Study On Primary Culture And Irritant Reaction Of Artificial Feed Of Chylostomach Cells From Bombyx Mori

Artificial feed sericulture is a major technical innovation in silk production. In the domestic and overseas, the living silkworm is mainly used for the material energy metabolization test and feeding trials of the artificial feed’s formula and ingredient at present. But living creature determination cycles are long, waste energy and take time. Moreover, the factors of season, temperature, strains have a great influence on the silkworm’s adapt to artificial feed. But, we can avoid the influence by adopt the enterocyte’s primary culture products to evaluate and study the formula and nutrition utility of the artificial feed. The silkworm chylostomach is the most developed silkworm digestive organs part, it is not only the digestion and absorption of food place, but also the important organs for the matter and energy metabolism. At present, the study of the silkworm is mostly on the material metabolism and cell chemistry, but intestinal epithelium cells in the response to food stimulates has not been involved. As silkworm is a model insect, culturing silkworm cells in vitro, researching the cultural cells theoretically and applying them to industry have been attaching great importance over decades. As early as the 1930s, William Trager began silkworm cell culture studies, but so far there has still been no intestinal cell lines reported in the silkworm, which is mainly the lack of an ideal medium and scientific and effective training methods. Especially successful method for primary culture.The experimental research purpose is to use silkworm’s digestive tract tissue for the materials to proceed the Primary culture, grope for the condition and method of the Primary culture. Examine the current silkworm main component of the artificial diet in intestinal cell proliferation, In order to provide cells level basis for silkworm fodder development research. And we expect to provide initial experiment matting for the building of the silkworm gastrointestinal cells. The main research content and results are as follows:1. Preparation of silkworm culture medium and additional fluidIn this experiment, centrifugation and filtration, dialysis and other methods to obtain silkworm acellular hemolymph, as well as Tyrosinase oxidation by adding vitamin C to prevent. Preparation of acellular hemolymph, light yellow, transparent, slightly viscous, the pH value of 6.3±0.3, protein concentration of 25.4mg/100mL. Silkworm by the removal of the head, digestive tract, Malpighian tubules, anal, the organization through the homogenate was mixed with PBS to get white, transparent silkworm of the homogenate, pH= 6.5±0.3, protein concentration of 69.6mg/100mL.2. Silkworm chylostomach cell culture and detection of the growth indexIn the present, we use 2-3 larvae from the silkworm of the gastrointestinal tract as the materials; use Grace’s culture medium and silkworm organization homogenate medium for basal medium; use the silkworm acellular hemolymph. Fetal bovine serum, silkworm organization homogenate as supplemented to make up different nutrients medium(a.Grace’s culture medium+20%(v/v)FBS; b.Grace’s culture medium +20%(v/v)acellular hemolymph; c. Grace’s culture medium+ 20%(v/v)silkworm organization homogenate; d.silkworm organization homogenate+20%(v/v)FBS; e. silkworm organization homogenate+20%(v/v)acellular hemolymph; f. silkworm organization homogenate); Take the silk fibroin from posterior silkgland cell to prepare the materials for cell growth adhesion; cultivate chylostomach cells for the Primary culture. And then test the related indexes of success passage cells’growing condition:the cell count, cell growth curve, the cell division index, cell vaccination the survival rate. Through comparing training silkworm intestinal cells in different media, we find that the experimental group using Grace’s culture medium as the foundation, added with 20%(v/v)silkworm acellular hemolymph to cells and 20%(v/v)FBS can stably grow in the 25 days of original generation cells inoculation training, and in 70-75 days, reaching the 80% spread rate. After continuous cell culture, in 45 days, reaching the 80% spread rate. Cells are blend composition of three kinds cells:the epithelial cells, the flat type of spindle, single growth, boundary clear; Another type is fibroblasts, this kind of cells are long spindle type, the central have ovoid nuclear, orderly rows; And there is a small amount of smooth muscle cells, like spindle. Through testing the passage cells’growth curve and vaccination survival rate from Grace’s culture medium+20%(v/v)FBS group and Grace’s culture medium+20%(v/v)acellular hemolymph group, we find that Intestinal cells grow slowly at the first time continuous cell culture, after inoculation there exist 7 to 10 days incubation period, from 22-45 days and 20-39 days is the logarithm of cell growth for there two group. At the 45 days and 39 days, cell concentration reaches to the top, and reaches the 80% spread rate. In the remaining three kinds of complete medium, not observed in cells.3. Stimulation of the artificial feed ingredients in the chylostomach cellsFor further study of artificial feed raw materials of silkworm safety index, growth period of the gastrointestinal cells for logarithmic object, with different food does stimulate cell, determine the poisonous force reaction. On the basis of success passage cells, according to adding different proportions of mulberry leaf juice (40%,20%,10%,5%), pupa protein (15%,10%,5%,2.5%, 1%), and defatted soybean meal (15%,10%,5%,2.5%,1%), the sorbic acid (10%,5%, 2.5%,1%,0.5%), calcium propionate (10%,5%,2.5%,1%,0.5%), chloromycetin (0.02%,0.01%,0.005%,0.002%,0.001%), terramycin (0.02%,0.01%,0.005%, 0.002%,0.001%),6 kinds of artificial feed raw materials to test its cytotoxic force. The results show that mulberry leaves without cell toxicity. Pupa protein and skimmed soybean meal’s concentration under 2.5% can keep the cells value activity; when the concentration above 2.5%, cells appear different degree of death. For the sorbic acid and the calcium propionate. when the concentration is above 2.5%, cell proliferation activity reduce greatly, cells turn up death. For chloramphenicol and Terramycin, when the concentration is above 0.005%, cell proliferation activity reduce greatly, cells turn up death. Cell poisonous force experiments provide a cellular level basis for silkworm feed exploitation research.