【作者】 申竑；

【导师】 李桂源；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 目的肝卵圆细胞(hepatic oval cell, HOC)是成体肝组织中主要的多能干细胞,它的增殖与分化是肝损伤后实现肝再生和肝组织自体修复的重要途径,并受到多种细胞生长因子的调控。骨形态发生蛋白-4 (bone morphogenetic protein-4, BMP-4)是参与多种固有干细胞和移植干细胞修复和治疗急慢性器官损伤的重要调控因子。我们前期研究结果发现BMP-4可诱导大鼠HOC细胞系WB-F344定向分化为肝细胞表型。但BMP-4对大鼠原代HOC增殖与分化的影响及其生物细胞信号传导机制尚不清楚。本课题的研究目的即在于明确BMP-4对大鼠原代HOC增殖和向肝细胞定向分化的调控作用及其细胞内信号传导因子和通路,加深我们对BMP-4调控HOC定向分化的生物学功能及其分子机理的全面了解,为利用BMP-4及其细胞信号传导机制,定向诱导HOC分化促进肝再生和肝修复、治疗肝脏损伤奠定理论和应用基础。方法采用2-芴基乙酰胺+2／3肝切除构建雄性SD大鼠HOC增殖动物模型,Collagenase IV/Pronase肝脏原位灌注消化肝组织+三密度Percoll细胞梯度离心分离高纯度大鼠原代HOC,体外培养和传代扩增大鼠HOC。以MTT法了解BMP-4对大鼠原代HOC增殖的影响,RT-PCR检测大鼠BMP-4的Ⅰ型和Ⅱ型细胞受体在大鼠HOC细胞膜上的mRNA表达情况。采用实时荧光定量PCR、Western杂交和免疫荧光染色等技术明确HOC中BMP-4的主要细胞转录因子Smad1和ERK 1／2的mRNA和蛋白表达水平,以及磷酸化蛋白表达及其向细胞核内转移和聚集情况,从而了解BMP-4调控HOC的细胞信号传导机制。然后以RT-PCR检测大鼠HOC、成熟肝细胞和胆管细胞等HOC分化细胞标志物的mRNA表达,全自动生化检测仪和ELISA技术分别检测HOC分化后细胞合成,并向细胞培养液中分泌的尿素和白蛋白浓度,透射电子显微镜观察BMP-4处理后大鼠HOC的细胞超微结构以及细胞间连接,从而了解BMP-4对大鼠HOC向肝细胞定向分化的诱导作用。并利用基因重组技术,构建大鼠BMP-4和BMP4 shRNA重组腺病毒,转染大鼠HOC后异体脾脏移植,观察其对大鼠CCl4急性肝损伤后肝组织修复与肝功能恢复的影响。结果成功建立大鼠HOC体内增殖模型,每只成年大鼠肝脏原位灌注分离可获得大约5.63±0.56×106个HOC,细胞活性率为90.32%+3.26%。BMP-4可呈剂量依赖性和时间依赖性促进培养大鼠HOC的增殖,且50 ng/ml是BMP-4理想的HOC作用浓度,以此浓度BMP-4干预6天后,HOC增殖可达到峰值。RT-PCR证实大鼠HOC上存在BMP-4的Ⅰ型细胞受体(BMPR-Ⅰ)的二个亚型BMPR-1A和BMPR-1B,以及BMP-4的Ⅱ型细胞受体BMPR-Ⅱ的表达。50 ng/ml的BMP-4作用对大鼠HOC的Smad1、ERK 1/2 mRNA及其蛋白表达水平无影响,但可促进蛋白的磷酸化及其向细胞核内转移和聚集。BMP-4(50ng/ml)处理大鼠HOC12天后,可观察到HOC干细胞标志物c-kit.胆管细胞标志物CK19和β4-integrin的mRNA表达显著降低或消失,而成熟肝细胞标志物TAT-1、A1b和G6Pase的mRNA表达则显著增强。同时,BMP-4处理组HOC培养液中尿素浓度(1.32±0.32 mmol/L)分别是Noggin+BMP-4组(0.61±0.09 mmol/L)和空白对照组(0.59±0.13 mmol/L)的2.16倍和2.24倍；BMP-4处理组HOC培养液中Alb浓度(68.256±13.256 ng/ml)分别是Noggin+BMP-4组(14.735±4.012 ng/ml)和空白对照组(15.897±4.283 ng/ml)的4.63倍和4.29倍,差异均具有统计学意义(p<0.05)。透射电子显微镜显示BMP-4诱导分化后HOC的超微结构以及相邻细胞间的良好连接,表明BMP-4可促进细胞向上皮细胞良好分化。BMP-4拮抗剂Noggin可显著抑制BMP-4诱导大鼠HOC体外增殖和向肝细胞分化的生物学作用。成功构建大鼠BMP-4和BMP4 shRNA重组腺病毒,HOC异体脾脏移植动物实验发现术后第12天,BMP-4腺病毒感染HOC移植组大鼠脾脏可见HOC分化后的肝细胞定置和“脾化肝”,肝组织修复明显,且该组的大鼠死亡率、毛发、精神、活动量、食欲等一般情况,以及体重、白蛋白和谷丙转氨酶(ALT)等肝功能指标明显优于HOC脾脏移植组、BMP4 shRNA腺病毒感染HOC脾脏移植组等五个实验对照组。结论BMP-4与大鼠HOC上BMPR-Ⅰ和BMPR-Ⅱ细胞受体结合后,可同时通过其细胞转录因子Smad1和ERK-1／2及其蛋白磷酸化实现细胞内信号传导,诱导大鼠HOC体外增殖并向肝细胞定向分化。BMP-4诱导分化的大鼠HOC具有成熟肝细胞的蛋白合成和分泌功能,以及细胞超微结构和细胞间连接,BMP-4可通过HOC异体脾脏移植促进大鼠急性肝损伤后的肝组织修复与肝功能恢复。更多还原

【Abstract】 Background & ObjectiveHepatic oval cell (HOC) is the major source of pluripotent stem cells in adult liver. The proliferation and differentiation of HOC, which is regulated by a series of cell growth factors, is one of the most important approaches and mechanisms of liver regeneration and tissue recovery following liver injury. Bone morphogenetic protein-4 (BMP-4) is a critical regulator involved in the treatment of different acute and chronic organs injury using several kinds of inherent and transplanted stem cells. WB-F344 cell, which was an adult rat HOC cell line, had been proved to be directionality differentiated into hepatic phenotype in our previous studies. However, the effects and the cellular signaling transduction mechanisms of BMP-4 on the proliferation and differentiation of the primary rat HOC remain unclear. The aims of the present stydy are to investigate the regulative effects and the intracellular signaling moleculars of BMP-4 on rat primary HOC proliferation and orientational differentiation into hepatocytes. The results will help us to have a better understanding for the biological functions and the molecular mechanisms of BMP-4 regualtion on HOC proliferation and orientational differentiation into hepatocytes, so as to provide the theoretical principle and clinical application basis for the treatment of liver injury by taking the advantages of BMP-4 and its cellular signaling transduction to promote liver regeneration and tissue revovery.MethodsThe animal models with proliferated HOC was set up by the methods of N-2-fluorenyl-acetamide gavage in combined with 2/3 hepatectomy in adult male Sprague-Dawley rats. Rat HOC was isolated and purified by in situ liver perfusion with Collagenase IV/ Pronase, which was followed by cellular gradient centrifugation using three density gradients of Percoll. The isolated primary rat HOC was cultured and sub-cultured to be amplified in vitro. The effects of BMP-4 on the proliferation of primary rat HOC was detected by MTT assay. RT-PCR was employed to determine mRNA expression of TypeⅠand TypeⅡBMP-4 receptors on rat HOC cytomembrane. To understand the intracellular signaling moleculars and transduction pathways of BMP-4 in rat HOC, the techniques of Real-time PCR, Western blot and immunofluorescent staining were used to explore mRNA and protein expression, as well as the phosphorylated protein expression and its accumulation in HOC nucleus of Smad1 and extracellular regulated protein kinases 1/2 (ERK 1/2), which were two major transcription factors of BMP-4. To approach the induction of BMP-4 on HOC orientational differentiation into hepatocytes, the cell markers of rat HOC, mature hepatocytes and biliary epithelia cell (BEC) were measured by RT-PCR for their mRNA expression. The concentrations of urea and albumin secreted by differentiated HOC into HOC culture medium were tested by automated biochemistry examination and ELISA assay. The HOC ultrastructures and cell-cell junctions were checked on a transmission electron microscope (TEM). Rat BMP-4 and BMP4 shRNA recombinant adenovirus were constructed applying gene recombination. The HOC infected by either BMP-4 or BMP4 shRNA recombinant adenovirus was applied in allograft models of rat spleen transplantation to observe the effects of BMP-4-carrying HOC transplantation on the recovery of liver tissue and functions following acute liver injury by carbon tetrachloride (CCl4) gavage.ResultsAbout 5.63±0.56 x 106 HOC with 90.32%±3.26% living cells were isolated from each Sprague-Dawley rat successfully established HOC proliferation in vivo. Recombinant BMP-4 protein could boost the proliferation of HOC in vitro in both dose-dependent and time-dependent manners. Comparing with either lower or higher BMP-4 concentrations. 50 ng/ml of BMP-4 was the optimal concentration on HOC proliferation promotion, which reached its peak at the sixth day of BMP-4 treatment at the concentration of 50 ng/ml. The existence and mRNA expression of BMPR-1A, BMPR-1B and BMPR-II on HOC cytomembrane were proved by RT-PCR. BMP-4 treatment at the concentration of 50 ng/ml could not influence mRNA and protein expression of both Smadl and ERK 1/2. However, BMP-4 intervention significantly enhanced phosphorylated Smad1 and ERK 1/2 proteins and its accumulation in HOC nucleus. Following 12-day treatment of BMP-4 at 50 ng/ml, the stem cell marker of c-kit and BEC markers of CK19 andβ4-integrin significantly faded or vanished, but the mRNA markers of matured hepatocytes including TAT-1、Alb and G6Pase were significantly increased in rat HOC. Meanwhile, the urea concentrations secreted by HOC into the cultured medium in BMP-4 treatment group (1.32±0.32 mmol/L) exhibited 2.16-fold and 2.24-fold of that in Noggin treament group (0.61±0.09 mmol/L) and the blank control (0.59±0.13 mmol/L), and the Alb concentrations in HOC cultured medium in BMP-4 group (68.256±13.256 ng/ml) were 4.63-fold and 4.29-fold of that in Noggin group (14.735±4.012 ng/ml) and the blank control (15.897±4.283 ng/ml), respectively (p< 0.05, respectively). The HOC ultra-structures and intercellular junctions observed under TEM indicated that BMP-4 induced rat primary HOC to differentiate into epithelium-like cells in vitro. Noggin, which acted as BMP-4 antagonist, could specifically block the regulative effects of BMP-4 on rat HOC proliferation and its differentiation into hepatocytes. The rat HOC infected with the constructed BMP-4 and BMP4 shRNA adenovirus were applied in rat spleen allografts. On the ninth day after intraspleenic HOC transplantation into homogenous rats receiving CCl4 gavage, the fixation of hepatocytes differentiated from transplanted HOC and the specific "hepaticized spleen", obviously recovered liver tissue, significanltly improved general conditions of rats’mortality, hair, spirit, movement and appetite, as well as the liver functional indices including total bilirubin (TBIL), albumin, ALT and AST were observed in spleen transplantation of the HOC infected with BMP-4 adenovirus, as compared with that of the other five experimental control groups including BMP4 shRNA adenovirus-infected HOC transplantation, etc.ConclusionsFollowing the binding to TypeⅠandⅡreceptors on rat HOC cytomembrane, BMP-4 transducts its intracellular signal through its transcription factors of Smadl and ERK-1/2 to induce HOC proliferation and orientational differentiation into hepatocytes with protein synthesis and secretion functions, as well as the ultrastructures and intercellular junctions of mature hepatocytes. Rat HOC spleen allografts regulated by BMP-4 will promote the liver tissue regeneration and liver function recovery in the rats with acute liver injury.更多还原