| IntroductionEarthworm, as one of precious Chinese drugs, can find all over the country, and have effects on many diseases. Earthworm pep tides are the general name of low molecular weight peptides in Earthworm. Its have many types and a lot of complicated biological functions. Researchers have separated many peptides from earthworm, such as Fi-brinolytic Enzyme, Annetocin, Myoactive peptides and so on. Earthworm peptides, which can regulate immunoresponsiveness, because its effects on anti - tumor and increase immunoreaction, now gain more and more concerned. The two representatives are G - 90 and 912. G -90 is a mixture that is composed of seventeen glyco - lipid -proteins, and can hold the growth of B16 cell and CMC1 Cell. 912 can suppress the proliferation of S180, H22 Hep-2 and MGc803. As immunoresponsiveness regulators, 912 can increase the weight of thymus and the amount of thymusic cell, and eliminate T cell immuno - deficiency that caused by radiotherapy. 912 also can raise the weight of spleen and stimulate the proliferation of spleen cell, and eliminate the deficiency of humoral immunity that caused by radiotherapy. To non ?specific immunity, it not only active macrophage , natural killer cell and lymphokine activated killer cell, but also enhance ADCC effect. But G -90 and 912 is high molecular weight proteins, and have many side effects. In our test, we separated and purified low molecular weight immuno - actived earthworm peptides, and research its effects on natural killer cell and macrophage.Materials and methods1. Materials: Earthworm, bought from culture plant of Beijing, is Lumbricidea Eiscmia foctide . SephadexG -25 and DEAE - Sephadex A - 25 is producted by pharmacia.2. Separation and purification of earthworm peptides: The separation of earthworm peptides are devided into three steps; crude separation , gel filtration chromatography, DEAE - sephadexA - 25 anion exchange chromatography.3. Quantity of protein; Act as Marions method of coomassie brilliant blue G-250.4. Detect the molecular weight of earthworm peptides; Act as Hermanin Schaggers method of Tricine - SDS - PAGE.5. Examining the active of NK cell: Use Lactate dehydrogenase method.6. Examining the active of macrophage: Use neutral red method.7. Statistics method: t - test .Results1. Production of earthworm peptides; Through SephadexG - 25 gel filtration chromatography, the sample is divided into three part. Collected peak one and peak two together called earthworm peptides T. As Tricine - SDS - PAGA test, earthworm peptides T are composed of serven component. Its molecular weight is all under 20,000. The result of E - RFC expressed that it can improve the E - RFC to 20%.Through DEAE - SephadexA - 25 anion exchange chromatogra-phy, earthworm peptides T are divided into two parts, called earthworm peptides A and earthworm peptides B. By analyzing of Tricine -SDS - PAGE, earthworm peptide B is a single component, and dyed by coomassie brilliant blue is one band, which molecular weight is 11, 000. Earthworm peptides A are consisted of five components, including four proteins whose molecular weight are fewer than 13,000. So earthworm peptides A, B and T are all low molecular weight peptides.2. Effect of earthworm peptides on NK cells active2. 1 Effect of each component of earthworm peptides on NK cells active: Earthworm peptides A and earthworm peptides T can obvious improve the active of NK cell at concentration of 100μg/ml and 10μg/ ml, and the effects is increasing as the concentration increasing. Especially when earthworm peptides A at concentration of 100μg/ml, the NK cytotoxicity is 24.5±1. 15%. This result is much higher than IL -2. But earthworm peptide B does not have such effect.2. 2 The effects of earthworm peptides on active NK cell by IL -2: IL -2 as a cytokin can active NK cell at concentration of 1000 -10U/ml. In our test, we mix different concentration of IL - 2 with earthworm peptides (10μg/ml) , but earthworm peptides do not work in c... |
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