本文選題：大氣壓低溫等離子體 + 質(zhì)粒構象�。� 參考：《華中科技大學》2011年博士論文

【摘要】：近年來,等離子體在生物醫(yī)學上的應用研究成為國內(nèi)外的熱點,等離子體在殺菌、控制腫瘤細胞增殖和促進凝血等方面已取得很好的效果。但至今人們對等離子體的研究大多數(shù)都集中在新型等離子體產(chǎn)生裝置的設計上,而對等離子體的生物活性機制的研究幾乎是空白。本文從細胞生物學和生物化學角度對等離子體誘導腫瘤細胞凋亡的作用機制進行了詳盡的研究,具體研究內(nèi)容和研究結果概括如下： 1.研究了等離子體射流裝置的放電特性,優(yōu)化了等離子體的產(chǎn)生條件和處理參數(shù)。并對等離子體進行了光譜診斷。在此基礎上,以質(zhì)粒pAHC25 DNA為研究對象,在經(jīng)過等離子體處理后,發(fā)現(xiàn)等離子體能夠破壞DNA雙鏈結構,從而改變質(zhì)粒DNA構象,超螺旋構象的質(zhì)粒逐漸減少,而線性和開環(huán)構象的質(zhì)粒逐漸增加；經(jīng)純化后對不同構象的質(zhì)粒所攜帶的基因進行PCR擴增檢測,結果顯示等離子體的處理不會明顯造成質(zhì)粒上所攜帶的基因的缺失。 2.采用MTT分析方法研究等離子體對肝癌細胞(HepG2)、黑素瘤細胞(B16)、前列腺癌細胞(PC3)、乳腺癌細胞(MDA-MB-231)和人正常肝臟細胞(L02)增殖的作用,發(fā)現(xiàn)等離子體處理對幾種腫瘤細胞系增殖都有明顯的抑制作用,但對正常細胞的作用比相應的腫瘤細胞作用要弱。等離子體處理對每種細胞系的EC 50值存在明顯的差異。 3.以HepG2細胞為主要研究對象,通過Hoechst 33342染色和AnnexinⅤ/PI雙染的方法觀察了等離子體處理對細胞凋亡的影響；采用DCFH探針和Griess比色法法分別檢測了細胞內(nèi)ROS水平和NO含量的變化；使用JC-1探針檢測細胞線粒體膜電位變化；在此基礎上進一步檢測分析caspase 3、8、9的酶活,并通過RT-PCR和Western-blot方法,從轉(zhuǎn)錄和翻譯水平分析Bcl-2/Bax表達的變化。結果表明,HepG2細胞經(jīng)等離子體處理后,細胞膜沒有受到明顯的損傷,細胞發(fā)生皺縮,AnnexinⅤ/PI和Hoechst 33342染色后熒光陽性細胞明顯增加,細胞內(nèi)線粒體凋亡通路被激活,細胞內(nèi)NO和ROS水平增高,caspase 3、9的活性明顯變強(caspase 8活性沒有明顯變化),對Bcl-2有明顯的下調(diào)作用,而上調(diào)了Bax水平,最終導致腫瘤細胞凋亡。 4.通過PI染色細胞、流式細胞儀觀察各個周期細胞的分布情況；采用RT-PCR和Western-blot的方法檢測細胞周期相關基因、蛋白的表達。結果表明,等離子體處理可以使HepG2細胞周期阻滯在G2/M期,同時伴隨有細胞凋亡的產(chǎn)生,并在mRNA水平和蛋白質(zhì)水平下調(diào)CDC2和cyclin B1水平,而上調(diào)p21和p53水平,表明等離子體通過降低細胞內(nèi)的CDC2和cyclin B1的表達水平使細胞周期阻滯在G2/M期,同時這個過程也受到p21和p53的調(diào)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，

本文編號：1934374