【作者】 柳欣欣；

【导师】 于健春； 刘玉琴； 康维明；

【作者基本信息】 中国协和医科大学 ， 外科学， 2010， 博士

【摘要】 [研究背景]二十二碳六烯酸(DHA)和二十碳五烯酸(EPA)属于ω-3多不饱和脂肪酸(ω-3 Poly Unsaturated Fatty Acids, co-3 PUFAs)家族成员。ω-3多不饱和脂肪酸在机体中有很多方面的有益作用,已在多种疾病的预防及治疗方面得到了广泛的应用。其主要有益的作用可概括为：(1)提供能量,作为脂肪乳剂提供机体需要的营养支持；(2)下调炎症细胞因子,减轻SIRS,降低IL-1、TNFα,启到免疫调节的作用；(3)抗炎、减少多器官创伤炎症应激；(4)心血管病的治疗中有降脂、抗凝等有益作用。近来,越来越多的实验证据表明ω-3多不饱和脂肪酸(DHA、EPA)在肿瘤治疗方面有着重要的有益作用。同时,我们前期的研究也从细胞培养及荷瘤小鼠水平证实,ω-3多不饱和脂肪酸(DHA、EPA)可以抑制肿瘤细胞增殖并联合化疗药物达到化疗增敏的作用。[研究目的]在细胞培养水平及荷瘤小鼠水平,研究co-3多不饱和脂肪酸(ω-3 PUFAs)单用及联合氟尿嘧啶(5-FU)对胃癌治疗作用的影响,在前期研究基础上进一步探讨ω-3PUFA对胃癌治疗作用的机制。第一部分体外实验：细胞培养水平探讨ω-3多不饱和脂肪酸单用及联合氟尿嘧啶对胃癌细胞系作用[细胞培养研究方法]体外实验方法：①首先采用96孔板体外培养细胞,MTT实验检测DHA及5-FU单用或联用对胃癌细胞SGC7901的抑制作用；②使用DCFH-DA活性氧探针检测胃癌细胞SGC7901经DHA及5-FU单用或联用干预后细胞内活性氧的变化情况；③使用荧光素酶双报告基因法检测经DHA及5-FU单用或联用干预后细胞内NF-κB信号通路的激活或抑制情况；④使用RT-PCR方法,检测经药物干预后胃癌细胞NF-κB信号通路下游与肿瘤增殖侵袭相关的基因MMP2、COX2、CyclinDl的mRNA表达的差异；⑤使用Westernblot方法,检测经药物干预后胃癌细胞NF-κB信号通路下游与肿瘤增殖侵袭相关的基因MMP2、COX2、CyclinDl的蛋白表达的差异；⑥使用MDA检测试剂盒检测经药物干预后胃癌细胞内脂质过氧化水平；⑦PI流式细胞分析方法检测DHA及5-FU单用或联用对胃癌细胞周期的影响。[体外实验结果]①MTT实验：DHA及5-FU均可达到抑制肿瘤细胞增殖的作用,DHA联合5-FU化疗可以进一步抑制肿瘤细胞增殖作用,达到化疗增敏的作用。②DCFH-DA活性氧测定：DHA及5-FU均可增加肿瘤细胞内的活性氧产生,DHA联合5-FU化疗可以进一步增加肿瘤细胞内的活性氧产生,达到化疗增敏作用。③NF-κB信号通路双荧光素酶报告基因检测：肿瘤细胞内NF-κB信号通路本身激活明显,DHA可抑制NF-κB信号通路,而5-FU化疗则可激活NF-κB信号通路。DHA联合5-FU化疗可以抑制化疗引起的NF-κB信号通路升高水平。④RT-PCR实验结果：5-FU化疗可激活NF-κB信号通路,并相应使得下游基因与肿瘤增殖侵袭相关的基因MMP2.COX2.CyclinDl的mRNA表达水平升高。联合DHA则可以抑制化疗引起的NF-KB信号通路下游基因MMP2.COX2.CyclinDl的mRNA表达升高水平。⑤Westernblot实验结果：与RT-PCR结果类似,5-FU化疗可激活NF-KB信号通路,并相应使得下游基因MMP2.COX2.CyclinDl的蛋白表达水平升高。联合DHA则可以抑制化疗引起的NF-κB信号通路下游基因MMP2.COX2.CyclinDl的蛋白表达升高水平。⑥MDA测定：DHA可增加肿瘤细胞内的脂质过氧化产物MDA的产生,5-FU化疗则对肿瘤细胞的脂质过氧化产物无明显影响。⑦DHA及5-FU对细胞周期的影响：PI流式细胞分析结果提示DHA能阻滞肿瘤细胞进入S期,5Fu使得S期细胞数量也明显减少,两药联合产生G0／G1期阻滞作用更加明显。各单药组均能降低胃癌细胞株SGC7901的增殖指数,联合用药组降低PI的程度显著强于其余各组(P<0.05)。DHA产生G0／G1期阻滞的作用可能与降低CvclinDl的表达有关。第二部分荷瘤小鼠实验：探讨ω-3多不饱和脂肪酸单用及联合氟尿嘧啶对小鼠前胃癌治疗作用[荷瘤小鼠研究方法]体内实验方法：小鼠前胃癌细胞系(MFC)皮下注射615小鼠构成荷瘤小鼠模型,将荷瘤小鼠分为四组：第1组无干预荷瘤小鼠组,第2组给予口服鱼油干预荷瘤小鼠组,第3组给予化疗干预荷瘤小鼠组,第4组给予口服鱼油及联合化疗干预荷瘤小鼠组。口服鱼油药物为北京百慧制药公司生产的高纯度(DHA、EPA>80%,乙酯型)软胶囊,按每天2g／kg给予小鼠灌胃。化疗方式为临床使用的5-FU静脉注射剂,自皮下种植瘤细胞后每隔两天(20mg／kg)给予一次小鼠腹腔注射治疗,干预14天后处死小鼠。在实验期间仔细记录及检测以下指标：①小鼠一般情况、饮食量及荷瘤小鼠体重的时间变化记录；②肿瘤体积及去瘤小鼠体重的时间变化；③实验结束后,提取各组小鼠肿瘤组织RNA,使用RT-PCR方法比较肿瘤侵袭及增殖相关基因MMP2、COX2、cyclinDl的mRNA表达差异情况：④提取各组小鼠肿瘤组织蛋白,使用Westernblot方法比较肿瘤组织CyclinDl基因蛋白表达差异情况；⑤使用RT-PCR方法比较各组荷瘤小鼠肿瘤组织炎性因子IL-1β. IL-6基因mRNA表达差异情况；⑥提取各组小鼠肿瘤组织蛋白,使用MDA检测试剂盒检测各组荷瘤小鼠肿瘤组织内脂质过氧化水平；⑦小鼠肿瘤组织及肺组织石蜡切片HE染色观察。[体内实验结果]①小鼠一般情况、饮食量及荷瘤小鼠体重的时间变化记录：鱼油干预的小鼠一般情况良好,无明显不适症状；5-Fu腹腔注射化疗本身可明显影响小鼠进食。各组荷瘤小鼠随着肿瘤不断的增长,饮食量也逐渐减少,一般状况也明显恶化,体重明显下降,出现恶液质症状。在给予鱼油的荷瘤小鼠组(第2、4组),较相应的无鱼油干预组(第1、3组),小鼠体重下降程度明显减轻。口服鱼油可以改善肿瘤恶液质。②肿瘤体积及去瘤小鼠体重的时间变化记录：有化疗干预的第3、4组荷瘤小鼠肿瘤体积较第1、2组小鼠肿瘤体积明显要小(P<0.05)。而给予鱼油干预的荷瘤小鼠组(第2组及第4组)相对于无鱼油干预的荷瘤小鼠组(第1组及第4组),小鼠肿瘤体积未见明显的降低(P>0.05)。去瘤小鼠体重与小鼠体重记录相似,在肿瘤明显增大后,各组小鼠的去瘤体重均出现明显降低。在给予鱼油的荷瘤小鼠组,较无鱼油给予组,小鼠去瘤体重明显较高(P<0.05)。③下游基因MMP2、COX2、cyclinDl基因mRNA表达情况：给予鱼油或者化疗干预均抑制了肿瘤组织中COX2、CyclinD1、MMP2的表达(第2、3、4组均较第1组明显降低,p<0.05)。④肿瘤组织CyclinDl基因蛋白表达情况,Westernblot检测：给予鱼油或者化疗干预均抑制了肿瘤组织中CyclinDl的表达(第2、3、4组均较第1组明显降低,p<0.05)。⑤肿瘤组织炎性因子IL-1β、IL-6基因mRNA表达情况：相对于对照组(第1组),在给予化疗或鱼油干预后(第2、3、4组)小鼠瘤组织的IL-1β、IL-6基因表达被强烈抑制(p<0.01)⑥脂质过氧化,MDA检测：给予鱼油明显增加了肿瘤组织中MDA的含量(第2、4组较第1、3组明显增高,p<0.05)。化疗干预组并未明显增加肿瘤组织的MDA含量(第1、3组比较无明显差异,p>0.05)⑦小鼠肿瘤组织及肺组织石蜡切片HE染色观察：各组间小鼠肿瘤组织及肺组织未见明显差异。[本研究结论]1.体外细胞培养水平：ω-3PUFA可以通过抑制NF-KB/IkB信号通路及下游与肿瘤增殖侵袭相关的基因MMP2、COX2、CyclinDl的表达,并可以通过增加细胞内过氧化应激反应达到化疗增敏作用,降低胃癌细胞株SGC7901的PI增殖指数。2.体内荷瘤小鼠水平：ω-3PUFA可以通过降低肿瘤组织表达炎性因子IL-1β、IL-6的表达而改善小鼠恶液质状况；co-3PUFA联合化疗可以在化疗同时,抑制肿瘤增殖侵袭相关的基因MMP2、COX2、CyclinDl的表达,联合co-3PUFA治疗可能对肿瘤化疗有一定的有益作用。3.相对于化疗药物的明显的副作用,ω-3多不饱和脂肪酸同时具有抑炎、调节血脂,以及几乎毫无副作用等优点,本实验结果为临床肿瘤病人应用ω-3PUFA(EPA／DHA)治疗提供了进一步的实验依据。更多还原

【Abstract】 [Background] Docosahexaenoic acid (DHA) and Eicosapentaenoic Acid (EPA) areω-3 polyunsaturated fatty acids (co-3PUFAs).ω-3PUFAs have many beneficial effects and have been widely used in prevention and treatment of various diseases. The main beneficial effects can be summarized as following:(1)Provide energy and nutrition support in fat emulsion; (2)Reduce inflammatory cytokines, such as IL-1,IL-6,TNFa, and regulate the immune effect; (3) Reduce inflammation in multiple organ trauma stress; (4)Treatment of cardiovascular disease by lipid-lowering, anticoagulant and other beneficial effects. Recently, more and more empirical evidences show thatω-3 polyunsaturated fatty acids have a significant beneficial effect in cancer treatment. Meanwhile, our preliminary studies in vitro and in vivo have further confirmed that co-3 polyunsaturated fatty acids can also sensitize effect of chemotherapy when combined with chemotherapy drugs.[Research Objective] In vitro cell culture level and in vivo tumor-bearing mice level, we investigate the anticancer mechanism of co-3PUFA and 5-Fuorouracil (5-FU) alone or in combination in treatment of gastric cancer, thus may provide new ideas for comprehensive treatment of gastric cancer.Part I:In vitro cell culture level, investigate the effect ofω-3 polyunsaturated fatty acid alone or combined with 5-FU chemotherapy to treat gastric cancer cell lines[Cell Culture Methods]In vitro method:①In vitro culturing cells in 96-well plates, MTT assay was used to investigate the inhibition effect of DHA and 5-FU alone or in combination to treat gastric cancer cells SGC7901;②Use DCFH-DA probe to detect ROS level of gastric cancer cell SGC7901 treated by DHA and 5-FU alone or in combination;③Use dual luciferase reporter gene assay to detect NF-κB signaling pathway changes of gastric cancer cell SGC7901 treated by DHA and 5-FU alone or in combination;④After gastric cancer cells treated by DHA and 5-FU alone or in combination, use RT-PCR method to detect mRNA expression of tumor proliferation and invasion-related gene MMP2, COX2, CyclinDl,which is the downstream gene of NF-κB signaling pathway;⑤After gastric cancer cells treated by DHA and 5-FU alone or in combination, use Westernblot method to detect protein expression of tumor proliferation and invasion-related gene MMP2, COX2, CyclinDl;⑥Use MDA assay kit to investigate the lipid peroxidation status of gastric cancer cells treated by DHA and 5-FU alone or in combination;⑦Use PI flow cytometry to detect gastric cancer cell cycle after treated by DHA and 5-FU alone or in combination.[In vitro Results]①MTT test:Both DHA and 5-FU can inhibit tumor cell proliferation, DHA combined 5-FU chemotherapy can further inhibit tumor cell proliferation and reach the enchancing chemotherapy sensitivity effect.②DCFH-DA Determination of Active Oxygen:Both DHA and 5-FU can increase reactive oxygen species, DHA combined 5-FU chemotherapy, can further increase the ROS level of tumor cells, and reach the enchancing chemotherapy sensitivity effect.③NF-κB signaling pathway by dual-luciferase reporter gene assay:Tumor cells’NF-κB signaling pathway can be significantly activated in itself, DHA inhibited NF-κB signaling pathway, and 5-FU chemotherapy can activate NF-κB signaling pathway. DHA combined 5-FU chemotherapy can inhibit the elevated levels of chemotherapy-induced NF-κB signaling pathway.④RT-PCR results:5-FU chemotherapy can activate NF-κB signaling pathway, and accordingly makes the downstream gene, which is associated with tumor proliferation and invasion, MMP2, COX2, CyclinDl elevated in mRNA expression level. Combining with DHA can inhibit the increased mRNA expression level of chemotherapy-induced NF-κB pathway downstream gene MMP2, COX2, CyclinDl.⑤Westernblot results:Similar with RT-PCR results,5-FU chemotherapy can activate NF-κB signaling pathway, and accordingly makes the downstream gene MMP2, COX2, CyclinDl elevated in protein expression level. Combining with DHA can inhibit the increased protein expression level of chemotherapy-induced NF-κB pathway downstream gene MMP2, COX2, CyclinDl.⑥MDA determination:DHA can increase lipid peroxidation of MDA production in gastric cancer cells SGC7901. However,5-FU chemotherapy has no significant effect in lipid peroxidation.⑦DHA and 5-FU on the cell cycle:PI flow cytometry results suggest that DHA can block tumor cells to enter S phase,5-Fu significantly reduce the number of S phase cells. In combination, two-drug produced the effect of G0/G1 phase arrest more significant. The DHA or 5Fu alone could reduce the proliferation index of SGC7901 gastric cancer cell line, while in combined group the PI was reduced more significantly than any other groups (P<0.05). DHA producing arrest in G0/G1 phase may be related with reducing the expression of CyclinDl.Part II:in vivo tumor-bearing mice level, investigate the effect of co-3 polyunsaturated fatty acid alone or combined with chemotherapy to treat the mice pre-gastric carcinoma[Tumor Bearing Mice Methods]In vivo Methods:Mice pre-gastric cancer cell line (MFC) subcutaneous injected into 615 mice to constitute a tumor-bearing mice model, the mice were divided into four groups: Group 1 tumor-bearing mice without intervention; Group 2 tumor-bearing mice were intervened with oral fish oil; Group 3 mice were intervened with 5FU chemotherapy; Group 4 tumor-bearing mice were intervened with oral fish oil and chemotherapy. Oral fish oil were pharmaceutical drugs made in Beijing BeiHui, which is high-purity (DHA, EPA> 80%, ethyl ester type) soft capsules, and the mice fed with 2g/kg per day. Chemotherapy is using clinical 5-FU intravenous injection, intraperitoneal injected into tumor bearing mouse every two days (20mg/kg), from the day of mice being subcutaneously injected with tumor cells to the day of treated mice being killed after 14 days’intervention. Carefully recorded the following index:①tumor-bearing mice’general status, food intake and body weight changes in time;②mice tumor volume and weight changes;③At the end of experiment, extract tumor tissue RNA of each group mice, using RT-PCR method, compare the mRNA expression of tumor invasion and proliferation related gene MMP2, COX2, cyclinDl;④Extract the tumor tissue protein of each group mice, using Westernblot method to detect CyclinDl expression in protein leverl;⑤sing RT-PCR method, compare the mRNA expression of tumor inflammatory factor related gene IL-1β,1L-6;⑥Extract tumor tissue protein of each group mice, use the MDA assay kit to detect the lipid peroxidation status;⑦make paraffin section of mouse lung and tumor tissue, observation the microscopic tissue with HE staining.[In vivo Results]①General status, food intake and body weight changes of tumor-bearing mice:the mice intervened with oral fish oil showed generally good, no obvious symptoms; Intraperitoneal injection of 5-Fu chemotherapy can significantly affect the food intake of the tumor bearing mice. With the growth of tumors, mice in each groups decreased food intake gradually, the mice’s general status deteriorated noticeably, body weight decreased, and turn out tumor cachexia symptoms. Mice intervened with oral fish oil (group 2,4), compared with the corresponding non-fish oil intervention groups (group 1,3), the body weight loss were reduced significantly. Oral fish oil can improve cancer cachexia.②Tumor volume and tumor weight changes of the tumor bearing mice:The tumor bearing mice intervened with chemotherapy (group3,4), compared with mice intervened without chemotherapy (group 1,2), tumor volumes were significantly smaller (P<0.05). The tumor bearing mice intervened with oral fish oil (group2,4), compared with mice intervened without oral fish oil (groupl,3), tumor volumes weren’t significantly different(P<0.05). Similar with body weight, the carcass weight decreased sharply with tumor volume increased. Mice intervened with oral fish oil, compared to group mice intervened without oral fish oil, carcass weight were significantly higher (P<0.05).③mRNA expression of downstream gene MMP2, COX2, cyclinDl:oral fish oil or 5FU chemotherapy inhibited tumor tissue COX2, CyclinDl, MMP2 in mRNA expression (three genes in 2,3,4 group were significantly lower than those in group 1, p< 0.05).④Tumor tissue gene CyclinDl protein expression by Westernblot test:oral fish oil or 5FU chemotherapy inhibited tumor tissue CyclinDl expression (Cyclin D1 in 2,3,4 group was significantly lower than that in group 1, p<0.05).⑤Tumor tissue inflammatory factor gene IL-1β, IL-6 mRNA expression:compared to the control group (group 1), intervened with chemotherapy or fish oil(2,3,4 group), tumor tissue gene IL-1β, IL-6 mRNA expression were strongly inhibited (p<0.01).⑥Lipid peroxidation by MDA test:oral fish oil significantly increased the MDA level in tumor tissue (group 2 and 4 were significantly higher than group 1 and 3, p<0.05). Chemotherapy did not increase the tumor tissue MDA level (no significant difference between group 1 vs.3, p>0.05).⑦Mice lung tumor tissue HE staining of paraffin sections:tumor and lung tissue in each group mice showed no significant difference. [Conclusion of this study]1. In vitro cell culture level:n-3PUFA can inhibit NF-KB/IkB cell signal pathway and the downstream proliferation and invasion-related gene MMP2, COX2, CyclinDl expression, n-3PUFA can also increase cellular oxidative stress response to enhance chemotherapy sensitizing effect, and reduce the proliferation index of SGC7901 gastric cancer cell line.2. In vivo tumor-bearing mice level:co-3PUFA can reduce the expression of tumor tissue inflammatory factors IL-1β, IL-6 and improve cancer cachexia;ω-3PUFA combined with chemotherapy can suppress tumor proliferation and invasion-related gene MMP2, COX2, CyclinDl expression. Combining withω-3PUFA has some beneficial effects in cancer chemotherapy.3. Compared to the significant side effects of chemotherapy drugs,ω-3PUFA has anti-inflammation, regulation of blood fat, all without side effects. This experimental result provides the lab basis for the further clinical application ofω-3PUFA to treatment of cancer patients.更多还原