CN110504017B - 一种示踪剂的制备和使用方法 - Google Patents
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Abstract
本发明公开一种评价ALPPS术后肝脏再生能力的分子影像学方法。包括以下步骤：1）制作VX2兔肝癌模型2）兔VX2肝癌模型ALPPS手术3）18F‑氟代甲基胆碱（18F‑methylcholine，18F‑FCH）合成。4）18F‑FCH PET/CT成像及数据处理。本发明开创性的首次提出使用18F‑FCH PET/CT监测残余肝脏的增殖能力，基于18F‑FCH在残余肝脏组织中具有较高的胆碱代谢，进而间接反映细胞膜合成能力增加，从而通过分子影像学方法评价ALPPS术后残余肝脏再生能力，为临床选择ALPPS选择二期手术的最佳时间提供了重要的新思路。
Description
技术领域
本发明涉及医学领域，具体为一种评价ALPPS术后肝脏再生能力的分子影像学方法。
背景技术
在现有技术中，联合离断和门静脉结扎的二步肝切除术(associating liverpartition and portal vein ligation for staged hepatectomy,ALPPS)是一种新兴的肝脏恶性肿瘤手术方式。它通过诱导残余肝脏组织(future liver remnant,FLR)迅速增殖，有效减少了肝癌切除术后由于残余肝脏体积不足造成的肝衰竭，从而增加了根治性切除的几率。ALPPS一期手术后残余肝脏再生能力和功能对于选择最佳的二期手术时间尤为重要。以往的临床研究通过传统影像学技术如CT或者MRI来测量术后残余肝脏的体积，间接预测患者术后肝功能情况来指导临床进一步行二期切除术。传统的影像学方法虽能测定残余肝脏体积，但残余肝脏体积并不完全等于肝功能，对改善ALPPS患者预后的评估具有很大的局限性。血清学方法检测肝脏功能往往不能及时反映肝脏功能变化，具有很大的误差。因此ALPPS一期术后残余肝脏再生能力的实时、动态、准确的监测和评估是个难题，需要一个新方法来解决。分子影像学技术可以通过评价增殖、代谢等多方面的功能学信息，来监测肿瘤或器官对治疗的反应。本发明主要通过18F-氟代甲基胆碱(18F-methylcholine，18F-FCH)PET/CT成像，通过反映肝脏细胞膜磷脂合成间接地反映肝脏再生能力。分子影像学在研究ALPPS术后病理和生理变化方面很有前景。
发明内容
本发明的目的针对现有的评估ALPPS术后残余肝脏体积和功能存在诸多缺点，通过PET 成像技术，从而实时、动态、准确的监测和评估ALPPS一期术后残余肝脏再生能力，为临床指导二期手术的开展提供更加科学合理的依据。
本发明技术解决方案是：一种评价ALPPS术后肝脏再生能力的分子影像学方法，其特征在于步骤如下：
步骤一制作VX2兔肝癌模型。
首先将VX2瘤株经传代后接种兔后腿外侧肌肉内，经3周肿瘤生长至直径大小2.0-3.0cm；将兔后腿肌肉内肿瘤取出，剔除坏死组织，选取肿瘤边缘的鱼肉样组织，并用眼科剪将肿瘤组织剪碎成1mm3组织块，用生理盐水反复清洗，于无菌培养皿中备用；将实验兔全麻，呈仰卧位固定于动物手术台上；对兔腹部手术区备皮，消毒，铺无菌手术洞巾；于剑突下1cm 处沿腹白线注射利多卡因行局部麻醉，并作上腹正中切口，逐层切开腹壁，暴露肝脏左外侧叶；于肝叶较厚处划开形成一个约3mm×3mm大小的孔道，将3-5个瘤块植入其中，接种后用明胶海绵颗粒填塞肝脏切口，并按压止血，缝合切口，关腹。
步骤二兔VX2肝癌模型ALPPS手术。
兔肝种植VX2瘤株两周后，荷瘤兔行ALPPS一期手术。
荷瘤兔随机分成ALPPS组和假手术组(Sham组)；将ALPPS组兔术前禁食12小时，全身麻醉后，仰卧位固定于兔台上，术区备皮；于剑突下偏左注射利多卡因，对切口周围进行麻醉后开腹，切口长5-7cm，暴露并分离门静脉左支，并用4-0手术缝线结扎，待左肝与右肝之间的缺血线形成，用手术剪沿着缺血线离断肝实质；止血，缝合腹壁切口，关闭腹腔，消毒。
步骤三18F-FCH合成。
采用GE Tracerlab FX-FN模块全自动化合成；具体步骤如下。
(1)[18F]由回旋加速器PETtrace通过18O(p，n)18F反应生产。
(2)将[18F]氟化物从回旋加速器递送并捕获在QMA-Light Sep-Pak除去[18O]H2O。
(3)[18F]然后使用K2 CO3溶液；将氟化物洗脱到反应容器中。
(4)将kryptofix2.2.2的溶液加入到反应容器中；加热至80℃并抽真空4分钟。
(5)将反应容器冷却至60±1℃，同时进行氦气流，4分钟真空吸附。
(6)将二甲基二甲苯磺酸7-8mg在无水乙腈和无菌水中的溶液加入到干燥的[18F]氟化物中，并将反应物加热至120℃，同时搅拌10分钟；随后将反应混合物冷却至50℃。
(7)然后加入胆碱前体DMAE，将其加热至120℃，再搅拌10分钟。
(8)然后将反应混合物冷却至60℃，并通过保持60℃进行反应溶剂的蒸发，并使反应连续进行氦气流并真空抽吸5分钟。
(9)将无菌水加入到干燥后的反应物中，通过C18-Plus Sep-Pak进入含有乙醇的圆底烧瓶中以捕获未反应的ditosylmethane和[18F]氟甲基甲苯磺酸盐，以及作为副产物生成的任何甲苯磺酰甲基胆碱。
(10)将水/乙醇混合物通过CM-Light Sep-Pak转移至所需的[18F]氟甲基胆碱。
(11)用乙醇洗涤CM-Light Sep-Pak以除去未反应的DMAE和水，以除去残留的乙醇至废物。
(12)随后将[18F]氟甲基胆碱洗脱到含有0.9％注射用生理盐水。
(13)然后将最终制剂通过0.22mm无菌过滤器进入无菌剂量小瓶中，以提供18F-FCH作为等渗溶液，获得产物；产物18F-FCH放化纯度大于90％，比活度>30GBq/μmol，可用于 PET成像。
步骤四18F-FCH PET/CT成像及数据处理。
ALPPS组和Sham组荷瘤兔分别在手术前0d、术后1d、3d、7d、14d，进行18F-FCH PET/CT扫描。
1)兔禁食6小时以上，全身麻醉。
2)每组荷瘤兔经耳源静脉通道分别注入37MBq/kg(1mci/kg)18F-FCH。
3)注射18F-FCH 1h后进行PET/CT扫描；将实验兔仰卧位固定在扫描床，先行低剂量螺旋CT轴位扫描，再行PET发射扫描；用低剂量CT做衰减校正、迭代重建得到PET图像，将图像数据传入GE AW4.6 PET后处理工作站，得到实验兔的CT、PET及PET/CT的融合图像；CT扫描参数为：电压120KV，电流10mA，层厚3.33mm；PET采集方式：3D模式，扫描床位数1个，每个床位3min。
4)兔18F-FCH PET/CT图像上，勾画出残余肝脏的兴趣区进而获得最大标准摄取值和平均标准摄取值；同时通过GE AW4.6工作站，勾勒出肝脏三维结构，测得肝脏功能性体积。
本发明的优点是：
1、本发明是首次通过分子影像学技术来评价肝癌ALPPS一期术后残余肝脏形态学和功能学的变化，揭示肝脏的再生能力，进而指导临床选择二期手术的最佳时间。
2、以往临床选择ALPPS手术最佳时间，主要通过常规影像学检查测量肝脏体积或者血清学检查检测肝脏功能，这些方法都存在诸多局限性。本项发明通过18F-FCH PET/CT成像及数据处理技术，将肝脏再生的过程进行可视化，实时、动态的监测到了肝脏体积和功能的变化。
3、以往18F-FCH主要应用于诊断癌症。本发明是将18F-FCH用于评价肝脏再生能力，这是该分子探针首次应用于评价组织再生。
4、本发明提出来一个新的概念，叫做有效容积功能(Valid volumetricfunction,VVF)来反映残余肝脏真实的功能，它等FLRSUVmean×FLRVFCH)。该指标可将肝脏的功能和体积相结合，作为反映肝脏真实再生能力的技术指标。
以下结合附图和具体实施例，对本发明作进一步说明。
附图说明
图1:兔VX2肝癌ALPPS模型18F-FCH PET/CT成像(横轴位)。
图2:兔VX2肝癌ALPPS模型18F-FCH PET/CT成像(冠状位)。
图3:通过PET后处理技术，将肝脏进行三维重建，可见18F-FCH在ALPPS组肝脏的摄取变化和肝脏的形态学变化。
图4:A，18F-FCH SUVmax在ALPPS组和假手术组残余肝脏的对比。
图5:B，肝脏功能性体积在术后各个时间点变化。
图6:C，有效容积功能在术后各个时间点变化。
具体实施方式
一种评价ALPPS术后肝脏再生能力的分子影像学方法：
步骤一制作VX2兔肝癌模型
采用肝脏切开癌组织碎块法。首先将VX2瘤株经传代后接种兔后腿外侧肌肉内。约经3 周肿瘤生长至直径大小2.0-3.0cm。将兔后腿肌肉内肿瘤取出，剔除坏死组织，选取肿瘤边缘的鱼肉样组织，并用眼科剪将肿瘤组织剪碎成1mm3组织块，用生理盐水反复清洗，于无菌培养皿中备用。将实验兔全麻(麻醉方法：肌肉注射舒泰1ml)，呈仰卧位固定于动物手术台上。对兔腹部手术区备皮，消毒手术区皮肤，铺无菌手术洞巾。于剑突下约1cm处沿腹白线注射利多卡因行局部麻醉，并作一长约4-5cm的上腹正中切口，逐层切开腹壁，暴露肝脏左外侧叶；于肝叶较厚处，用眼科镊子划开一长约3mm的小切口，形成一个约3mm×3mm大小的孔道，将3-5个瘤块植入其中，接种后用明胶海绵颗粒填塞肝脏切口，并按压止血，缝合切口，关腹。
步骤二兔VX2肝癌模型ALPPS手术
兔肝种植VX2瘤株两周后，荷瘤兔行ALPPS一期手术(即联合门静脉结扎和肝脏离断术)。
荷瘤兔随机分成ALPPS组(肝门静脉左支被结扎，沿着肝左中叶和肝右叶间的缺血线进行肝实质的离断)和Sham组(即假手术组，未进行门静脉结扎和肝脏离断)。将ALPPS组兔术前禁食12小时，全身麻醉后，仰卧位固定于兔台上，术区备皮。于剑突下偏左注射利多卡因，对切口周围进行麻醉后开腹，切口长约5-7cm，暴露并分离门静脉左支，并用4-0手术缝线结扎，待左肝与右肝之间的缺血线形成，用手术剪沿着缺血线离断肝实质。用凝胶海绵颗粒填充离断处进行止血，无明显出血后，逐层缝合腹壁切口，关闭腹腔，切口处用碘伏消毒。
步骤三18F-FCH合成
采用GE Tracerlab FX-FN模块全自动化合成。具体步骤如下：
(1)[18F]由回旋加速器PETtrace通过18O(p，n)18F反应生产。
(2)将[18F]氟化物从回旋加速器递送并捕获在QMA-Light Sep-Pak除去[18O]H2O。
(3)[18F]然后使用K2CO3溶液(3.0mg，在0.5mL水中)将氟化物洗脱到反应容器中。
(4)将kryptofix2.2.2(15mg在1mL乙腈中)的溶液加入到反应容器中。通过将反应容器加热至80℃并抽真空4分钟。
(5)将反应容器冷却至60±1℃，同时进行氦气流，4分钟真空吸附。
(6)将二甲基二甲苯磺酸(ditosylmethane，7-8mg)在无水乙腈(750μl)和无菌水(10μl)中的溶液加入到干燥的[18F]氟化物中，并将反应物加热至120℃，同时搅拌10分钟。随后将反应混合物冷却至50℃。
(7)然后加入胆碱前体DMAE(40μl，在350μlMeCN中)，将其加热至120℃，再搅拌 10分钟。
(8)然后将反应混合物冷却至60℃，并通过保持60℃进行反应溶剂的蒸发，并使反应连续进行氦气流并真空抽吸5分钟。
(9)将无菌水(11mL)加入到干燥后的反应物中，通过C18-Plus Sep-Pak进入含有乙醇 (10mL)的圆底烧瓶中以捕获未反应的ditosylmethane和[18F]氟甲基甲苯磺酸盐，以及作为副产物生成的任何甲苯磺酰甲基胆碱。
(10)将水/乙醇混合物通过CM-Light Sep-Pak转移至所需的[18F]氟甲基胆碱。
(11)用乙醇(15mL)洗涤CM-Light Sep-Pak以除去未反应的DMAE和水(20mL)以除去残留的乙醇至废物。
(12)随后将[18F]氟甲基胆碱洗脱到含有0.9％注射用生理盐水(10mL)。
(13)然后将最终制剂(10mL)通过0.22mm无菌过滤器进入无菌剂量小瓶中，以提供18F-FCH作为等渗溶液，获得产物。产物18F-FCH放化纯度大于90％，比活度>30GBq/μmol，可用于PET成像。
步骤四18F-FCH PET/CT成像及数据处理
ALPPS组和假手术组(Sham组)荷瘤兔分别在手术前0d、术后1d、3d、7d、14d，进行18F-FCH PET/CT扫描。
1)兔禁食6小时以上，肌注舒泰1ml全身麻醉。
2)每组荷瘤兔经耳源静脉通道分别注入37MBq/kg(1mci/kg)18F-FCH。
3)注射18F-FCH 1h后进行PET/CT扫描。将实验兔仰卧位固定在扫描床，先行低剂量螺旋CT轴位扫描，再行PET发射扫描。用低剂量CT做衰减校正、迭代重建得到PET图像，将图像数据传入GE AW4.6 PET后处理工作站，得到实验兔的CT、PET及PET/CT的融合图像。CT扫描参数为：电压120KV，电流10mA，层厚3.33mm。PET采集方式：3D模式，扫描床位数1个，每个床位3min。
4)兔18F-FCH PET/CT图像上，勾画出残余肝脏的兴趣区(region of interest，ROI)进而获得最大标准摄取值(Maximal Standardized uptake value，SUVmax)和平均标准摄取值(Mean Standardized uptake value，SUVmean)。同时通过GE AW4.6工作站，可勾勒出肝脏三维结构，可测得肝脏功能性体积(基于＞42％SUVmax)
数据处理的结果
在肝癌ALPPS一期手术模型中，18F-FCH在ALPPS组的残余肝脏组织，从术后第1d起，就呈现出明显的高摄取，说明胆碱代谢增强，肝细胞增殖活跃，这与ALPPS手术引起残余肝脏体积迅速增大的现象相符合。在术后前3d，18F-FCH摄取迅速增加，到7d左右达到顶峰，说明7d左右肝脏细胞增殖最活跃，这也提示在此时肝脏再生能力最强，在14d后18F-FCH摄取稍微下降，说明肝脏再生能力已经开始下降。同时，通过PET成像技术，我们可重建肝脏三维结构，进而实时、动态、直观看到肝脏真正的功能变化。尽管7d后，肝脏18F-FCH摄取下降，但是体积仍在增加。此时体积已经不再能够反映肝脏真实的功能。我们定义一个有效容积功能(Valid volumetric function,VVF)来反映残余肝脏真实的功能，它等于FLRSUV mean×FLRV FCH。在我们这项发明，我们成功发现从3d起VVF显著升高，7d时VVF达到最高值，说明此时肝脏再生能力最强，也提示临床选择二期手术的最佳时间可能是一期术后的3-7d(参见图1、2、3、4、5、6。图4中，A残余肝脏18F-FCH SUV max；图5中，B残余肝脏功能性体积；图6中，C残余肝脏有效容积功能。*ALPPS组与Sham组相比，P＜ 0.05)。
本发明开创性的首次提出使用18F-FCH PET/CT监测残余肝脏的增殖能力，基于18F-FCH 在残余肝脏组织中具有较高的胆碱代谢，进而间接反映细胞膜合成能力增加，从而通过分子影像学方法评价ALPPS术后残余肝脏再生能力。虽然这些结果目前处于临床前研究，在人体的ALPPS二期手术的时间窗可能会有所不同，但是为临床选择ALPPS选择二期手术的最佳时间提供了重要的新思路。
Claims (1)
1.一种用作示踪剂的18F-FCH的制备和使用方法，其特征在于，所述18F-FCH用作示踪剂；
(一)采用GE Tracerlab FX-FN模块全自动化合成18F-FCH；包括如下步骤：
(1)[18F]由回旋加速器PETtrace通过18O(p，n)18F反应生产；
(2)将[18F]氟化物从回旋加速器递送并捕获在QMA-Light Sep-Pak除去[18O]H 2O；
(3)[18F]然后使用K2CO3溶液；将氟化物洗脱到反应容器中；
(4)将kryptofix 2.2.2的溶液加入到反应容器中；加热至80℃并抽真空4分钟；
(5)将反应容器冷却至60±1℃，同时进行氦气流，4分钟真空吸附；
(6)将二甲基二甲苯磺酸7-8mg在无水乙腈和无菌水中的溶液加入到干燥的[18F]氟化物中，并将反应物加热至120℃，同时搅拌10分钟；随后将反应混合物冷却至50℃；
(7)然后加入胆碱前体DMAE，将其加热至120℃，再搅拌10分钟；
(8)然后将反应混合物冷却至60℃，并通过保持60℃进行反应溶剂的蒸发，并使反应连续进行氦气流并真空抽吸5分钟；
(9)将无菌水加入到干燥后的反应物中，通过C18-Plus Sep-Pak进入含有乙醇的圆底烧瓶中以捕获未反应的ditosylmethane和[18F]氟甲基甲苯磺酸盐，以及作为副产物生成的任何甲苯磺酰甲基胆碱；
(10)将水/乙醇混合物通过CM-Light Sep-Pak转移至所需的[18F]-FCH；
(11)用乙醇洗涤CM-Light Sep-Pak以除去未反应的DMAE和水，以除去残留的乙醇至废物；
(12)随后将[18F]-FCH洗脱到含有0.9％注射用生理盐水；
(13)然后将最终制剂通过0.22mm无菌过滤器进入无菌剂量小瓶中，以提供18F-FCH作为等渗溶液，获得产物；产物18F-FCH放化纯度大于90％，比活度>30GBq/μmol，可用于PET成像；
(二)18F-FCH PET/CT成像及数据处理；包括如下步骤：
1)注射18F-FCH后进行PET/CT扫描时，先行低剂量螺旋CT轴位扫描，再行PET发射扫描；用低剂量CT做衰减校正、迭代重建得到PET图像，将图像数据传入GE AW4.6 PET后处理工作站，得到CT、PET及PET/CT的融合图像；CT扫描参数为：电压120KV，电流10mA，层厚3.33mm；PET采集方式：3D模式，扫描床位数1个，每个床位3min；
2)在18F-FCH PET/CT图像上，勾画出残余肝脏的兴趣区进而获得最大标准摄取值和平均标准摄取值；同时通过GE AW4.6工作站，勾勒出肝脏三维结构，获得有效容积功能，测得肝脏功能性体积。
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Also Published As
| Publication number | Publication date |
|---|---|
| CN110504017A (zh) | 2019-11-26 |
| US11123033B2 (en) | 2021-09-21 |
| US20210015439A1 (en) | 2021-01-21 |
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