[摘要] 目的:在CT引導(dǎo)下建立兔VX2椎體腫瘤模型,觀察各時(shí)期的CT、MRI、PET-CT表現(xiàn)。方法:選取15只健康新西蘭大白兔,在CT引導(dǎo)下經(jīng)皮穿刺,將VX2瘤塊分別接種到兔L5椎體內(nèi),種植后7、14、21 d行CT、MRI、PET-CT檢查,觀察種植后影像學(xué)表現(xiàn),評(píng)估腫瘤生長情況。結(jié)果:CT示種植后7 d僅部分椎體見穿刺損傷;14 d時(shí)13.3%(2/15)椎體內(nèi)可見明顯骨質(zhì)破壞;21 d時(shí)73.3%(11/15)可見明顯骨質(zhì)破壞,部分伴成骨性改變。MRI示種植后7 d時(shí)86.7%(13/15)椎體內(nèi)未發(fā)現(xiàn)腫瘤信號(hào);14 d時(shí)80.0%(12/15)椎體內(nèi)形成明顯信號(hào)對(duì)比,可顯示腫瘤;21 d時(shí)部分較大瘤體可突破椎體后緣,可見脊髓受壓。PET-CT檢查示種植后7 d時(shí)80.0%(12/15)椎體內(nèi)可見小范圍明顯核素濃聚;14、21 d濃聚區(qū)逐漸擴(kuò)大,程度逐漸加深。14只實(shí)驗(yàn)兔建模成功,建模成功率為93.3%。結(jié)論:CT引導(dǎo)下經(jīng)皮穿刺注入瘤塊建立兔脊柱腫瘤模型,技術(shù)成功率高。CT對(duì)骨質(zhì)破壞顯示敏感性低;MRI對(duì)椎體周圍結(jié)構(gòu)顯示清楚,可顯示脊髓受壓和明確腫瘤范圍;PET-CT是早期評(píng)估種瘤成功最敏感的方法。兔椎體腫瘤模型無論從骨質(zhì)破壞性質(zhì)上還是從病程進(jìn)展上均與人類脊柱轉(zhuǎn)移存在一定相似性,可為脊柱轉(zhuǎn)移瘤臨床治療的隨訪及評(píng)價(jià)提供參考。
[關(guān)鍵詞] 脊柱轉(zhuǎn)移瘤;兔椎體腫瘤模型;診斷顯像
Construction and imaging features of rabbit vertebral tumor model
ZUO Taiyang1,2,WANG Zhilong2,LIANG Yining2,JIANG Fangzhou2,WEI Fengqin1
1TCM School,Shandong University of Traditional Chinese Medicine,Jinan 250355,China;2Department of Oncology Intervention,Central Hospital Affiliated to Shandong First Medical University,Jinan 250013,China.
[Abstract] Objective:To establish of rabbit VX2 vertebral tumor model under CT guidance,and to observe CT,MRI and PET-CT features in different periods. Methods:Fifteen healthy New Zealand white rabbits were selected and percutaneously punctured under CT guidance. VX2 tumors were inoculated into the L5 vertebra of rabbits. CT,MRI and PET-CT were performed on the 7th,14th and 21st days after implantation,and the imaging findings were observed to evaluate the growth of the tumor. Results:CT images displayed that the puncture injury in part of some vertebrae on the 7th day,the obvious bone destruction in 2 vertebrae (13.3%) on the 14th day,and the obvious bone destruction in 11 vertebrae (73.3%) on the 21th day. MRI images displayed no tumor signal in 13 vertebrae (86.7%) on the 7th day,the significant contrast signal and visible tumors in 12 vertebrae (80.0%) on the 14th day,and vertebrae edge bone destruction and the compressed spinal cord cause by large tumors on the 21st day. PET-CT images displayed the small area of obvious nuclide concentration in 12 vertebrae (80.0%) on the 7th day,and the larger and stronger nuclide concentration on the 14th,21th days. Fourteen rabbits were successfully modeled,with a success rate of 93.3%. Conclusions:The rabbit spinal tumor model can be established using percutaneous injection of tumor mass under CT guidance with a high technical success rate. CT shows a low sensitivity to bone destruction. MRI can show clearly the structures around the vertebrae,spinal cord compression and the extent of tumor. PET-CT is the most sensitive method for early evaluation in the success of the experiment. The rabbit vertebral tumor model is similar to human spinal metastasis in terms of bone destruction and disease progression,which can provide references for the follow-up and evaluation of clinical treatment of spinal metastasis.
[Key words] Spinal metastases;Rabbit vertebral tumor model;Diagnostic imaging
脊柱是惡性腫瘤最常見的骨轉(zhuǎn)移部位,當(dāng)發(fā)生脊柱轉(zhuǎn)移時(shí),患者會(huì)出現(xiàn)疼痛、神經(jīng)脊髓壓迫、活動(dòng)障礙、病理性骨折、截癱等癥狀[1-2]。早發(fā)現(xiàn)、早治療可改善患者預(yù)后。動(dòng)物模型是研究腫瘤發(fā)病機(jī)制、進(jìn)展和探討治療方案的重要工具,在腫瘤的研究中具有重要意義[3]。本研究通過建立兔椎體腫瘤模型,利用CT、MRI和PET-CT技術(shù)動(dòng)態(tài)觀察腫瘤影像學(xué)變化,為臨床治療的隨訪及評(píng)價(jià)提供參考。
1" 材料與方法
1.1" 實(shí)驗(yàn)動(dòng)物
荷瘤兔選擇健康新西蘭大白兔1只,雄性,體質(zhì)量2 kg,3~4周齡。脊柱VX2腫瘤模型構(gòu)建選擇健康新西蘭大白兔15只,雌兔8只、雄兔7只,體質(zhì)量3.0~3.5 kg,月齡3~4個(gè)月[購自濟(jì)南西嶺角養(yǎng)殖繁育中心,動(dòng)物實(shí)驗(yàn)許可證號(hào):SCXK(魯)2020 0004]。本研究經(jīng)濟(jì)南市中心醫(yī)院動(dòng)物研究委員會(huì)批準(zhǔn)(JNCHIACUC2022-4),并按照國際動(dòng)物保護(hù)委員會(huì)的指導(dǎo)方針進(jìn)行。
1.2" 模型制備與影像學(xué)檢查方法
有創(chuàng)操作或影像學(xué)檢查前禁食4 h,經(jīng)兔耳靜脈注射2%戊巴比妥鈉全身麻醉,劑量1.5~2.0 mL/kg體質(zhì)量。
1.2.1" 負(fù)荷瘤兔制備
在37 ℃水浴鍋中快速解凍VX2實(shí)體瘤組織,離心去除凍存液。將VX2實(shí)體瘤組織剪碎為0.5~1.0 mm3小塊,后用1 mL磷酸緩沖鹽溶液(PBS)重懸,采用無菌注射器將其全部注射到荷瘤兔(質(zhì)量2 000 g)的大腿肌肉里。2~3周后大腿肌肉可觸及質(zhì)韌腫塊,負(fù)荷瘤種植成功。
1.2.2" 兔脊柱VX2腫瘤模型構(gòu)建
取大腿荷瘤兔,大腿部備皮后處死。大腿外側(cè)用碘伏消毒,剪開局部皮膚,鈍性分離皮下組織,剜出完整腫瘤塊,清除多余血管和壞死組織,剪碎成1 mm3大小,置于裝有生理鹽水的燒杯中備用。以上所有操作均無菌條件下完成。
模型兔取俯臥位固定于操作臺(tái)上。采用大孔徑Philips CT機(jī)掃描定位,取L5椎體最大層面作為目標(biāo)穿刺層面。消毒穿刺點(diǎn)周圍皮膚后用17 G錐形穿刺針在CT引導(dǎo)下沿椎體側(cè)后緣與椎弓根交界處穿刺入椎體中心,用針芯推送3塊1 mm3的瘤塊,用1 cm明膠海綿條封堵針道。
1.2.3" 兔脊柱VX2腫瘤模型影像學(xué)檢查
接種術(shù)后7、14、21 d行MRI、PET-CT、CT檢查。①M(fèi)RI檢查采用Philips 3.0 T MRI掃描儀和膝關(guān)節(jié)線圈,層厚1.5 mm,行矢狀位與軸位T1WI(TR 500 ms,TE 12 ms)、矢狀位與軸位T2WI(TR 2 000 ms,TE 75 ms)、矢狀位與軸位脂肪抑制T2WI(TR 2 000 ms,TE 75 ms)。②在PET-CT前禁食4 h,18F-FDG經(jīng)兔耳靜脈注射(27.8 MBq/kg體質(zhì)量);30~40 min后俯臥位固定行PET-CT掃描(GE Discovery MI型PET-CT)。CT掃描參數(shù):80 kV,300 mA,掃描速度39.8 mm/s,螺距1,以體質(zhì)量為基礎(chǔ)的方案和每個(gè)床位2 min的采集時(shí)間獲得PET圖像。PET圖像采用迭代算法重建,并應(yīng)用基于CT的衰減校正。在后處理工作站中融合來自PET的代謝圖像和來自CT的解剖圖像。③使用Philips大孔徑CT行CT掃描,120 kV,200 mA,層厚1 mm,層距1 mm,螺距1。
2" 結(jié)果
CT平掃示:種植后7 d,15個(gè)椎體均未見較大骨質(zhì)破壞,僅部分椎體見種植時(shí)穿刺通道損傷;14 d時(shí)2個(gè)(13.3%)椎體內(nèi)可見較大骨質(zhì)破壞;21 d時(shí)11個(gè)(73.3%)椎體可見明顯骨質(zhì)破壞,部分伴成骨性改變(圖1),其中4個(gè)(26.7%)骨質(zhì)破壞仍不明顯。
MRI檢查示:種植后7 d,13個(gè)(86.7%)椎體內(nèi)未發(fā)現(xiàn)腫瘤信號(hào),余2個(gè)可見較小腫瘤信號(hào);14 d時(shí)12個(gè)(80.0%)椎體內(nèi)可見腫瘤,但瘤體較小,多局限在椎體骨髓腔內(nèi);21 d時(shí)14個(gè)(93.3%)椎體可明顯顯示腫瘤,且部分較大瘤體可突破椎體后緣,并見脊髓受壓(圖2)。
PET-CT檢查示:種植后7 d,12個(gè)(80.0%)椎體內(nèi)可見小范圍明顯核素濃聚;14、21 d時(shí)均可見14個(gè)(93.3%)椎體內(nèi)有明顯核素濃聚,且濃聚區(qū)擴(kuò)大、程度加深(圖3)。
僅1個(gè)(6.7%)椎體種植21 d時(shí)CT、MRI、PET-CT均未見腫瘤,但椎旁可見巨大腫塊,考慮種植失??;14只實(shí)驗(yàn)兔建模成功,成功率93.3%。種植后前2周實(shí)驗(yàn)兔的運(yùn)動(dòng)行為與狀態(tài)與健康兔差異不大,2周后逐漸出現(xiàn)運(yùn)動(dòng)功能障礙直至癱瘓,進(jìn)展迅速。隨機(jī)抽取2只癱瘓實(shí)驗(yàn)兔處死,解剖后行病理檢查,可見腫瘤細(xì)胞彌漫性生長,未見明顯壞死(圖4)。
3" 討論
兔VX2腫瘤具有種植簡單、成瘤率高、侵襲性強(qiáng)、倍增速度快等特點(diǎn),且生物學(xué)特點(diǎn)和影像表現(xiàn)與人類惡性腫瘤相似,是模擬惡性腫瘤脊柱轉(zhuǎn)移的理想動(dòng)物模型[4-9]。系統(tǒng)注射瘤細(xì)胞和直接種植是常用的椎體腫瘤模型種植方式,系統(tǒng)注射雖可模擬腫瘤細(xì)胞的血行轉(zhuǎn)移途徑,但成功率低,易造成實(shí)驗(yàn)動(dòng)物因全身轉(zhuǎn)移早期死亡;而直接種植具有種植部位固定、模型同質(zhì)化、成功率高等優(yōu)點(diǎn)。Amundson等[10-11]經(jīng)背部后入路暴露椎弓根,直視下將VX2腫瘤細(xì)胞懸液/瘤塊注入兔椎體內(nèi),成功建立了兔椎體腫瘤動(dòng)物模型。但外科種植對(duì)實(shí)驗(yàn)動(dòng)物創(chuàng)傷大,死亡率相對(duì)較高。而CT引導(dǎo)下穿刺種植創(chuàng)傷小,成像清晰且方便操作。本研究進(jìn)一步提出了一種改進(jìn)的兔脊柱VX2腫瘤模型構(gòu)建方法:①選取新鮮腫瘤組織,質(zhì)韌,通過穿刺針通道時(shí)不易破碎,且細(xì)胞活力較強(qiáng),荷瘤成活率較高;②采用多瘤塊種植,可增加存活率并加快腫瘤生長速度;③采用穿透力強(qiáng)的17 G穿刺針,可進(jìn)入髓腔深處,容納更多的腫瘤組織,且穿刺針套管直徑較大,可順暢植入瘤塊及明膠海綿條;④在推注瘤塊后用剪好的明膠海綿小條對(duì)穿刺處進(jìn)行封堵,可進(jìn)一步將髓腔內(nèi)瘤塊推向髓腔深處,降低瘤塊椎外種植風(fēng)險(xiǎn)。
種植成功后,動(dòng)物能否正常站立、移動(dòng)和奔跑是脊髓功能最簡單的評(píng)估標(biāo)準(zhǔn)[10-11]。Ikeda等[12]按癥狀程度不同,將兔后肢的功能分為0~5級(jí)對(duì)應(yīng)正常至截癱。有的小鼠椎體腫瘤模型甚至評(píng)估了運(yùn)動(dòng)損傷和更細(xì)微的傷害性癥狀。Basso小鼠運(yùn)動(dòng)量表(0~9分)從足、踝、膝和髖關(guān)節(jié)運(yùn)動(dòng),協(xié)調(diào)性、軀干穩(wěn)定性、尾部位置和爪子位置等方面對(duì)脊髓損傷進(jìn)行了詳細(xì)評(píng)估[13]。本研究中,種植2周后實(shí)驗(yàn)兔逐漸出現(xiàn)運(yùn)動(dòng)功能障礙直至癱瘓,這與患者臨床脊柱轉(zhuǎn)移病程發(fā)展所類似。
目前,X線平片、CT、MRI和PET在脊柱轉(zhuǎn)移瘤的臨床診斷與治療中發(fā)揮著重要作用。CT密度分辨力高,可清晰顯示骨皮質(zhì)和骨小梁,但敏感度較低,因此,早期骨轉(zhuǎn)移在CT上較難發(fā)現(xiàn)。另外,穿刺過程中損傷骨質(zhì),與早期骨質(zhì)破壞難以鑒別。本研究中73.3%(11/15)實(shí)驗(yàn)兔種植后21 d時(shí)CT上才出現(xiàn)較明顯的骨質(zhì)破壞,部分伴成骨性改變;這與臨床中骨轉(zhuǎn)移灶按病變特征可分為溶骨型、成骨型和混合型3種類型,而以溶骨性最多見一致。
MRI空間分辨力高,可清晰顯示椎體旁、硬膜外腫塊及神經(jīng)脊髓根受壓等,在評(píng)估骨髓腔浸潤范圍和周圍軟組織情況方面優(yōu)于其他影像檢查[14],這也在本研究中得到了印證。但其掃描時(shí)間長,易受運(yùn)動(dòng)的影響,需對(duì)實(shí)驗(yàn)動(dòng)物進(jìn)行良好的固定與麻醉。
PET主要依靠組織的葡萄糖代謝特性來診斷疾病,能早期顯示骨骼系統(tǒng)病變。本研究中,種植后7 d即可見80.0%(12/15)椎體內(nèi)小范圍明顯核素濃聚。Hu等[15]也在小鼠脊柱轉(zhuǎn)移的影像診斷中發(fā)現(xiàn),使用18F-FDG示蹤劑行PET-CT成像的敏感度、準(zhǔn)確性和陰性預(yù)測(cè)值均較MRI更高,與本研究一致。
綜上所述,在CT引導(dǎo)下穿刺構(gòu)建兔脊柱VX2腫瘤模型,成功率較高,是一種穩(wěn)定、便捷的建模方法。CT、MRI、PET-CT均對(duì)此模型有診斷價(jià)值;CT對(duì)骨質(zhì)破壞顯示敏感度低,MRI可清晰顯示椎體周圍結(jié)構(gòu)、脊髓受壓情況和腫瘤范圍;PET-CT是早期評(píng)估腫瘤最敏感的方法。兔椎體腫瘤模型無論從骨質(zhì)破壞性質(zhì)上還是從病程進(jìn)展上均與人類脊柱轉(zhuǎn)移存在一定相似性,可為脊柱轉(zhuǎn)移瘤臨床治療的隨訪及評(píng)價(jià)提供參考。
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(收稿日期" 2023-10-26)