胚胎干细胞分泌因子促进肝脏纤维化及其作用机制的研究

发布时间：2018-05-01 23:47

本文选题：肝纤维化 + 肝星状细胞　；参考：《浙江大学》2014年博士论文

【摘要】：第一部分胚胎干细胞分泌因子对肝纤维化相关细胞的作用目的：肝纤维化过程中,肝星状细胞(Hepatic stellate cell, HSC)和肝实质细胞在其中起着重要的作用,尤其是肝星状细胞,在肝脏损伤早期被激活,转变为成肌纤维样细胞,分泌细胞外基质蛋白,增加细胞外基质的沉积,抑制其降解。我们研究胚胎干细胞分泌因子对肝星状细胞的作用,有助于了解其对肝纤维化的影响。方法：由于直接移植胚胎干细胞有形成畸胎瘤等危险,我们采用含胚胎干细胞分泌因子的上清培养液,即胚胎干细胞条件培养基(ESC-S),研究其对对肝星状细胞和肝实质细胞的作用。用ESC-S处理细胞后,观察细胞的生长和形态的变化,检测其中基因的改变和蛋白表达的变化情况。结果：胚胎干细胞条件培养液可活化肝星状细胞,促进其显著增殖,细胞中的活化标志物蛋白显α-SMA和desmin著升高。RSC-S还可能通过上皮-间质转化(EMT)来促进肝星状细胞的激活。M1型巨噬细胞的分泌培养液对肝星状细胞有促进作用,而M2c型巨噬细胞的分泌培养液有抑制肝星状细胞活化的作用。ESC-S对肝实质细胞有抑制其生长,诱导其凋亡的作用。结论：胚胎干细胞分泌的细胞因子对肝细胞有正向调节作用,而对肝细胞有损伤作用。第二部分胚胎干细胞分泌因子对小鼠肝纤维化的影响目的：在前面的实验中我们可知ESC-S对肝星状细胞有促进和活化的作用,为进一步明确ESC-S对肝脏纤维化的作用,在小鼠体内研究其对肝脏功能的影响。方法：我们构建了四氯化碳肝纤维化模型,在注射四氯化碳的同时给予小鼠Con-M或ESC-S注射,共持续6周时间。6周后,对肝脏组织进行冰冻切片,同时对这些肝组织切片进行Sirius Red染色,观察两组肝组织中纤维化的程度。为进一步探讨ESC-S对小鼠肝脏的影响,我们将ESC-S通过腹腔注入小鼠体内,每周注射3次,每次注射1ml,持续6周时间。6周后,将小鼠予以安乐死,从下腔静脉取血,并获取肝脏标本。结果：在ESC-S与四氯化碳共同注射的小鼠肝脏中,HSC的活化标志物明显增多,胶原蛋白在细胞外基质中的沉积亦增加。在ESC-S独立处理组小鼠中,血清肝酶升高明显,细胞外基质的沉积也较对照组多。ESC-S处理的小鼠肝脏中有较多的巨噬细胞富集。结论： ESC-S能够启动并导致肝纤维化,富集M1型巨噬细胞到肝内,加速肝脏损伤,而且在原有肝脏损伤的基础上,辅助和加速了肝脏的纤维化。第三部分ESC-S导致肝纤维化的作用机制研究目的：在前期的实验研究表明,ESC-S在体外有促进肝纤维化的潜能,而在动物体内我们发现ESC-S确实有导致肝纤维化的作用。因此,我们研究其可能的作用机制。方法：在体外实验中我们发现,ESC-S可引起钙调磷酸酶(Calcineurin,CN)及相关通路蛋白的下调,我们对钙调磷酸酶基因敲除的小鼠和正常对照的小鼠,采用四氯化碳和胆总管结扎的方法建立两个肝纤维化的模型,研究CN在肝纤维化中的作用。同时我们分析了胚胎干细胞分泌的细胞因子,研究促进肝纤维化的细胞因子和可能的信号通路机制。结果：钙调磷酸酶基因敲除(CN-KO)小鼠比正常对照(WT)小鼠更容易导致肝纤维化,基因敲除小鼠中,细胞外基质中胶原蛋白的沉积明显增多,尤其是1型胶原蛋白,说明钙调磷酸酶在抑制肝纤维化中起着重要作用。通过体外实验,我们发现VEGF在促进肝星状细胞增殖和肝纤维化中起着主要的作用,其一方面通过上调肝星状细胞中TGF-β1的表达,而TGF-β1通过自分泌途径磷酸化星状细胞内的Smad2/3转录因子,活化肝星状细胞,分泌细胞外基质等促肝纤维化的因子；另一方面,肝星状细胞中的钙调磷酸酶下调,增加了STAT3转录因子的总量和磷酸化水平,通过STAT3信号通路激活肝星状细胞。结论：胚胎干细胞分泌因子中的血管内皮生长因子(VEGF)在促进肝纤维化中起着重要的作用,它通过TGF-β1-Smad2/3和Calcineurin-STAT3信号通路在肝脏中起作用。
[Abstract]:Role of first partial embryonic stem cell secretion factor on hepatic fibrosis - related cells

Purpose :

In the process of liver fibrosis , hepatic stellate cells ( HSC ) and liver parenchyma cells play an important role , especially hepatic stellate cells , which are activated in the early stage of liver injury , which are transformed into myofibroblast - like cells , secrete extracellular matrix proteins , increase the deposition of extracellular matrix and inhibit their degradation . We study the role of embryonic stem cell secretion factors on hepatic stellate cells and help to understand their effects on hepatic fibrosis .

Method :

By using ESC - S to treat the cells , the changes of the growth and morphology of the cells were observed , and the changes of the genes and the changes of protein expression were detected .

Results :

Embryonic stem cell conditioned medium can activate hepatic stellate cells and promote their proliferation . The activation of hepatic stellate cells is promoted by epithelial - mesenchymal transition ( EMT ) . The secretion culture medium of M1 type macrophages can promote the activation of hepatic stellate cells .

Conclusion :

The cytokines secreted by embryonic stem cells have a positive regulatory effect on liver cells , and have a damaging effect on liver cells .

Effect of second partial embryonic stem cell secretion factor on liver fibrosis in mice

Purpose :

In the previous experiments , we found that ESC - S plays a role in promoting and activating hepatic stellate cells . To further clarify the effect of ESC - S on liver fibrosis , the effect of ESC - S on liver function is studied in mice .

Method :

In order to further study the effect of ESC - S on the liver of mice , we injected 1 ml of ESC - S into mice by intraperitoneal injection for 6 weeks . After 6 weeks , the mice were euthanized and blood was taken from the inferior vena cava and liver specimens were obtained .

Results :

In the livers of mice co - injected with ESC - S and carbon tetrachloride , the activation markers of HSC increased significantly , and the deposition of collagen in extracellular matrix increased . In ESC - S independent treatment group , the serum hepatic enzymes increased significantly , and the deposition of extracellular matrix was much higher than that of control group .

Conclusion :

ESC - S can activate and cause liver fibrosis , enrich M1 - type macrophages into the liver , accelerate liver injury , and assist and accelerate fibrosis of the liver on the basis of the original liver injury .

Study on the Mechanism of Liver Fibrosis Induced by the Third Part of ESC - S

Purpose :

In previous experimental studies , ESC - S has the potential to promote liver fibrosis in vitro , whereas in animals we find that ESC - S does have the effect of liver fibrosis . Therefore , we study its possible mechanism of action .

Method :

In vitro experiments , we found that ESC - S could induce down - regulation of calcineurin ( CN ) and related pathway proteins . We established two models of hepatic fibrosis by using carbon tetrachloride and common bile duct ligation in mice and normal control mice , and studied the role of CN in liver fibrosis . At the same time , we analyzed the cytokines secreted by embryonic stem cells , and studied cytokines and possible signaling pathways .

Results :

In vitro experiments , we found that VEGF plays an important role in promoting the proliferation of hepatic stellate cells and liver fibrosis . In vitro experiments , we find that VEGF plays a major role in promoting the proliferation of hepatic stellate cells and liver fibrosis .
On the other hand , the downregulation of calcineurin in hepatic stellate cells increases the total amount and phosphorylation level of the transcription factor , and activates the hepatic stellate cells via the STATOR signal pathway .

Conclusion :

Vascular endothelial growth factor ( VEGF ) in the secretion factor of embryonic stem cells plays an important role in promoting liver fibrosis , which plays a role in the liver through the TGF - 尾1 - Smad2 / 3 and Calcineurin - STAT signaling pathways .

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R575.2

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