低浓度氧化铈纳米材料（CNPs）促进肝癌细胞增殖的机制研究

发布时间：2018-07-04 12:19

本文选题：氧化铈纳米材料 + 肝癌　；参考：《第三军医大学》2016年硕士论文

【摘要】：背景:氧化铈纳米材料(CNPs)因为其抗氧化性能,现目前已经有大量的研究用于各种疾病的治疗,特别是肿瘤的治疗。目前的研究主要是氧化铈纳米材料(CNPs)的细胞毒性,可以杀伤肿瘤细胞,限制肿瘤的侵袭,也可以增加肿瘤细胞对放疗的敏感性。一些高分子聚合物可以通过锚定在氧化铈的表面,增加氧化铈的生物学性能,提高杀伤肿瘤细胞的能力,而对正常的细胞和组织没有什么影响。但是以上研究均是在较高浓度氧化铈纳米材料(CNPs)(≥10μg/ml)作用下产生的生物学效应,主要是在动物体内得出的实验结果,对人体来说,浓度太高,可能会产生相应的一些副作用,很难应用于临床,然而低浓度的氧化铈纳米材料(CNPs)(≤1μg/ml)是否对肿瘤细胞产生影响,包括肿瘤细胞的增殖、侵袭和转移等,目前尚未有报道。在本研究中,我们发现通过阿伦磷酸钠将PEG600锚定在氧化铈纳米材料表面,可以增加氧化铈纳米材料的稳定性,而且低浓度(0.01μg/ml)的CNPs-AL-PEG600可以通过活化AKT/ERK信号通路,促进肝癌细胞的增殖,为氧化铈纳米材料治疗肿瘤的生长提供了新的有意义的信息。目的:1、低浓度氧化铈纳米材料(CNPs)可以促进肝癌细胞增殖;2、低浓度氧化铈纳米材料(CNPs)通过活化AKT/ERK信号通路促进肝癌细胞增殖。方法:1、细胞培养:将冻存在本实验室液氮罐中的肝癌细胞Hep G2、Huh7、7721、HCT116、MCF-7以及U2OS复苏,复苏后用含10%胎牛血清的DMEM在37℃、5%CO2的细胞培养箱培养;2、细胞增殖实验:用CCK-8法检测细胞增殖。将对数生长期的六种肿瘤细胞Hep G2、Huh7、7721、HCT116、MCF-7以及U2OS配制成1×104/ml单细胞悬浊液,将每种细胞分为六组,分别加入浓度为0、0.005、0.01、0.05、0.1、1μg/ml的纳米材料,接种于96孔板中,在37℃、5%CO2的细胞培养箱培养24小时。24小时后取出96孔板,每个孔中加入CCK-8试剂10μl,继续孵育2小时后,用酶标仪检测450nm处各孔的吸收度(OD值),测3次,取平均值;3、实时荧光定量PCR检测与凋亡相关的基因Bcl-2以及Bax m RNA的表达:肝癌细胞Hep G2、Huh7、7721经低浓度氧化铈纳米材料(CNPs)处理24小时后,用Trizol法提取细胞总RNA,用核酸浓度测量仪测浓度,根据Ta Ka Ra的RT-PCR试剂盒说明书,两步法逆转录合成c DNA,按组分配制PCR反应液,进行q RT-PCR反应,内参为GAPDH,Bcl-2基因上游引物:ATCg CCCTg Tgg ATg ACTg Ag T,下游引物:g CC Agg Ag AAATCAAACAg Agg C;Bax基因上游引物:TCAgg ATg Cg TCCACCAAg AAg,下游引物:Tg Tg TC CACgg Cgg CAATCATC;反应条件为:95℃,1min;95℃,10s;60℃,15s;72℃,30s,重复40次。最后进行统计学分析;4、基因微阵列分析:将Hep G2细胞接种于6孔板中,分为2组,每组3个复孔,分别为未给予氧化铈纳米材料处理的对照组和给予低浓度(0.01μg/ml)氧化铈纳米材料处理的实验组,在37℃、5%CO2的细胞培养箱培养24小时后,用PBS冲洗6孔板,每孔细胞用Trizol处理后,提取总RNA,转移到EP管中,在-80°C冰箱中保存。将标本送于上海其明信息技术有限公司做检测;5、基因分析和信号通路分析:由上海其明信息技术有限公司完成;6、免疫印迹实验:将浓度为0.01μg/ml氧化铈纳米材料与肝癌细胞Hep G2共孵育,分别提取共孵育0.5h、1h、2h的蛋白,以未用氧化铈纳米材料刺激的肝癌细胞Hep G2中提取的蛋白为对照,分别检测AKT,p AKT,ERK和p ERK抗体在不同时间段的表达量,内参为GAPDH;7、裸鼠皮下成瘤实验:购买10只6-7周龄裸鼠,随机分为2组并称体重,1组为对照组,1组为实验组。将1×106Hep G2细胞重悬于100μl PBS中,然后皮下注射两组裸鼠的右侧腹股沟。第二天开始,每隔一日在实验组裸鼠腹腔内注射剂量为0.01mg/kg的氧化铈纳米材料(CNPs),对照组注射同等剂量的PBS。30天后处死裸鼠,取下瘤体称重;8、统计学分析:用平均值±标准差表示计量资料数据,用Graph Pad Prism5软件进行统计学分析,两组间的差异比较采用t检验,差异性检验水准设为P0.05。结果:1、低浓度氧化铈纳米材料(CNPs)促进肝癌细胞增殖;2、低浓度氧化铈纳米材料(CNPs)减少肝癌细胞的凋亡;3、低浓度氧化铈纳米材料(CNPs)通过活化AKT/ERK信号通路促进肝癌细胞增殖;4、在体内低浓度氧化铈纳米材料(CNPs)促进肿瘤生长。结论:1、通过CCK-8实验证实,浓度为0.01μg/ml的CNPs-AL-PEG600促进肝癌细胞增殖;2、CNPs-AL-PEG600实验组细胞里凋亡相关基因Bcl-2/Bax比值较对照组升高,表明CNPs-AL-PEG600通过减少细胞凋亡途径来促进肝癌细胞增殖;3、基因芯片分析,CNPs-AL-PEG600可能是通过活化AKT/ERK信号通路促进肝癌细胞增殖;4、WB实验证实CNPs-AL-PEG600是通过活化AKT/ERK信号通路促进肝癌细胞增殖;5、在小鼠体内实验证实浓度为0.01mg/kg的CNPs-AL-PEG600促进肿瘤生长。
[Abstract]:Background: ceria nanomaterials (CNPs) have been widely studied for the treatment of various diseases, especially tumors, because of their antioxidant properties. The current research is mainly on the cytotoxicity of cerium oxide nanomaterials (CNPs), which can kill tumor cells, restrict tumor invasion, and increase tumor cells to radiotherapy. Sensitivity. Some polymers can be anchored to the surface of cerium oxide, increasing the biological properties of cerium oxide, improving the ability to kill tumor cells, and not affecting normal cells and tissues. But the above study is the biology produced under the action of high concentration of cerium oxide nanomaterials (CNPs) (> 10 mu g/ml). The effect, mainly the experimental results in animals, is too high for the human body, which may produce some side effects and is difficult to apply to the clinic. However, the low concentration of cerium oxide nanomaterial (CNPs) (less than 1 mu g/ml) affects the tumor cells, including the proliferation, invasion and metastasis of tumor cells and so on. In this study, we found that the stability of cerium oxide nanomaterials can be increased by anchoring PEG600 on the surface of ceria nanomaterials by Allen phosphate, and the low concentration (0.01 mu g/ml) of CNPs-AL-PEG600 can promote the proliferation of hepatoma cells by activating the AKT/ERK signaling pathway and the treatment of tumors for cerium oxide nanomaterials. Growth provides new and meaningful information. Objective: 1, low concentration cerium oxide nanomaterials (CNPs) can promote the proliferation of hepatoma cells; 2, low concentration cerium oxide nanomaterials (CNPs) promote the proliferation of hepatoma cells by activating AKT/ERK signaling pathway. Methods: 1, cell culture: Hep G2, Huh77721, frozen in the liquid nitrogen tank in our laboratory HCT116, MCF-7 and U2OS resuscitation, after resuscitation, DMEM in the cell culture box containing 10% fetal bovine serum was cultured at 37, 5%CO2 cell culture box; 2, cell proliferation experiment: CCK-8 method was used to detect cell proliferation. The six tumor cells of the logarithmic growth period Hep G2, Huh77721, HCT116, MCF-7, and U2OS were prepared into 1 x cell suspensions, and each cell was divided into six groups The nanomaterials with a concentration of 0,0.005,0.01,0.05,0.1,1 mu g/ml were inoculated in 96 Kong Banzhong, respectively, and 96 holes were taken out after 24 hours of.24 hours in the cell culture box of 5%CO2 at 37 degrees C. The CCK-8 reagent 10 micron l was added to each hole for 2 hours, and the absorbance (OD) of the pores in 450nm was detected by the enzyme labeling instrument, and the average value was measured for 3 times, and 3, real. The fluorescence quantitative PCR was used to detect the gene Bcl-2 and the expression of Bax m RNA: the hepatocellular carcinoma cell Hep G2, Huh77721 was treated with the low concentration of cerium oxide nanomaterial (CNPs) for 24 hours, and the cell total RNA was extracted by Trizol method, and the concentration was measured by the nucleic acid concentration measuring instrument. PCR reaction liquid is prepared according to the component, Q RT-PCR reaction, internal reference is GAPDH, Bcl-2 gene upstream primers: ATCg CCCTg Tgg ATg ACTg Ag T, upstream primers are primers: 95 degrees, 95 degrees, 60 degrees, 7; 2 degrees, 30s, repeated 40 times. Finally, statistical analysis was carried out; 4, gene microarray analysis: Hep G2 cells were inoculated into 6 orifice plates and divided into 2 groups, each group was treated with no cerium oxide nanomaterials and the experimental group treated with low concentration (0.01 mu g/ml) ceria nanomaterials, at 37, 5%CO2 cell culture box culture. After 24 hours, 6 holes were flushed with PBS. After each cell was treated with Trizol, the total RNA was extracted and transferred to the EP tube and stored in -80 C refrigerator. The specimens were sent to Shanghai its Ming Information Technology Co., Ltd. for testing; 5, gene analysis and signal pathway analysis were completed by Shanghai's Mingxin Technology Co., Ltd.; 6, immunoblotting experiment: the concentration of 0 .01 mu g/ml cerium oxide nanomaterials were incubated with Hep G2 of liver cancer cells, and the protein of 0.5h, 1H, 2h was incubated respectively. The proteins extracted from Hep G2 stimulated by ceria nanomaterials were compared. The expression of AKT, P AKT, and the expression of the antibody at different time periods were detected respectively, and 7, subcutaneous tumor formation experiment in nude mice: 10 6-7 week old nude mice were randomly divided into 2 groups and weighed in 2 groups. The 1 group was the control group and the 1 group was the experimental group. 1 x 106Hep G2 cells were suspended in 100 mu L PBS, and then the right groin of two groups of nude mice was injected subcutaneously. At the beginning of the day, the dose of cerium oxide nanomaterials (CNPs) in the peritoneal cavity of the experimental group nude mice were injected every other day, and the control group was compared to the control group every other day. The group was sacrificed to the nude mice after PBS.30 days of the same dose, and the weight of the tumor was weighed. 8, the statistical analysis was made by means of mean standard deviation. The statistical analysis was made with the Graph Pad Prism5 software. The difference of the difference between the two groups was compared with the t test. The difference test level was set as P0.05. results: 1, the low concentration cerium oxide nanomaterial (CNPs). Promote the proliferation of hepatoma cells; 2, low concentration cerium oxide nanomaterial (CNPs) reduces the apoptosis of hepatoma cells; 3, low concentration cerium oxide nanomaterial (CNPs) promotes the proliferation of hepatoma cells by activating the AKT/ERK signal pathway; 4, low concentration of cerium oxide nano material (CNPs) promotes tumor growth in the body. Conclusion: 1, the concentration is 0.01 um by CCK-8 test. The CNPs-AL-PEG600 of g/ml promoted the proliferation of hepatoma cells. 2, the apoptosis related gene Bcl-2/Bax ratio in the CNPs-AL-PEG600 experimental group was higher than that in the control group, indicating that CNPs-AL-PEG600 promoted the proliferation of hepatoma cells by reducing the apoptosis pathway; 3, gene chip analysis, CNPs-AL-PEG600 may promote the liver cancer by activating the AKT/ERK signaling pathway. Cell proliferation; 4, WB experiments confirmed that CNPs-AL-PEG600 promoted the proliferation of hepatoma cells by activating the AKT/ERK signaling pathway; 5, in mice, the experiment confirmed that the CNPs-AL-PEG600 concentration of 0.01mg/kg promoted the growth of the tumor.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7

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