CN1006331B - 提取细胞内环核苷酸的方法 - Google Patents

提取细胞内环核苷酸的方法

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CN1006331B
CN1006331B CN 87106203 CN87106203A CN1006331B CN 1006331 B CN1006331 B CN 1006331B CN 87106203 CN87106203 CN 87106203 CN 87106203 A CN87106203 A CN 87106203A CN 1006331 B CN1006331 B CN 1006331B
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cyclic nucleotide
glucose
suspension
nucleotide
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CN1032032A (zh
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楼兰花
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Zhejiang Chinese Medical College
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Zhejiang Chinese Medical College
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Abstract

一种提取细胞内环核苷酸的方法，不需要破膜，也不受温度和试剂的影响，而是通过加热，在终止酶反应的同时提取细胞内的环核苷酸，操作简便快速，提取结果准确，易于推广应用。

Description

本发明是关于小分子物质的提取方法，特别是关于提取细胞内环核苷酸的方法。
环核苷酸在细胞功能调节中起重要作用，是一项不可缺少的分子学指标。目前，对提取细胞内环核苷酸，常采用的是冻融裂解法、超声波破膜法、低渗破膜法和匀浆破膜法等。
冻融裂解法是通过将细胞反复冻融，使细胞膜破裂而释放出环核苷酸，采用此法时必须在细胞悬液中加磷酸二酯酶抑制剂（EDTA），以防止环磷酸腺苷（CAMP）和环磷酸乌苷（CGMP）被磷酸二酯酶（PDE）水解，酶抑制剂的存在，虽然PDE被抑制，但使细胞内其他酶类未被灭活，易受冻融温度和EDTA的交互影响，从而影响细胞内环核苷酸的含量，特别是对CGMP的影响，使其含量偏高。
超声波破膜法是通过超声波发生器机械性破细胞膜，释放出环核苷酸。该法需用三氯醋酸或高氯酸终止酶反应，但细胞的膜蛋白经酸固定后会影响破膜率，并且在破膜后须用水饱和乙醚提取三氯醋酸或用碱中和高氯酸。整个提取过程操作步骤多而复杂，易造成实验误差大，并且受到仪器设备限制，不易推广应用。
低渗破膜法采用细胞悬液中加水低渗、破膜，然后加热和加酸去蛋白，由于终止酶反应前细胞受到低渗刺激，影响环核苷酸的含量，加之须除去膜蛋白和提取酸，否则环核苷酸含量偏高。
匀浆破膜法是在自然沉降后的血浆中加蒸馏水低渗破坏红细胞，然后用氯化钠溶液恢复等渗，离心后的沉淀细胞用Tris缓冲液 （含EDTA）制成细胞悬液，其释放环核苷酸的方法是在上述细胞悬液中加过氯酸，再用匀浆器匀浆机械破细胞膜，离心去膜蛋白，其上清液再加碱中和酸，再离心去盐，然后取上清液经阴离子交换树脂分别洗脱收集环核苷酸。
用上述各方法提取细胞内环核苷酸，都是经过细胞分离处理，除去红细胞，制成细胞悬液后，采用不同手段破膜，以使细胞内环核苷酸释放出来，再经分离得到环核苷酸，其中都需要加一定的试剂。膜蛋白和试剂的存在，以及温度的变化，影响了环核苷酸的含量。由于提取过程操作步骤多而复杂，易造成实验系统误差大，而且所需仪器设备要求高，不易推广应用。
本发明的目的是建立一种操作简单，易推广，不受温度和试剂影响的提取细胞内环核苷酸的稳定方法。
本发明采用加热提取细胞内环核苷酸的方法，是将自然沉降后的血浆经低渗破坏红细胞后立即恢复等渗，离心去血红蛋白等，离心后的沉淀细胞以0.1%葡萄糖等渗离子液为基质，制成细胞悬液，将该细胞悬液置于80°～100℃水浴加热5～40分钟，冰浴冷却，经离心分离的上清液即含环核苷酸。
0.1%葡萄糖等渗离子液组成为：138mM NaCl 2.7mMKCl 8.1mM Na2HPO41.5mM KH2PO41mM MgCl20.6mM CaCl20.1%葡萄糖液PH7.4。
本发明是在加热终止酶反应的同时，提取细胞内的环核苷酸，提取时不需要加EDTA和其它试剂，可以消除酶抑制剂等试剂和温度变化对细胞内环核苷酸含量的影响;并且采用等渗离子液为基质以提高细胞存活率，特别是不经过破膜，其可溶性蛋白小于100微克/107细 胞，因此不需要除蛋白。整个操作简便，结果稳定，可以减少实验的系统误差，提高结果准确性，易于推广。并且还适用于提取血小板内的环核苷酸，以及作体外抗炎、免疫、药理等实验研究。
本发明的实施例是这样的：抗凝血置室温下自然沉降30～40分，吸出富有白细胞的血浆，根据标本要求分别分离白细胞、淋巴细胞、血小板等。若处理白细胞、淋巴细胞，则分别取富有白细胞的血浆沉淀物和淋巴细胞分离后的沉淀物，用少量生理盐水悬浮，加四倍体积的蒸馏水低渗30秒～2分钟（视标本对象而定）破坏红细胞，立即加3.6%氯化钠恢复等渗，再离心去血红蛋白等，取沉淀物加0.1%葡萄糖等渗离子液洗2次，取其细胞悬液分装试管内，置80°～100℃水浴，加热5～40分钟，取出试管，放入冰浴冷却，然后经3000rpm离心10分钟，上清液即含环核苷酸。可直接定量吸取上清液，加上一定量的氚标抗原和一定稀释量的抗体，作放射免疫竞争反应，测定环核苷酸的含量。也可以将上清液定量吸入小烧杯中，置65℃水浴蒸干，放入冰箱保存，待测环核苷酸，保存期可达2～3个月。
进行细胞分离处理所用的0.1%葡萄糖离子液的组成是：
138mM    NaCl    2.7mM    KCl
8.1mM Na2HPO41.5mM KH2PO4
1mM MgCl20.6mM CaCl2
0.1%葡萄糖液，PH7.4。
用该离子液做基质，可以提高细胞存活率。

Claims (1)

1、一种提取细胞内环核苷酸的方法，包括将自然沉降的血浆加蒸馏水低渗30～120秒，立即用氯化钠渗液恢复等渗，离心，取沉淀细胞制成细胞悬液，从悬浮细胞内释放出环核苷酸，分离得到环核苷酸，其特征在于：沉淀细胞用0.1%葡萄糖等渗离子液为基质制成细胞悬液，0.1%葡萄糖等渗离子液组成为：
138mM NaCl 2.7mM KCl 8.1mM Na2HPO4
1.5mM KH2PO41mM MgCl20.6mM CaCl2
0.1%葡萄糖液 PH7.4，然后将该细胞悬液置于80°～100℃水浴加热5～40分钟后，冰浴冷却，离心分离后的上清液即含环核苷酸。
CN 87106203 1987-09-05 1987-09-05 提取细胞内环核苷酸的方法 Expired CN1006331B (zh)

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CN1110567C (zh) * 2000-02-17 2003-06-04 北京太宫生物技术有限责任公司 脱氧核糖核苷三磷酸的制备方法
CN1322142C (zh) * 2004-10-22 2007-06-20 哈尔滨医科大学 提取dna的试剂及使用该试剂提取哺乳动物dna的方法
CN105647995B (zh) * 2016-02-29 2019-03-08 山东大学 一种提取2′，3′-环形核苷单磷酸的方法

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