肝卵圆细胞来源分化增殖的研究进展

卵圆细胞为肝内一种祖细胞，在肝细胞严重受损或分裂增生受抑制时可向肝细胞和胆管上皮细胞分化，是一种具有双向分化潜能的祖细胞。卵圆细胞的分化增殖受多种因素调控．新近研究认为SDF-1-CXCR4生物学轴在卵圆细胞增殖过程中起非常重要的作用。肝癌的发生与卵圆细胞的分化异常有一定的关系。卵圆细胞分化为肝细胞和胆管上皮细胞使其可能成为旰脏细胞移植的重要细胞来源，在治疗终末期肝病、生物人工肝及基因治疗的研究领域有着广阔的应用前景。     

c-fos基因在胆管细胞性肝癌中的表达及其预后关系的研究

ｃ－ｆｏｓ蛋白是Ｃ－ｆｏｓ原癌基因表达的一种ＤＮＡ结合蛋白,对细胞生长、分化与增殖具有重要调控作用,同时还能调节多种基因的转录,与某些肿瘤的发生、发展以及肿瘤的预后相关。目前已经发现在人骨肉瘤、结肠癌、肺癌、乳腺癌及肝细胞肝癌中有ｃ－ｆｏｓ基因的过度表达,但就胆管细胞性肝癌而言,国内尚未见报道。为此本研究应用免疫组织化学方法观察了ｃ－ｆｏｓ基因产物在胆管细胞性肝癌组织中的表达情况,并与癌旁胆管异型增生组织及正常肝内胆管上皮组织进行了对比,旨在探讨ｃ－ｆｏｓ基因与胆管细胞性肝癌的发生、发展及预后的…     

混合型肝癌的肝脏祖细胞起源研究

目的混合型肝癌同时具有肝细胞癌和胆管细胞癌的成分和特征,是较为少见的肝脏上皮性恶性肿瘤。研究肝脏祖细胞标志物在混合型肝癌中的免疫表型特征,以进一步了解混合型肝癌的细胞起源和发病机理。方法采用免疫组织化学和激光共聚焦免疫荧光双标染色研究HepParl,AFP,CK19,OV-6和c-kit在12例手术切除混合型肝癌标本组织中的表达。结果12例混合型肝癌均表达HepParl、CK19和OV-6,其中8例（8/12,66.7%）的移行区域肿瘤细胞共表达HepParl/CKl9,10例（10/12,83.3%）表达c-kit,其中7例（7/10,70%）共表达OV-6/c-kit。而作为对照的单纯肝细胞癌和肝内胆管细胞癌组织中免疫荧光双标染色均为阴性。结论混合型肝癌具有肝脏祖细胞的免疫表型特征,可能起源于肝脏祖细胞的恶性转化。     

混合型肝癌的临床病理分析

原发性肝癌可分为来自肝细胞的肝细胞型肝癌(hepatocellular carcinoma,HCC),来自肝内胆管上皮细胞的肝内胆管细胞癌(cholangiocarcinoma CC)以及同时含有肝细胞和肝内胆管上皮细胞的混合型肝癌(cHCC-CC).混合型肝癌临床较少见,大约占原发性肝癌的1.0%～3.5%.对其与肝细胞型和肝内胆管细胞型肝癌的联系及其在临床病理特征方面的报道亦甚少.     

p16抑癌基因在胆管细胞性肝癌中的表达与预后的关系

目的研究ｐ１６抑癌基因与胆管细胞性肝癌发生、发展及预后的关系。方法用免疫组织化学染色ＳＰ法检测胆管细胞性肝癌３８例、癌旁胆管异型增生组织２６例及正常肝内胆管组织中ｐ１６抑癌基因１６例的表达情况。结果ｐ１６抑癌基因在胆管细胞性肝癌组织中表达率为２６％,明显低于癌旁胆管异型增生组织（６９％,Ｐ＜００１）及正常肝内胆管组织（８１％,Ｐ＜００１）;中、低分化胆管细胞性肝癌ｐ１６抑癌基因表达率（１５％）显著低于高分化胆管细胞性肝癌的ｐ１６抑癌基因表达率（５４％,Ｐ＜００５）;在伴有淋巴结转移和门静脉癌栓的病例中,ｐ１６抑癌基因表达率明显低于相应的对照组（Ｐ＜００５）;ｐ１６抑癌基因阳性病人５年生存率为５６％,而ｐ１６抑癌基因阴性病人５年生存率仅为１４％,二者相比差异有显著意义（Ｐ＜００５）;ｐ１６抑癌基因的表达与癌灶体积、癌灶数目及ＨＢｓＡｇ无关。结论ｐ１６抑癌基因的失表达可能在胆管细胞性肝癌的发生发展中起重要作用;ｐ１６抑癌基因表达与胆管细胞性肝癌的分化程度及有无淋巴结转移和门静脉癌栓形成有关,是判定胆管细胞性肝癌恶性程度及估计预后的重要指标之一。     

肝内胆管细胞的分离与培养

肝内胆管细胞是许多肝胆疾病的攻击目标 ,如原发性硬化性胆管炎、原发性胆汁性肝硬化、肝移植物慢性排斥反应等 ;其生理功能和在肝再生、胆管癌发生过程中的变化也日益得到重视。与此相关的研究带动了胆管细胞分离与培养技术的发展。本文报道我们用免疫磁性分离方法分离肝内胆管细胞和其体外培养。资料与方法1.组织来源 :正常肝组织来源于 5例肝切除病人 ;病肝组织来源于肝移植病人 ,包括 2例原发性胆汁性肝硬化、1例自体免疫性肝炎和 1例Ｗｉｌｓｏｎ'ｓ病人。2 .胆管细胞分离 :将约 30 ｇ新鲜肝组织切成 1～ 2ｍｍ3大小碎片 ;用 1ｍｇ/ｍ…     

胆管细胞型肝癌误诊原因分析及经验总结

胆管细胞型肝癌也称肝内胆管细胞性肝癌(intrahepatic cholangiocarcinoma).该病缺乏特异性临床症状和体征,术前误诊率较高.该病有其特殊的临床特征,治疗也与肝细胞型肝癌不尽相同[1].降低术前误诊率、制定正确的治疗方案对改善预后意义重大.回顾分析我院收治的胆管细胞型肝癌患者,对术前误诊的29例患者分析误诊原因,并总结经验如下.     

自然杀伤细胞在乙型肝炎病毒感染性肝病和肝移植中的作用

由乙型肝炎病毒(HBV)引起的慢性乙型病毒性肝炎,是一种全球性常见感染性疾病,且慢性乙型肝炎可进展为肝纤维化、肝硬化,是肝癌发生的主要因素。肝切除、肝移植等治疗手段是治疗各种终末期肝病和原发性肝癌(肝癌)的有效措施。自然杀伤(NK)细胞是机体免疫重要组成部分,以非主要组织相容性复合体(MHC)限制的方式识别抗原,参与先天性免疫应答,且通过调控T细胞应答及对T细胞的直接作用,对特异性免疫反应有一定的调控作用。本文就NK细胞在HBV感染性肝病和肝移植术后宿主的排斥反应中的作用作一综述。     

胆管板畸形与先天性胆管发育异常

肝内胆管 ( intrahepatic bile ducts,IHBDS)来源于门静脉周围的肝祖细胞 ,这些细胞围绕门静脉形成胆管板 ,胆管板重塑进而形成胆管。如果胆管板在重塑过程中发生障碍 ,则形成胆管板畸形 ( ductal plate m alformation,DPM)。研究结果表明 ,胆管板畸形与大多数先天性胆管发育异常疾患如胆道闭锁、先天性肝纤化、Caroli' s病及肝囊肿的发生密切相关。对胆管板形成、胆管板重塑的分子机理以及胆管板畸形认识的逐步深入为进一步了解先天性肝内胆管畸形的发病理机开辟了新的领域。1　胆管板、胆管板重塑与胆管板畸形 ( DPM)的形成在胚胎发…     

原发性肝内胆管癌21例临床报告

胆系癌肿按其发生部位可分四类,即肝内胆管癌、肝门部胆管癌、肝外胆管癌及壶腹部癌。肝内胆管癌又称胆管细胞型肝癌,是原发性肝癌的一种较少见的类型。由于较少见,目前对     

骨质疏松小鼠模型骨髓间充质干细胞特性的研究

目的 探讨骨质疏松小鼠模型骨髓间充质干细胞（mesenchymal stem cells ,MSCs）生物学特性的改变。方法 通过皮下注射地塞米松构建小鼠骨质疏松动物模型（OP组）,同时设立对照（NC组）,分离OP组及NC组骨髓MSCs进行体外培养;通过流式细胞术检测细胞表面分化抗原;运用CKK-8实验检测细胞增殖活力;通过脱氧核苷酸末端转移酶介导的dUTP缺口末端标记法（TUNEL）检测细胞凋亡水平;运用碱性磷酸酶（ALP）活性检测、油红O染色及Western blot法检测两组细胞成骨分化及脂肪分化能力。结果 经过骨组织学检测证明模型建立成功,体外培养OP组及NC组骨髓MSCs细胞形态无差异。OP-MSCs细胞表面分化抗原Scal1及CD44为阳性, CD34及CD11b为阴性,与NC-MSCs相似,但OP-MSCs增殖活力下降（P<0.05）;此外两组细胞凋亡水平类似（P>0.05）。成骨分化诱导7 d后OP-MSCs的ALP活性显著低于对照组（P<0.01）,21 d后矿化小结明显减少（P<0.001）;脂肪分化诱导8 d后OP-MSCs形成更多脂滴（P<0.05）。Western blot结果显示OP-MSCs在成骨分化中低表达转录因子Runx2（P<0.05）及Osterix (P<0.001）,但在脂肪分化早期高表达PPARγ（P<0.001）及C/EBPα（P<0.01）。结论 骨质疏松小鼠骨髓MSCs的增殖及分化潜能显著下降。     

兔雪旺细胞的培养方法及形态学研究

利用兔坐骨神经组织进行体外细胞培养研究，获得了雪旺细胞。兔雪旺细胞的形态与其他文献报道的鼠或人的外周神经培养获得的雪旺细胞形态基本相似，典型的雪旺细胞形态特征为胞体较小、梭形、两侧突起纤长、核窄长。倒置显微镜下看，胞体四周常有亮边，细胞成极性生长排列。通过免疫组织化学染色证实具有典型形态的梭状细胞为雪旺细胞，而扁平状形态不规则的细胞为成纤维细胞     

尿嘧啶脱氧核苷及超顺磁性氧化铁双标记的神经干细胞活体移植后细胞迁徙及功能性分化的实验研究

目的观察尿嘧啶脱氧核苷(Brdu)及超顺磁性氧化铁(SPIO)双标记的神经干细胞活体移植后存活细胞的迁徙及分化。方法SPIO和Brdu对神经干细胞进行双标记,移植至大脑中动脉梗死模型的对侧脑组织,术后采用MRI监测。第6周MRI检查后取组织切片行免疫组织化学染色,观察神经干细胞的迁徙及分化。结果双标神经干细胞活体移植后第3周MRI开始显示细胞沿胼胝体迁徙,第6周脑组织免疫组织化学染色亦显示干细胞沿胼胝体向对侧迁徙,免疫组织化学荧光双标染色显示移植的神经干细胞可以分化为星形胶质细胞及神经元。结论移植存活的神经干细胞在迁徙过程中能够进行功能性分化。     

三维及二维培养大鼠胰腺导管来源干细胞的体外对比实验研究

目的观察在不同培养模式下大鼠胰腺导管来源干细胞(PDSC)的生物学特性。方法以大鼠胰腺组织为材料,采用动力性三维培养及传统二维培养方法进行细胞培养,获得原代PDSC,并连续传代,纯化PDSC,比较动力性三维培养及传统二维培养细胞间连接及细胞凋亡周期的差异。结果动力性三维培养的大鼠PDSC在培养第2天即可沿三维支架贴壁生长,在第7天即可形成细胞团,并沿三维支架多向贴壁生长;二维培养的PDSC在培养第5天贴壁生长。三维培养与二维培养相比,二维培养的细胞间连接少见,三维培养的细胞连接结构丰富。三维培养与二维培养相比,三维培养的G1期细胞增多,G2期细胞相对减少(P<0.01),三维培养早期自发凋亡细胞比例相对减少(P<0.05),晚期自发凋亡与坏死细胞比例两种培养方式间的差异无统计学意义(P>0.05)。结论动力性三维细胞培养较传统二维细胞培养,在细胞贴壁时间、细胞连接以及细胞凋亡方面具有明显的优势。     

外源性细胞因子对早孕蜕膜调节自然杀伤细胞活性的作用

为探索外源性细胞因子对妊娠的影响,本研究在培养的蜕膜细胞中加入不同浓度的干扰素 γ、表皮生长因子、白细胞介素 ２ 和白细胞介素 ６,培养 １２、２４ 和 ４８ 小时后取其上清液,５ １ Ｃｒ 掺入法检测其对人外周血自然杀伤细胞（ Ｎ Ｋ）的杀伤作用。结果发现,该上清液可以促进人外周血 Ｎ Ｋ 细胞的杀伤活性,这种作用与细胞因子的种类、浓度及作用时间无明显相关性。提示外源性细胞因子有可能通过破坏蜕膜细胞因子网络平衡状态对妊娠造成危害。     

大鼠肝窦内皮细胞分离、培养及其生物学特性的初步研究

目的　建立大鼠肝窦内皮细胞 (SEC)的原代分离、培养方法 ,并研究其生物学特性。方法　胶原酶灌注结合Percoll密度梯度离心加选择性贴壁的方法分离SEC ;用光镜、扫描电镜观察培养SEC的形态学变化及超微结构 ;用Ⅷ因子和CD14免疫细胞化学染色及RECA 1单抗间接免疫荧光法观察SEC表面分子的表达。结果　分离培养的SEC得率为 (2 .0 6± 0 .35 )× 10 7/只大鼠 ,活力≥ 92 % ,纯度达 90 % ;光镜下细胞形态典型 ,SEM下可观察到特征性的窗孔结构 ;Ⅷ因子染色阴性而CD14染色阳性 ,RECA 1单抗间接免疫荧光染色阳性。结论　分离培养的SEC细胞得率、活力及纯度较高 ,形态典型且具有一般细胞所没有的窗孔结构及表面标志 ,为其功能和作用机制的深入研究奠定了基础。     

Effects of fatty acids and eicosanoid synthesis inhibitors on the growth of two human prostate cancer cell lines

Dietary fatty acids (FAs) may be involved in the carcinogenic process within the prostate gland and progression to clinically manifest disease. We have shown that growth of the androgen-unresponsive PC-3 human prostate cancer cell line is stimulated in vitro by the presence of linoleic acid (LA), an omega-6 polyunsaturated FA. The response was positively related to the FA concentration over the entire range examined (5-750 ng/ml). Conversely, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), two omega-3 FAs present in fish oils, inhibited PC-3 cell growth in a dose-dependent manner; both were equally effective, with an approximately 65% reduction in growth occurring at a concentration of 2.0 micrograms/ml (P less than 0.001). The DU 145 human prostate cancer cell line, which is also androgen-unresponsive, showed no growth response to LA and was less susceptible to growth inhibition when cultured in the presence of omega-3 FAs. Growth experiments with indomethacin, esculetin, and piroxicam, pharmacological inhibitors of eicosanoid biosynthesis with differing sites of action, indicated that human prostate cancer cell growth requires intact metabolic pathways for both leukotriene and prostaglandin production.     

睾丸间质干细胞分化和移植

睾丸间质细胞是男性体内合成雄激素的主要细胞,胚胎发育期中肾胚的间质细胞及生精小管周成纤维样细胞可能是睾丸间质细胞的干细胞。在胚胎期间质干细胞分化为胎儿型间质细胞;出生后间质干细胞经间质祖细胞、未成熟间质细胞分化为成熟间质细胞。老年期间质细胞数量可能不变,但雄激素合成下降。间充质干细胞及脂肪干细胞等干细胞经诱导可分化为分泌雄激素的睾丸间质细胞,因此,间质干细胞移植可望成为治疗男性性腺功能不全和中老年雄激素缺乏的创新方法,本文对睾丸间质干细胞的分化及移植方面研究进行综述。     

Antitumor Killer Lymphocytes in the Peripheral Blood of a Patient with Transitional Cell Carcinoma of the Bladder

Background: Peripheral blood lymphocytes (PBL) from patients with bladder cancer also contain cells possessing cytotoxic activity against autologous tumor cells. These cells are phenotypically heterogenous and include natural killer (NK) and cytotoxic T cells. This study investigated the role of cytotoxic lymphocytes directed against autologous bladder cancer cells.
Methods: PBL were obtained at intervals before and after surgery and analyzed for cytotoxic activity against autologous bladder cancer cells in 4-hour⁵¹ Cr release assay. PBL stimulated with autologous tumor cells were also transformed with human T-lymphotropic virus type-1, establishing a cell line (KB31) which was analyzed for phenotype and cytotoxic activity against the autologous tumor cells.
Results: PBL preoperative cytotoxic activity was low, but increased after surgery. Cytotoxic activity was found not only against autologous bladder cancer cells, but also against heterologous bladder cancer (KK-47) and myeloid leukemia (K562) cells, with the highest activity against the heterologous cell lines. The cytotoxic activity of KB31 was 40|X% against autologous tumor cells 6 weeks after initiation of the cell line, but decreased to 5|X% by 6 months. This activity was lower than that against the other cell lines, and was similar to that of PBL in short-term culture. Fluorescence-activated cell sorter (FACS) analysis demonstrated that in KB31 cells at 6 weeks, CD8+ cells were dominant, but CD56+ cells predominated at 6 months.
Conclusion: These results suggest that the presence of cytotoxic activity in the peripheral blood of the patient was due to both cytotoxic T cells and NK cells. The cytotoxic activity was lowest prior to surgery and increased postoperatively.     

Evidence of pluripotent human prostate stem cells in a human prostate primary xenograft model

INTRODUCTION: The phenotypic plasticity of the human prostate stem cell within human prostate tissue was examined to determine the response of the stem cell to changes in the androgenic environment. METHODS: Prostate xenografts were transplanted into athymic nu/nu mice implanted with testosterone pellets, allowed to establish for 1 month time point, the hosts were castrated and pellets removed, and following 1 month of androgen deprivation, the hosts were stimulated with androgen for 2 days to induce proliferation of the residual population of stem cells (2-month time point). RESULTS: Glands in benign xenografts harvested at the 1- and 2-month time points contained basal cell layers that expressed p63 and high molecular weight cytokeratin, and in which essentially all of the cellular proliferation was localized, consistent with the proposed localization of the prostate stem cell. Benign glandular structures in the xenografts were populated by basal, secretory epithelial, neuroendocrine (NE), or squamous cells overlaying the basal cell layer, whereas, adenocarcinoma glands in the xenografts resembled the original prostate cancer (CaP) tissue. CONCLUSIONS: In this human prostate primary xenograft model, the residual stem cell population that survives transplantation, or androgen deprivation, maintains significant pluripotentiality as demonstrated by the capacity to generate progeny that differentiate along multiple lineages in response to microenvironmental signals, particularly along the secretory epithelial lineage in response to androgen, and along the NE cell lineage in response to androgen deprivation.