丙泊酚对肝细胞肝癌生长和转移的抑制作用及机制研究

发布时间：2018-03-02 18:09

  本文选题：肝细胞肝癌　切入点：丙泊酚　出处：《浙江大学》2015年博士论文　论文类型：学位论文

【摘要】：第一部分丙泊酚抑制肝癌细胞生长和转移的体外研究 研究背景： 丙泊酚是一种常用于肿瘤切除手术的静脉麻醉药,近年丙泊酚对肿瘤细胞生长和转移的抑制作用及对肿瘤免疫的影响逐渐受到关注。丙泊酚体外可以直接抑制人宫颈癌、纤维肉瘤、骨肉瘤等细胞系的侵袭能力首先被报道,随后,体内和体外实验均证明丙泊酚能抑制乳腺癌细胞系的生长和转移,关于食道癌、肺癌等离体细胞研究也证实了丙泊酚的抗癌作用。但是,丙泊酚对肝癌细胞的具体作用目前国内外尚无报道。 研究目的： 本研究的目的是明确丙泊酚对肝癌细胞的生长和侵袭能力的具体作用及初步机制。 研究方法： 1.不同浓度的丙泊酚与肝癌HepG2细胞、正常肝L-02细胞共培养。 2.MTT法测定肝癌HepG2细胞、正常肝L-02细胞的细胞活性。 3.酶联免疫吸附测定法检测Caspase8、Caspase9活性表达。 4. Transwell小室进行细胞侵袭试验。 5.明胶酶谱分析基质属蛋白酶-9的活性。 6. western blot测定MMP-9蛋白表达。 研究结果 1.丙泊酚抑制肝癌细胞HepG2(A)和正常肝细胞L02(B)的生长。其中,正常细胞表现出更强的丙泊酚抗性。 2.丙泊酚诱导肝癌细胞HepG2产生细胞凋亡,并伴随Caspase-8和Caspase-9活性的显著增强。而且,HepG2细胞凋亡率的增加与丙泊酚作用浓度呈正相关趋势。 3.5ug/ml和10ug/m丙泊酚与HepG2细胞共培养后均能显著降低HepG2细胞的附着力。 4.分别用5、10μg/ml的丙泊酚处理HepG2细胞16小时,MMP-9的活性呈剂量依赖性地降低,在HepG2细胞中的MMP-9的蛋白表达水平明显降低。 结论： 临床相关浓度的丙泊酚能够浓度依赖诱导肝癌细胞HepG2凋亡,可能与激活凋亡蛋白Caspase-8和Caspase-9有关。丙泊酚也能抑制HepG2的侵袭能力,明显抑制HepG2细胞MMP-9的活性。丙泊酚对肿瘤细胞的作用是多方面的,所以详细的机制还需要进一步的研究。 第二部分丙泊酚通过上调巨噬细胞及微泡中miR-142-3p发挥抗肝癌作用的研究 研究背景：本研究第一部分发现丙泊酚能够对离体肝癌细胞的生长和转移发挥抑制作用,但是这对指导临床还远远不够,我们进一步研究其作用机制,研究丙泊酚抑制肝癌细胞生长和转移的动物实验,特别是丙泊酚对机体免疫功能的影响。 巨噬细胞具有异质性和多样性,在不同微环境信号刺激下,能分化成为生物学功能相反的亚群。肿瘤相关巨噬细胞作为肿瘤免疫微环境中最丰富的免疫细胞,受到肿瘤细胞的募集与其他基质细胞一起共同构成肿瘤免疫微环境。肝癌患者体内巨噬细胞集落刺激因子水平升高可预测肝癌术后转移和复发情况。 对多种肿瘤的研究表明,miRNA在肿瘤致病过程中起着重要的调节作用,miR-142-3p在抑制肝癌细胞迁移和侵袭中起着重要的作用,而增强miR-142-3p的表达可以抑制肿瘤相关巨噬细胞的分化,发挥抗肿瘤作用。 微泡中负载包括miRNA在内的多中生物活性分子,共同组成“信号复合体”。巨噬细胞受到诱导后释放富含信息的微泡,通过释放到胞外环境的这些微泡,将信息物质传递给周围及远处的肿瘤细胞发挥相应的作用。 已有研究证明丙泊酚可以减少巨噬细胞释放的活性氧家族,发挥抗脑胶质瘤的作用,本部分研究假设丙泊酚能够活化肿瘤相关巨噬细胞,上调miR-142-3p表达并刺激肿瘤相关巨噬细胞分泌富含miR-142-3p的微泡,通过微泡发挥抑制肝癌细胞生长和侵袭的能力。 研究目的： 本研究假设丙泊酚能够活化肿瘤相关巨噬细胞,上调miR-142-3p的表达,并刺激肿瘤相关巨噬细胞分泌微泡,微泡运送miR-142-3p到肝癌细胞,从而抑制肝癌细胞的生长和转移。 实验方法： 1.建立肝癌细胞系Hepa1-6小鼠皮下移植瘤模型；差速离心分离血浆中MVs,根据处理药物的不同对荷瘤小鼠分组,共分8组： (1)20mg/kg丙泊酚； (2)100μg/kg clodrolip; (3)20mg/kg丙泊酚+100μg/kg clodrolip; (4)50mg/kg丙泊酚； (5)50mg/kg丙泊酚+100μg/kg clodrolip; (6)20μg MVs; (7) miR-142-3p抑制剂； (8)20mg/kg丙泊酚+miR-142-3p抑制剂。荷瘤小鼠处死后测量肿瘤体积和重量。 2.分离荷瘤小鼠TAMs并与肝癌Hepal-6细胞共培养,(?)ranswell小室进行细胞侵袭试验。 3.实时荧光定量PCR测定各组提取的巨噬细胞、微泡及肝癌Hepa1-6细胞内的miR-142-3p含量,使用LipofectamineTM2000转染miR-142-3p抑制剂给肝癌细胞荷瘤小鼠或巨噬细胞后测定miR-142-3p含量。 结果： 1.丙泊酚抑制肝细胞癌荷瘤小鼠肿瘤的生长。 2.丙泊酚处理后的肿瘤组织TAMs可上调Hepa1-6细胞miR-142-3p的表达。 3.丙泊酚刺激巨噬细胞miR-142-3p表达升高并作用于Hepa1-6细胞。 4.丙泊酚干预后的血浆MVs抑制荷瘤小鼠肿瘤的生长。 5. miR-142-3p参与丙泊酚的抗肿瘤作用。 结论： 总之,我们发现丙泊酚能通过上调肝癌相关的巨噬细胞及其分泌的微泡中的miR-142-3p抑制肝癌细胞的生长和转移。
[Abstract]:The first part of the study on the inhibition of the growth and metastasis of hepatoma cells by propofol
Research background:
Propofol is a commonly used tumor resection in propofol intravenous anesthetics, inhibition of tumor cell growth and metastasis and effect on tumor immunity has gradually attracted attention. Propofol can directly inhibit the in vitro human cervical cancer, fibrosarcoma, osteosarcoma cell line invasion was first reported, then, in vivo and in vitro studies that propofol can inhibit breast cancer cell growth and metastasis of esophageal cancer, lung cancer, and in vitro studies have also confirmed the antitumor effect of propofol. However, propofol on liver cancer cell specific role has not been reported at home and abroad.
The purpose of the study is:
The purpose of this study is to clarify the specific role and preliminary mechanism of propofol on the growth and invasion of hepatoma cells.
Research methods:
1. the different concentrations of propofol were co cultured with liver cancer HepG2 cells and normal liver L-02 cells.
2.MTT assay was used to determine the cell activity of liver cancer HepG2 cells and normal liver L-02 cells.
3. enzyme linked immunosorbent assay was used to detect the expression of Caspase8 and Caspase9.
Cell invasion test was carried out in 4. Transwell compartment.
5. the activity of matrix protease -9 was analyzed by the gelatinase spectrum.
The expression of MMP-9 protein was measured by 6. Western blot.
Research results
1. propofol inhibits the growth of HepG2 (A) and normal hepatocytes, L02 (B), in which normal cells show stronger propofol resistance.
2. propofol induced the apoptosis of HepG2 cells, and significantly enhanced the activity of Caspase-8 and Caspase-9. Moreover, the increase of apoptosis rate of HepG2 cells was positively correlated with the concentration of propofol.
After co culture of 3.5ug/ml and 10ug/m propofol and HepG2 cells, the adhesion of HepG2 cells was significantly reduced.
4., HepG2 cells were treated with propofol of 5,10 g/ml for 16 hours. The activity of MMP-9 decreased in a dose-dependent manner, and the expression level of MMP-9 protein in HepG2 cells was significantly reduced.
Conclusion:
Clinically relevant concentrations of propofol concentration dependent can induce the apoptosis of HepG2 cells may be related to the activation of apoptosis protein Caspase-8 and Caspase-9. Propofol could also inhibit the invasion ability of HepG2 and HepG2 cells was obviously inhibited the activity of MMP-9. The effect of propofol on tumor cells is various, so the detailed mechanism needs to be further investigated.
The second part of propofol plays an anti hepatoma effect by raising miR-142-3p in macrophages and microbubbles
Background: the first part of this study found that propofol can inhibit the in vitro liver cancer cell growth and metastasis, but the clinical guidance is not enough, we further study the mechanism of action, animal experiment study of propofol inhibits the growth and metastasis of hepatocellular carcinoma cells, especially the effects of propofol on immune function.
Macrophage heterogeneity and diversity in different micro environmental stimuli, can differentiate into the biological function of the opposite. A subgroup of tumor associated macrophages as the most abundant immune cells in the tumor microenvironment, tumor cells were raised with other stromal cells together constitute the tumor microenvironment in vivo macrophage colony in patients with hepatocellular carcinoma. Stimulating factor increased level of metastasis and recurrence after resection of hepatocellular carcinoma.
Study on tumor showed that miRNA plays an important role in tumor pathogenesis, miR-142-3p plays an important role in the inhibition of cell migration and invasion of hepatocellular carcinoma, and enhance the expression of miR-142-3p can inhibit the differentiation of tumor associated macrophages played an important role in anti-tumor.
Micro bubble load including miRNA of bioactive molecules, to form a signaling complex. By macrophages microbubble induced release of rich information, through the release to the extracellular environment of these microbubbles, the information of the material transfer to the surrounding and distant tumor cells play a relevant role.
Studies have demonstrated that propofol can reduce the ROS family release of macrophages, play the anti glioma effect, this part of the research hypothesis of propofol can activate tumor associated macrophages, microbubbles upregulate the expression of miR-142-3p and stimulate the secretion of tumor associated macrophages with miR-142-3p, through the micro bubble suppression ability of growth and invasion of hepatocellular carcinoma cells.
The purpose of the study is:
This study assumes that propofol can activate tumor associated macrophages, up regulate the expression of miR-142-3p, and stimulate tumor associated macrophages to secrete microbubbles. Microbubbles transport miR-142-3p to hepatocellular carcinoma cells, thereby inhibiting the growth and metastasis of hepatoma cells.
Experimental methods:
1., a subcutaneous transplantation tumor model of hepatocellular carcinoma cell line Hepa1-6 was established. The MVs of plasma was separated by differential centrifugation. The tumor bearing mice were grouped into 8 groups according to the different drugs.
(1) 20mg/kg propofol;
(2) 100 g/kg clodrolip;
(3) 20mg/kg propofol +100 g/kg clodrolip;
(4) 50mg/kg propofol;
(5) 50mg/kg propofol +100 g/kg clodrolip;
(6) 20 g MVs;
(7) miR-142-3p inhibitors;
(8) 20mg/kg propofol +miR-142-3p inhibitor. The tumor bearing mice were killed and the tumor volume and weight were measured.
2. TAMs was isolated from the tumor bearing mice and co cultured with Hepal-6 cells of liver cancer. The cell invasion test was carried out in the ranswell compartment.
3. real-time fluorescence quantitative PCR was used to measure the miR-142-3p content in macrophages, microbubbles and Hepa1-6 cells of each group. LipofectamineTM2000 was transfected with miR-142-3p inhibitor to detect miR-142-3p content in hepatoma cells, tumor bearing mice or macrophages.
Result锛，

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