本文選題：蓮房原花青素 + 氧化應(yīng)激��； 參考：《江蘇大學(xué)》2017年碩士論文

【摘要】：肝癌是我國最常見的惡性腫瘤之一,其死亡率位居全球第三,且仍呈上升趨勢,每年新增約60萬病例,依然嚴(yán)重威脅人類健康。大量研究報(bào)道已證實(shí)氧化應(yīng)激可作為一種有效的抗腫瘤手段,能夠通過多途徑多靶點(diǎn)誘導(dǎo)細(xì)胞死亡。本課題組發(fā)現(xiàn)蓮房原花青素(lotus seedpod procyanidins,LSPCs)具有抗氧化、抗輻射、抗腫瘤等多種生物活性。前期研究證實(shí),LSPCs能夠刺激人肝癌HepG2細(xì)胞產(chǎn)生大量活性氧(reactive oxygen species,ROS),誘導(dǎo)細(xì)胞氧化應(yīng)激損傷。但并未對ROS種類、作用途徑及機(jī)制進(jìn)行深入研究。基于此,本論文致力于研究LSPCs通過ROS積蓄介導(dǎo)HepG2細(xì)胞氧化應(yīng)激對線粒體功能、細(xì)胞內(nèi)鈣穩(wěn)態(tài)以及胞內(nèi)蛋白質(zhì)組的影響,力求闡明LSPCs通過誘導(dǎo)HepG2細(xì)胞氧化應(yīng)激的抗腫瘤機(jī)制。具體研究內(nèi)容和結(jié)果如下:1.LSPCs誘導(dǎo)ROS積蓄介導(dǎo)HepG2細(xì)胞氧化應(yīng)激對線粒體功能的影響(1)ROS的種類、分布和來源。利用H_2O_2敏感的熒光探針和O·2-的特異性熒光探針、線粒體特異性熒光探針以及線粒體呼吸鏈復(fù)合物(MRCC)抑制劑檢測給予LSPCs處理前后,胞內(nèi)ROS的種類、分布和主要來源。結(jié)果表明,LSPCs誘導(dǎo)HepG2細(xì)胞產(chǎn)生ROS的種類并非是單一的,至少存在H_2O_2和O·2-兩種,且ROS主要來源于MRCC I和III。(2)LSPCs對線粒體主要特性的影響。經(jīng)25、50和100μg/m L LSPCs處理HepG2細(xì)胞12 h后,線粒體膜電位水平、胞內(nèi)ATP水平分別下降到對照組的0.86-0.49倍和0.83-0.45倍;MPTP開放度增加到對照組的0.52-0.87倍;顯著降低了線粒體內(nèi)GSH、SOD、SDH、CAT四種抗氧化酶的活性,且呈現(xiàn)劑量依賴關(guān)系,四種酶活力分別下降到對照組的0.91-0.57倍、0.77-0.40倍、0.81-0.35倍和0.83-0.30倍;MDA含量升高到對照組的1.24-4.16倍;線粒體內(nèi)Ca~(2+)含量是對照組的1.21倍;MRCC I和III酶活性分別下降到對照組的0.80-0.37倍和0.78-0.33倍;MRCC I(subunit NDUFS1)和MRCC III(subunit UQCRC1)的蛋白表達(dá)量分別下降到對照組的0.84-0.37倍和0.86-0.35倍;Cyt-c釋放至胞漿的量逐漸增加。經(jīng)ROS抑制劑(NAC)預(yù)處理后,上述所有指標(biāo)均得到不同程度的改善。以上表明,LSPCs通過誘導(dǎo)HepG2細(xì)胞積蓄ROS嚴(yán)重?fù)p傷線粒體功能和特性,而NAC能夠有效改善上述現(xiàn)象,減輕線粒體損傷。2.LSPCs誘導(dǎo)ROS積蓄介導(dǎo)HepG2細(xì)胞氧化應(yīng)激對細(xì)胞鈣穩(wěn)態(tài)的影響經(jīng)50μg/m L LSPCs處理HepG2細(xì)胞12 h后,ROS水平達(dá)到最大值,熒光強(qiáng)度是對照組的1.22倍;處理24 h后,Ca~(2+)水平達(dá)到最大值,熒光強(qiáng)度是對照組的1.39倍,而且升高的Ca~(2+)是胞內(nèi)鈣庫釋放和胞外鈣內(nèi)流共同作用的結(jié)果。經(jīng)25、50、75、100和150μg/m L LSPCs處理HepG2細(xì)胞1、3、6、12、24和36 h后,ROS和Ca~(2+)相關(guān)系數(shù)r分別是0.862、0.937、0.968、0.940和0.931。以上表明,LSPCs能誘導(dǎo)HepG2細(xì)胞內(nèi)ROS積蓄和Ca~(2+)超載,而且兩者之間存在顯著的正相關(guān)性。3.LSPCs誘導(dǎo)ROS積蓄介導(dǎo)HepG2細(xì)胞氧化應(yīng)激對細(xì)胞蛋白質(zhì)組的影響采用蛋白質(zhì)組學(xué)技術(shù)對LSPCs處理HepG2細(xì)胞前后總蛋白表達(dá)譜進(jìn)行分析及鑒定。結(jié)果表明,LSPCs處理前后共鑒定出22個(gè)顯著差異表達(dá)蛋白,其中,17個(gè)蛋白為下調(diào)蛋白,5個(gè)蛋白在LSPCs處理組消失。這些蛋白主要與應(yīng)激反應(yīng)、氧化損傷、細(xì)胞骨架、細(xì)胞凋亡、遷移、細(xì)胞增殖分化、信號(hào)傳導(dǎo)等功能相關(guān)。因而推測LSPCs誘導(dǎo)HepG2細(xì)胞氧化應(yīng)激可能是通過抑制這些蛋白的表達(dá)水平而達(dá)到抑制腫瘤細(xì)胞增殖的目的。
[Abstract]:Liver cancer is one of the most common malignant tumors in China. Its mortality rate ranks third in the world, and it is still on the rise. About 600 thousand cases are added every year. It is still a serious threat to human health. A large number of research reports have proved that oxidative stress can be used as an effective anti-tumor means and can induce cell death through multiple channels and multiple targets. It was found that lotus seedpod procyanidins (LSPCs) has many biological activities, such as antioxidant, anti radiation, and anti-tumor. Earlier studies have confirmed that LSPCs can stimulate a large number of reactive oxygen species (reactive oxygen species, ROS) and induce oxidative stress damage in human hepatocellular carcinoma cells, but not to ROS species, the pathway and mechanism. Based on this, this paper aims to study the effect of LSPCs on mitochondrial function, intracellular calcium homeostasis and intracellular protein group through ROS accumulation in HepG2 cells, and to clarify the anti-tumor mechanism of LSPCs by inducing oxidative stress in HepG2 cells. The specific content and results are as follows: 1.LSPCs induced ROS accumulation The effects of oxidative stress on HepG2 cells (1) the species, distribution and origin of ROS, using H_2O_2 sensitive fluorescent probe and O 2- specific fluorescence probe, mitochondrial specific fluorescence probe and mitochondrial respiratory chain complex (MRCC) inhibitors detected by LSPCs treatment, the species, distribution and main of intracellular ROS The results showed that the type of ROS induced by LSPCs in HepG2 cells was not single, at least there were two kinds of H_2O_2 and O. 2-, and ROS mainly originated from the effect of MRCC I and III. (2) LSPCs on the main characteristics of mitochondria. After 25,50 and 100 micron, the level of the mitochondrial membrane potential decreased to the control, respectively. The 0.86-0.49 times and 0.83-0.45 times of the group increased to the 0.52-0.87 times of the control group; the activity of four antioxidant enzymes in the mitochondria, GSH, SOD, SDH, CAT, was significantly reduced, and the activity of the four enzymes was dosed, and the activity of the enzyme decreased to the 0.91-0.57 times of the control group, the 0.77-0.40 times, the 0.81-0.35 and the 0.83-0.30 times, and the content increased to the control. The content of Ca~ (2+) in mitochondria was 1.21 times as high as that of the control group; the activity of MRCC I and III enzyme decreased to 0.80-0.37 times and 0.78-0.33 times of the control group, and the expression of MRCC I (subunit NDUFS1) and the protein expression in the control group decreased respectively to the control group. Increase. After pretreatment with ROS inhibitor (NAC), all of the above indexes were improved in varying degrees. The above indicated that LSPCs could seriously damage the mitochondrial function and properties by inducing the accumulation of ROS in HepG2 cells, and NAC could effectively improve the above phenomenon and reduce the mitochondrial damage.2.LSPCs induced ROS accumulation to mediate the oxidative stress of HepG2 cells to cell calcium. After 50 g/m L LSPCs treatment of HepG2 cell 12 h, the level of ROS reached the maximum, the fluorescence intensity was 1.22 times that of the control group; after 24 h, the level of Ca~ (2+) reached the maximum, the fluorescence intensity was 1.39 times that of the control group, and the elevated Ca~ (2+) was the result of the interaction of intracellular calcium library release and extracellular calcium influx. Through 25,50,75100 and 15 After 0 g/m L LSPCs treatment of HepG2 cells 1,3,6,12,24 and 36 h, ROS and Ca~ (2+) correlation coefficient r are 0.862,0.937,0.968,0.940 and 0.931., which can induce accumulation and overload, and there is a significant positive correlation between them. The effect of the mass group was analyzed and identified by proteomics technology before and after LSPCs treatment of HepG2 cells. The results showed that 22 significant differentially expressed proteins were identified before and after LSPCs treatment, of which 17 proteins were down regulated proteins and 5 proteins disappeared in LSPCs treatment group. These proteins were mainly induced by stress reaction and oxidative damage. The functions of cytoskeleton, cell apoptosis, migration, cell proliferation and differentiation, and signal transduction are related. Therefore, it is presumed that LSPCs induced oxidative stress in HepG2 cells may inhibit the proliferation of tumor cells by inhibiting the expression level of these proteins.

【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R285

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