间充质干细胞：细胞替代治疗肝硬化

肝硬化是肝纤维化的晚期阶段,常引发致命性并发症,严重威胁人类健康。肝移植是目前治疗肝硬化的首选方法,但其治疗效果受多种因素限制。近几年,国内外研究者提出细胞替代治疗肝硬化的理念。其中,间充质干细胞(MSCs)具有来源广泛、抑制免疫、减少炎症浸润和促炎因子释放、可以经诱导分化为肝细胞等特点,是细胞替代治疗肝硬化的有效药物。本文对间充质干细胞的特点、治疗肝硬化的机制以及最新的实验研究和临床试验展开综述。     

Cellular and molecular functions of hepatic stellate cells in inflammatory responses and liver immunology

The liver is a central immunological organ. Liver resident macrophages, Kupffer cells (KC), but also sinusoidal endothelial cells, dendritic cells (DC) and other immune cells are involved in balancing immunity and tolerance against pathogens, commensals or food antigens. Hepatic stellate cells (HSCs) have been primarily characterized as the main effector cells in liver fibrosis, due to their capacity to transdifferentiate into collagen-producing myofibroblasts (MFB). More recent studies elucidated the fundamental role of HSC in liver immunology. HSC are not only the major storage site for dietary vitamin A (Vit A) (retinol, retinoic acid), which is essential for proper function of the immune system. This pericyte further represents a versatile source of many soluble immunological active factors including cytokines [e.g., interleukin 17 (IL-17)] and chemokines [C-C motif chemokine (ligand) 2 (CCL2)], may act as an antigen presenting cell (APC), and has autophagy activity. Additionally, it responds to many immunological triggers via toll-like receptors (TLR) (e.g., TLR4, TLR9) and transduces signals through pathways and mediators traditionally found in immune cells, including the Hedgehog (Hh) pathway or inflammasome activation. Overall, HSC promote rather immune-suppressive responses in homeostasis, like induction of regulatory T cells (Treg), T cell apoptosis (via B7-H1, PDL-1) or inhibition of cytotoxic CD8 T cells. In conditions of liver injury, HSC are important sensors of altered tissue integrity and initiators of innate immune cell activation. Vice versa, several immune cell subtypes interact directly or via soluble mediators with HSC. Such interactions include the mutual activation of HSC (towards MFB) and macrophages or pro-apoptotic signals from natural killer (NK), natural killer T (NKT) and gamma-delta T cells (γδ T-cells) on activated HSC. Current directions of research investigate the immune-modulating functions of HSC in the environment of liver tumors, cellular heterogeneity or interactions promoting HSC deactivation during resolution of liver fibrosis. Understanding the role of HSC as central regulators of liver immunology may lead to novel therapeutic strategies for chronic liver diseases.     

骨髓间充质干细胞促进大鼠小体积肝移植后肝再生的实验研究

目的 探讨骨髓间充质干细胞能否促进大鼠小体积肝移植后移植肝的再生.方法 体外分离、培养、鉴定大鼠骨髓间充质干细胞.在30％大鼠部分肝移植的模型基础上,实验分为对照组(30％ PLT)和骨髓间充质干细胞干预组(30％PLT+ MSC).比较两组肝移植术后的存活率,分析肝功能的变化,通过免疫组化来观察移植肝标本Cyclin D1和PCNA的表达,并对移植肝组织结构进行电镜观察.结果 骨髓间充质干细胞的干预,能够改善小体积肝移植术后肝功能状况,减轻组织学损伤,并能够提高存活率,30％ PLT组与30％ PLT+ MSC组一周存活率分别为16.7％和58.3％.而在Cyclin D1和PCNA的免疫组化表达中,30％ PLT组表达明显抑制,30％ PLT+ MSC组表达上调.结论 骨髓间充质干细胞可以存进大鼠小体积肝移植后移植肝的再生,改善肝功能,并提高存活率.     

mTOR/P70S6K信号通路活化在肝硬化门静脉高压症中意义的实验研究

目的 探讨mTOR/P70S6K信号通路在早期肝硬化门静脉高压症中的活化状态,证实mTOR信号通路的激活参与肝硬化门静脉高压症的发病,并探讨其可能机制.方法 本实验采用胆道结扎离断制备大鼠肝硬化门静脉高压症模型.雄性SD大鼠20只随机分为假手术组和模型组.通过组织病理学、形态学和血流动力学评估肝纤维化、炎症、和门静脉压力.采用RT-PCR和Western blot法分别测定肝纤维化标志物PC-αⅠ、α-SMA、TGFβ1及PDGF基因的转录和mTOR信号通路标志物P70S6K和磷酸化的P70S6K(P-P70S6K)的表达.结果 模型组大鼠胆道结扎离断3周后,P-P70S6K表达量明显增高而总蛋白P70S6K无显著差异,同时HSCs和胆管细胞活化增殖显著,肝脏促纤维化基因明显上调,间质胶原合成增加.结论 mTOR/P70S6K信号通路主要以磷酸化功能状态参与肝硬化门静脉高压症的形成,阻断该通路可能成为肝硬化门静脉高压症治疗的新靶向.     

胎盘源性干细胞在肝脏疾病中的研究进展

人类胎盘源性干细胞(hPDSCs)是干细胞的混合群.再生医学已将其用于某些功能衰竭和损伤器官的细胞再生、抗细胞凋亡、抗炎,抗肿瘤和细胞功能恢复研究.目前已有许多实验研究证明:胎盘间充质干细胞(PDMSCs)可以在体外分化为肝细胞样细胞,并于体内外促进干细胞增生和抗肝细胞凋亡,在动物肝损伤模型抑制肝纤维化.本文就胎盘干细胞的来源、分类、生物学特性以及胎盘干细胞在肝脏疾病中的治疗研究做一综述,以便为进一步探讨胎盘源性干细胞在肝脏疾病治疗中的应用提供新的思路.     

骨髓间充质干细胞输注治疗大鼠小体积肝移植术后肝衰竭

目的 探讨大鼠骨髓间充质干细胞输注治疗大鼠小体积肝移植术后肝衰竭的可行性.方法 体外分离、培养、鉴定大鼠骨髓间充质干细胞,采用细胞膜染料PKH26对骨髓间充质干细胞进行标记.将雄性SD大鼠随机分为对照组和治疗组.对照组于小体积(30 %)肝移植术中,在移植肝血供恢复时自尾静脉注入磷酸盐缓冲液(PBS) 0.5 ml,治...     

硫酸钙对小鼠骨髓间充质干细胞分化和迁移的影响

目的 分析不同浓度的硫酸钙（calcium sulfate , CaSO4）对小鼠骨髓间充质干细胞（mesenchymal stem cells , MSCs）分化和迁移的影响,探讨CaSO4促进骨修复的细胞机制。方法 分离小鼠骨髓MSCs,绘制细胞生长曲线,评价不同浓度的CaSO4对细胞生长的影响,用qRT-PCR方法评价CaSO4作用后的成骨相关基因Osterix因子和骨钙素的表达,通过刮痕合拢实验和琼脂糖斑点实验,评估CaSO4对MSCs迁移能力的影响。结果 中浓度（0.8 g/L）和低浓度（0.1 g/L）的CaSO4对细胞生长无明显影响,高浓度（1.5 g/L）的CaSO4抑制了细胞增殖;中浓度的CaSO4在作用了1~4 d后,显著提高了MSCs成骨基因Osterix和骨钙素的表达,也明显促进了细胞迁移;高浓度（1.5 g/L）的CaSO4反而抑制了MSCs分化和迁移。结论 一定浓度的CaSO4能够诱导MSCs成骨分化,使MSCs细胞迁移能力增强;成骨细胞的出现可能是细胞迁移能力提高的原因之一。关键词： 硫酸钙;间充质干细胞;迁移;骨再生     

人肝再生增强因子基因转导对大鼠肝硬化的保护作用

目的 探讨人肝再生增强因子(augmenter of liverregeneration，ALR)基因转导对肝硬化的保护作用。方法 构建hALR真核表达载体，并以肌肉注射方式转导至硫代乙酰胺诱导的大鼠肝硬化模型中，观察对肝硬化大鼠的代谢支持作用。结果 构建的hALR真核表达载体pchALR肌肉注射后显著降低了血清AST、ALT、ADH水平，延长了生存时间，提高了存活率。结论 人肝再生增强因子基因转导对肝硬化大鼠具有预防和治疗作用，可显著降低血清AST、ALT、ADH水平，延长生存时间，提高存活率。     

Role of Stem Cells Transplantation in Tissue Regeneration After Acute or Chronic Acetaminophen Induced Liver Injury

Purpose: Acetaminophen-induced liver injury (APAP) is recognized as a frequent etiologic factor responsible for hepatic damage in the developed world. Management remains still elusive as treatment options are limited and their results are inconclusive. Consequently new strategies are explored at the experimental level. Mesenchymal stem cells (MSCs) present a promising modality as they can promote liver regeneration (LG) and compensate acute liver injury (ALI).Materials and methods: Our research was focused on articles related to drug-induced liver injury, mechanisms of liver regeneration (LG) after Acute Liver Injury (ALI) and recent experimental protocols of Mesenchymal Stem Cells (MSCs) transplantation after chemical insult. All these studies are cited on Pubmed and MedLine. Results: This review has three distinct sections. First recent developments in ALI pathogenesis are presented. The second section covers cellular pathways and histological findings relevant to liver regeneration. The final chapter analyzes MSCs transplantation protocols after ALI and interrelation between liver regeneration and hepatic differentiation of MSCs. Conclusion: Adipose tissue stem cells (ADSCs) and (MSCs) transplantation represents a promising modality in severe ALI management although many aspects remain to be clarified.     

肝星状细胞对大鼠肝部分切除术后肝再生修复的影响

目的 探讨肝星状细胞（hepatic stellate cells,HSCs）在大鼠肝部分切除术后对肝损伤修复的影响.方法 体外实验部分：将大鼠肝星状细胞（HSCs）与大鼠肝细胞系（BRL-3A）共培养,CCK-8比色法检测细胞增殖情况;ELISA法检测培养肝星状细胞上清液中细胞因子,即肝细胞生长因子（hepatic growth factor,HGF）、胰岛素样生长因子（insulin growth factor,IGF）、表皮细胞生长因子（epidermal growth factor,EGF）、转化生长因子-α（transfactor growth factor-α,TGF-α）含量.体内实验部分;将45只260 ～330 g健康雄性SD大鼠随机分为三组：正常组（生理盐水组）、对照组（肝星状细胞培养液组）、实验组（肝星状细胞培养上清液组）.各组分别于肝大部切除术后第3、7、12天取血清检测肝功能情况并计算肝再生指数;HE染色切片显微镜下观察肝脏病理结构改变情况;免疫组织化学染色法检测增殖细胞核抗原（proliferating cell nucleus antigen,PCNA）、平滑肌肌动蛋白（α-smooth muscle actin,α-SMA）、甲胎蛋白（a1pha feta1 protein,AFP）及白蛋白（albumin,ALB）的表达情况.结果 ①CCK-8法检测肝星状细胞能促进肝细胞增殖（P＜0.05）;②上清液较培养液中细胞因子HGF、TGF-α含量多,差别有统计学意义（P＜ 0.05）;③实验组肝再生指数比对照组大,差别具有统计学意义（P＜0.05）;④实验组大鼠AST随着时间的推移,较对照组下降明显,差别有统计学意义（P＜0.05）;⑤实验组PCNA表达量较正常组、对照组增多,差别有统计学意义（P＜ 0.05）;⑥平滑肌肌动蛋白、白蛋白、甲胎蛋白三组间差别不显著（P＞ 0.05）.结论 ①体外肝星状细胞能促进肝细胞增殖;②肝星状细胞培养上清液能促进大鼠肝部分切除术后肝再生,其分泌的多种细胞因子在肝损伤再生修复中起了重要作用.     

间充质干细胞在肝再生研究中的应用

间充质干细胞(mesenchymal stem cells,MSCs)移植治疗肝损伤及肝衰竭已取得令人瞩目的成就.但是,细胞定植能力弱以及潜在致瘤性、促纤维化的可能,阻碍了MSCs的临床应用.因此,有必要探索更优化的治疗方式,以提高MSCs治疗的安全性及有效性.迄今,MSCs来源的肝细胞、基因修饰的MSCs及MSCs培养液成分运用促进肝再生已取得鼓舞人心的成果.本文就近些年MSCs在肝再生研究中的应用作一综述.     

脐带间充质干细胞旁分泌物质对暴发性肝衰竭大鼠肝功能及肝细胞增殖的影响

目的 探索人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,UC-MSCs)旁分泌物质对实验性暴发性肝衰竭大鼠的治疗作用,研究其对大鼠肝功能及肝细胞增殖的影响.方法 体外分离培养人脐带间充质于细胞,流式细胞仪检测UC-MSCs的表面标志,制备含有UC-MSCs旁分泌物质的条件培养基(MSC-CM),腹腔注射D-氨基半乳糖制备暴发性肝衰竭大鼠模型.实验分为3组:MSC-CM组、生理盐水(NS)组和促肝细胞生长素(PHGF)组.在模型制备后24 h从大鼠尾静脉分别注射三组治疗药物.每组8只大鼠治疗后12 h、24 h、36 h、60 h分别经内眦取血测定谷丙转氨酶(ALT)和总胆红素(TBIL)含量.每组另取5只大鼠在治疗后36 h取肝脏组织制备切片进行PCNA免疫组化染色,检测大鼠肝细胞增殖情况.观察并记录各组大鼠的生存状态及生存时间.结果 MSC-CM组及PHGF组大鼠治疗后24 h ALT及TBIL的含量均低于NS组(P＜0.05),MSC-CM组与PHGF组比较差异无统计学意义.大鼠治疗后36 h肝脏切片PCNA染色显示,MSC-CM组和PHGF组PCNA肝细胞阳性数显著高于NS组(P＜0.01),MSC-CM组与PHGF组比较差异无统计学意义.生存分析显示,MSC-CM组和PHGF组大鼠的生存率高于NS组(P=0.049),MSC-CM组与PHGF组比较差异无统计学意义.结论 人脐带间充质干细胞的旁分泌物质可以刺激暴发性肝衰竭大鼠肝细胞增殖,改善暴发性肝衰竭大鼠的肝功能,为暴发性肝衰竭的治疗提供了一种新途径.

Abstract：

Objective To investigate the therapeutic effect of human umbilical cord mesenchymal stem cell-paracrine substance on fulminant hepatic failure (FHF) rat, and to study the effect on liver function and hepatocyte proliferation. Methods Mesenchymal stem cells(MSCs)were separated from human umbilical cord, and surface makers of cells were detected by flow cytometry. Human umbilical cord mesenchymal stem cells-conditioned medium(MSC-CM) was prepared. FHF rat model was induced by intraperitoneal injection of D-galactosamine and they were randomly diveded into three groups: MSC-CM group, NS group, PHGF group. 24 h later, 1 ml MSC-CM, 1 ml 0. 9% NaCl solution and lml PHGF solution was injected into the tail vein of MSC-CM, NS, and PHGF rats, respectively. In each group (n=8 per group), blood samples were collected at 12, 24, 36, and 60 h after treatment from inner canthus for analysis of blood ALT and TBIL levels. We used five rats per group for tissue collection after sacrifice at 36 h after treatment and 10 animals per group for survival analysis. PCNA immunohistochemical staining was used in the sections of liver tissue to detect hepatocyte proliferation. Results 24 h after treatment, the levels of ALT and TBIL in the MSC-CM and PHGF groups were lower than those in the NS group(P＜0. 05), but there was no significant difference between the MSC-CM and PHGF groups. There were more PCNA-positive hepatocytes in the MSC-CM and PHGF groups than in the NS group(P＜0.01), but there was no significant difference between MSC-CM and PHGF group. Survival analysis found that the survival rate of rats in the MSC-CM and PHGF groups was higher than that of rats in the NS group (P=0. 049), but there was no significant difference between the MSC-CM and PHGF group. Conclusions The paracrine substance of human umbilical cord mesenchymal stem cells can stimulate hepatocyte proliferation and improve liver function of FHF rats, potentially creating a new avenue for the treatment of FHF.     

脂肪间质干细胞抑制肝星状细胞增殖活化及肝脏纤维化

目的 探讨脂肪间质干细胞(adipose tissue-derived mesenchymal stem cells,ADSCs)对活化态肝星状细胞(hepatic stellate cells,HSCs)增殖、活化的影响及其对活体肝硬化模型的治疗作用.方法 分别分离纯化培养HSCs及ADSCs,通过半透膜(transwell insert)建立HSCs与ADSCs的双层培养体系,大鼠正常肝细胞系((buffalo rat liver cells,BRLs)培养作为对照.通过CCK-8比色法检测ADSCs对HSCs增殖的影响;Western blot检测HSCs的α-肌动蛋白(α-SMA)表达;建立鼠肝硬化模型,经门静脉输注ADSCs或BRLs,检测肝组织肝硬化;通过检测培养液中细胞因子的含量探讨可能的作用机制.结果 成功分离获得原代HSCs与ADSCs,活化的HSCs与ADSCs共培养72 h,与对照组相比,ADSCs明显抑制HSCs的活化(各组灰度值分别为1.4±0.2,152±14,258±18,F=283.348,P＜0.05)与增殖(各组吸光度A值分别为2.172±0.107,1.424±0.013,1.209±0.117,F=90.605,P ＜0.05),活体移植显示ADSCs对肝硬化具有明显抑制作用(分别F=77.234、65.164、58.309,均P ＜0.05).培养液细胞因子检测显示ADSCs比BRLs分泌更多的肝细胞生长因子(hepatocyte growth factor,HGF)(F=0.005,P＜0.05),分泌较少的转化生长因子(transforming growth factor-β,TGF-β1)(F=1.767,P＜0.01)及神经生长因子(nerve growth factor,NGF)(F=2.301,P＜0.05). 结论 ADSCs具有分泌细胞因子抑制HSCs活化增殖的潜能,对活体肝硬化进程具有一定的抑制作用.     

转染大鼠白细胞介素10基因的骨髓源性肝干细胞移植治疗大鼠肝纤维化

目的 探讨转染大鼠白细胞介素10(IL-10)基因的β2m-/Thy-1+骨髓源性肝干细胞(BDLSC)移植对大鼠肝纤维化的治疗效果.方法 分选Wistar大鼠的β3m-/Thy-1+BDLSC,转染大鼠IL-10基因.Wistar大鼠皮下注射CCl4,制作肝纤维化模型,分为3组:(1)模型组,经大鼠门静脉分支输注无菌生理盐水1 ml;(2)BDSC组,经大鼠门静脉分支输注β2m-/Thy-1+BDLSC悬液1ml(含2×105个细胞);(3)IL-10组,经大鼠门静脉分支输注转染1L-10基因的β2m-/Thy-1+BDLSC悬液1 ml(含2×105个细胞);另以皮下注射橄榄油的Wistar大鼠为对照(正常组).用细胞核染色剂二脒基苯基吲哚(DAPI)标记β2m-/Thy-1+BDLSC,以观察该细胞在肝内的定位情况.观察各组大鼠肝组织病理学改变和胶原的沉积情况,检测其肝功能和凝血功能.结果 大鼠肝组织中可见DAPI标记的β2m-/Thy-1+BDLSC.模型组大鼠的肝组织病理学改变明显,BDLSC组病理学改变有所减轻,IL-10组的组织形态最接近正常组.模型组大鼠肝组织内胶原沉积明显增多,BDLSC组胶原沉积较少,IL-10组胶原沉积明显减少.IL-10组大鼠的血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、碱性磷酸酶(ALP)和胆红素总量(TBIL)以及全血凝血酶原时间(PT)和活化部分凝血激酶时间(APTT)均低于模型组,差异均有统计学意义(P<0.05),其中ALT、TBIL、PT和APTT均降至正常组水平,与BDLSC组相比较,差异有统计学意义(P<0.05).结论 转染大鼠IL-10基因的β2m-/Thy-1+BDLSC移植对大鼠肝纤维化有一定的治疗效果.     

Human Adipose-Derived Mesenchymal Stem Cells Promote Liver Regeneration

Background and Aims: Liver regeneration (LR) is of crucial importance to patients with acute liver failure, those undergoing live donor liver transplantations or extended liver resections. Effective treatment strategies aimed at accelerating liver regeneration could offer major benefits in these patients. Due to easy accessibility, human adipose-derived mesenchymal stem cells (HADMSC) are an attractive source for regenerative medicine. Herein, we investigated the effect of HADMSC on LR in a murine model. We hypothesized that HADMSC will promote LR. Methods: Mice were subjected to CCl₄-induced acute liver failure (ALF). Animals in the experimental arm were treated with HADMSC prior to CCl₄-induced ALF. Liver injury was evaluated using serum levels of alanine aminotransferase (ALT), serum interleukin-6 (IL-6), and histopathology. Liver samples were stained for a specific marker of regeneration, proliferating cell nuclear antigen (PCNA). Results: Histology, serum IL-6, and ALT release revealed that HADMSC treatment attenuated liver injury compared with control animals. In addition, animals treated with HADMSC were observed to have improved survival and increased number of PCNA positive cells on histology when compared with controls. Conclusion: HADMSCs represent a potential therapeutic strategy to promote liver regeneration.     

The effect of denervation on liver regeneration in partially hepatectomized rats

BACKGROUND/AIM: In a partial liver transplantation, the dissected hepatic nerves are left unrepaired during active liver regeneration. In fact, the pathophysiological influence of such hepatic denervation on liver regeneration has not yet been fully clarified. The aim of the present study is to elucidate the effect of total hepatic denervation on liver regeneration. METHODS: Experiment 1: To confirm the effect of hepatic denervation, the hepatic contents of norepinephrine were measured in both denervated (n = 5) and sham (n = 5) rats. The changes in the hepatic microcirculation were also measured in both denervated (n = 5) and sham (n = 5) rats. Experiment 2: The rats (n = 80) were randomly assigned to two groups: DN group (n = 40); hepatic denervation followed by a partial hepatectomy (PH). Control group (n = 40); sham hepatic denervation followed by PH. In both groups, the animals were killed at 12, 24, 36, 48, 72, 120, and 168 h after PH, respectively. The liver to body weight ratio and the proliferating cell nuclear antigen (PCNA) labeling index were measured at each time point. RESULTS: Experiment 1: Nearly a total depletion of norepinephrine (<99%) was observed in the DN rats. In addition, the hepatic tissue blood flow significantly increased in the DN rats. Experiment 2: The liver to body weight ratio of the DN group was also significantly higher than that of the control group at 168 h (P < 0.05). The PCNA labeling index peaked between 24 and 36 h in the control group, while that in the DN group showed a delayed peak. At 72 and 120 h, the PCNA labeling index was significantly higher in the DN group than in the control group (P < 0.05). CONCLUSION: Total hepatic denervation was thus found to enhance liver regeneration after a partial hepatectomy. This phenomenon is partially triggered by the increased hepatic blood flow to the remnant liver.     

骨髓间充质干细胞在肝脏疾病治疗中的应用

原发性肝癌大多于肝纤维化、肝硬化的基础上发生,最终导致肝功能衰竭和全身转移.手术切除是肝癌的首选治疗方式,介入治疗和肝移植也是治疗终末期肝癌的手段.然而,肝部分切除和介入治疗往往制约于肝功能的失代偿.肝移植由于缺乏供体和价格昂贵临床亦难以广泛开展.近十多年来,骨髓间充质干细胞(BMSC)研究在医学领域取得了令人瞩目的成就.本文就骨髓间充质干细胞在肝脏疾病治疗中的应用作一综述.     

大鼠小体积肝移植后肝细胞再生的研究

目的 探讨小体积肝移植术后肝再生的情况。方法 建立大鼠30％原位肝移植模型,实验分为肝切除（PH）组、全肝移植（0LT）组和30％小体积肝移植（30％POLT）组,观察1w生存率,并于术后1、2、3、7d检测肝细胞增殖活性、肝功能和肝组织学变化。结果 各组1w生存率均为100％;30％POLT组术后2d达到增殖高峰,峰值与PH组无差异（P〉0．05）;其肝功能酶学指标在术后1、3d明显升高,组织学见肝细胞核分裂极其活跃。结论 冷缺血1h的30％供肝和肝切除后肝脏具有同样的增殖活性,仅增殖高峰稍晚;较短的冷缺血时间以及熟练的手术技术可能对小体积供肝的再生起重要作用。