本文選題：亞硒酸化β-乳球蛋白 + K562細(xì)胞��； 參考：《天津科技大學(xué)》2015年碩士論文

【摘要】：本文以亞硒酸化p-乳球蛋白為研究對(duì)象對(duì)其體外抗腫瘤活性進(jìn)行測(cè)定,在與K562細(xì)胞共培養(yǎng)后,研究其對(duì)K562細(xì)胞增殖的抑制作用和誘導(dǎo)凋亡作用。通過(guò)MTT法、HE染色、流式細(xì)胞術(shù)、免疫熒光法和熒光定量PCR等方法從細(xì)胞形態(tài)學(xué)的變化、細(xì)胞周期的變化和相關(guān)周期蛋白及其mRNA的表達(dá)等方面考察了亞硒酸化β-乳球蛋白對(duì)K562細(xì)胞的影響。首先,對(duì)亞硒酸化p-乳球蛋白誘導(dǎo)K562細(xì)胞凋亡的現(xiàn)象進(jìn)行研究。MTT實(shí)驗(yàn)結(jié)果表明,隨著濃度和培養(yǎng)時(shí)間的升高,亞硒酸化p-乳球蛋白對(duì)K562細(xì)胞的抑制作用逐漸變強(qiáng),10μg/mL藥物作用K562細(xì)胞48 h后,抑制率達(dá)到90%,而正常小鼠成纖維細(xì)胞C2C12在經(jīng)過(guò)相同處理后仍然正常生長(zhǎng)；HE染色和PI/Hoechst雙染均觀(guān)察到亞硒酸化β-乳球蛋白使K562細(xì)胞出現(xiàn)體積縮小、核質(zhì)分離、出現(xiàn)凋亡小體等典型的凋亡現(xiàn)象；Annexin V-FITC/PI雙染結(jié)果表明,經(jīng)過(guò)48 h與亞硒酸化p-乳球蛋白共培養(yǎng)的K562細(xì)胞的凋亡率為18.75%,而共培養(yǎng)72 h后凋亡率上升為40.76%；流式細(xì)胞術(shù)結(jié)果表明亞硒酸化p-乳球蛋白能夠使細(xì)胞G1期和G2期發(fā)生顯著變化,細(xì)胞分別被阻滯于S→G2期和G2→M期。其次,研究了亞硒酸化p-乳球蛋白誘導(dǎo)K562細(xì)胞凋亡的信號(hào)途徑。免疫熒光實(shí)驗(yàn)和ELISA法共同確定了經(jīng)過(guò)亞硒酸化β-乳球蛋白處理的K562細(xì)胞,其cyclinB1蛋白含量顯著降低,p21蛋白含量顯著上升；亞硒酸化p-乳球蛋白還使K562細(xì)胞的cyclinB1 mRNA水平下調(diào),極顯著上調(diào)p21mRNA水平,是對(duì)照組的21倍,并且還顯著下調(diào)蛋白激酶CDK4mRNA的表達(dá)水平。綜上所述,亞硒酸化p-乳球蛋白能夠在體外有效的抑制白血病細(xì)胞K562的增殖并且誘導(dǎo)其凋亡,是一種很有潛力的抗癌活性物質(zhì)。
[Abstract]:In this paper, the antitumor activity of selenated p- lactoglobulin was determined in vitro. After co-culture with K562 cells, the inhibition of proliferation and the induction of apoptosis of K562 cells were studied. The changes of cell morphology were detected by MTT staining, flow cytometry, immunofluorescence and fluorescence quantitative PCR. The effects of 尾 -lactoglobulin selenite on K562 cells were investigated in terms of cell cycle changes and the expression of related cyclins and their mRNA. Firstly, the apoptosis of K562 cells induced by selenite p- lactoglobulin was studied. The results of MTT assay showed that the apoptosis of K562 cells increased with the increase of concentration and culture time. The inhibitory effect of selenated p- lactoglobulin on K562 cells increased gradually after treatment with 10 渭 g / mL of drugs for 48 h, the inhibition rate reached 90%, while the normal mouse fibroblasts C2C12 still grew normally after the same treatment. He staining and Pi / Hoechst double staining showed that 尾 -lactoglobulin selenite reduced K562 cell volume, separated nucleus and cytoplasm, and appeared apoptotic bodies. The results of Annexin V-FITC / Pi double staining showed that: 1. The apoptosis rate of K562 cells co-cultured with selenite p- lactoglobulin for 48 h was 18.75, while that of K562 cells co-cultured for 72 h was increased to 40.76.The results of flow cytometry showed that selenated p-lactoglobulin could significantly change the G1 and G2 phases of K562 cells. The cells were blocked in S G 2 phase and G 2 M phase, respectively. Secondly, the signaling pathway of selenated p- lactoglobulin induced apoptosis in K562 cells was studied. Immunofluorescence assay and Elisa combined to determine that the content of cyclin B1 in K562 cells treated with selenated 尾 -lactoglobulin significantly decreased the content of p21 protein, and the level of cyclin B1 mRNA in K562 cells was down-regulated by selenated p- lactoglobulin. The p21 mRNA level was significantly up-regulated, 21 times higher than that in the control group, and the expression level of protein kinase CDK4 mRNA was significantly down-regulated. In conclusion, selenated p- lactoglobulin can effectively inhibit the proliferation and induce apoptosis of leukemia cell line K562 in vitro, which is a potential anticancer active substance.
【學(xué)位授予單位】：天津科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R96

【相似文獻(xiàn)】

相關(guān)期刊論文 前3條

1 李慧,邵建新,范志濤,翟筱,李桂芝;乳汁中主要成分的含量測(cè)定[J];中國(guó)衛(wèi)生檢驗(yàn)雜志;2002年04期

2 唐俊典;牛奶蛋白過(guò)敏及耐受不良[J];國(guó)外醫(yī)學(xué)(兒科學(xué)分冊(cè));1980年05期

3 ;[J];;年期

相關(guān)博士學(xué)位論文 前1條

1 張業(yè)輝;蕓豆蛋白纖維狀聚集及凝膠機(jī)理研究[D];華南理工大學(xué);2010年

相關(guān)碩士學(xué)位論文 前3條

1 索佳麗;亞硒酸化β乳球蛋白誘導(dǎo)K562細(xì)胞凋亡及其機(jī)制研究[D];天津科技大學(xué);2015年

2 賈敏;加工食品中牛奶致敏原檢測(cè)方法的建立與應(yīng)用[D];江南大學(xué);2015年

3 王洋;蛋奶過(guò)敏原同位素稀釋質(zhì)譜定量方法研究及其應(yīng)用[D];北京化工大學(xué);2014年

，

本文編號(hào)：2045334