草鱼外周血淋巴细胞体外转化诸因素的研究

STUDIES ON SOME ELEMENTS OF TRANSFORMATION OF LYMPHOCYTES IN PERIPHERAL BLOOD FROM GRASS CARP (CTENOPHARYNGODEN IDELLUS)

于1993年3月-1993年11月，采用3H－TdR同位素掺入法，分别测定草鱼外周血中淋巴细胞在经两种特异有丝分裂原植物凝集素（PHA）与金黄色葡萄球菌蛋白A（SPA）刺激后发牛的T淋巴细胞与B淋巴细胞应答转化的平均刺激指数（SI）的值。经不同浓度的PHA与SPA刺激的T，B淋巴细胞转化实验表明，两种有丝分裂原促T，B淋巴细胞应答转化的最适剂量，PHA为500μg/ml，SPA为0.05％；用不同浓度的小牛血清培养试验表明，PHA组的最适浓度为10％，而SPA组的为15％；不同培养时间的试验显示，PHA最适刺激时间为1-2d，而SPA为2-3d.因此在采用鱼类淋巴细胞体外转化实验来进行鱼类的免疫机理等方面的研究中，必须考虑上述彼此相关的三个基本要素，即淋转刺激剂的最适剂量、培养液中的小牛血清的最佳浓度以及淋巴细胞转化应答所需的最适时间。

The mean stimulation index (SI) values of response transformation of T and B lymphocytes stimulated respectively by special mitogens, PHA and SPA in the Grass Chrp (Ctenopharyngoden idellus)'s peripheral blood were detected with incorpora-tion of 3H-TdR from March to November, 1993. The fish were bled (by syringe)from the tail aorta. Blood was collected into heparinized tubes to which had been added TC-199 medium solution containing 0.5ml of 20% calf serum on base.(1) The tests of effect on different concentrations of PHA and SPA were divided into two groups: final concentrations of PHA grouP were 0.0 (control), 62.5, 125,250, 500, 1000 and 1500μg /ml, to detect T lymphocyte transformation;concentrations for the SPA group were 0.0% (control),0.025%, 0.05%, 0.05%, 0.3%,0.4% and 0.5%, to detect B lymphocyte transformation. All the tubes were incubated in 20℃ for 1 - 2 days. Sixteen hours befor completion of culture, 2μCi/tube of 3H -TdR were put into the tubes. Finally, counts per minute (CPM) of all samples were detected in a TRI-CARB 2050 CA liquld scintillation counter.(2) The tests of effect on concentrations of calf serum were in two groups: the final concentration of PHA group was 500μg /ml, SPA group was 0.05%; concentrations of calf serum were 0% (control), 2%, 5%, 10%, 15%, 20% respectively. The processes were the same as those in (1).(3) The tests of effect on different cultural time were also in two groups: the fi-nal concentrations of PHA and SPA were the same as those in (2); concentrations of calf serum of the PHA group was 10%, of the SPA group was 15%; the cultural time was 0 (control), 1, 2, 3, 4, 5, 6, 7 days respectively. Other processes were also the same as those in (1).Every test was repeated 6 times and all numerical data were analyzedstatistically. Tests of different concentrations of PHA and SPA for stimulating T and B lymphocytes transformation of response showed that the best concentration was 500μg for PHA and 0.05% for SPA. Culture tests of different concentrations of calf serum showed that the best concentration for PHA stimulation was 10%, for SPA was 15%, and that the best stimulation time was 1-2days for PHA and 2-3days for SPA.