【作者】 黄家君；

【导师】 李少林；

【作者基本信息】 重庆医科大学 ， 药理学， 2002， 博士

【摘要】 目的：探讨受体介导的反义显像作为乳腺癌早期诊断新方法的可行性，使患者能够得到早期诊断、及时治疗，降低乳腺癌的死亡率。方法：采用自行合成的双功能螯合剂-巯基乙酰三甘氨酰-N-羟基琥珀酰亚胺酯；用临床最常用的、物理性能优良的^｛99m｝Tc标记乳腺癌的一个主要癌基因c-erbB2 mRNA的反义寡核苷酸；研究了标记产物的标记率、放射化学纯度、比活度、稳定性、血浆蛋白结合率、生物学分布、药代动力学特点、细胞的摄取率及返流比率、细胞的生长、癌蛋白的表达；对荷乳腺癌裸鼠进行显像。人工合成的3条15碱基的单链寡核苷酸，反义序列为:5’-NH₂-FTCCATGGTGCTCAC-3’，正义序列为：5’-NH₂-QTGAGCACCATGGAG-3’，无义序列为5’-NH₂-FGCCTTATCCGTAGC -3’（F，Q分别代表硫代磷酸化的碱基C、G）。反义寡核苷酸与c-erbB2癌基因 mRNA互补，正义、无义寡核苷酸作为对照。用^｛99m｝Tc标记后，以0.25M/L乙酸铵（pH=5.2）作为洗脱液，将标记混合物通过SephadexG25柱层析进行分离纯化。根据SephadexG25柱层析纯化结果，绘制淋洗曲线，计算标记率，收集第一个放射峰，用纸层析测定标记产物的放射化学纯度；分析标记产物室温放置4h后或与人血清孵育4h后的稳定性，用抽滤法测定标记的EGF-^｛99m｝Tc-ON的比活度。用50ml培养瓶90个，每个接种2.0×10⁵个<WP=7>细胞，移入二氧化碳孵箱培养2天。当细胞覆盖瓶壁50％-60％时，每个培养瓶分别转染2μg EGF-^｛99m｝Tc-ON或^｛99m｝Tc-ON，在20min、40min、60min、120min、180min时测定细胞的摄取率，18h后测定细胞的返流比率，并对三组的返流比率进行比较。采用96孔板，每孔接种5.0×103个细胞，培养48h后，分别将含0.5μCi、1.0μCi、2μCi、5μCi等不同浓度的三种EGF-^｛99m｝Tc-ON及0.2μg、0.4μg、0.8μg、1.6μgEGF-PL复合物转染癌细胞，每种浓度加入3个孔中，24h再加入四唑盐，处理后测定各孔的吸光度。用50ml培养瓶18个，每瓶接种5.0×10⁵个癌细胞，培养48h后，每瓶分别转染EGF-^｛99m｝Tc-ASON或三种^｛99m｝Tc-ON 2μg，或6μg EGF-PL复合物，24h后，测c-erbB2癌蛋白的表达水平。用三氯乙酸沉淀法测定EGF-^｛99m｝Tc-ON与家兔血浆蛋白结合率。以家兔为实验对象，研究EGF-^｛99m｝Tc-ASON和^｛99m｝Tc-ASON的药代动力学，绘制其药代动力学曲线，用3P97软件拟合药代动力学曲线方程。以雌性BALB/c小鼠为实验对象，每只经尾静脉注入约3μCi的EGF-^｛99m｝Tc-ASON或^｛99m｝Tc-ASON后，分别在0.5h、1h、2h、4h、8h、24h测定肝、脾、肾、心、血、肺、胃、肠、骨、脑、肌肉等组织器官的放射性计数，研究EGF-^｛99m｝Tc-ASON和^｛99m｝Tc-ASON在正常小鼠体内的分布。SKBR-3乳腺癌细胞1.0×10⁷个接种每只裸鼠，让癌细胞在裸鼠体内生长15天，待肿瘤直经约为1cm时，乳腺癌模型构建完毕。将EGF-^｛99m｝Tc-ASON和^｛99m｝Tc-ASON 3μCi/鼠经尾静脉注入体内，分别于0.5h、1h、2h、4h测定肝、脾、肾、心、血、肺、胃、肠、骨、脑、肌肉、肿瘤等组织器官的放射性计数，研究EGF-^｛99m｝Tc-ASON和^｛99m｝Tc-ASON在荷瘤裸鼠体内的分布。将EGF-^｛99m｝Tc-SON和<WP=8>EGF-^｛99m｝Tc-NON以相同的浓度和方式注入裸鼠体内，于2h时测定上述组织器官的放射性计数，并比较三种EGF-^｛99m｝Tc-ON之间的差异。将EGF-^｛99m｝Tc-ASON按0.1mCi/鼠经尾静脉注入体内，分别于注射后1h、2h、3h、4h用SPECT进行显像。结果：寡核苷酸的标记率分别为：反义寡核苷酸70.6％，正义寡核苷酸67.2％，无义寡核苷酸69.3％；分离纯化后放射化学纯度分别是97.8％、96.2％、97.1％；比活度分别为1.41MBq/μg、1.26MBq/μg、1.53MBq/μg；三种寡核苷酸的标记率、放射化学纯度和比活度相比较均无显著性差异（P>0.05）。EGF-^｛99m｝Tc-ON在室温放置4h，或与血清孵育4h后均相当稳定。血浆蛋白结合率分别为：反义寡核苷酸11.82％，正义寡核苷酸12.78％，无义寡核苷酸10.93％，三者之间无显著性差异（P>0.05）。细胞摄取率在20min-120min内，随时间的延长而增加，EGF-^｛99m｝Tc-ON细胞摄取率明显高于^｛99m｝Tc-ON，有显著性差异（P<0.05），在每一时间点前者的摄取率均比后者高3倍以上。细胞返流比率为：反义寡核苷酸21.81％，正义寡核苷酸39.28％，无义寡核苷酸50.76％，反义寡核苷酸明显低于正义和无义寡核苷酸（P<0.05），但三者的返流比率都较高。MTT实验，吸光度值：EGF-^｛99m｝Tc-ASON＜^｛99m｝Tc-ASON＜其余各组，用流式细胞仪测定癌蛋白的表达水平，EGF-^｛99m｝Tc-ASON组荧光强度最低，表明其能有效抑制癌基因的表达，使癌蛋白的表达量下降，与其它各组（^｛99m｝Tc-ASON、EGF- ^｛99m｝Tc-SON、EGF-^｛99m｝Tc-NON、EGF-PL及空白对照组）相比，有显著性差异（P<0.05）。EGF-^｛99m｝Tc-ASON和^｛99m｝Tc-ASON在体内的药代动力学符合二室模型，其曲线方程为：<WP=9>EGF-^｛99m｝Tc-ASON（KBq）：C=47.24351e^｛-0.16598t｝+1.83133e^｛-0.00651t｝^｛99m｝Tc-ASON（KBq）：C=78.43123e^｛-0.32872t｝+2.068347e^｛-0.00728t｝正常BALB/c小鼠体内放射性分布以肝、肾为最高，肺、脾、更多还原

【Abstract】 OBJECTIVE：To explore the possibility of receptor-mediated antisense imaging as a novel method for the earlier detection of breast cancer, we carry through this experiment to make the breast cancer patients to be diagnosed earlier and treated in time, and decrease mortality. METHODS: Antisense oligonucleotide for mRNA of c-erbB2 oncogene was labeled with ^｛99m｝-technetium, ^｛99m｝Tc, a common isotope whose physical performance is excellent, through a self-synthetic biofunctional chelator N-hydroxysuccinimide ester of mercaptoacetylglycylglycylglycine. Its labeling efficiency, radiochemical purity, specific activity, stability, biodistribution, rate of combination with plasma protein, pharmacokinetics, uptake rate, refluent rate, growth and oncoprotein expression of SKBR-3 cells were investigated. Then ^｛99m｝Tc-ON will be injected into tumor-bearing nude mice through their tail vein for imaging application.Three man-made synthetic single stranded oligonucleotides are antisense, sense and nonsense ones. Each contains 15 bases. Their sequences are antisense 5’-NH₂-FTCCATGGTGCTCAC-3’, sense 5’-NH₂-QTGAGCACCATGGAG-3’ and nonsense <WP=11>5’-NH2-FGCCTTATCCGTAGC-3’ （ F and Q represent phosphorothioate bases C and G, respectively）. Antisense oligonucleotides were targeted to the initial code of mRNA of c-erbB2, sense and nonsense ones for this mRNA acting as control. These oligonucleotiedes labeled with ^｛99m｝Tc were separated and purified through Sephadex G25 column chromatography by using 0.25M/L ammonium acetate （pH=5.2） as eluent. Then the leaching curve will be made, labeling efficiency calculated, the first radiation peak collected. The radiochemical purity of these labeled oligonucleotides will be measured by paper chromatography, and their stability after being put 4 hours at room temperature or incubation with serum analyzed. Ninety culture flasks with volume of 50ml were inoculated with 2.0×10⁵ cells for each one. Having been cultured in CO₂ incubator for 2 days, confluent cells being to 50-60 percent, these cells were transfected with 2μg EGF-^｛99m｝Tc-ASON or ^｛99m｝Tc-ASON respectively. The uptake rate of cells are tested at 20, 40, 60, 120, 240min, and refluent rate detected in 18h. Culture plate with 96 wells was inoculated with 5.0×10³ SKBR-3 cells for each one. Having been cultured for 48 hours with 50-60 percent of confluent cells, the plates were transfected with three types of EGF-^｛99m｝Tc-ON in different concentration 0.5, 1, 2, 5μCi and done with EGF-PL compound in different concentration 0.2, 0.4, 0.8, 1.6μg respectively. Each concentration was added to three wells. Then these plates were transferred to CO₂ incubator, after 24 hours MTT were added to these wells. The absorbency was tested by enzyme-linked immunoassay. Eighteen culture flasks with volume of 50ml were inoculated with 2.0×10⁵ SKBR-3 cells for each one. Having been cultured for 48 hours, each was transfected with 2μg EGF-^｛99m｝Tc-ASON or three types of ^｛99m｝Tc-ON or 6μg EGF-PL compound. Each group contains three<WP=12>flasks. The oncoprotein of c-erbB2 was detected at 24h after transfection. The rate of combination with plasma protein will be tested through trichloroacetic acid precipitation. EGF-^｛99m｝Tc-ASON and ^｛99m｝Tc-ASON are injected into rabbits through auricular marginal vein. Then the blood will be gotten at 1, 3, 5, 10, 20, 40, 60, 120, 240min, the radioactivity counts obtained through calibrator. The pharmacokinetics curves will be made according to the radioactivity counts of each gram blood, equations obtained through 3P97 software. Sixty female BALB/c mice classified into two groups were used to test the biodistribution in vivo. About 3μCi EGF-^｛99m｝Tc-ASON or ^｛99m｝Tc-ASON was injected into each mouse through its tail vein. Then the radioactivity counts of such organs or tissues as liver, spleen, kidney, heart, blood, lung, stomach, intestine, bone, muscle and brain were measured at 0.5, 1, 2, 4, 8 and 24h after injection. The cell line of breast cance更多还原