功能磁共振成像技术早期监测纳秒脉冲电场治疗宫颈癌荷瘤裸鼠模型疗效的研究

发布时间：2018-04-26 20:13

  本文选题：多b值弥散加权成像 + 体素内不相干运动　； 参考：《北京协和医学院》2014年博士论文

【摘要】：目的：利用不同强度纳秒脉冲电场(nsPEF)处理离体宫颈癌细胞株,初步探索nsPEF抑制肿瘤细胞增殖的特点和机制；建立小鼠功能磁共振成像技术——多b值弥散加权成像(DWI)及动态增强磁共振成像(DCE-MRI)扫描方案及后处理方法,在体定量评估不同类型宫颈癌荷瘤裸鼠模型的肿瘤弥散、灌注及新生血管功能等特点；利用多参数功能磁共振成像技术——多b值弥散加权成像(DWI)及动态对比剂增强磁共振成像(DCE-MRI)在体监测纳秒脉冲电场(nsPEF)治疗HeLa宫颈癌荷瘤裸鼠后肿瘤细胞及新生血管数量、功能等改变,以组织病理学结果为对照,探索nsPEF抗肿瘤机制。 材料与方法：分别利用三种不同强度(低强度20kv/cm,25个脉冲；中强度20kv/cm,40个脉冲；高强度25kv/cm40个脉冲)的纳秒脉冲电场处理离体人宫颈癌细胞株HeLa和SiHa。采用CCK-8实验观察处理后三个时间点(24小时、48小时及72小时)的细胞活性,计算细胞增殖抑制率；采用Annexin-FITC-PI双染色流式细胞仪技术测量处理后2小时细胞凋亡和坏死情况,并计算细胞凋亡和坏死百分比； 通过右背部皮下注射人宫颈癌细胞株HeLa和SiHa,分别构建宫颈腺癌和鳞癌荷瘤裸鼠模型,采用GE3.0T Signa750磁共振扫描仪和35mm直径小动物线圈进行MRI扫描。多b值DWI采用轴位自旋回波序列,选取11个b值(0,20,50,100,200,400,600,800,1000,1200,1500s/mm2),采用双指数模型进行后处理,认为b200s/mm2为快速衰减区,计算肿瘤区域纯分子弥散系数D (ADCslow)、假弥散系数D*(ADCfast)及灌注比例f(Perfusion fraction); DCE-MRI采取冠状位SPGR序列,时间分辨率为3s,共200期,在第10期通过尾静脉置管快速团注Gd-DTPA对比剂,根据Toft两室模型,计算对比剂增强比率CER、转运常数Ktrans,反向转移常数Kep,细胞外血管外组织间隙容积Ve,血浆容积比例fPV及曲线下面积AUC90等定量及半定量参数。完成扫描后处死裸鼠取肿瘤组织,进行HE病理检查； 选取HeLa宫颈癌皮下移植瘤裸鼠,随机分为2组,实验组进行100ns、1Hz、50kv/cm、200个脉冲的nsPEF治疗,对照组不处理。其中一组在治疗前、治疗后1天及14天分别行多b值DWI成像,应用双指数模型计算肿瘤区域纯分子弥散系数D (ADCslow)、假弥散系数D*(ADCfast)及灌注比例f (Perfusion fraction);另一组在治疗前及治疗后14天分别行DCE-MRI成像,根据Toft两室模型,计算对比剂增强比率CER、转运常数Ktrans,反向转移常数Kep,细胞外血管外组织间隙容积Ve,血浆容积比例fPV及曲线下面积AUC90等定量及半定量参数。在治疗前、治疗后1天及14天磁共振扫描完成后处死裸鼠取肿瘤组织,进行病理HE染色、免疫组化(CD31和CD34)及TUNEL染色。采用SPSS20.0软件对磁共振参数进行独立t检验,对荷瘤裸鼠3次多b值DWI成像参数进行重复测量多因素方差分析,认为双侧P0.05具有统计学显著性差异。 结果：CCK-8实验检测处理后HeLa细胞24h、48h及72h细胞增殖抑制率分别低强度47.55%、57.99%、39.71%；中强度87.64%、86.15%、76.80%；高强度89.55%、89.56%、79.79%。SiHa细胞24h、48h及72h细胞增殖抑制率分别低强度29.54%、43.75%、25.25%；中强度77.46%、77.72%、70.87%；高强度86.67%、88.61%、86.02%。流式细胞仪检测处理后HeLa细胞早前凋亡与坏死比例分别是低强度19.90%和54.30%；中强度11.40%和73.40%；高强度5.90%和81.00%；SiHa细胞早前凋亡与坏死比例分别是低强度68.5%和29.4%；中强度29.8%和69.4%；高强度14.5%和85.1%； 分别有10只HeLa宫颈腺癌和10只SiHa宫颈鳞癌裸鼠入组多b值DWI成像,两组体积无显著差异,HeLa裸鼠纯扩散系数明显低于SiHa,但不具有统计学差异(0.259＋0.031vs.0.363±0.064×10-3mm2/s, p=0.163); HeLa裸鼠灌注比例f显著高于SiHa裸鼠(0.211＋0.023vs.0.145＋0.012,p=0.022)。另外5只HeLa宫颈腺癌和4只SiHa宫颈鳞癌裸鼠入组DCE-MRI成像,HeLa裸鼠灌注相关参数均高于SiHa裸鼠,其中CER:1.892±0536vs.0.477±0.142, p=0.057; Ktrans:0.156±0.036vs.0.058±0.013min-1; fPV:0.126±0.038vs.0.022±0.007, p=0.050; AUC90:22.32±6.19vs.6.30±1.56, p=0.060; 17只进行多b值DWI及DCE-MRI扫描的HeLa宫颈癌裸鼠远期疗效观察实验,治疗后14天,实验组体积治疗前减小37%,对照组增加150%,差异具有显著性(p=0.025)。进行多b值DWI扫描实验组D值治疗后较治疗前显著升高(Dpre=0.331±0.166, D1day=0.463±0.193, D14day=0.438±0.161(×10-3mm2/s))且在治疗后1天升高明显；对照组D值随肿瘤进展持续降低(Dpre=0.398±0.116, D1day=0.236±0.046, D14day=0.145±0.064(×10-3mm2/s)),差异具有显著性(p=0.022)。进行DCE-MRI扫描HeLa宫颈癌裸鼠,治疗后14天实验组肿瘤Ktrans值较对照组明显降低(△Ktrans=Ktrans-14day-Ktranspre,△Ktranscontrol=0.049±0.018min-1vs.△KtransnsPEF=-0.058±0.006min-1p=0.005)。组织病理提示在治疗后1天,治疗组可见肿瘤坏死及TUNEL阳性凋亡细胞,提示nsPEF可在早期诱导细胞坏死与凋亡；在治疗后14天,治疗组肿瘤血管相关免疫组化(CD31与CD34)染色提示肿瘤轴位新生血管数目明显降低。 结论：纳秒脉冲电场(nsPEF)可通过诱导凋亡及坏死抑制宫颈癌细胞株HeLa和SiHa生长,呈现剂量依赖和时间依赖的特点；随着处理强度增大,肿瘤细胞晚期凋亡及坏死的比例增大,增殖抑制率升高,在处理后第48小时最为明显；HeLa较SiHa细胞对nsPEF处理更敏感。 多b值DWI成像及DCE-MRI显示HeLa宫颈腺癌模型较SiHa鳞癌模型肿瘤新生血管灌注、血管内皮通透性等功能明显增多；HeLa宫颈腺癌模型提示肿瘤体积在50-400mm3范围内,体积与DCE-MRI血管参数呈正相关,与多b值DWI弥散及灌注相关参数无明显相关性。 纳秒脉冲电场可通过早期诱导细胞坏死与凋亡,晚期抑制肿瘤新生血管来抑制肿瘤生长。多b值DWI成像可早期在体监测纳秒脉冲电场治疗宫颈癌裸鼠疗效,纯弥散系数D值是定量肿瘤凋亡与坏死的敏感指标；DCE-MRI可定量、无创、准确评估纳秒脉冲电场抑制宫颈癌裸鼠肿瘤血管生成,是一种良好影像学生物标志物。
[Abstract]:Objective : To investigate the characteristics and mechanism of nsPEF in inhibiting tumor cell proliferation by treating ex vivo cervical cancer cell line with different intensity nanosecond pulsed electric field ( nsPEF ) .
To establish the functional magnetic resonance imaging ( DWI ) and dynamic enhanced magnetic resonance imaging ( DCE - MRI ) scanning and post - treatment methods in mice , and to evaluate the characteristics of tumor diffusion , perfusion and neovascularization in nude mice with different types of cervical cancer .
Using multi - parameter functional magnetic resonance imaging ( DWI ) and dynamic contrast - enhanced magnetic resonance imaging ( DCE - MRI ) in the treatment of tumor cells and neovascularization in HeLa cervical cancer - bearing nude mice , the mechanism of nsPEF was explored with histopathological findings as control .

Materials and Methods : Three different strengths ( 20 kv / cm , 25 pulses ) were used .
Medium strength 20kv / cm , 40 pulses ;
The cell activity of human cervical cancer cell line HeLa and SiHa was treated by nanosecond pulsed electric field with high intensity of 25kv / cm40 pulses . The cell activity of three time points ( 24 hours , 48 hours and 72 hours ) was observed by CCK - 8 experiment , and the inhibition rate of cell proliferation was calculated .
Apoptosis and necrosis of cells were measured in 2 hours after treatment with annexin - FITC - PI double staining flow cytometry , and the percentage of apoptosis and necrosis was calculated .


A nude mouse model of cervical adenocarcinoma and squamous cell carcinoma was established by subcutaneous injection of human cervical cancer cell line HeLa and SiHa on the right back . Using GE3.0 T Signa750 magnetic resonance scanner and 35 mm diameter small animal coil for MRI scan .


A group of nude mice were randomly divided into 2 groups . The experimental groups were randomly divided into 2 groups . The experimental groups were divided into two groups : 100 ns , 1 Hz , 50 kv / cm , 200 pulses of nsPEF treatment .

DCE - MRI can be used for quantitative , non - invasive , accurate assessment of nanosecond pulse electric field to inhibit angiogenesis of cervical cancer in nude mice , which is a good imaging biomarker .
Middle strength 87.64 % , 86.15 % , 76.80 % ;
High strength 89.55 % , 89.56 % , 79.79 % . The inhibitory rates of proliferation of SiHa cells at 24 h , 48 h and 72 h were 29.54 % , 43.75 % and 25.25 % , respectively .
Middle strength 77.46 % , 77.72 % , 70.87 % ;
High strength 86.67 % , 88.61 % and 86.02 % . The percentage of apoptosis and necrosis of HeLa cells was 19.90 % and 54.30 % respectively after flow cytometry .
Middle strength 11.40 % and 73.40 % ;
High strength 5.90 % and 81.00 % ;
The percentage of early apoptotic and necrosis of SiHa cells was 68.5 % and 29 . 4 % , respectively .
The median strength was 29 . 8 % and 69.4 % ;
high strength 14.5 % and 85.1 % ;


There were no statistically significant differences between the two groups ( 0.259 + 0.031 vs . 0.363 卤 0.064 脳 10 - 3mm2 / s , p = 0.163 ) . In addition , 5 HeLa cervical adenocarcinoma and 4 SiHa cervical squamous cell carcinoma nude mice were injected with DCE - MRI , and the parameters of tumor perfusion in HeLa cells were higher than those of SiHa nude mice , of which CER : 1.892 卤 0536vs . 0.477 卤 0.142 , p = 0.057 ; Ktrans : 0.156 卤 0.036 vs . 0.058 卤 0.013min -1 ; fPV : 0.126 卤 0.038vs.0 . 022 卤 0.007 , p = 0.050 ; AUC 90 : 22.32 卤 6.19 vs . 6.30 卤 1 . 56 , p = 0.060 ;

17 patients were treated with DWI and DCE - MRI for the long - term follow - up observation . 14 days after treatment , the volume of the experimental group was decreased by 37 % and the control group increased by 150 % ( p = 0.025 ) . After treatment , the Dpre = 0.331 卤 0.166 , D14day = 0.438 卤 0.161 ( 脳 10 - 3 mm2 / s )) , and the elevation of the control group increased 1 day after treatment .
The D - value of the control group decreased with the progression of the tumor ( Dpre = 0.398 卤 0.116 , D14day = 0.236 卤 0.046 , D14day = 0.145 卤 0.064 ( 脳 10 - 3mm2 / s )) , and the difference was significant ( p = 0 . 022 ) . After the treatment , the Ktrans value of the experimental group was significantly lower than that in the control group ( 鈻，

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