【作者】 刘晓梅；

【导师】 孙志伟；

【作者基本信息】 吉林大学 ， 放射医学， 2007， 博士

【摘要】 氯化镉具有明显的抗肿瘤作用及良好的临床应用前景,但是其作用机制目前还不清楚。本研究在以往研究的基础上,以体外培养的人肝癌细胞株SMMC-7721为模型,采用形态学、生物学、免疫细胞化学及分子生物学方法,研究氯化镉诱导细胞线粒体损伤及细胞凋亡。结果表明:一定剂量的氯化镉可以抑制SMMC-7721细胞生长,存在着时间-效应、剂量-效应关系;氯化镉可以降低SMMC-7721细胞SOD、GSH-px活性,增加MDA的含量;氯化镉可以使线粒体膜电位降低,线粒体钙离子转移能力下降,损伤线粒体DNA;氯化镉还可以使bax mRNA表达上调、bcl-2 mRNA表达下降,凋亡相关蛋白Bax、Bcl-2、Caspase-9和Caspase-3表达增强,Bax/Bcl-2比值增加,胞核中AIF随剂量增加阳性表达增强,胞质中相关蛋白AIF和CytC表达增加。结论:氯化镉可以通过氧化应激造成线粒体的损伤;氯化镉可以通过Bax、Bcl-2 mRNA及蛋白比值的增加、膜通透性的改变,导致CytC、AIF从线粒体中释放出来,从而激活Caspase-9,进一步激活Caspase-3,导致线粒体通路的细胞凋亡。提示:线粒体是氯化镉致SMMC-7721细胞凋亡的重要靶点。本研究为研发高效低毒的治疗肝癌的药物及其治疗靶点提供理论依据。更多还原

【Abstract】 Cadmium chloride has a significant anti-cancer effect with a promising clinic therapy perspective; however, the mechanism is still unclear. In this study, the methods of morphology, biology, immuocytochemistry and molecular biology were used to detect mitochondria damage and cell apoptosis in SMMC-7721 cell lines induced by cadmium chloride in vitro to provide theoretical basis to develop medicines for hepatoma without significant side-effect and investigate its precise effect targets as well.1. Effects of cadmium chloride on survival rate of SMMC-7721 SMMC-7721 cells were exposed to cadmium chloride and observed by light inverted microscopy. In control group, cultured cells were in a good condition: grew to polygon with excellent refraction ability; while cells in administration group showed shrink, bad refraction, and more dead cells in culture medium.SMMC-7721 cells were exposed to cadmium chloride at 10, 20, 30 and 40μmol/L, and MTT assay was applied to observe the inhibitory effects of CdCl2 at 12, 24and 48 h. The results showed that the inhibitory rate of SMMC-7721 cells increased significantly with the increasing dosage and time of exposure compared with control group (P<0.01). There exists an obvious time-effect and dose-effect relationship.2. Effects of cadmium chloride on oxidase damage of SMMC-7721 Biochemical kits were used to study activities of SOD, GSH-px and content of MDA in SMMC-7721 cells 24 h after 10, 20, 30 and 40μmol/L of cadmium chloride treatment. The results showed that the activity of SOD wasdecreased, and it had a significant changes between CdC12 administration groupsand control group (P<0.05, P<0.01); the content of MDA was increasedsignificantly at different exposed groups compared with control group(P<0.01); The activity of GSH-px in all exposed groups were decreasedsignificantly compared with control group (P<0.01).3. Effects of cadmium chloride on mitochondria of SMMC-7721SMMC-7721 cells were treated with 10, 20, 30 and 40 ?mol/L cadmiumchloride for 24 h and then stained with Regand. The staining of mitochondriabecame light and the amount of cells was decrease with increasing dosage.The mitochondrion with Rhodamine 123 probes staining was detected byFCM. The results showed the content of the characteristic light fluorescence wasincreased and△Φm was decreased with increasing dosage. There was asignificant difference between the CdC12 groups and control group (P<0.01).Mitochondria were extracted from SMMC-7721 treated with CdC12 for 24 h. Spectrophotography was used to measure the transport ability of Ca2+ into mitochondria. The results showed that the ability was decreased with increasing dosage.Mitochondria DNA damage was observed with agarose gel electrophoresisin all administration groups. The result showed cadmium could causemitochondrial DNA damage.4. Effects of cadmium chloride on apoptosis of SMMC-7721SMMC-7721 cells were treated with 10, 20, 30 and 40 ?mol/L cadmiumchloride. Morphological features of apoptosis were stained by Giemsa, andapoptotic changes were observed by light microscopy. The results showedCdC12 could induce typical apoptosis: cell shrinkage, blebbing and chromatincondensation. The statistical analysis showed that the percentage of apoptoticcells was increased significantly at all exposed groups compared with control group (P<0.01); and there were significantly differences among the exposuregroups (P<0.01) with dose-time-effect relationship.AO/EB double fluorescent staining assay was used to observe cellapoptosis. The results showed the apoptotic cells appeared in all the CdClzgroups. Normal cell had a regular nuclear with green fluorimetric stain, whileapoptotic cell had unregular nuclear with yellowish green stain, and the capacityof cell became smaller. The amount of cell was decrease with increasing dosage,and necrosis cell appeared with red stained. Cells of 40 ?mol/L group werealmost dead.Apoptosis was measured by AnnexinV-FITC/PI double staining usingflow cytometry. The results showed that the percentage of apoptotic cells wasincreased significantly with increasing dosage (P<0.01), and there weresignificantly differences among the exposure groups (P<0.01).Diphenylamine spectrophotography was used to observe the rate of DNAfragmentation. The results showed the rate of DNA fragmentations in allexposed groups were increased compared with control group (P<0.01), andthere were significantly differences among the exposure groups (P<0.01).5. Changes in expression of Bax, Bel-2, Caspase-9, Caspase-3, CytC andAIF of SMMC-7721SMMC-7721 cells were treated with cadmium chloride for 24 h withdifferent concerntrations. Immunocytochemistry assay was used to observe thechanges of the Bax, Bcl-2, Caspase-9, Caspase-3, CytC and AIF proteinexpressions in SMMC-7721. Results showed that Bax expression was increasedwhile Bcl-2 expression was decreased with the increasing dosage. Caspase-9and Caspase-3 expressions in SMMC-7721 cells were increased significantlycompared with control group (P<0.01). CytC protein expression in theendochylema protein was increased significantly with the increasing of doses.AIF protein expression was increased significantly in the cellular nucleus withthe increasing dosage 6. Changes in mRNA level of bax and bcl-2 of SMMC-7721RT-PCR assay was used to measure the mRNA level of bax and bcl-2.Results showed that bcl-2 mRNA level was increased at first, then decreasedwith the increasing dosage, bax mRNA level was increased with the increasingdosage. Ratio of bax and bcl-2 was increased with the increasing dosage.7. Changes in protein expression of Bax, Bcl-2, CytC and AIF ofSMMC-7721The westem blot assay was used to detect the Bax and Bcl-2 expression oftotal protein in SMMC-7721 cells treated with CdCl2 for 24 h. Results showedthat the Bax and Bcl-2 expression and Ratio of Bax/Bcl-2 all increased with theincreasing of Cd.CytC both in the total protein and the endochylema protein of SMMC-7721cells treated with CdC12 for 24 h. Results showed that the content of CytC hadno significantly changes in total protein, while had an ncreasing trend with theincreasing dosage in endochylema.AIF both in the total protein and the endochylema protein of SMMC-7721cells treated with CdC12 for 24 h. Results showed that the content of AIF had nosignificantly changes in total protein, while had a increasing trend with theincreasing dosage of CdC12 in endochylema.更多还原