大鼠肝癌模型磁共振功能成像及HIFU术后差异蛋白组学
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摘要
第一部分二乙基亚硝胺诱导SD大鼠肝硬化肝癌模型的建立
目的：探讨二乙基亚硝胺诱导SD大鼠肝硬化、肝癌模型的成功率与特征。
材料与方法：SD大鼠128只,年龄4-5周,雄性,体重150-180g。采用纯度99.9%二乙基亚硝胺(DEN),每周以20mg/kg分3次腹腔注射。每周测大鼠体重并于Ow、8w、12w、14w进行超声检查,每期随机处死5只进行病理检查。采用SPSS19.0统计软件分析。
结果：14周建立肝癌模型72例,成功率56.25%。开始诱导后8w±1w、12w±1w、14w±1w基本达到肝硬化早期、晚期、肝癌阶段。诱导过程体重变化差异具统计学意义(F=54.79,P<0.001)。
结论：二乙基亚硝胺可以建立较稳定而理想的肝硬化及肝癌模型,且大鼠体重的变化在一定程度上反映了大鼠病程的发展。
第二部分SD大鼠肝癌模型磁共振DWI及SWI及HIFU术前后DWI特征
目的：探讨大鼠肝硬化早期、肝硬化晚期及肝癌模型DWI、SWI特征及在高强度聚焦超声(HIFU)前后评估中的价值。
材料与方法：采用二乙基亚硝胺(DEN)诱导SD大鼠128只建立肝癌模型72只,在开始诱导(Ow)、8w.12w、14w分别行磁共振常规序列及DWI、SWI动态检测,40只资料资料完整入组分析；23只大鼠模型HIFU术后24h DWI、SWI检查与非HIFU治疗组47只对照分析；10只大鼠诱导过程与HIFU后SWI资料进行动态分析。诱导过程以及HIFU术后10天分别处死部分大鼠进行病理研究。采用SPSS19.0统计软件分析。
结果：正常肝实质、肝硬化早期、肝硬化晚期、肝细胞癌DWI信号逐渐升高,肿瘤活性部分多为高信号,坏死区呈相对低信号,b=50s/mm2总体ADC有显著性意义(2645.18±369.97,2008.65±691.87,1679.50±206.16与2453.65±861.64,F=4.341,p<0.05),肝硬化晚期-肝癌ADC值变化有统计学意义(t=-2.82489, p<0.05); b=100s/mm2时总体ADC值变化没有显著性意义(2504.90±320.38,2185.90±455.91,1708.40±416.91与2284.30±223.18,Friedman检验,p=0.08)。b值分别为50s/mm2时或b=100s/mm2时,HIFU术前后肿瘤ADC值差异均没有显著性意义(2190.52±778.65,2115.39±633.08, t=0.35, p>0.05;2047.26±487.88,1889.33±547.17, Z=-0.882, p>0.05)。正常肝实质、肝硬化晚期、肝细胞癌SWI总体间差异没有显著性意义(14±2.9,25.6±6.0,34.6±±21.0,p>0.05)。
结论：大鼠肝硬化、肝细胞癌DWI成像具有特征性,DWI成像在发现、诊断与追踪肝细胞癌以及HIFU治疗前后评估、随访中中具有重要价值。
第三部分SD大鼠肝癌模型及HIFU术后流式细胞术和差异蛋白的检测
目的：探索大鼠肝癌模型HIFU治疗的免疫与蛋白机制。
材料与方法：SD大鼠肝癌模型72只,23只大鼠单结节模型接受1次或以上HIFU治疗并获得肿瘤标本、12只获血液标本,23只非HIFU治疗大鼠获肝细胞癌标本、6只获血液标本,分别进行流式细胞仪与差异蛋白组学检测,采用SPSS19.0统计软件行HIFU与非HIFU治疗组分析。
结果：HIFU与非HIFU治疗组外周血CD4、8、28、25、FoxP3、 r-IFN、IL4、CD4/8、Th1/Th2变化均无统计学意义(P>0.05)。差异蛋白组学发现了26个差异表达的蛋白质点,鉴定15种非冗余蛋白质,与肝癌模型细胞的蛋白质谱相比较有5个蛋白在细胞中表达下调,21个蛋白表达上调。
结论：HIFU治疗与不治疗组蛋白表达存在差异,上调、下调蛋白功能部分类似,提示HIFU治疗不完全可能导致残留肿瘤细胞侵袭转移活性增强,急需早期十预
PART ONE SD Rats Liver Cirrhosis, Hepatocellular Carcinoma Model Induced by Diethylnitrosamine(DEN)
OBJECTIVE:To study the success rate and characteristics of SD rats liver cirrhosis,hepatocellular carcinoma model induced by diethylnitrosamine(DEN).
MATERIALS AND METHODS:128SD rats of4-5weeks,male,150-180g were included. DEN,with99.9%Purity,was injected intraperitoneally with20mg/kg and3times every week. The weight of rats was measured every week.Every rat was carried out by Ultrasound(US) on beginning day and in8th,12th and14th week.Five rats were killed and rat liver was obtained for pathology after US.Data was analysed by SPSS19.0statistical software.
RESULTS:72rat liver tumor models were obtained after14weeks and the success rate was56.25%. Rat liver pathology achieved approximately prophase cirrhosis, advanced cirrhosis and hepatocellular carcinoma in8th±1week,12th±1week and14±1week.The change of rat weight was significant(F=54.79, P<0.001) between beginning day and in8th,12th and14th week.
CONCLUSION:Stable and ideal cirrhosis and hepatocellular carcinoma model can be establish by DEN and the development of rat liver pathology also can be reflected approximately by the change in body weight.
PART TWO Characteristics on Diffusion-weighted Imaging(DWI), Susceptibility Weighted Imaging(SWI) of SD Rats Liver Cancer Model before and after High Intensity Focused Ultrasound(HIFU)
OBJECTIVE:To study characteristics of SD rats prophase cirrhosis, advanced cirrhosis and hepatocellular carcinoma model and after HIFU on DWI and SWI and study the value of DWI after HIFU.
MATERIALS AND METHODS:72rat liver tumor models were obtained after14weeks by injecting DEN intraperitoneally for128SD rats with99.9%purity,20mg/kg and3times every week. Every rat was dynamically carried out by MRI,DWI and SWI on beginning day and in8th,12th and14th week and after HIFU.Data was analysed between40complete data on DWI before HIFU, between10complete data on SWI before and after HIFU and between23complete data on DWI after HIFU and47complete data on DWI non-HIFU.Some rats were killed and rat liver or tumor were obtained for pathology on beginning day and in8th,12th and14th week and10th after HIFU. Data was analysed by SPSS19.0statistical software.
RESULTS:The signal of liver normal parenchyma, prophase cirrhosis, advanced cirrhosis and hepatocellular carcinoma increased gradually on DWI. Part of viable cell tumor manifested high signal and zone of necrosis manifested low signal. ADC values of liver normal parenchyma, prophase cirrhosis, advanced cirrhosis and hepatocellular carcinoma were2645.18±369.97,2008.65±691.87,1679.50±206.16and2453.65±861.64mm2/s and the distinction was significant (F=4.34, p<0.05) when b value was50s/mm, which the distinction was significant (t=-2.82, p<0.05) between advanced cirrhosis and hepatocellular carcinoma. ADC values of liver normal parenchyma, prophase cirrhosis, advanced cirrhosis and hepatocellular carcinoma were2504.90±320.38,2185.90±455.91,1708.40±416.91and2284.30±223.18mm2/s and the distinction was not significant (F=3.23, p=0.08) when b value was100s/mm2.
ADC values of hepatocellular carcinoma were2190.52±778.65and2115.39±633.08mm2/s and the distinction was not significant (t=0.35, p>0.05) between before and after HIFU when b value was50s/mm2and its were2047.26±487.88,1889.33±547.17mm2/s and the distinction was not significant (Z=-0.88, p>0.05) when b value was100s/mm2. Data of blood vessel counting of liver normal parenchyma,advanced cirrhosis and hepatocellular carcinoma on SWI was14±2.9,25.6±6.0,34.6±21.0by turns and the distinction was not significant(p>0.05)
CONCLUSION:Manifestation of Rat liver cirrhosis and hepatocellular carcinoma is characteristic on DWI and it is important and potential for DWI to detect,diagnose, tracking hepatocellular carcinoma as well as to assess therapeutic effect after HIFU.
PART THREE Characteristics of Flow Cytometry (FCM) and Proteomics on SD Rats Liver Cancer Model after High Intensity Focused Ultrasound
OBJECTIVE:To study the immune and protein mechanism of rat hepatocellular carcinoma HIFU therapy.
MATERIALS AND METHODS:72rat hepatocellular carcinoma models were obtained after14weeks by injecting DEN intraperitoneally.23single neoplasms models were treated one or more by HIFU and all tumor samples were obtained on10day after HIFU and12rat blood preparation of them were obtained,while23tumor samples were obtained and6rat blood preparation of them from non-HIFU rat hepatocellular carcinoma models. All blood preparation were detected respectively by flow cytometry and all tumor samples were detected respectively by proteomics. Data was analysed by SPSS19.0statistical software.
RESULTS:The distinction of CD4,CD8,CD28,CD25of FoxP3r-IFN,IL4,CD4/8, Thl/Th2was not significant(P>0.05) between the factor expression of rat blood preparation after HIFU and non-HIFU.26protein spots of differential expression were detected by proteomics of them,15nonredundant protein were discriminated further. Compared with hepatocellular carcinoma protein spectrum,5proteins were down-regulated in cells and21proteins were up-regulated.
CONCLUSION:The distinction of protein expression is significant between HIFU hepatocellular carcinoma and non-HIFU,which its protein active components is similar either raised or lowered, and residual viable cell tumor must be accept further treatments as early as possible because its ability of invasion and metastasis rised.
目的：探讨二乙基亚硝胺诱导SD大鼠肝硬化、肝癌模型的成功率与特征。
材料与方法：SD大鼠128只,年龄4-5周,雄性,体重150-180g。采用纯度99.9%二乙基亚硝胺(DEN),每周以20mg/kg分3次腹腔注射。每周测大鼠体重并于Ow、8w、12w、14w进行超声检查,每期随机处死5只进行病理检查。采用SPSS19.0统计软件分析。
结果：14周建立肝癌模型72例,成功率56.25%。开始诱导后8w±1w、12w±1w、14w±1w基本达到肝硬化早期、晚期、肝癌阶段。诱导过程体重变化差异具统计学意义(F=54.79,P<0.001)。
结论：二乙基亚硝胺可以建立较稳定而理想的肝硬化及肝癌模型,且大鼠体重的变化在一定程度上反映了大鼠病程的发展。
第二部分SD大鼠肝癌模型磁共振DWI及SWI及HIFU术前后DWI特征
目的：探讨大鼠肝硬化早期、肝硬化晚期及肝癌模型DWI、SWI特征及在高强度聚焦超声(HIFU)前后评估中的价值。
材料与方法：采用二乙基亚硝胺(DEN)诱导SD大鼠128只建立肝癌模型72只,在开始诱导(Ow)、8w.12w、14w分别行磁共振常规序列及DWI、SWI动态检测,40只资料资料完整入组分析；23只大鼠模型HIFU术后24h DWI、SWI检查与非HIFU治疗组47只对照分析；10只大鼠诱导过程与HIFU后SWI资料进行动态分析。诱导过程以及HIFU术后10天分别处死部分大鼠进行病理研究。采用SPSS19.0统计软件分析。
结果：正常肝实质、肝硬化早期、肝硬化晚期、肝细胞癌DWI信号逐渐升高,肿瘤活性部分多为高信号,坏死区呈相对低信号,b=50s/mm2总体ADC有显著性意义(2645.18±369.97,2008.65±691.87,1679.50±206.16与2453.65±861.64,F=4.341,p<0.05),肝硬化晚期-肝癌ADC值变化有统计学意义(t=-2.82489, p<0.05); b=100s/mm2时总体ADC值变化没有显著性意义(2504.90±320.38,2185.90±455.91,1708.40±416.91与2284.30±223.18,Friedman检验,p=0.08)。b值分别为50s/mm2时或b=100s/mm2时,HIFU术前后肿瘤ADC值差异均没有显著性意义(2190.52±778.65,2115.39±633.08, t=0.35, p>0.05;2047.26±487.88,1889.33±547.17, Z=-0.882, p>0.05)。正常肝实质、肝硬化晚期、肝细胞癌SWI总体间差异没有显著性意义(14±2.9,25.6±6.0,34.6±±21.0,p>0.05)。
结论：大鼠肝硬化、肝细胞癌DWI成像具有特征性,DWI成像在发现、诊断与追踪肝细胞癌以及HIFU治疗前后评估、随访中中具有重要价值。
第三部分SD大鼠肝癌模型及HIFU术后流式细胞术和差异蛋白的检测
目的：探索大鼠肝癌模型HIFU治疗的免疫与蛋白机制。
材料与方法：SD大鼠肝癌模型72只,23只大鼠单结节模型接受1次或以上HIFU治疗并获得肿瘤标本、12只获血液标本,23只非HIFU治疗大鼠获肝细胞癌标本、6只获血液标本,分别进行流式细胞仪与差异蛋白组学检测,采用SPSS19.0统计软件行HIFU与非HIFU治疗组分析。
结果：HIFU与非HIFU治疗组外周血CD4、8、28、25、FoxP3、 r-IFN、IL4、CD4/8、Th1/Th2变化均无统计学意义(P>0.05)。差异蛋白组学发现了26个差异表达的蛋白质点,鉴定15种非冗余蛋白质,与肝癌模型细胞的蛋白质谱相比较有5个蛋白在细胞中表达下调,21个蛋白表达上调。
结论：HIFU治疗与不治疗组蛋白表达存在差异,上调、下调蛋白功能部分类似,提示HIFU治疗不完全可能导致残留肿瘤细胞侵袭转移活性增强,急需早期十预
PART ONE SD Rats Liver Cirrhosis, Hepatocellular Carcinoma Model Induced by Diethylnitrosamine(DEN)
OBJECTIVE:To study the success rate and characteristics of SD rats liver cirrhosis,hepatocellular carcinoma model induced by diethylnitrosamine(DEN).
MATERIALS AND METHODS:128SD rats of4-5weeks,male,150-180g were included. DEN,with99.9%Purity,was injected intraperitoneally with20mg/kg and3times every week. The weight of rats was measured every week.Every rat was carried out by Ultrasound(US) on beginning day and in8th,12th and14th week.Five rats were killed and rat liver was obtained for pathology after US.Data was analysed by SPSS19.0statistical software.
RESULTS:72rat liver tumor models were obtained after14weeks and the success rate was56.25%. Rat liver pathology achieved approximately prophase cirrhosis, advanced cirrhosis and hepatocellular carcinoma in8th±1week,12th±1week and14±1week.The change of rat weight was significant(F=54.79, P<0.001) between beginning day and in8th,12th and14th week.
CONCLUSION:Stable and ideal cirrhosis and hepatocellular carcinoma model can be establish by DEN and the development of rat liver pathology also can be reflected approximately by the change in body weight.
PART TWO Characteristics on Diffusion-weighted Imaging(DWI), Susceptibility Weighted Imaging(SWI) of SD Rats Liver Cancer Model before and after High Intensity Focused Ultrasound(HIFU)
OBJECTIVE:To study characteristics of SD rats prophase cirrhosis, advanced cirrhosis and hepatocellular carcinoma model and after HIFU on DWI and SWI and study the value of DWI after HIFU.
MATERIALS AND METHODS:72rat liver tumor models were obtained after14weeks by injecting DEN intraperitoneally for128SD rats with99.9%purity,20mg/kg and3times every week. Every rat was dynamically carried out by MRI,DWI and SWI on beginning day and in8th,12th and14th week and after HIFU.Data was analysed between40complete data on DWI before HIFU, between10complete data on SWI before and after HIFU and between23complete data on DWI after HIFU and47complete data on DWI non-HIFU.Some rats were killed and rat liver or tumor were obtained for pathology on beginning day and in8th,12th and14th week and10th after HIFU. Data was analysed by SPSS19.0statistical software.
RESULTS:The signal of liver normal parenchyma, prophase cirrhosis, advanced cirrhosis and hepatocellular carcinoma increased gradually on DWI. Part of viable cell tumor manifested high signal and zone of necrosis manifested low signal. ADC values of liver normal parenchyma, prophase cirrhosis, advanced cirrhosis and hepatocellular carcinoma were2645.18±369.97,2008.65±691.87,1679.50±206.16and2453.65±861.64mm2/s and the distinction was significant (F=4.34, p<0.05) when b value was50s/mm, which the distinction was significant (t=-2.82, p<0.05) between advanced cirrhosis and hepatocellular carcinoma. ADC values of liver normal parenchyma, prophase cirrhosis, advanced cirrhosis and hepatocellular carcinoma were2504.90±320.38,2185.90±455.91,1708.40±416.91and2284.30±223.18mm2/s and the distinction was not significant (F=3.23, p=0.08) when b value was100s/mm2.
ADC values of hepatocellular carcinoma were2190.52±778.65and2115.39±633.08mm2/s and the distinction was not significant (t=0.35, p>0.05) between before and after HIFU when b value was50s/mm2and its were2047.26±487.88,1889.33±547.17mm2/s and the distinction was not significant (Z=-0.88, p>0.05) when b value was100s/mm2. Data of blood vessel counting of liver normal parenchyma,advanced cirrhosis and hepatocellular carcinoma on SWI was14±2.9,25.6±6.0,34.6±21.0by turns and the distinction was not significant(p>0.05)
CONCLUSION:Manifestation of Rat liver cirrhosis and hepatocellular carcinoma is characteristic on DWI and it is important and potential for DWI to detect,diagnose, tracking hepatocellular carcinoma as well as to assess therapeutic effect after HIFU.
PART THREE Characteristics of Flow Cytometry (FCM) and Proteomics on SD Rats Liver Cancer Model after High Intensity Focused Ultrasound
OBJECTIVE:To study the immune and protein mechanism of rat hepatocellular carcinoma HIFU therapy.
MATERIALS AND METHODS:72rat hepatocellular carcinoma models were obtained after14weeks by injecting DEN intraperitoneally.23single neoplasms models were treated one or more by HIFU and all tumor samples were obtained on10day after HIFU and12rat blood preparation of them were obtained,while23tumor samples were obtained and6rat blood preparation of them from non-HIFU rat hepatocellular carcinoma models. All blood preparation were detected respectively by flow cytometry and all tumor samples were detected respectively by proteomics. Data was analysed by SPSS19.0statistical software.
RESULTS:The distinction of CD4,CD8,CD28,CD25of FoxP3r-IFN,IL4,CD4/8, Thl/Th2was not significant(P>0.05) between the factor expression of rat blood preparation after HIFU and non-HIFU.26protein spots of differential expression were detected by proteomics of them,15nonredundant protein were discriminated further. Compared with hepatocellular carcinoma protein spectrum,5proteins were down-regulated in cells and21proteins were up-regulated.
CONCLUSION:The distinction of protein expression is significant between HIFU hepatocellular carcinoma and non-HIFU,which its protein active components is similar either raised or lowered, and residual viable cell tumor must be accept further treatments as early as possible because its ability of invasion and metastasis rised.
引文
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