【作者】 狄军艳；

【导师】 余宏宇；

【作者基本信息】 第二军医大学 ， 病理学与病理生理学， 2011， 博士

【摘要】 成体肝内源性肝干细胞、肝外源性肝干细胞和肝细胞在肝再生中的相对角色是肝再生领域亟待阐明的核心命题之一,我们曾提出肝流域假设试图阐明之。本部分实验应用组织病理(肝细胞大核分裂相和小核分裂相、小胆管/赫令氏管/卵圆细胞增生等)、组织原位绿色荧光及免疫组化观察经致死剂量照射后性别交叉骨髓移植(或性别交叉性腺移植)小鼠不给予其它处理、给予假手术、给予野百合碱(Monocrotaline, MCT)抑制肝细胞分裂增殖、定期多次给予2/3切肝、给予MCT抑制肝细胞分裂增殖然后定期多次给予2/3切肝、给予反复CCl4损伤以及给予MCT抑制肝细胞分裂增殖的同时给予反复CCl4损伤。1.1性别交叉骨髓移植(♂GFP+ C57小鼠骨髓移植到经致死剂量照射的♀普通C57小鼠)后存活小鼠分入对照组,存活6周后开始观察,发现外周血内有核细胞基本上全部发绿色荧光,肝窦内单个核细胞基本上发绿色荧光,汇管区小胆管附近小细胞少数发绿色荧光,而肝板内肝细胞仅偶见个别发绿色荧光;这可能支持既往文献中两种争执观点中的一种:骨髓源肝干细胞在无明显肝损伤的情况下可以以极小的比例参与肝细胞的生理更新。假手术组(打开腹腔、不予2/3切肝、再关闭腹腔)与上述对照组所观察到的结果相似。1.2给予MCT抑制肝细胞分裂增殖者,发绿色荧光的肝板内肝细胞相对于上述对照组增多;此时MCT抑制成熟肝细胞分裂增殖,肝细胞的更新只能通过汇管区附近的肝内、外源性肝干细胞增生分化进行,此时能观察到汇管区附近少数小细胞(包括个别小胆管细胞)和与汇管区不直接邻接的肝板内甚至中央静脉附近的肝板内少数小细胞和肝细胞发绿色荧光,提示经血液循环而来的骨髓源性(肝外源性)肝干细胞可能有部分补充到汇管区附近肝内源性肝干细胞的行列中,也有部分在自小叶周边向中央静脉的肝窦血流路径上直接钻入肝板并可以增生分化为成熟肝细胞。1.3定期多次给予2/3切肝,肝再生时出现大量的大核分裂相,与对照组相比出现了轻微小胆管/COH细胞增生现象以及在肝窦部位之外偶尔出现少数小核分裂相;在成熟肝细胞分裂增生能力不被抑制的情况下,尽管肝再生主要由成熟肝细胞分裂增生完成,但肝内固有的肝内源性肝干细胞甚至肝外源性肝干细胞也在一定程度上参与了肝再生;并且肝内源性肝干细胞可以由肝外源性肝干细胞转变补充而来。1.4给予MCT抑制肝细胞分裂增殖者,再定期多次给予2/3切肝,出现明显的小胆管/COH细胞增生和卵圆细胞增生现象,大核分裂相难以观察到而小核分裂相明显增多;发绿色荧光的肝板内肝细胞、小胆管/COH细胞和卵圆细胞相对于上述对照组明显增多。这些观察结果提示:卵圆细胞既可来源于该鼠肝脏原来固有的小胆管/COH细胞的增生(不发绿色荧光者),也可来源于由骨髓源性(肝外源性)肝干细胞补充而来的小胆管/COH细胞及其附近小细胞的增生(发绿色荧光者)。增生小胆管上皮细胞免疫组化标记CK19阳性;卵圆细胞免疫组化标记AFP阴性或微弱阳性,体积较大者(即已转变为小肝细胞者)明显阳性,成熟肝细胞为阴性。1.5给予反复CCl4损伤、诱导肝纤维化并与对照组对比观察后发现,肝组织内发绿色荧光的细胞明显增多,其中大部分是肝细胞变性损伤所伴随的单个核小细胞的浸润,但发绿色荧光的肝细胞也逐渐增多;且在少数汇管区附近可见卵圆细胞增生;大核分裂相可以见到但明显少于不予MCT抑制肝细胞分裂增殖的单纯2/3切肝组,小核分裂相明显增多;提示CCl4肝损伤条件下,成熟肝细胞广泛变性甚至小灶坏死、分裂增生能力不如反复切肝组的肝细胞,肝内、外源性肝干细胞参与肝再生的程度明显高于反复2/3切肝组。1.6若给予反复CCl4损伤的同时用MCT抑制肝细胞分裂增殖,汇管区与小叶交界处小胆管和卵圆细胞增生反应趋于明显,部分小胆管/卵圆细胞增生灶不发绿色荧光而部分小胆管/卵圆细胞增生灶则发绿色荧光,肝组织内出现较多的肝细胞发绿色荧光;肝内炎症反应细胞样细胞和汇管区附近部分小细胞也可发绿色荧光。在CCl4损伤的情况下,肝内、外源性肝干细胞均能有效地参与肝再生,肝外源性肝干细胞还能补充到小胆管细胞中(原属于肝内源性肝干细胞存在的部位)。若停用CCl4,肝纤维化病变可逐步缓解消失,这与当前国外报道的现象相似。2.与肝流域假设同时期提出的假说还有干细胞池假说(包括性腺干细胞池假说),为此我们进行了GFP+性腺移植实验研究,结果发现性腺移植后存活小鼠给予反复2/3切肝或者CCl4损伤者外周血涂片极难找到发绿色荧光的单个核细胞;假如与此同时还加用了MCT抑制肝细胞分裂增殖,在出现明显的小胆管/COH细胞和卵圆细胞增生现象的同时,仅偶尔可在个别例小鼠的肝组织整张切片内找到极个别发绿色荧光的单个核小细胞或肝板内肝细胞。提示干细胞池中的性腺干细胞在肝细胞的生理更新或病理损伤后肝再生中基本上不起作用。本实验基本验证了肝流域假设的部分内容,肝内、外源性肝干细胞和肝细胞均参与肝细胞更新再生,尤其当肝细胞损伤、再生能力不足时,肝内、外源性肝干细胞就会在肝再生中发挥更重要作用(其中,骨髓源性<肝外源性>肝干细胞还可以补充到肝内源性肝干细胞中)。第二部分本课题组在进行第一部分实验的过程中,有几只照射后骨髓移植小鼠因故委托他人饲养照顾时误给了N-亚硝基二乙胺(N-nitrosodiethylamine,DEN)、18周后发现有肝肿瘤形成,并且在肿瘤与正常肝组织交界处观察到发绿色荧光的小细胞(骨髓来源细胞)。根据这个意外发现,我们考虑到干细胞在肿瘤细胞附近的出现对肿瘤细胞到底有何种影响?为了给出相应的答案,我们进行了第二部分的实验。第二部分实验通过共培养CBRH-7919大鼠肝癌细胞和WB-F344大鼠肝干细胞,发现了WB-F344大鼠肝干细胞在肝癌微环境中通过TGF-?/Smad途径促进CBRH-7919大鼠肝癌细胞凋亡进而抑制了CBRH-7919大鼠肝癌细胞的生长,此结果为临床诊治肝癌患者提供了新的理论依据。具体简要结果如下:流失细胞仪分析结果表明,随着共培养体系中WB-F344大鼠肝干细胞所占比例的提高,CBRH-7919大鼠肝癌细胞的凋亡率明显增加,说明CBRH-7919大鼠肝癌细胞的致瘤性可以被WB-F344大鼠肝干细胞抑制;软琼脂克隆形成实验的结果表明,随着共培养体系中WB-F344大鼠肝干细胞所占比例的提高,CBRH-7919大鼠肝癌细胞的克隆形成数量明显减少,说明CBRH-7919大鼠肝癌细胞的致瘤性可以被WB-344大鼠肝干细胞抑制;四组transwell细胞侵袭试剂盒检测细胞侵袭能力的结果均为阴性,表明WB-F344大鼠肝干细胞对CBRH-7919大鼠肝癌细胞的侵袭能力并没有明显的影响;半定量RT-PCR分析结果表明,随着共培养体系中WB-F344大鼠肝干细胞所占比例的提高,CBRH-7919大鼠肝癌细胞中的PTEN mRNA的表达水平增高,而BMP4,c-Myc和Bcl-2 mRNA的表达水平则降低,这更进一步证明了CBRH-7919大鼠肝癌细胞的致瘤性可以被WB-F344大鼠肝干细胞抑制;Western blot分析结果表明,随着共培养体系中WB-F344大鼠肝干细胞所占比例的提高,CBRH-7919大鼠肝癌细胞中的Smad 4和TGF-?RII蛋白表达水平上升,这则证明了WB-F344大鼠肝干细胞可能通过调节TGF-?/Smad途径促进CBRH-7919大鼠肝癌细胞的凋亡,进而抑制CBRH-7919大鼠肝癌细胞的致瘤性;在荷瘤小鼠模型建立第30天,随着共培养体系中WB-F344大鼠肝干细胞所占比例的提高,肝脏肿瘤体积明显减小,荷瘤小鼠的生存期明显延长,Kaplan Meier生存分析皆有统计学意义,这些结果表明WB-F344大鼠肝干细胞可抑制裸鼠体内CBRH-7919大鼠肝癌细胞的致瘤性;与实验组相比,对照组肿瘤恶性程度高,核/浆比例高,大片区域出现单核或者多核瘤巨细胞,并且在实验组中,随着共培养体系中起始WB-F344大鼠肝干细胞的比例增高,肿瘤细胞异型性降低,这表明在荷瘤小鼠模型中,CBRH-7919大鼠肝癌细胞的致瘤性可以被WB-F344大鼠肝干细胞抑制。更多还原

【Abstract】 That the relative roles of intrahepatic/extrahepatic stem cells and hepatocytes in liver regeneration in adult was still one core question urgently to answer in the liver regeneration field. And we had ever proposed liver valley hypothesis to elucidate it.Histiocytopathology(big hepatocyte karyomitoses,small karyomitoses and small bile ducts/Hering’s canal/oval cells proliferation and so on),frozen section green fluorescence and immunohistochemistry in situ were employed to dynamicly observe:the mouse survivals after cross-sex GFP+ bone marrow transplantation which were irradiated before transplantation, or cross-sex GFP+ gonad transplantation,were administrated without other treatment,with the sham operation,with monocrotaline to inhibit hepatocytes division and growth,with 2/3 partial hepatectomy repeatly,with monocrotaline and 2/3 partial hepatectomy repeatly,with CCl4 injury after injection of monocrotaline to inhibit hepatocytes division and growth or not.1.1 The mice were divided to the control group after cross-sex bone marrow transplantation that the bone marrow of GFP+ C57 male mice were transplanted into ordinary C57 female mice irradiated with lethal dose before transplanted.After survial for six weeks,we found that karyocytes in the peripheral blood had green fluorescene on the whole and mononuclearcells in the hepatic sinus had green fluorescene mainly.A few small cells around the small bile duct in the portal area had green fluorescene.And only few hepatocytes in the hepatic plate had green fluorescene by chance. These might support literatures that hepatic stem cells origing from bone marrow could participate in the physiological renewal of hepatocytes in very small ratio without obvious liver injury.The results from the sham operation group that was opened the abdominal cavity ,not given PH and closed the abdominal cavity were similar with those of the control group.1.2 The mice were given cross-sex bone marrow transplantation and monocrotaline which inhibited mature hepatocytes division and growth.Hepatocytes in the hepatic plate with green fluorescene were obviously more than those of the control group.Monocrotaline could inhibit division and growth of hepatocytes and the physiological renewal of hepatocytes was fulfilled by proliferation and differentiation of extrahepatic/intrahepatic stem cells in the portal area at this time.And we could find that a few small cells including the bile duct in or around the portal area with green fluorescence.And a few small cells and hepatocytes in the hepatic plate indirect approximation with the portal area and in the hepatic plate near the central veins were also with green fluorescence.These hinted that bone marrow (extrahepatic) stem cells origining from the blood circulation could replenish intrahepatic stem cells near the portal area,and some could go through the hepatic plate from the hepatic sinusoid bloodstream pathway from perilobule to the central vein and could proliferate and differentiate to mature hepatocytes.1.3 The mice were given PH many times regularly after cross-sex bone marrow transplantation and there were a lot of big karyomitosis with liver regeneration.But contrasting with the control group,there were a little proliferation of the small bile duct/COH and a few small karyomitosis besides the hepatic sinus by change.Our results hinted that not only intrahepatic stem cells but also extrahepatic stem cells participated in the liver regeneration in some degree,notwithstanding liver regeneration were accomplished by mature hepatocytes mainly when the capability of division and hyperplasia of mature hepatocytes was not inhibited.And intrahepatic stem cells could be recruited by extrahepatic stem cells.1.4 The mice were given monocrotaline which inhibited division and growth of hepatocytes after cross-sex bone marrow transplantation and given PH many times regularly.There were more obvious proliferation of the small bile duct/COH and OCs.We could nearly not find the big karyomotisis and the small karyomotisis were obviously more.There were more hepatocytes in the hepatic plate,the small bile duct/COH and OCs with green fluorescent than those of the control group.These observation hinted that OCs could origin from the resident small bile duct/COH without green fluorescence and from the small bile duct/COH and small cells nearby from bone marrow with green fluorescence.Cytokeratin 19 is positive in the small bile ducts with proliferation in immunohistochemisty.AFP is negative or weak positive in the oval cells,obviously positive in the bigger cells which had already changed to the small hepatocytes,and negative in the hepatocytes.1.5 The mice were given CCl4 after cross-sex bone marrow transplantation to induce hepatic fibrosis and contrasted with the control group.The cells with green fluorescence were more and majority were infiltration of the single nucleus small cells with degeneration and injury of hepatocytes.Hepatocytes with green fluorescence were more.There were more OCs regeneration around a few portal area.There were some big karyomitosis but obviously few than those of the PH group without monocrotaline which inhibited division and growth of hepatocytes.And there were obviously more small karyomitosis.These hinted that there were more extrahepatic/intrahepatic stem cells participating in the liver regeneration than those of the PH group,using CCl4 with more widespread degeneration of mature hepatocytes and even focus necrosis and the capability of division and regeneration of hepatocytes not more than that of the PH group.1.6 If we used monocrotaline which inhibited division and growth of hepatocytes at the same time,the proliferation reaction in the juncture of the lobule and the small bile duct was obvious.Some proliferation focus of the small bile duct/OC had not green fluorescence and some had.There were more hepatocytes with green fluorescence.The inflammatory reaction cells and some small cells near the portal area could have green fluorescence.Extrahepatic/intrahepatic stem cells could effectivly participate in liver regeneration with CCl4.And extrahepatic stem cells could replenish the small bile duct which were the position of intrahepatic stem cells.If we stoped CCl4,hepatic fibrosis could relieve step by step.If we gave these mice the GFP+ bone marrow cells in tail vein,hepatic fibrosis could also relieve step by step,which was similar with the correlated reports before.2. There were stem cell pool hypothesis including gonad stem cell pool hypothesis,raising about the same time with liver valley hypothesis.And we took research with GFP+ gonad transplantation.And we found that there were nearly not mononuclearcells with green fluorescent in the peripheral blood smear in mice with PH or CCl4 after gonad transplantation.If we took monocrotaline which inhibited hepatocyte division and growth,there were conspicuous regeneration of the small bile duct/COH and OCs and we could find very few mononuclearcells or hepatocytes with green fluorescent in the liver plate of the whole liver tissue section in a few mice.These hinted that gonad stem cells did nearly not play a part in the physiological renewal of hepatocytes or liver regeneration after pathological injury.The liver valley hypothesis was preliminary verificated based on the our experiment that both intrahepatic/extrahepatic stem cells and hepatocytes contributed to the liver regeneration,in which hepatic stem cells play a more impportant role when the hepatocytes were injured and their regeneration capacity decreased, especially the stem cells from bone marrow considering that extrahepatic stem cells could fill up intrahepatic liver stem cells.The Second PartWe entrusted other person to breed our mice,and he made a mistake to give our mice after bone marrow transplantation with lethal irradiation N-nitrosodiethylamine(DEN) in our first part experiment.And we found tumor in mice after 18 weeks,and there were some the small cells(cells from bone marrow) with the green fluorescence in the borderline of the tumor and the normal tissue.Based on this discovery by chance,we thought what role the stem cells were around the tumor?In order to get the answer,we did the second part experiment.We cultured the CBRH-7919 hepatic carcinoma cells and WB-344 hepatic stem cells of rat together,and we found that WB-344 rat hepatic stem cells promoted apoptosis of CBRH-7919 rat hepatic carcinoma cell and inhibited it grew with the TGF-?/Smad way in the microenvironment of the hepatic cancer.And this result gave new theory base to treat the hepatic cancer patients.The specific and concise results were as followed.The result from flow cytometer indicated that the apoptosis rate of CBRH-7919 rat hepatic carcinoma cell increased apparently with the increment proportion of the WB-344 rat hepatic stem cells in the co-culture system.This result indicated that WB-344 rat hepatic stem cells could inhibit the tumorigenicity of CBRH-7919 rat hepatic carcinoma cells.The soft agar clone formation experiment indicated that with the increment proportion of the WB-344 rat hepatic stem cells in the co-culture system,the amount of the CBRH-7919 rat hepatic carcinoma cells clones decreased apparently.This result indicated that WB-344 rat hepatic stem cells could inhibit the tumorigenicity of CBRH-7919 rat hepatic carcinoma cells.The four transwell cell infestation experiment indicated that the infestation ability were all negative.This result indicated WB-344 rat hepatic stem cells did not affect the infestation ability of CBRH-7919 rat hepatic carcinoma cells.Demi-quantitation RT-PCR analysis displayed that PTEN was upregulation and bone morphogenetic protein 4 (BMP4), Bcl-2, and c-Myc was downregulation in CBRH-7919 rat hepatic carcinoma cells with the increment proportion of the WB-F344 rat hepatic stem cells in the co-culture system.This result indicated that WB-344 hepatic stem cell could inhibit the tumorigenicity of CBRH-7919 rat hepatic carcinoma cells.The result from Western blot hybridization analysis indicated that with the increment proportion of the WB-344 rat hepatic stem cells in the co-culture system,the expression level of Smad 4 and TGF-?RII increased.This result indicated that WB-344 rat hepatic stem cells could inhibit the tumorigenicity of CBRH-7919 rat hepatic carcinoma cells regulating the TGF-?/Smad way to promote the apoptosis of CBRH-7919 rat hepatic carcinoma cells.In the thirtieth days,in the mice with the tumor with the increment proportion of WB-344 rat hepatic stem cells in the co-culture system,the volume of tumor decreased,life span increased conspicuously and Kaplan Meier survival analysis had statistics significance.This results indicated that WB-F344 rat hepatic stem cells could inhibit the tumorigenicity of CBRH-7919 rat hepatic carcinoma cells in nude mice in vivo.Contrasting with the experiment groups,the malignancy grade of tumor increased,the ration of nucleus to cytoplasm increased,there were some mononuclear or polynuclear cells in many area in the conrol group,and the heteromorphism of tumor cells degraded with the increment proportion of the WB-344 rat hepatic stem cells in the co-culture system.This results indicated that WB-F344 rat hepatic stem cells could inhibit the tumorigenicity of CBRH-7919 rat hepatic carcinoma cells.更多还原