大气压低温等离子体抑制HepG2细胞增殖的机制研究

发布时间：2018-05-25 19:21

  本文选题：大气压低温等离子体 + 质粒构象　； 参考：《华中科技大学》2011年博士论文

【摘要】：近年来,等离子体在生物医学上的应用研究成为国内外的热点,等离子体在杀菌、控制肿瘤细胞增殖和促进凝血等方面已取得很好的效果。但至今人们对等离子体的研究大多数都集中在新型等离子体产生装置的设计上,而对等离子体的生物活性机制的研究几乎是空白。本文从细胞生物学和生物化学角度对等离子体诱导肿瘤细胞凋亡的作用机制进行了详尽的研究,具体研究内容和研究结果概括如下： 1.研究了等离子体射流装置的放电特性,优化了等离子体的产生条件和处理参数。并对等离子体进行了光谱诊断。在此基础上,以质粒pAHC25 DNA为研究对象,在经过等离子体处理后,发现等离子体能够破坏DNA双链结构,从而改变质粒DNA构象,超螺旋构象的质粒逐渐减少,而线性和开环构象的质粒逐渐增加；经纯化后对不同构象的质粒所携带的基因进行PCR扩增检测,结果显示等离子体的处理不会明显造成质粒上所携带的基因的缺失。 2.采用MTT分析方法研究等离子体对肝癌细胞(HepG2)、黑素瘤细胞(B16)、前列腺癌细胞(PC3)、乳腺癌细胞(MDA-MB-231)和人正常肝脏细胞(L02)增殖的作用,发现等离子体处理对几种肿瘤细胞系增殖都有明显的抑制作用,但对正常细胞的作用比相应的肿瘤细胞作用要弱。等离子体处理对每种细胞系的EC 50值存在明显的差异。 3.以HepG2细胞为主要研究对象,通过Hoechst 33342染色和AnnexinⅤ/PI双染的方法观察了等离子体处理对细胞凋亡的影响；采用DCFH探针和Griess比色法法分别检测了细胞内ROS水平和NO含量的变化；使用JC-1探针检测细胞线粒体膜电位变化；在此基础上进一步检测分析caspase 3、8、9的酶活,并通过RT-PCR和Western-blot方法,从转录和翻译水平分析Bcl-2/Bax表达的变化。结果表明,HepG2细胞经等离子体处理后,细胞膜没有受到明显的损伤,细胞发生皱缩,AnnexinⅤ/PI和Hoechst 33342染色后荧光阳性细胞明显增加,细胞内线粒体凋亡通路被激活,细胞内NO和ROS水平增高,caspase 3、9的活性明显变强(caspase 8活性没有明显变化),对Bcl-2有明显的下调作用,而上调了Bax水平,最终导致肿瘤细胞凋亡。 4.通过PI染色细胞、流式细胞仪观察各个周期细胞的分布情况；采用RT-PCR和Western-blot的方法检测细胞周期相关基因、蛋白的表达。结果表明,等离子体处理可以使HepG2细胞周期阻滞在G2/M期,同时伴随有细胞凋亡的产生,并在mRNA水平和蛋白质水平下调CDC2和cyclin B1水平,而上调p21和p53水平,表明等离子体通过降低细胞内的CDC2和cyclin B1的表达水平使细胞周期阻滞在G2/M期,同时这个过程也受到p21和p53的调控。
[Abstract]:In recent years, the application of plasma in biomedical research has become a hot spot at home and abroad. Plasma has achieved good results in sterilization, tumor cell proliferation control and blood coagulation. But up to now, most of the researches on plasma are focused on the design of new plasma generator, but the research on the mechanism of plasma bioactivity is almost blank. In this paper, the mechanism of plasma-induced tumor cell apoptosis was studied in detail from the point of view of cell biology and biochemistry. The specific research contents and results are summarized as follows: 1. The discharge characteristics of the plasma jet device are studied, and the generation conditions and treatment parameters of the plasma are optimized. The spectrum diagnosis of the plasma was carried out. On this basis, the plasmids pAHC25 DNA were studied. After plasma treatment, it was found that plasma could destroy the double strand structure of DNA, thus changing the conformation of plasmid DNA and decreasing the superhelix conformation. The linear and open-ring conformation plasmids were gradually increased, and the genes carried by different conformation plasmids were detected by PCR amplification after purification. The results showed that the plasma-treated plasmids did not cause the deletion of the genes carried on the plasmids. 2. The effects of plasma on the proliferation of hepatoma cell line HepG2, melanoma cell line B16, prostate cancer cell line PC3, breast cancer cell line MDA-MB-231) and human normal liver cell line L02) were studied by MTT assay. It was found that plasma treatment could significantly inhibit the proliferation of several tumor cell lines, but the effect on normal cells was weaker than that on corresponding tumor cells. The EC50 values of each cell line were significantly different by plasma treatment. 3. The effect of plasma treatment on apoptosis of HepG2 cells was observed by means of Hoechst 33342 staining and Annexin 鈪，

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