| Bombyx mori is a kind of typical economic and complete metamorphism insect. Its life consists of four stages including egg, larvae, pupa and adult. The morphological and physiological function change significantly each period. It can be divided into univoltine, bivoltine and multivoltine type, which vary in body size, physique and silk quality. The ability to adverse environment from strong to weak is multivoltine, bivoltine, univoltine. Statistics from the General Office of the Ministry of Commerce shows that China issued 16.55 million pieces of silkworm egg, 650000 tons of silkworm cocoon silk production and 134000 tons of silk production in 2013. Larvae takes mulberry, stores nutrition, spins in the form of cocoon and turns into pupa finally. It mainly offers raw silk for human. In addition, the silkworm larvae have been used to study the biological reactor, genetic engineering, medicine. Compendium of Materia Medica written by Li Shizhen has recorded the medicinal function of bombyx batryticatus infected with Beauveria bassiana, bombyx batryticatus can dispel scar, treat the epilepsy specially in children, scrofula, head wind, the wind moaning worm tooth pain, dispelling wind and sputum tuberculosis. Larvae infected caterpillar fungus spore turning into caterpillar fungus with medicinal value gets more attention. In addition, larval hemolymph in anticancer, antibacterial, antiviral, antiapoptosis caused by apoptosis material, agglutination, insect cell culture medium have been reported. However, there is no research on the biomedical function of univoltine and bivoltine silkworm hemolymph stimulated by exogenous substances. Based on it, new sdudy will report on how Bombyx mori immuned by Escherichia coli, tumor cells and normal saline, collecting the immuned silkworm hemolymph and further analyzing their physical and chemical properties, studying the effects on bacteria, mammalian cell agglutination, tumor cells proliferation, the acute toxicity in mice to find the potential medicinal efficacy of silkworm hemolymph.The thesis mainly consists of two parts: collection, post-processing of immuned silkworm hemolymph and biomedical activity including physicochemical properties, agglutination, antibacterial, antitumor in vitro, acute toxicity in vivo.(1) The work carry out firstly by injecting bacteria, tumor cells and normal saline into hemocoel of univoltine and bivoltine silkworm and immuning for 24 hour, control group without any treatment. Collecting the silkworm hemolymph once immunity ended, and further centrifugate, heat treatment and vacuum freeze drying.(2) Secondly, testing the treated silkworm hemolymph of physicochemical properties including p H value, mass fraction, absorption spectrum and thermal stability. The absorption spectra from 190 nm to 900 nm of 7 kinds of silkworm hemolymph were measured by UV spectrophotometer. p H value were measured by p H meter. Mass fraction are calculated after vacuum freeze drying. Heating the silkworm hemolymph in 60℃ for 10 min, centrifugation and collecting supernatant. Results show that the hemolymph p H values are all weak acid though silkworm stimulated by different materials. Absorption peak appears in 250 nm, the smallest absorbance value about 1.3 belongs to bacteria treated group of bivoltine silkworm, the control group of univoltine silkworm is about 1.4, The absorbance values of the other 5 are larger and closer, reach approximately 1.6. However, the bacteria treated groups and cells treated group show new absorption peak in 430 nm.(3) Silkworm hemolymph agglutinate mammal red blood cells including mouse, rabbit, pig and human in vitro, respectively.7 types silkworm hemolymph all show agglutination in slide method. All silkworm hemolymph but UCT-SH have lectin activity in 96 well microtiter plate test.(4) The antibacterial circle test, broth dilution method and growth curve of bacteria test were qualitative and quantitative determination of bacteria inhibiting effect with silkworm hemolymph. Detection index mainly includes antibacterial circle diameter, minimum inhibitory concentration and bacteria growth curve. 7 kinds of silkworm hemolymph have no inhibitory effect on Staphylococcus aureus in antibacterial circle test. But UBT-SH and BBT-SH can inhibit Escherichia coli K12D31 and keep thermal stability. MIC of UBT-SH was less than 5.96 ng/m L from 50℃ to 100℃, MIC of BBT-SH decreased gradually insdead. All silkworm hemolymph reduce the growth curve of E.coli K12D31.(5) Silkworm hemolymph on proliferation of tumor cells in vitro carry out by static, dynamic MTT and flow cytometry apoptosis methods. Observe cell morphology, calculate cell proliferation inhibition rate and apoptosis rate. The results show that SW1990 and MCF7 tumor cells became round and shrinkage culturing with silkworm hemolymph, cell growth inhibited. UCT-SH shows stronger inhibition on tumor growth in the same condition.(6) Multicellular tumor spheroids(MTS)of MDA-MB-231 are cultured in agar plate in vitro and draw the MTS growth curve. Culturing formed MTS in 7 kinds of silkworm hemolymph to observe the volume and morphology.The results show that MTS of MDA-MB-231 forms successfully by agar culture method. They grow slowly in 0-8 h, increase rapidly during 16-20 h, keep stable in 20-38 h and reach 1.0 mm3 on average. MTS cultured in silkworm hemolymph all appear gossypine and translucent cells disappeare at their edges. On the other side, 7 kinds of silkworm hemolymph can slow volume growth rates of MTS compared with control. Volume growth rates of MTS in UBT-SH and BNS-SH are less than 1 which indicate that they could reduce tumor volume.(7) The univoltine and bivoltine silkworm hemolymph at the dose of 15000 mg/kg and 5001 mg/kg respectively are intragastric administration in 6 h to study the acute toxicity of the hemolymph in BALB/c mice. Observe the activities of mice, record weight for 14 day, liver and kidney function, blood routine test autopsy. The experimental results show that the experimental groups compared with the negative control group show no significant difference, in accordance with the compound can be determined by acute oral toxicity standard of silkworm hemolymph non-toxic. |
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