CN108318311B - 一种硬骨特异性钙质荧光染色方法及其应用 - Google Patents
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Abstract
本发明属生物技术领域,涉及一种硬骨特异性钙质荧光染色方法及其应用,该方法采用天然提取物红紫素(purpurin)、光泽汀(lucidin)或3‑羟基巴戟醌(3‑hydroxy‑morindone)作为荧光染色剂,可在活体脊椎动物(如斑马鱼和小鼠)中对硬骨进行荧光标记。该方法操作简单快捷,可被用于活体动态观察,硬骨荧光染色观察清晰。本方法为检验临床样本中硬骨和增生骨组织提供新的染色方案和工具。
Description
技术领域
本发明属生物技术领域,涉及一种硬骨特异性钙质荧光染色方法及其应用,尤其涉及一种可以特异性标记活体脊椎动物硬骨的荧光染色方法及其应用,该方法采用天然提取物红紫素(purpurin)、光泽汀(lucidin)或3-羟基巴戟醌(3-hydroxy-morindone)作为荧光染色剂,可在活体脊椎动物(如斑马鱼和小鼠)中对硬骨进行荧光标记。该方法可被用于活体动态观察。
背景技术
现有技术公开了红紫素(purpurin, CAS:81-54-9),又称1,2,4-三羟基蒽醌、尿红素、紫茜素、紫精,是茜草的天然成分,作为一种红/黄色染剂可用于棉布的快速染色。研究显示,红紫素可与多种金属离子形成复合物,可用于检测锆、硼、钙的试剂;此外,研究还显示,由于红紫素的分子六元(芳族的)环含有善于传递电子的羰基和羟基,富含电子,因此,很容易与锂离子结合,可作为原料制作红紫素电极生产新型的无毒持久的电池;
光泽汀(lucidin,CAS:478-08-0)同为蒽醌衍生物,也是一种茜草的天然成分,能诱导细菌和哺乳动物细胞的突变,同时具有抗菌、抗炎、抗癌等功能,其药用价值在于其根部包含的蒽醌类物质;有研究显示,天然的蒽醌类物质通常还具有良好的着色能力,被广泛用于食品、药品、化妆品、纺织品以及头发的染色;
3-羟基巴戟醌(3-hydroxy-morindone)是一种CAS号待定的新品小分子化合物,其作用尚未知。
目前通行的硬骨染色法为茜素红法钙质染色法(发表于1952年),此种染色方法的包括如下步骤:
1. 组织固定和/或切片:组织用10%甲醛或4%多聚甲醛固定,避免溶液含钙或有脱钙效果;小动物样本可直接继续染色,临床和厚组织可进行石蜡切片,并经脱蜡至水洗;
2. 染色:蒸馏水水洗5-10min,加入茜素红染液染色5-30min,再用蒸馏水速洗。茜素红染液配法如下:0.5g 茜素红粉末溶解于100ml蒸馏水中,使用Tris-HCL调节溶液PH至8.3。取10ul上述溶液加入40-60 mM MgCl2与70%乙醇混合液中即得到茜素红染液;
3. 漂白:用漂白液洗去样本表面色素和多余的染液。漂白液现配现用,由等量的3% H2O2和2%KOH混合组成,终浓度为1.5%H2O2和1%KOH溶液;
4. 清理或封片:对于小动物样本,去掉漂白液,加入20%甘油/0.25KOH室温振荡30min至10h,边振荡边观察胚胎清理状态,然后用50%甘油/0.25%KOH替换之前的清理液,并再次室温振荡2h,放置过夜,显微镜下观察;对于切片样本,用0.2%冰醋酸水溶液速洗,后用95%乙醇、无水乙醇迅速脱水,二甲苯透明后中性树胶封固。
由此可见,传统的茜素红法钙质染色法步骤复杂,且耗时久,在染色开始前,需对标本进行固定,无法活体实时观察骨质的变化过程。
基于现有技术的现状,本申请的发明人拟提供一种硬骨特异性钙质荧光染色方法,尤其是可以特异性标记活体脊椎动物硬骨的荧光染色方法,该方法采用天然提取物红紫素(purpurin)、光泽汀(lucidin)或3-羟基巴戟醌(3-hydroxy-morindone)作为荧光染色剂,可在对小动物的硬骨进行活体染色,且方法简便易行,可明显观察到样本处理过程中的变化。
发明内容
本发明的目的是为克服现有技术存在的缺陷,提供一种硬骨特异性钙质荧光染色方法,尤其涉及可以特异性标记活体脊椎动物硬骨的荧光染色方法,本方法采用三种小分子化合物作为荧光染料在脊椎动物活体中进行硬骨染色,该方法操作简单快捷,硬骨荧光染色观察清晰,并且可用于活体观察。
本发明中,采用的作为荧光染料的三种小分子化合物为,天然提取物红紫素(purpurin)、光泽汀(lucidin)或3-羟基巴戟醌(3-hydroxy-morindone)。
本发明中,所述的硬骨标本可取自活体脊椎动物硬骨或是小动物的硬骨标本。
本发明的硬骨特异性钙质荧光染色方法,其包括,获取硬骨标本,配制红紫素/光泽汀/3-羟基巴戟醌染液母液;染色后,进行荧光观察。本方法操作简单快捷,可用于活体观察且硬骨荧光染色观察清晰。
本发明的一个实施例中,以斑马鱼幼鱼为例,采用红紫素(purpurin)、光泽汀(lucidin)或3-羟基巴戟醌(3-hydroxy-morindone)作为荧光染料进行活体硬骨染色,该方法包括步骤:
1)获取斑马鱼幼鱼
斑马鱼胚胎由清晨雌雄鱼交配获得,将0dpf的胚胎置于E3培养液中,28℃培养48小时,喂养及产卵方案按照zebrafish(第四版)描述进行,每天更换E3培养液(E3 培养液为终浓度为290mg/L的NaCl,13.3mg/L的KCl,48.3mg/L的CaCl2·2H2O和81.5mg/L的MgCl2·6H2O混合而成,调节PH至7.2并加入百万分之一的亚甲基蓝溶液);
2)配制红紫素/光泽汀/3-羟基巴戟醌染液母液
称取红紫素/光泽汀/3-羟基巴戟醌粉末,溶解于DMSO溶液中,保存液浓度为10mM,4℃避光保存;
3)染色
将红紫素/光泽汀母液以1:1000加入E3 培养液中,将待染色斑马鱼养殖于此混合液中,28.5℃培养24小时或更久;
4)荧光观察
使用0.168mg/ml tricaine麻醉剂溶液麻醉斑马鱼,并置于荧光体式显微镜下观察。
结果如图1所示,采用本实验方法对幼体斑马鱼进行骨染色的结果显示出其中的斑马鱼为受精后14天,骨骼染色为图中红色荧光部分。
本发明的一个实施例中,以新生小鼠为例,采用红紫素(purpurin)作为荧光染料进行活体硬骨染色,其包括步骤:
1)幼鼠的获取
P0新生小鼠由C57品系内交获得;
2)配制红紫素牛乳液
将红紫素以100uM终浓度溶于新鲜牛乳中,预温待用;
3)人工喂食
连续三天通过移液枪喂食含有红紫素的牛乳,3-6h喂食一次, 每次喂食完毕将其放回母鼠笼中;
4)荧光观察
常规处理小鼠,置于荧光体式显微镜下观察。
结果如图2所示,采用红紫素对成体斑马鱼和P3初生小鼠进行骨染色的结果,并展示了3周10uM的抚育不会显著影响成体斑马鱼的硬骨骨量和骨密度。
本发明提供了一种硬骨特异性钙质荧光染色方法,该方法采用天然提取物红紫素(purpurin)、光泽汀(lucidin)或3-羟基巴戟醌(3-hydroxy-morindone)作为荧光染色剂在脊椎动物(如斑马鱼和小鼠)活体中对硬骨进行荧光标记进行硬骨染色,实验结果表明,该方法操作简单快捷,硬骨荧光染色观察清晰,并且可用于活体观察。本发明为检验临床样本中硬骨和增生骨组织提供新的染色方案和工具。
附图说明
图1显示了采用本实验方法对幼体斑马鱼进行骨染色的结果,其中斑马鱼为受精后14天,骨骼染色为图中红色荧光部分。
图2显示了采用红紫素对成体斑马鱼和P3初生小鼠进行骨染色的结果,并展示3周10uM的抚育不会显著影响成体斑马鱼的硬骨骨量和骨密度。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式,本领域技术人员可由本说明书所公开的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用,本说明书中的各项细节也可以基于不同观点加以应用,在没有背离本发明的精神下进行各种修饰或改变。
实施例1 野生型斑马鱼硬骨特异性钙质荧光染色
实验动物:野生型斑马鱼(AB品系)胚胎取自复旦大学代谢与分子医学教育部重点实验室模式生物斑马鱼研究中心,野生型斑马鱼从国际斑马鱼中心引进,喂养及产卵方案按照zebrafish(第四版)描述进行,受精卵由清晨雌雄鱼交配获得,然后将受精卵置于E3medium中进行孵化(28℃),每天更换E3medium,超过20小时的胚胎可以加入0.003%的苯硫脲(PTU)处理,以防止黑色素的生成。
主要设备:
1.水生生物养殖系统(美国aquatic Habitats公司);
2.MVX10荧光体式显微镜(日本奥林巴斯公司);
3.DP73数码相机(日本奥林巴斯公司);
主要试剂溶液
1. E3培养液
E3 培养液为终浓度为290mg/L的NaCl,13.3mg/L的KCl,48.3mg/L的CaCl2·2H2O和81.5mg/L的MgCl2·6H2O混合而成,调节PH至7.2并加入百万分之一的亚甲基蓝溶液。
2.红紫素(purpurin)、光泽汀(lucidin)或3-羟基巴戟醌(3-hydroxy-morindone)均来自复旦大学基础医学院小分子化合物库 或可通过市购渠道获得。
染色步骤:
1)获取斑马鱼幼鱼:斑马鱼胚胎由清晨雌雄鱼交配获得,将0dpf的胚胎置于E3培养液中,28℃培养48小时,喂养及产卵方案按照zebrafish(第四版)描述进行,每天更换E3培养液,E3 培养液为终浓度为290mg/L的NaCl,13.3mg/L的KCl,48.3mg/L的CaCl2·2H2O和81.5mg/L的MgCl2·6H2O混合而成,调节PH至7.2并加入百万分之一的亚甲基蓝溶液;
2)配制红紫素/光泽汀/3-羟基巴戟醌染液母液
称取红紫素/光泽汀/3-羟基巴戟醌粉末,溶解于DMSO溶液中,保存液浓度为10mM,4℃避光保存;
3)染色
将红紫素/光泽汀母液以1:1000加入E3 培养液中,将待染色斑马鱼养殖于此混合液中,28.5℃培养24小时或更久;
4)荧光观察
使用0.168mg/ml tricaine麻醉剂溶液麻醉斑马鱼,并置于荧光体式显微镜下观察;
观察结果如图1所示,其中显示了采用本实验方法对幼体斑马鱼进行骨染色的结果,显示出其中的斑马鱼为受精后14天,骨骼染色为图中红色荧光部分。
本实施例的结果表明,本发明的硬骨特异性钙质荧光染色方法,明显克服现有技术的缺陷,本方法操作简单快捷,硬骨荧光染色观察清晰,并且可用于活体观察。
Claims (2)
1.一种硬骨特异性钙质荧光染色方法,其特征在于,采用小分子化合物天然提取物光泽汀lucidin或 3-羟基巴戟醌3-hydroxy-morindone,作为荧光染料,对硬骨标本染色后,进行荧光观察;
其包括步骤:
获取硬骨标本,
配制光泽汀/3-羟基巴戟醌染液母液;
染色后,进行荧光观察。
2.按权利要求1所述的方法,其特征在于,所述的硬骨标本取自斑马鱼或小鼠。
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