胆管梗阻大鼠模型中卵原细胞的超微病理结构和时序性变化的研究
发布时间：2018-08-04 10:20
【摘要】：一、背景和目的 在肝胆外科临床中,胆管梗阻、胆汁淤积是常见的病理环境,较长时间梗阻,可诱发永久性肝损害,甚至发展成肝纤维化及肝硬化,是临床治疗的难题,相关发病机制基础研究有现实意义。 现已发现人类慢性肝病组织如肝硬化、亚急性肝坏死后肝再生、儿童先天性肝外胆管闭锁、遗传性血色病、慢性病毒性肝炎、原发性肝癌中都存在类似于卵圆细胞的肝前体细胞。这种肝前体细胞定位于汇管区、纤维间隔、汇管区旁肝实质、假小叶及炎症边界。随着肝脏卵圆细胞特异性标记物的发现,卵圆细胞相关研究成为热点,特别是肝脏卵圆细胞参与肝脏损伤修复、肝脏纤维化的相关研究。这些为研究肝脏损伤的保护、治疗及肝纤维化的形成机制提供了最新途径。 因此,本研究通过结扎Wistar大鼠胆总管,模拟胆管梗阻病理状态,通过免疫组化、PCR、透射电镜技术,探讨这一病理过程肝脏卵圆细胞在胆管梗阻肝脏纤维化过程中的变化与发挥的作用。 二、材料与方法 雄性Wistar大鼠60只,体重250-300克,健康,无明显畸形,将动物随机分成两组,一组(30只)作为实验组实施近肝门端胆总管单线结扎(BDL术)；另一组(30只)作为对照组实施假手术,开腹游离胆总管但不结扎。术后相同条件下饲养。分别于术后第1天、2天、3天、7天、14天、21天处死大鼠,每组各处死5只,取肝右叶应用4%多聚甲醛固定,留作免疫组化；取肝左叶-80℃冻存,留作RT-PCR检测；肝中叶小片肝组织,固定后留作电镜观察。 1、通过肉眼及HE染色观察胆管梗阻不同时间点肝脏形态改变。 2、通过免疫组化,OV-6标记卵圆细胞,观察不同时间点卵圆细胞的数目、分布。 3、通过投射电子显微镜观察卵原细胞的超微结构变化。 4、通过PCR检测各目的基因：肝细胞生长因子(HGF),胶原蛋白I (COL I),观察不同时间点各基因的表达情况。 三、结果 1、大体结果 Wistar大鼠胆管梗阻后可见肝脏黄染并肿胀,肝脏淤血、水肿。福尔马林固定后部分大鼠的肝叶标本,可见表面粗糙,颜色灰白等肝组织早期肝纤维化的表现。对照组：肝脏标本各肝叶色泽红润,表面光滑。 2、HE染色结果： 对照组：肝组织结构正常。实验组：术后第1天见少量嗜中性粒细胞和淋巴细胞浸润；术后第2天少许新增生无管腔的小胆管,少量嗜中性粒细胞和淋巴细胞浸润；术后第3天新增生的细小胆管和无管腔的小胆管,嗜中性粒细胞和淋巴细胞浸润较术后第2天明显增加,肝细胞轻度变性,可见灶状和片状坏死,肝组织汇管区可见胆管增生和纤维组织细胞增生,偶见胆管扩张；术后第7天肝细胞出现变性坏死发生率增加及程度加重,重者见片状出血和坏死,肝组织门区可见胆管增生和纤维细胞增生；术后14天小叶结构受到破坏,可见肝细胞空泡变性及坏死；术后21天小叶结构破坏更加严重,大量肝细胞坏死。随时间进展肝脏损伤、纤维化程度逐渐加重。 3、免疫组化结果 OV-6免疫组化阳性染色的细胞数目及分布范围随着梗阻时间的延长而增加,实验组和对照组有明显差异。OV-6阳性细胞表达部位主要位于汇管区胆管及肝间质新增生胆管及无管腔的小胆管细胞胞浆中。 4、电子显微镜观察结果 对照组：肝组织及肝细胞结构基本正常。实验组：术后1天血管旁可见少量卵圆形核细胞,有少量圆钝的胞质突起,核浆比大,胞质量少,细胞器数量少；术后2天肝细胞间也可找到一些卵圆形细胞,细胞切面呈钟形或胖橄榄形,核近似卵圆形,核大,核浆比大,胞质少,发育差；术后3天部分肝血窦内皮损伤,内皮细胞断裂崩解,内皮细胞核小,异染色质呈浓染的团块,边集于核膜下。枯否细胞内吞噬泡数量较多,细胞间隙内卵圆形核细胞多见；术后7天肝细胞板出现大的空隙,或仅残存细胞框架,一些肝细胞呈不可逆损伤,一些细胞呈凋亡样坏死,肝细胞间可见多形核白细胞存在,一些细胞仅可分辨核的大小差异,细胞无可分辨的细胞细节,在形态尚好的部位,仍可找到少量存活的卵圆核细胞,已向肝细胞方向分化；术后14天肝细胞间可见一些立方形细胞,4-5个细胞形成片块,细胞间可见小管样结构,两端的细胞间有紧密连接,小管内有微绒毛伸入,腔内无内容物,核形近似卵圆,但不甚规则,核膜有凹陷,细胞器中等发育,细胞片块与肝细胞有连接；术后21天一些胶原纤维束提示窦周间隙的位置。肝细胞间可见卵圆形核细胞片块,其中有的细胞含大量脂滴；有的细胞是成纤维细胞,细胞周围有大量胶原纤维存在；其余细胞性质不可辨。 5、RT-PCR结果 在对照组和实验组大鼠肝脏中均能检测出目的基因的表达。统计分析显示两组COLⅠ的表达差异从第1天开始有统计学意义,HGF的表达差异从第3天开始有统计学意义。对实验组不同天数组间的表达研究发现COLⅠ在3-7天无统计学意义,HGF在1-3天、7-21天无统计学意义,而其他相邻各组间均有统计学意义,COLⅠ指标1-3天增长趋势最明显,HGF指标3-7天增长趋势最明显。 四、结论 1、研究发现肝外胆管结扎造成胆管梗阻的大鼠模型,其肝脏呈现胆汁淤积性、进行性加重的肝脏受损表现。卵圆细胞参与胆管梗阻后损伤的修复过程,其时序性变化趋势与肝脏修复相关细胞因子变化趋势相似,可能参与肝脏受损后的修复过程。 2、利用电子显微镜观察到卵原细胞的超微结构形态,描述了其细胞特点及周围结构的形态特点,证实了卵原细胞的存在,直观的观察到其时序性过程中的变化。 3、研究发现实验组肝脏纤维化相关基因表达明显高于对照组,并且随着梗阻天数的增加表达量增加,其中COLⅠ可显示纤维化的进程,HGF与肝脏受损后的修复过程有关。 4、研究发现在肝纤维化过程中的3-7天是肝脏受损过程的特殊时期,第3天之前以肝脏受损过程为主要表现；第3天开始出现修复损伤表现,纤维化趋势减弱；第7天之后为持续的的肝脏损伤、纤维化进程。
[Abstract]:First, the background and the purpose
In the Department of hepatobiliary surgery, bile duct obstruction and cholestasis are common pathological conditions. Long time obstruction can induce permanent liver damage, and even develop into liver fibrosis and cirrhosis. It is a difficult problem in clinical treatment. The basic research of related pathogenesis is of practical significance.
It has been found that human chronic liver diseases such as liver cirrhosis, subacute hepatic necrosis after subacute hepatic necrosis, congenital extrahepatic bile duct atresia in children, hereditary hemochromatosis, chronic viral hepatitis, and primary liver cancer are all similar to oval cells in the hepatic precursor cells. False lobules and inflammatory boundaries. With the discovery of specific markers for oval cells in the liver, oval cells related research become hot spots, especially liver oval cells participate in the repair of liver injury and liver fibrosis, which provide the latest approach to study the protection of liver injury, treatment and the formation mechanism of liver fibrosis.
Therefore, by ligating Wistar rat common bile duct and simulating the pathological state of bile duct obstruction, the changes and roles of hepatic oval cells in the process of hepatic fibrosis in the bile duct obstruction were discussed by immunohistochemistry, PCR and transmission electron microscopy.
Two, materials and methods
60 male Wistar rats, weighing 250-300 grams, healthy and no obvious malformation, were randomly divided into two groups. One group (30) was used as the experimental group to carry out the unilateral ligation of the common bile duct of the liver (BDL); the other group (30) was used as a control group to carry out the sham operation, open the free common bile duct and no ligation after the operation. First The rats were killed in days, 2 days, 3 days, 7 days, 14 days and 21 days, and 5 rats were killed in each group. The right lobe of the liver was fixed with 4% polyformaldehyde and retained for immunohistochemical staining. The left lobe of the liver was stored at -80 for RT-PCR, and the liver tissue of the middle lobe of the liver was kept for electron microscopy after fixation.
1, the morphological changes of bile duct obstruction at different time points were observed by naked eye and HE staining.
2, by immunohistochemical staining, OV-6 labeled oval cells to observe the number and distribution of oval cells at different time points.
3, the ultrastructural changes of oocytes were observed by projection electron microscopy.
4. Hepatocyte growth factor (HGF) and collagen I (COL I) were detected by PCR, and the expression of each gene was observed at different time points.
Three, the result
1, gross results
Wistar rat bile duct obstruction can be seen after the liver yellow staining and swelling, liver congestion, edema. After formalin fixed part of the rat liver specimens, visible surface roughness, color gray and other liver tissue early liver fibrosis performance. Control group: liver specimens of the liver color red, surface smooth.
2, HE staining results:
In the control group, the liver tissue structure was normal. In the experimental group, a small amount of neutrophils and lymphocyte infiltration were seen on the first day after the operation, and a few small bile ducts with neutrophils and neutrophils and lymphocytes infiltrated on the second day after the operation, and the neutrophils and lymphatic cells and lymph nodes of newly proliferated small bile ducts and cavities, neutrophils and lymphatic lymph nodes on third days after the operation. The cell infiltration was significantly increased on the second day after the operation, the liver cells were slightly denatured, and the focal and flaky necrosis were seen. The bile duct hyperplasia and fibrous cell proliferation were seen in the liver tissue, and the bile duct dilatation was seen occasionally. The incidence of degeneration and necrosis was increased and the degree was aggravated on the seventh day after the operation, and the severe hemorrhage and necrosis were seen in the severe cases, and the portal area of the liver was visible. Bile duct hyperplasia and fibroblast proliferation; the structure of the lobule was damaged on the 14 day after the operation, and the vacuoles and necrosis of the liver cells were seen. The destruction of the structure of the lobule was more serious on the 21 day after the operation, and a large number of liver cells were necrotic.
3, immunohistochemical results
The number and distribution of OV-6 immunohistochemical positive staining cells increased with the prolongation of the obstruction time. There was a significant difference between the experimental group and the control group. The expression of.OV-6 positive cells was mainly located in the bile duct and the new cholangioplastic bile duct and the small bile duct cell cytoplasm without the lumen.
4, the observation result of electron microscope
In the control group, the liver tissue and the liver cell structure were basically normal. In the experimental group, a small number of oval nuclear cells were visible near the vessel 1 days after the operation, with a small number of blunt cytosolic protuberances, large nuclear pulp ratio, less cell mass and less organelles, and some oval cells could be found between the hepatocytes on the 2 day after the operation. The cells were bell shaped or fat olive shaped, and the nucleus approximated the egg. Round, large nuclear, large nuclear pulp ratio, less cytoplasm, poor development, 3 days after the operation, partial hepatic sinusoidal endothelial injury, endothelial cell rupture disintegration, small endothelial cell nucleus, heterochromatin under the dense mass, the edge of the nuclear membrane. The number of phagocytic vesicles in the Kupffer cell is more, and the oval cells in the cell space are more common; the liver cell board appears large empty after 7 days after the operation. Gap, or only residual cell frame, some liver cells are irreversible damage, some cells are apoptotic like necrosis, hepatic cells can be found to exist polymorphonuclear leukocytes, some cells can only distinguish the size difference between the nuclei, the cells have no discernible cell details, and a small number of surviving oval nuclear cells can still be found in the good shape of the cells, and the cells have been found to the liver cells. 14 days after the operation, there were cubic cells, 4-5 cells forming pieces, small tube like structures between the cells, close connections between the cells at both ends, microvilli in the canaliculus, no contents in the canaliculus, and nuclear shape similar to oval, but not very regular, the nuclear membrane was depressed, the organelles were moderately developed, cell block and liver thin. In the 21 day after the operation, some collagen fiber bundles suggest the location of the peri sinus space. The cells of the oval nucleus cells can be seen between the hepatocytes, some of them contain a large amount of lipid droplets, some cells are fibroblasts, and a large number of collagen fibers exist around the cells; the other cells are not distinguishes.
5, RT-PCR results
The expression of the target gene was detected in the liver of the control group and the experimental group. The statistical analysis showed that the difference in expression of the two groups of COL I was statistically significant from first days, and the difference in expression of HGF was statistically significant from third days. The expression of COL I between the different days of the experimental group found no statistical significance in the 3-7 days, HGF On the 1-3 day, the 7-21 days were not statistically significant, but the other adjacent groups were statistically significant. The growth trend of COL I index in 1-3 days was the most obvious, and the growth trend of the 3-7 days was the most obvious.
Four. Conclusion
1, the rat model of bile duct obstruction caused by extrahepatic bile duct ligation was found. The liver showed cholestasis and progressively aggravated liver damage. The repair process of oval cells involved in the injury after bile duct obstruction was similar to that of the liver repair related cytokines. It may be involved in the repair of liver damage. Complex process.
2, the ultrastructure of oocyte was observed by electron microscope, the characteristics of the cells and the morphological characteristics of the surrounding structure were described, and the existence of oocyte was confirmed, and the changes in the time sequence of the oocyte were observed.
3, the study found that the expression of liver fibrosis related genes in the experimental group was significantly higher than that in the control group, and the expression increased with the increase of the number of obstructive days, and COL I showed the process of fibrosis, and HGF was related to the repair process after liver damage.
4, the study found that the 3-7 day in the process of liver fibrosis was a special period of liver damage, and the liver damage was the main manifestation before third days, and the repair injury appeared at the beginning of the third day and the trend of fibrosis weakened; after seventh days, the liver injury and fibrotic process were continued.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575
本文编号：2163615
[Abstract]:First, the background and the purpose
In the Department of hepatobiliary surgery, bile duct obstruction and cholestasis are common pathological conditions. Long time obstruction can induce permanent liver damage, and even develop into liver fibrosis and cirrhosis. It is a difficult problem in clinical treatment. The basic research of related pathogenesis is of practical significance.
It has been found that human chronic liver diseases such as liver cirrhosis, subacute hepatic necrosis after subacute hepatic necrosis, congenital extrahepatic bile duct atresia in children, hereditary hemochromatosis, chronic viral hepatitis, and primary liver cancer are all similar to oval cells in the hepatic precursor cells. False lobules and inflammatory boundaries. With the discovery of specific markers for oval cells in the liver, oval cells related research become hot spots, especially liver oval cells participate in the repair of liver injury and liver fibrosis, which provide the latest approach to study the protection of liver injury, treatment and the formation mechanism of liver fibrosis.
Therefore, by ligating Wistar rat common bile duct and simulating the pathological state of bile duct obstruction, the changes and roles of hepatic oval cells in the process of hepatic fibrosis in the bile duct obstruction were discussed by immunohistochemistry, PCR and transmission electron microscopy.
Two, materials and methods
60 male Wistar rats, weighing 250-300 grams, healthy and no obvious malformation, were randomly divided into two groups. One group (30) was used as the experimental group to carry out the unilateral ligation of the common bile duct of the liver (BDL); the other group (30) was used as a control group to carry out the sham operation, open the free common bile duct and no ligation after the operation. First The rats were killed in days, 2 days, 3 days, 7 days, 14 days and 21 days, and 5 rats were killed in each group. The right lobe of the liver was fixed with 4% polyformaldehyde and retained for immunohistochemical staining. The left lobe of the liver was stored at -80 for RT-PCR, and the liver tissue of the middle lobe of the liver was kept for electron microscopy after fixation.
1, the morphological changes of bile duct obstruction at different time points were observed by naked eye and HE staining.
2, by immunohistochemical staining, OV-6 labeled oval cells to observe the number and distribution of oval cells at different time points.
3, the ultrastructural changes of oocytes were observed by projection electron microscopy.
4. Hepatocyte growth factor (HGF) and collagen I (COL I) were detected by PCR, and the expression of each gene was observed at different time points.
Three, the result
1, gross results
Wistar rat bile duct obstruction can be seen after the liver yellow staining and swelling, liver congestion, edema. After formalin fixed part of the rat liver specimens, visible surface roughness, color gray and other liver tissue early liver fibrosis performance. Control group: liver specimens of the liver color red, surface smooth.
2, HE staining results:
In the control group, the liver tissue structure was normal. In the experimental group, a small amount of neutrophils and lymphocyte infiltration were seen on the first day after the operation, and a few small bile ducts with neutrophils and neutrophils and lymphocytes infiltrated on the second day after the operation, and the neutrophils and lymphatic cells and lymph nodes of newly proliferated small bile ducts and cavities, neutrophils and lymphatic lymph nodes on third days after the operation. The cell infiltration was significantly increased on the second day after the operation, the liver cells were slightly denatured, and the focal and flaky necrosis were seen. The bile duct hyperplasia and fibrous cell proliferation were seen in the liver tissue, and the bile duct dilatation was seen occasionally. The incidence of degeneration and necrosis was increased and the degree was aggravated on the seventh day after the operation, and the severe hemorrhage and necrosis were seen in the severe cases, and the portal area of the liver was visible. Bile duct hyperplasia and fibroblast proliferation; the structure of the lobule was damaged on the 14 day after the operation, and the vacuoles and necrosis of the liver cells were seen. The destruction of the structure of the lobule was more serious on the 21 day after the operation, and a large number of liver cells were necrotic.
3, immunohistochemical results
The number and distribution of OV-6 immunohistochemical positive staining cells increased with the prolongation of the obstruction time. There was a significant difference between the experimental group and the control group. The expression of.OV-6 positive cells was mainly located in the bile duct and the new cholangioplastic bile duct and the small bile duct cell cytoplasm without the lumen.
4, the observation result of electron microscope
In the control group, the liver tissue and the liver cell structure were basically normal. In the experimental group, a small number of oval nuclear cells were visible near the vessel 1 days after the operation, with a small number of blunt cytosolic protuberances, large nuclear pulp ratio, less cell mass and less organelles, and some oval cells could be found between the hepatocytes on the 2 day after the operation. The cells were bell shaped or fat olive shaped, and the nucleus approximated the egg. Round, large nuclear, large nuclear pulp ratio, less cytoplasm, poor development, 3 days after the operation, partial hepatic sinusoidal endothelial injury, endothelial cell rupture disintegration, small endothelial cell nucleus, heterochromatin under the dense mass, the edge of the nuclear membrane. The number of phagocytic vesicles in the Kupffer cell is more, and the oval cells in the cell space are more common; the liver cell board appears large empty after 7 days after the operation. Gap, or only residual cell frame, some liver cells are irreversible damage, some cells are apoptotic like necrosis, hepatic cells can be found to exist polymorphonuclear leukocytes, some cells can only distinguish the size difference between the nuclei, the cells have no discernible cell details, and a small number of surviving oval nuclear cells can still be found in the good shape of the cells, and the cells have been found to the liver cells. 14 days after the operation, there were cubic cells, 4-5 cells forming pieces, small tube like structures between the cells, close connections between the cells at both ends, microvilli in the canaliculus, no contents in the canaliculus, and nuclear shape similar to oval, but not very regular, the nuclear membrane was depressed, the organelles were moderately developed, cell block and liver thin. In the 21 day after the operation, some collagen fiber bundles suggest the location of the peri sinus space. The cells of the oval nucleus cells can be seen between the hepatocytes, some of them contain a large amount of lipid droplets, some cells are fibroblasts, and a large number of collagen fibers exist around the cells; the other cells are not distinguishes.
5, RT-PCR results
The expression of the target gene was detected in the liver of the control group and the experimental group. The statistical analysis showed that the difference in expression of the two groups of COL I was statistically significant from first days, and the difference in expression of HGF was statistically significant from third days. The expression of COL I between the different days of the experimental group found no statistical significance in the 3-7 days, HGF On the 1-3 day, the 7-21 days were not statistically significant, but the other adjacent groups were statistically significant. The growth trend of COL I index in 1-3 days was the most obvious, and the growth trend of the 3-7 days was the most obvious.
Four. Conclusion
1, the rat model of bile duct obstruction caused by extrahepatic bile duct ligation was found. The liver showed cholestasis and progressively aggravated liver damage. The repair process of oval cells involved in the injury after bile duct obstruction was similar to that of the liver repair related cytokines. It may be involved in the repair of liver damage. Complex process.
2, the ultrastructure of oocyte was observed by electron microscope, the characteristics of the cells and the morphological characteristics of the surrounding structure were described, and the existence of oocyte was confirmed, and the changes in the time sequence of the oocyte were observed.
3, the study found that the expression of liver fibrosis related genes in the experimental group was significantly higher than that in the control group, and the expression increased with the increase of the number of obstructive days, and COL I showed the process of fibrosis, and HGF was related to the repair process after liver damage.
4, the study found that the 3-7 day in the process of liver fibrosis was a special period of liver damage, and the liver damage was the main manifestation before third days, and the repair injury appeared at the beginning of the third day and the trend of fibrosis weakened; after seventh days, the liver injury and fibrotic process were continued.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575
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