本文選題：宮頸腫瘤　切入點：-羥基-’-硝基--丙酰氧基異黃酮　出處：《腫瘤》2017年09期 　論文類型：期刊論文

【摘要】：目的:研究新型金雀異黃素衍生物5-羥基-4’-硝基-7-丙酰氧基異黃酮(5-hydroxy-4’-nitro-7-propionyloxy-isoflavone,HNPI)體外對人宮頸癌HeLa細胞增殖抑制及誘導凋亡的作用,并探討可能的分子生物學機制。方法:采用不同濃度的HNPI(5、10、20、40、60和80μmol/L)處理HeLa細胞48 h,MTT檢測HeLa細胞的增殖抑制率。原子力顯微鏡下觀測HeLa細胞表面形貌、細胞高度、細胞寬度、細胞均方根粗糙度(rootmean-squared roughness,Rq)及細胞平均粗糙度(average roughness,Ra)的變化。采用DAPI熒光染色法在激光掃描共聚焦顯微鏡下觀察HeLa細胞核形態(tài)的改變。FCM檢測HeLa細胞凋亡率、細胞周期、活性氧的生成及線粒體膜電位的變化。結(jié)果 :MTT法檢測結(jié)果顯示,當HNPI濃度10μmol/L后,細胞的增殖抑制率呈濃度依賴性增高,差異均有統(tǒng)計學意義(P值均0.05);HNPI對HeLa細胞的半數(shù)抑制濃度為46.83μmol/L。濃度為20和60μmol/L的HNPI處理HeLa細胞48 h后,原子力顯微鏡納米級超高分辨率成像結(jié)果顯示,細胞皺縮變圓,細胞質(zhì)發(fā)生濃縮,細胞核隆起,細胞膜表面的粗糙度降低,變得平整光滑,絲狀偽足減少、稀疏、變短、萎縮,甚至完全破壞;膜超微結(jié)構(gòu)成像結(jié)果顯示,HeLa細胞膜表面超微結(jié)構(gòu)突起稀疏,分布平整單一,匯合成較大的突起和隆起;對單細胞的定量分析結(jié)果顯示,細胞的高度呈濃度依賴性增加,而細胞的寬度、Rq和Ra均呈濃度依賴性降低,與0.9%氯化鈉溶液對照組比較,差異均有統(tǒng)計學意義(P值均0.05)。DAPI法檢測結(jié)果顯示,細胞核發(fā)生固縮、染色體聚集、凋亡小體形成。FCM法檢測結(jié)果顯示,HNPI可呈依賴性誘導HeLa細胞發(fā)生凋亡并阻滯HeLa細胞于G_0/G_1期,與0.9%氯化鈉溶液對照組比較,差異均有統(tǒng)計學意義(P值均0.05);HeLa細胞內(nèi)活性氧水平呈濃度依賴性升高,而線粒體膜電位呈濃度依賴性降低,與對照組比較,差異均有統(tǒng)計學意義(P值均0.05)。結(jié)論 :HNPI在體外可顯著抑制HeLa細胞增殖并誘導凋亡,其機制可能與HNPI改變細胞超微結(jié)構(gòu),引起細胞損傷,使細胞內(nèi)活性氧蓄積和線粒體膜電位降低,使細胞周期阻滯于G_0/G_1期進而發(fā)生凋亡相關。
[Abstract]:Aim: to study the inhibitory effect of a novel genistein derivative 5-hydroxy-4- nitro-7-propionyloxy-isoflavone HNPI on the proliferation and apoptosis of human cervical cancer HeLa cells in vitro, and to investigate the effects of 5-hydroxy-4-nitro-7-propionyloxy-isoflavone HNPI on the proliferation and apoptosis of human cervical cancer HeLa cells in vitro. Methods: HeLa cells were treated with different concentrations of HNPI51010C20C4060 and 80 渭 mol / L for 48 h to determine the inhibitory rate of HeLa proliferation. The surface morphology, cell height and cell width of HeLa cells were observed under atomic force microscope. The changes of root-squared roughnessen (RQ) and cell average average roughnessRa(). DAPI fluorescence staining was used to observe the morphologic changes of HeLa nuclei under laser scanning confocal microscope. FCM was used to detect the apoptosis rate and cell cycle of HeLa cells. Results the cell proliferation inhibition rate was increased in a dose-dependent manner when the concentration of HNPI was 10 渭 mol/L. The difference was statistically significant (P < 0.05). The median inhibitory concentration of HNPI on HeLa cells was 46.83 渭 mol / L. After 48 h treatment of HeLa cells with 20 and 60 渭 mol/L HNPI, the results of ultrahigh resolution atomic force microscopy (AFM) showed that the cells shrank and became round. The cytoplasm is concentrated, the nucleus bulges, the surface roughness of cell membrane is reduced, the surface of cell membrane becomes smooth and smooth, the filamentous pseudopodia is reduced, sparse, shorter, atrophic, even completely destroyed. The ultrastructural imaging results showed that the ultrastructural processes on the surface of HeLa cell membrane were sparse, distributed flat and single, and synthesized large protrusions and bulges, and the quantitative analysis of single cells showed that the cell height was increased in a concentration dependent manner. The width of RQ and Ra decreased in a concentration-dependent manner. Compared with the control group of 0.9% sodium chloride solution, the difference was statistically significant (P < 0.05). The results of DAPI method showed that the nuclei were pyknosis and chromosome aggregation. The results of apoptotic corpuscle formation. FCM assay showed that HNPI could induce apoptosis of HeLa cells in a dependent manner and block HeLa cells in the G _ 0 / G _ 1 phase, compared with the control group of 0.9% sodium chloride solution. The difference was statistically significant (P < 0.05). The level of reactive oxygen species in HeLa cells was increased in a concentration-dependent manner, while the mitochondrial membrane potential was decreased in a concentration-dependent manner, compared with the control group. Conclusion the proliferation and apoptosis of HeLa cells can be significantly inhibited and apoptosis induced by HNPI in vitro, and the mechanism may be similar to that of HNPI, which may change the ultrastructure of the cells and cause cell damage. The accumulation of reactive oxygen species in cells and mitochondrial membrane potential were decreased, and the cell cycle was blocked in the first phase of G _ (0) / G _ (1), which was related to apoptosis.
【作者單位】： 湖南省婦幼保健院婦一科;
【分類號】：R737.33

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