pertorin)、细胞毒因子(NKCF),对靶细胞起溶解杀伤作用,故认为是机体抗肿瘤,抗感染的第一道防线[17,18]。CD 57(I-INK一1,leu一7)是一种分子量为110000的糖蛋白,被认为是人自然杀伤细胞的特异性表面标记,也存在于T淋巴细胞上和一些肿瘤细胞表面[19,20],外周血中30%-80%的NK细胞可表达此抗原,故其阳性细胞数量水平在一定程度上能反映NK细胞的功能。方法:1.细胞和实验动物Hela/pcDNA3.1(+)–h/IL-8细胞、Hela/pcDNA3.1(+)–h细胞和Hela细胞由本室构建并保存。SPF级BALB/c-nu裸小鼠,21只,雌性,4-6周龄,体重16-18g,购自北京协和动物中心。2.Hela/pcDNA3.1(+)–h/IL-8细胞、Hela/pcDNA3.1(+)–h细胞和Hela细胞分别培养至对数生长期,取1.5×107个细胞(0.2ml),常规消毒皮肤后,接种于裸鼠右后背部皮下,当肿瘤长至直径约为5 mm时视为成瘤,并记录成瘤天数及成瘤率。3.成瘤后每周测量各组肿瘤的纵径和横径一次,通过公式V=π/6×长×宽2计算肿瘤体积(mm3),估算肿瘤体积。4.接种肿瘤细胞9周后颈椎脱臼处死小鼠,完整剥离肿瘤组织及肺组织。5.将小鼠成瘤组织及肺组织按病理学制样常规行福尔马林固定,石蜡包埋,切片,通过HE染色,观察肺组织的转移情况;通过免疫组化法观察CD34及CD57在三组瘤组织中的表达情况。结果:1.成功构建裸鼠移植瘤模型,各组成瘤率均为100%。两两比较结果显示,IL-8组成瘤时间与空质粒组及阴性对照组有明显差异(P<0.05),空质粒组与阴性对照组之间无明显差异(P>0.05)。2.7、8、9周IL-8组肿瘤生长活跃,体积明显大于空质粒组及阴性对照组,两两比较有统计学意义(P<0.05),前6周各组肿瘤体积比较无明显差别(P>0.05)。3.免疫组化结果MVD免疫组化结果: IL-8组肿瘤组织中CD34阳性表达明显增加,空质粒和阴性对照组的肿瘤组织内可见少量阳性反应。两两比较结果显示,IL-8组CD34平均光密度值(OD)值与空质粒组及阴性对照组均有明显差异(P<0.05),空质粒组与阴性对照组之间无明显差异(P>0.05)CD57免疫组化结果:空质粒和阴性对照组的肿瘤组织内可见大量阳性反应,IL-8组肿瘤组织中阳性表达明显少于空质粒和阴性对照组。两两比较结果显示,IL-8组CD57阳性细胞数与空质粒组及阴性对照组均有明显差异(P<0.05),空质粒组与阴性对照组之间无明显差异(P>0.05)。4.各实验组肺组织均未见癌组织转移。结论:1.成功构建IL-8基因裸鼠移植瘤模型,通过比较各组的成瘤率、成瘤时间及肿瘤体积证实IL-8能够促进肿瘤生长、增殖。2.动物实验研究发现IL-8促进肿瘤组织内毛细血管的生成,为宫颈癌组织生长侵袭和远处转移提供必要条件。3.CD57低表达可能与宫颈癌组织生长侵袭和远处转移有关。4.各组肺组织中无癌细胞的转移。更多还原

【Abstract】 Objective: Uterine cervix carcinoma is the second commonest female cancer worldwide, The Prevalence lies in first Place in our country (China) [1].The areas of its high occurrence are all connected with each other and people who developed it are at a much younger age than before[2]. It has become one of the main diseases which severely threaten the health of women in our country. The occurrence of cervical cancer development is a quantitative change to qualitative change, from gradient to the mutation process. Local infiltration and lymph node metastasis not only influence the operation effect but also correlate directly with the prognosis of patients. Lymph node metastasis is the main route of metastasis of uterine cervix carcinoma, and could take place in the early stage. It has been reported that the 5-year survival rate of the patients who had no lymph node metastases was 82.2% and who had lymph node metastasis was 50.8%[3].Angiogenesis is the process that from the previously blood capillary system to form new vessels, and plays important part in the physiology and pathology process. The regulation of angiogenesis depends on the balance between angiogenesis factors and vascular static factor factors, they discern to promote and restrain the tumor’s angiogenesis. If there is no blood vessel, the diffusion of oxygen and nutrient substance will be restrained, then, the tumor can not grow up. Angiogenesis process has many growth factors’trigger, These growth factors are from tumor cell-self or their peripheral matrix cell[4]. Along with the last years’investigation of angiogenesis,people discovered that all manners of growth factors are all can promote angiogenesis. Angiogenesis correction chemotatic factors and cytokine play important part in angiogenesis process, among these factors, IL-8 which has been described neutrophilia chemical inductor initial,is one of CXC chemotatic factors, today, more and more people pay attention to it.IL-8 was discovered in 1987, the first one, etc. Yoshimur[5] chemotactic factor, was then named monocyte-derived neutrophil chemotactic factor is associated with inflammation-related CXC chemokines. Along with the investigation functional mechanism of IL-8 and its acceptor, Strieter[6] discovered that NH2 termination of IL-8 had a ELR motif, playing an important role in angiogenesis. Murphy[7] also found that IL-8 involved in tumor angiogenesis, and tumor invasion, metastasis. Followed by researchers in small cell lung cancer[8] and stomach cancer[9] and ovarian cancer[10], and so have come to similar results, suggesting that IL-8 involved in tumor occurrence and development. IL-8 can promote the proliferation of tumor cells to tumor cells to endothelial cell [11] migration; regulating tumor cells and surrounding stromal cells secrete MMP-2, MMP-9 and to enhance its activity, thereby contributing to collagenase activity and increased in vitro invasion[12] of tumor cells. IL-8 can also promote vascular endothelial cell proliferation, migration, inhibit the spontaneous apoptosis and increased permeability of blood vessels[13] contribute to tumor cell metastasis. In addition, studies have shown that lymphatic endothelial cells through the secretion of chemokines in attracting tumor cells, and then take the initiative to promote lymph node metastasis of tumor cells. But until now, there has been little investigation of IL-8 in uterine cervix cancer. Tjiong[14] and others by comparing the cervical secretions and vaginal washing fluid levels of IL-8 that the cases of cervical cancer and CIN may have high levels of local vaginal IL-8 and IL-6, these cytokines may have local inflammatory response and tumor growth of a role. Japan’s Fujimot [15] and others studies have shown that IL-8 and cervical cancer angiogenesis and prognosis, but did not clarify the molecular mechanism of action. Suhui Wu[16], etc. carried out in early cervical squamous cell carcinoma cDNA microarray screening results show that IL-8 in a lymph node metastasis of cervical cancer tissue than those without lymph node metastasis was 3.4 times higher, suggesting it may at an early stage of cervical cancer invasion and metastasis play an important role. Although the research on the treatment of UCC has continued for nearly a hundred years and the result is very striking, the 5-year survival rate of early UCC hasn’t reached 90%-100%. To improve the treatment and promote the curative effect,in recent years, with the technological development of cell engineering and generic engineering, tumor immunotherapy is also developing rapidly. Recently cytokine gene therapy has become the focus of research. In this study, through animal experiments, immunohistochemical staining compared CD34 and CD57 in cervical tumor tissue expression was observed IL-8 in cervical cancer invasion and metastasis ability of and explore their mechanisms of action, with a view to cervical cancer The treatment and prognosis of new indicators. Now more than immunohistochemical techniques for quantitative microvessel count, that microvessel density count, this is MVD. Microvessel density, reflecting the markers are CD34, CD31, CD105, and vs factors, among which CD34 is a more sensitive and more specific for tumor angiogenesis endothelial cell surface marker, CD34 known as hematopoietic stem cell antigen targeted Wei Ren, in the hematopoietic stem cells, vascular endothelial cells, and others have expressed. In tumor tissues, its expression and tumor vascular endothelial cell proliferation, migration and related, and is considered to determine the prognosis of cancer patients as an independent factor. NK cell is one effector cell which has spontaneous cytototix reactive and effects on many kinds of target cells on immune function of organism. It play in the main anti-tumor non-specific immune function,It is neither need a complex antigen-presenting role nor restriction from the major histocompatibility complex (MHC), to release specific anti-protein -- pertorin (pertorin), cytotoxic factor (NKCF ) directly. It can dissolute and kill the target cell, so it is considered to be the body of anti-tumor, anti-infection of the first line of defense[17,18]. CD 57 (I-INK 1 1, leu-7) is a glycoprotein with a molecular weight of 110000, is considered the specificity of human natural killer cell surface markers. Also present in T lymphocytes and some tumor cell surface [19,20].The formerly study indicated that CD57 antigen can hold back the proliferation of homeocyte . In peripheral blood of 30% -80% of the NK cells can express views antigens, so the level of positive cells to some extent reflect the function of NK cells.Methods:1 Cells and experimental animalHela/pcDNA3.1(+)–h/IL-8 cell and Hela/pcDNA3.1(+)–h cell , these cells all come from laboratory.Nude-mice, 21, female, aged 4-6 week, body mass is 16-18g, buy from animal Center of Beijing Concord.2 Hela/pcDNA3.1 (+) -h/IL-8 cells, Hela/pcDNA3.1 (+)-h cells and Hela cells were cultured to logarithmic growth phase, taking 1.5×107 cells (0.2ml), conventional After skin disinfection and inoculated subcutaneously in nude mice, after the right, when the tumor grew to a diameter of about 5 mm were regarded as tumorigenicity, and record the animal tumorigenicity time and rate.3 After animal tumorigenicity, to measure every group tumor’s length and width, through formula V=π/6×a×b2 to calculate tumor volume(mm3).4 9 weeks after inoculation of tumor cells mice were sacrificed cervical dislocation, complete stripping tumor tissue and lung tissue.5 The nude mice tumor was paraffin-embedded and prepared into histologic section, and the tumor cells morphology was observed by HE staining, the expression of CD34 and CD57 in tumor tissue was detected by immunohistochemical staining.Result:1.The subcutaneously transplant nude mouse models were successfully established. The animal tumorigenicity rate are all 100%. Hela/pcDNA3.1(+)–h/IL-8 cell group’s tumorigenicity time is obviously lower than Hela/pcDNA3.1(+)–h cell group’s and Hela cell group’s(P<0.05),there are no obviously distinction between Hela/pcDNA3.1(+)–h cell group and Hela cell group(P>0.05).2.The gross tumor volume of the 7,8,9week,the Hela/pcDNA3.1(+)–h/IL-8 cell group’volume obviously bigger than Hela/pcDNA3.1(+)–h cell group and Hela cell group(P<0.05). After subcutaneously transplant,before sixth week the volume comparion has no significant differenct (P>0.05).3.The immunohistochemical resultThe immunohistochemical result of MVD: The CD34 expression of Hela/pcDNA3.1(+)–h/IL-8 cell group is obviously.Hela/pcDNA3.1(+)–h/IL-8 cell group obviously higher than Hela/pcDNA3.1(+)–h cell group and Hela cell group(P<0.05), there are no obviously distinction between Hela/pcDNA3.1(+)–h cell group and Hela cell group(P>0.05).The immunohistochemical result of CD57: Hela/pcDNA3.1(+)–h/IL-8 cell group obviously less than Hela/pcDNA3.1(+)–h cell group and Hela cell group(P<0.05). there are no obviously distinction between Hela/pcDNA3.1(+)–h cell group and Hela cell group(P>0.05).4.Lung tissue of each group have no cancer metastasis.Conclusion:1.IL-8 gene was successfully constructed in nude mice transplant model, by comparing each group into a tumorigenicity rate , time and tumor volume confirmed IL-8 can promote tumor growth.2.Animal study found that IL-8 can promote the generation of capillaries within the tumor tissue,and this provides the necessary conditions for the growth of cervical carcinoma invasion and distant metastasis .3.Low expression of CD57 may be related to the growth of cervical carcinoma invasion and distant metastasis.4.Lung tissue of each group have no cancer metastasis.更多还原