本文關(guān)鍵詞：骨形態(tài)發(fā)生蛋白BMP-4對(duì)大鼠肝卵圓細(xì)胞增殖和分化調(diào)控作用的實(shí)驗(yàn)研究　出處：《中南大學(xué)》2010年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 肝卵原細(xì)胞 BMP-4 干細(xì)胞 肝損傷 細(xì)胞分化

【摘要】： 目的 肝卵圓細(xì)胞(hepatic oval cell, HOC)是成體肝組織中主要的多能干細(xì)胞,它的增殖與分化是肝損傷后實(shí)現(xiàn)肝再生和肝組織自體修復(fù)的重要途徑,并受到多種細(xì)胞生長因子的調(diào)控。骨形態(tài)發(fā)生蛋白-4 (bone morphogenetic protein-4, BMP-4)是參與多種固有干細(xì)胞和移植干細(xì)胞修復(fù)和治療急慢性器官損傷的重要調(diào)控因子。我們前期研究結(jié)果發(fā)現(xiàn)BMP-4可誘導(dǎo)大鼠HOC細(xì)胞系WB-F344定向分化為肝細(xì)胞表型。但BMP-4對(duì)大鼠原代HOC增殖與分化的影響及其生物細(xì)胞信號(hào)傳導(dǎo)機(jī)制尚不清楚。本課題的研究目的即在于明確BMP-4對(duì)大鼠原代HOC增殖和向肝細(xì)胞定向分化的調(diào)控作用及其細(xì)胞內(nèi)信號(hào)傳導(dǎo)因子和通路,加深我們對(duì)BMP-4調(diào)控HOC定向分化的生物學(xué)功能及其分子機(jī)理的全面了解,為利用BMP-4及其細(xì)胞信號(hào)傳導(dǎo)機(jī)制,定向誘導(dǎo)HOC分化促進(jìn)肝再生和肝修復(fù)、治療肝臟損傷奠定理論和應(yīng)用基礎(chǔ)。 方法 采用2-芴基乙酰胺+2／3肝切除構(gòu)建雄性SD大鼠HOC增殖動(dòng)物模型,Collagenase IV/Pronase肝臟原位灌注消化肝組織+三密度Percoll細(xì)胞梯度離心分離高純度大鼠原代HOC,體外培養(yǎng)和傳代擴(kuò)增大鼠HOC。以MTT法了解BMP-4對(duì)大鼠原代HOC增殖的影響,RT-PCR檢測(cè)大鼠BMP-4的Ⅰ型和Ⅱ型細(xì)胞受體在大鼠HOC細(xì)胞膜上的mRNA表達(dá)情況。采用實(shí)時(shí)熒光定量PCR、Western雜交和免疫熒光染色等技術(shù)明確HOC中BMP-4的主要細(xì)胞轉(zhuǎn)錄因子Smad1和ERK 1／2的mRNA和蛋白表達(dá)水平,以及磷酸化蛋白表達(dá)及其向細(xì)胞核內(nèi)轉(zhuǎn)移和聚集情況,從而了解BMP-4調(diào)控HOC的細(xì)胞信號(hào)傳導(dǎo)機(jī)制。然后以RT-PCR檢測(cè)大鼠HOC、成熟肝細(xì)胞和膽管細(xì)胞等HOC分化細(xì)胞標(biāo)志物的mRNA表達(dá),全自動(dòng)生化檢測(cè)儀和ELISA技術(shù)分別檢測(cè)HOC分化后細(xì)胞合成,并向細(xì)胞培養(yǎng)液中分泌的尿素和白蛋白濃度,透射電子顯微鏡觀察BMP-4處理后大鼠HOC的細(xì)胞超微結(jié)構(gòu)以及細(xì)胞間連接,從而了解BMP-4對(duì)大鼠HOC向肝細(xì)胞定向分化的誘導(dǎo)作用。并利用基因重組技術(shù),構(gòu)建大鼠BMP-4和BMP4 shRNA重組腺病毒,轉(zhuǎn)染大鼠HOC后異體脾臟移植,觀察其對(duì)大鼠CCl4急性肝損傷后肝組織修復(fù)與肝功能恢復(fù)的影響。 結(jié)果 成功建立大鼠HOC體內(nèi)增殖模型,每只成年大鼠肝臟原位灌注分離可獲得大約5.63±0.56×106個(gè)HOC,細(xì)胞活性率為90.32%+3.26%。BMP-4可呈劑量依賴性和時(shí)間依賴性促進(jìn)培養(yǎng)大鼠HOC的增殖,且50 ng/ml是BMP-4理想的HOC作用濃度,以此濃度BMP-4干預(yù)6天后,HOC增殖可達(dá)到峰值。RT-PCR證實(shí)大鼠HOC上存在BMP-4的Ⅰ型細(xì)胞受體(BMPR-Ⅰ)的二個(gè)亞型BMPR-1A和BMPR-1B,以及BMP-4的Ⅱ型細(xì)胞受體BMPR-Ⅱ的表達(dá)。50 ng/ml的BMP-4作用對(duì)大鼠HOC的Smad1、ERK 1/2 mRNA及其蛋白表達(dá)水平無影響,但可促進(jìn)蛋白的磷酸化及其向細(xì)胞核內(nèi)轉(zhuǎn)移和聚集。BMP-4(50ng/ml)處理大鼠HOC12天后,可觀察到HOC干細(xì)胞標(biāo)志物c-kit.膽管細(xì)胞標(biāo)志物CK19和β4-integrin的mRNA表達(dá)顯著降低或消失,而成熟肝細(xì)胞標(biāo)志物TAT-1、A1b和G6Pase的mRNA表達(dá)則顯著增強(qiáng)。同時(shí),BMP-4處理組HOC培養(yǎng)液中尿素濃度(1.32±0.32 mmol/L)分別是Noggin+BMP-4組(0.61±0.09 mmol/L)和空白對(duì)照組(0.59±0.13 mmol/L)的2.16倍和2.24倍；BMP-4處理組HOC培養(yǎng)液中Alb濃度(68.256±13.256 ng/ml)分別是Noggin+BMP-4組(14.735±4.012 ng/ml)和空白對(duì)照組(15.897±4.283 ng/ml)的4.63倍和4.29倍,差異均具有統(tǒng)計(jì)學(xué)意義(p0.05)。透射電子顯微鏡顯示BMP-4誘導(dǎo)分化后HOC的超微結(jié)構(gòu)以及相鄰細(xì)胞間的良好連接,表明BMP-4可促進(jìn)細(xì)胞向上皮細(xì)胞良好分化。BMP-4拮抗劑Noggin可顯著抑制BMP-4誘導(dǎo)大鼠HOC體外增殖和向肝細(xì)胞分化的生物學(xué)作用。成功構(gòu)建大鼠BMP-4和BMP4 shRNA重組腺病毒,HOC異體脾臟移植動(dòng)物實(shí)驗(yàn)發(fā)現(xiàn)術(shù)后第12天,BMP-4腺病毒感染HOC移植組大鼠脾臟可見HOC分化后的肝細(xì)胞定置和“脾化肝”,肝組織修復(fù)明顯,且該組的大鼠死亡率、毛發(fā)、精神、活動(dòng)量、食欲等一般情況,以及體重、白蛋白和谷丙轉(zhuǎn)氨酶(ALT)等肝功能指標(biāo)明顯優(yōu)于HOC脾臟移植組、BMP4 shRNA腺病毒感染HOC脾臟移植組等五個(gè)實(shí)驗(yàn)對(duì)照組。 結(jié)論 BMP-4與大鼠HOC上BMPR-Ⅰ和BMPR-Ⅱ細(xì)胞受體結(jié)合后,可同時(shí)通過其細(xì)胞轉(zhuǎn)錄因子Smad1和ERK-1／2及其蛋白磷酸化實(shí)現(xiàn)細(xì)胞內(nèi)信號(hào)傳導(dǎo),誘導(dǎo)大鼠HOC體外增殖并向肝細(xì)胞定向分化。BMP-4誘導(dǎo)分化的大鼠HOC具有成熟肝細(xì)胞的蛋白合成和分泌功能,以及細(xì)胞超微結(jié)構(gòu)和細(xì)胞間連接,BMP-4可通過HOC異體脾臟移植促進(jìn)大鼠急性肝損傷后的肝組織修復(fù)與肝功能恢復(fù)。
[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.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 陳利鋒;益氣解毒法對(duì)大鼠肝干細(xì)胞系WB-F344作用機(jī)制的研究[D];湖北中醫(yī)藥大學(xué);2012年

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本文編號(hào)：1366911