摘要
龙须菜(Gracilaria lemaneiformis)是江蓠属中重要的产琼胶的大型经济海藻。具有适温范围较广、生长快、适应环境能力强等优点。它不仅是改善生态环境的理想生物修复材料,而且因其营养丰富,药用价值高,还可作为海洋药物资源,用来辅助治疗多种疾病,具有广泛的应用前景。
本论文在实验条件下,研究了环境物理因子(温度、盐度、光照),化学因子(氮磷化合物-N、P、重金属镉-Cd、有机物邻苯二甲酸二甲酯-DMP)胁迫对藻龙须菜生理生化特性及藻体细胞超微结构的影响;运用流式细胞技术研究了龙须菜粗提物的免疫调节及抑制肿瘤活性的能力。
结果显示,温度22-25℃、盐度30‰、光照2000-25001x物理条件适于龙须菜生长,叶绿体及线粒体超微结构显示出良好的功能状态;在温度30℃、盐度22‰、光照50001x时龙须菜藻体细胞出现明显的质壁分离、类囊体膨胀变形、嗜饿颗粒沉积及线粒体部分溶解等特征,龙须菜生长受到明显抑制。
当加富N浓度为200～600μmol/L或者N、P浓度分别为50～400μmol/L、3.13～25μmol/L时有利于龙须菜生长,而当N浓度超过600μmol/L或者N、P浓度分别超过400μmol/L、25μmol/L尤其是达到600μmol/L、37.5μmol/L时龙须菜生长受到明显抑制。此时龙须菜藻体硝酸还原酶(NR)活性受到抑制,藻体叶绿体数量增多但多数发生溶解,类囊体肿胀、排列混乱,线粒体数量减少,且有部分发生分解,线粒体横切面的嵴突增粗、膨胀。表明龙须菜在高N或高N、P污染环境均可能存在修复临界点(N临界浓度为600μmol/L;N、P临界浓度分别为400μmol/L、25μmol/L),超过此临界点,龙须菜生长将受到抑制。研究还发现在高浓度N环境,龙须菜对N具有选择吸收特点,优先选择吸收NH_4~+-N,对三种形态N的吸收速率关系为:NH_4~+-N＞NO_3~--N＞NO_2~--N。
Cd和DMP对龙须菜的胁迫均受污染物浓度和暴露时间的影响,过高浓度Cd(≥4.5mg/L)及DMP(≥0.2mg/L)会逐渐破坏龙须菜藻体酶活性,降低可溶性蛋白、叶绿素a及藻红素含量,藻体细胞出现严重的质壁分离,类囊体条数增多且膨胀,线粒体多数溶解,并出现较多的嗜饿小体及Cd颗粒沉积物等,龙须菜生长受到明显抑制。但在接近自然近海环境Cd浓度(1.0mg/L左右)或者稍高于养殖污染区Cd浓度(＜3.0mg/L)以及当DMP浓度≤0.1mg/L时,龙须菜对Cd、DMP胁迫均表现出一定的抵抗能力。
龙须菜粗提物的免疫调节及抑制肿瘤活性实验研究结果显示龙须菜粗提物不同浓度处理条件下BALB/c小鼠脾脏淋巴细胞中CD45~+ CD83~+/CD45~+及CD45~+CD80~+/CD45~+细胞百分率与对照组比较无显著性差异(P＞0.05),但CD45~+TGF-β/CD45~+细胞百分率与龙须菜粗提物反应液浓度则呈明显的线性剂量依赖关系(R~2=0.8145,P＜0.05),最适反应相对百分浓度为2%。TGF-β主要来源于CD3~+T细胞(约占35.4%)和CD49b~+NK细胞(约占46.6%)。研究结果表明龙须菜有效成分具有通过TGF-β进行免疫抑制或生物调节的功能。
Gracilaria lemaneiformis(Gracilaria,Rhodophyta) is one of the important economic seaweeds.Featuring advantages such as rapid growth,high uptake of the nutrients nitrogen(N) and phosphorus(P),Gracilaria lemaneiformis has been suggested to be a promising plant to remediate the ecological environment in eutrophic waters.Besides,G.lemaneiformis has been suggested to be a potential marine drug in assisted treatment for diseases and therefore,has variable applications and market values.
In this paper,the response of seaweed G.lemaneiformis to environmental stress was studied in indoor seawater culture system.The impact of environmental factors including changes in temperature,salinity,light intensity,nitrogen,phosphorus, cadmium and dimethly phthalate on the growth of G.lemaneiformis were studied.In addition,the immunomodulatory and antitumour effects of the seaweed extraction were analysed using flow cytometry.
The results were summarized as follows:
(1) G.lemaneiformis showed high growth rate and normal ultrastructures of both chloroplast and mitochondrion at 22～25℃with 30‰salinity and with light intensity of 2000～2500 lx.Plasmolysis,disordered grana structure with loosen layer of thylakoid and irregular arrangement,vacuoles,blurry mitochondrion cista and inhibited growth of G.lemaneiformis were observed at 30℃with 22‰saltinity and with light intensity of 5000lx.
(2) G.lemaneiformis showed increased growth rate with the concentration of N increased from 200μmol/L to 600μmol/L,as well as with the N/P concentrations increased from 50/3.13μmol/L to 400/25μmol/L.The growth rate decreased significantly with＞600μmol/L N or with＞400/25μnol/L N/P.The activity of nitrate reductase was inhibited at the above indicated concentrations.Increased number of swollen,irregularly aligned,ruptured and partly dissolved thylakoids have been observed in some cases.Also,increased volume of chloroplasts,significant accumulations of starch grains in chloroplasts and the reduced mitochondrion were observed.Given these results,a remediation critical point for the growth of G. lemaneiformis under high N or N/P concentrations was proposed.The critical concentration of N was 600μmol/L,and of N/P were 400/25μmol/L.The result also indicated that the seaweed possessed the capability of assimilating nitrogen selectively, and the sequencing of assimilating rate was NH_4~+-N,NO_3~--N and NO_2~--N.
(3) The growth of G lemaneiformis was influenced by changes in concentrations of pollutants and by the changes of exposure time in cadmium(Cd) or dimethly phthalate(DMP) treatment.The activities of enzyme of the seaweed were destroyed and contents of the soluble protein,chlorophyll a and phycoerythrin were decreased when the concentrations of Cd and DMP exceeded 10mg/L,0.2mg/L,respectively.At these concentrations,the ultrastructures of the seaweed cells were severely damaged including the occurring of marked plasmolysis,dissolved crista,disappearing of the chloroplast envelope,disturbed thylakoid and Cd precipitation near the cell wall, which resulted in remarkably inhibited growth of G.lemaneiformis.On the other hand, the Gracilaria has a certain resistance capability against Cd and DMP stress when the concentrations of Cd and DMP were lower than 3.0mg/L,0.1mg/L,respectively.
(4) No statistical differences in CD45~+ CD83~+ / CD45~+ and CD45~+ CD80~+ / CD45~+ cell population were observed between BALB/c mice treated with different Gracilaria extraction and the control group(P>0.05).However,dose-dependent increasing in CD45~+ TGF-β/ CD45~+ cells was observed with the treatment of the seaweed extraction at different concentrations(R~2=0.8145,P＜0.05).The optimized concentration of Gracilaria extraction was 2%.The research indicats the effective components of the seaweed have different immunomodulatory effects and merit further investigation.
引文
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