活化肝星状细胞促进大鼠肝细胞去分化为肝前体细胞

发布时间：2018-04-19 21:23

本文选题：肝前体细胞（HPC） + 肝星状细胞（HSC）　；参考：《第二军医大学》2014年硕士论文

【摘要】：【研究背景及目的】肝前体细胞（Hepatic progenitor cell, HPC）是损伤肝脏中出现的一种具有双向分化潜能的细胞，可向肝细胞和胆管上皮细胞分化参与肝再生。在慢性肝病状态下，HPC可被激活参与肝损伤修复，而且HPC的数量与疾病的严重程度及肝癌发生的风险指数相关。HPC既表达胆管上皮细胞标志物（OV-6、PCK和Sox9等）和成熟肝细胞标志物（白蛋白等），同时也表达肝母细胞标志物（AFP和Dlk1等）。目前，HPC来源尚不明确，传统观点认为HPC来自Hering管，也有研究显示肝细胞、肝星状细胞（Hepaticstellate cell, HSC）及骨髓干细胞在肝损伤环境下可转化为HPC。因此，有学者认为HPC可能是一种来源不同的异质细胞群。 HSC位于Disse间隙，与肝细胞和肝窦内皮细胞直接接触。正常情况下，HSC处于静止状态，肝损伤时，HSC可激活，产生细胞外基质（Extracellular matrix, ECM），并分泌大量的细胞因子和生长因子。HSC的激活是肝纤维化发生发展的中心环节。近年来不断有证据提示HSC还具有促进肝组织再生的作用，同时也有研究表明HPC可来源于HSC。这些研究均表明HSC在肝脏再生过程中扮演着重要角色。基于以上背景，本课题旨在通过肝损伤动物模型明确活化HSC对HPC产生的影响，并利用体外共培养实验进一步明确HPC的细胞来源。【实验方法】一、探讨HSC活化与HPC产生的关系 1、收集人急性肝炎、肝纤维化、肝硬化组织标本，免疫荧光检测HPC标志物PCK及活化HSC标志物α-SMA表达情况。 2、构建2-乙酰氨基芴（2-acetylaminofluorene,2-AAF）/肝大部切除术（Partialhepatectomy, PH）动物模型诱导HPC活化。予2-AAF以10mg/kg体重的剂量对Sprague-Dawley（SD）大鼠进行灌胃，每天1次，持续5天，于第6天行肝大部切除术，手术当日停止灌胃2-AAF，术后第2天继续予2-AAF灌胃，并持续1周。分别取肝大部切除术后0天、4天、6天、9天的大鼠肝脏组织，免疫荧光检测PCK及α-SMA表达情况。 3、构建对乙酰氨基酚（N-acetyl-paraaminophen, APAP）肝损伤模型。按照300mg/kg体重的剂量对SD大鼠腹腔注射APAP，仅注射1次，注射24小时后处死大鼠，取新鲜肝脏组织，免疫荧光检测PCK及α-SMA表达情况。二、明确活化HSC对HPC产生的影响（一）鉴定氯化钆（Gadolinium chloride, GdCl3）和胶霉毒素（Gliotoxin）对Kupffer细胞和活化HSC的作用 1、以2ml/kg体重的剂量对SD大鼠腹腔注射20%四氯化碳（Carbon tetrachloride,CCl4），隔日注射1次，共注射2次，末次注射后48小时，予GdCl3以10mg/kg体重的剂量尾静脉注射1次。注射GdCl324小时后取大鼠肝脏组织。免疫荧光检测Kuppfer细胞标志物CD68和活化HSC标志物α-SMA的表达情况。 2、对SD大鼠腹腔注射CCl4，隔日注射1次，共注射2次，在末次注射后48小时，予Gliotoxin以3mg/kg体重的剂量腹腔注射1次。注射Gliotoxin24小时后处死大鼠，取肝脏组织。免疫荧光检测CD68和α-SMA的表达情况。（二）观察抑制HSC活化对HPC产生的影响 1、分离SD大鼠原代HSC，在体外予DMEM培液培养。收集体外培养后第3天（静息）和第12天（活化）的HSC培养上清。 2、在2-AAF/PH模型肝大部切除术或APAP模型APAP注射前1天，予模型大鼠尾静脉注射GdCl3抑制Kupffer细胞活性及HSC活化。分别于肝大部切除术或APAP注射72小时后处死大鼠，取肝脏组织，免疫荧光检测PCK及α-SMA表达情况，对比观察抑制HSC活化对HPC产生的影响。 3、在以上模型注射GdCl3抑制HSC活化后，于肝大部切除术或APAP注射的次日，腹腔注射不同活化状态的原代HSC培养上清4ml1次。48小时后取大鼠肝脏组织，免疫荧光检测PCK及α-SMA表达变化情况，对比观察补充HSC培养上清对HPC产生的影响。（三）观察清除活化HSC对HPC产生的影响 1、在2-AAF/PH模型中，在灌胃2-AAF的第1、3天，同时以2ml/kg体重的剂量对SD大鼠腹腔注射20%CCl4以刺激HSC活化，并于肝大部切除术前1天，按3mg/kg体重的剂量对大鼠腹腔注射Gliotoxin清除活化HSC，于肝大部切除术后3天取大鼠肝脏组织，免疫荧光检测PCK及α-SMA表达情况，比较清除活化HSC前后HPC产生数量的变化。 2、在2-AAF/PH+Gliotoxin处理的基础上，于肝大部切除术后24小时腹腔注射不同活化状态的原代HSC培养上清4ml1次。48小时后处死大鼠，免疫荧光检测肝脏组织中PCK及α-SMA表达情况，明确活化HSC对HPC产生的影响。三、明确活化HSC是否可促进大鼠肝细胞去分化为HPC 1、分离SD大鼠原代肝细胞和原代HSC，利用可使两种细胞不直接接触的双层细胞共培养体系，将肝细胞分别与不同活化状态HSC共培养，光镜下观察肝细胞的形态变化。在共培养的第0、3、5天，细胞免疫荧光检测肝细胞标志物HNF4α及HPC标志物PCK、Sox9表达变化情况。2、为进一步明确与活化HSC共培养的肝细胞中出现的PCK阳性细胞具有双向分化潜能，将其置于胆管上皮细胞分化诱导液（含丁酸钠）中培养，8天后细胞免疫荧光检测胆管上皮细胞标志物CK19表达情况；将其置于肝细胞分化诱导液（含HGF、胰岛素、转铁蛋白、地塞米松）中培养，11天后细胞免疫荧光检测肝细胞标志物白蛋白（Albumin, Alb）表达情况。【实验结果】一、活化HSC促进HPC产生（一）HPC产生与HSC活化相关 1、免疫荧光检测人急性肝炎、肝纤维化、肝硬化组织中PCK及α-SMA的表达情况，结果显示仅在与α-SMA阳性细胞毗邻区域存在PCK阳性细胞。 2、在2-AAF/PH模型中，于肝大部切除术后0天、4天、6天、9天取肝脏组织，，免疫荧光结果显示第0天和第4天时PCK阳性细胞及α-SMA阳性细胞数量均较少，但第6天和第9天时PCK阳性细胞及α-SMA阳性细胞数量明显增加，且两者紧密相联，交织存在。 3、取APAP注射24小时后肝脏组织，免疫荧光结果显示PCK阳性细胞被大量α-SMA阳性细胞紧密围绕，提示HPC产生与HSC活化相关。（二）活化HSC促进HPC产生 1、GdCl3可抑制Kuppfer细胞活性， Gliotoxin可清除活化HSC （1）CCl4造成急性肝损伤过程中，尾静脉注射GdCl3，取肝组织，免疫荧光结果显示与单纯CCl4急性肝损伤标本相比，CD68及α-SMA表达均明显减少。（2）在CCl4造成急性肝损伤后，腹腔注射Gliotoxin，免疫荧光检测肝组织中α-SMA及CD68表达情况，结果发现与单纯CCl4处理组相比，α-SMA阳性细胞明显减少，而CD68阳性细胞却没有明显变化。 2、抑制HSC活化对HPC产生的影响（1）在2-AAF/PH及APAP造模过程中，利用GdCl3抑制Kuppfer细胞活性及HSC活化，免疫荧光结果显示抑制HSC活化后，PCK阳性细胞及α-SMA阳性细胞数量均明显减少。（2）抑制HSC活化后，再补充不同活化状态HSC培养上清，免疫荧光结果显示补充活化HSC培养上清后，PCK阳性细胞及α-SMA阳性细胞数量增加，且两者交织存在。而补充静息HSC培养上清后，肝组织PCK及α-SMA表达没有明显变化。 3、清除活化HSC对HPC产生的影响（1）与单纯2-AAF/PH模型组织中存在大量PCK阳性细胞和α-SMA阳性细胞不同，腹腔注射Gliotoxin清除活化HSC后，免疫荧光结果显示肝组织中仅存在极少量PCK阳性细胞和α-SMA阳性细胞。（2）清除活化HSC后，再补充不同活化状态HSC培养上清，结果显示补充活化HSC培养上清后，PCK阳性细胞数量增加。而补充静息HSC培养上清后，PCK阳性细胞数量却没有明显变化。二、活化HSC促进大鼠肝细胞去分化为HPC 1、将肝细胞与不同活化状态HSC共培养，细胞免疫荧光结果显示共培养5天时，与活化HSC共培养的肝细胞中可出现PCK阳性细胞，而与静息HSC共培养或单独培养的肝细胞中并没有PCK阳性细胞的出现。在肝细胞与活化HSC共培养的第0、3、5天细胞免疫荧光检测Sox9及HNF4α的表达情况，结果显示第0天时Sox9表达极弱，而HNF4α大量表达，至第5天时，Sox9表达量明显增多，而HNF4α表达减弱。 2、将PCK阳性细胞置于胆管上皮细胞分化诱导液中培养8天后，细胞免疫荧光显示PCK阳性细胞分化为CK19阳性细胞。将PCK阳性细胞置于肝细胞分化诱导液中培养11天后，细胞免疫荧光结果显示PCK阳性细胞中出现Alb阳性细胞。【结论】一、肝前体细胞的产生伴随肝星状细胞的活化。二、活化肝星状细胞促进肝前体细胞的产生。三、活化肝星状细胞促进大鼠肝细胞去分化为肝前体细胞。四、肝细胞来源的肝前体细胞可向肝细胞和胆管上皮细胞分化。
[Abstract]:Background and purpose of research

In chronic liver disease , HPC can be activated to participate in liver injury repair , and the number of HPC is associated with the severity of the disease and the risk index of liver cancer . HPC is not yet clear . Traditional views suggest that HPC comes from Hering tube , and it also shows that the liver cells , hepatic stellate cells ( HSC ) and bone marrow stem cells can be transformed into HPC in the environment of liver injury . Therefore , it is thought that HPC may be a heterogeneous population of different sources .

HSC is located in the Disse Gap and is in direct contact with liver cells and endothelial cells . In normal circumstances , HSC can be activated to produce extracellular matrix ( ECM ) and secrete a large amount of cytokines and growth factors . The activation of HSC is a central link in the development of liver fibrosis . In recent years , it has been shown that HSCs also have the role of promoting the regeneration of liver tissue . These studies have shown that HSCs play an important role in liver regeneration .

Based on the above background , the aim of this study was to clarify the effect of activated HSC on HPC in animal model of liver injury , and to use in vitro co - culture experiment to further clarify the cell source of HPC .

EXPERIMENTAL METHOD

I . To explore the relationship between HSC activation and HPC production

1 . Collect human acute hepatitis , liver fibrosis , liver cirrhosis tissue specimen , immunofluorescence assay HPC marker PCK and activated HSC marker 伪 - SMA expression .

2 - Acetamidofluorene ( 2 - AAF ) / partial hepatectomy ( PH ) was used to induce HPC activation . 2 - AAF was administered to Sprague - Dawley ( SD ) rats at a dose of 10 mg / kg for 5 days .

3 . The model of hepatic injury of acetaminophen ( N - acetyl - parainfluenza , APAP ) was established . APAP was injected intraperitoneally with a dose of 300 mg / kg body weight . The rats were sacrificed only once . After 24 hours of injection , the rats were sacrificed , fresh liver tissue and immunofluorescence were used to detect PCK and 伪 - SMA expression .

II . Identify the effects of activated HSC on HPC

( 1 ) To identify the effects of gadolinium chloride ( GdCl3 ) and Gliotoxin on kupffer cells and activated HSC

1 . The rats were injected intraperitoneally with 20 % carbon tetrachloride ( CCl _ 4 ) at a dose of 2 ml / kg body weight for 2 times . After the last injection for 48 hours , GdCl3 was injected intravenously once a dose of 10 mg / kg body weight . The rat liver tissues were taken after the injection of GdCl324 hours . The expression of CD68 and activated HSC marker 伪 - SMA was detected by immunofluorescence .

The rats were sacrificed at 3 mg / kg body weight at 3 mg / kg after injection of Gliotoxin for 24 hours . The expression of CD68 and 伪 - SMA was detected by immunofluorescence assay .

( 2 ) To observe the effect of inhibiting HSC activation on HPC production

1 . The primary HSC of SD rats was isolated and cultured in vitro in DMEM culture . The culture supernatant was cultured on days 3 ( resting ) and 12 days ( activated ) after in vitro culture .

2 . After 2 - AAF / PH model hepatectomy or APAP model APAP injection , GdCl3 was injected into the tail vein of model rats to inhibit the activity of kupffer cells and the activation of HSC . After 72 hours of liver resection or APAP injection , the rats were sacrificed , liver tissues were sacrificed , PCK and 伪 - SMA expression were detected by immunofluorescence , and the effects of HSC activation on HPC were observed .

3 . After injection of GdCl3 by the above model , HSC activation was inhibited after HSC activation . After 48 hours , the changes of the expression of PCK and 伪 - SMA were detected in rat liver tissues and immunofluorescence assay , and the effect of supernatant on HPC was observed .

( iii ) Observe the effect of removing activated HSC on HPC

1 . In the 2 - AAF / PH model , the rats were injected intraperitoneally with 20 % carbon tetrachloride at 2 ml / kg for 1 day at the same time with 2 ml / kg body weight to stimulate the activation of HSC , and the rats were injected intraperitoneally with Gliotoxin for 1 day before hepatectomy .

2 . On the basis of 2 - AAF / PH + Gliotoxin treatment , the expression of PCK and 伪 - SMA in liver tissues was detected after 4 ml of HSC culture at 24 hours after hepatectomy , and the effects of activated HSC on HPC were clearly defined .

III . It is clear whether activated HSC can promote rat liver cells to differentiate into HPC .

1 . The primary hepatocytes and the primary HSC were isolated from SD rats . The hepatocytes were co - cultured with HSC of different activation states , and the morphological changes of hepatocytes were observed under light microscope . The expression of the hepatocyte marker HNF4 伪 and HPC markers PCK and Sox9 were observed under light microscope .
It was cultured in hepatocyte differentiation inducing solution ( including HGF , insulin , transferrin , dexamethasone ) , and the expression of liver cell marker albumin ( Albumin , albumin ) was detected after 11 days .

Results of experiment

I . Activate HSC to promote HPC production

( i ) HPC production related to HSC activation

1 . The expression of PCK and 伪 - SMA in acute hepatitis , liver fibrosis and liver cirrhosis were detected by immunofluorescence , and the results showed that PCK - positive cells were only in the area adjacent to 伪 - SMA positive cells .

2 . In the 2 - AAF / PH model , the number of PCK - positive cells and 伪 - SMA - positive cells were significantly increased at 0 days , 4 days , 6 days and 9 days after hepatectomy , but the number of PCK - positive cells and 伪 - SMA - positive cells increased significantly on the 6th and 4th days , and the number of PCK - positive cells and 伪 - SMA - positive cells increased significantly at the 6th and 9th days , and both were closely related and interleaved .

3 . After 24 - hour injection of APAP , the results showed that PCK - positive cells were closely surrounded by a large number of 伪 - SMA positive cells , suggesting that HPC production was related to HSC activation .

( 2 ) Activated HSC promotes HPC production

1 . GdCl3 can inhibit the activity of kuppfer cells . Gliotoxin can remove activated HSC .

( 1 ) In the course of acute liver injury , the expression of CD68 and 伪 - SMA was significantly decreased compared with the acute liver injury .

( 2 ) The expression of 伪 - SMA and CD68 in liver tissues was detected by intraperitoneal injection of Gliotoxin after the acute liver injury .

2 . Inhibition of HSC Activation on HPC Production

( 1 ) In the process of 2 - AAF / PH and APAP modeling , the cell activity and HSC activation were inhibited by GdCl3 . The results showed that the number of PCK - positive cells and 伪 - SMA positive cells decreased significantly after HSC activation .

( 2 ) After inhibiting HSC activation , HSC culture supernatants were supplemented with different activation states , and the results showed that the number of PCK - positive cells and 伪 - SMA positive cells increased and the number of PCK - positive cells and 伪 - SMA positive cells increased , and the expression of PCK and 伪 - SMA in liver tissue was not significantly changed after the culture supernatant of HSC .

3 . Effect of Removal of Activated HSC on HPC

( 1 ) There was a large number of PCK positive cells and 伪 - SMA positive cells in the tissues of the pure 2 - AAF / PH model , but only a small amount of PCK positive cells and 伪 - SMA positive cells were found in the liver tissues after intraperitoneal injection of Gliotoxin .

( 2 ) After removing activated HSCs , HSC culture supernatants were supplemented with different activation states . The results showed that the number of PCK - positive cells increased after the culture supernatant was supplemented with activated HSC , while the number of PCK - positive cells did not change significantly after the culture supernatant was supplemented .

2 . Activation of HSC promotes rat hepatocytes to differentiate into HPC

The expression of Sox9 and HNF4 伪 in hepatocytes co - cultured with activated HSC showed that Sox9 expression was very weak at day 0 , while the expression of HNF4 伪 increased significantly , while the expression of HNF4 伪 decreased .

2 . The PCK - positive cells were cultured for 8 days in the differentiation - inducing solution of bile duct epithelial cells . The cells of PCK - positive cells were differentiated into CK19 - positive cells .

Conclusion

First , the production of hepatic progenitor cells is accompanied by activation of hepatic stellate cells .

secondly , activating the hepatic stellate cells to promote the generation of the liver precursor cells .

thirdly , activating hepatic stellate cells to promote the dedifferentiation of rat liver cells into liver precursor cells .

4 . The liver precursor cells derived from hepatocytes can differentiate into hepatocytes and bile duct epithelial cells .

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575

【共引文献】