CN113045599B - 一种高对比度区分癌细胞/组织的方法及荧光探针的制备 - Google Patents
一种高对比度区分癌细胞/组织的方法及荧光探针的制备 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及荧光探针领域,具体涉及一种高对比度区分癌细胞/组织的方法及荧光探针的制备。
背景技术
肿瘤细胞的快速增殖是一个极其耗能的过程,肿瘤细胞主要以糖酵解方式提供能量,这种产能方式一方面为肿瘤的快速生长提供了大量能量,同时也为癌细胞造成了一个特殊的微环境,如缺氧、细胞内外pH值降低、活性硫(RSS)和活性氧(ROS)水平升高、极性降低等,因此癌细胞特殊的微环境为广谱型肿瘤荧光诊断试剂的设计提供了一个新思路。然而,由于癌细胞和正常细胞的pH和RSS差异不大,开发能精确区分这种差异的肿瘤诊断试剂有较大的难度,因此报道的实例并不多见。相反,癌细胞内本底的活性氧化物(ROS)浓度大约是正常细胞的10倍,因此,利用癌细胞比正常细胞过量表达ROS的特点,可以对癌细胞/组织与正常细胞/组织进行区分,且具有广谱性。
醌氧化还原酶(NQO1)是体内一种重要的Ⅱ相反应酶,主要存在于胞浆和细胞核中,在电子供体NAD(P)H存在下,可以催化醌类物质发生还原反应,继而在氧气的氧化下生成自由基等氧化产物。研究表明,NQO1在多种实体瘤组织中过表达,包括非小细胞肺癌、胰腺癌、乳腺癌、肝癌、胃癌、肾癌、结肠癌、卵巢癌和头颈癌等,其表达水平比正常细胞/组织高5-200倍,因此被认为是治疗多种肿瘤的潜在分子靶标。β-拉帕醌是一种天然的萘醌类化合物,具有独特的醌结构,可以在NQO1的催化下,通过氧化还原循环反应,快速产生大量的活性氧化物(ROS),导致氧化应激,诱导NQO1+的癌细胞程序性坏死。由于异常旺盛的代谢速度,癌细胞内本底的ROS浓度大约是正常细胞的10倍,利用ROS含量的差异,可以实现对二者的区分。尽管如此,活性氧在癌细胞内的含量仍然较低,导致迄今报道的基于细胞内本底的活性氧含量来区分癌细胞和正常细胞的探针对二者的区分度并不明显。受上述研究的启发,我们推测,由于癌细胞/组织NQO1的表达量远大于正常细胞,若用β-拉帕醌处理后,癌细胞/组织产生的ROS的含量将进一步提升。利用该特点,再结合一个对ROS敏感的荧光探针,就可以实现对癌细胞/组织与正常细胞/组织的高对比度区分。
发明内容
基于此,本发明提出了一种基于β-拉帕醌特异性诱发癌细胞中ROS扩增的策略实现癌细胞/组织的高对比度荧光诊断的方法,该方法基于β-lapachone(β-Lap)在癌细胞中过量表达的 NQO1酶的催化下,选择性的放大肿瘤细胞内的ROS水平,再结合本发明开发的高活性氧( hROS)荧光探针PSiR3,从而实现肿瘤细胞/组织的高对比度荧光区分。本发明可以实现对癌细胞、老鼠肿瘤组织和病人肿瘤组织的高对比度诊断。此外,利用本发明提出的PSiR3和 PSiR3/β-Lap双重组合方式,还可以实现对肿瘤细胞/组织和炎性细胞/组织的区分。
实现本发明的技术方案是:
利用荧光探针PSiR3/β-Lap组合实现癌细胞/组织和正常细胞/组织的区分,癌细胞与正常细胞的平均荧光密度比例为15,癌组织与正常组织的平均荧光密度比例为24。
所述的荧光探针的制备方法,步骤如下:
(1)在N2保护下,将4,4'-亚甲基双(3-溴-N,N-二甲基苯胺)溶于干燥的THF中,溶液冷却至-78℃并逐滴加入正丁基锂溶液,反应液在此温度下搅拌反应2小时后,继续加入二氯二烷基硅烷,室温搅拌反应2小时;反应结束后,向上述反应液中加入1N的HCl至反应液呈中性,在旋转蒸发仪上蒸干THF,剩余水溶液用EtOAc萃取,收集有机相,有机相经洗涤、干燥、蒸发后得中间体,将该中间体与KMnO4粉末溶于丙酮中,-15℃下反应2小时,反应液经过滤、蒸发后得到硅氧杂蒽酮化合物;
(2)在N2保护下,将硅氧杂蒽酮化合物溶于干燥的CH3CN中,向该溶液中逐滴滴入Tf2O ,反应液在0℃搅拌反应10分钟,然后继续加入2,4-二甲基-3-乙基吡咯,室温搅拌反应10分钟,减压除去溶剂,所得固体经柱色谱纯化得PSiRs。
所述步骤(1)中二氯二烷基硅烷的结构式为SiR2Cl2,其中R为-CH3、-CH2CH3、-CH(CH3)2或-Ph。
所述步骤(1)中4,4'-亚甲基双(3-溴-N,N-二甲基苯胺)和二氯二烷基硅烷的摩尔比为1:1.8 ,中间体与KMnO4的摩尔比为1:2.5;步骤(2)中Tf2O、硅氧杂蒽酮化合物和2,4-二甲基-3- 乙基吡咯的摩尔比为2.2:1:3。
本发明反应过程如下:
荧光探针PSiRs对ROS的传感机制如下:
本发明的有益效果是:本发明利用β-Lap选择性诱发癌细胞内ROS扩增的策略,再结合本发明合成的荧光探针PSiR3,实现肿瘤细胞/组织的高对比度荧光诊断。癌细胞与正常细胞的平均荧光密度比例为15,癌组织与正常组织的平均荧光密度比例为24,远超出了临床可接受的阈值2.0。重要的是,考虑到炎性细胞本身就具有较高的本底ROS水平,利于本发明提出的 PSiR3和PSiR3/β-Lap双重组合策略还可以对肿瘤组织和炎症组织进行区分。具体操作如下,取相邻的两个待检测的切片,分别用荧光探针PSiR3和PSiR3/β-Lap组合处理,然后在激光共聚焦显微镜上观测上述两个切片的荧光密度,如果出现弱荧光/弱荧光,则判断为正常/良性组织;如果出现弱荧光/强荧光,则判断为癌性组织;如果出现强荧光/强荧光,则判断为炎性组织。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为PSiR1的1H NMR图(600MHz,CDCl3)。
图2为PSiR1的13C NMR图(150MHz,CDCl3)。
图3为PSiR1的HRMS图。
图4为PSiR2的1H NMR图(600MHz,CDCl3)。
图5为PSiR2的13C NMR图(150MHz,CDCl3)。
图6为PSiR2的HRMS图。
图7为PSiR3的1H NMR图(600MHz,CDCl3)。
图8为PSiR3的13C NMR图(150MHz,CDCl3)。
图9为PSiR3的HRMS图。
图10为PSiR4的1H NMR图(600MHz,CDCl3)。
图11为PSiR4的13C NMR图(150MHz,CDCl3)。
图12为PSiR4的HRMS图。
图13为在PBS(50mM,pH=7.4)中PSiR1-4与不同ROS反应的荧光光谱图,包括ClO-(2 μM)、ONOO-(2μM)、HO·(40μM)、NO(40μM)、1O2(40μM)、H2O2(40μM)和O2 ·- (40μM)。λex:620nm;λem:680nm左右;狭缝:5/10nm;电压:700V;T=25℃。
图14为在PBS(50mM,pH=7.4)中PSiR3分别与OCl-(4μM),OH.(40μM)和ONOO- (4μM)反应后680nm处的荧光光谱随时间的变化。
图15(A)为向探针PSiR3(2μM)中加入ClO-(0-2μM)的荧光光谱变化图及PSiR3对ClO-的工作曲线;(B)为向探针PSiR3(2μM)中加入ONOO-(0-2μM)的荧光光谱变化图及PSiR3对ONOO-的工作曲线;(C)为向探针PSiR3(2μM)中加入HO·(0-40μM)的荧光光谱变化图及PSiR3对HO·的工作曲线。
图16为正常细胞(Cos7、HUVEC、BEAS-2B细胞)(A)和癌症细胞(A549、HepG2、 MCF-7细胞)(B)分别用PSiR3(5μM,20min)处理或先用PSiR3(5μM,20min)处理,再用β-Lap(10μM,60min)处理后的荧光共聚焦成像图;(C)为(A)和(B)的平均荧光密度图。发射波长为650–750nm(λex=633nm);标尺:20μm。
图17(A)为A549细胞的荧光共聚焦成像图。(i)空白;(ii)细胞用PSiR3(5μM,20min )处理;(iii)细胞先用PSiR3(5μM,20min)处理,再用β-Lap(10μM,60min)处理;(iv) 细胞先用DIC(40μM,60min)处理,再用PSiR3(5μM,20min)/β-Lap(10μM,60min )处理;(v)细胞先用NAC(1mM,60min)处理,再用PSiR3(5μM,20min)/β-Lap(10μM ,60min)处理。(B)为(A)的平均荧光密度图。发射波长为650–750nm(λex=633nm) ;标尺:20μm。
图18(A)为正常细胞(Cos7和HUVEV细胞)和癌细胞(A549和HepG2细胞)先用PSiR3(5μM,20min)处理,再用β-Lap(10μM,60min)处理后的流式细胞术分析图。(B)为 A549和HepG2细胞先用PSiR3(5μM,20min)处理,再用β-Lap(10μM,60min)或DIC( 40μM,60min)/β-Lap(10μM,60min)处理后的流式细胞术分析图。
图19为A549(A)、HeLa(B)和U89(C)肿瘤鼠模型的肿瘤组织切片和正常右下肢组织切片的荧光共聚焦成像图。组织用PSiR3(5μM,20min)处理;或用PSiR3(5μM,20min )/β-Lap(10μM,60min)组合处理;或先用DIC(40μM,60min)处理,再用PSiR3(5μM ,20min)/β-Lap(10μM,60min)组合处理。(D)为(A–C)的平均荧光密度图。发射波长为650–750nm(λex=633nm);标尺:20μm。
图20为从手术组织获取的肺癌组织/良性肺组织切片(A)和甲状腺癌组织切片/良性甲状腺组织切片(B)分别用PSiR3(5μM,20min)/β-Lap(10μM,60min)组合处理后的荧光共聚焦成像图以及平均荧光密度图。发射波长为650–750nm(λex=633nm);标尺:20μm。
图21为用LPS/IFN-γ处理或不处理的Raw264.7细胞分别用PSiR3(5μM,20min)处理或用PSiR3(5μM,20min)/β-Lap(10μM,60min)组合处理后的荧光共聚焦成像图以及平均荧光密度图。发射波长为650–750nm(λex=633nm);标尺:20μm。
图22(A)为老鼠的正常左下肢组织切片、A549肿瘤组织切片和炎性组织切片分别用PSiR3 (5μM,20min)处理或用PSiR3(5μM,20min)/β-Lap(10μM,60min)组合处理后的荧光共聚焦成像图以及平均荧光密度图;(B)为病人的良性子宫组织切片、恶性肺组织切片和炎症甲状腺组织切片分别用PSiR3(5μM,20min)处理或用PSiR3(5μM,20min)/β-Lap (10μM,60min)组合处理后的荧光共聚焦成像图以及平均荧光密度图。发射波长为650–750 nm(λex=633nm);标尺:20μm。
具体实施方式
下面将结合本发明实施例,对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有付出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例
高活性氧化物(hROS)荧光探针PSiR1-4制备方法为:在N2保护下,将4,4'-亚甲基双( 3-溴-N,N-二甲基苯胺)(6.00g,14.6mmol)溶于干燥的THF(200mL)中,溶液冷却至-78℃并逐滴加入正丁基锂溶液n-BuLi(4当量),反应液在此温度下搅拌反应2小时后,继续加入二氯二烷基硅烷(26.28mmol,1.8当量),室温搅拌反应2小时。反应结束后,向上述反应液中加入1N的HCl至反应液呈中性,在旋转蒸发仪上蒸干THF,剩余水溶液用EtOAc萃取,收集有机相,有机相经洗涤、干燥、蒸发后得中间体,将该中间体与KMnO4粉末(5.75g)溶于丙酮(30mL)中,-15℃下反应2小时,反应液经过滤、蒸发后得到硅氧杂蒽酮化合物,该化合物无需提纯可直接用于下一步反应。在N2保护下,将硅氧杂蒽酮化合物(0.541mmol)溶于干燥的CH3CN中,向该溶液中逐滴滴入Tf2O(200μL,1.188mmol),反应液在0℃搅拌反应 10分钟,然后继续加入2,4-二甲基-3-乙基吡咯(200mg,1.623mmol),室温搅拌反应10分钟,减压除去溶剂,所得固体经柱色谱(MeOH/CH2Cl2=30/1)纯化得PSiRs。
PSiR1:(322mg,90.1%yield).1H NMR(600Hz,CDCl3)δ10.33(s,1H),7.63(d,J=9.6Hz, 2H),6.96(d,J=3.0Hz,2H),6.78(dd,J1=3.0Hz,J2=9.6Hz,2H),3.22(s,12H),2.47(s,3H), 2.44(t,J=7.8Hz,2H),1.79(s,3H),1.11(t,J=7.8Hz,3H),0.47(s,6H);13CNMR(150MHz, CDCl3)δ173.9,160.6,157.2,151.8,142.6,137.9,131.1,123.5,115.7,115.5,115.2,98.7,48.4, 17.1,16.5,16.3,15.5;ESI-MS[M]+:calcd for 430.2673,Found 430.2672.
PSiR2:(237mg,63.2%yield).1H NMR(600Hz,CDCl3)δ10.14(s,1H),7.67(d,J=9.6Hz, 2H),6.99(s,2H),6.82(d,J=7.8Hz,2H),3.26(s,12H),2.47(m,5H),1.79(s,3H),1.13(t,J=7.8 Hz,3H),1.00(q,J=6.6Hz,4H),0.93(t,J=6.6Hz,6H);13C NMR(150MHz,CDCl3)δ160.5, 152.5,143.1,141.3,139.5,131.4,129.3,129.2,128.3,127.7,123.9,121.8,119.7,118.5,117.5, 113.6,40.4,31.8,29.3,25.7,23.5,22.6,17.7,15.6,12.4,11.9,7.3,5.7;ESI-MS:[M+]calcd for 458.2986,Found 458.2990.
PSiR3:(229mg,58.4%yield).1H NMR(600Hz,CDCl3)δ10.31(s,1H),7.71(d,J=7.8Hz, 2H),7.04(s,2H),6.88(s,2H),3.28(s,12H),2.47(m,5H),1.78(s,3H),1.31(m,2H),1.13(t,J= 7.8Hz,3H),1.04(d,J=7.2Hz,12H);13C NMR(150MHz,CDCl3)δ175.7,160.6,160.5,152.2, 142.1,129.7,123.9,121.7,119.6,117.5,113.8,113.7,68.2,40.6,39.4,17.7,15.0,14.1,14.0,12.7, 11.9;ESI-MS:[M+]calcd for 486.3299,Found486.3298.
PSiR4:(293mg,67.7%yield).1H NMR(600Hz,CDCl3)δ10.77(s,1H),7.75(d,J=9.0Hz, 2H),7.56(d,J=7.2Hz,4H),7.47(t,J=7.2Hz,2H),7.39(t,J=7.2Hz,4H),6.94(d,J=1.8Hz, 2H),6.88(d,J=9.0Hz,2H),3.12(s,12H),2.54(s,3H),2.43(q,J=7.2Hz,2H),1.71(s,3H), 1.09(t,J=7.2Hz,3H);13C NMR(150MHz,CDCl3)δ159.4,151.9,139.9,139.4,135.9,131.9, 130.5,128.3,119.9,113.8,40.2,17.6,14.7,14.1,13.1,12.3;ESI-MS:[M+]calcd for 554.2986, Found 554.2994.
性能测试
(1)溶液配制
探针PSiR1-4先用乙腈配成2mM的储存液,再根据测试需要用PBS(50mM,pH 7.4)稀释成相应的工作浓度。
β-Lap和DIC用干燥的二甲亚砜配成10mM的储存液。
代表性活性氧化物(ROS)配置如下:O2 ·-溶液是将KO2和18-Crown-6(1当量)溶于二甲基亚砜(DMSO)中制得。HO·是通过Fenton反应(Fe2++H2O2)制得,其浓度等于Fe2+的浓度;1O2溶液是通过次氯酸钠溶液(NaClO)与双氧水溶液(H2O2)的反应制得,其浓度等于 NaClO的浓度。NO是将商业化NO供体NOC-9溶解于0.1M的NaOH中制得。H2O2溶液是通过稀释商业化高浓度双氧水制得,其浓度由240nm处的吸光度值计算确定(摩尔消光系数为43.6 M-1cm-1)。ClO-溶液是通过稀释商业化高浓度NaClO制得,其浓度由292nm处吸光度值计算确定(摩尔消光系数为350M-1cm-1)。ONOO-根据文献中报道的方法制得(R.M.Uppu,W.A.Pryor,Synthesis of peroxynitrite in a two-phase system using isoamyl nitrite andhydrogen peroxide,Anal. Biochem.1996,236,242-249.),其浓度由302nm处吸光度值计算确定(摩尔消光系数为1670 M-1cm-1);进行细胞实验时,ONOO-是将商业化ONOO-供体SIN-1溶解于0.1M的NaOH中制得。
(2)细胞培养和荧光成像
所有细胞系均购自GeneFull生物技术有限公司(中国)。
所有细胞均培养在37℃、含5%的二氧化碳的培养箱中,其中Raw 264.7细胞和Cos-7细胞培养在含有10%的胎牛血清、100U/mL青霉素G钠和100μg/mL链霉素的RPMI 1640培养基中;A549细胞、HepG2细胞、MCF-7细胞、BEAS-2B细胞和HUCEC细胞培养在含有10%的胎牛血清、100U/mL青霉素G钠和100μg/mL链霉素的DMEM(高糖)培养基中;进行细胞影像实验前,预先将细胞置于30mm的玻底细胞培养皿上,静置12小时待细胞贴壁。细胞分别用PSiR3(5μM,20min)处理或者先用PSiR3(5μM,20min)处理,再用β-Lap(10μM,60 min)处理,磷酸缓冲液(PBS)洗涤细胞3次,用Ceiss LMS 710共聚焦显微镜进行荧光成像,收集波长为650-750nm(λex=633nm)。
(3)组织切片成像
BALB/c雄性裸鼠(6-8周龄)购自北京维通利华实验动物技术有限公司,所有的动物实验都是按照山西大学伦理委员会颁布的相关法律和指南进行的。将A549细胞、HeLa细胞或 U89细胞(1×106个细胞)经皮下注射于裸鼠左腋(或左腿)处,接种15天后,获得荷瘤小鼠;向荷瘤鼠右下肢注射LPS(1mg/mL,200μL)诱导发炎。注射LPS15小时后,荷瘤鼠经过度麻醉处死,取五脏、肿瘤、左下肢正常组织及右下肢发炎组织,在冰冻切片机上制成10μm 厚度的切片。病人的良性组织切片、癌组织切片及炎性组织切片均取自山西省肿瘤医院。上述切片分别用PSiR3(5μM,60min)处理或者用PSiR3/β-Lap(5μM/10μM,60min)组合处理,然后在激光共聚焦上观测上述切片的荧光密度,如果出现弱荧光/弱荧光,则判断为正常/良性组织;如果出现弱荧光/强荧光,则判断为癌性组织。
(4)流式细胞分析
将A549细胞、HepG2细胞、Cos7细胞和HUVEC细胞悬浮在1.5mL的PBS中,细胞浓度为1.0×106个/mL。每组细胞分成3份:1、空白组,细胞不做任何处理;2、药物组,细胞用PSiR3/β-Lap(5μM/10μM,60min)组合处理;③抑制组,细胞先用DIC(40μM,60min )处理,再用PSiR3/β-Lap(5μM/10μM,60min)组合处理。细胞经离心、PBS洗涤后,重悬在包含1%血清的PBS中,立即用流式细胞仪分析。
测试结果:
(1)探针的光物理性质
在PBS(50mM,pH=7.4)体系中,分别测试了PSiR1-4与不同ROS(包括ClO-、ONOO-、HO·、NO、1O2、H2O2和O2 ·-)的响应情况。如图13所示(图中如箭头所示,最上面三条曲线从下到上依次为ONOO-、HO·、ClO-),由于吡咯基团对荧光团的PET作用,PSiR1-4具有极低的背景荧光,当向探针中逐渐加入10当量常见的活性氧化物后,包括ClO-、ONOO-、HO·、NO、1O2、H2O2和O2 ·-,PSiR1-4均对ClO-、ONOO-、HO·(hROS)表现出off-on的荧光响应,即680nm左右处的荧光强度明显增强,而其他ROS仅引起轻微的荧光变化。经过比较,探针PSiR3对hROS荧光off-on响应最明显,因此选取PSiR3作为ROS探针进行后续的细胞及组织实验。进一步,测试了PSiR3与hROS反应的动力学及荧光滴定实验,如图14所示,PSiR3 与hROS的反应非常快,可以在10秒内完成。图15的滴定实验所示,当向探针(2μM)中加入1当量的ClO-、ONOO-和20当量的HO·时,探针在680nm处的荧光强度达到饱和;探针在680 nm处的荧光强度与ClO-、ONOO-和HO·的浓度呈良好的线性关系,最低检出限(S/N=3)分别为5.39nM、6.86nM、0.12μM。
(2)细胞成像
如图16(A)所示,正常细胞Cos-7、BEAS-2B细胞和HUCEC细胞用PSiR3(5μM,20min )处理,或先用PSiR3(5μM,20min)处理,再用β-Lap(10μM,60min)处理后,细胞内均未观测到明显的荧光信号。如图16(B)所示,癌细胞A549、HepG2、MCF-7用PSiR3(5μM ,20min)处理后,细胞内的平均荧光密度是正常细胞的2.8倍(T/N=2.8),这是由于癌细胞有比正常细胞更高的本底ROS水平;癌细胞先用PSiR3(5μM,20min)处理,再用β-Lap( 10μM,60min)处理后,细胞内的平均荧光密度是正常细胞的15倍(T/N=15),远远超过了临床可接受的阈值2.0,说明β-Lap在癌细胞内过量表达的NQO1催化下诱发癌细胞进一步产生了大量的ROS。
为了验证癌细胞内过量产生的ROS是由β-Lap诱导产生的,随后在A549细胞内进行了抑制实验。如图17所示,A549细胞预先用40μM的双香豆素(DIC,一种有效的NQO1抑制剂)或 1mM的N-乙酰半胱氨酸(NAC,一种ROS清除剂)处理60分钟后,再用PSiR3/β-Lap(5μM/10μM)组合处理细胞,细胞内未观察到明显的荧光增强,该实验结果说明细胞内的ROS确实是由β-Lap诱导产生的。为验证实验结果具有统计学意义,选取两种正常细胞和两种癌细胞进行了流式细胞分析,如图18所示,细胞用PSiR3/β-Lap(5μM/10μM)组合处理细胞后,癌细胞(A549和HepG2细胞)的荧光强度明显强于正常细胞(Cos-7和HUVEV细胞);当癌细胞A549 和HepG2细胞预先用DIC(40μM,60min)处理后,再用PSiR3/β-Lap(5μM/10μM)组合处理后,荧光强度受到了极大的抑制。
细胞实验说明,利用ROS含量不同来区分正常细胞和癌细胞时,本发明提出的用PSiR3/β-Lap组合方式比仅用PSiR3更具优势。
(3)癌组织和正常组织切片成像
如图19所示,与细胞实验类似,老鼠正常组织切片用PSiR3(5μM,20min)处理,或先用PSiR3(5μM,20min)处理,再用β-Lap(10μM,60min)处理后,荧光信号未发生明显变化。A549、HeLa、U89癌组织切片用PSiR3(5μM,20min)处理后,平均荧光密度比正常组织略有增强;癌组织先用PSiR3(5μM,20min)处理,再用β-Lap(10μM,60min )处理后,观察到了明显的荧光信号,平均荧光密度分别是正常组织的15、30、27倍,此T/N 比值比细胞水平上观察到的要高,主要是由于体内和体外生长的癌细胞内NQO1活性不同导致的。上述结果说明本发明提出的用PSiR3/β-Lap组合方式可以高对比对区分老鼠的肿瘤组织和正常组织。
为了验证本发明提出的策略的临床应用价值,进一步在病人组织切片上验证了该策略的可行性。如图20所示,从山西省肿瘤医院获取的病人组织切片分别用PSiR3/β-Lap(5μM/10μM )组合处理60分钟,其中四个肺组织样本和四个甲状腺组织样本在红色通道中显示了明亮的荧光信号,说明它们可能是癌组织;其中一个肺组织样本和一个甲状腺组织样本在红色通道中显示了微弱的荧光信号,说明它们可能是良性组织。该推测也通过HE染色结果得到了验证。上述结果说明本发明提出的用PSiR3/β-Lap组合方式可以高对比对区分病人的肿瘤组织和良性组织。
(4)癌细胞/组织切片和炎性细胞/组织切片成像
由于巨噬细胞或其他吞噬细胞在免疫呼吸爆发时会产生高水平的ROS,也就是说炎性细胞本底ROS含量较高,而正常细胞和肿瘤细胞本底ROS含量较低,因此利用发明提出的PSiR3 和PSiR3/β-Lap双重组合方式可以区分癌细胞/组织和炎性细胞/组织。首先在细胞水平验证其可能性,如图21所示,RAW264.7细胞分别用PSiR3(5μM,20min)和PSiR3/β-Lap(5μM/10 μM,60min)组合处理后,红色通道均未观测到明显的荧光信号,说明RAW264.7细胞内本底的ROS含量和NQO1酶活性都较低;当RAW264.7细胞预先用LPS处理4小时诱导发炎,再用 PSiR3和PSiR3/β-Lap组合处理,红色通道观测到相似的荧光信号增强,该结果说明LPS可诱导RAW264.7细胞内ROS上调,β-Lap并不能诱导RAW264.7细胞内ROS上调。然而,与炎性细胞不同,癌细胞经PSiR3和PSiR3/β-Lap组合处理后,后者的平均荧光密度是前者的15倍。因此可以利用细胞经PSiR3和PSiR3/β-Lap双重组合处理后荧光密度增强的倍数不同实现对癌细胞和炎性细胞的区分,即细胞分别被PSiR3和PSiR3/β-Lap组合处理,若出现弱荧光/弱荧光,可判断为正常细胞;若出现强荧光/强荧光,可判断为炎性细胞;若出现弱荧光/强荧光,可判断为癌细胞。
最后,在老鼠和病人组织水平验证该策略的可行性。如图22(A)所示,老鼠正常组织切片、癌组织切片和炎性组织切片分别用PSiR3和PSiR3/β-Lap组合处理,正常组织切片中后者和前者在红色通道的荧光信号均可忽略不计;癌组织切片中后者在红色通道的荧光信号是前者的5.4倍;炎性组织切片中后者和前者在红色通道均显示出类似强烈的荧光信号。如图22 (B)所示,病人的良性子宫组织切片、肺癌组织切片和炎性甲状腺组织切片分别用PSiR3和 PSiR3/β-Lap组合处理,正常组织切片中后者和前者在红色通道的荧光信号均可忽略不计;癌组织切片中后者在红色通道的荧光信号是前者的11.2倍;炎性组织切片中后者和前者在红色通道均显示出类似强烈的荧光信号。上述结果说明本发明提出用PSiR3和PSiR3/β-Lap双重组合方式可以区分肿瘤组织和炎症组织。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (3)
2.权利要求1所述的荧光探针的制备方法,其特征在于,步骤如下:
(1)在N2保护下,将4,4'-亚甲基双(3-溴-N,N-二甲基苯胺)溶于干燥的THF中,溶液冷却至-78oC并逐滴加入正丁基锂溶液n-BuLi,反应液在此温度下搅拌反应2小时后,继续加入二氯二烷基硅烷,室温搅拌反应2小时;
反应结束后,向上述反应液中加入1 N的HCl至反应液呈中性,在旋转蒸发仪上蒸干THF,剩余水溶液用EtOAc萃取,收集有机相,有机相经洗涤、干燥、蒸发后得中间体,将该中间体与KMnO4粉末溶于丙酮中,-15oC下反应2小时,反应液经过滤、蒸发后得到硅氧杂蒽酮化合物;
(2)在N2保护下,将硅氧杂蒽酮化合物溶于干燥的CH3CN中,向该溶液中逐滴滴入Tf2O,反应液在将0oC搅拌反应10分钟,然后继续加入2,4-二甲基-3-乙基吡咯,室温搅拌反应10分钟,减压除去溶剂,所得固体经柱色谱纯化得PSiR。
3.根据权利要求2所述的制备方法,其特征在于:所述步骤中4,4'-亚甲基双(3-溴-N,N-二甲基苯胺)和二氯二烷基硅烷的摩尔比为1:1.8;中间体与KMnO4的摩尔比为1:2.5;Tf2O、硅氧杂蒽酮和2,4-二甲基-3-乙基吡咯的摩尔比为2.2:1:3。
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