小鼠成体肝脏干细胞体外培养模型的建立

目的 观察正常成体小鼠肝细胞的增殖能力和分化潜能,分离成体小鼠肝脏内可能存在的干细胞或祖细胞,建立细胞培养模型.方法 应用改良的Seglen二步法灌注和离心分离肝脏细胞,用含血清的改良DMEM培养基进行培养,持续观察超过60 d.应用免疫荧光技术对肝细胞及其形成的克隆进行Albumin、AFP和CKl9染色.结果 部分肝脏细胞培养第2～3天后活化,迅速增殖并形成细胞克隆,培养30 d后克隆内出现类似成熟的肝细胞,细胞克隆持续扩增超过60d.该类细胞培养第1天强阳性表达肝细胞标记物Albumin,培养第5天细胞克隆开始表达肝脏干细胞标记物AFP,第55天表达胆管细胞标记物CKl9.结论 在成体小鼠未损伤肝脏内存在一种成体肝脏祖细胞(adult hepatic progenitor cells,AHPCs),该细胞体外培养具有较强的增殖能力,可分化为肝细胞和胆管细胞,并成功建立了AHPCs的体外培养模型.     

经肝动脉灌注平阳霉素碘油乳剂对兔正常肝脏组织的影响

目的：探讨平阳霉素碘油乳剂（PLE）经肝动脉灌注对兔正常肝脏组织的影响。方法：14只4～5个月龄日本大耳白兔，体重（2.5±0.2）kg，按注入生理盐水或PLE的量分为假手术组、实验A组（低剂量组）和实验B组（高剂量组）。各组兔均开腹穿刺肝动脉，按分组剂量注入PLE。术后1，2，4，6周取病理切片，HE染色，光镜下观察肝脏组织学改变;免疫组织化学（免疫组化）染色标记血小板衍化生长因子B链（PDGF-B），并行图象分析。结果：A组HE染色肝细胞呈一过性水样变，变性在2周时最重，至6周已明显减轻。B组HE染色2周时肝细胞明显水样变，4周时可见汇管区纤维组织增生，6周时部分肝组织出现明显假小叶结构。免疫组化染色显示，PDGF-B在肝细胞胞膜及纤维间隔中有明显表达。结论：经肝动脉灌注PLE可导致正常肝脏组织产生不同程度的肝纤维化;PDGF-B参与了肝纤维化的病理过程。     

羟基荧光素二醋酸盐琥珀酰亚胺脂标记大鼠肝脏NK细胞体内转输示踪

目的 利用羟基荧光素二醋酸盐琥珀酰亚胺脂(CFSE)标记大鼠肝脏NK细胞进行体内示踪,观察其在受鼠体内的存活.方法 经SD大鼠门静脉转输1×106个/ml CFSE体外标记新鲜分离的SD供鼠肝脏NK细胞,于输人后1、3、7、15 d分别切取肝脏和脾脏行冰冻切片免疫荧光观察,并收集门静脉血监测淋巴细胞和中性粒细胞的比例变化.结果 CFSE对大鼠肝脏NK细胞的标记率为98.63%;转输NK细胞后第1天受鼠肝脏内CFSE荧光最强,7 d时荧光变微弱,15 d时荧光基本消失;脾脏在第1、3天时可见较弱荧光,7 d时荧光消失.转输NK细胞后相同时间点肝脏内CFSE荧光均明显强于脾脏.转输肝脏NK细胞的受鼠门静脉血中性粒细胞比例第1天时最高,随时间延长逐渐下降,第15天时基本回复正常水平,淋巴细胞比例仅一过性降低.结论 转输的肝脏NK细胞在受体内的数量随时间延长而减少,在受体内的存活时间约为15 d.     

大鼠胚胎来源肝干细胞分离纯化方法的比较

目的 观察比较机械分离法和酶消化法分离纯化孕鼠胚胎来源肝干细胞的优劣.方法 取孕13.5 d SD大鼠胚胎肝后分别用机械分离法和酶消化法分离纯化肝干细胞,比较两种方法所得细胞总量和存活率的差异,用免疫荧光染色法鉴定细胞,并比较原代培养前后肝干细胞存活率的变化.结果两种方法分离培养出的细胞形似卵圆形,经免疫荧光染色法检测表达干细胞标志物CD34和胆管细胞标志物CK19,机械分离法分离的肝干细胞数量(57.49±14.25)×106与酶消化法(63.36±12.67)× 106相当,两者比较差异无统计学意义(P>0.05),但机械分离法所得肝干细胞的存活率(96.37±0.82)%高于酶消化法(93.53±0.63)%(P<0.05),并且培养前后细胞存活率也明显高于酶消化法,两两比较差异有统计学意义(P<0.05).结论 机械分离法是一种简单、经济、实用的胚胎来源肝干细胞分离纯化方法.     

HNF4α在胎肝干细胞促肝组织损伤修复中的作用

目的探讨肝细胞核因子(HNF)4α在胎肝干细胞促大鼠肝组织损伤修复中的作用。方法胶原酶结合机械消化法制备14 d胎龄大鼠胎肝干细胞悬液并进行体外长期培养。采用四氯化碳制作SD大鼠急性化学性肝损伤模型,造模成功后将肝损伤大鼠随机分为移植组(20只)和对照组(20只),移植组将胎肝干细胞经门静脉移植至大鼠肝脏,对照组注射等容计量的生理盐水,分别于注射前6 h,注射后1,3,5周检测大鼠血清谷氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST),并检查肝组织病理学变化,观察肝脏修复情况。采用免疫组化及Western-blot方法检测肝脏治疗前后HNF4α表达情况。结果对照组大鼠肝功能改善缓慢,ALT,AST明显高于移植组(P0.05),移植组大鼠损伤的肝组织逐渐修复,肝功能恢复正常。第5周移植组大鼠术后存活率显著高于对照组(P=0.002)。肝组织损伤后,HNF4α在肝组织的表达明显升高;随着肝组织的逐渐修复,HNF4α表达逐渐下降。western-blot及免疫组化检测亦证实此结果。结论HNF4α在肝脏损伤初期起保护作用。可能系通过促进胎肝干细胞向正常肝细胞分化增殖,并迁移至损坏的肝小叶从而替代损伤的肝实质,从而提高肝损伤后的恢复能力。     

3D细胞培养和类器官在骨髓源性间充质干细胞成骨分化中的研究进展

类器官（organoids）来源于自体的组织及干细胞,是通过体外3D培养后形成的细胞团块,这种团块具有原始组织及器官的三维结构,并保留了相对应的功能和遗传特征。由于其具有模拟特定机体器官的发育和疾病发生发展的潜能,这一模型在多种药物的筛选和分子机制研究中拥有更多的优势。近年来,已有实验表明骨髓源性间充质干细胞（BMSCs）通过3D培养及成骨分化可以形成骨的类器官,并可以植入机体发挥特定的作用。这种骨类器官模型的构建,不仅可以为骨质疏松等相关疾病研究提供更多方法,还在骨组织移植及修复等组织工程学中发挥重要作用。本文就BMSCs成骨分化的类器官相关3D模型研究进展作一综述,为BMSCs成骨分化的类器官的基础和临床研究提供更多理论依据和思路。     

肝动脉缺血对供肝及胆道组织损伤的实验研究

通过简易犬肝自体原位移植模型，研究了肝动脉缺血（ＨＡＩ）对供肝及胆道组织的损伤作用。结果发现，随着ＨＡＩ时间的延长，肝及胆道细胞超微结构出现明显的病理改变，肝脏组织内氧自由基含量增多，术后血清胆红素和丙氨酸转氨酶含量亦随之增高。当ＨＡＩ３小时，胆道上皮细胞线粒体出现不可逆性病理改变，即絮状电子致密斑。说明ＨＡＩ能对肝脏及胆道组织产生损伤作用，是肝移植术后胆道并发症的主要致病因素之一。     

肝星状细胞在肝脏疾病中的作用

肝星状细胞（HSC）是肝脏内重要的非实质细胞之一,可分泌、释放多种胶原纤维和细胞骨架蛋白参与肝脏疾病的病理生理过程。正常状态下,HSC通过调节细胞外基质蛋白的合成和降解维持肝脏正常的组织结构;肝脏损伤时,HSC被激活,活化的HSC导致细胞外基质的增加是肝纤维化形成并最终导致肝硬化、肝衰竭的主要原因。因此,深入研究HSC在肝脏疾病发生与发展中的作用和机制,并研究与HSC相关的治疗策略,对于提高患者生存率具有一定意义。     

活化的肝星状细胞在肿瘤微环境中的免疫调节作用

目的 研究活化的肝星状细胞(hepatic stellate cells,HSCs)在小鼠肝细胞癌发生发展中的免疫调节作用及肿瘤微环境的免疫功能变化.方法 于体外,用Brdu细胞增殖实验检测HSCs上清对肿瘤增殖的影响,用混合淋巴实验检测HSCs的免疫调节作用.体内实验采用皮下移植瘤模型,分为H22单纯注射组与H22+HSCs联合注射组.20 d后对荷瘤小鼠行瘤组织解剖测量移植瘤大小;用免疫组化法观察两组肿瘤中T细胞亚群浸润变化情况和接种肿瘤PD-L1的表达变化;用流式细胞术分析脾脏中T细胞亚群变化.然后,通过Tunel检测肿瘤组织单核细胞凋亡情况.结果 在体外实验,Brdu检测显示HSC:条件培养基(HSCs-CM)能明显诱导H22细胞的增殖,而混合淋巴实验则显示HSCs对T细胞增殖有明显的抑制作用.在体内实验,HSCs+H22组小鼠皮下移植瘤的形成时间与生长速度明显快于单纯接种H22组;肿瘤组织免疫组化检测显示HSCs+H22组浸润的T细胞及其亚型较单纯H22组有不同程度降低;HSCs+H22组脾脏亦显示T细胞及其亚型出现不同程度降低.TUNEL试剂盒检测显示HSCs+H22组肿瘤组织中单核细胞的凋亡数量增加,免疫组化分析显示HSCs+H22组肿瘤组织中PD-L1表达增加.结论 HSCs可通过免疫抑制作用在肿瘤微环境中促进肿瘤的发生、发展,这一发现可能为肝癌的治疗提供新的思路.

Abstract：

Objective To determine immune modulatory activity of activated hepatic stellate cells( HSCs) in hepatocellular carcinoma and immune response in tumor microenvironment. Methods Cell proliferation was measured by BrdU incorporation with a microtiter plate reader at 450 nm. The effect of HSCs on T cell proliferation was measured by MLR. Mouse hepatic cancer cell line H22 were implanted on the backs of BALB/c mice to establish the subcutaneous transplanted tumor model. Then the mice were sacrificed after 20 days for anatomical and size determination. Furthermore, Paraffin-embedded tissue was removed by serial tissue sectioning and immunohistochemically examined for expression of T lymphocyte subsets. T lymphocyte subsets in splenocytes were detected by FCM. Apoptotic mononuclear cells were evaluated by FITC-labeled Tunel assay. Results We determined that HSCsCM promoted hepatocellular carcinoma(HCC) cell line proliferation and HSCs inhibit T cell proliferation by MLR in vitro. We also examined normal immune mice to assess the immunosuppression of HSCs in the development of HCC. In the co-transplantation with HSCs group, T cells and their subtypes decreased obviously in the tumors and the spleen. The results showed that co-transplanted HSCs can induce more PD-L1 expression and more mononuclear cell apoptosis in tumor tissue. Conclusion Our results demonstrated that HSCs promote HCC progression partially because of their immune regulatory activity. Our data supply new information to support an integral role for HSCs in promoting HCC progression and suggest that HSCs may serve as a therapeutic target for HCC.     

In vitro functionality of human fetal liver cells and clonal derivatives under proliferative conditions

Mature human hepatocytes are not suitable for large-scale in vitro applications that rely on hepatocyte function, due to their limited availability and insufficient proliferation capacity in vitro. In contrast, human fetal liver cells (HFLC) can be easily expanded in vitro. In this study we evaluated the hepatic function of HFLCs under proliferative conditions, to determine whether HFLCs can replace mature hepatocytes for in vitro applications. HFLCs were isolated from fetal livers of 16 weeks gestation. Hepatic functions of HFLCs were determined in primary culture and after expansion in vitro. Clonal derivatives were selected and tested for hepatic functionality. Results were compared to primary mature human hepatocytes in vitro. No differences were observed between primary HFLCs and mature human hepatocytes in albumin production and mRNA levels of various liver-specific genes. Ureagenesis was 4.4-fold lower and ammonia elimination was absent in HFLCs. Expanding HFLCs decreased hepatic functions and increased cell stretching. In contrast, clonal derivatives had stable functionality and morphology and responded to differentiation stimuli. Although their hepatic functions were higher than in passaged HFLCs, functionality was at least 20 times lower compared to mature human hepatocytes. HFLCs cannot replace mature human hepatocytes in in vitro applications requiring extensive in vitro expansion, because this is associated with decreased hepatic functionality. Selecting functional subpopulations can, at least partly, prevent this. In addition, defining conditions that support hepatic differentiation is necessary to obtain HFLC cultures suitable for in vitro hepatic applications.     

肠上皮干细胞分离培养方法研究进展

肠上皮干细胞是位于肠黏膜陷窝内的具有自我更新和增殖分化为成熟肠上皮细胞功能的细胞。近年来,肠上皮干细胞的研究越来越受到重视,通过分离SP细胞、培养隐窝器官样组织和利用组织工程等方法研究肠上皮干细胞的分裂、分化过程,加深了对肠上皮干细胞的生物学特征的了解。但肠上皮干细胞的分离、培养和鉴别等研究方法尚未取得突破性进展。     

骨髓基质干细胞作软骨组织工程种子细胞研究

目的　综述近年来骨髓基质干细胞体外培养、定向分化为软骨细胞的条件及相关研究进展。方法　广泛查阅相关文献 ,对上述研究进展进行整理、综合和分析。结果　骨髓基质干细胞易于分离培养 ,体外增殖能力强 ,传代多次后仍保持有多分化潜能 ;体内成软骨能力明确 ;但骨髓基质干细胞定向分化的软骨细胞 ,不是终末分化阶段 ,而只是一个中间阶段。结论　骨髓基质干细胞以其自身多方面的特点已成为软骨组织工程的另一种可选择的种子细胞 ,但其进一步分化为成熟软骨细胞的条件尚需进一步研究。     

慢性肝损伤时肝内干细胞的增殖

目的：采用免疫组织化学的方法研究慢性肝损伤时肝干细胞在肝脏内的分布和增值,初步探讨肝脏的损伤修复与肿瘤发生的细胞生物学机制。方法：3,-me-DAB喂食大鼠四周,建立应激模型刺激大鼠肝内干细胞的迅速增殖,取材、固定制成石蜡切片,ABC法检测肝干细胞特异性的表面标记蛋白CD34、C-11、CK19、OV6和神经前体细胞的特异性表面标记Nestin在大鼠肝脏内的表达。结果：大鼠肝实质结构改变显著,汇管区内结缔组织细胞大量增生,浸润周围肝组织形成假小叶。靠近界板处断续的排列着卵圆样细胞分别表达CD34、CK19、OV6和Nestin,阳性细胞的形态和分布特点相似,而且具有典型的干细胞的特点。此外,肝实质内可见大量的单个核样干细胞分别表达OV6、CD34、Nestin和CK19。这些细胞类似造血系统来源的单核细胞分布在肝索、肝血窦、肝内血管、增生的小叶间胆管和胆管之间,多见于汇管区的结缔组织内。结论：3’-me-DAB作用后,肝内存在着多种干细胞,而且具有空间分布的规律性。增生的干细胞在损伤应激下继续增殖分化为肝实质细胞,参与肝脏的组织修复,同时成为引发肝脏肿瘤的可能原因之一,具体机制还需要进一步的深入研究。     

Construction and transplantation of an engineered hepatic tissue using a polyaminourethane-coated nonwoven polytetrafluoroethylene fabric

BACKGROUND: Acute liver failure (ALF) is a serious condition that has a high mortality rate. Construction of an efficient culture and transplantation engineering system of hepatic tissue is an important approach to treat patients suffering from ALF to provide short-term hepatic support until the damaged liver spontaneously recovers or a donor liver becomes available for transplantation. Here, we evaluate the construction and transplantation of an engineered hepatic tissue (EHT) using primary isolated hepatocytes cultured onto polyaminourethane (PAU)-coated, nonwoven polytetrafluoroethylene (PTFE) fabric. METHODS: The isolated hepatocytes cultured onto PAU-coated PTFE fabric were able to adhere and spread over the individual fibers of the net and formed hepatic clusters after 3 days, such clusters revealed Gap junctions and well-developed bile canaliculi. RESULTS: When PAU-coated PTFE was utilized, ammonia-, and diazepam- metabolizing capacities and albumin production ability were significantly increased compared with collagen control. To test the function of this hepatic tissue in vivo, we transplanted a nonwoven PAU-coated PTFE fabric inoculated with one million hepatocytes on the surface of the spleen of Balb/c mice suffering from ALF induced by 90% hepatectomy, and found that this EHT prolonged the survival of liver failure-induced mice without adverse effects. Ultrastructure analyses showed good attachment of the cells on the surface of PTFE fabric and strong albumin expression seven days after the newly formed hepatic tissue was transplanted. CONCLUSION: We have here demonstrated the efficient construction and transplantation of hepatic tissue using primary hepatocytes and PAU-coated PTFE fabric.     

Mechanical Dissociation of Swine Liver to Produce Organoid Units for Tissue Engineering and In Vitro Disease Modeling

The complex intricate architecture of the liver is crucial to hepatic function. Standard protocols used for enzymatic digestion to isolate hepatocytes destroy tissue structure and result in significant loss of synthetic, metabolic, and detoxification processes. We describe a process using mechanical dissociation to generate hepatic organoids with preserved intrinsic tissue architecture from swine liver. Oxygen-supplemented perfusion culture better preserved organoid viability, morphology, serum protein synthesis, and urea production, compared with standard and oxygen-supplemented static culture. Hepatic organoids offer an alternative source for hepatic assist devices, engineered liver, disease modeling, and xenobiotic testing.     

In vitro production of functionally mature hepatocytes from prospectively isolated hepatic stem cells

Hepatocyte transplantation and artificial organ hepatic support require a number of functionally mature hepatocytes. However, their growth activity and functional behaviors are much smaller in culture after isolation from the liver. We examined whether continuously differentiating hepatocytes from multipotent hepatic stem cells that were isolated by using flow cytometry and propagated clonally in culture could be a source of clinical application. They actually gave rise to cells that were functionally equal to mature hepatocytes found in the adult liver, which secreted albumin into culture medium and metabolized harmful ammonium into urea. These data suggest that stem cell-derived hepatocytes are a useful cell source for developing therapeutic strategies, such as cell transplantation, gene therapy, and artificial liver organ to treat various liver disorders.     

Differential nitric oxide synthase expression during hepatic ischemia-reperfusion

BACKGROUND: In recent years the important role of nitric oxide in hepatic ischemia-reperfusion injury has been increasingly recognised. The prevailing consensus is that reperfusion injury may be partly the result of decreased production of nitric oxide from endothelial nitric oxide synthase and excessive production of nitric oxide from the inducible isoform. We therefore undertook this study to characterize the expression of different nitric oxide synthase isoforms during hepatic reperfusion. METHODS: Male Wistar rats (n = 6) were subjected to 45 minutes of partial hepatic ischemia (left lateral and median lobes) followed by 6 hours of reperfusion. Control animals (n = 6) were subjected to sham laparotomy. The expression of endothelial and inducible nitric oxide synthase was examined using immunohistochemistry and Western blotting. Liver sections were also stained with nitrotyrosine antibody, a specific marker of protein damage induced by peroxynitrite (a highly reactive free radical formed from nitric oxide). RESULTS: Liver sections from all the control animals showed normal expression of the endothelial isoform and no expression of inducible nitric oxide synthase. Livers from all the animals subjected to hepatic ischemia showed decreased expression of endothelial nitric oxide synthase, and all but one animal from this group showed expression of the inducible isoform both in inflammatory cells and in hepatocytes. Western blotting confirmed these findings. Staining with the antinitrotyrosine antibody was also confined to five liver sections from animals subjected to hepatic ischemia. CONCLUSIONS: During the reperfusion period after hepatic ischemia, endothelial nitric oxide synthase is downregulated while inducible nitric oxide synthase is expressed in both hepatocytes and inflammatory cells. The presence of nitrotyrosine in livers subjected to hepatic ischemia-reperfusion suggests that the expression of inducible nitric oxide synthase plays an important role in mediating reperfusion injury in this model.     

主肝静脉急性阻断后引流肝段保留价值的研究

目的观察主肝静脉阻断后保留肝段的病理形态学变化。方法７８只大鼠随机分为对照组、肝段静脉结扎组、左主肝静脉缩窄组与结扎组,动态观测受累肝叶的病理学,肝脏微循环与血流动力学变化。结果主肝静脉结扎后２４小时即发生肝细胞坏死,门静脉血内毒素与ＴＸＢ２／６－Ｋｅ－ｔｏ－ＰＧＦ１α明显升高,主肝静脉缩窄组受累肝叶边缘大量肝静脉与门静脉侧支形成,门静脉血内毒素与ＴＸＢ２／６－Ｋｅｔｏ－ＰＧＦ１α也发生不同程度升高,两组均明显高于肝段静脉结扎组与对照组。结论正常肝组织不能耐受主肝静脉急性阻断,无肝静脉引流的肝组织不但完全丧失功能,而且引起内毒素血症与肝脏微循环障碍,主肝静脉结扎应同时将引流肝段切除。     

Transplantation of liver organoids in the omentum and kidney

Liver organoids were reconstructed by mouse-immortalized hepatocytes and nonparenchymal cells (sinusoidal endothelial cells and hepatic stellate cells) in a radial-flow bioreactor (RFB). A biodegradable apatite-fiber scaffold (AFS) was used as a scaffold packed in the RFB, which enables three-dimensional cell cultures. The organoids cocultured in the RFB showed a liver-like structure with high-density layers of hepatocytes and the formation of vessel-like structures. A liver organoid consisting of three cocultured cells was transplanted under the kidney capsule (kidney group) or into the omentum (omentum group) using BALB/c nude mice. Transplanted liver organoids survived in the kidney or omentum. The expression of mRNAs of albumin, connexin 26 and 32, hepatocyte nuclear factor 4α, and glucose-6-phosphatase was increased in both groups at 8 weeks after transplantation in comparison to the pretransplant status. Tyrosine aminotransferase appeared only in the omentum group. The results suggested that the functions of liver organoids differed depending on the transplanted site in the recipient animals.     

联合应用SSH和cDNA表达谱芯片筛选肝星状细胞早期活化的相关基因

目的利用抑制消减杂交(suppressionsubtractivehybridization,SSH)和cDNA表达谱芯片筛选早期活化的肝星状细胞(hepaticstellatecell,HSC)和大鼠正常肝脏组织、轻度肝纤维化组织、重度肝纤维化组织中差异表达的基因。方法从SSH构建的大鼠轻度肝纤维化、重度肝纤维化2个差异cDNA文库中挑选1000条上调显著的基因,与正常大鼠4136条基因克隆制作成一张芯片,筛选早期活化的肝星状细胞相关基因。结果获得与HSC早期活化相关的上调基因有202条,下调基因有80条,其中血清和糖皮质激素调节蛋白激酶(serumandglucocorticoidsensitivekinase,SGK)在正常大鼠基因克隆和2周及8周SSH上调差异基因中均呈上调信号。结论联合应用SSH和cDNA表达谱芯片是筛选和鉴定不同样本中差异表达基因的快速、经济和有效的方法;SGK可能作为多种细胞信号传导通路和细胞磷酸化级联反应的一个功能性交汇点,参与了肝星状细胞的早期活化和信号传导。