Evaluating Effect Of Immune Polysaccharide In Vitro

The experiment was conducted to found a system of crusian carp (Carassius auratus)lymphocyte transformation test in vitro upon studying a series of experimental conditionssuch as optimum conditions of crusian carp lymphocyte separation, optimal wavelengthof MTT test and optimum conditions of crusian carp lymphopoiesis. In this system addeddifferent concentration immune polysaccharide (yeast-cell-wall), we could quicklyevaluate effect of immune polysaccharide (yeast-cell-wall) by rule and line according tosignificant level of crusian carp lymphocyte transformation rates. So this experimentalsystem could be regarded as a new method of evaluating immune polysaccharide(yeast-cell-wall). The experiment included following contents:The separation of lymphocyte from peripheral blood of crusian carp was performedwith Ficoll-Urografin solution adjusted with Urografin. The results showed thatlymphocytes can be isolated from peripheral blood of crusian carp effectively at 800 g for25 min at 20℃with Ficoll-Urografin solution at density 1.085, average of 4.88×10~6lymphocytes were isolated from 1 mL peripheral blood of crusian carp, recovery rate oflymphocytes was 76.21%～93.79% and the average was 85.02%. Isolation technics oflymphocyte from peripheral blood of crusian carp has been founded successfully.Detection of optimal wavelength was conducted in MTT test. As a result, maximalabsorbing wavelength of crusian carp lymphocyte suspension was at 560 nm in the rangeof 450～700 nm, and the next was 570 nm. Optimal wavelength detecting lymphocytetransformation rates in crusian carp lymphocyte transformation test has been found.Orthogonal experiment to affectoi such as density of cell, concentration of PHA-P,influx of newborn cattle serum (NCS) and time on incubation of crusian carp lymphocytein vitro was conducted and analysis of variance was performed. The results indicated thatthe effects of 4 factors on transformation rates of crusian carp lymphocyte had significantdiversity (P＜0.01), the levels of transformation rates of crusian carp lymphocyte fromhead kidney and blood were similar, and optimal levels combination were all density oflymphocyte at 2.5×10~6 cells/mL, influx of NCS at 25%, concentration of PHA-P at 12.5μg/mL and time on incubation at 72 h. Illuminated that crusian carp lymphocytes comingfrom different source have same reactivity to mitogen and the same conditions werepracticable.Different concentrations immune polysaccharide (yeast-cell-wall) were added toculture medium of crusian carp lymphocyte from head kidney and blood for 72 h anddetected the effects of immune polysaccharide (yeast-cell-wall) on transformation rates making use of MTT test. The results illustrated that 70% and 90% immunepolysaccharide (yeast-cell-wall) all had influences evidently on transformation rates ofcrusian carp lymphocyte from head kidney, maximal transformation rates presented to 20μg/mL immune polysaccharide (yeast-cell-wall), and the effect of 90% immunepolysaccharide (yeast-cell-wall) was more remarkably; And on transformation rates ofcrusian carp lymphocyte from blood showed that the effects were evidently also, maximaltransformation rates presented to 40μg/mL, and the effect of 70% immunepolysaccharide (yeast-cell-wall) was more remarkably. Transformation rates of crusiancarp lymphocyte from head kidney and blood would fall little by little when theconcentration of immune polysaccharide (yeast-cell-wall) were upper or lower than theforegoing value. Illuminated that immune polysaccharide (yeast-cell-wall) by way ofimmunostimulant was capable of stimulating lymphocyte differentiation and proliferationjust when the concentration of immune polysaccharide (yeast-cell-wall) was in rightamount.