本文關(guān)鍵詞： 兔VX2肝癌模型 能譜CT CT灌注成像 碘含量 血管內(nèi)皮抑制素 腫瘤血管生成 成纖維細(xì)胞生長因子　出處：《鄭州大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：實(shí)驗(yàn)?zāi)康模?1分析兔VX2肝臟移植瘤在能譜CT灌注掃描的時(shí)間密度曲線、灌注參數(shù)特點(diǎn)。 2分析能譜成像掃描的單能量圖像特點(diǎn)，能譜衰減曲線、碘含量的特點(diǎn)。 3分析重組入血管內(nèi)皮抑制素（恩度）治療后兔VX2肝移植瘤在灌注參數(shù)及能譜掃描碘含量的變化特點(diǎn)。 4用Western-blot檢測所取組織的FGF2蛋白的表達(dá)并與CT灌注參數(shù)、標(biāo)準(zhǔn)化碘含量（NIC）進(jìn)行相關(guān)性分析。 材料和方法： 由河南省實(shí)驗(yàn)動(dòng)物中心提供健康新西蘭大白兔52只（體重2.6～2.9Kg），實(shí)驗(yàn)得到鄭州大學(xué)實(shí)驗(yàn)動(dòng)物倫理委員會(huì)許可，采用開腹種植新鮮瘤組織塊法建立肝移植瘤模型。 1抗血管生成治療方案 模型建立中，由于麻醉意外，死亡1只。在移植瘤術(shù)后7天、14天經(jīng)CT檢查1只模型兔未見成瘤，剔除后續(xù)試驗(yàn)。從48只成功VX2肝臟移植瘤模型隨機(jī)挑選2只，做病理檢查HE染色。剩余46只進(jìn)行分組，每組23只，對(duì)照組（A組）于移植瘤術(shù)后14天開始，每天靜脈注射和治療組相同劑量安慰劑，生理鹽水，連續(xù)注射7天。治療組(B組)于移植瘤術(shù)后第14天開始，每天靜脈注射重組入血管內(nèi)皮抑制素（恩度），（劑量為0.3mg/kg），連續(xù)注射7天。 2CT影像學(xué)檢查 選用GE公司Discovery CT750HD對(duì)建模成功的兔VX2肝移植瘤在種植后7天、14天、21天（用藥后7天）均行CT灌注掃描和能譜掃描（gemstone spectralimaging）。將重建圖像數(shù)據(jù)傳輸?shù)接跋窈筇幚砉ぷ髡?Advantage Windows4.6, GEHealthcare)進(jìn)行分析。 ①用CT灌注功能分析軟件進(jìn)行分析CT灌注成像：選擇主動(dòng)脈作為輸入動(dòng)脈，門靜脈為輸入靜脈。在軸位上劃感興趣區(qū)，第一個(gè)感興趣區(qū)選擇整個(gè)腫瘤組織，第二個(gè)感興趣區(qū)選擇遠(yuǎn)離腫瘤的無瘤肝實(shí)質(zhì)內(nèi)。分別獲得時(shí)間密度曲線（Time density cure，TDC）。自動(dòng)生成灌注參數(shù)：血流量BF（blood flow）（ml/min/100mg），血容量BV（blood volume）（ml/100mg），平均通過時(shí)間MTT（mean transit time）（s），毛細(xì)血管表面通透性PS(permeability of the capillaryvessel surface)（ml/min/100mg）和肝動(dòng)脈分?jǐn)?shù)HAF（Hepatic arterial fraction），并得到相應(yīng)的偽彩圖。 ②用能譜成像（gemstone spectral imaging，GSI）后處理軟件分析GSI圖像：獲得常規(guī)CT平掃圖像，動(dòng)脈期常規(guī)混合能量圖像（conventional polychromaticimage，QC）并重建出單能量圖像（monochromatic image，Mono）。對(duì)40keV水平、最佳信噪比（optimal CNR，OP）keV水平、常規(guī)混合能量（QC）水平三組圖像的信噪比(contrast-to-noise ratio，，CNR)，圖像噪聲（niose）進(jìn)行定量比較，并對(duì)三組圖像的病灶顯著性和總體圖像質(zhì)量進(jìn)行評(píng)分（lesion conspicuity scores，LCS；overall image quality scores，OQS）；分析腫瘤區(qū)域以及無瘤肝組織的能譜曲線特征；在碘基圖上定量測量腫瘤區(qū)域、無瘤肝組織、腹主動(dòng)脈的碘含量，分別計(jì)算標(biāo)準(zhǔn)化碘含量（normalized iodine concentrations，NIC），比較治療前后，腫瘤區(qū)域之間標(biāo)準(zhǔn)化碘含量的差異。 3灌注參數(shù)及標(biāo)準(zhǔn)化碘含量與分子生物學(xué)的相關(guān)性分析 每次CT掃描結(jié)束后，用CT引導(dǎo)下穿刺的方法取活體組織（腫瘤組織和遠(yuǎn)處無瘤肝組織）存放在-196℃的液氮中冷藏。western-blot檢測組織內(nèi)成纖維細(xì)胞生成因子（FGF1）蛋白的表達(dá)。按照蛋白樣品的制備→SDS-PAGE→轉(zhuǎn)膜→封閉→一抗雜交→二抗雜交→底物顯色的實(shí)驗(yàn)流程，測量組織內(nèi)FGF2蛋白的表達(dá)，分析灌注參數(shù)及標(biāo)準(zhǔn)化碘含量與FGF2表達(dá)的相關(guān)性。 結(jié)果 1兔VX2肝移植模型的CT表現(xiàn)及大體病理 ①正常兔肝臟，肝左葉大于肝右葉，增強(qiáng)掃描動(dòng)脈期呈輕度強(qiáng)化，門靜脈期強(qiáng)化程度增強(qiáng)。大體觀：正常兔肝臟，位于腹腔前部，從肝臟腹面的裂溝可以將肝臟分為4部分，左側(cè)兩個(gè)肝葉肥大，右側(cè)肝葉較小，左葉和右葉中間有狹窄的中央葉，在中央葉前方的肝門處還有尾狀葉和乳頭竇。新鮮的兔肝臟呈紅褐色。重量約85～130g。顯微鏡下，可見肝細(xì)胞排列規(guī)整，細(xì)胞核形態(tài)規(guī)則，大小均勻，肝小葉、肝竇結(jié)構(gòu)清晰。 ②種植成功的兔VX2肝移植瘤，CT平掃呈類圓形低密度影，增強(qiáng)掃描動(dòng)脈期呈環(huán)形強(qiáng)化，環(huán)形壁較厚，門靜脈期呈相對(duì)低密度，隨著腫瘤增長，中心液化壞死增多。大體觀：移植瘤呈類圓形，灰白色，包膜完整，中心切面可見液化壞死。低倍鏡下可見瘤細(xì)胞呈巢狀分布，腫瘤間質(zhì)內(nèi)有大量新生的毛細(xì)血管，高倍鏡下，瘤細(xì)胞體積大，細(xì)胞形態(tài)不規(guī)則，大小不均，細(xì)胞核大、深染，可見異性型細(xì)胞核，核分裂像多見。 2兔VX2移植瘤灌注結(jié)果 ①腫瘤區(qū)域BF、BV、PS、HAF高于無瘤肝組織，腫瘤區(qū)域MTT低于無瘤肝組織。分別進(jìn)行統(tǒng)計(jì)學(xué)分析具有統(tǒng)計(jì)學(xué)意義（p＜0.05）。②術(shù)后7天到14天隨著腫瘤的生長，腫瘤組織CT灌注參數(shù)BF、BV、HAF呈增高的趨勢，MTT、PS呈減低的趨勢，其中，BF、MTT、PS差異具有統(tǒng)計(jì)學(xué)意義（p＜0.05），而BV、HAF差異沒有統(tǒng)計(jì)學(xué)意義（p＞0.05）。無瘤肝組織從術(shù)后7天到14天各個(gè)灌注參數(shù)值隨腫瘤生長無明顯變化（p＞0.05）。③重組人血管內(nèi)皮抑制素（恩度）治療后，治療組BF、BV、PS、HAF較對(duì)照組減低，MTT較對(duì)照組增高，分別進(jìn)行統(tǒng)計(jì)學(xué)分析，BF具有統(tǒng)計(jì)學(xué)意義（P＜0.05），其余參數(shù)無統(tǒng)計(jì)學(xué)意義（p＞0.05）。④各灌注參數(shù)與血管生成因子FGF2蛋白進(jìn)行相關(guān)分析，BF與FGF1蛋白之間有相關(guān)性，相關(guān)系數(shù)為r=0.932其他各灌注參數(shù)與FGF1蛋白之間無相關(guān)性。 3能譜CT掃描結(jié)果 ①三組圖像評(píng)估：OP組CNR（2.63±1.59）低于40keV（2.81±1.74）兩者進(jìn)行統(tǒng)計(jì)學(xué)分析無統(tǒng)計(jì)學(xué)意義（p＞0.05），OP組CNR高于QC組（1.95±1.55）進(jìn)行統(tǒng)計(jì)學(xué)分析有統(tǒng)計(jì)學(xué)意義（p＜0.05），40keV組和QC組之間的差異有統(tǒng)計(jì)學(xué)意義（p＜0.05）。圖像噪聲，OP組（7.89±1.35）低于40keV組（9.12±1.28）高于QC組（6.67±1.36）分別進(jìn)行統(tǒng)計(jì)學(xué)分析差異有統(tǒng)計(jì)學(xué)意義（p＜0.05）。病灶顯著性評(píng)分（LCS），OP組（3.86±0.68）與40keV組（3.77±0.57）之間的差異沒有統(tǒng)計(jì)學(xué)意義（p＞0.05），兩組分別都高于QC組（3.29±0.49）之間的差異有統(tǒng)計(jì)學(xué)意義（p＜0.05）�？傮w質(zhì)量評(píng)分（OQS），OP組（3.85±0.58）高于40keV組（3.10±0.25）和QC組（3.38±0.55）之間的差異有統(tǒng)計(jì)學(xué)意義（p＜0.05）。 ②能譜曲線分析：計(jì)算能譜曲線上不同單能點(diǎn)的斜率，計(jì)算公式為Kx=(HU40keV-HUXkeV)/(X-40)，其中Kx為不同單能量點(diǎn)所對(duì)應(yīng)的斜率，HU40keV為40keV的CT值，HUXkeV為不同單能量所對(duì)應(yīng)的CT值。在各個(gè)單能量點(diǎn)，腫瘤組織的斜率均大于無瘤肝組織，進(jìn)行統(tǒng)計(jì)學(xué)分析，有統(tǒng)計(jì)學(xué)意義（p＜0.05）。 ③標(biāo)準(zhǔn)化碘含量（NIC）：7天、14天腫瘤組織的NIC大于無瘤肝組織，之間的差異具有統(tǒng)計(jì)學(xué)意義（p＜0.05）。重組人血管內(nèi)皮抑制素恩度治療后，治療組腫瘤區(qū)域NIC小于對(duì)照組，二者之間的差異有統(tǒng)計(jì)學(xué)意義（p＜0.05）。NIC與FGF2蛋白進(jìn)行先關(guān)性分析有良好的相關(guān)性，相關(guān)系數(shù)為r=0.957。 結(jié)論： 1采用開腹種植瘤塊法建立兔VX2肝移植瘤模型，成瘤率高，成瘤時(shí)間短，移植瘤形態(tài)規(guī)則，轉(zhuǎn)移率低，其病理學(xué)表現(xiàn)及生物學(xué)行為接近人類原發(fā)性肝細(xì)胞肝癌，該模型可廣泛應(yīng)用于相關(guān)的影像學(xué)及血流動(dòng)力學(xué)的基礎(chǔ)研究。 2CT灌注成像及能譜成像影像，分別通過生成灌注參數(shù)和碘基值可以提供肝移植瘤的血流灌注及能譜成像信息，反映腫瘤的血流動(dòng)力學(xué)。能譜成像在最佳單能量水平更有助于對(duì)腫瘤的檢出。 3FGF2蛋白在腫瘤組織中處于高表達(dá)狀態(tài)，可以反映腫瘤的血供，在臨床上，F(xiàn)GF2可以反映提供有關(guān)腫瘤的血管生成的相關(guān)信息。為觀察腫瘤生成及治療提供可參考的生物學(xué)指標(biāo)。 4重組人血管內(nèi)皮抑制素（恩度）表現(xiàn)出明顯的抑制腫瘤血管生成作用，在化療的過程可用于CT灌注成像及能譜成像觀察腫瘤血管生成�？赏ㄟ^CT灌注參數(shù)及能譜掃描評(píng)價(jià)抗血管治療的效果，這項(xiàng)無創(chuàng)檢查技術(shù)值得進(jìn)一步推廣應(yīng)用于臨床的肝癌的診斷及療效判定的研究。
[Abstract]:Objective:
1 the time density curve and perfusion parameters of VX2 liver transplantation tumor in rabbit were analyzed by CT perfusion scan.
2 the characteristics of the single energy image, the spectral attenuation curve and the iodine content of the spectral imaging scan are analyzed.
3 the changes of the perfusion parameters and the content of iodine in the rabbit VX2 liver transplantation tumor after recombinant endostatin (endostatin) were analyzed.
4 Western-blot was used to detect the expression of FGF2 protein in the tissue and to analyze the correlation between the CT perfusion parameters and the standardized iodine content (NIC).
Materials and methods:
52 healthy New Zealand white rabbits (weighing 2.6 to 2.9Kg) were provided by Henan experimental animal center. The experiment was approved by the laboratory animal ethics committee of Zhengzhou University, and a liver transplantation tumor model was established by planting fresh tumor tissue in the open abdomen.
1 antiangiogenic treatment scheme
In the model, because of anesthesia accident death of 1. In the transplanted tumor after 7 days, 14 days CT 1 rabbit models were tumorigenic, excluding the follow-up test. From 48 successful VX2 liver tumor model of randomly selected 2 rats for pathological examination of HE staining. The remaining 46 groups, each group of 23 only, the control group (A group) in the beginning 14 days of transplantation tumor after operation, daily intravenous injection and treatment group the same dose of placebo, normal saline, continuous injection of 7 days. The treatment group (B group) on the fourteenth day after transplanted, every day into the intravenous injection of recombinant endostatin (Endostar), dose (0.3mg/kg), continuous injection of 7 days.
2CT imaging examination
The GE Discovery CT750HD for modeling the success of rabbit VX2 liver tumor in the field after 7 days, 14 days, 21 days (7 days after treatment) underwent CT perfusion scan and EDS (gemstone spectralimaging). The reconstructed image data to the image postprocessing workstation (Advantage Windows4.6, GEHealthcare) were analyzed.
The CT perfusion software to analyze CT perfusion imaging: the aorta as input artery, portal vein. The input for ROI in the axial region of interest, the first choice of the tumor, second regions of interest from tumor tumor free liver parenchyma. Obtain time density curve respectively (Time density cure, TDC). The blood flow perfusion parameters: automatic generation of BF (blood flow) (ml/min/100mg), blood volume (BV blood volume) (ml/100mg), mean transit time MTT (mean transit time) (s), permeability surface (permeability PS of the capillaryvessel (ml/min/100mg) and surface) hepatic arterial fraction HAF (Hepatic arterial fraction), and get the corresponding pseudo color.
The spectral imaging can be used (gemstone spectral imaging, GSI) analysis software GSI image postprocessing: conventional CT scan image, the arterial phase conventional mixed energy images (conventional polychromaticimage, QC) and reconstruct the single energy image (monochromatic image, Mono). The level of 40keV, the best signal-to-noise ratio (optimal CNR, OP the level of keV), conventional mixed energy (QC) SNR level of three sets of images (contrast-to-noise ratio, CNR), image noise (niose) for quantitative comparison, and were significant and the overall image quality of three groups of image score (lesion conspicuity scores overall image quality scores, LCS; OQS); analysis of tumor area and tumor free liver tissue spectrum curve characteristics; quantitative measurement of tumor in iodine kitu region, tumor free liver tissue, the iodine content of the abdominal aorta, the iodine content of standard were calculated (normalized iodine concentrat Ions, NIC), compare the difference of the standard iodine content between the tumor regions before and after the treatment.
The correlation analysis between 3 perfusion parameters and the standard iodine content and molecular biology
Every time after CT scan, and living tissue by CT method under the guidance of puncture (tumor tissues and distant tumor free liver tissue) stored in liquid nitrogen frozen -196 degrees of detection of.Western-blot in fibroblast growth factor (FGF1) protein expression. According to the experimental process of protein sample preparation, SDS-PAGE, transfer film, a closure, anti - two hybrid, hybrid anti chromogenic substrate, FGF2 protein expression measurement within the organization, analysis of the correlation between perfusion parameters and expression of normalized iodine content and FGF2.
Result
The CT manifestation and general pathology of 1 rabbit VX2 liver transplantation model
The normal rabbit liver, the left hepatic lobe is larger than the right lobe of the liver, enhanced scan and slight enhancement in the arterial phase, portal venous phase enhancement enhancement. General observation: normal rabbit liver, located in the abdominal part from the liver, ventral fissure can be divided into 4 parts on the left side of the liver, two hepatic lobe hypertrophy, right lateral lobe of the liver., with a narrow central lobe between right and left lobe, in front of the door at the central lobe of liver and caudate lobe and papillary sinus. Fresh rabbit liver is red brown. The weight is about 85 ~ 130g. under the microscope, visible liver cells arranged in neat nuclei in regular shape, uniform size, hepatic lobule, hepatic sinus clear structure.
The success of rabbit VX2 liver tumor. CT scan showed circular low density, enhanced scan showed ring like enhancement, the annular wall is thick, the portal venous phase showed relatively low density, with tumor growth, central liquefaction and necrosis increased. General observation: transplanted tumors were round, gray white, complete capsule the center section, visible necrosis. Under low magnification microscope nests of tumor cells, tumor stroma within a large number of new capillaries, at high magnification, the tumor cells were large and irregular in shape, uneven size, big nucleus anachromasis, visible type specific nuclei and mitotic more common.
The results of 2 rabbit VX2 transplanted tumor
鈶犺偪鐦ゅ尯鍩烞F,BV,PS,HAF楂樹簬鏃犵槫鑲濈粍緇

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