摘要
海带(Laminaria japonica Aresch)是一种营养丰富的食物，海带多糖(LaminariaJaponica polysaccharides)是海带的主要提取物．近年来研究发现海带多糖具有多方面的药理作用和生物活性功能，其中有关褐藻糖胶的研究倍受国内外学者关注。本课题采用分子生物学前沿技术研究了褐藻糖胶抗辐射和抗白血病作用与细胞凋亡关系，并从凋亡相关基因表达的角度探讨其可能机制。完成工作主要包括以下方面：
将Wistar雄性清洁级大鼠随机分为正常对照组，模型组和4个不同剂量褐藻糖胶实验组(100mg／kg、200mg／kg、300mg／kg、400mg／kg)，每组6只。对照组，模型组给予生理盐水2ml／只，实验组给予不同浓度等量褐藻糖胶溶液。灌胃10d后模型组和实验组行一次性全身~(60)Co γ射线照射，剂量9.0Gy／只，18h后测定各组大鼠脾淋巴细胞增殖反应；腹腔巨噬细胞数目及吞噬功能；血清溶血素；迟发性变态反应及脾淋巴细胞凋亡率和凋亡相关基因Bcl-2和Bax蛋白的表达。
模型组免疫指标均明显低于对照组，证明辐射模型是成功的。褐藻糖胶各实验组与模型组比较，均能对抗辐射对大鼠相关免疫指标的抑制作用，并且在实验浓度范围内存在量效关系。脾淋巴细胞凋亡指标包括TUNEL实验；流式细胞术分析；TUNEL结果显示对照组少见TUNEL阳性细胞，模型组和各实验组大鼠在脾脏可见有大量TUNEL阳性细胞，并且随着褐藻糖胶量的增加TUNEL阳性细胞相应减少，流式细胞术结果显示模型组凋亡率35.48％较对照组13.53％明显增高；各实验组脾淋巴细胞凋亡率分别为28.39％，24.71％，20.97％和18.68％，明显高于对照组。两项指标均说明褐藻糖胶对照射诱导的大鼠脾淋巴细胞凋亡具有一定的拮抗作用，并呈现浓度的依赖效应。
通过免疫组化的方法研究凋亡相关基因Bcl-2、Bax蛋白的表达。结果显示模型组与对照组比较，Bcl-2蛋白低表达而Bax蛋白高表达，Bcl-2／Bax降低。随着褐藻糖胶含量的增加，Bcl-2蛋白表达增加而Bax蛋白表达下降，Bcl-2／Bax上升。褐藻糖胶抗辐射作用的可能机理之一是使凋亡抑制基因Bcl-2表达上调、促凋亡基因Bax蛋白表
达下调，Bcl一2旧ax比值明显增大，防止脾淋巴细胞凋亡，促进免疫功能恢复。
本研究应用细胞生长曲线、MTT法、单细胞凝胶电泳、DNA电泳、TUNEL法、
流式细胞术和免疫化学法探讨了褐藻糖胶对人白血病细胞株K562的影响，并探讨其
可能机制。以0.0、0.5、1.0、2.0、3.omg/ml的褐藻糖胶配成5个浓度组，分别处理细
胞。24h后分别按以上方法检测。
结果发现，K562细胞经褐藻糖胶作用24h后，以上各剂量褐藻糖胶均能抑制K562
细胞的生长，且使K562细胞DNA受损，其中以lmg/ml组效果最佳。DNA琼脂糖凝
胶电泳可见明显的梯装条带，TUNEL方法检测呈阳性，流式细胞仪分析图上出现典型
的凋亡细胞峰，各浓度组凋亡率分别为3.98%，9.56%，29.86%，20.960/0和12.98%。
均显示1拟g/ml组效果最佳。提示诱导白血病细胞凋亡是褐藻糖胶抗白血病的可能机
制之一。
进一步通过免疫细胞化学的结果显示:在褐藻糖胶0.0、0.5、1.。、2.0、3omg/ml
培养24h后。凋亡抑制基因Bcl一2和促凋亡基因Bax的表达比(Bcl.2旧ax)分别为2.22、
1.15、0.43、0.69和1.02，其中lm岁ml组效果最明显。提示褐藻糖胶是通过调节K562
凋亡相关基因Bcl一2和Bax的表达影响其凋亡进而起到抑制白血病细胞生长的作用。
Laminaria japonica Aresch is a food which is full of nutrition. It is widely recognized that the extract of Laminaria japonica Aresch, Laminaria Japonica polysaccharides, plays multiple roles in pharmacological and biological functions in recent studys. This paper was performed on fucoidan , one of Laminaria Japonica polysaccharides, antiradiation and antitumor mechanism by apoptosis, and investigating the reasons by study the expression of some relative-apoptosis genes. The main results are as follows:
The 60 Wistar rats were randomly divided into 6 groups (n=6): normal control group model group and 4 group with different dosage of fucoidan (100 mg/ kg 200 mg/ kg 300 mg/ kg, 400 mg/ kg). All the 60 rats were administered by gavage for 10 d , then whole-body irradiation with 9.0Gy 60Co Y -ray.
The indexes of T,B lymphocyte proliferation responses, the number and phagocytosis of macrophage, serum hemolysin contents, the delayed type hypersensitivity response (DTH)of rats exposed to y -ray were measured 18h later, and TUNEL (TdT-mediated dUTP Nick End Labelling) and flow cytometry were used to investigate the effects of fucoidan on splenic lymphocyte apoptosis. Immunohistochemical technique was used to detect the expression of bcl-2 and bax. The relative immune indexes of model groups were obviously lower than those of the control. Fucoidan significantly modulated the function of immune system in irradiated rats and there was a dose-effect relationship within a certain range of dosage. The TUNEL and flow cytometry show that apoptosis ratio of splenic lymphocyte of the model groups were higher than those of the control.
Fucoidan could markedly inhibit the effects of irradiation on apoptosis and the ratio of bcl-2/bax, and in a dose-effect manner. It was suggested that fucoidan could inhibit rat splenic lymphocyte apoptosis induced by irradiation, and its mechanism is associated with regulating the expression of bcl-2 and bax.
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We want to study the influence and the possible mechanisms of fucoidan on tumor cells in vitro. We use the growth curve, MTT assay, Single cell gel electrophoresis assay (SCEG), agarose gel electrophoresis of DNA, TUNEL, flow cytometry and immunohistochemical staining, and K562 cell line was selected as target cell line. K562 were treated with fucoidan in different dosage (0.0, 0.5, 1.0, 2.0 and 3.0 mg/ml) for 24 h.
The results were fucoidan could markedly inhibit the growth of K562 and induced DNA damages, and the effect of 1 mg/ml is the best. Agarose gel electrophoresis of treated groups showed typical "DNA ladder" and TUNEL positive cells were detectable. We could also detect apoptotic peaks by flow cytometry in model groups. The percentages of apoptosis cells were 3.98%, 9.56%, 29.86%, 20.96% and 12.98% in turn, and the best effect of fucoidan is 1 mg/ml. Inducing apoptosis in leukemia cells is the important anti-leukemia mechanism of fucoidan.
The ratio of apoptosis relative gene Bcl-2/Bax is 2.22 > 1.15, 0.43, 0.69 and 1.02, and the best effect of fucoidan is also 1 mg/ml. It revealed that fucoidan perhaps through modulating the expression of Bcl-2 and Bax to induce apoptosis
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