文章快速检索     高级检索
   复旦学报(医学版)  2022, Vol. 49 Issue (3): 326-331      DOI: 10.3969/j.issn.1672-8467.2022.03.002
0
Contents            PDF            Abstract             Full text             Fig/Tab
负荷试验CT灌注成像结合HIF1α、MMP2及VEGF评价大鼠C6胶质瘤侵袭力
李竹君1 , 陆娜2 , 于洋2 , 刘颖1     
1. 复旦大学基础医学院病理学系 上海 200032;
2. 复旦大学附属华山医院放射科 上海 200040
摘要目的 探寻负荷试验CT灌注成像中可以无创评估神经胶质瘤侵袭性的影像学生物标志物。方法 取20只雄性大鼠随机分为4组,其中3组分别在原位种植C6胶质瘤10、14、18天后行CT灌注成像,1组注射生理盐水作为对照。在乙酰唑胺负荷前后进行CT灌注,收集并分析各项灌注生物标志物。再取各组鼠脑开展病理学检验,进行缺氧诱导因子1α(hypoxia-inducible factor-1 α,HIF1α)、基质金属蛋白酶2(matrix metalloproteinase 2,MMP2)及血管内皮生长因子(vascular endothelial growth factor,VEGF)免疫组织化学染色。结果 乙酰唑胺负荷后脑血流量(cerebral blood flow,CBF)、脑血容量(cerebral blood volume,CBV)和表面通透性(surface-permeability,PS)明显高于负荷前;随肿瘤种植天数的增加,CBF、CBV和PS百分比变化率随之降低,尤其是18天组与对照组之间比较:对照组分别为119.5%±21.2%、125.6%±9.0%、816.2%±116.4%,而18天组分别为54.6%±2.0%、24.5%±4.3%、4.5%±0.8%;HIF1α、MMP2及VEGF阳性率和CBF、CBV、PS百分比变化率之间呈显著负相关(P均 < 0.01)。结论 负荷试验CT灌注成像的影像学生物标志物CBF、CBV和PS百分变化率可以无创评价大鼠C6胶质瘤的侵袭性。
关键词胶质瘤    灌注成像    肿瘤侵袭性    大鼠    
Stress CT perfusion imaging combined with HIF1α, MMP2 and VEGF in evaluating the invasiveness of rat with C6 glioma
LI Zhu-jun1 , LU Na2 , YU Yang2 , LIU Ying1     
1. Department of Pathology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China;
2. Department of Radiology, Huashan Hospital, Fudan University, Shanghai 200040, China
Abstract: Objective To look for imaging biomarkers that can evaluate the invasiveness of gliomas in non-invasive way in stress CT perfusion imaging. Methods Twenty male Sprague-Dawley rats were randomized into four groups: five rats with orthotopically implanted C6 gliomas at 10-day old, five with gliomas at 14-day old, five with gliomas at 18-day old and five with orthotopically injected normal saline served as controls. CT perfusion was performed pre- and post-acetazolamide challenge.Then changes of perfusion parameters were collected and analyzed.The mouse brains of each group were taken for pathological examination, and immunohistochemical staining for hypoxia-inducible factor-1α (HIF1α), matrix metalloproteinase 2 (MMP2) and vascular endothelial growth factor (VEGF) was performed. Results The differences in cerebral blood flow (CBF), cerebral blood volume (CBV) and surface-permeability (PS) between the pre- and post-challenge were statistically significant, with the values were higher post acetazolamide than pre-challenge. CBF, CBV and PS percentage changes decreased markedly as the number of days of tumor implantation increased, especially between 18-day group and control: The percentage change rates of CBF, CBV, and PS in the control group were 119.5%±21.2%, 125.6%±9.0% and 816.2%±116.4%, while the percentage change rates of CBF, CBV, and PS in 18-day group were 54.6%±2.0%, 24.5%±4.3% and 4.5%±0.8%, respectively.Negative correlations were detected between HIF1α, MMP2, VEGF and CBF pre-challenge, CBV pre-challenge, PS pre-challenge.Negative correlations were found between HIF1α, MMP2, VEGF positive rate and CBF, CBV, PS percentage changes (All P values less than 0.01). Conclusion Imaging biomarkers of CT perfusion with challenge can reflect the invasiveness of rat with C6 glioma tumor in non-invasive way.
Key words: glioma    perfusion imaging    neoplasm invasiveness    rat    

胶质母细胞瘤(glioblastoma,GBM)是星形细胞胶质瘤中恶性程度最高的脑肿瘤。大部分患者的预后不佳,中位生存期一般低于1年[1]。侵袭力在胶质瘤进展及其恶性程度评价中发挥着重要作用[2],且已成为治疗神经胶质瘤的重要靶向[3-5],因而评价胶质瘤的侵袭力在临床上有较高的应用价值。

评价胶质瘤侵袭力的金标准是免疫组织化学染色,主要有基质金属蛋白酶2(matrix metalloproteinase 2,MMP2)、缺氧诱导因子1α(hypoxia inducible factor-1α,HIF1α)、血管内皮生长因子(vascular endothelial growth factor,VEGF)等。MMP2可以降解Ⅳ型胶原,即降解基膜可反映肿瘤侵袭力[6-7];HIF1α反映组织中的氧稳态,在胶质瘤的微血管生成中担任重要角色[8-9];VEGF在肿瘤组织血管生成中发挥核心作用,指征胶质瘤侵袭力[10]。但免疫组化所用的组织标本一般通过手术、穿刺、活检等有创方式获取且周期较长,因此探寻一种无创活体评价胶质瘤侵袭力的方法有助于简化诊疗过程,减少患者负担。

乙酰唑胺是临床上常用来扩张血管的药物,在正常情况下会使脑阻力血管扩张进而引发脑血流量上升[11]。乙酰唑胺负荷常被用来检测脑血管异常区域,有用于诊断慢性脑供血不足、颈动脉狭窄等疾病的潜力[12-13],但尚未应用到肿瘤诊断层面,而神经胶质瘤也无法耐受乙酰唑胺的舒血管作用。因此,通过乙酰唑胺负荷前后脑部CT灌注生物标志物的变化可以了解胶质瘤的生长状况。本研究旨在寻找可以无创评价胶质瘤侵袭力的CT灌注生物标志物。

材料和方法

大鼠胶质瘤模型建立  20只雄性清洁级SD(Sprgue-Dawley)大鼠(上海斯莱克实验动物有限公司),重约250~300 g,随机分成4组,每组5只,分别为胶质瘤原位植入10、14、18天组以及对照组。接种前,每只大鼠用2%异氟烷麻醉,再将其固定于立体定位仪上,沿头皮中缝行纵线切口。在颅骨上钻1 mm直径圆孔,孔洞位置在前囟前方1 mm,偏右3 mm。用微量进样针缓慢推入10 μL的C6胶质瘤PBS混悬液,注射速度约为1 μL/min,深度在颅骨下方5 mm,留置5 min后慢速抽出,用少量骨蜡轻擦孔洞,再缝合头皮。约10天后大鼠C6胶质瘤动物模型可建成。

SD大鼠饲养均在清洁标准环境中,12 h/12 h光暗周期。实验操作符合复旦大学附属华山医院动物实验伦理要求(2020华山医院JS-057)。

负荷试验与CT灌注成像  CT扫描在256层CT机型(Philips Brilliance iCT 256,美国Cleveland公司)上开展,试验前大鼠先用2%异氟烷麻醉,将静脉留置针插入大鼠阴茎背静脉中以进行对比剂给药。首次扫描时确保整个鼠脑处于可扫描范围之内。用高压注射器以1mL/s的流速注射对比剂欧乃派克(2 mL/kg,Omnipaque®300,美国GE公司)。CT灌注成像流程见本团队文献[14]

CT数据处理采用Philips自带软件,流入动脉为大脑中动脉,基于去卷积运算,生成伪彩图。在每张彩图上划定3个感兴趣区(region of interest,ROIs),取平均值。在乙酰唑胺负荷前后记录脑血流量(cerebral blood flow,CBF)、脑血容量(cerebral blood volume,CBV)、表面通透性(surface-permeability,PS),并计算百分比变化率。

免疫组织化学染色  每组大鼠于对应天数进行CO2吸入型安乐死,然后用4%多聚甲醛进行灌注固定。所有大鼠脑组织石蜡切片均经标准化流程进行脱腊,抗原修复以及封闭后敷一抗。所用一抗为HIF1α(A16873,中国爱博泰克公司)、MMP2(NB200-193,美国Novus Biologicals公司)和VEGF(9698,美国CST公司)。使用DAKO En Vision系统(peroxidase/DAB+,兔/鼠)在标准方案下检测抗体结合情况,苏木精衬染。

染好的组织切片由两名病理医师分析,一致性达100%。采用半定量方法,每张切片挑选3~5个视野,在光学显微镜下拍照(Stemi 305,德国Carl Zeiss公司),使用I-solution软件(I-Solution,加拿大IMT公司)计算阳性率。

统计学分析  采用GraphPad Prism(version 8.0)统计学软件进行分析。独立样本配对t检验统计乙酰唑胺负荷前后影像学生物标志物之间的关系。ANOVA检验不同肿瘤种植组间的差异。灌注生物标志物与HIF1α、MMP2、VEGF之间的关系用Pearson相关系数进行分析。经Bonferroni校正后,P < 0.05为差异有统计学意义。

结果

负荷试验前后组内灌注成像生物标志物对比  与对照组相比,乙酰唑胺负荷前生物标志物CBF、CBV、PS值均随种植天数的增加而上升(P < 0.01)。施加负荷之后,CBF、CBV、PS数值全部大于负荷之前的生物标志物值(P < 0.01,表 1)。14天组别大鼠乙酰唑胺负荷前后CT灌注彩图见图 1

表 1 乙酰唑胺负荷前后CBF,CBV和PS水平 Tab 1 Levels of CBF, CBV, PS pre- and post-acetazolamide challenge  
(x±s)
Group n CBFpre-challenge(mL·100 g-1·min-1 CBF post-challenge
(mL·100 g-1·min-1
CBV pre-challenge
(mL/100 g)
CBV post-challenge
(mL/100 g)
PS pre-challenge
(mL·100 g-1·min-1
PS post-challenge
(mL·100 g-1·min-1
Control 5 53.7±4.7 118.4±18.3(1) 8.6±0.8 19.3±1.5(1) 1.2±0.1 11.2±0.8(1)
Day10 5 63.1±2.0(2) 163.5±3.0(1) 38.2±0.9(2) 57.3±1.3(1) 10.9±0.8(2) 31.0±0.8(1)
Day14 5 77.4±14.0(2)(3) 154.7±6.9(1) 46.0±1.2(2) 57.3±7.7(1) 15.5±0.6(2)(3) 27.0±0.2(1)
Day18 5 95.0±1.4(2)(3)(4) 146.9±2.5(1) 50.1±1.2(2)(3) 62.3±2.3(1) 18.2±0.3(2)(3)(4) 19.0±0.2(1)
(1) vs. pre-challenge,(2) vs. control,(3) vs. day 10,(4) vs. day 14,P < 0.01.
A-D: Maximum intensity projection image (A), perfusion maps for cerebral blood flow (B), cerebral blood volume (C) and surface-permeability (D) pre-acetazolamide challenge; E-H: Maximum intensity projection image (E), perfusion maps for cerebral blood flow (F), cerebral blood volume (G) and surface-permeability (H) post acetazolamide challenge. 图 1 大鼠C6胶质瘤乙酰唑胺负荷前后CT灌注彩图 Fig 1 CT perfusion color maps in a rat with C6 gliomas pre and post acetazolamide challenge

负荷后不同组间灌注成像生物标志物对比  与对照组相比,18天组CBF百分比变化率显著降低(P < 0.01),10天组、14天组与对照组之间无明显差异。相对于对照组,10天组、14天组、18天组CBV及PS百分比变化率显著降低,且随着肿瘤种植天数的增加,CBV及PS百分比变化率随之降低(P < 0.01,表 2)。

表 2 乙酰唑胺负荷前后CBF,CBV,PS百分比变化率 Tab 2 Percentage changes of CBF, CBV, PS pre- and post-acetazolamide challenge  
(x±s)
Group n CBF percentage change(%) CBV percentage change(%) PS percentage change(%)
Control 5 119.5±21.2 125.6±9.0 816.2±116.4
Day 10 5 127.5±5.2 50.2±5.8(1) 185.5±19.8(1)
Day 14 5 105.0±36.2 24.6±15.1(1)(2) 74.6±6.0(1)(2)
Day 18 5 54.6±2.0(1) 24.5±4.3(1)(2) 4.5±0.8(1)(2)(3)
(1) vs. Control,(2) vs. day 10,(3) vs. day 14,P < 0.01.

免疫组织化学染色结果分析  14天组和18天组的VEGF、MMP2和HIF1α高于10天组(P < 0.01)。18天组的VEGF、MMP2和HIF1α高于14天组(P < 0.01)。10天、14天和18天组中所有免疫组化病理学标志物阳性率见表 3。10天、14天和18天组的镜下免疫组化结果见图 2

表 3 大鼠C6胶质瘤HIF1α、MMP2和VEGF免疫组织化学染色结果 Tab 3 Histopathological values of HIF1α, MMP2, VEGF in rats with C6 gliomas  
(x±s)
Group n HIF1α MMP2 VEGF
Day 10 5 2.82±0.9 1.8±0.5 3.9±0.3
Day 14 5 5.7±0.7(1) 3.9±0.8(1) 7.5±1.7(1)
Day 18 5 9.2±1.1(1) (2) 7.2±1.1(1) (2) 12.4±1.3(1)(2)
(1) vs. day 10;(2) vs. day 14,P < 0.01.
Scale bar=20 μm. 图 2 大鼠C6胶质瘤HIF1α、MMP2、VEGF免疫组织化学染色图 Fig 2 Histologic images of HIF1α, MMP2 and VEGF staining in rats with C6 gliomas

灌注生物标志物与HIF1α,MMP2,VEGF之间相关性分析  HIF1α、MMP2、VEGF与负荷前CBF、CBV、PS呈正相关。HIF1α、MMP2、VEGF和CBF、CBV、PS百分比变化率之间呈负相关(P < 0.01,表 4图 3)。

表 4 大鼠C6胶质瘤HIF1α,MMP2,VEGF与CT灌注生物标志物之间Pearson相关性分析 Tab 4 Pearson correlation of PCT with challenge parameters and histopathological values in rats with C6 gliomas
Items r(CBFpercentage change) r(CBVpercentage change) r(PSpercentage change) r(CBFpre-challege r(CBVpre-challege r(PSpre-challege
HIF1α -0.748 -0.685 -0.952 0.797 0.919 0.949
MMP2 -0.751 -0.653 -0.929 0.783 0.919 0.936
VEGF -0.888 -0.653 -0.911 0.911 0.872 0.886
Correlation was significant at the 0.05 level.
A-C: Correlation between HIF1α and CBF, CBV, PS percentage changes; D-F: Correlation between MMP2 and CBF, CBV, PS percentage changes; G-I: Correlation between VEGF and CBF, CBV, PS percentage changes. 图 3 大鼠C6胶质瘤HIF1α、MMP2、VEGF与CT灌注生物标志物之间Pearson相关性分析图 Fig 3 Pearson correlation diagrams between HIF1α, MMP2, VEGF and PCT with challenge parameters in rats with C6 glioma
讨论

CT灌注成像在脑肿瘤诊断中应用广泛,检查速度快、结果直观准确,可无创地评价微循环血流动力学变化。研究显示256层CT灌注成像生物标志物CBV、CBF和PS能有效反映人脑胶质瘤的血管生成状况,证明CT灌注成像在评估肿瘤生长上有很大潜力[15]。评价肿瘤进展必须考虑多个因素,已有研究指出抗血管生成治疗会诱发肿瘤侵袭力增强进而导致其恶性进展[16],因此,多维度评价胶质瘤进展至关重要。

HIF1α、MMP2和VEGF作为常见的组织病理学生物标记物在胶质瘤评价中有重要意义。胶质瘤内部通常是缺氧环境,肿瘤细胞为了降低氧消耗会抑制HIF-1降解,进一步活化HIF1α亚基信号通路,刺激其下游因子如VEGF、MMP2的表达,促进肿瘤血管生成,调节细胞代谢,增强肿瘤的生长与侵袭[17]。另一方面,研究指出VEGF/VEGF2信号通路能增强血管通透性,帮助肿瘤细胞渗出和转移,VEGF本身也可以降低肿瘤细胞免疫应答,促进肿瘤细胞的浸润,且VEGF与MMP2通常在肿瘤中的表达水平成正相关[18-19]。由此可见,HIF1α、MMP2和VEGF 3种因子的免疫组化染色结果能为评价大鼠C6胶质瘤的侵袭能力提供有力证据。

本研究发现CBF百分比变化率、CBV百分比变化率和PS百分比变化率与标记肿瘤侵袭力的组织病理学生物标志物间呈现负相关,即HIF1α、MMP2、VEGF免疫组化阳性率越高,肿瘤的侵袭能力越强,病变血管对乙酰唑胺的反应也越微弱,表现在CBF、CBV和PS的百分比变化率越小。本研究注意到PS百分比变化率相关性尤其显著,这一发现与先前研究结果吻合,即血脑屏障(blood brain barrier,BBB)完整性的破坏与胶质瘤的侵袭能力之间联系紧密,而血脑屏障完整性一般是用CT灌注成像技术计算对比剂从血管内到血管外组织间隙的单向传输速率进而获得表面通透性(PS)的定量数据来表示[20-21]

与磁共振(MRI)灌注成像相比,CT灌注成像成本更低,检测方便,获得的是完全定量的数据,且CT灌注成像不容易受钙化、出血、金属等影响,因此目前应用更加广泛[22]。本研究通过对比标准免疫组织化学诊断标记物,进一步明确CT灌注生物标志物与肿瘤侵袭力的相关性,可能为临床医师提供更直观可靠的影像学依据。

本研究的局限性在于缺乏直接的临床试验证据,人与大鼠之间确实存在诸多差异。考虑到试验中辐射剂量随采集次数线性增加,确实不宜招募志愿患者在临床实践中进行该试验。但无论如何,本研究希望其结果可以为胶质母细胞瘤的临床影像学检测提供更多的解读思路。

综上所述,本研究证实CT灌注成像影像学生物标志物CBF百分比变化率、CBV百分比变化率和PS百分比变化率作为胶质瘤侵袭力预测指标有一定意义,乙酰唑胺负荷CT灌注成像在无创评价胶质瘤侵袭力的过程中具有潜在的应用价值。

作者贡献声明  李竹君  动物和细胞实验,文章撰写。陆娜  CT影像学部分实验。于洋  影像学实验数据分析。刘颖  免疫组织化学结果分析。

利益冲突声明  所有作者均声明不存在利益冲突。

参考文献
[1]
LUBIN E. Brain tumors[J]. N Engl J Med, 2001, 344(19): 1478-1479.
[2]
SICA G, LAMA G, ANILE C, et al. Assessment of angiogenesis by CD105 and nestin expression in peritumor tissue of glioblastoma[J]. Int J Oncol, 2011, 38(1): 41-49.
[3]
BIKFALVI A, MOENNER M, JAVERZAT S, et al. Inhibition of angiogenesis and the angiogenesis/invasion shift[J]. Biochem Soc Trans, 2011, 39(6): 1560-1564. [DOI]
[4]
ARBAB AS. Activation of alternative pathways of angiogenesis and involvement of stem cells following anti-angiogenesis treatment in glioma[J]. Histol Histopathol, 2012, 27(5): 549-557.
[5]
LEMASSON B, SERDUC R, MAISIN C, et al. Monitoring blood-brain barrier status in a rat model of glioma receiving therapy: dual injection of low-molecular-weight and macromolecular MR contrast media[J]. Radiology, 2010, 257(2): 342-352. [DOI]
[6]
CARMELIET P, JAIN RK. Molecular mechanisms and clinical applications of angiogenesis[J]. Nature, 2011, 473(7347): 298-307.
[7]
YAMAMOTO M, MOHANAM S, SAWAYA R, et al. Differential expression of membrane-type matrix metalloproteinase and its correlation with gelatinase A activation in human malignant brain tumors in vivo and in vitro[J]. Cancer Res, 1996, 56(2): 384-392.
[8]
VAUPEL P. The role of hypoxia-induced factors in tumor progression[J]. Oncologist, 2004, 95: 10-17. [URI]
[9]
KEITH B, SIMON MC. Hypoxia-inducible factors, stem cells, and cancer[J]. Cell, 2007, 129(3): 465-472. [DOI]
[10]
PLATE KH, BREIER G, WEICH HA, et al. Vascular endothelial growth-factor is a potential tumor angiogenesis factor in human gliomas in vivo[J]. Nature, 1992, 359(6398): 845-848. [DOI]
[11]
LU N, DI Y, FENG X, et al. Comparison between acetazolamide challenge and 10% carbon dioxide challenge perfusion CT in rat C6 glioma[J]. Acad Radiol, 2012, 19(2): 159-165. [DOI]
[12]
WONG TH, SHAGERA QA, RYOO HG, et al. Basal and acetazolamide brain perfusion SPECT in internal carotid artery stenosis[J]. Nucl Med Mol Imaging, 2020, 54(1): 9-27.
[13]
LEE YB, JUNG BK, KANG CK. Automated quantitative analysis of cerebrovascular reactivity measured by acetazolamide spect for detection of abnormal cerebrovascular territories[J]. J Neurol Sci, 2021, 429(S): 31.
[14]
LU N, DI Y, FENG X, et al. CT perfusion with acetazolamide challenge in C6 gliomas and angiogenesis[J]. PLoS One, 2015, 10(3): e01216313.
[15]
陆娜, 冯晓源, 邸悦, 等. CT灌注成像评价人脑胶质瘤肿瘤血管生成[J]. 中国医学计算机成像杂志, 2014, 20(6): 481-484. [CNKI]
[16]
PAEZ-RIBES M, ALLEN E, HUDOCK J, et al. Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis[J]. Cancer Cell, 2009, 15(3): 220-231.
[17]
周炳娟, 孙吉瑞, 赵文明, 等. 乳腺癌分子亚型中HIF-1α和Glut-1表达及其与基底细胞样型乳腺癌的关系[J]. 临床与试验病理学杂志, 2015, 31(9): 991-995. [CNKI]
[18]
陈东, 冯林森, 王羽丰. VEGF/VEGFR2信号通路调控机制的研究进展[J]. 癌症进展, 2019, 17(7): 748-751. [CNKI]
[19]
WANG Y, MI Y, TIAN J, et al. Intermedin alleviates renal ischemia-reperfusion injury and enhances neovascularization in Wistar rats[J]. Drug Des Devel Ther, 2020, 14: 4825-4834.
[20]
XIAO J, LIU L, ZHONG Z, et al. Mangiferin regulates proliferation and apoptosis in glioma cells by induction of microRNA-15b and inhibition of MMP-9 expression[J]. Oncol Rep, 2015, 33(6): 2815-2820.
[21]
OZAWA A, KADOWAKI E, HAGA Y, et al. Acetylcholine esterase is a regulator of GFAP expression and a target of dichlorvos in astrocytic differentiation of rat glioma C6 cells[J]. Brain Res, 2013, 1537: 37-45.
[22]
MILES KA, CHAMSANGAVEJ C, LEE FT, et al. Application of CT in the investigation of angiogenesis in oncology[J]. Acad Radiol, 2000, 7(10): 840-850.

文章信息

李竹君, 陆娜, 于洋, 刘颖
LI Zhu-jun, LU Na, YU Yang, LIU Ying
负荷试验CT灌注成像结合HIF1α、MMP2及VEGF评价大鼠C6胶质瘤侵袭力
Stress CT perfusion imaging combined with HIF1α, MMP2 and VEGF in evaluating the invasiveness of rat with C6 glioma
复旦学报医学版, 2022, 49(3): 326-331.
Fudan University Journal of Medical Sciences, 2022, 49(3): 326-331.
Corresponding author
LIU Ying, E-mail: yliu@shmu.edu.cn.
基金项目
国家自然科学基金(81701686)
Foundation item
This work was supported by the National Natural Science Foundation of China (81701686)

工作空间