二乙基亚硝胺诱发大鼠原发性肝癌过程中肝脏胶原的动态观察
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摘要
研究背景
肝细胞癌(hepatocellular carcinoma, HCC,以下简称肝癌)是全球最常见的恶性肿瘤之一,世界上每年新诊断的病例约60万例,肝癌引起的死亡位居所有癌症相关死亡的第三位。乙肝病毒慢性感染是肝癌形成的重要因素之一,由于我国有超过1亿的乙肝感染者,所以原发性肝癌更是高发的重大疾病。据卫生部统计,2007年我国城市居民肝癌死亡率占恶性肿瘤死因的第2位(25.47/10万)。在农村居民中,肝癌死亡率甚至达到了28.72/10万。虽然肝癌切除技术日益完善,但许多肝癌患者就诊时病情已进展,导致肝癌可切除率不高、术后复发率居高不下,患者的预后严重受严重影响。因此,肝癌的预防和早期诊断至关重要。目前,临床诊断手段缺乏,早期诊断率低,许多肝癌一经发现己至进展期。尽早发现肝癌的癌前病变是早期诊断和改善预后的关键,但在临床上尚缺乏诊断肝癌前期病变的可靠指标。尽快阐明肝癌癌前病变,从而建立针对这些高危人群的癌症预防战略,是医学工作者的一项迫切任务。肝硬化作为肝癌的癌前期病变,其组织内最大的病理特征改变是胶原纤维的沉积,本实验通过二乙基亚硝胺(DEN)诱癌,模拟动态观察肝硬化肝癌发生过程中肝组织胶原的变化特点,初步探索其中规律,以期为寻找肝癌癌前期特异性病理变化提供参考。
目的
1.建立改良型伴发肝硬化肝癌大鼠模型,为研究肝癌发病特点和药物干预提供较理想动物模型。
2.动态观察大鼠自肝炎、肝硬化发展为肝癌过程中胶原纤维的变化特点,探讨肝癌前期病变的病理特征,为肝癌预防和肝癌预后提供参考。
材料和方法
1.动物分组和模型制备：50只Wistar大鼠(100-120g)随机分为模型组30只、对照组20只。模型组以二乙基亚硝胺(DEN)腹腔注射,50mg/Kg体重,每周2次,连续4周后改为每周一次腹腔注射DEN,50mg/Kg体重,至14周停止；对照组以生理盐水腹腔注射,0.1ml/次,每周2次,连续4周后改为每周一次腹腔注射生理盐水,0.1ml/1次,至14周停止。于诱癌开始后第2、4、8、12、14、18周,分批处死大鼠,每次处死模型组大鼠3只,对照组大鼠3只。持续乙醚吸入麻醉,心脏取血5ml,制备血清,置-20℃储存。出现癌灶者取肉眼癌灶及距癌灶边缘1cm癌旁组织,大小约0.5cmx0.5cm,用10%中性福尔马林固定,未出现癌灶者,随机取部分肝组织,大小约0.5cm×0.5cm,用10%中性福尔马林固定,用于病理石蜡切片制备。其余组织-80℃冰箱冷冻保存。
2.血清生化指标测定：使用Aeroset全自动生化分析仪进行血清谷丙转氨酶、总胆红素、γ-谷氨酰转肽酶检测。
3. Western-blot蛋白印迹技术：进行肝组织内基质金属蛋白酶MMP-2、MMP-9的测定。
4.PCR技术：进行肝组织内Ⅰ、Ⅲ型胶原mRNA的检测。
5.常规病理检测和特殊染色：对不同时期肝组织切片进行HE、Masson三色、网状染色等。并采用Knodell分级、Ishak分期及Metavir分期对标本进行分级分期。
6.荧光免疫染色：检测肝组织内CD31的表达量。
7.数据处理：利用SPSS13.0统计软件对各数据进行统计分析。
结果
1.诱癌14周以后成功诱导出肝硬化伴发的原发性肝癌,14周时成癌情况为3/5,18周时成癌情况为3/3,成癌率为75%,总体死亡率33%(10/30)；
2.模型组大鼠体重在诱癌2周以后与对照组相比减轻,且具有统计学意义,p<0.05；
3.模型组大鼠血清丙氨酸氨基转移酶(ALT)第2周开始升高,第14周达高峰,第18周时下降。第2周以后模型组与对照组间均有统计学差异(p<0.05)；
4.模型组大鼠血清总胆红素(TBIL)第4周开始升高,第14周达高峰,第18周时下降。第4周以后(除12周外)模型组与对照组间均有显著性统计学差异(p<0.01)；
5.模型组大鼠血清γ-谷氨酰转肽酶随时间逐渐升高,12周以后变化不明显。诱癌第12周以后模型组GGT含量与对照组比较有统计学差异(p<0.05)；
6.DEN诱导的伴发肝硬化的大鼠肝癌模型中,肝硬化时,肝组织内胶原沉积明显,窦周胶原沉积增加；肝癌组织胶原纤维明显减少；
7.CD31在大鼠肝癌组织内表达强于在肝硬化肝组织内表达；
8.Ⅰ、Ⅲ型胶原分布、沉积特点与总体胶原变化一致；
9.TPEF、SHG技术分析肝脏胶原结果与传统病理技术分析较一致；
结论
1.改良型二乙基亚硝胺诱导的伴发肝硬化肝癌模型是较理想的疾病研究模型。
2.DEN诱导的大鼠肝脏癌变过程中,胶原纤维在硬化肝组织中沉积明显增加,而在肝癌组织中明显被降解减少,且随着肝癌的进展,癌组织内胶原进一步减少,提示胶原纤维过度沉积发生在肝脏癌变前,而癌变的肝组织内胶原纤维含量则跟癌灶的进展呈负相关；
3.CD31在大鼠肝硬化后期、肝癌组织内均有表达,且肝癌组织中的表达要高于肝硬化肝组织,提示肝硬化肝组织内CD31的表达可能跟肝硬化后期的肝窦毛细血管化有关,而在肝癌组织中的进一步高表达则可能肝癌的新生血管有关；
4.随着肝硬化的进展,肝组织内Ⅰ、Ⅲ型胶原与总体胶原沉积均明显增加,在肝癌形成后均明显减少,与基质金属蛋白酶变化呈负相关,提示Ⅰ、Ⅲ型胶原在该疾病模型中能代表肝脏胶原变化趋势,可能与肝癌的预后有关；
5.经初步探索,TPEF联合SHG技术跟传统病理技术相比,在判定不同肝纤维化分期上与其较一致,且更省时、省力,减少了人为影响因素,是一种值得推广的肝脏胶原分析手段。
应用价值
1.改良型二乙基亚硝胺诱导的伴发肝硬化肝癌模型较传统诱导模型,具有重复性好、能精确控制药物摄入量、成癌时间短、成癌率高、死亡率低、与人类疾病模型相似等特点,是一种理想的用于人类疾病研究的动物模型。
2.进一步阐明了大鼠肝脏癌变前后组织中胶原纤维的沉积特点,为临床诊断肝癌癌前病变及判定肝癌患者预后提供了参考。
初步展示了一种新技术-SHG联合TPEF技术在肝组织内胶原纤
维观察中的应用价值,可望促进肝组织胶原分析技术的发展。
Hepatocellular carcinoma (HCC) is of the most common malignancies throughout the world, and there are about600,000new cases every year. The mortality caused by HCC is in the third of that caused by all carcinoma. HBV infection is one of the important risk factors. There are more than100,000,000carriers of HBV in China. So HCC is a multiple critical illness in our country. According to Chinese Ministry of Health statistics, in2007mortality caused by HCC in China urban residents is in the second of that caused by all malignancies(25.47/100000), and the mortality in rural residents is28.72/100000. Although the technology of hepatectomy is progressing, but many patients have poor prognosis because of delay in diagnosis and recurrence after operation. So prevention and early diagnosis of HCC is essential. Currently, because of lack of clinical diagnostic indicators, the rate of early diagnosis is low, many patients are diagnosed at an advanced stage. Early detection of precancerous lesions of HCC is key to early diagnosis and prognosis, currently the clinical diagnosis of precancerous lesions of HCC is still in lack of reliable indicators. As soon as possible to clarify the precancerous lesions of HCC and establish prevention strategies for hige-risk groups, improve prognosis, is an urgent task for the medical workers. Cirrhosis as a precancerous lesions of HCC, one of the pathological features in its organization change is the deposition of collagen fibers. We dynamic observed the characteristic changes of collagen fibers in rat HCC induced by DEN, in order to find the precancerous lesions of HCC and provide a pathological reference for clinical diagnosis and treatment.
Objective
1.Establish improved concurrency model of cirrhosis of HCC in rats, provide an ideal animal model for the research of characteristics of HCC and drug intervention.
2. To observe the characteristic changes of collagen fibers in rat HCC induced by DEN dynamically, explore the pathological features of HCC precursor lesions, provide a reference for prevention and prognosis of HCC.
Materials and methods
1.Experimental protocol:50male Wistar rats of about100-120g were randomly divided into normal group(20rats) and model group(30rats). The model group was administered a dose of50mg DEN per kilogram intraperitoneal injection, twice a week, for4weeks, then once a week, for another10weeks. The normal group was given saline instead of DEN. Three rats of model and normal group were killd at2,4,8,12,14, weeks,respectively. Operation is under continuous ether inhalation anesthesia,5ml blood were taken for all animals before killed, blood serum stored at-20℃. Take foci of tumor and adjacent organization, which is lcm far from the foci edge, and every organization block size is of about0.5×0.5cm. Cut part of liver tissue of rats in normal group and in model group without tumor, randomly. Tissue were fixed in10%neutral formalin, then for pathological paraffin sections. The remaining liver stored at-80℃.
2.Serum biochemistry:the serum gamma-glutamy transpeptidase, total bilirubin, alanine aminotransferase were tested by automatic-biochemical analyser.
3. Western-blot was performed to evaluate the content of matrix metalloproteinase-2and matrix metalloproteinase-9in liver.
4.PCR was performed to evaluate the content of collagen type Ⅰ and Ⅲ in liver.
5. Histopathological analysis:paraffin sections w ere cut to stained by H.E., Masson's Trichrome, argyrophilic fiber.The degree of necrosis, fibrosis, cirrhosis were analyzed by Knodell, Ishak, Metavir.
6.Fluorescent immunostaining is performed observe the change of CD31in liver.
7.Statistical analysis:use spss13.0to analyse results.
Results
1.The rat model both with cirrhosis and HCC was established successfully after14weeks inducing. At14th week, there were3rats checked with cirrhosis and HCC in5rats, and3rats in3at18th week. The carcinogenesis rate was75%, and overall mortality was33%.
2.Body weight of Model rats were lighter than the control group after2weeks of medicine inducing, and the result had a statistical significance.
3.In model group, ALT in serum began to increase at2th week after inducing, reached a peak at14th week, and decreased from18th week. After2weeks of inducing, comparision between the two groups was statistically significant, P<0.05.
4. In model group, TBIL in serum began to increase at4th week after inducing, reached a peak at14th week, and decreased from18th week. After4weeks of inducing, comparision between the two groups was statistically significant, except of12th week, P<0.01.
5. In model group, y-GGT in serum began to increase at2th week after inducing, reached a peak at12th week, then at a steady level. Comparision between the two groups was statistically significant after12weeks of inducing, P<0.01.
6.In the model group, when rats were cirrhosis, collagen deposition in liver tissue was significantly increased, in the time, perisinusoidal collagen deposition together. When rats geted HCC, collagen deposition was reduced in the foci.
7. CD31expression in rat hepatoma tissue is stronger than in the cirrhotic liver tissue.
8. Changes of typeⅠ and type Ⅲ collagen deposition in tissue was consistent with overall collagen.
9.The result was similar in analysis of liver collagen, either using SHG and TPEF or traditional pathological techniques.
conclusion
1.The improved model is better for HCC research than traditional methods.
2.In the progress of HCC in this improved model, collagen fiber deposition in cirrhotic liver tissue was significantly increased, and decreased in HCC tissue.The result indicates that excessive collagen fiber deposition may occurrence before HCC formation, collagen fiber of HCC is negatively correlated with the progress of the foci.
3.There was expression of CD31both in cirrhosis and HCC, and expression of CD31in HCC is higher than in cirrhotic liver tissue. This result indicates that CD31expression in cirrhosis may be related with hepatic sinusoidal capillarization, and related with neovascularization in HCC.
4.In the progress of cirrhosis, type Ⅰ, type Ⅲ, and all collagen increased significantly, then decreased in HCC. It was negative related with MMP. This result indicates thatchanges of type Ⅰ, type Ⅲ are consistent with overall collagen in HCC formation, and may be related with prognosis.
5. With preliminary exploration, we think the technology of TPEF united SHG is consistent with traditional pathology, and may be more effective, and reduce the human impact factors. It may be a better tool for liver collagen analysis.
Application value
1.Improved HCC model is an ideal tool for human desease research, because it is better reproducible,precise control of medicine intake, short time to carcinogenesis,lower mortality and similar to human HCC.
2.This experiment confirm the characteristics of collagen deposition, provide a reference for clinical diagnosis and prognosis.
3.Demonstrate a new technology of SHG united TPEF which can be used in the liver collagen fiber analysis. Hope to promote the development of analytical techniques in liver tissue collagen.
肝细胞癌(hepatocellular carcinoma, HCC,以下简称肝癌)是全球最常见的恶性肿瘤之一,世界上每年新诊断的病例约60万例,肝癌引起的死亡位居所有癌症相关死亡的第三位。乙肝病毒慢性感染是肝癌形成的重要因素之一,由于我国有超过1亿的乙肝感染者,所以原发性肝癌更是高发的重大疾病。据卫生部统计,2007年我国城市居民肝癌死亡率占恶性肿瘤死因的第2位(25.47/10万)。在农村居民中,肝癌死亡率甚至达到了28.72/10万。虽然肝癌切除技术日益完善,但许多肝癌患者就诊时病情已进展,导致肝癌可切除率不高、术后复发率居高不下,患者的预后严重受严重影响。因此,肝癌的预防和早期诊断至关重要。目前,临床诊断手段缺乏,早期诊断率低,许多肝癌一经发现己至进展期。尽早发现肝癌的癌前病变是早期诊断和改善预后的关键,但在临床上尚缺乏诊断肝癌前期病变的可靠指标。尽快阐明肝癌癌前病变,从而建立针对这些高危人群的癌症预防战略,是医学工作者的一项迫切任务。肝硬化作为肝癌的癌前期病变,其组织内最大的病理特征改变是胶原纤维的沉积,本实验通过二乙基亚硝胺(DEN)诱癌,模拟动态观察肝硬化肝癌发生过程中肝组织胶原的变化特点,初步探索其中规律,以期为寻找肝癌癌前期特异性病理变化提供参考。
目的
1.建立改良型伴发肝硬化肝癌大鼠模型,为研究肝癌发病特点和药物干预提供较理想动物模型。
2.动态观察大鼠自肝炎、肝硬化发展为肝癌过程中胶原纤维的变化特点,探讨肝癌前期病变的病理特征,为肝癌预防和肝癌预后提供参考。
材料和方法
1.动物分组和模型制备：50只Wistar大鼠(100-120g)随机分为模型组30只、对照组20只。模型组以二乙基亚硝胺(DEN)腹腔注射,50mg/Kg体重,每周2次,连续4周后改为每周一次腹腔注射DEN,50mg/Kg体重,至14周停止；对照组以生理盐水腹腔注射,0.1ml/次,每周2次,连续4周后改为每周一次腹腔注射生理盐水,0.1ml/1次,至14周停止。于诱癌开始后第2、4、8、12、14、18周,分批处死大鼠,每次处死模型组大鼠3只,对照组大鼠3只。持续乙醚吸入麻醉,心脏取血5ml,制备血清,置-20℃储存。出现癌灶者取肉眼癌灶及距癌灶边缘1cm癌旁组织,大小约0.5cmx0.5cm,用10%中性福尔马林固定,未出现癌灶者,随机取部分肝组织,大小约0.5cm×0.5cm,用10%中性福尔马林固定,用于病理石蜡切片制备。其余组织-80℃冰箱冷冻保存。
2.血清生化指标测定：使用Aeroset全自动生化分析仪进行血清谷丙转氨酶、总胆红素、γ-谷氨酰转肽酶检测。
3. Western-blot蛋白印迹技术：进行肝组织内基质金属蛋白酶MMP-2、MMP-9的测定。
4.PCR技术：进行肝组织内Ⅰ、Ⅲ型胶原mRNA的检测。
5.常规病理检测和特殊染色：对不同时期肝组织切片进行HE、Masson三色、网状染色等。并采用Knodell分级、Ishak分期及Metavir分期对标本进行分级分期。
6.荧光免疫染色：检测肝组织内CD31的表达量。
7.数据处理：利用SPSS13.0统计软件对各数据进行统计分析。
结果
1.诱癌14周以后成功诱导出肝硬化伴发的原发性肝癌,14周时成癌情况为3/5,18周时成癌情况为3/3,成癌率为75%,总体死亡率33%(10/30)；
2.模型组大鼠体重在诱癌2周以后与对照组相比减轻,且具有统计学意义,p<0.05；
3.模型组大鼠血清丙氨酸氨基转移酶(ALT)第2周开始升高,第14周达高峰,第18周时下降。第2周以后模型组与对照组间均有统计学差异(p<0.05)；
4.模型组大鼠血清总胆红素(TBIL)第4周开始升高,第14周达高峰,第18周时下降。第4周以后(除12周外)模型组与对照组间均有显著性统计学差异(p<0.01)；
5.模型组大鼠血清γ-谷氨酰转肽酶随时间逐渐升高,12周以后变化不明显。诱癌第12周以后模型组GGT含量与对照组比较有统计学差异(p<0.05)；
6.DEN诱导的伴发肝硬化的大鼠肝癌模型中,肝硬化时,肝组织内胶原沉积明显,窦周胶原沉积增加；肝癌组织胶原纤维明显减少；
7.CD31在大鼠肝癌组织内表达强于在肝硬化肝组织内表达；
8.Ⅰ、Ⅲ型胶原分布、沉积特点与总体胶原变化一致；
9.TPEF、SHG技术分析肝脏胶原结果与传统病理技术分析较一致；
结论
1.改良型二乙基亚硝胺诱导的伴发肝硬化肝癌模型是较理想的疾病研究模型。
2.DEN诱导的大鼠肝脏癌变过程中,胶原纤维在硬化肝组织中沉积明显增加,而在肝癌组织中明显被降解减少,且随着肝癌的进展,癌组织内胶原进一步减少,提示胶原纤维过度沉积发生在肝脏癌变前,而癌变的肝组织内胶原纤维含量则跟癌灶的进展呈负相关；
3.CD31在大鼠肝硬化后期、肝癌组织内均有表达,且肝癌组织中的表达要高于肝硬化肝组织,提示肝硬化肝组织内CD31的表达可能跟肝硬化后期的肝窦毛细血管化有关,而在肝癌组织中的进一步高表达则可能肝癌的新生血管有关；
4.随着肝硬化的进展,肝组织内Ⅰ、Ⅲ型胶原与总体胶原沉积均明显增加,在肝癌形成后均明显减少,与基质金属蛋白酶变化呈负相关,提示Ⅰ、Ⅲ型胶原在该疾病模型中能代表肝脏胶原变化趋势,可能与肝癌的预后有关；
5.经初步探索,TPEF联合SHG技术跟传统病理技术相比,在判定不同肝纤维化分期上与其较一致,且更省时、省力,减少了人为影响因素,是一种值得推广的肝脏胶原分析手段。
应用价值
1.改良型二乙基亚硝胺诱导的伴发肝硬化肝癌模型较传统诱导模型,具有重复性好、能精确控制药物摄入量、成癌时间短、成癌率高、死亡率低、与人类疾病模型相似等特点,是一种理想的用于人类疾病研究的动物模型。
2.进一步阐明了大鼠肝脏癌变前后组织中胶原纤维的沉积特点,为临床诊断肝癌癌前病变及判定肝癌患者预后提供了参考。
初步展示了一种新技术-SHG联合TPEF技术在肝组织内胶原纤
维观察中的应用价值,可望促进肝组织胶原分析技术的发展。
Hepatocellular carcinoma (HCC) is of the most common malignancies throughout the world, and there are about600,000new cases every year. The mortality caused by HCC is in the third of that caused by all carcinoma. HBV infection is one of the important risk factors. There are more than100,000,000carriers of HBV in China. So HCC is a multiple critical illness in our country. According to Chinese Ministry of Health statistics, in2007mortality caused by HCC in China urban residents is in the second of that caused by all malignancies(25.47/100000), and the mortality in rural residents is28.72/100000. Although the technology of hepatectomy is progressing, but many patients have poor prognosis because of delay in diagnosis and recurrence after operation. So prevention and early diagnosis of HCC is essential. Currently, because of lack of clinical diagnostic indicators, the rate of early diagnosis is low, many patients are diagnosed at an advanced stage. Early detection of precancerous lesions of HCC is key to early diagnosis and prognosis, currently the clinical diagnosis of precancerous lesions of HCC is still in lack of reliable indicators. As soon as possible to clarify the precancerous lesions of HCC and establish prevention strategies for hige-risk groups, improve prognosis, is an urgent task for the medical workers. Cirrhosis as a precancerous lesions of HCC, one of the pathological features in its organization change is the deposition of collagen fibers. We dynamic observed the characteristic changes of collagen fibers in rat HCC induced by DEN, in order to find the precancerous lesions of HCC and provide a pathological reference for clinical diagnosis and treatment.
Objective
1.Establish improved concurrency model of cirrhosis of HCC in rats, provide an ideal animal model for the research of characteristics of HCC and drug intervention.
2. To observe the characteristic changes of collagen fibers in rat HCC induced by DEN dynamically, explore the pathological features of HCC precursor lesions, provide a reference for prevention and prognosis of HCC.
Materials and methods
1.Experimental protocol:50male Wistar rats of about100-120g were randomly divided into normal group(20rats) and model group(30rats). The model group was administered a dose of50mg DEN per kilogram intraperitoneal injection, twice a week, for4weeks, then once a week, for another10weeks. The normal group was given saline instead of DEN. Three rats of model and normal group were killd at2,4,8,12,14, weeks,respectively. Operation is under continuous ether inhalation anesthesia,5ml blood were taken for all animals before killed, blood serum stored at-20℃. Take foci of tumor and adjacent organization, which is lcm far from the foci edge, and every organization block size is of about0.5×0.5cm. Cut part of liver tissue of rats in normal group and in model group without tumor, randomly. Tissue were fixed in10%neutral formalin, then for pathological paraffin sections. The remaining liver stored at-80℃.
2.Serum biochemistry:the serum gamma-glutamy transpeptidase, total bilirubin, alanine aminotransferase were tested by automatic-biochemical analyser.
3. Western-blot was performed to evaluate the content of matrix metalloproteinase-2and matrix metalloproteinase-9in liver.
4.PCR was performed to evaluate the content of collagen type Ⅰ and Ⅲ in liver.
5. Histopathological analysis:paraffin sections w ere cut to stained by H.E., Masson's Trichrome, argyrophilic fiber.The degree of necrosis, fibrosis, cirrhosis were analyzed by Knodell, Ishak, Metavir.
6.Fluorescent immunostaining is performed observe the change of CD31in liver.
7.Statistical analysis:use spss13.0to analyse results.
Results
1.The rat model both with cirrhosis and HCC was established successfully after14weeks inducing. At14th week, there were3rats checked with cirrhosis and HCC in5rats, and3rats in3at18th week. The carcinogenesis rate was75%, and overall mortality was33%.
2.Body weight of Model rats were lighter than the control group after2weeks of medicine inducing, and the result had a statistical significance.
3.In model group, ALT in serum began to increase at2th week after inducing, reached a peak at14th week, and decreased from18th week. After2weeks of inducing, comparision between the two groups was statistically significant, P<0.05.
4. In model group, TBIL in serum began to increase at4th week after inducing, reached a peak at14th week, and decreased from18th week. After4weeks of inducing, comparision between the two groups was statistically significant, except of12th week, P<0.01.
5. In model group, y-GGT in serum began to increase at2th week after inducing, reached a peak at12th week, then at a steady level. Comparision between the two groups was statistically significant after12weeks of inducing, P<0.01.
6.In the model group, when rats were cirrhosis, collagen deposition in liver tissue was significantly increased, in the time, perisinusoidal collagen deposition together. When rats geted HCC, collagen deposition was reduced in the foci.
7. CD31expression in rat hepatoma tissue is stronger than in the cirrhotic liver tissue.
8. Changes of typeⅠ and type Ⅲ collagen deposition in tissue was consistent with overall collagen.
9.The result was similar in analysis of liver collagen, either using SHG and TPEF or traditional pathological techniques.
conclusion
1.The improved model is better for HCC research than traditional methods.
2.In the progress of HCC in this improved model, collagen fiber deposition in cirrhotic liver tissue was significantly increased, and decreased in HCC tissue.The result indicates that excessive collagen fiber deposition may occurrence before HCC formation, collagen fiber of HCC is negatively correlated with the progress of the foci.
3.There was expression of CD31both in cirrhosis and HCC, and expression of CD31in HCC is higher than in cirrhotic liver tissue. This result indicates that CD31expression in cirrhosis may be related with hepatic sinusoidal capillarization, and related with neovascularization in HCC.
4.In the progress of cirrhosis, type Ⅰ, type Ⅲ, and all collagen increased significantly, then decreased in HCC. It was negative related with MMP. This result indicates thatchanges of type Ⅰ, type Ⅲ are consistent with overall collagen in HCC formation, and may be related with prognosis.
5. With preliminary exploration, we think the technology of TPEF united SHG is consistent with traditional pathology, and may be more effective, and reduce the human impact factors. It may be a better tool for liver collagen analysis.
Application value
1.Improved HCC model is an ideal tool for human desease research, because it is better reproducible,precise control of medicine intake, short time to carcinogenesis,lower mortality and similar to human HCC.
2.This experiment confirm the characteristics of collagen deposition, provide a reference for clinical diagnosis and prognosis.
3.Demonstrate a new technology of SHG united TPEF which can be used in the liver collagen fiber analysis. Hope to promote the development of analytical techniques in liver tissue collagen.
引文
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