P-糖蛋白对~(18)F-FDG、~(99m)Tc-MIBI摄取影响的实验研究
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摘要
第一部分P-糖蛋白功能验证及与18F-FDG摄取相关蛋白检测
目的利用Western Blot法测定Bcap37/MDR1与Bcap37细胞中P-糖蛋白（P-glycoprotein，P-gp）、葡萄糖转运蛋白1（Glucose transporter1，Glut-1）及己糖激酶-II（Hexokinase-II，HK-II）表达，利用罗丹明123荧光染色评价P-gp功能。方法提取Bcap37/MDR1及Bcap37细胞蛋白，采用Western Blot法测定细胞中P-gp、Glut-1及HK-II的表达情况，在有或无P-gp抑制剂维拉帕米（Verapamil，VER）存在的情况下，分别将Bcap37/MDR1与Bcap37细胞与罗丹明123共孵育60min，然后吸去上清液，再用冰冷的PBS液洗脱3次，用荧光显微镜观察细胞内罗丹明123荧光强度。
结果P-gp在Bcap37/MDR1细胞中有高水平表达，而Bcap37细胞几乎无P-gp表达；Bcap37/MDR1细胞有较高水平的Glut-1表达，而Bcap37细胞仅表现为非常弱的Glut-1的表达； HK-II在两种细胞中均有较高水平表达。Bcap37、Bcap37/MDR1细胞与罗丹明123共孵育，Bcap37细胞内有明显荧光染色，Bcap37/MDR1细胞内仅有弱的荧光染色。加入VER后，Bcap37/MDR1细胞内的荧光染色明显增强。VER对Bcap37细胞荧光染色无明显影响。
结论P-gp、Glut-1在Bcap37/MDR1细胞中的表达明显强于Bcap37细胞；Bcap37/MDR1所表达的P-gp具有相应的外排功能，该细胞能够用来评价P-gp与18F-FDG、99mTc-MIBI摄取之间的关系。
第二部分99mTc-MIBI在Bcap37、Bcap37/MDR1细胞中的聚集实验
目的通过99mTc-MIBI细胞内摄取动力学研究99mTc-MIBI能否评价Bcap37/MDR1细胞P-gp功能及在评价P-gp抑制剂抑制效能中的作用。方法培养Bcap37及Bcap37/MDR1细胞，将Bcap37及Bcap37/MDR1细胞接种于6孔细胞培养板上，接种密度为1×106细胞/孔，将99mTc-MIBI、VER及GF120918用不含胎牛血清的RPMI1640培养基分别配成74KBq/mL99mTc-MIBI溶液、74KBq/mL99mTc-MIBI+100μmol/L VER溶液及74KBq/mL的99mTc-MIBI+50μmol/L GF120918溶液。用上述配制好的溶液孵育Bcap37及Bcap37/MDR1细胞，分别于10min、30min、60min及120min吸取孵育液，置于试管内，用冰冷的PBS液洗脱3次，洗脱液一并放置相应的试管内。然后采用胰酶消化细胞，消化完成后吸出细胞置于试管内，再分别用1mL PBS液洗脱3次，洗脱液置于放置细胞的试管内，采用γ计数仪测量各个试管内的放射性计数，计算Bcap37、Bap37/MDR1细胞中99mTc-MIBI的摄取率。摄取率=细胞内放射性计数/（细胞内放射性计数+上清液放射性计数）×100%，实验重复三次。计算结果用x±s表示，两种细胞间及同种细胞不同处理组间99mTc-MIBI摄取率比较行配对t检验。
结果Bcap37细胞在有或无抑制剂存在的情况下，99mTc-MIBI摄取率之间无统计学差异；Bcap37/MDR1细胞在有抑制剂存在的情况下99mTc-MIBI摄取率在孵育30min、60min及120min要明显高于无抑制剂存在时的摄取率（P<0.05）；在无抑制剂存在的情况下，Bcap37细胞99mTc-MIBI摄取率在孵育30min、60min及120min要明显高于Bcap37/MDR1细胞（P<0.05）；在有抑制剂存在的情况下，Bcap37/MDR1细胞的99mTc-MIBI摄取率略低于Bcap37细胞，但两者无统计学差异。
结论99mTc-MIBI在Bcap37细胞与Bcap37/MDR1细胞中的摄取率有显著差异，P-gp抑制剂能够增加Bcap37/MDR1细胞的99mTc-MIBI摄取率，表明可以用99mTc-MIBI摄取动力学变化评价P-gp的功能；同时多药耐药细胞系99mTc-MIBI摄取率的变化能够用于评价P-gp抑制剂的抑制效率。
第三部分18F-FDG在Bcap37、Bcap37/MDR1细胞中的聚集实验
目的测定18F-FDG在Bcap37与Bcap37/MDR1细胞的摄取动力学变化，评价P-gp抑制剂存在时Bcap37与Bcap37/MDR1细胞对18F-FDG摄取的变化，在细胞水平上探讨18F-FDG摄取与P-gp表达之间的关系。方法将Bcap37及Bcap37/MDR1细胞分别接种于6孔细胞培养板上，细胞密度为1×106细胞/孔，将18F-FDG、VER及GF120918用不含胎牛血清的RPMI1640培养基分别配成37KBq/mL18F-FDG溶液、37KBq/mL18F-FDG+100μmol/L VER溶液及37KBq/mL的18F-FDG+50μmol/L GF120918溶液。分别用上述配制好的溶液孵育Bcap37及Bcap37/MDR1细胞，分别于10min、30min、60min及120min吸取孵育液置于试管内，再用冰冷的PBS液洗脱3次，洗脱液一并放置相应的试管内。细胞用胰酶消化，消化完成后吸出细胞置于试管内，用1mL PBS液洗脱3次，洗脱液置于放置细胞的试管内，用γ计数仪测量各个试管内的放射性计数，计算Bcap37、Bap37/MDR1细胞的18F-FDG摄取率，摄取率=细胞内放射性计数/（细胞内放射性计数+上清液放射性计数）×100%，实验重复三次。计算结果用x±s表示，两种细胞间及同种细胞不同处理组间18F-FDG摄取率比较行配对t检验。
结果无抑制剂存在的情况下，孵育10min时Bcap37/MDR1细胞中18F-FDG摄取率显著高于Bcap37细胞，其摄取率分别为1.88%±0.19%和1.37%±0.18%（P<0.05）；孵育60min和120min时Bcap37细胞中18F-FDG摄取率显著高于Bcap37/MDR1细胞，其摄取率分别为2.29%±0.23%、2.34%±0.15%和1.47%±0.14%、1.53%±0.22%（P<0.05）。VER或GF120918共孵育后，Bcap37/MDR1细胞在60min和120min时18F-FDG摄取率分别为2.45%±0.21%、2.46%±0.25%和2.50%±0.24%、2.48%±0.27%，较无抑制剂存在时明显增加（P<0.05）。Bcap37细胞在有无抑制剂存在的情况下，18F-FDG摄取率无明显变化，差异无统计学意义。
结论细胞实验结果表明18F-FDG是P-gp的底物之一，Bcap37/MDR1细胞与Bcap37细胞间18F-FDG摄取率的差异是由于P-gp所介导的外排引起的，可以用18F-FDG动力学变化评价肿瘤细胞的P-gp功能，细胞内试验结果显示18F-FDG有可能作为活体内无创性评价肿瘤细胞的P-gp功能的显像剂。
第四部分99mTc-MIBI在Bcap37、Bcap37/MDR1荷瘤小鼠显像研究
目的利用荷Bcap37/MDR1与Bcap37细胞的BALB/c裸鼠模型，研究99mTc-MIBI在荷瘤小鼠体内显像，探讨99mTc-MIBI显像可否用于活体内评价肿瘤细胞的多药耐药。方法取处于对数生长期的Bcap37及Bcap37/MDR1细胞，用无血清的RPMI1640培养基制成细胞悬液，细胞计数后用无血清的RPMI1640培养基将细胞浓度稀释为1×10~7/mL，按0.2mL/只接种于4~6周龄的BALB/c裸鼠右侧腋窝皮下，5只裸鼠接种Bcap37细胞，5只裸鼠接种Bcap37/MDR1细胞。待肿瘤长至直径1.0~1.5cm时进行99mTc-MIBI SPECT显像。将荷Bcap37细胞裸鼠及荷Bcap37/MDR1细胞裸鼠通过尾静脉给予37MBq（1mCi，0.2mL）配制好的99mTc-MIBI，分别于给药后10min及60min行平面静态显像，隔日在相同采集条件下，尾静脉给予37MBq（1mCi，0.2mL）配制好的含有0.5mg/Kg VER的99mTc-MIBI溶液，分别于给药后10min及60min行平面静态显像。显像结束后，处死荷瘤小鼠后迅速取出肿瘤组织及肝脏组织，制成电镜标本，观察荷瘤小鼠肿瘤组织及肝脏组织线粒体情况。
结果99mTc-MIBI生理性摄取可见于肝脏、肠道、肾脏及膀胱，部分荷瘤小鼠尾静脉注射部位可见轻度显影。荷Bcap37或Bcap37/MDR1肿瘤细胞裸鼠肿瘤部位在给予99mTc-MIBI后10min未见明显显影，60min后肿瘤部位同样未见明显显影。荷Bcap37或Bcap37/MDR1肿瘤细胞裸鼠在给予含0.5mg/Kg VER的99mTc-MIBI后10min及60min肿瘤部位均未见明显显影。荷Bcap37及Bcap37/MDR1肿瘤细胞裸鼠肿瘤组织细胞内线粒体数量较少，肝脏组织细胞内存在较多的线粒体，且线粒体体积明显大于肿瘤细胞内线粒体。
结论荷Bcap37与Bcap37/MDR1肿瘤细胞裸鼠肿瘤组织99mTc-MIBI未见显影，同时给予P-gp抑制剂后肿瘤组织仍未见显影，表明选用荷瘤小鼠行99mTc-MIBI显像评价P-gp功能时要筛选肿瘤细胞株，应挑选99mTc-MIBI摄取率高且肿瘤细胞内线粒体较丰富的细胞株。
第五部分18F-FDG在Bcap37、Bcap37/MDR1荷瘤小鼠显像研究
目的研究荷Bcap37/MDR1及Bcap37肿瘤细胞荷瘤小鼠在有/无VER存在情况下肿瘤组织的18F-FDG摄取，在活体内评价P-gp与18F-FDG摄取的关系。方法荷瘤小鼠禁食6h，将需要动态采集的荷瘤小鼠置于小动物PET床板上，异氟烷吸入麻醉，胶带固定，身体置于视野中心，维持异氟烷麻醉浓度为2%。床前经尾静脉给予7.4MBq（0.2mCi，0.2mL）18F-FDG，给药后立即采集，共采集90min，得到各个时间段的动态显像图像，绘制感兴趣区（region of interest, ROI）的时间放射性曲线（time activity curve，TAC）。比较荷Bcap37肿瘤细胞裸鼠与荷Bcap37/MDR1肿瘤细胞裸鼠肿瘤组织18F-FDG摄取的TAC曲线。相同采集及处理方法，尾静脉给予含有0.5mg/Kg VER的7.4MBq（0.2mCi，0.2mL）18F-FDG，获得VER治疗后肿瘤组织18F-FDG摄取的TAC曲线。观察VER治疗后其18F-FDG TAC曲线，并与未行VER治疗时进行比较。另取10只荷瘤小鼠（5只荷Bcap37肿瘤细胞，5只荷Bcap37/MDR1肿瘤细胞），采用静态采集方法行18F-FDG Micro PET显像。荷瘤小鼠禁食6h，吸入异氟烷预麻醉，尾静脉给予7.4MBq（0.2mCi，0.2mL）18F-FDG，60min后行静态采集。在相同的采集处理条件下，尾静脉给予含有0.5mg/Kg VER的7.4MBq（0.2mCi，0.2mL）18F-FDG，获得VER治疗后肿瘤组织18F-FDG的静态图像，计算肿瘤组织的SUVmean。比较VER治疗前后肿瘤组织18F-FDG SUVmean的变化。显像结束后，处死荷瘤小鼠后将肿瘤组织制成蜡块，肿瘤标本行P-gp、Glut-1及HK-II免疫组化染色。
结果18F-FDG Micro-PET动态显像显示Bcap37肿瘤组织18F-FDG摄取渐进性增高，在10~15min内达峰值水平，然后维持在平台水平；Bcap37/MDR1肿瘤组织18F-FDG摄取同样渐进性增高，在大约9min时达到峰值，然后18F-FDG摄取渐进性下降，在25~30min时达到平台，然后维持在平台水平。Bcap37/MDR1肿瘤组织18F-FDG摄取在0~9min要高于Bcap37肿瘤组织，9min后Bcap37肿瘤组织18F-FDG摄取水平要高于Bcap37/MDR1肿瘤组织。有或无VER存在情况下Bcap37肿瘤组织18F-FDG摄取动态曲线无明显差异；Bcap37/MDR1肿瘤组织在有或无VER存在的情况下，其0~15min18F-FDG摄取动态曲线无明显差异，但在15min下降到最低点后，18F-FDG摄取开始渐进性升高，约30min达到最高水平，然后维持在平台水平。VER显著增加Bcap37/MDR1肿瘤组织平台期18F-FDG摄取水平。荷瘤小鼠静态Micro PET显像，在无VER存在情况下Bcap37肿瘤组织SUVmean要显著高于Bcap37/MDR1肿瘤组织（1.00±0.06、0.66±0.11，P<0.05）；Bcap37肿瘤组织18F-FDG摄取水平在有或无VER存在情况下无明显差异（1.00±0.06、1.09±0.22，P>0.05）；Bcap37/MDR1肿瘤组织在VER存在情况下，18F-FDG摄取水平要明显高于无VER存在时（1.01±0.16、0.66±0.11，P<0.05）。Bcap37/MDR1肿瘤标本细胞膜P-gp免疫组化强染色，Bcap37肿瘤组织细胞膜几乎无P-gp免疫组化染色；Bcap37/MDR1肿瘤组织细胞膜可见Glut-1免疫组化强染色，Bcap37肿瘤组织细胞膜仅有轻度Glut-1免疫组化染色；Bcap37/MDR1与Bcap37肿瘤组织细胞浆内见轻-中度HK-II免疫组化染色。结论动物实验与细胞实验结果相吻合，提示18F-FDG是P-gp的底物。18F-FDG结合VER可能是一种有效、无创检测肿瘤多药耐药的方法。荷Bcap37/MDR1肿瘤细胞裸鼠肿瘤组织18F-FDG在有/无VER存在时呈特征性改变，提示18F-FDG PET或PET/CTList Mode采集在探测肿瘤多药耐药方面可能具有潜在的临床应用价值。
Part Ⅰ Verification of P-glycoprotein functional and detection of someprotein related to18F-FDG uptake expressed in cell lines
Objective The expression of P-glycoprotein (P-gp), Glut-1and HK-II in Bcap37/MDR1and Bcap37cells were detected using Western Blot. The function of P-gp was evaluatedusing Rhodamine123.
Methods The proteins for Western Blot assay were extracted in Bcap37/MDR1andBcap37cells. For the function evaluation, Bcap37/MDR1and Bcap37cells were seeded insix-well plates. Those cells were incubated using serum-free RPMI1640mediumcontaining5μmoL/L Rhodamine123with or without100μmol/L verapamil (VER) for1hat37℃. Then the cells were washed with ice-cold phosphate buffered saline (PBS) forthree times and analyzed by the fluorescent microscopy.
Results P-gp and Glut-1were strongly expressed in Bcap37/MDR1cells, but weaklyexpressed in Bcap37cells. HK-II was expressed strongly both in Bcap37/MDR1andBcap37cells. Bcap37cells exhibited bright fluorescent staining in the presence ofRhodamine123. In contrast, Bcap37/MDR1cells showed only very weak fluorescence.Additionally, inhibition of the P-gp function by adding VER resulted in a marked increaseof the fluorescence in Bcap37/MDR1cells. There was no significant difference in thefluorescence of Bcap37cells in the presence or absence of VER.
Conclusions Functional P-gp was strongly expressed in Bcap37/MDR1cells.Bcap37/MDR1and Bcap37cells can be used to evaluate the relationship between P-gp and18F-FDG and99mTc-MIBI uptake.
Part Ⅱ The uptake of99mTc-MIBI in Bcap37and Bcap37/MDR1cells
Objective To evaluate the uptake kinetics change of99mTc-MIBI in Bcap37andBcap37/MDR1cells using the classical inhibitors of P-gp: VER or GF120918.Methods Bcap37and Bcap37/MDR1were plated in six-well plates3days prior to theexperiments at a density of1×106/well. On the day of experiment,74KBq/mL99mTc-MIBI,74KBq/mL99mTc-MIBI+100μmoL/L VER or74KBq/mL99mTc-MIBI+50μmoL/LGF120918was added to each well. After an incubation period of10min,30min,60minand120min (37℃,5%CO2), the medium was removed and immediately washed threetimes with1mL ice-cold PBS. The cells were collected from the wells usingtrypsin-ethylenediaminetetraacetic acid (EDTA) treatment. The radioactivity of99mTc-MIBI was immediately determined using a gamma counter. The uptake of99mTc-MIBI was expressed at overall99mTc-MIBI radioactivity in Bcap37orBcap37/MDR1cells to the overall radioactivity added to the cells. Three independentexperiments were performed.
Results The uptake of99mTc-MIBI had no significantly difference with or without VER orGF120918in Bcap37cells. VER or GF120918significantly increased the uptake of99mTc-MIBI in Bcap37/MDR1cells after an incubation period of30min,60min and120min (P<0.05). The uptake of99mTc-MIBI was significantly higher in Bcap37cells than inBcap37/MDR1cells after incubation for30min,60min and120min (P<0.05). The uptakeof99mTc-MIBI in Bcap37/MDR1was a little lower than that in Bcap37cells in thepresence of VER or GF120918.
Conclusions The difference in the uptake of99mTc-MIBI between Bcap37andBcap37/MDR1reflected the expression level of P-gp. The uptake kinetics of99mTc-MIBImay be used to evaluate the function of P-gp. The inhibitors of P-gp (VER and GF120918)can significantly increase the uptake of99mTc-MIBI in Bcap37/MDR1cells. The change ofthe uptake of99mTc-MIBI in multidrug resistant cell lines can be used to evaluate theinhibitor efficiency of P-gp inhibitors.
Part Ⅲ The uptake of18F-FDG in Bcap37and Bcap37/MDR1cells
Objective To evaluate the uptake kinetics change of18F-FDG in Bcap37and Bcap37/MDR1cells in the presence or absence of VER or GF120918, and to elucidate therelationship of18F-FDG uptake and P-gp expression at the cellular level.
Methods Bcap37and Bcap37/MDR1cells were plated at a density of1×106/well insix-well plates3days prior to the experiments. On the day of experiment,37KBq/mL18F-FDG,37KBq/mL18F-FDG+100μmol/L VER or37KBq/mL18F-FDG+50μmoL/LGF120918was added to each well. After an incubation period of10min,30min,60minand120min (37℃,5%CO2), the medium was removed and the cells were washed threetimes with1mL ice-cold PBS immediately. The cells were collected from the wells bytrypsin-EDTA treatment. The radioactivity of18F-FDG was determined immediately usinga gamma counter. The uptake of18F-FDG was expressed at overall18F-FDG radioactivityin Bcap37or Bcap37/MDR1cells to the overall radioactivity added to the cells. Threeindependent experiments were performed.
Results18F-FDG uptake was higher in Bcap37/MDR1cells than that in Bcap37cells afterincubation of10min. The uptake rate of18F-FDG was1.88%±0.19%in Bcap37/MDR1cells and1.37%±0.18%in Bcap37cells (P<0.05). In contrast,18F-FDG uptake wassignificantly higher in Bcap37cells than that in Bcap37/MDR1cells after60min and120min incubation. The uptake rate of18F-FDG was2.29%±0.23%,2.34%±0.15%inBcap37/MDR1cells and1.47%±0.14%,1.53%±0.22%in Bcap37cells (P<0.05).18F-FDG uptake was significantly higher in the presence of VER or GF120918inBcap37/MDR1cells than that in the absence of VER or GF120918after60min and120min of incubation (P<0.05), the uptake rate of18F-FDG in the presence of VER orGF120918was2.45%±0.21%,2.46%±0.25%and2.50%±0.24%,2.48%±0.27%.VER or GF120918did not influence the uptake of18F-FDG in Bcap37cells.
Conclusions18F-FDG was a substrate of P-gp in cellular experiments. P-gp may act as anefflux pump to reduce18F-FDG uptake in Bcap37/MDR1cells. The kinetics of the uptakeof18F-FDG can be used to evaluate the function of P-gp.18F-FDG may have potentialvalue for detecting P-gp in vivo.
Part Ⅳ The uptake of99mTc-MIBI in Bcap37and Bcap37/MDR1tumors
Objective Planar scintigraphic images of99mTc-MIBI were acquired in BALB/c severecombined immune deficiency (SCID) mice carrying Bcap37or Bcap37/MDR1tumor. Itwas to be evaluated whether99mTc-MIBI imaging could be used to reflect the function ofP-gp in vivo.
Methods Bcap37or Bcap37/MDR1cells (1×107cells/mL diluted using RPMI1640culture media,0.2mL) were injected into the subcutaneous tissue of the right axillary fossain SCID mice. The animals underwent99mTc-MIBI SPECT imagine when the solid tumorshad grown to a size of1~1.5cm (after3weeks for Bcap37cells and4weeks forBcap37/MDR1cells). Volumes of200μL99mTc-MIBI, dissolved in0.9%NaCl to a finalconcentration of37MBq/200μL, was injected into mice via the tail vein using a1mLLuer syringe. Five mice carried Bcap37tumors, and five carried Bcap37/MDR1tumors.Image acquisition was performed using a three-head gamma camera equipped with alow-energy high-resolution parallel-hole collimator. Planar scintigraphic images wereacquired at10min and60min after the injection of99mTc-MIBI. The VER-treated animalswere co-injected with0.5mg/Kg VER and37MBq99mTc-MIBI intravenously as a bolus.Image acquisition was performed at10min and60min after injection of99mTc-MIBI. Afterthe scintigraphy were completed, the animals were sacrificed by cervical dislocation.Tissue specimens of tumor and liver were taken from the animals for electron microscopy.Mitochondria was compared between tumor cells and liver cells using electron microscopy.
Results Planar scintigraphic images of SCID mice showed that the physiologic distributionof99mTc-MIBI uptake was in the liver, intestine, and urinary bladder. In addition, theinjection site at the tail was visible in several animals. There was no significant differenceof99mTc-MIBI uptake between the mice carrying Bcap37or those carrying Bcap37/MDR1tumors. The tumors were not visible in all mice. In addition, no significant differenceswere observed in the images obtained at10min and60min after injection. The tumorswere not visible in the mice treated with VER. Electron microscopy revealed that Bcap37and Bcap37/MDR1tumor cells showing almost no99mTc-MIBI uptake in vivo containedonly a few mitochondria, whereas hepatocytes, displaying high99mTc-MIBI uptake in vivo, were packed densely with mitochondria.
Conclusions Neither Bcap37nor Bcap37/MDR1tumors could be detected in the SCIDmice with or without VER-treated. The cell lines, with high99mTc-MIBI uptake and richmitochondria, should be selected to evaluate the function of P-gp in vivo.
Part Ⅴ The uptake of18F-FDG in the Bcap37and Bcap37/MDR1tumorsObjective To evaluate the relationship between18F-FDG uptake and P-gp expression usingBALB/c the SCID mice carrying Bcap37or Bcap37/MDR1tumors.
Methods The mice were fasted for6h before the trace injection and maintained underisoflurane anesthesia during the injection and scanning periods. After anesthesia, theintravenous injection of0.2mL7.4MBq was completed as a bolus. The dynamic PETscans were carried out for90min. On the PET images, the tumors were covered with oneregion of interest (ROI), and the time-activity curves were obtained. The kinetics of18F-FDG uptake in mice carrying Bcap37or Bcap37/MDR1tumors in the presence orabsence of VER was compared. Ten mice, five with Bcap37tumors and five withBcap37/MDR1tumors, were used, and approximately7.4MBq/0.2mL18F-FDG with orwithout0.5mg/Kg VER was administered via the tail vein. The Micro-PET images wereacquired60min after injection. To quantify the uptake of18F-FDG by the tumors, ROIswere drawn over the tumors and SUVmean was obtained. After the micro-PET imaginewas completed, the animals were sacrificed by cervical dislocation. The tumors were cutand embedded in10%paraffin. P-gp, Glut-1and HK-II were detected usingimmunohistochemical stain.
Results18F-FDG uptake in Bcap37tumors gradually increased to a peak level at10min to15min and then remained at a constant level. VER did not significantly alter the18F-FDGaccumulation curve in Bcap37tumors.18F-FDG uptake in Bcap37/MDR1tumorsgradually increased until9min and then decreased to a constant level at25min to30min.18F-FDG uptake in Bcap37/MDR1tumors treated with VER increased to a peak level at9min, decreased to a minimum level at15min and then increased again to a peak level atapproximately30min and remained constant level. Bcap37/MDR1tumors showed higher 18F-FDG accumulation than Bcap37tumors from0to9min. In static micro-PET imaging,the mean standardized uptake value (SUVmean) of18F-FDG was significantly higher inBcap37tumors than that in Bcap37/MDR1tumors (1.00±0.06、0.66±0.11，P<0.05). VERdid not influence18F-FDG uptake in Bcap37tumors (1.00±0.06、1.09±0.22，P>0.05).VER significantly increased the SUVmean in Bcap37/MDR1tumors (1.01±0.16、0.66±0.11, P<0.05). Bcap37/MDR1tumors showed strong P-gp and Glut-1immunoreaction,whereas Bcap37tumors showed almost complete absence of P-gp and Glut-1immunoreaction. Both Bcap37/MDR1and Bcap37tumors showed moderate HK-IIimmunoreaction.
Conclusions It was indicated that18F-FDG was a substrate of P-gp in vivo experiments.18F-FDG combined with VER may be an effective noninvasive method for the diagnosis ofP-gp expression in tumors. In particular, the18F-FDG uptake kinetics curves hadcharacteristic shapes for the mice carrying Bcap37/MDR1tumors. It indicated that18F-FDG PET or PET/CT using the ListMode acquisition mode may have potential clinicalvalue for detecting P-gp in vivo.
目的利用Western Blot法测定Bcap37/MDR1与Bcap37细胞中P-糖蛋白（P-glycoprotein，P-gp）、葡萄糖转运蛋白1（Glucose transporter1，Glut-1）及己糖激酶-II（Hexokinase-II，HK-II）表达，利用罗丹明123荧光染色评价P-gp功能。方法提取Bcap37/MDR1及Bcap37细胞蛋白，采用Western Blot法测定细胞中P-gp、Glut-1及HK-II的表达情况，在有或无P-gp抑制剂维拉帕米（Verapamil，VER）存在的情况下，分别将Bcap37/MDR1与Bcap37细胞与罗丹明123共孵育60min，然后吸去上清液，再用冰冷的PBS液洗脱3次，用荧光显微镜观察细胞内罗丹明123荧光强度。
结果P-gp在Bcap37/MDR1细胞中有高水平表达，而Bcap37细胞几乎无P-gp表达；Bcap37/MDR1细胞有较高水平的Glut-1表达，而Bcap37细胞仅表现为非常弱的Glut-1的表达； HK-II在两种细胞中均有较高水平表达。Bcap37、Bcap37/MDR1细胞与罗丹明123共孵育，Bcap37细胞内有明显荧光染色，Bcap37/MDR1细胞内仅有弱的荧光染色。加入VER后，Bcap37/MDR1细胞内的荧光染色明显增强。VER对Bcap37细胞荧光染色无明显影响。
结论P-gp、Glut-1在Bcap37/MDR1细胞中的表达明显强于Bcap37细胞；Bcap37/MDR1所表达的P-gp具有相应的外排功能，该细胞能够用来评价P-gp与18F-FDG、99mTc-MIBI摄取之间的关系。
第二部分99mTc-MIBI在Bcap37、Bcap37/MDR1细胞中的聚集实验
目的通过99mTc-MIBI细胞内摄取动力学研究99mTc-MIBI能否评价Bcap37/MDR1细胞P-gp功能及在评价P-gp抑制剂抑制效能中的作用。方法培养Bcap37及Bcap37/MDR1细胞，将Bcap37及Bcap37/MDR1细胞接种于6孔细胞培养板上，接种密度为1×106细胞/孔，将99mTc-MIBI、VER及GF120918用不含胎牛血清的RPMI1640培养基分别配成74KBq/mL99mTc-MIBI溶液、74KBq/mL99mTc-MIBI+100μmol/L VER溶液及74KBq/mL的99mTc-MIBI+50μmol/L GF120918溶液。用上述配制好的溶液孵育Bcap37及Bcap37/MDR1细胞，分别于10min、30min、60min及120min吸取孵育液，置于试管内，用冰冷的PBS液洗脱3次，洗脱液一并放置相应的试管内。然后采用胰酶消化细胞，消化完成后吸出细胞置于试管内，再分别用1mL PBS液洗脱3次，洗脱液置于放置细胞的试管内，采用γ计数仪测量各个试管内的放射性计数，计算Bcap37、Bap37/MDR1细胞中99mTc-MIBI的摄取率。摄取率=细胞内放射性计数/（细胞内放射性计数+上清液放射性计数）×100%，实验重复三次。计算结果用x±s表示，两种细胞间及同种细胞不同处理组间99mTc-MIBI摄取率比较行配对t检验。
结果Bcap37细胞在有或无抑制剂存在的情况下，99mTc-MIBI摄取率之间无统计学差异；Bcap37/MDR1细胞在有抑制剂存在的情况下99mTc-MIBI摄取率在孵育30min、60min及120min要明显高于无抑制剂存在时的摄取率（P<0.05）；在无抑制剂存在的情况下，Bcap37细胞99mTc-MIBI摄取率在孵育30min、60min及120min要明显高于Bcap37/MDR1细胞（P<0.05）；在有抑制剂存在的情况下，Bcap37/MDR1细胞的99mTc-MIBI摄取率略低于Bcap37细胞，但两者无统计学差异。
结论99mTc-MIBI在Bcap37细胞与Bcap37/MDR1细胞中的摄取率有显著差异，P-gp抑制剂能够增加Bcap37/MDR1细胞的99mTc-MIBI摄取率，表明可以用99mTc-MIBI摄取动力学变化评价P-gp的功能；同时多药耐药细胞系99mTc-MIBI摄取率的变化能够用于评价P-gp抑制剂的抑制效率。
第三部分18F-FDG在Bcap37、Bcap37/MDR1细胞中的聚集实验
目的测定18F-FDG在Bcap37与Bcap37/MDR1细胞的摄取动力学变化，评价P-gp抑制剂存在时Bcap37与Bcap37/MDR1细胞对18F-FDG摄取的变化，在细胞水平上探讨18F-FDG摄取与P-gp表达之间的关系。方法将Bcap37及Bcap37/MDR1细胞分别接种于6孔细胞培养板上，细胞密度为1×106细胞/孔，将18F-FDG、VER及GF120918用不含胎牛血清的RPMI1640培养基分别配成37KBq/mL18F-FDG溶液、37KBq/mL18F-FDG+100μmol/L VER溶液及37KBq/mL的18F-FDG+50μmol/L GF120918溶液。分别用上述配制好的溶液孵育Bcap37及Bcap37/MDR1细胞，分别于10min、30min、60min及120min吸取孵育液置于试管内，再用冰冷的PBS液洗脱3次，洗脱液一并放置相应的试管内。细胞用胰酶消化，消化完成后吸出细胞置于试管内，用1mL PBS液洗脱3次，洗脱液置于放置细胞的试管内，用γ计数仪测量各个试管内的放射性计数，计算Bcap37、Bap37/MDR1细胞的18F-FDG摄取率，摄取率=细胞内放射性计数/（细胞内放射性计数+上清液放射性计数）×100%，实验重复三次。计算结果用x±s表示，两种细胞间及同种细胞不同处理组间18F-FDG摄取率比较行配对t检验。
结果无抑制剂存在的情况下，孵育10min时Bcap37/MDR1细胞中18F-FDG摄取率显著高于Bcap37细胞，其摄取率分别为1.88%±0.19%和1.37%±0.18%（P<0.05）；孵育60min和120min时Bcap37细胞中18F-FDG摄取率显著高于Bcap37/MDR1细胞，其摄取率分别为2.29%±0.23%、2.34%±0.15%和1.47%±0.14%、1.53%±0.22%（P<0.05）。VER或GF120918共孵育后，Bcap37/MDR1细胞在60min和120min时18F-FDG摄取率分别为2.45%±0.21%、2.46%±0.25%和2.50%±0.24%、2.48%±0.27%，较无抑制剂存在时明显增加（P<0.05）。Bcap37细胞在有无抑制剂存在的情况下，18F-FDG摄取率无明显变化，差异无统计学意义。
结论细胞实验结果表明18F-FDG是P-gp的底物之一，Bcap37/MDR1细胞与Bcap37细胞间18F-FDG摄取率的差异是由于P-gp所介导的外排引起的，可以用18F-FDG动力学变化评价肿瘤细胞的P-gp功能，细胞内试验结果显示18F-FDG有可能作为活体内无创性评价肿瘤细胞的P-gp功能的显像剂。
第四部分99mTc-MIBI在Bcap37、Bcap37/MDR1荷瘤小鼠显像研究
目的利用荷Bcap37/MDR1与Bcap37细胞的BALB/c裸鼠模型，研究99mTc-MIBI在荷瘤小鼠体内显像，探讨99mTc-MIBI显像可否用于活体内评价肿瘤细胞的多药耐药。方法取处于对数生长期的Bcap37及Bcap37/MDR1细胞，用无血清的RPMI1640培养基制成细胞悬液，细胞计数后用无血清的RPMI1640培养基将细胞浓度稀释为1×10~7/mL，按0.2mL/只接种于4~6周龄的BALB/c裸鼠右侧腋窝皮下，5只裸鼠接种Bcap37细胞，5只裸鼠接种Bcap37/MDR1细胞。待肿瘤长至直径1.0~1.5cm时进行99mTc-MIBI SPECT显像。将荷Bcap37细胞裸鼠及荷Bcap37/MDR1细胞裸鼠通过尾静脉给予37MBq（1mCi，0.2mL）配制好的99mTc-MIBI，分别于给药后10min及60min行平面静态显像，隔日在相同采集条件下，尾静脉给予37MBq（1mCi，0.2mL）配制好的含有0.5mg/Kg VER的99mTc-MIBI溶液，分别于给药后10min及60min行平面静态显像。显像结束后，处死荷瘤小鼠后迅速取出肿瘤组织及肝脏组织，制成电镜标本，观察荷瘤小鼠肿瘤组织及肝脏组织线粒体情况。
结果99mTc-MIBI生理性摄取可见于肝脏、肠道、肾脏及膀胱，部分荷瘤小鼠尾静脉注射部位可见轻度显影。荷Bcap37或Bcap37/MDR1肿瘤细胞裸鼠肿瘤部位在给予99mTc-MIBI后10min未见明显显影，60min后肿瘤部位同样未见明显显影。荷Bcap37或Bcap37/MDR1肿瘤细胞裸鼠在给予含0.5mg/Kg VER的99mTc-MIBI后10min及60min肿瘤部位均未见明显显影。荷Bcap37及Bcap37/MDR1肿瘤细胞裸鼠肿瘤组织细胞内线粒体数量较少，肝脏组织细胞内存在较多的线粒体，且线粒体体积明显大于肿瘤细胞内线粒体。
结论荷Bcap37与Bcap37/MDR1肿瘤细胞裸鼠肿瘤组织99mTc-MIBI未见显影，同时给予P-gp抑制剂后肿瘤组织仍未见显影，表明选用荷瘤小鼠行99mTc-MIBI显像评价P-gp功能时要筛选肿瘤细胞株，应挑选99mTc-MIBI摄取率高且肿瘤细胞内线粒体较丰富的细胞株。
第五部分18F-FDG在Bcap37、Bcap37/MDR1荷瘤小鼠显像研究
目的研究荷Bcap37/MDR1及Bcap37肿瘤细胞荷瘤小鼠在有/无VER存在情况下肿瘤组织的18F-FDG摄取，在活体内评价P-gp与18F-FDG摄取的关系。方法荷瘤小鼠禁食6h，将需要动态采集的荷瘤小鼠置于小动物PET床板上，异氟烷吸入麻醉，胶带固定，身体置于视野中心，维持异氟烷麻醉浓度为2%。床前经尾静脉给予7.4MBq（0.2mCi，0.2mL）18F-FDG，给药后立即采集，共采集90min，得到各个时间段的动态显像图像，绘制感兴趣区（region of interest, ROI）的时间放射性曲线（time activity curve，TAC）。比较荷Bcap37肿瘤细胞裸鼠与荷Bcap37/MDR1肿瘤细胞裸鼠肿瘤组织18F-FDG摄取的TAC曲线。相同采集及处理方法，尾静脉给予含有0.5mg/Kg VER的7.4MBq（0.2mCi，0.2mL）18F-FDG，获得VER治疗后肿瘤组织18F-FDG摄取的TAC曲线。观察VER治疗后其18F-FDG TAC曲线，并与未行VER治疗时进行比较。另取10只荷瘤小鼠（5只荷Bcap37肿瘤细胞，5只荷Bcap37/MDR1肿瘤细胞），采用静态采集方法行18F-FDG Micro PET显像。荷瘤小鼠禁食6h，吸入异氟烷预麻醉，尾静脉给予7.4MBq（0.2mCi，0.2mL）18F-FDG，60min后行静态采集。在相同的采集处理条件下，尾静脉给予含有0.5mg/Kg VER的7.4MBq（0.2mCi，0.2mL）18F-FDG，获得VER治疗后肿瘤组织18F-FDG的静态图像，计算肿瘤组织的SUVmean。比较VER治疗前后肿瘤组织18F-FDG SUVmean的变化。显像结束后，处死荷瘤小鼠后将肿瘤组织制成蜡块，肿瘤标本行P-gp、Glut-1及HK-II免疫组化染色。
结果18F-FDG Micro-PET动态显像显示Bcap37肿瘤组织18F-FDG摄取渐进性增高，在10~15min内达峰值水平，然后维持在平台水平；Bcap37/MDR1肿瘤组织18F-FDG摄取同样渐进性增高，在大约9min时达到峰值，然后18F-FDG摄取渐进性下降，在25~30min时达到平台，然后维持在平台水平。Bcap37/MDR1肿瘤组织18F-FDG摄取在0~9min要高于Bcap37肿瘤组织，9min后Bcap37肿瘤组织18F-FDG摄取水平要高于Bcap37/MDR1肿瘤组织。有或无VER存在情况下Bcap37肿瘤组织18F-FDG摄取动态曲线无明显差异；Bcap37/MDR1肿瘤组织在有或无VER存在的情况下，其0~15min18F-FDG摄取动态曲线无明显差异，但在15min下降到最低点后，18F-FDG摄取开始渐进性升高，约30min达到最高水平，然后维持在平台水平。VER显著增加Bcap37/MDR1肿瘤组织平台期18F-FDG摄取水平。荷瘤小鼠静态Micro PET显像，在无VER存在情况下Bcap37肿瘤组织SUVmean要显著高于Bcap37/MDR1肿瘤组织（1.00±0.06、0.66±0.11，P<0.05）；Bcap37肿瘤组织18F-FDG摄取水平在有或无VER存在情况下无明显差异（1.00±0.06、1.09±0.22，P>0.05）；Bcap37/MDR1肿瘤组织在VER存在情况下，18F-FDG摄取水平要明显高于无VER存在时（1.01±0.16、0.66±0.11，P<0.05）。Bcap37/MDR1肿瘤标本细胞膜P-gp免疫组化强染色，Bcap37肿瘤组织细胞膜几乎无P-gp免疫组化染色；Bcap37/MDR1肿瘤组织细胞膜可见Glut-1免疫组化强染色，Bcap37肿瘤组织细胞膜仅有轻度Glut-1免疫组化染色；Bcap37/MDR1与Bcap37肿瘤组织细胞浆内见轻-中度HK-II免疫组化染色。结论动物实验与细胞实验结果相吻合，提示18F-FDG是P-gp的底物。18F-FDG结合VER可能是一种有效、无创检测肿瘤多药耐药的方法。荷Bcap37/MDR1肿瘤细胞裸鼠肿瘤组织18F-FDG在有/无VER存在时呈特征性改变，提示18F-FDG PET或PET/CTList Mode采集在探测肿瘤多药耐药方面可能具有潜在的临床应用价值。
Part Ⅰ Verification of P-glycoprotein functional and detection of someprotein related to18F-FDG uptake expressed in cell lines
Objective The expression of P-glycoprotein (P-gp), Glut-1and HK-II in Bcap37/MDR1and Bcap37cells were detected using Western Blot. The function of P-gp was evaluatedusing Rhodamine123.
Methods The proteins for Western Blot assay were extracted in Bcap37/MDR1andBcap37cells. For the function evaluation, Bcap37/MDR1and Bcap37cells were seeded insix-well plates. Those cells were incubated using serum-free RPMI1640mediumcontaining5μmoL/L Rhodamine123with or without100μmol/L verapamil (VER) for1hat37℃. Then the cells were washed with ice-cold phosphate buffered saline (PBS) forthree times and analyzed by the fluorescent microscopy.
Results P-gp and Glut-1were strongly expressed in Bcap37/MDR1cells, but weaklyexpressed in Bcap37cells. HK-II was expressed strongly both in Bcap37/MDR1andBcap37cells. Bcap37cells exhibited bright fluorescent staining in the presence ofRhodamine123. In contrast, Bcap37/MDR1cells showed only very weak fluorescence.Additionally, inhibition of the P-gp function by adding VER resulted in a marked increaseof the fluorescence in Bcap37/MDR1cells. There was no significant difference in thefluorescence of Bcap37cells in the presence or absence of VER.
Conclusions Functional P-gp was strongly expressed in Bcap37/MDR1cells.Bcap37/MDR1and Bcap37cells can be used to evaluate the relationship between P-gp and18F-FDG and99mTc-MIBI uptake.
Part Ⅱ The uptake of99mTc-MIBI in Bcap37and Bcap37/MDR1cells
Objective To evaluate the uptake kinetics change of99mTc-MIBI in Bcap37andBcap37/MDR1cells using the classical inhibitors of P-gp: VER or GF120918.Methods Bcap37and Bcap37/MDR1were plated in six-well plates3days prior to theexperiments at a density of1×106/well. On the day of experiment,74KBq/mL99mTc-MIBI,74KBq/mL99mTc-MIBI+100μmoL/L VER or74KBq/mL99mTc-MIBI+50μmoL/LGF120918was added to each well. After an incubation period of10min,30min,60minand120min (37℃,5%CO2), the medium was removed and immediately washed threetimes with1mL ice-cold PBS. The cells were collected from the wells usingtrypsin-ethylenediaminetetraacetic acid (EDTA) treatment. The radioactivity of99mTc-MIBI was immediately determined using a gamma counter. The uptake of99mTc-MIBI was expressed at overall99mTc-MIBI radioactivity in Bcap37orBcap37/MDR1cells to the overall radioactivity added to the cells. Three independentexperiments were performed.
Results The uptake of99mTc-MIBI had no significantly difference with or without VER orGF120918in Bcap37cells. VER or GF120918significantly increased the uptake of99mTc-MIBI in Bcap37/MDR1cells after an incubation period of30min,60min and120min (P<0.05). The uptake of99mTc-MIBI was significantly higher in Bcap37cells than inBcap37/MDR1cells after incubation for30min,60min and120min (P<0.05). The uptakeof99mTc-MIBI in Bcap37/MDR1was a little lower than that in Bcap37cells in thepresence of VER or GF120918.
Conclusions The difference in the uptake of99mTc-MIBI between Bcap37andBcap37/MDR1reflected the expression level of P-gp. The uptake kinetics of99mTc-MIBImay be used to evaluate the function of P-gp. The inhibitors of P-gp (VER and GF120918)can significantly increase the uptake of99mTc-MIBI in Bcap37/MDR1cells. The change ofthe uptake of99mTc-MIBI in multidrug resistant cell lines can be used to evaluate theinhibitor efficiency of P-gp inhibitors.
Part Ⅲ The uptake of18F-FDG in Bcap37and Bcap37/MDR1cells
Objective To evaluate the uptake kinetics change of18F-FDG in Bcap37and Bcap37/MDR1cells in the presence or absence of VER or GF120918, and to elucidate therelationship of18F-FDG uptake and P-gp expression at the cellular level.
Methods Bcap37and Bcap37/MDR1cells were plated at a density of1×106/well insix-well plates3days prior to the experiments. On the day of experiment,37KBq/mL18F-FDG,37KBq/mL18F-FDG+100μmol/L VER or37KBq/mL18F-FDG+50μmoL/LGF120918was added to each well. After an incubation period of10min,30min,60minand120min (37℃,5%CO2), the medium was removed and the cells were washed threetimes with1mL ice-cold PBS immediately. The cells were collected from the wells bytrypsin-EDTA treatment. The radioactivity of18F-FDG was determined immediately usinga gamma counter. The uptake of18F-FDG was expressed at overall18F-FDG radioactivityin Bcap37or Bcap37/MDR1cells to the overall radioactivity added to the cells. Threeindependent experiments were performed.
Results18F-FDG uptake was higher in Bcap37/MDR1cells than that in Bcap37cells afterincubation of10min. The uptake rate of18F-FDG was1.88%±0.19%in Bcap37/MDR1cells and1.37%±0.18%in Bcap37cells (P<0.05). In contrast,18F-FDG uptake wassignificantly higher in Bcap37cells than that in Bcap37/MDR1cells after60min and120min incubation. The uptake rate of18F-FDG was2.29%±0.23%,2.34%±0.15%inBcap37/MDR1cells and1.47%±0.14%,1.53%±0.22%in Bcap37cells (P<0.05).18F-FDG uptake was significantly higher in the presence of VER or GF120918inBcap37/MDR1cells than that in the absence of VER or GF120918after60min and120min of incubation (P<0.05), the uptake rate of18F-FDG in the presence of VER orGF120918was2.45%±0.21%,2.46%±0.25%and2.50%±0.24%,2.48%±0.27%.VER or GF120918did not influence the uptake of18F-FDG in Bcap37cells.
Conclusions18F-FDG was a substrate of P-gp in cellular experiments. P-gp may act as anefflux pump to reduce18F-FDG uptake in Bcap37/MDR1cells. The kinetics of the uptakeof18F-FDG can be used to evaluate the function of P-gp.18F-FDG may have potentialvalue for detecting P-gp in vivo.
Part Ⅳ The uptake of99mTc-MIBI in Bcap37and Bcap37/MDR1tumors
Objective Planar scintigraphic images of99mTc-MIBI were acquired in BALB/c severecombined immune deficiency (SCID) mice carrying Bcap37or Bcap37/MDR1tumor. Itwas to be evaluated whether99mTc-MIBI imaging could be used to reflect the function ofP-gp in vivo.
Methods Bcap37or Bcap37/MDR1cells (1×107cells/mL diluted using RPMI1640culture media,0.2mL) were injected into the subcutaneous tissue of the right axillary fossain SCID mice. The animals underwent99mTc-MIBI SPECT imagine when the solid tumorshad grown to a size of1~1.5cm (after3weeks for Bcap37cells and4weeks forBcap37/MDR1cells). Volumes of200μL99mTc-MIBI, dissolved in0.9%NaCl to a finalconcentration of37MBq/200μL, was injected into mice via the tail vein using a1mLLuer syringe. Five mice carried Bcap37tumors, and five carried Bcap37/MDR1tumors.Image acquisition was performed using a three-head gamma camera equipped with alow-energy high-resolution parallel-hole collimator. Planar scintigraphic images wereacquired at10min and60min after the injection of99mTc-MIBI. The VER-treated animalswere co-injected with0.5mg/Kg VER and37MBq99mTc-MIBI intravenously as a bolus.Image acquisition was performed at10min and60min after injection of99mTc-MIBI. Afterthe scintigraphy were completed, the animals were sacrificed by cervical dislocation.Tissue specimens of tumor and liver were taken from the animals for electron microscopy.Mitochondria was compared between tumor cells and liver cells using electron microscopy.
Results Planar scintigraphic images of SCID mice showed that the physiologic distributionof99mTc-MIBI uptake was in the liver, intestine, and urinary bladder. In addition, theinjection site at the tail was visible in several animals. There was no significant differenceof99mTc-MIBI uptake between the mice carrying Bcap37or those carrying Bcap37/MDR1tumors. The tumors were not visible in all mice. In addition, no significant differenceswere observed in the images obtained at10min and60min after injection. The tumorswere not visible in the mice treated with VER. Electron microscopy revealed that Bcap37and Bcap37/MDR1tumor cells showing almost no99mTc-MIBI uptake in vivo containedonly a few mitochondria, whereas hepatocytes, displaying high99mTc-MIBI uptake in vivo, were packed densely with mitochondria.
Conclusions Neither Bcap37nor Bcap37/MDR1tumors could be detected in the SCIDmice with or without VER-treated. The cell lines, with high99mTc-MIBI uptake and richmitochondria, should be selected to evaluate the function of P-gp in vivo.
Part Ⅴ The uptake of18F-FDG in the Bcap37and Bcap37/MDR1tumorsObjective To evaluate the relationship between18F-FDG uptake and P-gp expression usingBALB/c the SCID mice carrying Bcap37or Bcap37/MDR1tumors.
Methods The mice were fasted for6h before the trace injection and maintained underisoflurane anesthesia during the injection and scanning periods. After anesthesia, theintravenous injection of0.2mL7.4MBq was completed as a bolus. The dynamic PETscans were carried out for90min. On the PET images, the tumors were covered with oneregion of interest (ROI), and the time-activity curves were obtained. The kinetics of18F-FDG uptake in mice carrying Bcap37or Bcap37/MDR1tumors in the presence orabsence of VER was compared. Ten mice, five with Bcap37tumors and five withBcap37/MDR1tumors, were used, and approximately7.4MBq/0.2mL18F-FDG with orwithout0.5mg/Kg VER was administered via the tail vein. The Micro-PET images wereacquired60min after injection. To quantify the uptake of18F-FDG by the tumors, ROIswere drawn over the tumors and SUVmean was obtained. After the micro-PET imaginewas completed, the animals were sacrificed by cervical dislocation. The tumors were cutand embedded in10%paraffin. P-gp, Glut-1and HK-II were detected usingimmunohistochemical stain.
Results18F-FDG uptake in Bcap37tumors gradually increased to a peak level at10min to15min and then remained at a constant level. VER did not significantly alter the18F-FDGaccumulation curve in Bcap37tumors.18F-FDG uptake in Bcap37/MDR1tumorsgradually increased until9min and then decreased to a constant level at25min to30min.18F-FDG uptake in Bcap37/MDR1tumors treated with VER increased to a peak level at9min, decreased to a minimum level at15min and then increased again to a peak level atapproximately30min and remained constant level. Bcap37/MDR1tumors showed higher 18F-FDG accumulation than Bcap37tumors from0to9min. In static micro-PET imaging,the mean standardized uptake value (SUVmean) of18F-FDG was significantly higher inBcap37tumors than that in Bcap37/MDR1tumors (1.00±0.06、0.66±0.11，P<0.05). VERdid not influence18F-FDG uptake in Bcap37tumors (1.00±0.06、1.09±0.22，P>0.05).VER significantly increased the SUVmean in Bcap37/MDR1tumors (1.01±0.16、0.66±0.11, P<0.05). Bcap37/MDR1tumors showed strong P-gp and Glut-1immunoreaction,whereas Bcap37tumors showed almost complete absence of P-gp and Glut-1immunoreaction. Both Bcap37/MDR1and Bcap37tumors showed moderate HK-IIimmunoreaction.
Conclusions It was indicated that18F-FDG was a substrate of P-gp in vivo experiments.18F-FDG combined with VER may be an effective noninvasive method for the diagnosis ofP-gp expression in tumors. In particular, the18F-FDG uptake kinetics curves hadcharacteristic shapes for the mice carrying Bcap37/MDR1tumors. It indicated that18F-FDG PET or PET/CT using the ListMode acquisition mode may have potential clinicalvalue for detecting P-gp in vivo.
引文
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