| The disorders in the amount of total protein(amino acid) in circulating blood are common metabolic diseases, including hyperproteinemia and hypoalbuminemia. More than 100 kinds of genetic disease are founded, which are caused by an unnomarl raise in amino acid levels because of amino acid metabolic disorders, but lack of the report purely about pathogenesis and treatment of hyperproteinemia.The main reason is that there is no effective animal model of hyperproteinemia. This is reason why we carried out the related research about the model of invertebrate animal, silkworm.Furthermore, silk gland, as the most characteristic organ, degenerates by programmed cell death during the larval-pupal metamorphosis, which is always paid attention. Based on the basis of morphologica l parameters being measured, general protein concentration in silkworm hemolymph is measured by BC A method and 21 kinds of amino acids relative content are measured by HPLC(High Performance Liquid Chromatography). Genes, related to apoptosis and autophagy, are investigated to determine the expression differences in silk gland of hyperproteinemia silkwarm by PT-PCR, q RT-PCR. The expression of Caspase-3, one of apoptosis proteins, is detected through immunofluorescence, which acts a preliminary sudy for deteriorated programmed cell death in silk gland of hyperproteinemia silkworm. The main results are as follows:1. Silkworm model of hyperproteinemia is built.After several attempts, it is better that orifice of the silkworm is sealed by low melting-point wax to build silkworm model of hyperproteinemia. After hyperproteinemia model of silkworm had been built, general protein concentration and free amino acids in hemolymph are measured, whose results showed that the concentration of total protein and total free amino acid content are significantly higher than that of wild type and almost maintained for whole pupation. The methods to block the orifice of silkworm include high or low temperatures of laser handling, high temperature burnt, low melting wax seal and non-toxic glue seal coatingThe results through BCA method show that the concentration of total protein in hemolymph of hyperproteinemia silkworm is 150-600% higher than that of the control group during pupation(9d-11d). The total amino acid level measured by HPLC in the hemolymph of hyperproteinemia silkworm during pupation(9d-11d) is 130-200% higher than that of the control group, among which eight kinds of amino acids(Ser, Gln, Thr, Arg, Val, Trp, Phe and Lys) are significantly higher and the content of histidine(His) is significantly lower than that of the control group. Several amino acids(alanine, serine and tyrosine), abundant in silk proteins, are reasonably higher than that of the control group in hemolymph of hyperproteinemia silkworm, while glycine(33% of silk proteins) in hyperproteinemia silkworm has no significantly difference from that of the control group.2. The characteristics of silk gland degradation in model silkworm are investigated.Degradation process of silk gland in hyperproteinemia silkworm is 120 h longer than that of control group, showed by the research. The result from HE staining shows the signal of PCD. The results from genes expression related to a utophagy and apoptosis signaling pathway show that the expression of Atg6 and Atg8, genes related to autophagy, in the silk gland cells of hyperproteinemia silkworm upregulates significantly slower than that of the control group during pupation, while Dronc, apoptosis-related gene, in the silk gland cells of hyperproteinemia silkworm shows no significant difference during pupation. Immunohistochemical result shows that Caspase-3, a key protein to initiate apoptosis in the silk gland, is expressed throughout whole process of silk gland degradation in both of hyperproteinemia silkworm and the control group, indicating that the time delay occurred during the degradation of silk gland in hyperproteinemia silkworm is mainly due to the postponed startup of autophagy related genes. |
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