Hepatic non-parenchymal cells and extracellular matrix participate in oval cell-mediated liver regeneration

AIM： To elucidate the interaction between non- parenchymal cells, extracellular matrix and oval cells during the restituting process of liver injury induced by partial hepatectomy （PH）. METHODS： We examined the localization of oval cells, non-parenchymal cells, and the extracellular matrix components using immunohistochemical and double immunofluorescent analysis during the proliferation and differentiation of oval cells in N-2- acetylaminofluorene （2-AAF）/PH rat model. RESULTS： By day 2 after PH, small oval cells began to proliferate around the portal area. Most of stellate cells and laminin were present along the hepatic sinusoids in the periportal area. Kupffer cells and fibronectin markedly increased in the whole hepatic Iobule. From day 4 to 9, oval cells spread further into hepatic parenchyma, closely associated with stellate cells, fibronectin and laminin. Kupffer cells admixed with oval cells by day 6 and then decreased in the periportal zone. From day 12 to 15, most of hepatic stellate cells （HSCs）, laminin and fibronectin located around the small hepatocyte nodus, and minority of them appeared in the nodus. Kupffer cells were mainly limited in the pericentral sinusoids. After day 18, the normal liver Iobule structures began to recover.CONCLUSION： Local hepatic microenvironment may participate in the oval cell-mediated liver regeneration through the cell-cell and cell-matrix interactions.

Activation of canonical Wnt signaling pathway promotes proliferation and self-renewal of rat hepatic oval cell line WB-F344 in vitro

AIM： To investigate the effect of activation of canonical Wnt signaling pathway on the proliferation and differentiation of hepatic oval cells in vitro. METHODS： WB-F344 cells were treated with recombinant Wnt3a （20, 40, 80, 160, 200 ng/mL） in serum-free medium for 24 h. Cell proliferation was measured by Brdu incorporation analysis; untreated WB-F344 cells were taken as controls. After treatment with Wnt3a （160 ng/mL） for 24 h, subcellular localization and protein expression of p-catenin in WB-F344 cells treated and untreated with Wnt3a were examined by immunofluorescence staining and Western blot analysis. CyclinD1 mRNA expression was determined by semi-quantitative reverse-transcript polymerase chain reaction （RT-PCR）. The mRNA levels of some phenotypic markers （AFP, CK-19, ALB） and two hepatic nuclear factors （HNF-4, HIVF-6） were measured by RT-PCR. Expressions of CK-19 and AFP protein were detected by Western blot analysis. RESULTS： Wnt3a promoted proliferation of WB-F344 cells. Stimulation of WB-F344 cells with recombinant Wnt3a resulte^l in accumulation of the transcriptional activator β-catenin, together with its translocation into the nuclei, and up-regulated typical Wnt target gene CyclinD1. After 3 d of Wnt3a treatment in the absence of serum, WB-F344 cells retained their bipotential to express several specific phenotypic markers of hepatocytes and cholangiocytes, such as AFP and CK-19, following activation of the canonical Wnt signaling pathway. CONCLUSION： The canonical Wnt signaling pathway promotes proliferation and self-renewal of rat hepatic oval cells.

Intrahepatic transplantation of hepatic oval cells for fulminant hepatic failure in rats

AIM： To evaluate the effect of intrahepatic transplantation of hepatic oval cells （HOC） on fulminant hepatic failure （FHF） in rats.METHODS： HOC obtained from rats were labeled with green fluocescent protein （GFP） or 5, 6- carboxyfluorescein diacetate succinmidyl ester （CFDASE）. Cell fluorescence was observed under fluorescent microscope at 6, 24, 48 and 72 h after labeling. CFDA- SE labeled HOC （5 × 10^6 cells each rat） were injected into livers of rats with FHF induced by D-galactosamine. Serum albumin （ALB）, alanine aminotransferase （ALT）, aspartate aminotransferase （AST） and total bilirubin （TBil） levels were measured at different time points. Liver function of rats was examined on days 3, 7, 14 and 21 after HOC transplantation.RESULTS： The positive rate of GFP and CFDA-SE labeled HOC was 10% and 90%, respectively, with no significant change in cell viabilities. The survival rate was higher in HOC transplantation group than in control group, especially 48 （9/15 vs 6/15） and 72 h （9/15 vs 4/15） after HOC transplantation. The serum ALT, AST and TBil levels were decreased while the serum AIb level was increased after HOC transplantation. Fluorescence became faded and diffused in liver tissues, suggesting that proliferation and differentiation occur in transplanted HOC.CONCLUSION： CFDA-SE is superior to GFP in labeling HOC, although fluorescence intensity is decreased progressively with cell division. HOC transplantation can improve the liver function and increase the survival rate of recipients.

The expression of c-kit and proliferating cell nuclear antigen in oval cells of rats with hepatocellular carcinoma

OBJECTIVE: To study the relationship between oval cells and primary hepatocarcinoma and the expression of c-kit and proliferating cell nuclear antigen (PCNA) in oval cells of rats with hepatocellular carcinoma. METHODS: A hundred and twenty clean SD rats were divided into three groups: normal group, cancer-induction group and intervention group. The normal group was fed with standard forage while the rest two groups were fed with 3'-methyl-2-methylamino-azobenzene (DAB) to induce carcinoma for 14 weeks and then fed with standard forage and water. Uscharidin was injected abdominally to the intervention group from the first week to the 14th week. All rats were killed and biopsy specimens were taken from the left and right liver lobes for immunohistochemical staining of c-kit and PCNA on the 2nd, 4th, 6th, 8th, 10th, 12th, 14th, 16th, 18th, 20th, 22nd, and 24th week. RESULTS: From the 2nd to 14th week after liver infection, c-kit positive cells, mainly oval cells were found in the portal area in the carcinoma-induction group and dotted positive pigmentations in liver lobules. In the 22nd week, a large number of cancerous nodes occurred and nuclei heteromorphi-m was apparent; the number of positive cell decreased but positive cells could be sparsely observed in cancerous nodes. In the 2nd week of the carcinoma-induction process, PCNA positive cells were oval cells in the portal area. In the 4th week, a lot of hepatic cells were positively stained, especially in the central vein area. In the 6th week, PCNA positive cells could be seen in the lobules of the liver. In the 8th week, the number of PCNA cells decreased comparatively. From the 10th to 14th week, oval cells in the portal area were still over-expressed. From the 16th to 24th week, a large number of cancerous nodes occurred and PCNA was over-expressed in some of them. In necrotic cancerous nodes, the para-cancerous PCNA positive cells were sparsely distributed and their number was less than that of PCNA positive cells of cancerous tissues. CONCLUSIONS: Hepatic stem cells originating from the terminal biliary plexus of the portal area are involved in the development of hepatocarcinoma because c-kit positive cells expressed in cancerous nodes, accompany the whole process of the development. In the middle inflammatory period of carcinoma-induction, the expression of PCNA in hepatic cells peaked, but the index decreased in the late inflammatory period and in the proliferated fibrosis stage. The expression of PCNA is a tortuous process, going up, down, then up again from normal tissues to cancerous tissues. Combined with pathological findings, PCNA can be considered as a warning index for carcinomatous cells.

Distribution of oval cells and c-myc mRNA expression in mouse hepatocarcinogenesis

BACKGROUND: This study was designed to assess theroles of oval cells and c-myc mRNA in the process of hepa-tocarcinogenesis and to clarify the function of carcinogenec-myc in the development of hepatocellular carcinoma( HCC) and the mechanism of inhibitory function of uscha-ridin on HCC in mouse hepatocarcinogenesis.METHODS: A total of 120 clean SD mice were divided intonormal group, cancer induction group, and interventiongroup. The normal group was fed with standard foragewhile the rest two groups were given p-dimethylaminoazo-benzene (DAB) to induce cancer. Thirteen weeks after in-duction of cancer, the two groups were fed with standardforage and water. Once the pattern was set up, the inter-vention group was given uscharidin injection into the ab-dominal cavity from the first week to the 14th week. Onthe 2nd, 4th, 6th, 8th, 10th, 12th, 14th, 16th, 18th, 20th,22nd, and 24th week, all mice were killed and biopsiedfrom the liver lobe for pathological analysis. At the sametime, the number of tumor nodes was counted and the ex-pression of c-myc mRNA was tested by RT-PCR.RESULTS: Since the 2nd week after cancer induction, pro-liferated oval cells could be seen in the portal area. Initially,the oval cells appeared in the cortical layer of the portalarea, then proliferated gradually and immigrated into theliver parenchyma. In the period of fibrosis after liver proli-feration, proliferated heaps of oval cells were noted in bothportal and peripheral areas. In the period of carcinomatouschange, oval cells could be seen both outside and inside ofcancer nodes, but most of them were distributed outside.The c-myc gene was expressed negatively in the liver tissueof mice. The quantity of the expression began to increaseat the time of infection of the liver and tended to increasewith the degree of hepatic injury. In the period of cancera-tion, the expression level of c-myc mRNA increased gra-dually. The intervention of uscharidin could not inhibit butdelay the increase of the expression of c-myc mRNA.CONCLUSION: Oval cells are closely related to hepatocar-cinoma cells, which play an important role in the occur-rence and development of hepatocarcinogenesis. Uschari-din can inhibit the occurrence of hepatocarcinogenesis orlocal spreading at the early stage of cancer induction byDAB, but it cannot inhibit the expression of c-myc.

Ultrastructure of oval cells in children with chronic hepatitis B,with special emphasis on the stage of liver fibrosis:The first pediatric study

AIM:To investigate the ultrastructure of oval cells in children with chronic hepatitis B,with special emphasis on their location in areas of collagen fibroplasia. METHODS:Morphological investigations were conducted on biopsy material obtained from 40 children,aged 3-16 years with chronic hepatitis B. The stage of fibrosis was assessed histologically using the arbitrary semiquantitative numerical scoring system proposed by Ishak et al. The material for ultrastructural investigation was fixed in glutaraldehyde and paraformaldehyde and processed for transmission-electron microscopic analysis. RESULTS:Ultrastructural examination of biopsy specimens obtained from children with chronic hepatitis B showed the presence of two types of oval cells,the hepatic progenitor cells and intermediate hepatic-like cells. These cells were present in the parenchyma and were seen most commonly in areas of intense periportal fibrosis (at least stage 2 according to Ishak et al) and in the vicinity of the limiting plate of the lobule. The activated nonparenchymal hepatic cells,i.e. transformed hepatic stellate cells and Kupffer cells were seen in close proximity to the intermediate hepatic-like cells. CONCLUSION:We found a distinct relationship between the prevalence of oval cells (hepatic progenitor cells and intermediate hepatocyte-like cells) and fibrosis stage in pediatric patients with chronic hepatitis B.

ISOLATION OF HEPATIC OVAL CELLS FROM DIFFERENT MODEL RATS INCLUDING DIABETIC RATS

Objective To acquire oval cells （progenitor stem cells ） from adult rat liver of different models including diabetic rats. Methods Thirty Sprague-Dawley （ SD ） rats were divided into 5 groups randomly： control, 2-acetylaminofluorene （ 2-AAF ）, 2-AAF ＋ partial hepatectomy （ PH ）, 2-AAF ＋ carbon tetrachloride （ CCl4 ）, and diabetic groups. As two-step collagenase perfusion protocol of Seglen, oval cells were isolated by Percoll density gradient centrifugation. Thy1. 1 positive cells were sorted by flow cytometry, and then cultured in Dulbecco＇s minimum Eagle＇s medium （DMEM）. Immunofluorescence staining was applied to labelling Thyl. 1. Results Different rates of Thy1.1 positive oval cells were found in different rat model groups ： 0. 5 % in 2-AAF, 0. 3% in 2-AAF ＋ PH, 0. 2% in 2-AAF ＋ CCl4, 0. 1% in diabetic, and 0. 0% in control. Isolated cells adhered to plate with fusiform or polygon as epithelial cells. Conclusion Progenitor stem cells exist in injured liver tissue including those from diabetic rats.

Mouse A6-positive Hepatic Oval Cells Derived from Embryonic Stem Cells

Summary： Oval cells have a potential to differentiate into a variety of cell lineages including hepatocytes and biliary epithelia. Several models have been established to activate the oval cells by incorporating a variety of toxins and carcinogens, alone or combined with surgical treatment. Those models are obviously not suitable for the study on human hepatic oval cells. It is necessary to establish a new and efficient model to study the human hepatic oval cells. In this study, the hepatocyte growth factor（HGF） and epidermal growth factor（EGF） were used to induce differentiation of mouse embryonic stem（ES） cells into hepatic oval cells. We first confirmed that hepatic oval cells derived from ES cells, which are bipotential, do exist during the course of mouse ES cells＇ differentiation into hepatic parenchymal cells. RT-PCR and transmission electron microscopy were applied in this study. The ratio of Sca-1＋/CD34＋ cells sorted by FACS in the induction group was increased from day 4 and reached the maximum on the day 8, whereas that in the control group remained at a low level. The differentiation ratio of Sca-1＋/CD34＋ cells in the induction group was significantly higher than that in the control group. About 92.48% of the sorted Sca-1＋/CD34＋ cells on the day 8 were A6 positive. Highly purified A6＋/Sca-1＋/CD34＋ hepatic oval cells derived from ES cells could be obtained by FACS. The differentiation ratio of hepatic oval cells in the induction group（up to 4.46%） was significantly higher than that in the control group. The number of hepatic oval cells could be increased significantly by HGF and EGF. The study also examined the ultrastructures of ES-derived hepatic oval cells＇ membrane surface by atomic force microscopy. The ES-derived hepatic oval cells cultured and sorted by our protocols may be available for the future clinical application.

Immunohistochemical study of hepatic oval cells in human chronic viral hepatitis

AIM: To detect immunohistochemically the presence of oval cells in chronic viral hepatitis with antibody against c-kit. METHODS: We detected oval cells in paraffin embedded liver sections of 3 normal controls and 26 liver samples from patients with chronic viral hepatitis, using immunohistochemistry with antibodies against c-kit, piclass glutathione S-transferase (pi-GST) and cytokeratins 19 (CK19). RESULTS: Oval cells were not observed in normal livers. In chronic viral hepatitis, hepatic oval cells were located predominantly in the periportal region and fibrosis septa,characterized by an ovoid nucleus, small size,and scant cytoplasm. Antibody against stem cell factor receptor, c-kit, had higher sensitivity and specificity than pi-GST and CK19. About 50%-70% of c-kit positive oval cells were stained positively for either pi-GST or CK19. CONCLUSION: Oval cells are frequently detected in human livers with chronic viral hepatitis, suggesting that oval cell proliferation is associated with the liver regeneration in this condition.

Stimulation of oval cell and hepatocyte proliferation by exogenous bombesin and neurotensin in partially hepatectomized rats

AIM:To investigate the effect of the neuropeptides bombesin(BBS)and neurotensin(NT)on oval cell proliferation in partially hepatectomized rats not pretreated with a known hepatocyte inhibitor.METHODS:Seventy male Wistar rats were randomly divided into five groups:Ⅰ=controls,Ⅱ=sham operated,Ⅲ=partial hepatectomy 70%(PHx),Ⅳ=PHx+ BBS(30μg/kg per day),Ⅴ=PHx+NT(300μg/kg per day).Forty eight hours after liver resection,portal en-dotoxin levels and hepatic glutathione redox state were determined.α-fetoprotein(AFP)mRNA(in situ hybridisation),cytokeratin-19 and Ki67 antigen expression (immunohistochemistry)and apoptosis(TUNEL)were evaluated on liver tissue samples.Cells with morphological features of oval cells that were cytokeratin-19 (+)and AFP mRNA(+)were scored in morphometric analysis and their proliferation was recorded.In addition,the proliferation and apoptotic rates of hepatocytes were determined.RESULTS:In the control and sham operated groups,oval cells were significantly less compared to groups Ⅲ,ⅣandⅤ(P<0.001).The neuropeptides BBS and NT significantly increased the proliferation of oval cells compared to groupⅢ(P<0.001).In addition,BBS and NT induced a significant increase of hepatocyte proliferation(P<0.001),whereas it decreased their apoptotic activity(P<0.001)compared to groupⅢ.BBS and NT significantly decreased portal endotoxemia (P<0.001)and increased the hepatic GSH:GSSG ratio (P<0.05 and P<0.001,respectively)compared to groupⅢ.CONCLUSION:BBS and NT stimulated oval cell proliferation in a model of liver regeneration,without use of concomitant suppression of hepatocyte proliferation as oval cell activation stimuli,and improved the hepatocyte regenerative response.This peptides-induced combined stimulation of oval cell and hepatocyte proliferation might serve as a possible treatment modality for several liver diseases.

CCl4诱导的肝纤维化大鼠模型肝小叶内卵圆细胞总体积与轮廓数密度变化的体视学分析

目的 定量研究CCl4诱导的肝纤维化大鼠肝小叶内卵圆细胞(HOC)总体积和轮廓数密度的变化。方法 将11只雄性健康SD大鼠随机分为对照组(n=5)、肝纤维化组(n=6),每周2次皮下注射CCl4与橄榄油混悬液,每次3 mL/kg。在肝纤维化造模5周后取材,从每只大鼠肝脏随机抽选5个大小约1 mm3的肝组织块分别制作1张Epon812环氧树脂包埋超薄切片,运用体视学方法结合透射电子显微镜技术,对大鼠肝小叶内的HOC总体积和轮廓数密度进行定量研究,另从每只大鼠剩余肝脏等距随机抽选4个2 mm厚的肝组织块并分别制作2张石蜡包埋的Masson染色切片,按照肝纤维化Metavir分期标准定性评估每只大鼠的肝纤维化程度。计量资料两组间比较采用成组t检验。结果 体视学定量研究显示,对照组肝小叶内HOC总体积为(15.40±7.63) mm3,肝纤维化组肝小叶内HOC总体积为(146.80±114.00) mm3,与对照组比较,肝纤维化组大鼠肝小叶内HOC总体积显著增加了8.53倍(t=-2.551,P=0.031);对照组肝小叶内HOC轮廓数密度为(56.20±40.40),肝纤维化组肝小叶内HOC轮廓数密度为(566.50±317.00),与对照组比较,肝纤维化组大鼠肝小叶内轮廓数密度显著增加了9.08倍(t=-3.539,P=0.006);定性观察研究结果显示,肝纤维化大鼠肝纤维化分期按照Metavir评分标准达到Ⅱ~Ⅲ期,大鼠窦周隙内肝星状细胞增生部位周围伴随着HOC的大量增生。结论 CCl4诱导肝纤维化大鼠肝小叶内HOC显著增生。

Isolation, characterization and culture of Thy1-positive cells from fetal rat livers

AIM： To investigate whether Thyl recognizes oval cells in the fetal liver and to characterize the cultured Thy1 selected cells from E14 rat livers. METHODS： Thyl populations were analyzed by fluorescence activated cell sorter analysis. Thyl positive cells were isolated using magnetic beads. Hepatic markers were detected by Western blotting, immunocytochemistry and RT-PCR. RESULTS： The percentage of Thyl-positive cells decreased during early development of fetal rat liver （E13-E16）. E14 fetal livers contained 7.8% Thy1 positive cells, of which 61% were positive for α-fetoprotein （AFP） and 25% expressed albumin. The Thy1＋ population expressed oval cell markers c-Kit and CXCR4, liver enriched-transcription factors HNF1α and HNF6, hepatocytic markers albumin, AFP and cytokeratin 18, and biliary marker cytokeratin 19. Thy1- selected cells formed only mesenchymal colonies when plated on collagen and in serum-containing media. Thyl selected cells were able to form hepatic colonies positive for HNF1α, HNF6, albumin, AFP, cytokeratin 18, cytokeratin 19 and glycogen, when grown on STO feeder layers in serum free-media. CONCLUSION： Oval cells positive for Thyl are present in early liver embryonic stages.

裂果薯总皂苷对大鼠肝卵圆细胞系WB-F344上皮间质转化的影响

目的:探讨裂果薯总皂苷(SSPHs)对转化生长因子-β1(TGF-β1)诱导的大鼠肝卵圆细胞系WB-F344上皮间质转化(EMT)的影响及作用机制。方法:将WB-F344细胞分为对照组、模型组,SSPHs低、中、高剂量组和PI3K/AKT信号通路抑制剂LY294002组。除对照组外,其余各组均以10μg/L TGF-β1诱导WB-F344构建EMT模型。采用MTT法、细胞划痕实验分别检测细胞增殖、迁移能力,实时荧光定量PCR(RT-qPCR)、western blotting法分别检测E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(Ncadherin)、波形蛋白(Vimentin)的m RNA和蛋白表达,western blotting法检测PI3K/AKT信号通路关键蛋白表达。结果:SSPHs可抑制TGF-β1诱导的WB-F344细胞的增殖,24 h的细胞半数抑制浓度(IC50)为3.02μg/mL。与对照组比较,模型组N-cadherin、Vimentin的mRNA及蛋白表达水平显著升高,E-cadherin mRNA及蛋白表达水平显著降低,p-PI3K/PI3K、p-AKT/AKT比值显著升高(均P<0.05)。与模型组比较,SSPHs中、高剂量组N-cadherin和Vimentin mRNA及蛋白表达下调,E-cadherin mRNA及蛋白表达上调,p-PI3K/PI3K、p-AKT/AKT比值降低(均P<0.05),结果与LY294002组相似。结论:SSPHs可抑制TGF-β1诱导的WB-F344细胞EMT进程,其机制可能与抑制PI3K/AKT信号通路有关。

Therapy with bone marrow cells reduces liver alterations in mice chronically infected by Schistosoma mansoni

AIM： To investigate the potential of bone marrow mononuclear cells （BM-MCs） in the regeneration of hepatic lesions induced by Schistosoma mansoni （S.mansoni） chronic infection. METHODS： Female mice chronically infected with S.rnansoni were treated with BM-MCs obtained from male green fluorescent protein （GFP） transgenic mice by intravenous or intralobular injections. Control mice received injections of saline in similar conditions, Enzyme-linked immunosorbent assay （ELISA） assay for transforming growth factor-beta （TGF-β）, polymerase chain reaction （PCR） for GFP DNA, immunofluorescence and morphometric studies were performed. RESULTS： Transplanted GFP^＋ cells migrated to granuloma areas and reduced the percentage of liver fibrosis. The presence of donor-derived cells was confirmed by Fluorescence in situ hybridization （FISH） analysis for detection of cells bearing Y chromosome and by PCR analysis for detection of GFP DNA. The levels of TGF-β, a cytokine associated with fibrosis deposition, in liver fragments of mice submitted to therapy were reduced. The number of oval cells in liver sections of S.mansoni-infected mice increased 3-4 fold after transplantation. A partial recovery in albumin expression, which is decreased upon infection with S.mansoni, was found in livers of infected mice after cellular therapy. CONCLUSION： In conclusion, transplanted BMCs migrate to and reduce the damage of chronic fibrotic liver lesions caused by S.mansoni.

硫代乙酰胺诱导慢性肝损伤小鼠肝组织YAP信号对胆管反应的影响及其分子机制

目的:探讨Yes相关蛋白(YAP)信号对硫代乙酰胺(TAA)诱导慢性肝损伤模型小鼠肝组织胆管反应(DR)的影响及其可能的分子机制。方法:选择野生型C57BL/6小鼠为实验对象,将TAA 300 mg/L喂养6周并每周予0.9%氯化钠溶液尾静脉注射小鼠作为TAA组,将TAA喂养6周并每周予YAP活性抑制剂维替泊芬(VP)尾静脉注射小鼠作为VP组,另设蒸馏水喂养的Control组。HE染色和天狼猩红染色观察小鼠肝组织形态学变化。采用Western blot检测肝组织YAP和肝细胞核因子4α(HNF4α)蛋白表达水平,采用qPCR检测YAP、HNF4α、卵圆细胞(OCs)标志分子A6、细胞增殖标志分子Ki-67、肝细胞标志分子清蛋白(ALB)和胆管上皮细胞(BECs)标志分子SOX9的mRNA表达水平;采用电镜观察肝组织中紧密连接的形成,采用免疫组织化学法检测肝组织细胞角蛋白19(CK19)表达水平。结果:TAA组肝组织DR较对照组明显,YAP蛋白和mRNA表达明显增加,HNF4α蛋白和mRNA显著降低。同时,A6和Ki-67的mRNA表达均明显增加,ALB mRNA表达明显降低,SOX9 mRNA表达明显增加。抑制YAP活性后,HNF4α蛋白和mRNA明显增加,A6 mRNA表达无明显改变,Ki-67 mRNA表达下降,但ALB mRNA表达明显增加,SOX9 mRNA表达明显降低。结论:TAA诱导小鼠慢性肝损伤模型中,YAP可能通过下调HNF4α的表达来抑制OCs分化为肝细胞,且促进OCs分化为BECs,进而诱导DR的发生而促进肝纤维化形成。

SOX9在小鼠肝卵圆细胞定向分化为胆管上皮细胞过程中的表达

目的:研究转录因子SOX9在小鼠肝卵圆细胞(HOC)定向分化为胆管上皮细胞(BEC)过程中的表达变化。方法:通过2-乙酰氨基芴灌胃联合2/3肝切除术构建小鼠HOC活化模型,利用二步酶消化法及Percoll密度梯度离心法提取HOC后于体外条件下联合利用表皮生长因子(EGF)、干细胞生长因子(SCF)、白血病抑制因子(LIF)诱导HOC定向分化为BEC。于倒置显微镜下观察分化前后细胞形态变化,利用流式细胞术对BEC进行鉴定,免疫荧光法检测分化前后SOX9的表达。结果:小鼠原代HOC在EGF、SCF以及LIF联合作用下分化至第8代可获得较为纯化的BEC。在原代HOC中SOX9主要表达于胞质,随着分化的进展逐渐表达于胞核,同时平均光密度值在分化过程中显著上调。结论:SOX9在小鼠HOC定向分化为BEC过程中由胞质进入胞核并且表达量显著升高,SOX9可能参与调控小鼠HOC向BEC的分化过程。

大鼠肝卵圆细胞的诱导、分离及鉴定

目的建立大鼠肝卵圆细胞的增殖模型,并探索其分离及鉴定方法。方法雄性Wistar大鼠每天1次连续灌胃给予不同剂量二乙酰氨基芴(2-AAF熏5、10、15、20、25mg/kgBW),第5天行标准的2/3肝切除术,术后按各自剂量继续给予11天,不同时间取肝脏组织,行甲胎蛋白、细胞角蛋白18及19染色并观察。以确定的2-AAF最佳剂量制备大鼠肝干细胞增殖模型,Seglen胶原酶原位灌注结合Percoll密度梯度离心分离纯化大鼠肝卵圆细胞,光镜、电镜下观察细胞特点,并进行上述细胞表型标志免疫组化染色。结果2-AAF15mg/kgBW能建立较理想的肝卵圆细胞增殖模型。HE染色可见汇管区及中央静脉周围大量增殖的嗜碱性小细胞,电镜下观察此种细胞具有卵圆形细胞核、细胞质少而淡、核/浆比例较大等特点,免疫组化染色证实甲胎蛋白、细胞角蛋白18和19染色阳性,白蛋白及白细胞共同抗原(LCA)染色阴性。分离所得底层细胞,光镜下表现大小不等、不规则圆形细胞,体积较小,细胞核/浆比例较大,电镜下细胞表面可见少量短而小的微绒毛状突起,余同增殖细胞特点,免疫组化染色与增殖细胞表现相同细胞表型特点。结论本方法可成功诱导、分离、纯化大鼠肝卵圆细胞,符合肝卵圆细胞的形态特点、超微结构及细胞表型标志特点。

华支睾吸虫溶血磷脂酶对大鼠肝星状细胞和卵圆细胞的作用

目的:观察华支睾吸虫溶血磷脂酶(CslysoPLA)重组蛋白体外对大鼠肝星状细胞和卵圆细胞的作用。方法:应用免疫荧光方法,观察CslysoPLA重组蛋白是否可与大鼠肝星状细胞和卵圆细胞膜结合;应用MTT方法,测定CslysoPLA重组蛋白对大鼠肝星状细胞和卵圆细胞的促增殖作用;应用流式细胞术(FCM)检测CslysoPLA重组蛋白对大鼠卵圆细胞细胞周期的影响。结果:CslysoPLA重组蛋白可与大鼠肝星状细胞和卵圆细胞膜结合,MTT方法测定结果表明低浓度重组蛋白对大鼠肝星状细胞和卵圆细胞有一定促增殖作用(P<0.05),20mg/L重组蛋白可促进大鼠卵圆细胞进入G2期,但高浓度重组蛋白可导致大鼠肝星状细胞和卵圆细胞死亡。结论:低浓度的CslysoPLA重组蛋白在体外可刺激大鼠肝星状细胞和卵圆细胞增殖,表明CslysoPLA可能是华支睾吸虫导致胆管上皮增生、腺瘤样病变和肝纤维化的效应分子之一。而高浓度的CslysoPLA重组蛋白可引起肝星状细胞和卵圆细胞死亡,表明CslysoPLA亦可能是华支睾吸虫引起的化学损伤的毒力因子之一。

卵圆细胞在实验性肝癌发生过程中的演变特征

背景与目的:卵圆细胞在肝癌发生过程中的作用至今还不十分明了。本研究拟通过动态的方法观察卵圆细胞在肝癌发生过程中的演变规律,揭示卵圆细胞与肝癌发生之间的关系。方法:构建实验性肝癌的大鼠诱癌模型,运用常规HE染色、免疫组织化学和爱新蓝特殊染色等方法,动态观察卵圆细胞在肝癌发生过程中的演变规律。结果:HE和免疫组化结果显示,在诱癌的第4周即可见散在的卵圆细胞在门管区附近出现,卵圆细胞OV-6免疫染色呈阳性。在诱癌的第8周和第14周,OV-6阳性细胞逐渐增多,并向肝小叶实质内深入,将肝组织分割成假小叶状。到诱癌的第17周和第24周,多个癌灶出现,同时OV-6阳性细胞的总体数量下降,癌灶内可观察到OV-6阳性细胞。爱新蓝特殊染色显示,诱发的肿瘤属混合性肝癌,其中胆管上皮细胞癌爱新蓝染色呈阳性,肝细胞癌染色呈阴性。结论:卵圆细胞在肝癌的发生过程中可能扮演重要的角色。

大鼠肝脏再生过程中细胞标志物演变与骨髓细胞增生

目的:为研究大鼠在部分肝切除术后肝再生过程中是否存在骨髓细胞的增生与动员以支持肝脏再生过程,以及肝卵圆细胞(hepatic oval cells,HOC)在体内细胞标志物的演变过程. 方法:SD(Sprague-Dawley)大鼠81只,随机分为3组: 2/3部分肝切除组(partial hepatectomy,PHx)、2/3部分肝切除加2-乙酰氨基芴(2-acetylaminofluorene, 2AAF)预处理组(PHx+2AAF)及假手术组,每组分为9小组(n=3),分别于术后1,2,4,6,8,12,16, 20,24 d采集大鼠骨髓细胞及再生肝组织;采用percoll密度梯度液分离骨髓细胞中的单个核细胞,并用流式细胞分析技术检测单个核细胞中CD34+,CD45+细胞比例; 采用冰冻组织切片技术进行再生肝组织切片,并用免疫荧光组织化学染色检测再生组织中与CD34共表达的部分造血干细胞、肝卵圆细胞及肝细胞的细胞标志物Thy1.1,CD45,CK19,vimentin,AFP,Alb. 结果:在PHx模型中,流式细胞检测结果显示大鼠骨髓单个核细胞中CD34+及CD45+细胞在术后2,4, 6 d与对照组相比增高(分别为2.774±0.166 vs 1.903±0.044,P=0.016<0.05;3.164±0.056 vs 1.862±0.057, P=0.002<0.01;2.708±0.160 vs 1.897±0.149,P= 0.032<0.05),免疫荧光组织化学染色结果示在肝组织中第4 d发现少量CD34,CD45共表达细胞,但未检出其他共表达的细胞标志物.在PHx+2AAF模型中, 流式细胞仪检测结果示CD34+及CD45+细胞于术后2, 4 d与对照组相比增高(分别为2.472±0.141 vs 1.903±0.044,P=0.020<0.05;2.985±0.120 vs 1.862±0.057, P=o.008<0.01),荧光免疫组织化学染色显示其再生肝组织中与CD34共表达的抗原Thy1.1,CK19,Alb, AFP,vimentin等存在动态演变:在标志物出现过程中,Thy1.1最早出现,随后可检出AFP,vimentin, CK19,而Alb最后检出;在标志物的消失过程,则Thy1.1, Alb,CK19最早消失,随后AFP,vimentin未检出. 结论:大鼠在急性肝损时的肝再生过程中存在骨髓CD34+与CD45+的细胞增生现象,再生肝组织中与CD34共表达抗原的动态演变可能反映肝卵圆细胞在体内的产生与分化的演变过程.