【作者】 张爱荣；

【导师】 孔北华；

【作者基本信息】 山东大学 ， 妇产科， 2004， 博士

【摘要】 腹腔镜手术是近年发展起来的微创技术,与传统的开腹手术比较,具有手术效果好、并发症率低、术后疼痛轻、恢复快等优点,目前已经逐步成为良性妇科疾病重要的手术方式。随着腹腔镜技术的发展及患者对生存质量要求的提高,腹腔镜这种微创手术在妇科恶性肿瘤领域中的应用逐渐得以开展,文献报道腹腔镜肿瘤外科手术后引起的切口肿瘤种植率高于开腹手术,因此,腹腔镜在恶性肿瘤领域的应用尚存在争议,其争议的焦点在于腹腔镜手术中所使用的人工气腹介质CO₂是否促进腹腔恶性肿瘤细胞的生长和转移。 本研究模拟CO₂气腹环境,通过MTT、细胞计数、细胞周期比率变化及PCNA蛋白的表达观察暴露于纯CO₂气腹环境后人卵巢癌细胞系SKOV-3、F344大鼠来源的低分化卵巢腺癌细胞NuTu-19体外生长情况。应用划痕实验观察SKOV-3细胞随机运动性变化;应用Boyden小室实验、琼脂糖悬滴法、细胞粘附实验及流式细胞术测定CD44v6、nm23蛋白的表达观察纯CO₂气腹环境对体外培养的F344大鼠来源的低分化卵巢腺癌NuTu-19细胞体外侵袭能力的影响。通过建立F344大鼠卵巢癌动物模型,模拟CO₂气腹腹腔镜手术过程,并与开腹手术组对照,观察CO₂气腹对动物体内肿瘤生长和转移性的影响。 研究发现纯CO₂气腹环境对SKOV-3和NuTu-19的体外生长均有促进作用,并且使NuTu-19细胞的体外侵袭能力增强;其促肿瘤细胞生长作用与暴露于纯CO₂环境的时间成正比。CO₂气腹环境对动物模型体内肿瘤的生长转移亦有促进作用。更多还原

【Abstract】 Laparoscopy is a minimally invasive surgical techniques and steadily becoming the predominant techniqe for various surgical interventions. Among the advantages of this technique is the marked reduction in healing time and significant decrease in postoperative pain and postsurgery adhesion formation. Recently laparoscopy is used in cancer surgery. In laparoscopy the abdominal cavity is artificially inflated with carbon dioxide. Numerous clinical reports have raised the possibility that laparoscopic cancer surgery , in contrast to conventional surgery, has an inherently detrimental effect on tumor growth. Laparoscopy used in cancer surgery has been contradictory. The aim of this study was to assess the effect of CO2 pneumoperitoneum on the growth and metastasis of ovarian carcinoma in vitro and in vive.Immortalized ovarian epithelial carcinoma cell (SKOV-3 cell line and NuTu-19 cell) cultures were exposed to carbon dioxide. Cell growth was determined with the use of a spectrophotometric assay ( MTT) and cell counting . The cell cycle fraction of NuTu-19 cells were measured with flow cytometer method ( FCM ). The expression of PCNA gene of NuTu-19 cells were detected by useing of FCM too. SKOV-3 Cell movement was determined with the use of Wound-assay. To measure the adhesive rate , invasive ability and motility of rat ovarian carcinoma NuTu-19 cell after exposed to carbon dioxide. The expression of CD44v6 and nm23 gene of NuTu-19 cells were detected by useing of FCM.To establish an animal model of epithelial ovarian cancer in immunocompetent rat Fischer 344. Twenty rats were randomized to two groups (10 rats /group): CO2 insufflation or sham laparotomy. The insufflation group underwent CO2 pneumoperitoneum 8 mmHg for 30 min via a 20-gauge angiocatheter.CO2 pneumoperitoneum significantly promotes the growth of SKOV-3 cells and NuTu-19 cells ,and promotes the adhesive rate, invasive ability and motility of NuTu-19 cells . The increased levels had a linear relationship to the length of carbon dioxide exposure. CO2 pneumoperitoneum laparoscopy has effects on growth and spread of intraperitoneal tumor compared with laparotomy in an animal model. PART ITHE EFFECTS OF CARBON DIOXIDE ON OVARIAN CARCINOMA CELL GROWTH IN VITROABSTRACTObjective: To determine whether carbon dioxide exposure increases the growth of ovarian cancer cells in vitro.Methods: Immortalized ovarian epithelial carcinoma cell ( SKOV-3cell line ) were cultured in RPMI-1640 medium with 5% fetal bovine serum. The cells were grown to 100% confluence in a standard culture atmosphere of 95% room air and 5% carbon dioxide at 37°C. To determine the effect of carbon dioxide exposure on cell growth, individual groups of cells were exposured to 100% carbon dioxide for time periods of 1.0 , 2.0 and 3.0 hours( Al, Bl, and Cl group respectively ). Control SKOV-3 cells ( Dl ) were incubated in 95% room air and 5% carbon dioxide for similar time periods , and cells were allowed to grow for 0, 1, 2, 3, 4, or 5 days befor cell growth was measured . Cell growth was determined with the use of a spectrophotometric assay ( MTT) and cell counting. The results were compared with control cells by paired t tests and liner regressions analysis. Rat ovarian carcinoma NuTu-19 cells were exposed to carbon dioxide. Groupe A was exposed to carbon dioxide for one hours; Groupe B for three hours; Groupe C was control groupe ( was cultured in a standard culture atmosphere of 95% room air and 5% carbon dioxide at 37°C ). Cell growth wasdetermined with the use of cell counting . The cell cyclin rate of NuTu-19 cell in three groups were measured with flow cytometer method (FCM). The expression of PCNA gene of rat ovarian carcinoma NuTu-19 cells were detected by use of FCM too. Results: The levels of MTT of SKOV-3 cells that were exposed to carbon dioxide had increased after 4 days in culture. The increased cell growth had a linear relationship to the length of carbon dioxide exposure ( r = 0.899). The doubling times of carcinoma cells in group Al, Bl and Cl was 22.80 ±1.25h, 21.49 ±1.09h and 17.27±1.15h respectively, and were shorter than that in control group Dl (23.15 ±1.97d ). The doubling times of NuTu-19 cells in group A, B was 18.2±2.4h> 16.5±2.1h respectively , and were shorter than that in control group C ( 20.2±2.8h). There were significant difference between group B and group C. The S-Phase of carcinoma cells were 18.36±10.37 % in groupe A, 25.02±12.81% in groupe B, and were higer than that in groupe C (15.44±9.25%) (P<0.05,P<0.01). The G2/M of carcinoma cells were not different among three groupes. The expression of PCNA was higer in groupe A and groupe B than that in groupe (P value all<0.05).Conclusion: Carbon dioxide exposure increases the growth of ovarian carcinoma cells in vitro. PART IITHE EFFECTS OF CARBON DIOXIDE ON INVASION OF RAT OVARIAN CARCINOMA CELLS IN VITROABSTRACTObjective:To observe the effects of carbon dioxide on movement of human ovarian cancer cells and rat ovarian carcinoma cells NuTu-19 in terms of adhesion, motility and invasion in vitro. Such study will provide a primary evidence for the safety of CO2 pneumoperitoneum laparoscopy surgery in malignancies.Methods: SKOV-3 cells were exposured to 100% carbon dioxide for time periods of 1.0, 2.0 and 3.0 hours (group Al, Bl, and Cl respectively ). Control SKOV-3 cells ( Dl ) were incubated in 95% room air and 5% carbon dioxide for similar time periods . Cell movement was determined with the use of Wound-assay.Rat ovarian carcinoma NuTu-19 cells were exposed to carbon dioxide. Groupe A was exposed to carbon dioxide for one hours-, Groupe B was exposed to carbon dioxide for three hours; Groupe C was control groupe. To measure the adhesive rate , invasive ability , motility of rat ovarian carcinoma NuTu-19 cell after exposed to carbon dioxide. The expression of CD44v6 and nm23 gene of rat ovarian carcinoma NuTu-19 cells were detected by use of FCM. The experiments were repeated 4 times. The results were compared with control cells by paired t tests and liner regressions analysis.Results: The number of migrating cells (SKOV-3) into the wound surface in group Al,Bl,Cland Dl were 21.61 ±5.21, 19.24 ±4.51, 20.45 ±4.611 and 22.35±5.52 respectively at 24 h after exposed to carbon dioxide ; at 72h after exposed to carbon dioxide the number of migrating cells in group Al, Bl, Cl and Dl were 19.26 ± 4.37 , 17.21 ±3.61 , 18.34 ± 3.95 and 21.71 ± 5.62 respectively; at 96h were 22.35 ± 6.53 , 20.58 ± 5.41 ,19.26 ± 4.57 and 23.21 ± 6.16respectively. There were no significant difference to be found among four groups (P value all >0.05).The adhesive rate of rat ovarian carcinoma NuTu-19 cells after exposed to carbon dioxide in group A, B and C were 10.24±4.17%, 11.61±6.43%, 8.25±2.15% at 30 min; 35.36±7.25%, 40.27±11.36%, 20.25±8.52% at 60min; 68.23±15.34, 79.16±16.32, 55.29± 15.40 at 90min respectively. The adhesive rate of cells were no significant difference among three groups at 30 min after exposed to carbon dioxide ( P>0.05 ) . The adhesive rate of cells in group A, group B were higher than that in group C at 60 min and 90 min after exposed to carbon dioxide ( PO.05, PO.01) . The number of rat ovarian carcinoma NuTu-19 cells invading through Matrigel filter was 7.12±2.18cells/HP in group A, 20.45±4.35 cells /Hp in group B ,and significantly increased than that in group C (3.18±1.15cells /HP ) ( PO.05, PO.001). The motility of rat ovarian carcinoma NuTu-19 cells in group A , group B were more increased than that in control groupe C. The increased levels had a linear relationship to the length of carbon dioxide exposure. The expression of CD44v6 was higer in groupe A and groupe B than that in groupe C (P=0.003, PO.001) . The expression of nm23protein was less in groupe A and groupe B than that in groupe C( P = 0.004, P < 0.001). Conclusion: There is no effect on movement of SKOV-3 cell after exposed to carbon dioxide. The adhesive rate, invasive ability and motility of rat ovarian NuTu-19 cells were increased after exposed to carbon dioxide. Carbon dioxide exposure increases the invasive ability of rat ovarian carcinoma NuTu-19 cells in vitro. PART IIITHE EFFECTS OF CO2 PNEUMOPERITONEUM ON TUMOR GROWTH AND METASTASIS IN VIVOABSTRACTObjective: The aim of this study was to evaluate the effects of CO2 pneumoperitoneum laparoscopy on growth and spread of intraperitoneal tumor in an animal model. Methods: To establish an animal model of epithelial ovarian cancer in immunocompetent rat Fischer 344. NuTu-19, a cell line derived from a poorly differentiated adenocarcinoma of Fischer 344 rat, was cultured in vitro. Cells were injected into the peritoneal cavity of female Fischer 344 rats (lxlO6 cells/rat). Two weeks later, twenty rats were randomized to two groups (10 rats /group): CO2 insufflation or sham laparotomy control. The insufflation group underwent CO2 pneumoperitoneum 8 mmHg for 30 min via a 20-gauge angiocatheter. The laparotomy group underwent a midline incision from xiphoid to pubis, which was closed after 30 min. To weight and observe condition of rats once a week. Tumors were excised from half the mice in each group at autopsy on postoperative day 28. Tumor mass, local regional invasion incidence, lymph node involvement, and liver and lung metastases were evaluated. Sections of tumors were made then cell cycle fraction was measured by quantitating DNA in individual cells using flow cytometry analysis. The expression of Ki-67, VEGF, and CD44v6 protein were measured using immunocytochemistry by flow cytometry too. To observe animal survival time from the remaining rats.更多还原