【作者】 孙锡同；

【导师】 安伟德；

【作者基本信息】 大连医科大学 ， 外科学， 2010， 硕士

【摘要】 目的：胃癌是常见的恶性肿瘤,其发病率和死亡率都很高,而腹膜转移是其重要的转移途径。过去认为腹膜转移是由于瘤细胞本身通过重力作用,散落在低位脏器浆膜面,形成新的继发肿瘤。现有研究表明乳斑在肿瘤腹膜转移过程中起着重要作用。腹膜转移的最主要途径为种植性转移,腹腔内存在有生物学活性的癌细胞的胃癌患者73.9%将发生腹膜转移。患者术后多死于转移或复发,微转移则是肿瘤转移、复发的主要根源。乳斑是由巨噬细胞聚集为主伴随少量淋巴细胞、肥大细胞围绕着血管网构成的,在腹腔内主要集中在大网膜。本部分实验主要是通过建立胃癌腹膜转移模型,动态观察腹腔游离细胞发生腹膜乳斑转移的特点,探讨大网膜乳斑在胃癌腹膜转移过程中的作用及胃癌乳斑微转移病灶形成的特点,为进一步了解腹膜转移的特点和机制奠定基础。方法：将30只615小鼠分成3组,向小鼠腹腔内打入104经Dil荧光化学染料标记的615小鼠近端胃癌来源的胃癌细胞株MFC,3组小鼠分别于24、48、72小时被杀死取其大网膜,用FITC直接染色大网膜乳斑中的巨噬细胞,在免疫荧光显微镜下观察乳斑区胃癌细胞的变化。结果：1.在透射电镜下观察乳斑中主要是大量的巨噬细胞,巨噬细胞的大小和形状各种各样并且有许多的微绒毛。在巨噬细胞中间有少量的淋巴细胞,呈圆形或椭圆形,细胞表面有少量短小微绒毛。胞核大,占整个细胞大部分,呈圆形或椭圆形,可有浅的凹陷。乳斑中还含有少量中性粒细胞。2.24小时处死的小鼠的大网膜在免疫荧光显微镜下看到在有大量巨噬细胞聚集的区域里,有肿瘤细胞Dil-MFC的出现,而在其他区域肿瘤细胞碎片的出现。乳斑区与非乳斑区胃癌细胞数比率168：1(p<0.01)。腹腔注射48小时,在巨噬细胞作用下,巨噬细胞聚集的区域的Dil-MFC细胞减少,大部分胃癌细胞消除,仅有少量细胞存活,散在分布在乳斑的周围。乳斑区与非乳斑区胃癌细胞数比率60：1(p<0.01)。72小时后,Dil-MFC细胞进一步减少,可见增殖细胞团,形成微转移增殖病灶。乳斑区与非乳斑区胃癌细胞数比率(P<0.05)。3.乳斑区在24、48、72小时三个时间点胃癌细胞数差异有统计学意义p<0.05)。结论：1.电子显微镜揭示了乳斑是以巨噬细胞为主,包括少量的淋巴细胞和中性粒细胞共同组成的。2.胃癌细胞在腹腔转移早期阶段选择性地侵犯乳斑。腹腔注射72小时后,乳斑内存活Dil-MFC细胞开始增殖,形成微转移增殖灶。更多还原

【Abstract】 Objective:Gastric cancer is a common malignant tumor with high incidence and mortality rate,and peritoneal metastasis is a key channel for metastasis.The transcoelomic dissemination of tumor was reckoned as a result of the gravity of tumor cells that falls on serous membrane of low viscera and forms the new secondary tumor. The current study shows that milky spots play an important role in the process of tumor peritoneal dissemination. The main channel of peritoneal metastasis is transcoelomic metastasis, and 73.9% of patients who suffered from gastric cancer with biological activity cancer cell in abdominal cavity will have peritoneal metastasis. Their deaths are mostly caused by metastasis or relapse, and micro metastasis is the main source of metastasis and relapse. Milky spots are formed by macrophages gathering with few lymphocytes and mast cells around the vascular network, which is mainly at great omentum of abdominal cavity.This experiment is to discuss the role of milky spots in peritoneal metastasis of gastric cancer and features of formation focus caused by micro-metastasis of gastric cancer milky spots, through the establishment of peritoneal metastasis model and dynamic observation of features of peritoneal free cells’ peritoneal milky spots metastasis, which lay the foundation for further features and mechanism understanding of peritoneal metastasis.Method:30 615 mice were divided into 3 groups and 104 gastric cancer cell line MFC which is the source of proximal gastric cancer of 615 mice marked by Di fluorescent chemical dye, and 3 groups of mice were killed in 24,48 and 72 hours respectively and their greater omentum were taken out. The macrophages in omentum milky spots were directly stained with FITC to observe gastric cancer cell changes under immune fluorescence microscope in milky spot area.Result: 1. Under transmission electron microscope observation, it can be found that the milky spots mainly consist of macrophages, with a variety of sizes and shapes and many microvilli. There are a few round or ovalymphocytes in the middle of macrophages, with a small amount of short microvilli on cell surface. The cell nucleus is relative large, which occupied the most of the cell in a round or oval shape and shallow depression. Also, there is a small amount of neutrophil in milky spots.2. Under immunofluorescence microscope observation of omentum in mice that were killed in 24 hours, there were tumor cells Dil-MFC in area where a large number of macrophages gathered and tumor cell debris was found in other areas. The gastric cancer cells ratio of milky spots and non-milky spots area was 168:1(p<0.01).After 48 hours of intraperitoneal injection, the Dil-MFC cells decreased in macrophage gathering area under the action of macrophages, and the majority of cancer cells were eliminated with only a few cells survived by scattering around the milky spots. Gastric cancer cells ratio of milky spots and non-milky spots was 60:1(p<0.01).After 72 hours, Dil-MFC cells further decreased, and the proliferation of cell clusters and the formation of the proliferation of tumor micrometastase could be seen. The gastric cancer cells ratio of milky spots and non-milky spots was(p<0.05).3. The gastric cancer cells variation in the three time points of 24 hours,48 hours,and 72 hours in milky spot area has statistical significance (p<0.05).Conclusion:1. Through electron microscope observation, it reveals that milky spots are mainly composed of with a few lymphocytes and neutrophils.2. The cells selectively violate milky spots in the early stages of peritoneal metastasis. In 72h after intraperitoneal injection, the Dil-MFC cells that survive in the milky spots begin to proliferate and form micrometastasis proliferation.更多还原