摘要
目的
盐亭是我国食管癌高发区之一，盐亭食管癌死亡率一直位居当地恶性肿瘤之首。盐亭居民具有独特的饮食习惯，饮食因素在食管癌发生发展中占重要地位，然而盐亭饮食与当地食管癌之间的关系迄今仍不明确。
近年来抗氧化剂及对食管上皮具有保护作用的维生素与食管癌关系倍受关注，维生素A、核黄素及叶酸均可保护组织上皮完整性，否则受损的上皮组织易受到致癌物的攻击；维生素E与维生素C有协同抗氧化作用，二者可阻断亚硝胺类化学致癌物的合成。已有确凿证据表明维生素C与核黄素可降低食管癌发生的危险性；而维生素A、E、叶酸与食管癌关系的证据尚不充分，目前尚无较一致的结论。
现有的饮食、营养素与食管癌研究多限于流行病学调查、动物诱癌实验及细胞生物学研究，但它们不能全面、真实反映饮食与肿瘤之间的关系。血清生理学方法通过借鉴中药血清药理学方法，将这几种传统的方法相结合，避免它们单独使用时的不足，更全面、深入探讨饮食在肿瘤发生发展中的作用。
近年来细胞周期调控因子与食管癌的关系也备受关注，但盐亭食管癌分子水平的研究较少，而且相对滞后。
本研究首先观察模拟成都成人饮食喂饲大鼠对其生长的影响，初步探讨用成人饮食喂饲大鼠的可行性；用喂饲常规饲料的大鼠血清培养人食管癌细胞，探讨大鼠血清取代小牛血清培养细胞的可行性；从而建立饮食与食管癌研究的血清生理学方法。本研究采用血清生理学方法分别观察成都成人饮食、盐亭食管癌高发区病人饮食以及添加几种维生素(组合1：维生素A、E、叶酸；组合2：维生素A、E、叶酸、核黄素、维生素C)的盐亭饮食喂饲大鼠血清对人食管癌细胞生长增殖的影响，为探讨饮食营养因素在癌症发生发展中的作用提供新技术，为食管癌高发区居民调整饮食结构和食管癌高危人群制定营养干预措施提供实验依据。本研究采用RT-PCR定量技术及细胞免疫荧光法，分别观察盐亭饮食及添加几种维生素的盐亭饮食喂饲大鼠血清对人食管癌细胞周期调控因子表达的影响，探讨盐亭饮食及维生素在食管癌发展中的分子机制，为寻找新的基因治疗靶位提供实验依据。
方法
(1)模拟成都成人饮食(以下简称成都饮食)喂饲大鼠，同时设立大鼠常规饲料对照组，观察成都饮食对大鼠采食量及体重的影响，探讨成人饮食喂饲大鼠的可行性。
(2)采用MTT法绘制细胞生长曲线，筛选不同浓度、灭活与否的大鼠空白血清(喂饲常规饲料的大鼠血清)对人食管癌细胞的最佳作用条件；通过设立小牛血清对照以及人正常肝上皮细胞对照，进一步完善血清生理学方法。
(3)通过MTT法绘制细胞生长曲线、计算细胞群体倍增时间、~3H-TDR掺入实验测定细胞DNA合成及流式细胞术分析细胞周期，分别观察喂饲成都饮食、盐亭饮食及添加几种维生素(组合1：维生素A、E、叶酸；组合2：维生素A、E、叶酸、核黄素、维生素C，各设高低两个剂量)盐亭饮食的大鼠血清对人食管癌细胞生长增殖的影响。
(4)采用RT-PCR定量技术及细胞免疫荧光法，分别探讨盐亭饮食及添加几种维生素的盐亭饮食喂饲大鼠血清对人食管癌细胞中参与细胞周期调控的原癌基因CyclinD1、细胞周期素依赖性激酶CDK4及抑癌基因P16、Rb在mRNA、蛋白水平表达的影响。
结果
(1)本实验模拟成都饮食喂饲大鼠，其采食量明显低于大鼠常规饲料组，但其体重增长与大鼠常规饲料组无明显差异，表明成都饮食对大鼠生长无明显影响。
(2)本实验用不同浓度、灭活与否的大鼠空白血清(喂饲常规饲料的大鼠血清)取代细胞培养液中传统的小牛血清来培养人食管癌细胞，筛选出5％未灭活大鼠空白血清最适合癌细胞生长。5％未灭活大鼠空白血清对人正常肝上皮细胞生长的影响与小牛血清对照作用相近，提示该血清同样适合正常细胞生长。
(3)细胞生长曲线、细胞群体倍增时间、DNA合成及细胞周期分布结果表明，与小牛血清对照相比，盐亭饮食喂饲大鼠血清明显促进人食管癌细胞生长增殖、却抑制人正常肝上皮细胞生长增殖；而成都饮食喂饲大鼠血清对两株细胞的作用均与小牛血清对照相近。添加几种维生素的盐亭饮食喂饲大鼠血清则逆转了盐亭饮食喂饲大鼠血清对两株细胞的作用，尤以两个维生素组合的高剂量作用较明显。
(4)喂饲盐亭饮食的大鼠血清处理人食管癌细胞后，细胞中原癌基因CyclinD1及CDK4在mRNA及蛋白水平均异常高表达，同时还伴有抑癌基因P16低表达，抑癌基因Rb有低表达趋势，但变化不明显。而添加几种维生素的盐亭饮食喂饲大鼠血清则逆转了盐亭饮食喂饲大鼠血清的作用，尤以两个维生素组合的高剂量作用较明显。
结论
(1)模拟成人饮食喂饲大鼠是可行的。
(2)大鼠血清取代小牛血清培养人食管癌细胞Eca-109是可行的。
(3)与成都成人饮食喂饲大鼠血清相比，盐亭食管癌高发区病人饮食喂饲大鼠血清明显促进人食管癌细胞增殖、却抑制人正常肝上皮细胞增殖，提示盐亭饮食中可能存在促癌因素。
(4)添加几种维生素(组合1：维生素A、E、叶酸；组合2：维生素A、E、叶酸、核黄素、维生素C)的盐亭饮食喂饲大鼠血清明显抑制人食管癌细胞增殖、却促进人正常肝上皮细胞增殖，逆转了盐亭饮食喂饲大鼠血清对两株细胞增殖的作用，提示维生素充足时可保护上皮细胞免于致癌物攻击，而缺乏时则可能成为促癌因素。
(5)盐亭饮食喂饲大鼠血清激活人食管癌细胞中原癌基因、使抑癌基因失活等针对细胞周期的调控，可能是其促进癌细胞增殖的机制之一；添加几种维生素盐亭饮食喂饲大鼠血清下调人食管癌细胞中原癌基因表达、上调抑癌基因表达等调控可能是其抑制癌细胞增殖的机制之一。
Objectives:
Esophageal cancer (EC) is prevalent in China where Yanting (YT) country is one of the regions with the highest incidence, and EC has been the leading cause of cancer deaths in YT region for more than thirty years. It is suggested that diet has a profound effect on susceptibility to EC. Studies on the etiology of esophageal cancer have been conducted in Yanting region since the 1970s. Little evidence, however, is available on the potential role of the local diet in Yanting region (YT diet) on the progression of esophageal cancer.
The relationship between low intakes of vitamins and EC has attracted great interest worldwide; especially in those antioxidants that protect epithelial tissue. Vitamin A, riboflavin and folic acid play crucial roles in maintaining epithelial tissue, otherwise carcinogens could attack the damaged epithelial cells more easily. As indispensable anti-oxidants, vitamin E and vitamin C cooperate with each other in inhibiting carcinogenesis. It is confirmed that riboflavin and vitamin C are capable of lowering the risk of EC. Other vitamins such as vitamin A, E and folic acid have uncertain roles in the pathogenesis of EC.
Recent research on diet and cancers has mainly focused on epidemiological studies, in vivo and in vitro experiments. Combining these methods together, could provide greater insight into the etiology of EC in Yanting region. Therefore, in the present study, a sero-physiology method, similar to the sero-pharmacology, was applied to avoid the shortcomings of each of the above methods when used independently.
Nowadays, the roles of cell-cycle-regulating-gene on the etiology of EC have attracted increasing attentions, while few studies conducted in Yanting region focused on this field.
In the present study, to assess the feasibility of feeding the rat with human adult diet, we observed effects of the human adult diet in Chengdu region on growth of the rats, which lay foundation for further study on the relationship between the Yanting diet and EC. A sero-physiology method was used to examine for possible effects of the sera of rats fed the Yanting diet and the Yanting diet supplemented with vitamins ( Mix.1, vit.A, E and folic acid; Mix.2 was Mix.1 plus riboflavin and vit.C ) at two doses on the proliferation of the human esophageal cancer cell line Eca-109. That will not only offer a new way to study the roles of the diet and nutrients on the development of cancer, but also provide evidence for regulation of the Yanitng diet and chemoprevention of EC in future human trials in the Yanting region. The third objective was to explore effects of the sera of rats fed the Yanting diet and the Yanting diet supplemented with vitamins on expression of cell-cycle-regulating-gene in human esophageal cancer cell line Eca-109, by means of real-time polymerase chain reaction (RT-PCR) and immunofluorescence methods, which will give insight into the mechanisms of the Yanting diet and vitamins on the progression of EC and maybe provide evidence for gene-therapy in clinic.
Methods:
(1) To assess the feasibility of feeding the rat with human adult diet, we observed effects of the healthy human adult diet in Chengdu region (Chengdu diet) on diet intakes and body weights of the rats, and a standard diet for rat was set as the control.
(2) MTT cell growth assay was used to select the optimal conditions of rat's serum, such as different concentrations and deactivated or not, for cultivating the human esophageal cancer cell line Eca-109. In addition to this, 10% FBS (fetal bovine serum) and human normal liver epithelial cell line HL7702 were set as serum control and normal cell line control, respectively, which made the sero-physiology method more perfect.
(3) The effects of sera of rats fed the Chengdu diet, the Yanting diet and the Yanting diet supplemented with vitamins (Mix. 1, vit. A, E and folic acid; Mix.2 was Mix.1 plus riboflavin and vit.C) at two doses on proliferation of human esophageal cancer cell line Eca-109 were assessed by means of MTT assay, doubling time, DNA synthesis, and flow cytometry assays.
(4) Real-time polymerase chain reaction (RT-PCR) and immunofluorescence methods were adopted to explore effects of the rats' sera fed the Yanting diet and the Yanting diet supplemented with vitamins on expression of cell-cycle-regulating-gene at mRNA and protein level of human esophageal cancer cell line.
Results:
(1) In the animal trial, the mean daily food intakes were significantly lower in the Chengdu diet group than the standard diet group. However, no obvious differences of body weight gains were found between the two groups.
(2) Non-deactivated rat serum (serum of rats fed the standard diet) at a concentration of 5% provided the optimal conditions for cultivating the Eca-109 cell, and such condition had exactly the same effect on the proliferation of human normal liver epithelial cell line HL7702 as that of FBS.
(3) MTT assay, DNA synthesis, and flow cytometry assays showed that rats' sera fed the YT diet significantly accelerated Eca-109 cell proliferation, while inhibited the proliferation of HL7702 cell in contrast with the control (FBS). These changes, however, were reversed by supplementation with two vitamin mixtures, and especially in the groups with high doses. Rats' sera fed the Chengdu diet had similar effects on the proliferation of two cell lines as those of the control.
(4) Rats' sera fed the Yanting diet abnormally increased the expression of oncogenes CyclinD1 and CDK4 at mRNA and protein level of human esophageal cancer cell line, whereas the sera had the contrary effects on those of anti-oncogene P16. The sera showed the tendency to lower the expression of anti-oncogene Rb, but there were no obvious differences between it and the control. These changes, however, were reversed by supplementation with two vitamin mixtures, and especially in the groups with high doses.
Conclusions:
(1) It is feasible to feed the rat with human adult diet without affecting its' growth.
(2) It is feasible to cultivate the human esophageal cancer cell line Eca-109 with rat serum instead of the conventional fetal bovine serum (FBS).
(3) Compared to sera of rats fed the Chengdu diet, sera of rats fed the Yanting diet significantly accelerated Eca-109 cell proliferation, while inhibited the proliferation of HL7702 cell, which indicates that there maybe exist cancer-promoting factors in the Yanting diet.
(4) The effects of sera of rats fed the Yanting diet on the proliferation of two cell lines were reversed by sera of rats fed the Yanting diet supplemented with vitamin mixture, which shows that an abundance of dietary vitamins may protect the epithelial cell against carcinogens, while vitamin deficiency has the opposite effect.
(5) Sera of rats fed the Yanting diet made the oncogenes abnormally highly expression, while lowered the expression of anti-oncogenes in human esophageal cancer cell line, which maybe one of the mechanisms that the sera could promote esophageal cancer cell proliferation. However, vitamin mixtures having opposite effects on those genes maybe contribute to the inhibitory effects on the proliferation of the cancer cells.
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