骨形态发生蛋白BMP-4对大鼠肝卵圆细胞增殖和分化调控作用的实验研究

发布时间：2018-01-02 00:28

本文关键词：骨形态发生蛋白BMP-4对大鼠肝卵圆细胞增殖和分化调控作用的实验研究　出处：《中南大学》2010年博士论文　论文类型：学位论文

【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(p0.05)。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。
[Abstract]:objective
Hepatic oval cells (hepatic oval cell, HOC) is a main body in the liver tissue of pluripotent stem cell proliferation and differentiation, it is an important way to repair liver regeneration and liver tissue of autologous liver injury, which is regulated by a variety of cell growth factors. Bone morphogenetic protein -4 (bone morphogenetic protein-4, BMP-4) is involved in a variety of natural stem cells and transplantation of stem cells to repair the important regulatory factors and treatment of acute and chronic organ damage. The results of our previous study found that BMP-4 can induce the differentiation of rat WB-F344 HOC cells for the liver cell phenotype. But the effect of BMP-4 on the proliferation and differentiation of primary HOC rats and its biological cell signaling mechanism is not clear. The objective of this research is to clear BMP-4 in primary cultured rat HOC proliferation and signal transduction pathway and its effect factor and hepatocyte differentiation, and I We have a comprehensive understanding of the biological function and molecular mechanism of BMP-4 regulating HOC directional differentiation. It will lay a theoretical and applied foundation for the utilization of BMP-4 and its cellular signal transduction mechanism, the induction of HOC differentiation, the promotion of liver regeneration and liver repair, and the treatment of liver injury.
Method
Using 2- N-2-fluorenylacetamide +2 / 3 hepatectomy construct male SD rats HOC proliferation animal model of liver Collagenase perfusion digestion in situ liver IV/Pronase + three Percoll cell density gradient centrifugation of high-purity primary rat HOC cultured in vitro and cultured rat HOC. by MTT method the effect of BMP-4 on rat primary solution generation of HOC proliferation, type I and type II cell receptor RT-PCR in rats were detected the expression of BMP-4 in the cell membrane of HOC rats on mRNA. By real-time quantitative PCR, the main cell transcription of BMP-4 Western hybridization and immunofluorescence staining technique in HOC clear factor Smad1 and ERK 1 / 2 mRNA and protein expression and phosphorylation of protein expression in the nucleus transfer and aggregation, so as to understand the signal transduction mechanism of BMP-4 regulation of HOC. Then RT-PCR was used to detect the HOC of rat hepatocytes and bile duct cells HOC differentiation Cell marker expression of mRNA synthesis of HOC cells after differentiation were detected by automatic biochemical detector and ELISA technology, and cultivate the secretion of urea and albumin concentration to cells were observed by transmission electron microscopy BMP-4 cell ultrastructure of rats after HOC and the connections between cells, so as to understand the BMP-4 of HOC rats to induce effect of liver cell differentiation. And the use of recombinant DNA technology, construction of rat BMP-4 and BMP4 shRNA recombinant adenovirus transfection allogeneic spleen transplantation in rats after HOC, to observe its effect on the recovery of liver function and liver tissues of rats with acute liver injury after CCl4.
Result
The successful establishment of rat HOC proliferation in vivo model of liver perfusion in situ each adult rat separation can get about 5.63 + 0.56 * 106 HOC, rate of 90.32%+3.26%.BMP-4 cell activity in a dose and time dependent manner and promote the cultivation of rat HOC proliferation and the concentration of HOC is 50 ng/ml to BMP-4, then the concentration of BMP-4 after 6 days, the proliferation of HOC can reach the peak of.RT-PCR cell receptor type I confirmed the existence of BMP-4 rats on HOC (BMPR- I) of the two subtypes of BMPR-1A and BMPR-1B, Smad1 and ng/ml expression of.50 cell receptor BMPR- type II BMP-4 the effect of BMP-4 on rat HOC, ERK 1/2 and mRNA the protein expression level has no effect, but can promote protein phosphorylation and to the nucleus transfer and aggregation of.BMP-4 (50ng/ml) treatment of rats after HOC12 days, observed HOC stem cell marker c-kit. bile duct cell marker CK19 and beta 4-integrin The expression of mRNA was significantly reduced or disappeared, and mature liver cell markers TAT-1, A1b and G6Pase mRNA expression was significantly enhanced. At the same time, the BMP-4 treatment group HOC liquid urea concentration in culture (1.32 + 0.32 mmol/L) were Noggin+BMP-4 group (0.61 + 0.09 mmol/L) and control group (0.59 + 0.13 mmol/L). 2.16 times and 2.24 times; BMP-4 treatment group HOC Alb concentration in medium (68.256 + 13.256 ng/ml) were Noggin+BMP-4 group (14.735 + 4.012 ng/ml) and control group (15.897 + 4.283 ng/ml) 4.63 times and 4.29 times, the differences were statistically significant (P0.05). Transmission electron microscopy showed that BMP-4 induced differentiation ultrastructure of HOC and the good connection between adjacent cells, showed that BMP-4 can promote the differentiation of.BMP-4 cells to epithelial cells with good antagonist Noggin can significantly inhibit the proliferation of rat BMP-4 induced by HOC in vitro and to the biological function of liver cell differentiation. Power construction of rat BMP-4 and BMP4 shRNA recombinant adenovirus HOC allogeneic spleen transplantation animal experiment found twelfth days after operation, BMP-4 adenovirus infection HOC transplantation group rat spleen visible after differentiation of HOC liver cells and spleen - liver, liver tissue repair is obvious, and the rats death rate. Hair, spirit, activity, appetite and weight, etc. in general, albumin and alanine aminotransferase (ALT) and liver function index was significantly higher than that of HOC spleen transplantation group, BMP4 shRNA adenovirus infection HOC spleen transplantation group and five in the control group.
conclusion
BMP-4 and HOC of rat BMPR- I and BMPR- II cell receptor binding, and through its cell transcription factor Smad1 and ERK-1 / 2 and protein phosphorylation of signal transduction in the cell, inducing the proliferation of rat HOC in vitro and differentiation into hepatocyte differentiation of.BMP-4 rats with HOC protein synthesis of mature liver cells and secretion, and cell ultrastructure and connections between cells, BMP-4 through HOC allogeneic spleen transplantation promotes rat liver tissue after acute liver injury repair and recovery of liver function.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329

【引证文献】