CN107827854B - 一种标记恶性肿瘤细胞的生物标志物,制备方法及其用途 - Google Patents
一种标记恶性肿瘤细胞的生物标志物,制备方法及其用途 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及生物医药领域,特别是指一种标记恶性肿瘤细胞的生物标志物的制备方法和用途。
背景技术
多胺是维持细胞生长分化等生命活动的必需物质,肿瘤的增殖,侵袭及其恶性程度与细胞内多胺水平密切相关。多胺是广泛存在于生物体内的脂肪族有机阳离子化合物。天然多胺包括腐胺(Putrescine,PUT),亚精胺(Spermidine,SPD) 和精胺(Spermine,SPM),其对细胞增殖、分化、染色质构象维持、离子通道调节和细胞膜稳定性维持等具有重要作用。哺乳动物细胞通过鸟氨酸-尿素循环生成多胺,鸟氨酸脱羧酶(Ornithinedecarboxylase,ODC)和S-腺苷甲硫氨酸脱羧酶(S-adenosylmethionine decarboxylase,S-AdeMetDC)是多胺合成的限速酶。在自身合成多胺不足的情况下,哺乳动物细胞也可从外界摄取多胺。
在生理条件下,细胞内多胺水平受其生物合成、代谢和细胞膜上多胺转运系统(Polyamine transport system,PTS)的精密调控,维持细胞周期的正常运转。在病理条件下,特别是肿瘤的发生发展中,常伴随多胺代谢异常。肿瘤细胞的增殖需要细胞内高多胺水平促进DNA复制、蛋白质合成和肿瘤组织血管生成,因而多胺合成酶活性上调,细胞膜上PTS高表达。
PTS是一种特殊结构的膜蛋白,肿瘤细胞膜上过表达的PTS可特异性地将外源多胺转运入胞,以满足肿瘤生长对多胺的旺盛需求。PTS不仅能转运多胺物质,还能转运很多基于多胺结构的分子。利用PTS对底物分子泛宿主性的特点,设计合成结构多样的多胺类似物和缀合物。这些化合物不具备天然多胺的生理功能,而是用于评价PTS的转运效率,抑制多胺合成酶的表达、诱导多胺代谢酶的活性或竞争性阻碍癌细胞对外源性多胺的摄取,耗竭细胞内多胺而诱导细胞凋亡,是一种具有重要临床应用前景的恶性肿瘤标记与检测手段,能够促进临床抗肿瘤治疗。
发明内容
有鉴于此,本发明的目的在于提出一种标记肿瘤细胞的生物标志物的制备方法和用途,腐胺-FITC缀合物能够有效通过PTS被肿瘤细胞摄取,肿瘤细胞内FITC的荧光强度与细胞所摄取的腐胺-FITC化合物以及肿瘤细胞的增殖程度成正相关。
基于上述目的、本发明提供一种标记肿瘤细胞的生物标志物,具有式I所示结构:
式I所示化合物的制备方法包括如下:
物质的合成:向反应瓶中加入荧光素(1mmol)、1,4-丁二胺(1mmol),乙醇(25mL),在90-100℃下加热回流5小时。冷却至室温后,红色固体析出,抽滤得粗产物,经冷乙醇洗涤三次,得目标产物。
附图说明
图1为本发明实施例式I所示化合物的H1核磁图。
图2为本发明实施例式I所示化合物的C13核磁图。
图3为本发明实施例式I所示化合物被肿瘤细胞吸收后的镜下荧光图像。a: A549细胞内的腐胺-FITC富集效应;b:A549细胞对腐胺-FITC的摄取作用量化分析。
具体实施方式
实施例1
物质的合成:向反应瓶中加入荧光素(1mmol)、1,4-丁二胺(1mmol),乙醇(25mL),在90℃下加热回流5小时,冷却至室温后,红色固体析出,抽滤得粗产物,经冷乙醇洗涤三次,得目标产物。化合物式I终产率为72%。
实施例2
1)将式I所示化合物溶解在50%DMSO中,配置成浓度10mM的母液;
2)在pH7.4的无菌磷酸盐缓冲液中加入式I所示化合物母液,配制成0.5μM, 1μM,5μM,10μM的溶液。
3)传代培养人非小细胞肺癌A549细胞到6孔细胞培养板,于含10%血清的DMEM培养基中常规培养(95%湿度,37℃,5%CO2)至细胞生长达到70%融合。然后在细胞培养液中加入式I所示化合物(即:腐胺-FITC),混匀并继续培养24小时,使用倒置荧光显微镜(于450nm波长激发)观察A549细胞内腐胺-FITC的富集。然后采用荧光酶标仪检测荧光值,根据荧光值的大小反映A549 细胞对腐胺-FITC的摄取作用和A549细胞内腐胺-FITC含量与处理浓度的关系。
阴性对照组:相同条件培养的A549细胞,在其培养液中加入等体积磷酸盐缓冲液,其它操作条件均与实验组相同。
根据上述实验组和阴性对照组获得的荧光强度和荧光图片反映A549细胞对腐胺-FITC的摄取作用和该含量与处理浓度的关系
由图3a所示的荧光显微镜图像可以看出,随着式I所示化合物(腐胺-FITC) 浓度的增加,A549细胞内的绿色荧光相应增强,显示A549细胞对该化合物具有富集效应。进一步通过荧光酶标仪检测各浓度梯度处理组和对照组的A549细胞内荧光值,然后分别与对照组比较得出相对荧光强度值;该量化分析结果与荧光显微镜图片所示一致(图3b)。上述结果证实A549细胞能够有效摄取式I所示化合物(腐胺-FITC),并且其摄取作用随着外源性添加的化合物浓度增加而逐渐加强。
因此,式I所示化合物对A549肿瘤细胞具有显著的标记效果。
由上述描述可以看出,本发明提供的一种标记肿瘤细胞的生物标志物的制备方法简单易行,能够有效检测非小细胞肺癌肿瘤细胞对多胺的摄取,从而标记恶性肿瘤细胞,为临床检测非小细胞肺癌提供新的途径。
Claims (5)
3.根据权利要求2所述的制备方法,其特征在于,荧光素、1,4-丁二胺的摩尔比为1:1-3。
4.根据权利要求2所述的制备方法,其特征在于,荧光素、1,4-丁二胺的摩尔比为1:1。
5.根据权利要求1所述的标记非小细胞肺癌肿瘤细胞A549的生物标志物在制备检测非小细胞肺癌疾病上的药物上的应用。
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CN105368853A (zh) * | 2015-11-30 | 2016-03-02 | 成都山权江生物科技有限公司 | 一种与非小细胞肺癌辅助诊断相关的标志物及其应用 |
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CN105368853A (zh) * | 2015-11-30 | 2016-03-02 | 成都山权江生物科技有限公司 | 一种与非小细胞肺癌辅助诊断相关的标志物及其应用 |
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