本文選題：肝細胞肝癌　切入點：丙泊酚　出處：《浙江大學》2015年博士論文　論文類型：學位論文

【摘要】：第一部分丙泊酚抑制肝癌細胞生長和轉(zhuǎn)移的體外研究 研究背景： 丙泊酚是一種常用于腫瘤切除手術的靜脈麻醉藥,近年丙泊酚對腫瘤細胞生長和轉(zhuǎn)移的抑制作用及對腫瘤免疫的影響逐漸受到關注。丙泊酚體外可以直接抑制人宮頸癌、纖維肉瘤、骨肉瘤等細胞系的侵襲能力首先被報道,隨后,體內(nèi)和體外實驗均證明丙泊酚能抑制乳腺癌細胞系的生長和轉(zhuǎn)移,關于食道癌、肺癌等離體細胞研究也證實了丙泊酚的抗癌作用。但是,丙泊酚對肝癌細胞的具體作用目前國內(nèi)外尚無報道。 研究目的： 本研究的目的是明確丙泊酚對肝癌細胞的生長和侵襲能力的具體作用及初步機制。 研究方法： 1.不同濃度的丙泊酚與肝癌HepG2細胞、正常肝L-02細胞共培養(yǎng)。 2.MTT法測定肝癌HepG2細胞、正常肝L-02細胞的細胞活性。 3.酶聯(lián)免疫吸附測定法檢測Caspase8、Caspase9活性表達。 4. Transwell小室進行細胞侵襲試驗。 5.明膠酶譜分析基質(zhì)屬蛋白酶-9的活性。 6. western blot測定MMP-9蛋白表達。 研究結果 1.丙泊酚抑制肝癌細胞HepG2(A)和正常肝細胞L02(B)的生長。其中,正常細胞表現(xiàn)出更強的丙泊酚抗性。 2.丙泊酚誘導肝癌細胞HepG2產(chǎn)生細胞凋亡,并伴隨Caspase-8和Caspase-9活性的顯著增強。而且,HepG2細胞凋亡率的增加與丙泊酚作用濃度呈正相關趨勢。 3.5ug/ml和10ug/m丙泊酚與HepG2細胞共培養(yǎng)后均能顯著降低HepG2細胞的附著力。 4.分別用5、10μg/ml的丙泊酚處理HepG2細胞16小時,MMP-9的活性呈劑量依賴性地降低,在HepG2細胞中的MMP-9的蛋白表達水平明顯降低。 結論： 臨床相關濃度的丙泊酚能夠濃度依賴誘導肝癌細胞HepG2凋亡,可能與激活凋亡蛋白Caspase-8和Caspase-9有關。丙泊酚也能抑制HepG2的侵襲能力,明顯抑制HepG2細胞MMP-9的活性。丙泊酚對腫瘤細胞的作用是多方面的,所以詳細的機制還需要進一步的研究。 第二部分丙泊酚通過上調(diào)巨噬細胞及微泡中miR-142-3p發(fā)揮抗肝癌作用的研究 研究背景：本研究第一部分發(fā)現(xiàn)丙泊酚能夠?qū)﹄x體肝癌細胞的生長和轉(zhuǎn)移發(fā)揮抑制作用,但是這對指導臨床還遠遠不夠,我們進一步研究其作用機制,研究丙泊酚抑制肝癌細胞生長和轉(zhuǎn)移的動物實驗,特別是丙泊酚對機體免疫功能的影響。 巨噬細胞具有異質(zhì)性和多樣性,在不同微環(huán)境信號刺激下,能分化成為生物學功能相反的亞群。腫瘤相關巨噬細胞作為腫瘤免疫微環(huán)境中最豐富的免疫細胞,受到腫瘤細胞的募集與其他基質(zhì)細胞一起共同構成腫瘤免疫微環(huán)境。肝癌患者體內(nèi)巨噬細胞集落刺激因子水平升高可預測肝癌術后轉(zhuǎn)移和復發(fā)情況。 對多種腫瘤的研究表明,miRNA在腫瘤致病過程中起著重要的調(diào)節(jié)作用,miR-142-3p在抑制肝癌細胞遷移和侵襲中起著重要的作用,而增強miR-142-3p的表達可以抑制腫瘤相關巨噬細胞的分化,發(fā)揮抗腫瘤作用。 微泡中負載包括miRNA在內(nèi)的多中生物活性分子,共同組成“信號復合體”。巨噬細胞受到誘導后釋放富含信息的微泡,通過釋放到胞外環(huán)境的這些微泡,將信息物質(zhì)傳遞給周圍及遠處的腫瘤細胞發(fā)揮相應的作用。 已有研究證明丙泊酚可以減少巨噬細胞釋放的活性氧家族,發(fā)揮抗腦膠質(zhì)瘤的作用,本部分研究假設丙泊酚能夠活化腫瘤相關巨噬細胞,上調(diào)miR-142-3p表達并刺激腫瘤相關巨噬細胞分泌富含miR-142-3p的微泡,通過微泡發(fā)揮抑制肝癌細胞生長和侵襲的能力。 研究目的： 本研究假設丙泊酚能夠活化腫瘤相關巨噬細胞,上調(diào)miR-142-3p的表達,并刺激腫瘤相關巨噬細胞分泌微泡,微泡運送miR-142-3p到肝癌細胞,從而抑制肝癌細胞的生長和轉(zhuǎn)移。 實驗方法： 1.建立肝癌細胞系Hepa1-6小鼠皮下移植瘤模型；差速離心分離血漿中MVs,根據(jù)處理藥物的不同對荷瘤小鼠分組,共分8組： (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抑制劑； (8)20mg/kg丙泊酚+miR-142-3p抑制劑。荷瘤小鼠處死后測量腫瘤體積和重量。 2.分離荷瘤小鼠TAMs并與肝癌Hepal-6細胞共培養(yǎng),(?)ranswell小室進行細胞侵襲試驗。 3.實時熒光定量PCR測定各組提取的巨噬細胞、微泡及肝癌Hepa1-6細胞內(nèi)的miR-142-3p含量,使用LipofectamineTM2000轉(zhuǎn)染miR-142-3p抑制劑給肝癌細胞荷瘤小鼠或巨噬細胞后測定miR-142-3p含量。 結果： 1.丙泊酚抑制肝細胞癌荷瘤小鼠腫瘤的生長。 2.丙泊酚處理后的腫瘤組織TAMs可上調(diào)Hepa1-6細胞miR-142-3p的表達。 3.丙泊酚刺激巨噬細胞miR-142-3p表達升高并作用于Hepa1-6細胞。 4.丙泊酚干預后的血漿MVs抑制荷瘤小鼠腫瘤的生長。 5. miR-142-3p參與丙泊酚的抗腫瘤作用。 結論： 總之,我們發(fā)現(xiàn)丙泊酚能通過上調(diào)肝癌相關的巨噬細胞及其分泌的微泡中的miR-142-3p抑制肝癌細胞的生長和轉(zhuǎn)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錛，

本文編號：1557605