中华绒螯蟹eriochier sinensis血细胞组成 功能及体外培养的分析-analysis of blood cell composition, function and in vitro culture of erioc hier sinensis, erioc, erioc, erioc, erioc, erioc, erioc, hier, sinensis.docxVIP

上海海洋大学硕士学位论文 上海海洋大学硕士学位论文 PAGE PAGE IV 质的入侵。 2. 比较了几种常见血细胞培养基（ L-15 、 2×L-15 、 3×L-15 、 M199 和 RMPI-1640）对中华绒螯蟹血细胞离体培养中细胞形态以及存活率的影响，以及在 筛选获得的最佳培养基中添加不同比例胎牛血清(FBS) ( 0% 、5% 、10% 和 15%)， 进一步观察了血清对中华绒螯蟹血细胞培养效果的比较。结果表明，3×L-15 培养 基培养效果较好，所培养的细胞形态相对完整，数量较多，培养至 96 h 时血细胞 存活率仍大于 60% ; 而其他 4 种培养基效果较差，培养 12 h 存活率均低于 50%， 且细胞形态结构变化明显。以 3×L-15 培养基为基础，添加不同比例胎牛血清后发 现，对细胞存活有显著影响，存活率明显降低。因此，不添加血清的 3×L-15 培养 基对中华绒螯蟹血细胞的生长较为适宜。 3. 研究了不同 pH（6.4、6.8、7.2、7.6 和 8.0）、温度（26、28 和 30℃）和渗 透压（500、700、900、1100 和 1300 mOsM kg-1）对中华绒螯蟹血细胞离体培养中 细胞存活率和细胞形态的影响，并比较了在不同培养基下（L-15 和 3×L-15）改变 渗透压（1100 和 1300 mOsM kg-1）对其离体培养的影响。结果表明，3×L-15 培养 基调整渗透压至 1100 mOsM kg-1，pH 6.8，28℃为中华绒螯蟹血细胞离体培养的最 佳条件，能提供血细胞＞40%存活率达 7d。通过不连续梯度密度离心，初步分离 了富含三种类型细胞的细胞带。将各类型细胞分别在最佳条件下培养，使小颗粒 细胞和透明细胞的存活时间显著增加，保持＞50%存活率 15d 以上。相反，分离后 的大颗粒细胞在 3d 内迅速死亡。 关键词： 中华绒螯蟹，血细胞，组成，功能，体外培养，培养条件 ABSTRACT As an undertaker in cellular immunity and a provider in humoral immunity, hemocytes play important roles in crustacean immune defense. Different types of hemocytes are responsible for various denfense mechanisms including phagocytosis, encapsulation, melanization, cytotoxicity, cell-cell communication and activation of proPO system. Chinese mitten crab, Eriocheir sinensis, is an economically important freshwater species for aquaculture in China and was chosen as our experimental subject. To investigate the hemocyte components, classification and role in innate immunity, we studied the morphology and enzyme content of hemocytes, together with the change of THC, DHC during the infection of Aeromonas hydrophilia; To investigate the optimal culture conditions about medium, serum, pH, temperature and Osmolarity, a primary hemocytes culture was performed in vitro; We developed the survival of semigranulocytes and hyalinocytes up to 15d by separating different type of hemocytes. Our work may lay a foundation for the further researches in the cytogenesis, mechanism of innate immunity and establishment of a cell line. The results are listed below: