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Study on Anti-Leukemia Cell Effect in Vitro of MITC from Mustard Seed

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Tutor: CaoXianYing
School: Hainan University
Course: Biochemical Engineering
Keywords: MITC,chronic myelogenous leukemia cell k562,mustard seed
CLC: R733.7
Type: Master's thesis
Year:  2014
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Abstract:
Objective:Natural plant is a huge resource, looking for active components from natural plants is one of the strategic focus to develop anticancer drug. Cruciferous vegetable mustard growed in Hainan province contains rich isothiocyanates, the extraction of isothiocyanates and active research has been focused. Four isothiocyanates were extract from mustard seed, this research was to screen the compound that have the higher effect on the proliferation of leukemia cell k562and preliminary study the mechanism of action.Methods:the compound that have the higher effect on the proliferation of leukemia cell k562was screened from four isothiocyanates,3-methyl sulfonium phenyl isothiocyanate,3-cyano phenyl isothiocyanate,4-cyanophenyl isothiocyanate and methallyl isothiocyanate (MITC) determined by MTT. The effect of MITC on k562leukemia cells in vitro determined by cell colony forming experiment and cell growth rate experiment。cell morphology observation,agarose gel electrophoresis,cell aptosis rate and the cell cycle was determined to anlysis the mechanism.Results:MITC was the compound that had the higher effect on the proliferation of leukemia cell k562determined by MTT. Anti-leukemia effect of MITC in vitro was determined by MTT method,cell colony forming and cell growth rate experiment, it is concluded that IC50of MITC effect on k562cells for24h,48h,72h,96h,120h were250.0,100.5,39.0,54.7,88.7μM, IC50for72h was39.0μM<100μM, the highest inhibition rate was87.5±1.4%.The proliferation of human kidney epithelial cell293T and lekemia cell k562was inhibited selectively by MITC.The inhibition rate of human kidney epithelial cell293T was lower than lekemia cell k562.Wright-Gimsa stain, DAPI staining and rhodamine123staining suggested that MITC made k562cell shrinkage, cell nucleus concetrate and inward concave,mitochondria potential decrease. The DNA ladder was not appeared in the agarose gel electrophoresis of DNA lekemia cell k562.The aptosis rate determined by flow cytometry suggested that leukemia cell k562was not induced aptosis by MITC.Flow cytometry measure showed that the percent of G1phase decreased, the percent of G2phase increased.Conclusion:Leukemia cell k562proliferation was inhibited by MITC. According to agarose gel electrophoresis and cell aptosis rate, leukemia cell k562was not induced aptosis by MITC. MITC induced G2phase arrest of k562cells by flow cytometry instrument.
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