双TOR抑制剂AZD2014对体外细粒棘球绦虫原头蚴活性影响的研究
发布时间：2020-07-31 18:31
【摘要】：目的:由于AZD2014是TOR蛋白激酶的抑制剂,因此通过观察不同浓度AZD2014在体外培养的细粒棘球蚴原头蚴的活性,从而得到AZD2014作用于原头蚴的相对最适浓度,进一步验证了原头蚴体内存在TOR蛋白激酶;并通过阻断原头蚴体内的TOR蛋白激酶活性,从而阻断PI3K-AKT-TOR细胞信号通路,进而杀灭囊泡内的原头蚴,为靶向药物治疗包虫病提供理论和研究基础。方法:1.不同浓度的AZD2014在体外培养细粒棘球蚴原头蚴,通过0.1%的伊-红染色后观察原头蚴的存活率,并连续观察15天同时统计其存活率;2.不同浓度AZD2014体外培养原头蚴4天后,取其蛋白上清液,用Western-blot验证TOR下游蛋白因子的表达情况,用ELISI试剂盒检测Caspase-3酶活性的表达;3、用100μM的AZD2014培养原头蚴1天,4天,7天后,取其蛋白上清液,用Western-blot验证TOR下游蛋白因子的表达情况,用ELISI试剂盒检测Caspase-3酶活性的表达。结果:1、随AZD2014培养浓度的增大而原头蚴的存活率逐渐下降,随AZD2014培养时间的延长而原头蚴的存活率逐渐下降;2、Western-blot验证TOR蛋白下游蛋白因子p-AKT,p-S6K1,p-4EBP1的表达量随AZD2014浓度的增大而下降,且随AZD2014培养时间的延长而下降(P㩳0.05);3、ELISI验证Caspase-3的活性随AZD2014培养浓度的增大而升高,且随AZD2014培养时间的延长而升高(P㩳0.05)。结论:1、AZD2014通过抑制原头蚴体内的TOR蛋白而具有杀灭原头蚴的作用;2、AZD2014杀灭体外培养的细粒棘球蚴原头蚴具有时间和浓度的依赖性。
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2018
【分类号】：R383.33
【图文】：
17光学显微镜观察不同浓度 AZD2014 作用下细粒棘球蚴原头蚴的形态学变化。（A）BDMSO 组;（C）25uM 组;（D）50uM 组;（E-G）100uM 组;（H）200uM 组；（A-D， 天的原头蚴;（E）培养 1 天的原头蚴;（F）培养了 7 天的原头蚴。注：图中箭头所指该头节活性弱或者已死亡。he morphological changes of E.granulosus protoscoleces which cultured in different conceD2014 by using optical microscopy.(A)cultured protoscoleces in Blank group;(Boleces in DMSO group;(C)cultured protoscoleces in 25uM group;(D)cultured protoscgroup;(E-G)cultured protoscoleces in 100uM group;(H)cultured protoscoleces inA-D,F,H )keeped protoscoleces for 4 days;(E)keeped protoscoleces for 1 day;(oleces for 7 days。Note: Those protoscoleces which are indicated by the arrow in the figury are weak or dead.
图 2.AZD2014 在体外作用于原头蚴后，用 0.1%的伊-红通过对原头蚴的染色来检测原头蚴的存活率。通过观察发现原头蚴存活率对 AZD2014 具有作用浓度和时间依赖性。Fig.2. The survival rate of E.granulosus protoscoleces which were cultured by AZD2014 in vivro anddetected by using 0.1% eosin staining.Results dependented on the culure time and- dose of CDCA.
19如图 3. 不同浓度的 AZD2014 培养原头蚴 4 天后 Western-blot 检测其蛋白因子的表达情况。（A）p-AKT1，p-4EBP1 和 p-S6K1 为检测的目的蛋白因子，GPDH 为内部参照。（B-D）p-AKT1，p-4EBP1和 p-S6K1 在不同组别中的表达。结果采用均数±标准差进行统计学分析。采用单因素方差分析（ANOVA）。P <0.05 时差异具有统计学意义（*P <0.05，** P <0.01）。Fig.3. Western blot analysised the protein samples of E.granulosus protoscoleces which were cultured bydifferent doses of AZD2014 for 4days respectively in vivro. (A)p-AKT1, p-4EBP1 and p-S6K1 were thedetected purpose. GPDH was used as loading control. (B-D)The expression of p-AKT1, p-4EBP1 andp-S6K1 in different groups. All the results were presented as the mean±SD(standard deviation). In thefigurs, the differences of statistically significance were detected by one-way analysis of variance (ANOVA).P-values <0.05 was authenticated statistically significant(*P < 0.05, **P < 0.01)。
本文编号：2776842
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2018
【分类号】：R383.33
【图文】：
17光学显微镜观察不同浓度 AZD2014 作用下细粒棘球蚴原头蚴的形态学变化。（A）BDMSO 组;（C）25uM 组;（D）50uM 组;（E-G）100uM 组;（H）200uM 组；（A-D， 天的原头蚴;（E）培养 1 天的原头蚴;（F）培养了 7 天的原头蚴。注：图中箭头所指该头节活性弱或者已死亡。he morphological changes of E.granulosus protoscoleces which cultured in different conceD2014 by using optical microscopy.(A)cultured protoscoleces in Blank group;(Boleces in DMSO group;(C)cultured protoscoleces in 25uM group;(D)cultured protoscgroup;(E-G)cultured protoscoleces in 100uM group;(H)cultured protoscoleces inA-D,F,H )keeped protoscoleces for 4 days;(E)keeped protoscoleces for 1 day;(oleces for 7 days。Note: Those protoscoleces which are indicated by the arrow in the figury are weak or dead.
图 2.AZD2014 在体外作用于原头蚴后，用 0.1%的伊-红通过对原头蚴的染色来检测原头蚴的存活率。通过观察发现原头蚴存活率对 AZD2014 具有作用浓度和时间依赖性。Fig.2. The survival rate of E.granulosus protoscoleces which were cultured by AZD2014 in vivro anddetected by using 0.1% eosin staining.Results dependented on the culure time and- dose of CDCA.
19如图 3. 不同浓度的 AZD2014 培养原头蚴 4 天后 Western-blot 检测其蛋白因子的表达情况。（A）p-AKT1，p-4EBP1 和 p-S6K1 为检测的目的蛋白因子，GPDH 为内部参照。（B-D）p-AKT1，p-4EBP1和 p-S6K1 在不同组别中的表达。结果采用均数±标准差进行统计学分析。采用单因素方差分析（ANOVA）。P <0.05 时差异具有统计学意义（*P <0.05，** P <0.01）。Fig.3. Western blot analysised the protein samples of E.granulosus protoscoleces which were cultured bydifferent doses of AZD2014 for 4days respectively in vivro. (A)p-AKT1, p-4EBP1 and p-S6K1 were thedetected purpose. GPDH was used as loading control. (B-D)The expression of p-AKT1, p-4EBP1 andp-S6K1 in different groups. All the results were presented as the mean±SD(standard deviation). In thefigurs, the differences of statistically significance were detected by one-way analysis of variance (ANOVA).P-values <0.05 was authenticated statistically significant(*P < 0.05, **P < 0.01)。
【参考文献】
相关期刊论文 前1条
1 ;Up-regulation of PIK3CA promotes metastasis in gastric carcinoma[J];World Journal of Gastroenterology;2010年39期
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