大鼠肝卵圆细胞增殖模型建立及其表面标记检测

本文拟建立大鼠卵圆细胞增殖模型,分离纯化卵圆细胞并对其特性进行研究。给SD大鼠喂饲2-乙酰氨基芴(2-AAF),剂量分别为5、10、15、20mg／kg,连续给药6d,第7天行三分之二肝切除术,术后继续给药1周,并于术后每隔3d取肝组织行常规组织学观察、细胞增殖试验及免疫组化染色。经免疫磁珠标记C-kit阳性细胞以纯化卵圆细胞,再经免疫细胞化学及RT-PCR对其进行鉴定。结果显示,10、15、20mg／kg三种剂量2-AAF均可成功建立卵圆细胞增殖模型,卵圆细胞增殖于第8天较明显,至第11天达高峰,第14天有所减少。卵圆细胞胞核Brdu染色阳性,卵圆细胞表达特异性抗原OV6、肝细胞标记白蛋白、胆管细胞标记CK19、甲胎蛋白以及连接蛋白43,同时还表达造血干细胞标记C-kit。实验表明,2-AAF剂量为10～20mg／kg时可获得较理想的大鼠卵圆细胞增殖模型,增生的卵圆细胞为具有双向分化潜能的肝干细胞,前体细胞。     

连接蛋白43在病理性瘢痕成纤维细胞中的表达

目的:探讨连接蛋白43(Cx43)及其构成的缝隙连接细胞间通讯(GJIC)在病理性瘢痕中的调控作用.方法:选择临床上不同病理分类的瘢痕组织(包括瘢痕疙瘩、增生性瘢痕)和正常修复组织,以正常皮肤为对照,应用免疫组织化学检测Cx43在成纤维细胞中的表达.结果:Cx43在增生性瘢痕和瘢痕疙瘩成纤维细胞中的表达明显少于正常修复组织及正常皮肤.结论:成纤维细胞Cx43的表达下调可能是造成病理性瘢痕组织中成纤维细胞间GJIC异常,从而导致病理性瘢痕发生的因素之一.     

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。     

黄芪甲苷减轻镉致TM3细胞Cx43表达下降及缝隙连接细胞间通讯功能减退

目的探讨黄芪甲苷(As)对镉(Cd)致TM3细胞连接蛋白43(Cx43)表达改变及由Cx43介导的缝隙连接细胞间通讯(GJIC)功能改变的保护作用。方法用TM3细胞系作为研究对象,设对照组、As(20 mg/L)组、Cd(10μmol/L)组、Cd加As组,免疫组织化学方法分析Cx43阳性产物表达,荧光定量PCR检测Cx43 mRNA表达,Western blot检测Cx43蛋白表达,划痕标记染料示踪技术(SLDT)检测GJIC功能。结果与对照组相比,Cd组TM3细胞Cx43阳性产物表达的平均吸光度值(A_(OD))显著降低(P0.01),Cx43 mRNA和蛋白表达下降(P0.01),TM3细胞GJIC功能降低(P0.01);Cd+As组TM3细胞Cx43阳性产物表达虽较对照组减弱但明显高于相应Cd组(P0.01),Cx43 mRNA、蛋白表达及GJIC功能虽比对照组降低,但较Cd组高(P0.01)。结论黄芪甲苷对镉致TM3细胞Cx43表达下降及GJIC功能减退有明显的拮抗作用。     

C-kit+细胞体外增殖、分化的研究

分离纯化卵圆细胞并对其体外增殖、分化进行研究。SD大鼠喂饲2-乙酰氨基芴(2-acetainofluorene,2-AAF)10mg/kg,以促进肝脏干细胞增殖,通过免疫磁珠法(Magnetic activated cell sorting,MACS)标记C-kit阳性细胞以纯化卵圆细胞,进行体外培养,采用免疫细胞化学、逆转录聚合酶链反应(RT-PCR)等方法观察其体外增殖、分化特性。以MACS纯化的C-kit阳性卵圆细胞,90%以上为卵圆细胞抗原(OV6)阳性,大部分甲胎蛋白(AFP)阳性。C-kit阳性细胞在体外增殖能力较强,能形成集落样生长,并向胆管细胞及肝细胞分化,表达角蛋白19(CK19)和/或白蛋白(ALB)标记。细胞集落RT-PCR结果显示AFP、CK19及ALB基因均有表达。以上结果表明利用C-kit标记通过MACS纯化的C-kit 细胞为具有双向分化潜能的肝脏干细胞。     

重组肝刺激因子对肝脏卵圆细胞增殖的作用

目的观察重组人肝刺激因子(hHSS)对肝脏卵圆细胞增殖的影响。方法分离大鼠肝卵圆细胞，加以培养，并进行细胞学鉴定。将重组的hHSS作用于肝脏卵圆细胞，以MTT法、流式细胞术观察其对卵圆细胞的增殖作用。结果实验分离的卵圆细胞同时表达肝细胞和胆管细胞特异性标志物。给予重组hHSS作用12～24h后，细胞增殖速度减缓，细胞DNA合成减弱，其作用的最有效浓度为240mg/L。结论重组hHSS能抑制卵圆细胞的增殖。     

三七总皂苷对神经胶质瘤细胞增殖、迁移及凋亡的影响

目的:探讨三七总皂苷(Panax notoginseng saponins,PNS)对神经胶质瘤U251和U87细胞增殖、迁移和凋亡的影响及其作用机制。方法:将U251和U87细胞各分为两组:对照(control)组和三七总皂苷(PNS)组。CCK-8检测细胞增殖水平;划痕实验检测细胞迁移能力;流式细胞仪检测细胞凋亡水平;Western Blot检测Caspase-3、Bax、Bcl-2、p-AKT、AKT、p-mTOR和mTOR的蛋白表达水平。结果:U251和U87细胞中的检测结果一致。与control组相比,PNS组的细胞增殖能力和迁移能力均显著下降(P0.05);细胞凋亡率显著升高(P0.05);Bcl-2的蛋白表达水平显著下调(P0.05),而Bax和Caspase-3的表达水平显著上调(P0.05);p-Akt和p-mTOR的蛋白表达水平下降,且差异具有显著性(P0.05)。结论:PNS能够抑制U251和U87细胞的增殖和迁移,促进细胞凋亡,这可能与PI3K-Akt-mTOR信号通路的抑制有关。     

CX26/CX32缝隙连接蛋白的Tet-on HeLa细胞模型的建立及鉴定

目的 体外建立一种特异性快速有效观察药物对细胞缝隙连接(GJ)作用的特异性细胞模型,为发现作用于GJ的药物以及GJ的特异性研究提供一种有力的技术手段.方法 构建同时双向表达2种不同连接蛋白(CXs)的质粒;用表达CX26/CX32的质粒转染Tet-on HeLa细胞,建立稳定表达CX26/CX32并形成异质性GJ的HeLa细胞模型.设置多西环素(Dox)(1μg/mL)处理组和未处理组,分别用RT-PCR和Western blot法检测细胞CX26 mRNA和蛋白质表达;细胞接种荧光示踪法检测细胞GJ功能;丽丝胺罗丹明B(SRB)法检测细胞的增殖.在上述细胞模型上,用GJ抑制剂2-氨基乙酯二苯基硼酸(2-APB)和GJ激活剂维甲酸(RA)干预.结果 HeLa细胞无内源性CX的表达,Dox可诱导Tet-on HeLa细胞CX26 mRNA和蛋白质表达,并且被诱导表达的CX可形成有效的GJ.2-APB(50 μmol/L)减少稳定表达CX26/Cx32的HeLa细胞间的荧光传递数目,GJ抑制率为51.3％ (P ＜0.01);RA(10 μmol/L)增多细胞间的荧光传递数目,GJ增强率为60.3％ (P ＜0.01).结论 成功建立Dox调控的、稳定表达CX26/Cx32的Tet-on HeLa细胞模型,为寻找可调节GJ功能的药物、观察药物对GJ的特异性作用提供了有力的实验手段.     

人神经胶质瘤组织中连接蛋白43的表达

目的　为了研究人神经胶质瘤发生中连接蛋白 43的作用。方法　本实验用免疫组化的方法 ,检测了 2 6例不同级别的脑瘤组织中连接蛋白 43的蛋白表达。结果　在正常脑组织星形胶质细胞和良性的脑质细胞瘤连接蛋白 43表达阳性反应 ,在恶性的脑胶质细胞瘤 ,连接蛋白 43明显减弱 ,呈弱性或阴性。结论　人脑胶质细胞肿瘤的发展及恶化程度与其连接蛋白 43蛋白表达有关 ;在癌的多阶段学说 (起始阶段、促癌变阶段、进展阶段 )中 ,连接蛋白 43表达的减少被认为是促瘤变阶段的重要机制。     

伊贝沙坦和培垛普利对左心室肥大时心肌连接蛋白43、结蛋白与肌钙蛋白T表达的实验研究

目的：探讨左室肥大时血管紧张素Ⅱ受体Ⅰ型拮抗剂伊贝沙坦和血管紧张素转换酶抑制剂培垛普利对心肌连接蛋白43（CX43）、结蛋白与肌钙蛋白T（cTnT）表达的影响。 方法： 分假手术组、SD大鼠腹主动脉部分结扎的手术组及腹主动脉部分结扎的处理组即伊贝沙坦组（20 mg·kg-1·d-1 ig）、培垛普利组（2 mg·kg-1·d-1 ig）、两药联用组（伊贝沙坦组20 mg·kg-1·d-1 ig+培垛普利组2 mg·kg-1·d-1 ig），术后次周起干预8周，观察左室重量指数（LVMI）和心肌细胞横径（TDM），免疫组织化学检测心肌CX43、结蛋白与cTnT 表达。 结果： 培垛普利组、伊贝沙坦组和联合用药组LVMI和TDM均显著低于手术组（P<0.05），手术组心肌细胞闰盘区域和侧侧连接的胞膜上均有不规则CX43表达；伊贝沙坦组、培垛普利和其联用组的CX43表达分布较有规律，主要以带状表达于闰盘；手术组心肌CX43、结蛋白、cTnT表达量明显少于伊贝沙坦组、培垛普利组和其联用组（P<0.05）。 结论： 左室肥大时伊贝沙坦和培垛普利有利于心肌CX43、结蛋白与cTnT的表达与分布，能减轻左室心肌肥大，减少心肌细胞损伤，促进心肌细胞骨架结构与缝隙连接的基本正常恢复。     

大鼠肝癌前病灶与卵圆细胞的关系

目的探讨肝脏癌前病灶的细胞学起源。方法用雄性Ｆｉｓｈｅｒ３４４大鼠，在两个不同的化学致癌模型中，用放射自显影方法追踪癌前病灶的发生与卵圆细胞（ｏｖａｌｃｅｌ）或肝细胞的关系。模型Ⅰ动物连续饲喂含００２％二乙酰胺基芴（２ＡＡＦ）共２８天，第７天作２／３肝大部切除（２／３ＰＨ）以刺激卵圆细胞增生，第１２天用３ＨＴｄｒ腹腔注射以选择性标记卵圆细胞；模型Ⅱ在２８天连续饲喂００２％２ＡＡＦ之前第９天，先进行２／３ＰＨ，术后２２小时用３ＨＴｄｒ腹腔注射以标记代偿性增生的肝细胞。结果模型Ⅰ与模型Ⅱ在癌前病灶出现之前分别获得了对卵圆细胞和肝细胞的选择性标记，而癌前病灶细胞核内的示踪银颗粒只出现在卵圆细胞被选择性标记的模型Ⅰ中。结论提供了大鼠以２ＡＡＦ为致癌物诱发的癌前病灶来自卵圆细胞的直接证据。     

In vivo cell lineage analysis during chemical hepatocarcinogenesis in rats using retroviral-mediated gene transfer: evidence for dedifferentiation of mature hepatocytes

Feeding adult rats with a diet containing 2-acetylaminofluorene (2-AAF) results in suppression of hepatocyte proliferation and stimulation of oval cell proliferation. Although oval cells may be facultative liver stem cells, the actual relationship between oval cells and liver cancer has not been clearly established in vivo. Our goal was to label hepatic cells in vivo using retroviral vectors and follow their fate during the early steps of chemically induced hepatocarcinogenesis. Oval cell proliferation was induced by continuous feeding with a carcinogenic diet containing 2-AAF. We used two different strategies to genetically label hepatic cells: (a) labeling of proliferating cells in rats fed 2-AAF by injecting recombinant retroviral vectors containing the beta-galactosidase gene either in a peripheral vein or in the common bile duct at the peak of oval cell proliferation and (b) prelabeling of hepatocytes by intravenously injecting recombinant vectors 1 day after partial hepatectomy and 1 week before subsequent administration of 2-AAF. Using the first strategy, transgene expression occurred in both oval cells and hepatocytes. Using the second strategy, we could selectively label, and hence study the fate of, differentiated hepatocytes. In the latter case, we observed clusters of beta-galactosidase-positive hepatocytes, some of them also expressing preneoplastic markers such as gamma-glutamyl transpeptidase as well as the placental form of glutathione-S-transferase. These results demonstrate that preneoplastic foci can originate from mature hepatocytes and are consistent with the hypothesis that dedifferentiation of mature hepatocytes may occur during the course of carcinogenic regimen.     

Sequential analysis of hepatic carcinogenesis. Regeneration of liver after carbon tetrachloride-induced liver necrosis when hepatocyte proliferation is inhibited by 2-acetylaminofluorene

The effect of inhibition of hepatocyte proliferation by dietary 2-acetylaminofluorene (2-AAF) on the restoration of liver in rats after a necrogenic dose of carbon tetrachloride has been studied. The liver weights remained low during the entire feeding period of the 2-AAF, and virtually no hepatocyte proliferation was seen, as determined autoradiographically after thymidine incorporation and by the absence of mitotic figures. Oval cell proliferation was extensive. Morphometric analysis showed (a) equal and maximum liver cell necrosis by 24 hours in both the experimental and control groups, (b) similar kinetics of removal of dead liver cells, and (c) similar values for the mean liver cell area. The distance between the portal triad and terminal hepatic vein in animals on the dietary 2-AAF was considerably reduced. Massive hepatocyte proliferation began after termination of the 2-AAF diet, and the liver returned to normal appearance within 14 days. The oval cells disappeared during this period of liver cell restoration. A new hypothesis for oval cell proliferation based on differential inhibition of hepatocyte proliferation resulting in unbalanced growth of ductular cells is presented.     

Transforming growth factor-beta1 protein, proliferation and apoptosis of oval cells in acetylaminofluorene-induced rat liver regeneration

Administering of 2-acetylaminofluorene (2-AAF) before a two-thirds partial hepatectomy (PHx) results in suppression of hepatocyte proliferation and stimulation of oval cell proliferation. The objectives of this study was to examine the oval cell behaviour and associated transforming growth factor-beta1 (TGF-beta1) protein expression by combining 2-AAF with selective hepatic damage caused by PHx. We also studied the temporal relationship between TGF-beta1 expression, and proliferation and apoptosis of oval cells. Oval cells emerged from the portal areas and became more numerous with time fanning out into the periportal and midzonal hepatic parenchyma. Both smooth muscle actin (SMA) and TGF-beta1 immunostain revealed that TGF-beta1-positive cells were SMA-positive hepatic stellate cells (HSCs). Coinciding with the proliferation of oval cells, an increase expression of TGF-beta1 produced by SMA-positive HSCs was observed, thereafter apoptosis of oval cells reached its peak. This result implicated that TGF-beta1 produced by HSCs is intimately associated with proliferation and apoptosis of oval cells, and plays a role in the cessation of oval cell activation and remodeling of liver parenchyma in 2-AAF induced liver regeneration.     

Bone marrow progenitors are not the source of expanding oval cells in injured liver

Liver progenitor/oval cells differentiate into hepatocytes and biliary epithelial cells, repopulating the liver when the regenerative capacity of hepatocytes is impaired. Recent studies have shown that hematopoietic bone marrow (BM) stem/progenitor cells can give rise to hepatocytes in diseased/damaged liver. One study has reported that BM cells can transdifferentiate into liver progenitor/oval cells, but it has not been proven that the latter can repopulate the liver. To answer this question, we have lethally irradiated female DPP4(-) mutant F344 rats and transplanted them with 50 million wild-type male F344 BM cells. One month after transplantation, the recipient BM was reconstituted with male hematopoietic cells, determined by quantitative polymerase chain reaction using primers for Y chromosome-specific sry gene. In addition, DPP4(+) cells, single or in clusters and predominantly in the periportal region, were detected in all liver sections of recipient rats. Animals were subjected to the following three different liver injury protocols for activation and expansion of oval cells: D-galactosamine, retrorsine/partial hepatectomy (Rs/PH), and 2-acetylaminofluorene/partial hepatectomy (2-AAF/PH). In all three models, prominent expansion and accumulation of cytokeratin 19-positive (CK-19(+)) oval cells was observed. However, most of the DPP4(+) clusters dispersed over time, and their total number decreased. Very few oval cells (less than 1%) showed double DPP4/CK-19 labeling. None of the small hepatocytic clusters in the Rs/PH or 2-AAF/PH model were comprised of DPP4(+) cells. These data demonstrate that the sources of oval cells and small hepatocytes in the injured liver are endogenous liver progenitors and that they do not arise through transdifferentiation from BM cells.     

Studies on the proliferation and fate of oval cells in the liver of rats treated with 2-acetylaminofluorene and partial hepatectomy.

The kinetics of oval cell proliferation in the liver and their fate were studied by combined autoradiography and immunohistochemical staining for epidermal prekeratin and epoxide hydrolase (EH). The oval cell proliferation was induced in rats by exposure to dietary 2-acetylaminofluorene (2-AAF) for 2 weeks with the midway performance of partial hepatectomy (PH). The labeling with 3H-thymidine [3H-TdR] was done in different groups of rats by two procedures: continuous exposure for 1 week with the aid of a minipump and brief exposure by the administration of a single dose. The livers of groups of animals were examined from 1 to 10 weeks after PH. Oval cells and duct epithelium showed positive staining for prekeratin and negative for EH, whereas hepatocytes showed the reverse pattern of staining. A critical finding was the observation that the exposure to the 2-AAF inhibited virtually completely the labeling of hepatocytes with [3H]-TdR in the caudate lobe and incompletely in the right lobe without interfering with the labeling of the oval cells in either lobe. This made it possible to study the fate of the oval cells vis-à-vis hepatocytes. This qualitative-quantitative study of oval cells and hepatocytes clearly indicates that oval cells under these experimental conditions do not become hepatocytes within 10 weeks. Over 80% of oval cells disappear within this period, and the remainder persist as such. These results indicate that under one set of experimental conditions related to hepatocarcino-genesis in the rat, no evidence for the conversion of oval cells to hepatocytes was obtained.     

Bile ductular damage induced by methylene dianiline inhibits oval cell activation.

Administration of 2-acetylaminofluorene (2-AAF) given before a two-thirds partial hepatectomy (PHx), results in suppression of hepatocyte proliferation and stimulation of oval cell proliferation. Our objective in this study was to examine the oval cell response and associated alpha-fetoprotein (AFP) gene expression by combining 2-AAF with selective hepatic damage caused by either carbon tetrachloride (CCl4) exposure or by PHx. We also examined oval cell response with the above two protocols (2-AAF/CCl4 and 2-AAF/PHx) as affected by previous bile ductular damage caused by 4,4'-methylene dianiline (4,4'-diaminodiphenylmethane, DAPM) exposure. DAPM is an aromatic diamine, known to cause bile ductular damage in both humans and animals. Using the protocols of 2-AAF/ CCl4 and 2-AAF/PHx, when DAPM was given 24 hours before the hepatic injury, no oval cell proliferation was seen (histological) and AFP expression was not detected by Northern blot analysis. These results provide direct evidence that oval cells are closely associated with the biliary epithelial cells and supports the theory that hepatic oval cells may originate from cells derived from either intraportal or periportal ductules.     

急性肝功能衰竭模型大鼠缝隙连接蛋白的表达

背景：缝隙连接蛋白是组成相邻细胞间通道的主要结构,承担着细胞间的多种物质传输和信息交流的作用,可协助调节细胞的生长和分化。 目的：观察急性肝功能衰竭大鼠缝隙连接蛋白32表达与肝细胞增生的关系。 方法：采用乳果糖+庆大霉素灌胃和四氯化碳+橄榄油腹腔注射法建立大鼠急性肝功能衰竭模型。造模前7 d,苯巴比妥组大鼠用含体积分数0.08%苯巴比妥的水喂养,直至取材。对照组大鼠不造模,仅腹腔注射橄榄油与生理盐水的混合物。分别于造模后1,3,7,10,14 d取材。 结果与结论：大鼠肝功能衰竭后,部分大鼠出现死亡,存活大鼠肝细胞出现变性坏死,谷丙转氨酶明显升高,肝细胞间缝隙连接蛋白32 mRNA及蛋白表达明显降低。苯巴比妥可降低肝功能衰竭大鼠的死亡率,同时在一定程度上降低肝功能衰竭大鼠谷丙转氨酶水平及肝细胞间缝隙连接蛋白32 mRNA及蛋白的表达。说明通过苯巴比妥预先下调肝细胞间的缝隙连接蛋白32水平可以减轻急性肝功能衰竭大鼠急性期的肝脏损害,促进残存肝细胞增生和肝功能的好转,降低急性肝功能衰竭大鼠的病死率。