【摘要】 通过体外及体内实验2种途径,探讨了壳聚糖对异育银鲫(Carassius auratus gibelio)和中华绒螯蟹(Eriocheir sinensis)的血淋巴细胞部分特性的影响。体外实验通用3H-TdR、14C-UR、3H-Val掺入法研究了不同剂量[0μg/mL(对照)、5μg/mL、10μg/mL、20μg/mL、50μg/mL和100μg/mL]的壳聚糖对异育银鲫PHA和LPS刺激的淋巴细胞DNA、RNA和蛋白质增殖的影响。结果表明,随着蟹血淋巴中壳聚糖剂量的增加,检测到3H-TdR、14C-UR、3H-Val的放射强度逐渐增强,当壳聚糖终质量浓度达20μg/mL时,PHA和LPS刺激的DNA、RNA、蛋白质增殖作用达到最大值,而壳聚糖的终质量浓度达到100μg/mL时,放射强度低于不添加壳聚糖时,表明此时的壳聚糖剂量抑制了淋巴细胞DNA、RNA和蛋白质的增殖。同样,随着鲫血淋巴中壳聚糖剂量的增加,检测到3H-TdR、14C-UR、3H-Val的放射强度也逐渐增强,当壳聚糖终质量浓度达10μg/mL时,经PHA和LPS刺激的放射强度达峰值,其每分钟放射性计数(cpm)分别为11133、8882,8457、6726,5964、4286,而壳聚糖的终质量浓度达到100μg/mL时放射强度甚至低于不添加壳聚糖时,表明此时的壳聚糖剂量抑制了淋巴细胞DNA、RNA和蛋白质的增殖。同时,利用流式细胞仪技术,检测了以壳聚糖(100mg/kg)投喂鲫和蟹不同时间(7～30d)后淋巴细胞的细胞周期(Cellscycle)、增殖指数(PI)和凋亡指数(AI)的变化情况。结果表明,各采样时间蟹淋巴细胞的细胞周期、增殖指数(PI)和凋亡指数(AI)与对照组无显著差异(P>0.05);日粮中添加壳聚糖后对鲫淋巴细胞AI没有显著影响(P>0.05),但对PI则显著提高(P<0.05),对鲫淋巴细胞周期中的G2/M和S时相的比例显著或极显著提高。实验结果提示,壳聚糖可促进鲫和蟹淋巴细胞的增殖,从而增强其免疫机能,但不同水产动物对壳聚糖的免疫刺激作用敏感性不同。更多还原

【Abstract】 3H-thymidine(3H-TdR),14C-uracil(14C-UR)and 3H-Valine(3H-Val)incorporation assay have been used to measure the proliferation of DNA,RNA and protein of lymphocyte in Carassius auratus gibelio and Eriocheir sinensis, and chitosan with different concentration(5μg/mL,10μg/mL,20μg/mL,50μg/mL and 100μg/mL)was added into lymphocyte to detect the synergism with stimulation factor,such as lipopolysaccharide(LPS)and phatohemagglutinin (PHA).Data were collected with liquid-sparkling counter.The result indicated that radioactivity of 3 H-TdR, 14 C-UR and 3 H-Val enhanced gradually as the chitosan dose increased,and the radioactivity peak was reached when 20μg/mL chitsan was added into lymphocyte of Eriocheir sinensis,the counts per minute(cpm)was 8 499,6 496;6 924,5 013; 4 228,2 664,respectively.When the dose of chitosan amouted to 100μg/mL,the radioactivity decreased to the lowest level,it showed that high dose chitosan inhibited the proliferation of DNA,RNA and protein.Similarly,radioactivity of 3 H-TdR, 14 C-UR, 3 H-Val enhanced gradually as the chitosan dose increased,and the radioactivity peak was reached when 10μg/mL chitsan was added into lymphocyte of Carassius auratus gibelio,the counts per minute(cpm)is 11 133, 8 882;8 457,6 726;5 964,4 286,respectively.When the dose of chitosan amouted to 100μg/mL,the radioactivity decreased to the lowest level.The other experiment was conducted with the objective of examining the effect of dietary chitosan on cell cycle,productive index(PI)and apoptosis(AI)of the lymphocyte in C.auratus gibelio and E.sinensis by the methods of flow cytometry(FCM).One hundred and eighty healthy C.auratus gibelio(body weight about 10 g)and 120 E.sinensis(body weight about 4.5 g)were randomly divided into two groups separately based on diets as follows:control group(0 mg/kg chitosan in feed)and experimental group(100 mg/kg chitosan in feed),and 3 repeats in each group.All groups were treated for 30 days.After the experimental diets with chitosan(100 mg/kg)were supplied, samples were collected at 4 time points(7 d,14 d,21 d and 30 d)on schedule,the results showed that no significant difference detected about the cell cycles,PI and AI of lymphocyte,E.sinensis,comparing experimental group with the control group(P>0.05).But in same conditions,the chitosan added to C.auratus gibelio can boost the PI significantly (P<0.05),despite of its no effect on AI(P>0.05),and the G 2 /M&S phase in the cell cycles were enhanced significantly or very significantly.In conclusion,chitosan can contribute as immune intensifier in E.sinensis and C.auratus gibelio, but a noticeable thing should be put forward is that the appended dosage is different in various species.更多还原