CN107446012B - 一类肿瘤荧光显像剂及制备和应用 - Google Patents
一类肿瘤荧光显像剂及制备和应用 Download PDFInfo
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Abstract
本发明提供了一类肿瘤荧光显像剂，是一类Cy3,Cy3.5,Cy5,Cy5.5,Cy7或者Cy7.5荧光标记的DNA或者PNA化合物，通过将5’‑氨基修饰或者3’‑氨基修饰的DNA或者PNA在碱性条件下与荧光基团Cy3,Cy3.5,Cy5,Cy5.5,Cy7,Cy7.5反应，然后通过Glen‑Pak DNA纯化柱纯化分离得到产物。实验证明该类荧光标记的化合物以MALAT1基因为靶点，可以用于肿瘤尤其是MALAT1基因表达高的肿瘤的体内外显像，在制备肿瘤显像剂中的应用。所述化合物结构式如下：
Description
技术领域
本发明属于医药领域，涉及一类荧光标记的DNA或者PNA的化合物，尤其涉及一类荧光基团Cy3,Cy3.5,Cy5,Cy5.5,Cy7,Cy7.5荧光标记的DNA或者PNA化合物，及其制备方法，以及在制备肿瘤荧光显像剂中的应用。
背景技术
人类基因组研究计划的成果证实：人类30多亿个碱基对的基因序列中2/3的序列被反转录，而最终仅有不到2％的核酸序列用于编码蛋白，大部分基因不表达蛋白质，这一类基因被称为非编码RNA(non-coding RNA,ncRNA)，其所占全基因组的比例与生物种间的复杂等级有着更密切的相关性。lncRNA起初被认为是基因组转录的“垃圾”，是RNA聚合酶II转录的副产物，不具有生物学功能。2011年《Cancer Research》上发表文章,认为长链非编码RNA有助于肿瘤的早期诊断、预后判断和分子靶向治疗，具有较好的临床应用价值，这些非编码RNA和编码基因(mRNA)一样可以作为特定恶性肿瘤的分子诊断标记，也是潜在的分子靶向治疗靶点。MALAT1基因(metastasis associated in lung denocarcinomatranscript 1)即人肺腺癌转移相关转录本1基因，其过表达与多种肿瘤息息相关，目前研究已经证实MALAT1在多种肿瘤中表达升高，其异常表达改变了肿瘤细胞的生物学表型，使肿瘤细胞增殖能力提高，转移能力和侵袭能力增强，促进了肿瘤的发生发展。
肽核酸(PNA)是具有类多肽骨架的DNA类似物，PNA的主链骨架是由N(2-氨基乙基)-甘氨酸与核酸碱基通过亚甲基羰基连接而成的。PNA可以特异性地与DNA或RNA杂交，形成稳定的复合体。PNA由于其自身的特点可以对DNA复制、基因转录、翻译等进行有针对的调控，同时作为杂交探针大大提高了遗传学检测和医疗诊断的效率和灵敏度。
目前特异靶向MALAT1治疗药物目前主要是基于靶向RNA的核酸或者肽核酸(PNA)，通过其调节癌细胞中长链RNA，PNA水平表达以及结构序列改变。研究显示抑制癌相关的RNA如反义核酸(ASO)，核酶及适配子显示优于siRNAs，且独具特性。
发明内容
本发明的目的是提供一类荧光标记的DNA或者PNA化合物，是一类Cy3,Cy3.5,Cy5,Cy5.5,Cy7或者Cy7.5荧光标记的脱氧核糖核酸(DNA)或者肽核酸(PNA)化合物，结构式如下：
结构式的特征为5’或者3’位荧光基团Cy3,Cy3.5,Cy5,Cy5.5,Cy7,Cy7.5结构，DNA或者PNA的序列为：AS1:GGGAGTTACTTGCCAACTTG，AS2：ATGGAGGTATGACATATAAT，AS3:TGCCTTTAGGATTCTAGACA。
本发明的另一个目的是提供荧光基团Cy3,Cy3.5,Cy5,Cy5.5,Cy7,Cy7.5标记DNA或者PNA的核酸的标记方法，该标记方法的特征是标记序列(反应底物序列)为AS1:GGGAGTTACTTGCCAACTTG，AS2：ATGGAGGTATGACATATAAT，AS3:TGCCTTTAGGATTCTAGACA。通过以下方法实现：将5’-氨基修饰或者3’-氨基修饰的DNA(或者PNA)溶解在15uL的0.1M的NaHCO3里面，在碱性条件下(PH＝9)与荧光基团Cy3,Cy3.5,Cy5,Cy5.5,Cy7,Cy7.5的琥珀酰亚胺酯的荧光化合物的DMSO溶液，混合溶液在室温下避光反应12小时，反应完毕后加入40uL的水，然后反应物用Glen-Pak DNA纯化柱纯化得到产物，或者HPLC分离方法得到荧光标记产物。
本发明的第三个目的是提供一类荧光标记的DNA或者PNA化合物在制备肿瘤显像剂中的应用，该化合物以MALAT1基因为靶点。实验证明该类荧光标记的化合物可以用于肿瘤尤其是MALAT1基因表达高的肿瘤的体内外显像。
本发明提供了一类新型荧光标记的DNA或者PNA化合物，以及制备方法，该类化合物可以实现活体内肿瘤显像，尤其对肿瘤MALAT1基因表达高的肿瘤均有高选择性，由于其标记制备方法简单，靶向明确，可以制备为显像剂，应用到临床体内肿瘤显像或者体外探测MALAT1基因的表达。
附图说明
图1：5’(Cy5.5)-MALAT1 ASO特异性结合MALAT1 MHCC-LM3细胞核转染实验；其中a：MALAT1表达，b：细胞核，c:merged图像。
图2：Cy5.5标记DNA在荷瘤鼠体内显像。
图3：Cy7标记DNA在荷瘤鼠体内显像。
图4：Cy7标记PNA在荷瘤鼠体内显像。
图5：Cy5.5标记DNA在荷瘤鼠的脏器显像。其中1血,2骨3肌肉4肿瘤5肾,6肺，7脾,8肝,9心。
具体实施方式
本发明结合附图与实例做进一步说明，但本发明并不受其限制。
实施例1:Cy5.5标记5’-氨基修饰长链非编码RNA的合成，
将10nmol的5’-氨基修饰的长链MALAT1 ASO溶解在15μL 0.1M的NaHCO3里面，向该溶液里加入7μL 49mM Cy5.5-NHS的DMSO溶液，混合溶液在室温下避光反应12小时，反应完毕后加入40uL的水，然后反应物用Glen-Pak DNA纯化柱纯化得到产物。
实施例2:Cy5.5标记3’-氨基修饰长链非编码RNA的合成，
将10nmol的3’-氨基修饰的长链MALAT1 ASO溶解在15μL 0.1M的NaHCO3里面，向该溶液里加入7μL 49mM Cy5.5-NHS的DMSO溶液，混合溶液在室温下避光反应12小时，反应完毕后加入40uL的水，然后反应物用Glen-Pak DNA纯化柱纯化得到产物。
实施例3:Cy3,Cy3.5,Cy5,Cy7,Cy7.5标记5’-氨基修饰的长链DNA的合成，
将10nmol的5’-氨基修饰的长链DNA溶解在15μL 0.1M的NaHCO3里面，向该溶液里加入7μL 49mM Cy3,Cy3.5,Cy5,Cy7,或者Cy7.5的琥珀酰亚胺酯的荧光化合物的DMSO溶液，混合溶液在室温下避光反应12小时，反应完毕后加入40uL的水，然后反应物用Glen-PakDNA纯化柱纯化得到产物。
实施例4:Cy3,Cy3.5,Cy5,Cy7,Cy7.5标记3’-氨基修饰的长链DNA的合成，
将10nmol的3’-氨基修饰的长链DNA溶解在15μL 0.1M的NaHCO3里面，向该溶液里加入7μL 49mM Cy3,Cy3.5,Cy5,Cy7,或者Cy7.5的琥珀酰亚胺酯的荧光化合物的DMSO溶液，混合溶液在室温下避光反应12小时，反应完毕后加入40uL的水，然后反应物用Glen-PakDNA纯化柱纯化得到产物。
实施例5:Cy3,Cy3.5,Cy5,Cy5.5,Cy7,Cy7.5标记5’-氨基修饰长链PNA的合成
将10nmol的5’-氨基修饰肽核酸(PNA)溶解在15μL 0.1M的NaHCO3里面，向该溶液里加入7μL 49mM Cy3,Cy3.5,Cy5,Cy5.5,Cy7,或者Cy7.5的琥珀酰亚胺酯的荧光化合物的DMSO溶液，混合溶液在室温下避光反应12小时，反应完毕后加入40uL的水，然后反应物用Glen-Pak DNA纯化柱纯化得到产物。
实施例6:Cy3,Cy3.5,Cy5,Cy5.5,Cy7,Cy7.5标记3’-氨基修饰长链PNA的合成
将10nmol的3’-氨基修饰肽核酸(PNA)溶解在15μL 0.1M的NaHCO3里面，向该溶液里加入7μL 49mM Cy3,Cy3.5,Cy5,Cy5.5,Cy7,或者Cy7.5的琥珀酰亚胺酯的荧光化合物的DMSO溶液，混合溶液在室温下避光反应12小时，反应完毕后加入40uL的水，然后反应物用Glen-Pak DNA纯化柱纯化得到产物。
实施例7:应用HPLC产品的提纯
离子对反相HPLC色谱法由于分离与纯化修饰好的DNA，用C-18柱(Zorbax ODS4.6*25mm)，HPLC的梯度缓冲液为：A：2％的乙腈的0.1M三乙基乙酸铵缓冲液，PH＝7，B：50％的乙腈的0.1M三乙基乙酸铵缓冲液缓冲溶液，梯度条件为：0-100％的B在30min内，1mL/min。收集的液体约6mL在110度吹He气的浓缩到100uL，再加入300UL的水稀释，通过NAP-5柱子(GE Healthcare，Mississauge，ON,Canada)除去盐，得到产品。
实施例8:应用阴离子交换柱分离
应用阴离子交换柱子分离产品，所用的柱子为The Thermo ScientificTMDNAPacTM PA200(250x 4mm)，缓冲溶液A:25mM三(羟甲基)氨基甲烷,PH＝8,5％的乙腈；缓冲溶液B：25mM三(羟甲基)氨基甲烷,PH＝8,5％的乙腈,1.0M NH4Cl,pH 8.梯度条件为：0-90％的B在30min内，1mL/min。
实施例9:Cy5.5标记5’-位长链非编码RNA的质量控制，
标记后的产品应用ESI测量，5’(Cy5.5)-(CH2)12-MALAT1 ASO，AS1:GGGAGTTACTTGCCAACTTG，(M+H+)的值为7226.1(calculated,7229)。
实施例10：Cy5.5标记3’-位长链非编码RNA的质量控制，
标记后的产品应用ESI测量，3’(Cy5.5)-(CH2)12-MALAT1 ASO,AS1:GGGAGTTACTTGCCAACTTG，(M+H+)的值为7225.1(calculated,7229)。
实施例11：探针高表达MALAT1 MHCC-LM3细胞核转染实验
5*105cells/well(24-well culture plates)100nmol AS用lipofectamine2000转染，转染后4-6小时后收集细胞，在荧光聚焦显微镜下，DPI染核，然后融合，图1显示AS可特异性聚集于细胞核内。
实施例12：Cy5.5标记DNA在肿瘤体内的分布。
将：Cy5.5标记DNA(GGGAGTTACTTGCCAACTTG)通过尾静脉注入到MHCC-LM3的荷瘤鼠体内，48小时候显像，发现肿瘤位置有浓聚(图2)，说明该显像剂可以很好的显示肿瘤。
实施例13：Cy7标记DNA在肿瘤体内的分布。
将Cy7标记DNA(ATGGAGGTATGACATATAAT)通过尾静脉注入到癌症的荷瘤鼠体内，48小时候显像，发现肿瘤位置有浓聚(图3)，说明该显像剂可以很好的显示肿瘤。
实施例14：Cy7标记PNA在肿瘤体内的分布。
将：Cy7标记PNA(TGCCTTTAGGATTCTAGACA)通过尾静脉注入到癌症的荷瘤鼠体内，48小时候显像，发现肿瘤位置有浓聚(图4)，说明该显像剂可以很好的显示肿瘤。
实施例15：Cy5.5标记DNA(GGGAGTTACTTGCCAACTTG)在老鼠的脏器显像。
Cy5.5标记DNA(GGGAGTTACTTGCCAACTTG)通过尾静脉注入到癌症的荷瘤鼠体内，48小时后将老鼠解剖，取各个脏器，显像见图5。
<110> 浙江大学
<120>一类肿瘤荧光显像剂及制备和应用
<160> 3
<210> 1
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 肿瘤荧光显像剂制备的荧光探针序列
<400> 1
GGGAGTTACTTGCCAACTTG 20
<210> 2
<211> 20
<212> DNA
<213> 人工序列
<220>
<223>肿瘤荧光显像剂制备的荧光探针序列
<400> 2
ATGGAGGTATGACATATAAT 20
<210> 3
<211> 13
<212> DNA
<213> 人工序列
<220>
<223>肿瘤荧光显像剂制备的荧光探针序列
<400> 3
TGCCTTTAGGATTCTAGACA 20
Claims (4)
1.一类荧光标记的DNA化合物，其特征在于，结构式如下：
R1=GGGAGTTACTTGCCAACTTG；
ATGGAGGTATGACATATAAT；
或TGCCTTTAGGATTCTAGACA；
R2= Cy3.5, Cy5.5,Cy7或Cy7.5；
n=1-100。
2.根据权利要求1所述的一类荧光标记的DNA化合物的制备方法，其特征在于，通过以下步骤实现：将5’-氨基修饰或者3’-氨基修饰的DNA溶解在15uL的0.1M的NaHCO3里面，在碱性条件下与荧光基团Cy3.5, Cy5.5,Cy7或Cy7.5的琥珀酰亚胺酯的荧光化合物的DMSO溶液混合，混合溶液在室温下避光反应12小时，反应完毕后加入40uL的水，然后反应物用Glen-Pak DNA 纯化柱纯化得到产物，或者HPLC分离方法得到最终产品，其中标记序列为AS1:GGGAGTTACTTGCCAACTTG，AS2：ATGGAGGTATGACATATAAT，AS3:TGCCTTTAGGATTCTAGACA 。
3.根据权利要求1所述的一类荧光标记的DNA化合物在制备肿瘤显像剂中的应用。
4.根据权利要求3所述的一类荧光标记的DNA化合物在制备肿瘤显像剂中的应用，其特征在于，所述化合物以MALAT1基因为靶点。
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