| In this experiments, we study the main biological characters of lymphocytes of bullfrogs, such as the rate of Lymphocyte Colony forming, the capability of proliferation, the toxicity of different medicines to them, the rate of its distribution in immune organs by fostering lymphocytes in the outer environment of bullfrogs and dying.The first part: Added 0.30 milliliter liquid which contains Penicillin and Streptomycin to 60 milliliters cultivation liquid and adjusted the pH to 7.3 with 5.6% NaHCO3 with the end density of the cells is 2 × 105/ml, then we put the composite cultivation liquid into cultivation plank with 4 holes to foster in the surroundings of 28℃ and saturated humidity. In the first week, the rate of Lymphocyte Colony forming is 147.33±2.52/(2×105), two weeks later,it is 224.00 ±3.61/(2×105) and three weeks later , it is 192.33 ± 4.04/(2×105).The second part: The study of bullfrogs' lymphocytes surface marks shows T-lymphocytes contain sheep red blood cell receptor and the total number of garlands is 36.25±3.10%,the live garland count is 22.50 ±1.92%,the stable garlands count is 6.75 ± 0.50%;The B-lymphocytes have complement C3 receptor and Fc receptor,the contains is 45.33 ± 3.51% and 55.67 + 3.54% separately.The third part: Taking out the immune organs of bone marrow, thymus gland, spleen and kidney of bullfrogs,some for drawing slice and dying in the ANAE dye,while the others we put them in the liquid of Carnoy for a week,embody them in paraffin,cut them into slices and dye them with the means of H.E and MG-P.Through these studies we know that Bone marrow, as one of central immune organs and hcmopoietic organs contains 77.20 ± 2.02% B-lymphocytes,which distribute primarily in the red marrow,but there are few lymphocytes in the white marrow;The thymus gland contains the most T-lymphocytes among these immune organs,the contain is 74.88 ± 9.58%,there are more T-lymphocytes in the medullas than that of in the cortexes,while B-lymphocytes mainly distribute in the cortexes;The spleen has a great number of B-lymphocytes and most of the B-lymphocytesdistribute in the white pulp, while the T-lymphocytes mainly cover in the red pulp andits contains is 16.33 ± 1.44%; The kindey,as a immune organ,its T-lymphocytes mainly cover in the cortexes of kindey,which contains is 28.50 ± 0. 29%, while most B-lymphocytes distribute in the medullas.The fourth part: Matching percoll with 0.67% normal saline into separation liquid by different ratio of volume and quality to separate outside blood of bullfrogs,we find out that the separation liquid(1.060~1.065)can separate lymphocytes from outside blood of the bullfrogs effectively.Then we separate T-lymphocytes and B-lymphocytes with nylon cotton and foster them separately in costar plank to propagate with the mitogens of phytoagglutinin and staphylococcus protein A at 22℃ and 28℃. The results shows the transformation rate of T-lymphocytes in matched control with the rising of the temperature, runs high 2.00%, whereas the transformation rate of B-lymphocytes in match control only increases 0.66%; And the transformation rate of T-lymphocytes is higher than that of B-lymphocyte under the same density, but different mitogens, using t- test to analyse the data, we find that the difference is obvious.The fifth part: Put the separated T-lymphocytes and B-lymphocytes into the costar plank in different density of the medicines and temperature.The results of the experiment is that the toxic value to T-lymphocytes and B-lymphocytes is 69.94 ± 4.51% and 72.36±1.89% separately in the group of Gentamycin(1000μg/ml)at 22℃, while the results of all other groups is lower than 50.00%.When the temperature increases to 28℃,the toxicity of Gyclophosphamide to T-lymphocytes becomes more serious,but it is invalid to the B-lymphocytes;In addition we also know that the groups of Gentamycin(≥500Mg/ml) and the groups of Streptomycin(≥ 250Hg/ml) are valid to T-lymphocytes and B-lymphocytes.
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