本文選題：多b值彌散加權(quán)成像 + 體素內(nèi)不相干運動�。� 參考：《北京協(xié)和醫(yī)學院》2014年博士論文

【摘要】：目的：利用不同強度納秒脈沖電場(nsPEF)處理離體宮頸癌細胞株,初步探索nsPEF抑制腫瘤細胞增殖的特點和機制；建立小鼠功能磁共振成像技術(shù)——多b值彌散加權(quán)成像(DWI)及動態(tài)增強磁共振成像(DCE-MRI)掃描方案及后處理方法,在體定量評估不同類型宮頸癌荷瘤裸鼠模型的腫瘤彌散、灌注及新生血管功能等特點；利用多參數(shù)功能磁共振成像技術(shù)——多b值彌散加權(quán)成像(DWI)及動態(tài)對比劑增強磁共振成像(DCE-MRI)在體監(jiān)測納秒脈沖電場(nsPEF)治療HeLa宮頸癌荷瘤裸鼠后腫瘤細胞及新生血管數(shù)量、功能等改變,以組織病理學結(jié)果為對照,探索nsPEF抗腫瘤機制。 材料與方法：分別利用三種不同強度(低強度20kv/cm,25個脈沖；中強度20kv/cm,40個脈沖；高強度25kv/cm40個脈沖)的納秒脈沖電場處理離體人宮頸癌細胞株HeLa和SiHa。采用CCK-8實驗觀察處理后三個時間點(24小時、48小時及72小時)的細胞活性,計算細胞增殖抑制率；采用Annexin-FITC-PI雙染色流式細胞儀技術(shù)測量處理后2小時細胞凋亡和壞死情況,并計算細胞凋亡和壞死百分比； 通過右背部皮下注射人宮頸癌細胞株HeLa和SiHa,分別構(gòu)建宮頸腺癌和鱗癌荷瘤裸鼠模型,采用GE3.0T Signa750磁共振掃描儀和35mm直徑小動物線圈進行MRI掃描。多b值DWI采用軸位自旋回波序列,選取11個b值(0,20,50,100,200,400,600,800,1000,1200,1500s/mm2),采用雙指數(shù)模型進行后處理,認為b200s/mm2為快速衰減區(qū),計算腫瘤區(qū)域純分子彌散系數(shù)D (ADCslow)、假彌散系數(shù)D*(ADCfast)及灌注比例f(Perfusion fraction); DCE-MRI采取冠狀位SPGR序列,時間分辨率為3s,共200期,在第10期通過尾靜脈置管快速團注Gd-DTPA對比劑,根據(jù)Toft兩室模型,計算對比劑增強比率CER、轉(zhuǎn)運常數(shù)Ktrans,反向轉(zhuǎn)移常數(shù)Kep,細胞外血管外組織間隙容積Ve,血漿容積比例fPV及曲線下面積AUC90等定量及半定量參數(shù)。完成掃描后處死裸鼠取腫瘤組織,進行HE病理檢查； 選取HeLa宮頸癌皮下移植瘤裸鼠,隨機分為2組,實驗組進行100ns、1Hz、50kv/cm、200個脈沖的nsPEF治療,對照組不處理。其中一組在治療前、治療后1天及14天分別行多b值DWI成像,應(yīng)用雙指數(shù)模型計算腫瘤區(qū)域純分子彌散系數(shù)D (ADCslow)、假彌散系數(shù)D*(ADCfast)及灌注比例f (Perfusion fraction);另一組在治療前及治療后14天分別行DCE-MRI成像,根據(jù)Toft兩室模型,計算對比劑增強比率CER、轉(zhuǎn)運常數(shù)Ktrans,反向轉(zhuǎn)移常數(shù)Kep,細胞外血管外組織間隙容積Ve,血漿容積比例fPV及曲線下面積AUC90等定量及半定量參數(shù)。在治療前、治療后1天及14天磁共振掃描完成后處死裸鼠取腫瘤組織,進行病理HE染色、免疫組化(CD31和CD34)及TUNEL染色。采用SPSS20.0軟件對磁共振參數(shù)進行獨立t檢驗,對荷瘤裸鼠3次多b值DWI成像參數(shù)進行重復測量多因素方差分析,認為雙側(cè)P0.05具有統(tǒng)計學顯著性差異。 結(jié)果：CCK-8實驗檢測處理后HeLa細胞24h、48h及72h細胞增殖抑制率分別低強度47.55%、57.99%、39.71%；中強度87.64%、86.15%、76.80%；高強度89.55%、89.56%、79.79%。SiHa細胞24h、48h及72h細胞增殖抑制率分別低強度29.54%、43.75%、25.25%；中強度77.46%、77.72%、70.87%；高強度86.67%、88.61%、86.02%。流式細胞儀檢測處理后HeLa細胞早前凋亡與壞死比例分別是低強度19.90%和54.30%；中強度11.40%和73.40%；高強度5.90%和81.00%；SiHa細胞早前凋亡與壞死比例分別是低強度68.5%和29.4%；中強度29.8%和69.4%；高強度14.5%和85.1%； 分別有10只HeLa宮頸腺癌和10只SiHa宮頸鱗癌裸鼠入組多b值DWI成像,兩組體積無顯著差異,HeLa裸鼠純擴散系數(shù)明顯低于SiHa,但不具有統(tǒng)計學差異(0.259＋0.031vs.0.363±0.064×10-3mm2/s, p=0.163); HeLa裸鼠灌注比例f顯著高于SiHa裸鼠(0.211＋0.023vs.0.145＋0.012,p=0.022)。另外5只HeLa宮頸腺癌和4只SiHa宮頸鱗癌裸鼠入組DCE-MRI成像,HeLa裸鼠灌注相關(guān)參數(shù)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只進行多b值DWI及DCE-MRI掃描的HeLa宮頸癌裸鼠遠期療效觀察實驗,治療后14天,實驗組體積治療前減小37%,對照組增加150%,差異具有顯著性(p=0.025)。進行多b值DWI掃描實驗組D值治療后較治療前顯著升高(Dpre=0.331±0.166, D1day=0.463±0.193, D14day=0.438±0.161(×10-3mm2/s))且在治療后1天升高明顯；對照組D值隨腫瘤進展持續(xù)降低(Dpre=0.398±0.116, D1day=0.236±0.046, D14day=0.145±0.064(×10-3mm2/s)),差異具有顯著性(p=0.022)。進行DCE-MRI掃描HeLa宮頸癌裸鼠,治療后14天實驗組腫瘤Ktrans值較對照組明顯降低(△Ktrans=Ktrans-14day-Ktranspre,△Ktranscontrol=0.049±0.018min-1vs.△KtransnsPEF=-0.058±0.006min-1p=0.005)。組織病理提示在治療后1天,治療組可見腫瘤壞死及TUNEL陽性凋亡細胞,提示nsPEF可在早期誘導細胞壞死與凋亡；在治療后14天,治療組腫瘤血管相關(guān)免疫組化(CD31與CD34)染色提示腫瘤軸位新生血管數(shù)目明顯降低。 結(jié)論：納秒脈沖電場(nsPEF)可通過誘導凋亡及壞死抑制宮頸癌細胞株HeLa和SiHa生長,呈現(xiàn)劑量依賴和時間依賴的特點；隨著處理強度增大,腫瘤細胞晚期凋亡及壞死的比例增大,增殖抑制率升高,在處理后第48小時最為明顯；HeLa較SiHa細胞對nsPEF處理更敏感。 多b值DWI成像及DCE-MRI顯示HeLa宮頸腺癌模型較SiHa鱗癌模型腫瘤新生血管灌注、血管內(nèi)皮通透性等功能明顯增多；HeLa宮頸腺癌模型提示腫瘤體積在50-400mm3范圍內(nèi),體積與DCE-MRI血管參數(shù)呈正相關(guān),與多b值DWI彌散及灌注相關(guān)參數(shù)無明顯相關(guān)性。 納秒脈沖電場可通過早期誘導細胞壞死與凋亡,晚期抑制腫瘤新生血管來抑制腫瘤生長。多b值DWI成像可早期在體監(jiān)測納秒脈沖電場治療宮頸癌裸鼠療效,純彌散系數(shù)D值是定量腫瘤凋亡與壞死的敏感指標；DCE-MRI可定量、無創(chuàng)、準確評估納秒脈沖電場抑制宮頸癌裸鼠腫瘤血管生成,是一種良好影像學生物標志物。
[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 鹵 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 鹵 0536vs . 0.477 鹵 0.142 , p = 0.057 ; Ktrans : 0.156 鹵 0.036 vs . 0.058 鹵 0.013min -1 ; fPV : 0.126 鹵 0.038vs.0 . 022 鹵 0.007 , p = 0.050 ; AUC 90 : 22.32 鹵 6.19 vs . 6.30 鹵 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 鹵 0.166 , D14day = 0.438 鹵 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 鹵 0.116 , D14day = 0.236 鹵 0.046 , D14day = 0.145 鹵 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，

本文編號：1807485