CAJ | 학술논문

目的探讨特异靶向人表皮生长因子受体2（ HER2）七肽分子LTVSPYW偶联超顺磁性氧化铁（ SPIO）纳米颗粒分子探针的构建方法及其表征，以及对体外乳腺癌细胞行靶向MR成像的可行性。方法（1）通过共沉淀法合成表面包被聚乳酸的 SPIO （ PS ）；将 PS 与异硫氰酸荧光素（FITC）标记的七肽分子LTVSPWY（FITC-LTVSPWY）偶联构建分子靶向探针（FITC-LTVSPWY-PS），用透射电子显微镜分别测量PS和FITC-LTVSPWY-PS粒径大小，动态光散射仪检测其粒径分布及表面电位，3.0 T MRI检测弛豫率。（2）制备高表达HER2的人乳腺癌细胞株MCF-7爬片，与FITC-LTVSPWY-PS进行孵育，荧光显微镜观察细胞与探针的结合情况，并行普鲁士蓝染色验证。（3）将合成的2种探针分别与HER2高表达的MCF-7细胞及HER2阴性的MDA-MB-231细胞孵育，消化离心后置入1.5 ml Eppendorf管。 FITC-LTVSPWY-PS与MCF-7孵育作为实验组，PS与MCF-7孵育作为对照组，不添加探针的MCF-7细胞为空白组，每种处理设立3管，采用3.0 T MR扫描1次，采集T2 WI序列图像，观察各组细胞MR信号并测出T2信号值，检测分子探针的靶向增强作用。各组间T2信号值的比较采用单因素方差分析法。结果分子靶向探针构建成功，电子透射显微镜测得PS纳米粒子的核心粒径大小为（13.9±1.6） nm，动态光散射仪测得PS的表面粒径为（122.0±5.5） nm。 FITC-LTVSPWY-PS探针的表面电位与弛豫率分别为（-30.7±2.2） mV和70.7 m· M-1· s-1，PS的表面电位与弛豫率分别为（28.1±2.8） mV和72.1 m· M-1· s-1。25μg/ml FITC-LTVSPWY-PS与MCF-7细胞孵育1 h后体外细胞MRI显示，实验组呈短T2低信号，对照组与空白细胞组呈等信号。实验组、对照组和空白组T2信号值分别为（61.8±5.7）、（101.6±2.5）和（103.5±1.9）ms，差异有统计学意义（ F＝355.698，P＜0.05）。结论针对HER2与聚乳酸修饰的SPIO耦联后获得的MR靶向探针具有良好的物理学表征与磁学特性，可与HER2阳性细胞特异性结合，满足靶向成像的基本要求。目的探討特異靶嚮人錶皮生長因子受體2（ HER2）七肽分子LTVSPYW偶聯超順磁性氧化鐵（ SPIO）納米顆粒分子探針的構建方法及其錶徵，以及對體外乳腺癌細胞行靶嚮MR成像的可行性。方法（1）通過共沉澱法閤成錶麵包被聚乳痠的 SPIO （ PS ）；將 PS 與異硫氰痠熒光素（FITC）標記的七肽分子LTVSPWY（FITC-LTVSPWY）偶聯構建分子靶嚮探針（FITC-LTVSPWY-PS），用透射電子顯微鏡分彆測量PS和FITC-LTVSPWY-PS粒徑大小，動態光散射儀檢測其粒徑分佈及錶麵電位，3.0 T MRI檢測弛豫率。（2）製備高錶達HER2的人乳腺癌細胞株MCF-7爬片，與FITC-LTVSPWY-PS進行孵育，熒光顯微鏡觀察細胞與探針的結閤情況，併行普魯士藍染色驗證。（3）將閤成的2種探針分彆與HER2高錶達的MCF-7細胞及HER2陰性的MDA-MB-231細胞孵育，消化離心後置入1.5 ml Eppendorf管。 FITC-LTVSPWY-PS與MCF-7孵育作為實驗組，PS與MCF-7孵育作為對照組，不添加探針的MCF-7細胞為空白組，每種處理設立3管，採用3.0 T MR掃描1次，採集T2 WI序列圖像，觀察各組細胞MR信號併測齣T2信號值，檢測分子探針的靶嚮增彊作用。各組間T2信號值的比較採用單因素方差分析法。結果分子靶嚮探針構建成功，電子透射顯微鏡測得PS納米粒子的覈心粒徑大小為（13.9±1.6） nm，動態光散射儀測得PS的錶麵粒徑為（122.0±5.5） nm。 FITC-LTVSPWY-PS探針的錶麵電位與弛豫率分彆為（-30.7±2.2） mV和70.7 m· M-1· s-1，PS的錶麵電位與弛豫率分彆為（28.1±2.8） mV和72.1 m· M-1· s-1。25μg/ml FITC-LTVSPWY-PS與MCF-7細胞孵育1 h後體外細胞MRI顯示，實驗組呈短T2低信號，對照組與空白細胞組呈等信號。實驗組、對照組和空白組T2信號值分彆為（61.8±5.7）、（101.6±2.5）和（103.5±1.9）ms，差異有統計學意義（ F＝355.698，P＜0.05）。結論針對HER2與聚乳痠脩飾的SPIO耦聯後穫得的MR靶嚮探針具有良好的物理學錶徵與磁學特性，可與HER2暘性細胞特異性結閤，滿足靶嚮成像的基本要求。
목적탐토특이파향인표피생장인자수체2（ HER2）칠태분자LTVSPYW우련초순자성양화철（ SPIO）납미과립분자탐침적구건방법급기표정，이급대체외유선암세포행파향MR성상적가행성。방법（1）통과공침정법합성표면포피취유산적 SPIO （ PS ）；장 PS 여이류청산형광소（FITC）표기적칠태분자LTVSPWY（FITC-LTVSPWY）우련구건분자파향탐침（FITC-LTVSPWY-PS），용투사전자현미경분별측량PS화FITC-LTVSPWY-PS립경대소，동태광산사의검측기립경분포급표면전위，3.0 T MRI검측이예솔。（2）제비고표체HER2적인유선암세포주MCF-7파편，여FITC-LTVSPWY-PS진행부육，형광현미경관찰세포여탐침적결합정황，병행보로사람염색험증。（3）장합성적2충탐침분별여HER2고표체적MCF-7세포급HER2음성적MDA-MB-231세포부육，소화리심후치입1.5 ml Eppendorf관。 FITC-LTVSPWY-PS여MCF-7부육작위실험조，PS여MCF-7부육작위대조조，불첨가탐침적MCF-7세포위공백조，매충처리설립3관，채용3.0 T MR소묘1차，채집T2 WI서렬도상，관찰각조세포MR신호병측출T2신호치，검측분자탐침적파향증강작용。각조간T2신호치적비교채용단인소방차분석법。결과분자파향탐침구건성공，전자투사현미경측득PS납미입자적핵심립경대소위（13.9±1.6） nm，동태광산사의측득PS적표면립경위（122.0±5.5） nm。 FITC-LTVSPWY-PS탐침적표면전위여이예솔분별위（-30.7±2.2） mV화70.7 m· M-1· s-1，PS적표면전위여이예솔분별위（28.1±2.8） mV화72.1 m· M-1· s-1。25μg/ml FITC-LTVSPWY-PS여MCF-7세포부육1 h후체외세포MRI현시，실험조정단T2저신호，대조조여공백세포조정등신호。실험조、대조조화공백조T2신호치분별위（61.8±5.7）、（101.6±2.5）화（103.5±1.9）ms，차이유통계학의의（ F＝355.698，P＜0.05）。결론침대HER2여취유산수식적SPIO우련후획득적MR파향탐침구유량호적물이학표정여자학특성，가여HER2양성세포특이성결합，만족파향성상적기본요구。
Objective To develop a superparamagnetic iron oxide nanoparticles ( SPIO ) based on MRI probe specifically targeting human epidermal growth factor receptor 2 (HER2) and explore its value as MRI positive contrast agents in vitro.Methods (1) The superparamagnetic iron oxide ( PS) was obtained by means of classical coprecipitation in polylactic acid solution , then coupled with fluorescein isothiocyanate (FITC) labeled LTVSPYW to develop the targeted probe ( FITC-LTVSPWY-PS).The particle size was measured under transmission electron microscope.Relaxation rate was detected by 3.0 T MR scanner.(2) Climbing films of human breast cancer cell MCF-7 were prepared and incubated with FITC-LTVSPWY-SPIO, then fluorescence distribution was observed under inverted microscope.And distribution of iron particles was confirmed by prussian blue staining.(3) MCF-7 and MDA-MB-231 cells were incubated with FITC-LTVSPWY-SPIO and PS, respectively.MCF-7 incubated with FITC-LTVSPWY-PS were used as experimental group, MCF-7 treated with PS as control group , and cells added with nothing as blank group.There were 3 samples in each group.The MR imaging was performed only once and T 2 WI signal intensity of cells was recorded.The comparison of T 2 signal intensity among groups was conducted by using one-way ANOVA.Results The core and surface size of nanoparticles were (13.9 ±1.6) nm and (122.0 ±5.5) nm respectively.Zeta potential and relaxation rate of the FITC-LTVSPWY-PS were ( -30.7 ±2.2 ) mV and 70.7 m· M-1 · s-1 respectively, and the PS were (28.1 ±2.8) mV and 72.1 m· M-1 · s-1 respectively.The fluorescence could be seen on the surface of MCF-7 cells, and the prussian blue staining showed that FITC-LTVSPWY-PS could specifically target HER 2-positive cells.The low signal on T 2 WI was observed in MCF-7 cells incubated with FITC-LTVSPWY-PS, whereas cells treated with PS and blank group showed equal signals , the T2 values were ( 61.8 ±5.7 ) , ( 101.6 ±2.5 ) and ( 103.5 ±1.9 ) ms respectively.Significant difference existed among these groups ( F =355.698, P <0.05 ).Conclusions The MR targeting probe FITC-LTVSPWY-PS was prepared successfully , its physical characterization and magnetic properties could target the HER 2 highly expressing on the surface of breast cancer cells and meet the need of targeted imaging.It provides an important tool for MR molecular imaging.