摘要
细胞凋亡(apoptosis)，是细胞在一系列内源性基因的调控下发生的自然或生理性死亡过程。细胞凋亡过程阻滞与细胞增殖异常一样，是肿瘤发生的主要机制之一。因而，肿瘤细胞凋亡是肿瘤研究发展的新领域，寻找高效低毒的能诱导肿瘤细胞凋亡的药物有可能成为肿瘤治疗的新热点。
叶酸(folic acid，folate，FA)是一种水溶性B族维生素，参与腺嘌呤、鸟嘌呤及胸腺嘧啶的合成以及DNA、tRNA复合物等多种蛋白质的合成，同时叶酸又是多种氨基酸转换酶和多种维生素转换过程中的主要底物之一，其辅酶形式是四氢叶酸，在酶促反应中参与一碳单位的转移。因而，叶酸在细胞代谢和细胞增殖分化中起重要作用。叶酸缺乏可以引起DNA损伤、不稳定及甲基化异常，这些变化均可引起肿瘤的发生。研究表明，叶酸对肿瘤细胞有诱导凋亡作用。饮食中缺乏叶酸与结直肠肿瘤发生的高危险性有相关关系，在饮食中补充叶酸可以起到化学预防作用，而且叶酸的细胞保护作用可能有剂量依赖性。
大肠癌是常见的恶性肿瘤之一，近年发病率迅速增长。我国大肠癌的发病率已从30年前的10/10~5上升到现在的30/10~5，在恶性肿瘤的排名中，从第6位上升到第
3、4位。有大量的临床流行病学及临床干预实验表明，叶酸在结直肠肿瘤发生中有
化学性预防作用，但叶酸的确切作用机制仍不十分明确。本课题采用体外细胞培养
人结肠癌细胞株HCT一8，通过观察叶酸对HCT一8细胞增殖及凋亡的影响，用MTT
方法选择接近ICS。的浓度来诱导细胞凋亡。运用免疫组化、原位杂交以及琼脂糖凝
胶电泳分析DNA变化等方法观察叶酸诱导HCT一8细胞的凋亡，并探讨其诱导细胞
凋亡的机制，从而为叶酸用于肿瘤的预防性干预治疗提供理论依据。
方法
1.不同浓度的叶酸处理体外培养的HCT一8细胞72h后，用Ml，T方法测定叶酸对
结肠癌细胞的生长抑制率，选择50 pg/m1浓度的叶酸来研究细胞凋亡。
2.叶酸诱导凋亡的检测
取对数生长期的HcT一8细胞，细胞浓度调整至7 x 1 04/ml，接种于25而的培养
瓶中，24h后随机分组，分别为叶酸实验组和对照组，每组5瓶，对照组加入不含叶
酸的完全培养液。
2.1.用原位杂交方法，检测50pg/inl叶酸作用 72h后实验组与对照组细胞凋亡调
控基因bcl-2及bax的mRNA表达的变化。
2.2，用免疫组化sP法，检测50 p g/ml叶酸作用72h后实验组与对照组细胞凋亡
调控基因Bcl-2及刀以蛋白表达的变化。
2.3.叶酸组分别加入50“g/ml和100pg/ml的含药物培养液，对照组加入完全培
养基。用酚和氯仿提取DNA技术，提取叶酸作用72h后实验组与对照组细胞的DNA，
琼脂糖凝胶电泳分析DNA断裂的情况。
结果
1.MTT结果显示，不同浓度的叶酸对HCT一8细胞作用72h后，细胞生长和增殖
受到不同程度的抑制。随叶酸浓度的增加，叶酸对HCT一8细胞生长的抑制作用逐渐
郑州大学硕士学位论文(2003届)叶酸诱导人结肠癌细胞株凋亡的实验研究
增加，5 pg/ml、lopg/ml、Zopg/ml、sopg/ml、100协岁血1、Zoopg/inl的抑制率分
别为7.4%、20.4%、28.5%、41.7%、67.7%、73.1%。
2.原位杂交检测bcl-肠ax的mRNA的结果显示，50协留ml的叶酸作用于HCT一8
细胞72h后，细胞内凋亡调控基因bcl-2的mRNA阳性率明显减少(0.582士0.049)，
bax的mRNA阳性率明显增加(0.582士0.045)，与对照组相比差异均有统计学意义
(P(0.05)。
3.免疫组化SP法结果显示，50p留ml的叶酸作用HCT-8细胞72h后，凋亡调
控基因bcl-2的蛋白阳性表达率明显降低(0.566士0.059)，bax基因的蛋白阳性表达
率明显增高(0.557士0.035)，与对照组相比差异均有统计学意义(P<0 .05)。
4.DNA抽提及琼脂糖凝胶电泳结果提示，50p岁nil及loop岁ml的叶酸诱导细胞
凋亡时，可以出现明显的DNA“梯状”断裂条带，而对照组无此现象。
结论
1.FA可以抑制人结肠癌细胞株HCT一8的生长，随着叶酸浓度的增加，对细胞生
长的抑制作用明显增强;对于不同的肿瘤细胞来说，FA的最适剂量各不相同。
2.本研究结果提示，叶酸作用HCT-8后bel一2的mRNA和蛋白表达明显降低，
而bax的mRNA和蛋白表达明显增高，琼脂糖凝胶电泳可出现凋亡“梯状”DNA条
3.FA可望作为诱导肿瘤细胞凋亡的抗肿瘤药物应用于肿瘤的预防性化学治疗。
Background and Objective: Cell apoptosis, is a natural process controlled by a
series of endogenous genes. The block of cell apoptosis is one of the important mechanisms as the same as the abnormality of cell proliferation in carcinogenesis.
Therefore, the apoptosis of tumor cell may be a new area in tumor research. It would be a
new hot spot to find some drugs, which could induce some malignant cells apoptosis with high efficacy and low toxicity in tumor therapy.
Folic acid is one of the dissolvable vitamins Bs. It is associated with the synthesis of adenine, guanine, thymine and proteins including DNA, tRNA compound. What's more, it is also one of the main substrates in some transforming processes of some amino acids convertase and vitamins. The coenzymes of folate are tetrahydrofolate that act as
acceptors or donors of one-carbon units during enzyme-promoting rection. Hence, folic
acid takes a major part in the cellular metabolism, cellular proliferation and differentiation. Deficiency of folic acid may cause damage, instability and abnormal methylation of DNA, which could bring about in carcinogenesis. Some researches support that folic acid may induce cell apoptosis. Some evidence suggested there was an inverse association between folate intake and colorectal cancer incidence. Folate supplementation protects against subsequent neoplasia. Moreover, folate-mediated cytoprotection may be dose-dependent.
Colorectal carcinoma is one of the most malignant tumors. In recent years the incidence of the disease is rising rapidly. In our country its incidence has increased from
10/105 to 30/105 comparing with that of thirty years ago. In the range of malignant tumors it has raised from the NO.6 to the NO.3. Many clinic epidemiological studies and human intervention trials have suggested that folic acid may be a cancer chemopreventive agent in the colorectal carcinogenesis. But the firmly mechanism of folic acid is not clarity.In this paper, Human colon cancer cell line, HCT-8, was cultured in vitro and the cell growth and apoptosis were observed with the concentration approaching IC50 detected by MTT method. We investigated the effect of HCT-8 cell apoptosis was examined by hybridization in situ, immunochemistry and gel electrophoresis in order to probe the mechanism of cell apoptosis induced by folic acid may provide theoretical basis for interventional therapy of tumor.
Methods:
1. The HCT-8 cells cultured in vitro were treated with the different concentration of folic acid for 72 hours. The proper concentration of folic acid was selected by means of MTT method and used to induce the cells apoptosis for assessing the changes of the cell growth suppression.
2. Investigation on cell apoptosis by folic acid:
The concentration of HCT-8 cell in logarithm growth stage were adjusted to 7X
104/ml and cells were incubated in 25ml cultivate bottles. After 24 hours they were matched two groups: one was the experiment group, the other was the control group.
2.1. The mRNA expression of cell apoptosis associated bcl-2 and box was examined by hybridization in situ in the depend on dealing group (adding folic acid) and control group (without folic acid).
2.2. The protein expression of cell apoptosis associated genes bcl-2 and box was checked with immunochemistry in the depend on dealing group (adding folic acid) and.
2.3. Three groups (folic acid-no added, 50μg/ml and 100μg/ml FA) were used to
examine DNA changes of cells after adding folic acid for 72 hours. DNA of's cell(adding folic acid) and contral group (without folic acid) were extracted after folic acid was added for 72 hours. Agarose gel electrophoresis was carried out to analyze the state of DNA fragmentation.
Results:
1. The result of MTT suggested the adding different concentration of folic acid for 72
hours inhibited the cell growth and proliferation in some degree. The folic acid cytotoxicity was increased more obviously with the concentration of folic acid increasing gradually. Its degree was asso
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