改良原代大鼠肝细胞的体外分离培养和功能研究

目的建立经济有效且功能良好的原代肝细胞体外分离培养系统。方法采用0．02％胶原酶和Percoll分离液分离纯化大鼠肝细胞．光镜和电镜下观察HepatoZYME-SFM培养的肝细胞形态，自动生化仪定期检测培养肝细胞功能，RT—PCR半定量方法检测白蛋白mRNA，高效液相色谱法（high performance liquid chromatography，HPLC）分析安定代谢能力。结果分离纯化的大鼠肝细胞总数（2-3）×10^8 cells／整肝，活力和纯度大于95％，生长良好形态正常。培养3d后ALT、AST显著下降，加入NH4Cl后8d内BUN保持相对稳定高水平，白蛋白合成在第3-4天达到高峰，培养的2～5d内肝细胞可有效清除安定。结论通过使用低浓度胶原酶灌流、Pereoll分离液纯化以及HepatoZYME—SFM无血清培养基培养，肝细胞可有效保持良好形态结构和一定的生物合成代谢能力。     

胎肝滤液诱导骨髓间充质干细胞向肝细胞分化

目的 探讨胚胎肝组织滤液定向诱导大鼠骨髓间充质干细胞(BMSCs)分化为肝细胞的诱导条件,为肝组织工程提供新的种子细胞来源。 方法 在体外培养体系中加入胎肝滤液,模拟体内肝脏微环境,诱导BMSCs向肝细胞定向分化,以免疫细胞化学检测肝细胞标志物;PAS检测糖原表达;靛青绿染色检测转化细胞的分化程度;测定细胞培养上清液中丙氨酸氨基转移酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)的含量以检测其功能状态。 结果 BMSCs经胎肝滤液诱导14d时细胞呈现多角形、卵圆形或圆形细胞的特征性改变;甲胎蛋白(AFP)和白蛋白(ALB)免疫反应、PAS反应和吲哚靛青绿(ICG)摄入实验及上清液中ALT、AST、ALP等酶均在诱导的第7天开始出现,AFP和ALB免疫反应在21d时达高峰;PAS反应和ICG摄入实验随着时间的延长而增强;上清液中的各个酶在14d达高峰,之后呈下降趋势。 结论 胎肝滤液可诱导BMSCs形成具有肝细胞形态和功能特点的细胞。     

以胶原水凝胶为支架构建肝细胞三维培养系统CSCD

背景:肝细胞体外培养时快速失去功能限制了肝细胞疗法的发展。目的:以胶原水凝胶为支架建立能够长时间有效维持肝细胞功能的肝细胞三维培养系统。方法:将SD大鼠肝细胞与预混肝细胞生长因子及DMEM的液态Ⅰ型胶原混合,形成肝细胞/胶原凝胶复合物,将该复合物接种至培养板,待形成胶冻状凝胶后再加培养液进行培养。通过光学显微镜、苏木精-伊红染色及透射电子显微镜对肝细胞的形态学特征及超微结构进行观察;并通过糖原染色、免疫荧光染色、实时定量PCR进一步对肝细胞特异表型及功能进行检测。收集培养上清,检测细胞白蛋白及尿素合成能力。结果与结论:①肝细胞/胶原水凝胶复合物形成后,可见肝细胞呈圆形分布于水凝胶中,培养14d后仍保持肝细胞形态并呈类似肝脏结构的肝索样聚集排列,超微结构观察显示肝细胞之间形成紧密连接。②培养14d后,糖原染色、白蛋白及肝细胞核因子4α免疫荧光染色呈阳性,证实肝细胞具有糖原及白蛋白合成能力并仍具有分化潜能。③三维培养中,肝细胞的白蛋白及尿素合成能力及分泌水平明显高于二维培养,且至少能够在较高水平维持15d。④培养7d后,Albumin、HNF-4α、Claudin-3、CYP1A1、CYP3A1以及G6P等肝细胞特异性基因的表达水平明显高于二维培养。以上结果证实,以胶原水凝胶为支架构建的三维培养系统使肝细胞在结构上更类似肝脏组织,并能在更长时间内有效的维持肝细胞合成代谢等各方面功能。     

生长抑素及奥曲肽的肝细胞保护作用及其机制研究

目的： 探讨生长抑素（SST）及其类似物奥曲肽（OCT）对大鼠肝细胞的保护作用及其机制。方法: 以原代培养肝细胞建立无水乙醇/四氯化碳（CCl4）细胞损伤模型，观察SST及OCT预处理对培养上清液中丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）含量的影响。此外，将75只SD大鼠随机分为正常对照组、肝纤维化模型组及大、中、小剂量SST治疗组。除正常对照组外均以40％CCl4皮下注射8周，期间各SST治疗组分别给予SST 200 μg·kg-1·d-1、100 μg·kg-1·d-1、50 μg·kg-1·d-1。采用酶试剂法、末端核苷酸转移酶介导的脱氧三磷酸尿苷原位缺口末端标记法（TUNEL）分别检测肝功能及肝细胞凋亡指数。结果: 经SST（10-8-10-6 mol/L）及OCT（10-7-10-5 mol/L）预处理后，损伤模型组肝细胞的培养上清液中ALT、AST水平显著下降。不同剂量SST治疗还能明显降低肝纤维化大鼠的血清ALT、AST、碱性磷酸酶（ALP）及总胆红素（TBIL）水平，提高血清清蛋白（ALB）水平，抑制肝细胞凋亡，其中小剂量SST治疗组最佳。结论: SST及OCT可减轻CCl4引起的肝细胞损伤，改善肝功能，并抑制肝细胞凋亡，可能在肝纤维化的防治中发挥重要作用。     

两种示踪方式的骨髓间充质干细胞在促进肝缺血再灌注损伤修复中的对比研究

目的　比较慢病毒转染绿色荧光蛋白(GFP)的骨髓间充质干细胞(BMSCs)和GFP转基因的BMSCs在大鼠肝缺血再灌注损伤修复中修复时效性及荧光稳定性的差异。 方法　常规体外培养2种BMSCs,MTT法检测二种细胞生长曲线间的异同;将40只SD大鼠随机分为假手术组、模型组、慢病毒转染GFP组(LV-GFP组)和GFP转基因组(GFP-BMSCs组),造模后LV-GFP组及GFP-BMSCs组于门静脉立即注入相应细胞悬液200 μl (数量约1×106个),模型组注入等体积的PBS溶液。于术后1、2、3、4周检测4组大鼠天门冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)及血清白蛋白(ALB)水平;于术后1~5 d切取实验组肝脏组织检测BMSCs入肝情况;于术后4周切取实验组肝脏组织检测BMSCs的荧光稳定性及其肝角蛋白18(CK18)的表达情况。 结果　GFP转基因大鼠的BMSCs在对数期的增殖能力明显强于慢病毒转染GFP的BMSCs(P<0.05);术后1、2周 GFP-BMSCs组AST及ALT水平明显低于 LV-GFP组 (P<0.05),术后2、3周 GFP-BMSCs组ALB水平明显高于 LV-GFP组(P<0.05);GFP-BMSCs组与LV-GFP组分别于术后3、5 d在肝区内见到GFP标记的BMSCs细胞;GFP-BMSCs及LV-GFP组都可于术后4周在肝区内见到已分化为肝细胞的BMSCs细胞,但GFP-BMSCs组BMSCs的荧光强度明显优于LV-GFP组。 结论　GFP转基因大鼠的BMSCs在大鼠肝缺血再灌注损伤修复中较慢病毒转染GFP的BMSCs展现出较好的修复时效性及荧光稳定性。     

miR-30a-5p启动子区DNA甲基化在同型半胱氨酸致肝损伤中的作用

目的:探讨微小RNA-30a-5p(miR-30a-5p)启动子区DNA甲基化在肝损伤中的作用。方法:随机选取4周龄胱硫醚β-合成酶(CBS)基因正常(CBS^+/+)小鼠(n=12)及单基因敲除(CBS^+/-)小鼠(n=12),均给予高蛋氨酸饮食8周。HL-7702细胞体外常规培养,分为对照(control)组、同型半胱氨酸(Hcy)组和Hcy+5-氮杂胞苷(AZC)组。全自动生化分析仪检测小鼠血清Hcy、丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)水平;微板法测定肝细胞ALT和AST水平;小鼠肝脏石蜡切片行HE染色观察肝脏损伤情况;细胞活力染色检测肝细胞活力;RT-qPCR法检测小鼠肝脏组织和肝细胞中miR-30a-5p的表达;Pearson相关性分析肝脏miR-30a-5p表达与血清ALT和AST水平的相关性;巢式降落式甲基化特异性PCR (nMS-PCR)检测小鼠肝脏组织以及肝细胞中miR-30a-5p启动子区DNA甲基化水平的变化。结果:与CBS^+/+对照组相比,CBS^+/-组...     

肝卵圆细胞对肝纤维化大鼠肝组织TGF-β/Smad信号通路的影响

目的:探讨肝卵圆细胞(HOCs)对肝纤维化(HF)大鼠肝组织TGF-β/Smad信号通路蛋白表达的影响。方法:采用CCl4和复方因素制备HF大鼠模型,取模型组大鼠分离纯化HOCs,从门静脉植入HF大鼠肝组织内,连续观察30d,同时以五灵胶囊为阳性对照。在植入后8d、15d、23d、30d各组大鼠尾静脉采血,酶法测定血清天冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT),实验结束取肝组织Masson染色观察肝组织形态学变化,Western blotting检测肝组织Ⅰ型胶原(Col-Ⅰ)、丝氨酸/苏氨酸蛋白激酶(ERK)、磷酸化丝氨酸/苏氨酸蛋白激酶(p-ERK)、转化生长因子β受体Ⅰ(TβRI)、转化生长因子β受体Ⅱ(TβRⅡ)、果蝇MAD类似基因2/3(Smad2/3)、果蝇MAD类似基因7(Smad7)蛋白的表达。结果:HOCs植入组与五灵胶囊组在植入后15d、23d、30dAST、ALT水平显著降低;肝组织胶原纤维增生程度明显减轻;肝组织表达ERK、p-ERK、TβRI、TβRⅡ蛋白作用显著降低,表达Smad7的作用显著增加。结论:植入HOCs可阻止大鼠HF的进展,其作用机制可能与其抑制肝组织内TGF-β/Smad信号通路p-ERK、TβRⅠ、TβRⅡ蛋白的表达有关。     

体外诱导脂肪源性干细胞向类肝细胞的定向分化

背景：用脂肪源性干细胞治疗肝脏疾病之前,如何建立有效稳定的肝细胞分化诱导方案,纯化并快速扩增性能稳定的类肝细胞等问题亟待解决。 目的：建立大鼠脂肪源性干细胞转化为类肝细胞的程序化诱导体系。 方法：分离纯化Lewis大鼠脂肪源性干细胞,流式细胞仪鉴定其表面标志,分3个阶段加入含有肝细胞生长因子、成纤维细胞生长因子4、酸性成纤维细胞生长因子、制瘤素M细胞因子的诱导培养体系,使脂肪源性干细胞向肝细胞转化。 结果与结论：大鼠脂肪源性干细胞诱导7,14,21 d后,细胞阳性表达 ALB、AFP、CK18mRNA,表达量随诱导时间延长而增强,类肝细胞具有白蛋白合成功能。氨代谢和尿素的合成功能在9~12 d出现并持续存在。结果表明脂肪源性干细胞体外分段诱导可成功转化为类肝细胞。     

胰岛素对大鼠肝细胞损伤的保护及其抗炎机制

目的观察胰岛素对大鼠继发性肝细胞损伤是否具有保护作用并探讨其机制。方法胶原酶原位灌流分离大鼠枯否细胞(Kupffercell,KC)及肝细胞并原代培养,分别用脂多糖(LPS)及胰岛素处理KC4h,ELISA法检测KC培养上清液中肿瘤坏死因子-α(TNF-α)和白介素-10(IL-10)水平;将不同处理的KC培养上清液分别作用肝细胞,12h后检测肝细胞损伤及存活率,并给予TNF-α单克隆抗体(TNF-α-mAb)阻断TNF-α,观察胰岛素对肝细胞作用的变化。结果(1)LPS处理的KC培养上清液可引起肝细胞损伤,使丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)增高;胰岛素可减轻上述肝细胞损伤(ALT活性降低30.1%,AST活性降低21.2%)(P<0.01,n=8),提高肝细胞存活率(P<0.01,n=8);(2)胰岛素降低KC培养上清液中的TNF-α水平(P<0.01,n=8),同时增加了IL-10的水平(P<0.05,n=8);(3)LPS激活的KC培养上清中加入TNF-α-mAb(中和TNF-α),亦可减轻肝细胞损伤,而此时胰岛素对肝细胞的保护效应未见叠加增强。结论首次发现胰岛素可通过抑制KC分泌促炎性细胞因子TNF-α,同时促进其释放抗炎性细胞因子IL-10,从而减轻肝细胞损伤,促进肝细胞存活。     

一种分离新生小鼠肝细胞的简单方法

目的建立新生小鼠肝细胞体外分离培养方法。方法采用胶原酶消化组织块法分离新生小鼠肝细胞，倒置显微镜动态观察细胞形态特征，并进行鉴定：PAS（periodieacid-Schiff，PAS）染色观察细胞肝糖元的含量；免疫组化SP法检测细胞甲胎蛋白AFP（alpha fetal protein，AFP）的表达；生化法检测肝细胞培养上清白蛋白的含量。结果肝组织块经用胶原酶直接消化，获得的肝细胞成活率达80％以上。肝细胞培养一周后长满瓶底，呈集落生长，融合成片。培养的肝细胞PAS糖原染色阳性；AFP表达阳性；培养上清白蛋白含量至培养5～7d时迭峰值，随后又逐渐下降。结论胶原酶消化组织块法分离新生小鼠肝细胞，方法简单、高效，培养的原代肝细胞符合新生肝细胞的生物学特性。     

壳聚糖球形多孔微载体支持下培养的肝细胞功能及其代谢活性检测

背景：在肝细胞移植及生物型人工肝研究中,肝细胞培养仍然是其关键,如何方便地获取足够数量、活性较好、功能良好的肝细胞已经成为其首要问题。微载体培养技术作为一项体外高密度细胞培养技术,近年来已在肝细胞的体外培养中得到应用。 目的：对壳聚糖球形多孔微载体培养的人肝细胞L-02进行定时的细胞功能和代谢活性检测。 方法：对以自制的壳聚糖球形多孔微载体样本为支架培养的人肝细胞L-02进行定时的细胞功能检测(包括测定谷草转氨酶,谷丙转氨酶和乳酸脱氢酶质量浓度)和代谢活性检测(包括检测培养基中白蛋白、尿素、葡萄糖含量),其中实验组为壳聚糖球形多孔微载体支持下培养人肝细胞L-02,对照组为无壳聚糖球形多孔微载体支持下培养人肝细胞L-02,检测时间点为人肝细胞L-02培养的第1,2,3,4,5天。 结果与结论：实验组和对照组测定的谷草转氨酶、谷丙转氨酶和乳酸脱氢酶活性在前3 d持续下降,第3天降到最低,而从第4天开始重新反弹上升;但白蛋白、尿素和葡萄糖水平在前3 d持续上升,第3天升到最高值,而从第4天开始逐渐下降,实验组每天测定上述各项水平始终明显高于对照组(P < 0.05),提示实验组中的人肝细胞L-02代谢活性较对照组增强。     

Well maintained expression of CYP genes in sandwich-culturing hepatocytes: quantitative analysis using real-time PCR method

Sandwich-culturing is an excellent hepatocyte culturing method in drug metabolism studies, however, its advantages for gene expression of cytochrome P450 (CYP) have not been evaluated so far. The present study was undertaken to determine the utilities of sandwich-culturing hepatocytes for evaluation of CYP genes expression. Hepatocytes from male rats were cultured for 5 days between two layers of type-I collagen gel (sandwich-culturing) or over type-I collagen gel (single gel culturing). To determine the expression of CYP genes rapidly and accurately, the time course study using real-time RT-PCR quantification was conducted in the present study, and CYP2B1, CYP2B2, CYP3A2, CYP3A9 and CYP3A23 genes were measured. Albumin secretion was also measured by ELISA to evaluate cell viability. Higher expression and excellent maintenance of all CYP genes were confirmed in sandwich-culturing hepatocytes than those in single gel culturing. Particularly, significant difference in the amounts of CYP genes expression was observed between both methods after 3 days culturing. Albumin secretion was also higher in sandwich-culturing after 3 days culturing, suggesting that the cell viability of hepatocytes was maintained. These results indicate that sandwich-culturing method is much more advantageous than the ordinate method in maintaining the CYP gene expression and cell viability.     

CYP3A5基因多态性与双环醇保肝降酶效应的关系

目的 研究CYP3A5酶基因多态性与双环醇保肝降酶作用临床效应的关系.方法 34例慢性乙型肝炎患者在常规治疗的基础上予双环醇片治疗24周;治疗前后检测肝功能指标(ALT、AST);采用PCR-RFLP法对患者CYP3A5酶基因型进行检测.结果 将检测结果基因型为CYP3A5*l(共16例,包括CYP3A5*1/*1型2例和CYP3A5*1/*3型14例)作为观察组,基因型为CYP3A5*3组(即CYP3A5*3/*3型,18例)作为对照组.治疗后,CYP3A5*1组和CYP3A5*3组患者ALT、AST水平均有显著下降(P＜0.05);CYP3A5*3组患者的ALT和AST下降幅度分别为79.73%和74.76%,显著大于CYP3A5*1组的65.90%和49.63%(P＜0.05);治疗后,两组之间的ALT及AST相比差异有统计学意义(P＜0.05),CYP3A5*3组ALT复常率和AST复常率均高于CYP3A5*1组,差异有统计学意义(P＜0.05).结论 CYP3A5酶基因型对双环醇治疗慢性乙肝的临床疗效抗炎保肝降酶效应有明显影响,基因型为CYP3A5*3的慢性乙肝患者双环醇疗效反应较强,该酶基因型可能成为双环醇疗效(尤其是降酶效应)的预测参照因子.     

Rat small hepatocytes for liver support: growth and metabolic activities in culture

It has been reported that small hepatocytes need to be cocultivated with nonparenchymal cells for proliferation in vitro. The purpose of this study was to investigate whether purified small hepatocytes would proliferate in the absence of nonparenchymal cells and to determine the their metabolic activities in culture. Purified small hepatocytes were obtained by centrifugation in Percoll density gradients. Small hepatocytes were seeded on collagen-coated dishes at a cell density of 4 × 10⁴ cells/cm² and were cultured in a hormonally modified Williams' medium E with 10 mM nicotinamide (WE-HN: group I). Comparision of 1 mM dimethyl sulfoxide (DMSO) (group II) or 5 mM retinoic acid (group III) in WE-HN was performed. Cell proliferation, as indicated by DNA content, and metabolic function, as assessed by urea synthesis, gluconeogenesis, and albumin production, were measured for 14 days. In each group, DNA content increased significantly during 14 days in culture. An increase of approximately 400% was observed in group II. The cell proliferation of group II was confirmed by microscopic observations. Urea synthesis and gluconeogenesis in each group declined gradually from days 1 to 14. Albumin production also fell, but to a less extent. We found that the purified small hepatocytes could proliferate without cocultivation of nonparenchymal cells. Further, they expressed metabolic activities of mature hepatocytes. Received: October 10, 2000 / Accepted: May 10, 2001     

Cryopreservation of suckling pig hepatocytes.

To determine the best and simplest method for cryopreservation of pig hepatocytes, we compared immediate cryopreservation with cryopreservation after short-term culture. Suckling pig hepatocytes were isolated by a modified 2-step in situ collagenase perfusion method, suspended in serum-free medium, and preserved for 10 da by two cryopreservation methods. Serial measurements were made of cell viability, LDH release, synthesis of protein, urea and glucose, glucose-6-phosphatase (G-6-Pase) activity, and diazepam transformation after thawing. These measurements were performed on both groups of cultured hepatocytes, and on freshly isolated hepatocytes, which served as a control. High viability (>95%)of thawed hepatocytes was obtained and maintained in both cryopreservation groups. There were no significant differences in cell viability, protein synthesis, glucose synthesis, G-6-Pase activity, or diazepam transformation between the two cryopreservation groups. In the immediate cryopreservation group, urea synthesis was less than in the group with cryopreservation after short-term culture. Protein synthesis, glucose synthesis, and diazepam transformation were lower in both cryopreserved groups than in the controls. The results showed that a protocol of immediate cryopreservation of hepatocytes in RPMI-1640 medium containing 10% DMSO, hormones, growth factors, and 10% newborn bovine serum, together with rate-controlled freezing and rapid thawing, provides indices of cell viability and function during subsequent serum-free culture that are comparable to hepatocytes cryopreserved after short-term culture, except for lower urea production. This simple procedure can be used in studies of bioartificial liver and hepatocyte transplantation.