【作者】 马望；

【导师】 樊青霞； 陆士新； 王瑞林；

【作者基本信息】 郑州大学 ， 肿瘤学， 2004， 硕士

【摘要】 恶性肿瘤严重威胁着人类的健康和生命，目前化学药物治疗仍是治疗恶性肿瘤的主要手段之一。但是化疗药物具有很大的毒性，在杀伤肿瘤细胞的同时也损伤了正常细胞，有些肿瘤又对化疗药物不敏感或有耐药性，导致肿瘤治疗失败。目前急需寻找和发现能够增加肿瘤对化疗药物敏感性的化疗增敏剂，使化疗药物能够更大程度的杀伤肿瘤细胞而尽量减少对正常细胞的损伤，提高肿瘤治疗水平。这是临床肿瘤化疗和肿瘤综合治疗迫切需要解决的重要问题。 在肿瘤治疗实践中发现：生长慢、恶性度低的肿瘤对化疗敏感性差；而生长快、恶性度高的肿瘤却有较好的化疗敏感性。其主要原因是恶性度高的肿瘤细胞多处于增殖周期，分裂增殖活跃，代谢旺盛，对周围环境敏感，容易遭受细胞毒药物的损伤，最终导致细胞死亡。恶性度低的肿瘤细胞多处于G。期，没有进入增殖周期，代谢不活跃，对周围环境敏感性差，故受细胞毒药物的影响也较小。那么，利用代谢促进剂修饰肿瘤细胞，提高肿瘤细胞的代谢水平，继之给予化疗能否在一定程度上提高化疗的疗效呢? 胰岛素是一种代谢促进剂。它最显著的功能是提高组织摄取葡萄糖的能力，促进肝脏、肌肉和脂肪组织中的合成代谢。研究表明：胰岛素可以提高正常细胞和肿瘤细胞的代谢水平。 本实验以代谢促进剂胰岛素为诱导剂，修饰肿瘤细胞，然后给予化疗药物5-氟尿嘧啶(5-Fu)，利用体外培养的大肠癌HCT-8细胞和食管癌Eca-109细胞株进行试验。观察胰岛素能否增加5-Fu对两种细胞株的抑制作用，并从胰岛素 胰岛素增效5一氟尿嗜吮对人结肠癌HCI书细胞株郑州大学2()04届硕士研究生论文和食管癌Eca一109细胞株的抗瘤作用对细胞周期的影响和其相应受体等方面对胰岛素化疗增效作用的机制进行研究。 材料与方法: (l)以食管癌Eca一109细胞和结肠癌HCT一8细胞为实验材料。食管癌组织及癌周正常组织蜡块取自郑州大学第二附属医院病理科。化疗药物选用斤氟尿嚓咤(5一厂、，)，以代谢促进剂胰岛素为诱导剂。(2)采用M’rT比色分析法检测不同药物处理组的细胞抑制率。(3)流式细胞仪检测不同加药处理组的肿瘤细胞周期时相变化及凋亡情况。(4)采用细胞免疫化学的方法检测胰岛素作用前后细胞周期蛋白D(cyolinD)的表达情况。(5)采用免疫组织化学的方法检测胰岛素样生长因子一l受体(IGF一IR)在食管癌组织和正常食管组织中的表达。(6)实验数据经51，55 10.0统计软件处理，采用单因素方差分析及xZ检验，以I)<0.()5为差异显著性标准。结果: (l)对5一Fu设定一定范围的浓度梯度进行MTT实验，选用对细胞生长抑制率接近50%的药物浓度为实验用药浓度。即5一Fu作用于HCT一8细胞的实验浓度为10林g/耐，5一Fu作用于Eca一1的细胞的实验浓度为50林g/耐。 佗)在寻找胰岛素最佳诱导时间的实验中，胰岛素分别与加入化疗药物5一F:}前24卜、12h、sh、4h、Oh和加入化疗药物后4h、sh加入。三次实验结果经统计，两种肿瘤细胞前811、前4h、0h组与5一I7u对照组相比抑制率明显增高 (尸《).01)。其它时间组与5一Fu对照组相比抑制率无显著差异(P>0.05)甚至低一于5一F。对照组。单用胰岛素组00值高于空白对照组，差异有显著性(P<0.05)，说明单独使用胰岛素有促进肿瘤细胞增殖的作用。 ‘:均在寻找胰岛素最佳诱导浓度的实验中，胰岛素分别设立0.08皿」/耐、().8;nL;/rr]1、1 .6 mu/ml、8 mu/ml、16mu/ml、SOmu/ml、160mu/mJ、20()m。/n}l浓度组。三次实验结果经统计，两种肿瘤细胞1.6mu/ml、smu/m土、16nl。/ml、8001，工/。1、16()mu/fnl、ZOOmu/ml各组与5一l了u对照组相比抑制率显著升高(尸《).05)。胰岛素浓度在0.8一sm叮ml之间时，细胞抑制率随着胰岛素剂量的增加而增加。但当胰岛素剂量在8一200m。/m1之间时，细胞抑制率并未随胰岛素剂量的增加而增加，而是稳定在一定的水平。 胰岛素增效5一氟尿哦睫对人结肠癌HCT-8细胞株郑州大学2004届硕士研究生论文和食管癌Eca一109细胞株的抗瘤作用 (4)利用IGF一lR的单克隆抗体阻断工GF一IR与胰岛素的结合观察胰岛素是否还能够发挥化疗增效作用，结果细胞抑制率与5一Fu+胰岛素阳性对照组相比非但没有降低反而明显增高(P<0.01)，说明阻断IGF一IR信号转导途径本身即可明显抑制肿瘤生长，与化疗药物联合应用可以提高对肿瘤的抑制作用。 (5)通过流式细胞仪对细胞周期时相的分析:胰岛素作用于两种肿瘤细胞6h、12h、24h、48h各时间组与空白对照组相比，G，期细胞所占百分数显著降低 (P<0.01)，6h、12h、24h时间组s期细胞所占百分数明显增加(P<0.01)，其中以6h组S期细胞百分数最高，以后随着胰岛素作用时间的延长S期细胞百分数逐渐降低，48h时甚至低于对照组。5一Fu作用于两种肿瘤细胞6h、12h、24h、48h各时间组与空白对照组相比S期细胞所占百分数明显降低且呈递减趋势 (P<0 .01)。 (6)细胞免疫化学对胰岛素作用前后Eca一109细胞cyClinD蛋白的表达进行分析，结果经胰岛素作用的更多还原

【Abstract】 The malignant tumor has badly been threatening the health and life of human beings. At present, chemotherapy is one of the main ways to the treatment of cancer. But the chemotherapeutic drugs have so much toxicity that it kills normal cells as well as tumor cells. What’s more, tumors are not sensitive to the chemotherapeutic drugs or have a drug resistance which usually makes the chemotherapy fail. Now, it is urgent to find potentiators which can improve the cancer treatment by making the chemotherapeutic drugs kill cancer cells more effectively and decrease the damnification to normal cells. It’s an urgent problem to settle in clinical oncology and cancer comprehensive treatment.During the clinical practice, people have found that rapidly growing tumors are often responsive to chemotherapy, whereas slowly growing tumors respond less favorably to chemotherapy. One reason is that the cells in rapidly growing tumors have better metabolism and are subject to be attacked, whereas the cells in slowly growing tumors have a lower metabolism level and are not easy to be attacked. So, can we improve the chemotherapeutic effect after increasing the tumor metabolic level?Insulin is a metabolism accelerant. Its prominent function is to promote the tissue incepting more glucose and to promote complex metabolism in liver, muscle and fat. Investigations show that insulin can increase the metabolic level of normal cell and malignant cells.We use 5-Fliorouracil (5-Fu), a chemotherapeutic drug, after modificating caner cell with insulin and observe whether the insulin can induce an enhancement of chemotherapeutic response of 5-Fliorouracil to HCT-8 human colon cancer cell and Eca-109 human esophageal cancer cell. Material and method:(1) Eca-109 human esophageal cancer cell line and HCT-8 human colon cancer cell line were chosen as the trial material with 5-Fliorouracil as a chemotherapeutic drug and insulin as a metabolism accelerant. Esophageal carcinoma tissue and normal Esophagus tissue were obtained from Pathology Department of the Second Affiliated Hospitol of Zhengzhou University. (2)MTT assay was used to examine the suppressive rate of cell growth dealt with different drugs. (3) Cell cycle and cell metabolism were examined by PI fluorescence flow cytometry. (4)The expression of cyclinD protein in Eca-109 cell before and after affected by insulin were tested by immunocytochemistry. (5)The expression of insulin-like growth factors-1 receptor (IGF-1R) on malignant and normal esophagus tissue were tested by immunohistochemistry. (6)A11 experimental data were processed by statistical package for social and sicence 10.0(SPSS 10.0). There was a statistical significant when PO.05 occurred. Results:(1) 5-Fliorouracil with different concentration act on two cancer cell line. Select the concentration of 5-Fliorouracil with 50% inhibitory rate as the experimental concentration. Namely, the concentration of 5-Fliorouracil act on HCT-8 cell line was 10 u g/ml and on Eca-109 cell line was 50 u g/ml.(2) To find the best inducement time of insulin, the inhibitory rates of different time groups were tested including the 24h, 12h, 8h, 4h, Oh before 5-Fu and the 4h, 8h after 5-Fu time groups. Statistical results of three times test showed, for both cancer cells, the inhibitory rates of the former 8h, 4h and Oh groups were significant higher than that of 5-Fu control groups in three times test(P<0.01). Other time groups were no significant with 5-Fu control groups(P>0.05), further more, some groups were even lower than 5-Fu control groups. The OD numerical value ofinsulin control group were higher than that of vacant control group with a significant difference (P<0.05). It showed that insulin can induce a faint proliferation of cancer cell.(3) To find the best inducement concentration of insulin, the inhibitory rates of different concentration groups were tested including 0.08mu/ml, 0.8mu/ml, 1.6mu/ml, 8mu/ml, 16mu/ml, 80mu/ml, 160mu/ml, 200mu/ml groups. Statistical results of three times test showed, fo更多还原