【摘要】 背景:肝脏干细胞的多能性在肝脏移植方面具有重要价值。目前已经成功地鉴定并分离出动物肝脏干细胞,但人肝脏组织是否存在干细胞、如何对其进行分离鉴定的报道不多。目的:尝试从人类肝癌旁组织中分离和体外培养肝脏干细胞,并对其表面标志物进行鉴定,寻找肝脏干细胞新的来源。设计:细胞观察实验。单位:吉林大学第一医院普外科,东丰县中医院普外科。材料:①组织来源:10例标本来自2005-10/2006-06吉林大学第一临床学院肝胆外科收治的肝细胞癌患者,年龄45～58岁,在行肝叶切除术时切取距癌巢边2cm处的肝组织,经病理证实无癌细胞,对本实验均签署知情同意书。②主要试剂:细胞培养用DMEM/F12干粉由美国Hyclone公司提供,新鲜胎牛血清由天津联星生物技术有限公司配制,各种细胞生长因子为美国Cytolay公司产品。方法:将肝脏癌旁组织剪碎,Hank’s液冲洗,Ⅳ型胶原酶溶液消化后将细胞悬浮在含体积分数为0.15胎牛血清的DMEM/F12培养液中,加入肝细胞生长因子、表皮生长因子、α-成纤维细胞生长因子,浓度均为25μg/L。待培养细胞约铺满2/3瓶底后,胰蛋白酶消化传代,按2×10⁷L^-1密度接种继续培养。细胞传至3～4代时,加入胰酶制成浓度为2.60×10⁹L^-1细胞悬液,依次加入抗人C-kit抗体、免疫磁珠、Buffer液,免疫磁珠法初步分离C-kit⁺细胞。HE染色和免疫荧光细胞化学染色检测癌旁组织中是否存在肝脏干细胞。制备C-kit⁺细胞爬片,免疫荧光化学染色鉴定肝脏干细胞表面标志物CK19及AFP的表达。主要观察指标:①细胞形态观察。②癌旁组织肝脏干细胞的鉴定。③C-kit⁺细胞免疫荧光化学鉴定。结果:①原代培养2周,贴壁细胞呈集落样生长,换液后成熟肝细胞逐渐破碎、消失,小圆形细胞增殖成片,多位于集落中央,呈簇状排列,多数细胞只有1个核,胞核较大,胞浆较少,细胞边界清楚。传至1～2代时仍可见细胞呈集落样生长,生长速度加快,经免疫磁珠分离获得的C-kit⁺细胞呈类圆形,胞核较大,胞浆较少,体外培养1周后呈栅栏状、漩涡、片状或不规则样生长。②癌旁组织HE染色后汇管区内可见不典型增生的小胆管,其内部存在小圆形细胞;免疫荧光细胞化学染色后癌旁组织汇管区内增生的小胆管内的小圆形细胞共表达红色荧光AFP和绿色荧光CK19,二者叠加部分呈黄色。③C-kit⁺细胞免疫荧光化学后,胞浆内表达AFP红色颗粒与CK19绿色颗粒,二者重叠部分即AFP⁺/CK19⁺双阳性细胞,呈黄色荧光。结论:人类肝癌旁组织中存在肝脏干细胞,可联合其表面标志物的表达,将C-kit⁺/AFP⁺/CK19⁺作为鉴定分离肝脏干细胞的一种方法。体外分离培养得到的小圆形细胞即卵圆细胞,具备向肝细胞及胆管上皮细胞双向分化的潜能。更多还原

【Abstract】 BACKGROUND:Multipotency of hepatic stem cells is of important value in liver transplantation.Stem cells have been successfully identified and isolated from the animal livers.However,reports on whether stem cells exist in human hepatic tissue and how to isolate and identify them are few. OBJECTIVE:This study was in attempt to isolate hepatic stem cells from human para-cancerous tissues of hepatoma and in vitro culture them,also to identify the stem cell surface marker,in order to find a new source of heptatic stem cells. DESIGN:Cell observation experiment. SETTING:Department of Common Surgery,First Hospital,Jilin University;Department of Common Surgery, Dongfeng Hospital of Traditional Chinese Medicine. PARTICIPANTS:Samples were harvested from 10 patients with hepatoma admitted to Department of hepatobiliary surgery,First Clinical College,Jilin University between October 2005 and June 2006,with age of 45 to 58 years. Hepatic tissue 2cm away from cancer nest was cut when patients underwent hepatectomy,and it was pathologically confirmed as carcinoma-free tissue.Written informed consents were obtained from each patient.DMEM/F12 dry powder used for cell culture was provided by Hyclone Company,USA.Fresh fetal bovine serum was prepared by Lianxing Biotech Co.,Ltd,Tianjin.Various cell growth factors were the products of Cytolay Company,USA. METHODS:Para-cancerous tissues of hepatoma was cut into pieces,rinsed with Hank’s solution and digested with typeⅣcollagenase.Then the isolated cells were re-suspended in the DMEM/F12 medium supplemented with 0.1 volume fraction of fetal bovine serum,and hepatocyte growth factors,epidermal growth factors andα-fibroblast growth factors of 25μg/L each were added in the above medium.When the cultured cells covered 2/3 of bottom, they were digested with trypsinase for passage and inoculated at 2×10⁷ L^-1.When cells propagated to the 3^rdand 4^th generations,2.60×10⁹ L^-1cell suspension prepared with trypsinase was added,and subsequently,anti-human C-kit antibody,immunomagnetic beads and Buffer solution were added in order.C-kit⁺ cells were preliminarily isolated by immunomagnetic bead separation.Haematoxylin-eosin staining and immunofluorescent histochemical double-staining were used for detecting the hepatic stem cells in para-cancerous tissues. MAIN OUTCOME MEASURES:①Observation of cell morphology.②Identification of hepatic stem cells from para-cancerous tissues.③Identification of C-kit⁺ cells by immunofluorescent histochemical double-staining. RESULTS:①After primarily cultured for 2 weeks,the adherent cells grew in colony.After one half of culture medium was renewed,mature hepatocytes were gradually broken and disappeared.Small round cells propagated,and most of them were located in the center and arranged in cluster.Most cells were found with one big nucleus in each,less cytoplasm and clear cell boundary.When cells propagated to the 1^stand 2^ndgenerations,they still grew in colony,but fast.Each C-kit⁺ cell isolated by immunomagnetic bead separation presented a spherical cell body with a very big nucleus and less cytoplasm.After in vitro cultured for 1 week,it presented broken pieces and apoptotic symptoms.②After para-cancerous tissue was stained by haematoxylin-eosin,atypically proliferated biliary tracts with small round cells could be seen in the portal area.After para-cancerous tissue was stained by immunofluorescent histochemical double-staining,small round cells in the biliary tracts proliferated in the portal area co-expressed red fluorescence AFP and green fluorescence cytokeratin（CK）19 with yellow superposition area.③After C-kit⁺ cells were stained by fluorescence immunocytochemisty,cytoplasm expressed alpha-fetoprotein（AFP）red granules and CK19 green granules.The superposition area of both presented yellow fluorescence of AFP⁺/CK19⁺-positive cells. CONCLUSION:Hepatic stem cells exist in human para-cancerous tissues of hepatoma.Therefore,expressions of C-kit⁺/AFP⁺/CK19⁺,the surface markers of hepatic stem cells,can be used for identifying and isolating hepatic stem cells.Small round cells obtained by in vitro isolation and culture,Le.hepatic oval cells possess bipotential differentiation of hepatocyte and hepatobiliary epithelial cells.更多还原