摘要
研究背景:间皮细胞覆盖在浆膜腔及腔内脏器的表面,具有屏障、免疫、物质转运等功能。淋巴孔是毛细淋巴管在浆膜间皮细胞之间的微小开口,通过淋巴孔,浆膜腔与淋巴管系直接相通。淋巴孔具有物质吸收、免疫等功能。肿瘤腹膜转移一般经历细胞游离、粘附、侵袭、生长四个阶段。在肿瘤腹膜转移时,间皮细胞和淋巴孔形态会发生改变。
研究目的:研究腹腔接种肿瘤细胞后,膈腹膜、肠系膜上的间皮细胞及淋巴孔在肿瘤腹膜转移过程中不同时期的变化,探讨间皮细胞及淋巴孔在肿瘤腹膜转移过程中发挥的作用。
研究方法:小鼠腹腔接种HeA-H细胞后,每天记录体重增长状况,以确定收集腹水的时间点。接种后每两天取一次膈腹膜及肠系膜,扫描电镜观察腹腔接种肿瘤细胞后膈腹膜和肠系膜上间皮细胞及淋巴孔在不同时期的变化。扫描电镜观察用腹水连续处理后膈腹膜和肠系膜间皮细胞及淋巴孔的变化。以Boyden小室法研究HepA-H细胞侵袭能力。以ELISA法测定腹水中VEGF含量。肠系膜消化法培养间皮细胞,建立间皮细胞体外培养体系,为肿瘤转移过程中间皮细胞和淋巴孔的体外研究建立基础。
研究结果:小鼠腹腔接种HepA-H肿瘤细胞后,体重较对照组有明显增长,第11天后体重增长减缓,第14天后出现死亡。研究发现肿瘤腹膜转移过程中间皮细胞发生明显肿胀,淋巴孔开放数目增加,孔中可见肿瘤细胞。腹水连续处理后,较之对照组,膈腹膜及肠系膜上淋巴孔开放明显增加,膈腹膜间皮细胞肿胀,绒毛增生,肠系膜间皮细胞边界出现明显微绒毛。Boyden小室测定HepA-H细胞侵袭能力为23.4±1.5%。腹水中VEGF含量为5.43±0.23ng/ml。从细胞形态及免疫荧光两方面的检测,确定肠系膜消化法培养之细胞为间皮细胞。
结论:膈腹膜间皮细胞在HepA-H肝癌细胞腹膜转移过程中,细胞体肿胀,间皮细胞间隙增大,微绒毛增生,肿瘤细胞种植于间皮细胞之间。肠系膜间皮细胞形态变化不大,后期可见结节状肿瘤细胞。肿瘤细胞可通过淋巴孔传播。
Background:Mesothelial cells cover the entire surface of the three serosal cavities and the internal organs.The mesothelial cells have many functions including protection,immune response,transportation and so on.Lymphatic stomata are small openings of lymphatic capillary on the free surface of mesothelium.The peritoneal cavity and pericardial cavity are connected with lymphatic system via these small openings which have the function of active absorption.Dissemination of tumors requires four steps:detachment of cancer cells from the primary tumor,attachment to distant peritoneum,invasion into subperitoneal space,and proliferation.Changes of mesothelial cells happen during the dissemination of tumor cells.
Objective:To study the morphological changes of peritoneal mesothelial cells and lymphatic stomata on the diaphragm and mesentery during the dissemination of HepA-H cells.
Methods:After injection of HepA-H ascites tumor to the peritoneal cavity, diaphragm and mesentery were observed by SEM at different time point.The fluid of ascites tumor was injected to the peritoneal cavity for 3 days.Then the two tissues were examined by SEM.Migration ability of HepA-H cells were tested by Boyden chamber.VEGF level was assayed with ELISA kit.Mesothelial cells from mesentery were cultured in vitro.
Results:Compared to the control,the weight of mice showed an obvious growth. The growth speed decreased after the 11~(th) day since injection of HepA-H.Death occurred in the 14~(th) day.Morphological changes was observed during dissemination of ascites tumor.The stomata showed larger in size and quantity both on diaphragm and on mesentery.Tumor cells were also seen in the stomata.The migration ability tested by Boyden chamber was 23.4±1.5%.VEGF level in ascites was 5.43±0.23ng/ml.The morphological observation and immunofluorescence staining tests proved the cells collected from mesentery were mesothelial cells.
Conclusion:Mesothelial cells on diaphragm showed tumid cell bodies,enlarged spaces between mesothelial cells,and increased microvilli during metastasis of tumor cells.Tumor cells were also observed planting between the mesothelial cells.Little morphological change were shown in mesenteric mesothelial cell.Nodular tumor cells can be seen in the late.Tumor cells were also seen getting through the lymphatic stomata.
引文
[1]Whitaker,D,Papadimitriou JM,Walters MN,The mesothelium and its reactions:a review.Crit Rev Toxicol,1982.10(2):p.81-144.
[2]Mustsaers SE.The mesothelial cell.IJBCB,2004.36:9-16.
[3]Mutsaers,SE,Mesothelial cells:their structure,function and role in serosal repair.Respirology,2002.7(3):p.171-91.
[4]Liberek T,Topley N,Luttmann W,et al.Adherence of neutrophils to human peritoneal mesothelial cells:Role of intercellular adhesion molecule-1.Journal of American Society of Nephrology,1996.7:208-217.
[5]Hausmann,MJ,et al.Accessory role of human peritoneal mesothelial cells in antigen presentation and T-cell growth.Kidney Int,2000.57(2):p.476-86.
[6]Baradi AF,Rao SN.A scanning electron microscope study of mouse peritoneal mesothelium.Tissue Cell.1976.8(1):159-162.
[7]Li JC,Yu SM.Study on the ultrastructure of the peritoneal stomata in humans.Acta Anat(Basel).1991.141(1):26-30.
[8]Tesch C,Henne-Burns D,Holstein AF.Open lymph vessels of the ligamentum falciforme hepatis in the human.Langenbecks Arch Chir.1990.375(3):130-134.
[9]Li JC,Zhou JL,Gao YS.The ultrastructure and computer imaging of the lymphatic stomata in the human pelvic peritoneum.Ann Anat.1997 Jun.179(3):215-220.
[10]Li JC,Chen XB.The discovery of the human fetus cerebral meningeal stomata and their image processing.Acta Anatomica Sinica.1998.29(1):98-101.
[11]Li JC,Tota,B.Discovery of frog pericardial lymphatic stomata and its significavce.Acta Zoologica Sinica.2000.46(4):372-375.
[12]Li YY,Li JC.Ultrastructure and three-dimensional study of the lymphatic stomata in the costal pleura of the rabbit.Microsc Res Tech.2003 Oct 15.62(3):240-246.
[13]Sui MH,Li JC.The discovery of ovary lymphatic stomata in human and its significance.Acta Anatomica Sinica.2001.32(3):220-222.
[14]Leak LV,Rahil K.Permeability of the diaphragmatic mesothelium:the ultrastructural basis for "stomata".Am J Anat.1978 Apr.151(4):557-593.
[15]Shimotsuma M,Hagiwara A,Takahashi T,Kawata M,Shields JW.Surface structure and cell zonation in human omental milky spots.Lymphology.1990 Dec. 23(4):207-208.
[16]Shimotsuma M,Takahashi T,Kawata M,Dux K.Cellular subsets of the milky spots in the human greater omentum.Cell Tissue Res.1991 Jun.264(3):599-601.
[17]Li J,Zhou J,Tota B,Scalia G,Gattuso A.Regulating effects of vascular endothelial growth factor and ANG Ⅱ on frog's pericardial stomata,mesothelium and angiogenesis.Chin Med Sci J.2001 Mar.16(1):23-28.
[18]Sui MH,Li JC.The influence of pregnancy and hormone on the lymphatic stomata in guinea pig's ovary bursa.Shi Yan Sheng Wu Xue Bao.2003 Feb.36(1):43-48.
[19]Yonemura Y,Endou Y,Fujita H,et al.Role of MMP-7 in the formation of peritoneal dissemination in gastric cancer.Gastric Cancer,2000.3(2):p.63-70
[20]Guyot F,Faivre J,Manfredi S,et al.Time trends in the treatment and survival of recurrence from colorectal cancer.Ann Oncol,2005.16:756-761
[21]Meyerhardt JA,Mayer RJ.Follow-up strategies after curative resection of colorectal cancer.Semin Oncol,2003.3:349-360
[22]van Grevenstein WM,Hofland LJ,van Rossen ME,et al.Inflammatory cytokines stimulate the adhesion of colon carcinoma cells to mesothelial monolayers.Dig Dis Sci,2007.52(10):p.2775-83
[23]Yoshioka M,Yasuda M,Tahira K,et al.Experimental study of the mechanism of peritoneal dissemination—with special reference to scanning electron microscopic observations.Nippon Sanka Fujinka Gakkai Zasshi,1986.38(10):p.1683-91
[24]Kiyasu Y,Kaneshima S,Koga S.Morphogenesis of peritoneal metastasis in human gastric cancer.Cancer Res,1981.41(3):p.1236-9
[25]刘伏友,段绍斌,龙志高,等。人腹膜间皮细胞的培养及特征。湖南医科大学报,2001.26(4):321-324.
[26]Carpenter PM,Dao AV.The role of hyaluronan in mesothelium-induced motility of ovarian carcinoma cells.Anticancer Res,2003.23(5A):p.3985-90.
[27]Jones LM,Gardner MJ,Catterall JB,et al.Hyaluronic acid secreted by mesothelial cells:a natural barrier to ovarian cancer cell adhesion.Clin Exp Metastasis,1995.13(5):p.373-80
[28]Namba Y.An electron microscopic demonstration of the invasion of tumor cells into the diaphragm.Nippon Geka Gakkai Zasshi.1989 Nov.90(11):1915-1921.
[29]Tsujimoto H,Hagiwara A,Shimotsuma M,et al.Role of milky spots as selective implantation sites for malignant cells in peritoneal dissemination in mice.J Cancer Res Clin Oncol,1996.122(10):p.590-5
[30]Tsilibary EC,Wissig SL.Lymphatic absorption from the peritoneal cavity:regulation of patency of mesothelial stomata.Microvasc Res.1983 Jan.25(1):22-39.
[31]Li YY,Li JC.Cell signal transduction mechanism for nitric oxide regulating lymphatic stomata and its draining capability.Anat Rec(Hoboken).2008 Feb.291(2):216-223.
[32]Rothen-Rutishauser B,Riesen FK,Braun A,et al.Dynamics of tight and adherens junctions under EGTA treatment.J Membr Biol,2002.188:151-162.
1.Whitaker D,Papadimitriou JM,Waiters MN.The mesothelium and its reactions:a review.Crit Rev Toxicol,1982.10(2):p.81-144
2.Yonemura Y,Endou Y,Fujita H,et al.Role of MMP-7 in the formation of peritoneal dissemination in gastric cancer.Gastric Cancer,2000.3(2):p.63-70
3.Mutsaers SE.Mesothelial cells:their structure,function and role in serosal repair.Respirology,2002.7(3):p.171-91
4.Hausmann MJ,Rogachev B,Weiler M,et al.Accessory role of human peritoneal mesothelial cells in antigen presentation and T-cell growth.Kidney Int,2000.57(2):p.476-86
5.Namba Y.An electron microscopic demonstration of the invasion of tumor cells into the diaphragm.Nippon Geka Gakkai Zasshi,1989.90(11):p.1915-21
6.Yoshioka M,Yasuda M,Tahira K,et al.Experimental study of the mechanism of peritoneal dissemination—with special reference to scanning electron microscopic observations.Nippon Sanka Fujinka Gakkai Zasshi,1986.38(10):p.1683-91
7.Kiyasu Y,Kaneshima S,Koga S.Morphogenesis of peritoneal metastasis in human gastric cancer.Cancer Res,1981.41(3):p.1236-9
8.Tsujimoto H,Hagiwara A,Shimotsuma M,et al.Role of milky spots as selective implantation sites for malignant cells in peritoneal dissemination in mice.J Cancer Res Clin Oncol,1996.122(10):p.590-5
9.Mochizuki Y,Nakanishi H,Kodera Y,et al.TNF-alpha promotes progression of peritoneal metastasis as demonstrated using a green fluorescence protein(GFP)-tagged human gastric cancer cell line.Clin Exp Metastasis,2004.21(1):p.39-47
10. van Grevenstein WM,Hofland LJ,van Rossen ME,et al.Inflammatory cytokines stimulate the adhesion of colon carcinoma cells to mesothelial monolayers.DigDis Sci,2007.52(10):p.2775-83
11. Burleson KM,Casey RC,Skubitz KM,et al.Ovarian carcinoma ascites spheroids adhere to extracellular matrix components and mesothelial cell monolayers.Gynecol Oncol,2004.93(1):p.170-81
12. Takatsuki H,Komatsu S,Sano R,et al.Adhesion of gastric carcinoma cells to peritoneum mediated by alpha3betal integrin(VLA-3).Cancer Res,2004.64(17):p.6065-70
13. Carpenter PM,Dao AV.The role of hyaluronan in mesothelium-induced motility of ovarian carcinoma cells.Anticancer Res,2003.23(5A):p.3985-90.
14. Jones LM,Gardner MJ,Catterall JB,et al.Hyaluronic acid secreted by mesothelial cells:a natural barrier to ovarian cancer cell adhesion.Clin Exp Metastasis,1995.13(5):p.373-80
15. Mizutani K,Kofuji K,Shirouzu K.The significance of MMP-1 and MMP-2 in peritoneal disseminated metastasis of gastric cancer.Surg Today,2000.30(7):p.614-21
16. Yonemura Y,Endou Y,Fujita H,et al.,Inhibition of peritoneal dissemination in human gastric cancer by MMP-7-specific antisense oligonucleotide.J Exp Clin Cancer Res,2001.20(2):p.205-12
17. Ren J,Xiao YJ,Singh LS,et al,Lysophosphatidic Acid Is Constitutively Produced by Human Peritoneal Mesothelial Cells and Enhances Adhesion,Migration,and Invasion of Ovarian Cancer Cells.Cancer Res,2006.66(6):p.3006-3014
18. Hiroaki S,Shunichi T,Shinichi O,et al.The expression of transforming growth factor-betal is significantly correlated with the expression of vascular endothelial growth factor and poor prognosis of patients with advanced gastric carcinoma.Cancer,1999.86(8):p.1455-1462
19. Akihiro S,Joji K,Dai S,et al.,Lysophosphatidic Acid(LPA)-Induced Vascular Endothelial Growth Factor(VEGF)by Mesothelial Cells and Quantification of Host-Derived VEGF in Malignant Ascites.The Journal of surgical research,2006.130(1):p.94-101