【作者】 于歌；

【导师】 余佩武；

【作者基本信息】 第三军医大学 ， 外科学， 2010， 硕士

【摘要】 背景和目的:胃癌是消化系统常见的恶性肿瘤,死亡率居恶性肿瘤第二位。腹膜转移是胃癌转移的常见方式,也是胃癌术后复发、死亡的主要原因之一。胃癌腹腔转移的发生机制目前尚不清楚,根据经典的“种子-土壤”学说,胃癌腹腔转移包括如下三个重要环节:1.胃癌细胞从原发灶脱落;2.腹腔游离胃癌细胞与腹膜间皮细胞粘附;3.胃癌细胞突破腹膜间皮屏障进入间皮下基质。任何影响上述三个环节的因素都将对胃癌腹腔转移产生影响。其中在适宜的局部微环境下,游离癌细胞与腹膜间皮细胞相互粘附是其发生的基础。而在众多影响肿瘤局部微环境的因素中,炎症与肿瘤的关系目前已得到国内外学者的广泛认同,近年已将炎症视为肿瘤所具有的七类重要特征之一。手术创伤会刺激机体发生反应性炎症应答,进而引起腹腔内多种细胞释放一系列炎症因子和生长因子,其中最常见的为肿瘤坏死因子-α(TNF-α)和白介素-1β(IL-1β)。这些炎症因子通过趋化巨噬细胞、中性粒细胞向创伤处迁移从而有利于创伤的愈合。然而目前的研究也发现TNF-α和IL-1β参与肿瘤生长、侵袭以及转移等多个过程。TNF-α和IL-1β作为体内两类多功能细胞因子均可激活细胞内核转录因子-κB(NF-κB)信号通路,而该通路活化后可促进不同的基因转录。其中细胞间粘附分子-1(ICAM-1)、血管细胞粘附分子-1(VCAM-1)等粘附分子的启动子区既有NF-κB的结合位点,因此TNF-α和IL-1β可能具有调节ICAM-1、VCAM-1等粘附分子表达的作用。粘附分子的异常或过量表达能促进肿瘤细胞与其它的肿瘤细胞、正常细胞以及细胞外基质成分粘附从而有利于游离癌细胞转移,然而关于TNF-α和IL-1β是否会促进胃癌细胞与腹膜间皮细胞粘附,以及它们对胃癌腹腔转移是否有影响目前尚未见报道。目前根治性手术切除仍然是胃癌患者获得治愈的惟一途径,腹腔镜手术治疗恶性肿瘤能达到开腹手术的根治效果,且具有腹壁切口小、术中失血少、术后恢复快等微创优势,因此是近年来微创胃肠外科研究热点。在日本早期胃癌行腹腔镜手术治疗已成为标准术式之一,但是对于进展期胃癌行腹腔镜手术治疗是否会促进胃癌术后腹膜转移仍是目前外科医师关注的焦点。TNF-α和IL-1β在由创伤刺激引起的急性期反应中发挥重要作用,并且这两种炎症因子的表达改变与手术创伤程度的大小及术后相关并发症的发生具有密切联系。因而目前普遍认为TNF-α和IL-1β可以作为评估手术创伤程度的两个重要指标。那么腹腔镜手术的微创特点是否具有对因创伤引起的炎症因子释放影响小的优势,进而可能有利于减轻炎症因子所介导的胃癌腹膜转移呢?为此本课题首先体外原代培养人腹膜间皮细胞,利用不同浓度的TNF-α或IL-1β分别处理腹膜间皮细胞,然后观察其对腹膜间皮细胞与胃癌细胞粘附的影响,并采用Real-time PCR方法检测TNF-α和IL-1β对腹膜间皮细胞不同粘附分子mRNA表达的影响。为进一步阐述粘附分子及其配体对胃癌腹膜转移过程的影响,本研究采用免疫组化染色方法检测胃癌组织和腹膜组织中粘附分子及其配体表达情况。同时通过ELISA法检测腹腔镜胃癌手术和开腹胃癌手术患者血液及腹腔冲洗液中TNF-α和IL-1β浓度的变化。期望通过以上研究初步探讨TNF-α和IL-1β对胃癌细胞腹膜粘附的影响,进而为临床防治胃癌腹膜转移的发生提供新的理论和实验依据。方法:1.以体外原代培养腹膜间皮细胞为实验对象,实验分为处理组和对照组,其中处理组又分为炎症因子处理组和抗炎症因子处理组。在炎症因子处理组中人腹膜间皮细胞经不同浓度TNF-α或IL-1β处理后与胃癌细胞共培养,通过流式细胞仪检测细胞间黏附率。在抗炎症因子处理组中不同浓度的TNF-α或IL-1β抑制剂分别阻断TNF-α或IL-1β后再处理人腹膜间皮细胞,处理后的人腹膜间皮细胞与胃癌细胞共培养并通过流式细胞仪检测细胞间黏附率。对照组中以PBS代替TNF-α或IL-1β。2.人腹膜间皮细胞经不同浓度TNF-α或IL-1β处理后,采用Real-time PCR方法检测腹膜间皮细胞中CD44、ICAM-1以及VCAM-1mRNA表达情况。3.采用Real-time PCR方法检测人胃腺癌细胞株中ICAM-1配体:淋巴细胞功能相关抗原-1(LFA-1)、VCAM-1配体:极迟抗原-4(VLA-4)以及CD44mRNA的表达;4.收集本中心胃癌手术患者的腹膜组织及胃癌组织。通过免疫组化染色检测腹膜组织中ICAM-1、VCAM-1、CD44v6的表达;同时检测胃癌组织中LFA-1、VLA-4以及CD44v6的表达。5.以本中心57例行胃癌根治术患者为研究对象。其中32例患者行腹腔镜手术治疗(腹腔镜手术组),同期25例患者行开腹手术治疗(开腹手术组)。通过ELISA法检测两组手术患者腹腔冲洗液及血液中TNF-α和IL-1β浓度的变化。两组病人的腹腔冲洗液均收集3次,即开腹后或建立气腹后和术后24、48小时;血液标本均收集5次,即麻醉诱导后和术后2、4、24及48小时。结果:1.原代培养人腹膜间皮细胞经不同浓度TNF-α或IL-1β处理后,其与胃癌细胞的黏附率逐渐升高,与对照组比较差异有统计学意义(P<0.05)。并且这种作用与炎症因子的浓度之间呈明显量效关系,当TNF-α或IL-1β浓度达到10ng/ml时细胞间粘附率达到最大值,随后粘附率并不因炎症因子浓度的增加而升高。同时这种作用可以分别被TNF-α或IL-1β的抑制剂所阻断。2. TNF-α和IL-1β均可促进人腹膜间皮细胞中ICAM-1、VCAM-1及CD44mRNA表达上调,并且呈明显量效关系即随着TNF-α或IL-1β浓度增加,这些粘附分子mRNA表达水平逐渐增高。与对照组比较差异有统计学意义(P<0.01)。3.人胃腺癌细胞株AGS、SGC-7901、MKN-45中CD44、LFA-1和VLA-4mRNA均呈阳性表达。4.免疫组化染色结果表明人腹膜组织中ICAM-1、VCAM-1及CD44v6表达阳性;胃癌组织中LFA-1、VLA-1及CD44v6表达阳性。5.手术后两组患者腹腔冲洗液中TNF-α和IL-1β浓度均显著升高,在术后24小时达到高峰,其中开腹组患者腹腔冲洗液中IL-1β浓度高于腹腔镜组,两组比较差异有统计学意义(P<0.05)。而两组患者腹腔冲洗液中TNF-α浓度比较无明显差异(P>0.05)。随后两组患者腹腔冲洗液中TNF-α和IL-1β浓度逐渐下降,在术后48小时开腹组患者腹腔冲洗液中TNF-α和IL-1β浓度均高于腹腔镜组,两组比较差异有统计学意义(P<0.05)。6.手术后两组患者血液中TNF-α和IL-1β浓度均显著升高,在术后4小时达到高峰,然后开始逐渐下降。其中开腹组患者血液中IL-1β浓度高于腹腔镜组,两组比较差异有统计学意义(P<0.05)。而两组患者血液中TNF-α浓度比较无明显差异(P>0.05)。结论:1.炎症因子TNF-α和IL-1β通过上调人腹膜间皮细胞粘附分子ICAM-1、VCAM-1及CD44的表达,促进胃癌细胞与腹膜间皮细胞间相互黏附,从而在胃癌细胞腹腔种植转移中发挥正性促进作用。这可能是炎症因子促进胃癌腹腔转移的重要机制之一。2. TNF-α和IL-1β抑制剂可阻断TNF-α和IL-1β对腹膜间皮细胞与胃癌粘附的促进作用,因而可能具有预防胃癌术后发生腹膜转移的作用。3.与传统开腹胃癌手术相比,腹腔镜胃癌手术具有腹壁切口小、术中出血少、术后恢复快等优势。并且其对腹腔局部和机体系统炎症反应影响小,炎症因子分泌水平较传统开腹手术低,因此腹腔镜胃癌手术相关炎症因子可能对胃癌腹膜转移的影响小于传统开腹手术。更多还原

【Abstract】 Background and objectives:Gastric cancer is one of the most common digestive malignant tumors and its mortality is in the second rank of the malignant diseases. Peritoneal metastasis is a major route after radical gastrectomy for the treatment of gastric cancer, which is the leading cause of recurrence and death in patients with gastric cancer. However, there still remain unknown about the exact mechanism of peritoneal metastasis. According to“seeds and soil”theory, the mechanism of peritoneal metastasis may include three steps: 1. gastric cancer cell spill from the original organ; 2. free gastric cancer cells in the peritoneal cavity adhere to peritoneal mesothelial cells; 3.gastric cancer cell transmigrate through the peritoneal mesothelial layer, and subsequently colonize in the submesothelial extracellular matrix. Therefore, any pathogenic factors which have an effect on the above process will affect peritoneal metastasis. While under a suitable local environment, tumor spill and subsequent adhesion of these cells in the peritoneal cavity is an important mechanism accounting for tumor metastasis. The association of inflammation and cancer has been well recognized in many types of cancer and inflammation has been regarded as the‘seventh hallmark of cancer’.After abdominal surgery, there is a reactive inflammatory response, during which cytokines and growth factors are produced, such as Tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β). These inflammatory cytokines are known to be conducive to wound healing by initiating the cellular cascade in the course of which macrophages and leukocytes migrate to the injured site. Meanwhile, they construct the first line of defense within the abdominal cavity to prevent bacterial infection. But, many recent studies have confirmed that these inflammatory mediators are also beneficial for tumor cells proliferation, infiltration and metastasis. TNF-αand IL-1βare both key cytokines involved in inflammation and have multifunction though convergence of the nuclear factor (NF)-kB signaling pathways, and thereby enhance different genes translation by translocation of NF-kB from cytoplasm to nucleus. Meanwhile, several adhesion molecules such as intercellular adhesion molecule-1(ICAM-1) and vascular cell adhesion molecule-1(VCAM-1), have been found that their promoter have the binding site of NF-kB. Therefore TNF-αand IL-1βhave the potential ability to stimulate a sort of adhesion molecules genes translation. While the overproduction or dysfunction of adhesion molecules is associated with tumor cell adhere to other tumor cell, surrounding normal cells and component of extracellular matrix. However whether TNF-αand IL-1βhave the effect on promoting spill gastric cancer cell to mesothelial cells, and therefore may support peritoneal metastasis of gastric cancer are still unknown.At present, distal gastrectomy is still the only way for curing gastric cancer patients. Laparoscopy-assisted distal gastrectomy (LADG) for gastric cancer has the same effect comparing with conventional open distal gastrectomy (CODG). Moreover, LADG for gastric cancer has the advantage of less invasive trauma such as less abdominal scar, reduced blood loss and faster postoperative recovery. Therefore, there is uncontroversial in the treatment of early gastric cancer by laparoscopy-assisted gastrectomy which has been regarded as a standard procedure in Japan. With regard to advanced gastric cancer, one of the greatest concerns is whether performing laparoscopic-assisted gastrectomy will enhance the possibility of peritoneal metastasis, as compared with conventional open gastrectomy. Thus, the role of laparoscopy surgery remains controversial, and this controversy is highlighted by the issue of tumor dissemination and recurrence. TNF-αand IL-1βare the major mediators of the acute phase response in humans. The post-operative levels of these cytokines have been found to correlate with the magnitude of the surgery and the presence of complications. Owing to less impact on the postoperative abdominal regional and systemic immune responses, whether laparoscopic surgery may not only shows clinically relevant advantages but also causes less effect of inflammatory factors on peritoneal metastasis of gastric cancer than conventional operations?Therefore, in this study primary cultured peritoneal mesothelial cells(HPMCs) were used to investigate the effects of the different concentration of inflammatory cytokines, TNF-αor IL-1β, on the interaction between gastric cancer cells and mesothelial cells. Moreover, after incubation with TNF-αor IL-1β, several adhesion molecules mRNA in mesothelial cells were detected by Real-time PCR. In order to further elucidate the influence by adhesion molecules and their ligand on the process of peritoneal metastasis for gastric cancer, immunohistochemistry staining were used to detect these adhesion molecules and ligand in peritoneal or gastric cancer tissue. Finally, ELISA immunoassay were used to evaluate differences in both the peritoneal and systemic cytokine (TNF-αand IL-1β) concentrations after laparoscopic and conventional surgical approaches. Thus, we hope to offer a novel theoretical and experimental basis for clinical prevention and treatment by the consequences of above experiments.Methods:1. A reproducible human in vitro assay was developed to study the adhesion of human gastric cancer cells to monolayers of primary cultured peritoneal mesothelial cells. Tumor cell adhesion to a mesothelial monolayer was assessed after preincubation of the monolayer with TNF-αand IL-1βby using flow cytometry. To inactivate TNF-αand IL-1β, preincubation was performed with anti-human TNF-αor anti-human IL-1βhomeochronous prior to the adhesion assay. In negative control, PBS was used to substitute inflammatory cytokines.2. After incubation with TNF-αor IL-1β, several adhesion moleculesCD44, ICAM-1 amd VCAM-1 mRNA in mesothelial cells were detected by Real-time PCR.3. mRNA of lymphocyte function-associated antigen-1(LFA-1), very late antigen-4(VLA-4), and CD44 in different gastric cancer cell lines were detected by Real-time PCR.4. The expression of adhesion molecules (ICAM-1, VCAM-1, and CD44v6) and their counterparts (LFA-1, VLA-4 and CD44v6) in peritoneal and gastric cancer tissue were investigated by means of immunocytochemical staining.5. 57 patients with gastric cancer in our center were selected as objects. Radical gastrectomy was performed for 32 cases by LADG and 25 cases by CODG. In both groups, the peritoneal fluid was collected at the beginning and at 24 h and 48 h after surgery from a suction drain, while venous blood samples were collected from a central venous catheter after the induction of anesthesia (baseline) and at 2, 4, 24, and 48 h perioperatively. Then the proinflammatory cytokines TNF-αand IL-1βwere measured by enzyme immunoassay.Results: 1. The percentage of gastric cancer cells adhering to mesothelial cells that were preincubated with TNF-αor IL-1βwas significantly higher than that to non-preincubated mesothelial cells (p<0.05). Moreover, this effect was dose-dependent, and maximum stimulation was achieved at 10ng/ml, meanwhile this effect was completed inhibited by stimulation with a hundred-fold excess of anti-TNF-αand. anti-IL-1βrespectively.2. As HPMCs were preincubated with increasing concentrations of TNF-αand IL-1β, the mRNA expression of ICAM-1, VCAM-1, and CD44 increased, and this effect was dose-dependent. This represented a significant difference compared with the group that did not receive preincubation (p<0.01).3. ICAM-1, LFA-1, and VLA-4 mRNA expression in AGS, SGC-7901 and MKN-45 human gastric cancer cells were observed by Real-time reverse transcriptase polymerase chain reaction.4. The results of immunohistochemical staining show that peritoneal tissues stained positive for ICAM-1, VCAM-1, and CD44v6, and the gastric cancer tissues were positive for LFA-1, VLA-4, and CD44v6.5. Peritoneal fluid levels of TNF-αand IL-1βincreased rapidly after operation in both groups, peaking for both at 24 hours. The level of IL-1βin the peritoneal fluid from the CODG group was significantly higher than that in the LADG group (p<0.05), whereas there was no significant difference in TNF-αlevels in the peritoneal fluid in both groups (p>0.05). And then levels of TNF-αand IL-1βgradually decreased. At 48 hours after operations, the level of TNF-αand IL-1βin the peritoneal fluid from the CODG group was significantly higher than that in the LADG group (p<0.05).6. After gastrectomy TNF-αand IL-1βlevels in serum increased rapidly and peaked at 4 h in both groups. IL-1βlevels were significantly higher after CODG than after LADG (p<0.05). TNF-αlevels did not change significantly in both groups (p>0.05).Conclusion:1. The presented results prove that TNF-αand IL-1βare significant stimulating factors in gastric cancer cells adhesion in vitro and may, therefore, partly account for peritoneal metastasis in vivo because they promote several adhesion molecules expression in peritoneal mesothelial cells, including ICAM-1, VCAM-1 and CD44.2. Anti-TNF-αand Anti-IL-1βcan completely inhibit the effect of TNF-αor IL-1βon the adhesion of gastric cancer cell to mesothelial cells respectively and thus may have the ability to prevent peritoneal metastasis after gastrectomy.3. Owing to less impact on the postoperative abdominal regional and systemic inflammatory responses, laparoscopic surgery may not only shows clinically relevant advantages such as less abdominal scar, reduced blood loss and faster postoperative recovery, but also causes less effect of inflammatory factors on peritoneal metastasis of gastric cancer than conventional operations.更多还原