醫(yī)學(xué)免費(fèi)論文:蝙蝠葛酚性堿誘導(dǎo)多藥耐藥細(xì)胞系K562/MDR1凋亡及逆轉(zhuǎn)耐藥性的研究

醫(yī)學(xué)免費(fèi)論文:蝙蝠葛酚性堿誘導(dǎo)多藥耐藥細(xì)胞系K562/MDR1凋亡及逆轉(zhuǎn)耐藥性的研究

【摘要】 目的： 探討蝙蝠葛酚性堿(phenolic alkaloids from Menispermum dauricum,PAMD)誘導(dǎo)多藥耐藥(multidrug resistance,MDR)細(xì)胞系K562/MDR1凋亡及逆轉(zhuǎn)耐藥性的作用。方法: 四甲基偶氮唑藍(lán)(MTT)比色法檢測(cè)K562/S及K562/MDR1細(xì)胞對(duì)不同濃度PAMD的敏感性,并計(jì)算半數(shù)抑制濃度(IC50)。膜聯(lián)蛋白V (Annexin V)�伯惲蚯杷釤晒馑�(FITC)+碘化丙啶(PI)雙參數(shù)檢測(cè)細(xì)胞凋亡百分率變化,分析在PAMD的作用下,兩種細(xì)胞對(duì)伊馬替尼(IM,STI571)敏感性的變化。結(jié)果: PAMD可誘導(dǎo)兩種細(xì)胞凋亡,其低劑量72 h時(shí)對(duì)K562/S和K562/MDR1細(xì)胞的凋亡率分別為(10.92±1.03)%和(8.12±0.98)%,并可提高伊馬替尼對(duì)K562/MDR1的凋亡率。PAMD可顯著逆轉(zhuǎn)K562/MDR1細(xì)胞對(duì)伊馬替尼的耐藥性,其逆轉(zhuǎn)倍數(shù)為2.22。結(jié)論: PAMD對(duì)K562/S和K562/MDR1細(xì)胞具有誘導(dǎo)凋亡作用,同時(shí)具有逆轉(zhuǎn)白血病細(xì)胞株K562/MDR1多藥耐藥性、回歸靶位的作用。

【關(guān)鍵詞】 蝙蝠葛酚性堿; 慢性粒細(xì)胞白血病; 多藥耐藥細(xì)胞系; 凋亡

Effects of phenolic alkaloids from Menispermum dauricum on inducing

the multidrug resistance cell line K562/MDR1 to apoptosis and reversing

their multidrug resistance

HE Zhi�瞴i, LIU Xiang�瞙ui, GANG Hong�瞝in

(Harbin Food and Drug Inspection Center, Harbin Heilongjiang 150525, China)

[Abstract] Objective: To study the effects of phenolic alkaloids from Menispermum dauricum(PAMD) on inducing the multidrug resistance (MDR) cell line K562/MDR1 to apoptosis and reversing their MDR. Methods: MTT colorimetric assay was employed to detect the sensitivity of K562/S and K562/MDR1 cell lines treated with PAMD of different concentration for 72 h. IC50 values of PAMD were analyzed by MTT assay. The apoptosis rates of the two cell lines were detected by Annexin V/PI to analyze the sensitivity of two cell lines treated with PAMD on imatimib mesylate (IM,STI571). Results: PAMD can induce the apoptosis of the two kinds of cells and the apoptosis rates of the two cell lines were (10.92±1.03)% and (8.12±0.98)% respectively. It was also able to enhance the apoptosis rates of K562/MDR1 on IM. PAMD could reverse MDR of K562/MDR1 on IM and its reversal multiple was 2.22. Conclusion: PAMD could induce K562/S and K562/MDR1 to apoptosis and reverse MDR of K562/MDR1 on IM and recur its target醫(yī).學(xué).全.在.線m.f1411.cn.

[Key words] phenolic alkaloids from Menispermum dauricum(PAMD); chronic myelogenic leukemia; multidrug resistance cell line; apoptosis

多藥耐藥(multidrug resistance,MDR)是指腫瘤細(xì)胞對(duì)一種化療藥物產(chǎn)生耐藥的同時(shí),對(duì)其他結(jié)構(gòu)和作用機(jī)制不同的化療藥物也產(chǎn)生交叉耐藥的現(xiàn)象,它是導(dǎo)致腫瘤化療失敗的原因之一[1]�？朔﨧DR的方法之一是使用逆轉(zhuǎn)劑,一些具有鈣拮抗作用的逆轉(zhuǎn)劑(異搏定、維拉帕米)作用較溫和,能在逆轉(zhuǎn)MDR中提高化療效果,是一類(lèi)具有很好應(yīng)用和開(kāi)發(fā)前景的藥物。本實(shí)驗(yàn)選用具有鈣通道阻滯作用的中藥蝙蝠葛酚性堿(phenolic alkaloids from Menispermum dauricum,PAMD)進(jìn)行逆轉(zhuǎn)耐藥性的研究,以期尋找克服MDR、提高化療療效和對(duì)耐藥腫瘤細(xì)胞進(jìn)行更有效殺傷作用的新型藥物。

1 材料與方法

1.1 材 料

1.1.1 細(xì)胞培養(yǎng) 人紅白血病敏感細(xì)胞株K562/S購(gòu)自中科院上海細(xì)胞所,多藥耐藥細(xì)胞株K562/MDR1由江蘇血液研究所陳子興教授惠贈(zèng)。兩種細(xì)胞于含10%小牛血清、100 U/ml青霉素和100 μg/ml鏈霉素的RPMI1640培養(yǎng)基中,在37 ℃、5%CO2飽和濕度培養(yǎng)箱內(nèi)傳代培養(yǎng)。

1.1.2 主要試劑與儀器 蝙蝠葛酚性堿由黑龍江中醫(yī)藥大學(xué)藥學(xué)院王棟教授提供,RPMI1640培養(yǎng)基(Gibco公司),小牛血清(杭州四季青生物材料工程公司),四甲基偶氮唑藍(lán)(MTT,Sigma公司),二甲基亞砜(DMSO,北京化工廠);酶標(biāo)儀(DG3022,華東電子管廠),EPICS�瞂L型流式細(xì)胞儀(Beckman Coluter公司);膜聯(lián)蛋白V (Annexin V)-異硫氰酸熒光素(FITC)和碘化丙啶(PI)試劑盒購(gòu)于BD Pharmingen公司。

1.2 方 法

1.2.1 流式細(xì)胞術(shù)(FCM)檢測(cè)細(xì)胞的凋亡百分率 取對(duì)數(shù)生長(zhǎng)期的K562/S和K562/MDR1細(xì)胞,調(diào)整細(xì)胞的初濃度,實(shí)驗(yàn)組分別加入PAMD至終濃度為40,20,10,5,2.5,1.25,0.625 mg/L,陰性對(duì)照組加入等體積的RPMI 1640培養(yǎng)基。置37 ℃、5%CO2培養(yǎng)箱培養(yǎng)72 h,收集細(xì)胞,用預(yù)冷的1×PBS洗2次,每管用1×結(jié)合緩沖液把細(xì)胞調(diào)整成1×106濃度,每管取100 μl加入5 μl的AnnexinV�睩ITC溶液進(jìn)行染色,輕輕混勻,再加入5 μl的PI溶液染色,雙染后在室溫避光靜止15 min后每管再加入1×結(jié)合緩沖液400 μl,在1 h內(nèi)上流式細(xì)胞儀進(jìn)行凋亡檢測(cè)。凋亡后繼發(fā)壞死細(xì)胞既有膜生化改變又有膜通透性改變,AnnexinV�睩ITC+,PI+;壞死細(xì)胞呈AnnexinV�睩ITC-,PI+;正常細(xì)胞呈AnnexinV�睩ITC-,PI-;AnnexinV�睩ITC+,PI-為早期凋亡細(xì)胞。每份標(biāo)本設(shè)一個(gè)復(fù)管,重復(fù)1次,計(jì)算凋亡細(xì)胞百分?jǐn)?shù)。

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