Molecular imaging of a cell-penetrating peptide labeled Fluorescein-5-isothiocyanate and MR contrast agents: Gadopentetate dimeglumine
- Author:
- No author available
- Journal Title:
- CHINESE JOURNAL OF RADIOLOGY
- Issue:
- 2
- DOI:
- 10.3760/j.issn:1005-1201.2006.02.006
- Key Word:
- 肽类;磁共振成像;分子显像;造影剂;荧光素-5-异硫氰酸盐
Abstract： 目的选择穿膜肽的基本序列之一,构建目的多肽序列标记异硫氰酸荧光素(FITC)及钆喷替酸葡甲胺(Gd-DTPA),探讨穿膜肽携带FITC及Gd-DTPA跨膜转运性能及MR分子显像的价值.方法在穿膜肽9聚精氨酸[arginine(9)]基础上,构建新目的多肽序列CPP13: LAGRRRRRRRRRK,固相合成多肽后标记FITC(记为CPP13-FITC)和Gd-DTPA(记为CPP13-DTPA-Gd);分别取CPP13-FITC和FITC 溶于无血清Dulbecco最低必须培养液(DMEM)培养基中,待HEPG2细胞及鼠骨髓干细胞爬片上生长至80%～90%汇合时,取2只30 mm2皿,弃去培养皿中培养液,一皿中加入CPP13-FITC/DMEM溶液,另一皿中加入FITC/DMEM溶液,37℃ CO2孵箱中抚育10、30、60 min时依次取出1片, 磷酸平衡盐溶液(PBS)冲洗爬片后置于倒置荧光显微镜上观察细胞内出现荧光素分布的时间和部位.CPP13-DTPA-Gd作用肝癌细胞株HEPG2后,MRI研究CPP13-DTPA-Gd在细胞内转运性能及MRI的特点,将CPP13-DTPA-Gd 溶于无血清DMEM培养基中,浓度为3 mg/ml.待HEPG2细胞在100 mm2培养皿中长至80%～90%汇合时,取3只皿分别加入CPP13-DTPA-Gd的DMEM溶液、 DTPA-Gd/DMEM溶液和 DMEM溶液,孵育30 min 后弃去培养液,以0.1 M PBS反复冲洗,胰酶消化,加入2 ml 1%琼脂糖/PBS溶液混匀,装入2 ml离心管中,将3管固定于装有150 ml 1%琼脂糖/PBS溶液的微量加样枪头盒中,待凝固后测定MR信号.结果固相合成多肽成功,测定分子量为1792.78,与理论值接近.纯度达到95%以上;标记荧光素后,倒置荧光显微镜观察细胞摄取显示10 min时肝癌细胞株HEPG2和鼠骨髓干细胞胞质与细胞核内均出现荧光分布;CPP13标记的Gd-DTPA,作用细胞30 min后MRI显示CPP13-DTPA-Gd作用HEPG2细胞组呈短T1、短T2信号.3层面内3组细胞感兴趣区T1信号强度(Ii)与琼脂糖T1信号强度(Io)的比值及统计学分析如下:(1)号管3层面内Ii1/Io(为CPP-DTPA-Gd作用cell组管内信号值与管外烧杯内琼脂糖背景信号值的比值)分别为:2.84、2.60、2.48;(2)号管3层面内Ii2/Io(为Gd-DTPA作用cell组管内信号值与管外烧杯内琼脂糖背景信号值的比值)分别为1.15、1.11、1.12;(3)号管3层面内Ii3/Io(为空白cell对照组管内信号值与管外烧杯内琼脂糖背景信号值的比值)分别为1.13、1.11、1.11.两两组间F检验:(1)与(2):F(1,2)=201.88(P<0.001);(1)与(3):F(1,3)=206.37(P<0.001);(2)与(3): F(2,3)=0.529(P=0.507).说明T1WI信号强度与Gd-DTPA作用组及与单纯细胞组信号强度比较差异有统计学意义;HEPG2细胞不摄取Gd- DTPA,30 min时信号强度与单纯细胞组信号强度间差异无统计学意义.结论在穿膜肽基本序列基础上成功地重新构建的多肽能够携带荧光素和MR对比剂,并有效地跨膜转运进入细胞,为MR分子显像提供了1种新的重要手段.
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