DATS和辛伐他汀對骨肉瘤細胞的作用及機制研究
發(fā)布時間：2018-08-07 22:03
【摘要】：第一部分DATS對骨肉瘤細胞的作用及機制研究研究背景骨肉瘤是最常見的起源于骨骼系統(tǒng)的原發(fā)性惡性骨腫瘤,主要發(fā)生于兒童和青少年中。肺轉(zhuǎn)移是骨肉瘤最常見的早期轉(zhuǎn)移部位,也是導(dǎo)致死亡的重要原因。目前骨肉瘤的標準治療方式是術(shù)前的新輔助化療,手術(shù)切除,術(shù)后輔助化療。然而,即使采用了廣泛的外科手術(shù)切除及高強度的輔助化療方案,大約35-55%的初診局限腫瘤患者會出現(xiàn)腫瘤復(fù)發(fā)或轉(zhuǎn)移。而化療耐藥的出現(xiàn)和化療的副作用進一步影響了骨肉瘤治療的效果。因此,需要繼續(xù)開發(fā)新的治療藥物來進一步提高骨肉瘤治療的效果并改善預(yù)后。大蒜素是從大蒜的鱗莖中提取的天然有機硫化物(organosulfur compounds, OSCs),是多種具有揮發(fā)性的烯丙基硫化物的混合物,主要成分包括二烯丙基一硫化物(diallyl sulfide, DAS)、二烯丙基二硫化物(diallyl disulfide, DADS)、二烯丙基三硫化物(diallyl trisulfide, DATS)和大蒜烯(Ajoene)。近年來,流行病學(xué)研究中發(fā)現(xiàn)大蒜的攝入量與某些腫瘤的發(fā)生呈負相關(guān),很多體內(nèi)和體外實驗也證實了大蒜素具有明顯的抗腫瘤活性。在大蒜素的各種活性成分中,DATS顯示出了相比其他幾種成分更強的抗腫瘤特性,是一種潛在的抗癌藥物。既往研究顯示DATS對胃癌、結(jié)腸癌、肺癌、乳腺癌、前列腺癌具有明顯得抗腫瘤作用。然而,DATS對骨肉瘤的作用及機制研究尚不充分。本研究中我們應(yīng)用骨肉瘤細胞系MG63和MNNG/HOS兩種細胞作為研究對象,研究DATS對兩種細胞的作用及可能涉及的分子機制,以期為DATS用于治療骨肉瘤提供了理論依據(jù)。研究目的1.觀察DATS對骨肉瘤細胞的作用。2.探尋DATS抑制骨肉瘤生長的可能作用機制。研究方法1.應(yīng)用不同濃度的DATS處理骨肉瘤細胞MG63和MNNG/HOS 24h,48h,72h后,用CCK-8法檢測骨肉瘤細胞細胞活性,分析DATS對骨肉瘤細胞增殖的影響。2.應(yīng)用不同濃度DATS孵育MG63和MNNG/HOS細胞48h后,倒置顯微鏡下觀察細胞的形態(tài)學(xué)變化,觀察DATS對骨肉瘤細胞的形態(tài)學(xué)影響。3.應(yīng)用細胞周期檢測試劑盒檢測DATS處理骨肉瘤細胞MG63和MNNG/HOS后的細胞周期分布變化,觀察DATS對骨肉瘤細胞周期分布的影響。應(yīng)用Western Blot檢測細胞周期相關(guān)蛋白cyclin D1、p21、p27的表達,分析DATS誘導(dǎo)骨肉瘤細胞G0/G1周期阻滯的可能分子機制。4.應(yīng)用細胞凋亡檢測試劑盒檢測DATS處理骨肉瘤細胞MG63和MNNG/HOS后的細胞細胞凋亡比率,觀察DATS對骨肉瘤細胞凋亡的影響。5.應(yīng)用熒光探針DCFH-DA通過熒光顯微鏡和流式細胞儀檢測DATS誘導(dǎo)MG63和MNNG/HOS細胞內(nèi)活性氧(ROS)變化水平。6.應(yīng)用熒光探針JC-1檢測DATS處理后的骨肉瘤細胞MG63和MNNG/HOS的線粒體膜電位變化。7. DATS處理MG63和MNNG/HOS細胞后,提取總蛋白,應(yīng)用Western Blot檢測PI3K/Akt信號通路中PI3Kp110β、PI3Kp85α、Akt、phosoho-Akt的表達及下游分子Bad、Bax、Bcl-2、Bcl-XL的表達,檢測線粒體凋亡通路中cytochrome c、caspase-9、caspase-3、cleaved PARP蛋白表達變化。分析DATS誘導(dǎo)骨肉瘤細胞凋亡的可能分子機制。實驗結(jié)果1. DATS對骨肉瘤細胞MG63和MNNG/HOS的活性抑制作用隨藥物濃度和處理時間的增加而增強。DATS對MG63細胞活性有著更強的抑制作用。2. DATS處理細胞48h后,導(dǎo)致骨肉瘤細胞發(fā)生明顯的形態(tài)變化。3. DATS誘導(dǎo)MG63和MNNG/HOS細胞發(fā)生G0/G1周期阻滯,抗氧化劑NAC能夠逆轉(zhuǎn)DATS誘導(dǎo)的G0/G1周期阻滯。DATS處理骨肉瘤細胞后cyclin D1蛋白表達下調(diào),而p21和p27蛋白的表達明顯上調(diào)。4. DATS能以劑量和時間依賴的方式誘導(dǎo)骨肉瘤細胞發(fā)生凋亡。NAC預(yù)處理細胞后可逆轉(zhuǎn)DATS誘導(dǎo)的凋亡。5. DATS作用MG63和MNNG/HOS 16h后,細胞內(nèi)ROS水平明顯增加。DATS隨著濃度增加和作用時間延長而明顯增加骨肉瘤細胞內(nèi)ROS水平。NAC則完全消除了ROS的增加。6. DATS作用骨肉瘤細胞后,線粒體膜電位水平發(fā)生明顯下降,NAC對抗DATS對線粒體膜電位的破壞作用。7. DATS作用后骨肉瘤細胞內(nèi)PI3Kp11oβ, PI3Kp85a, Akt和p-Akt的蛋白表達呈現(xiàn)與劑量相關(guān)的下降。同時Bad和Bax表達上調(diào),而Bcl-2和Bcl-XL表達下調(diào),細胞內(nèi)cytochrome c及活化的caspase-9和caspase-3水平,cleaved PARP蛋白表達明顯增加。說明DATS通過下調(diào)PI3K/Akt通路和激活線粒體凋亡通路誘導(dǎo)骨肉瘤細胞凋亡。同時,DATS對PI3K/Akt信號通路起到了PI3K/Akt信號通路抑制劑LY294002的類似作用,而LY294002和DATS共同處理細胞時起到了明顯的協(xié)同抑制作用。NAC完全逆轉(zhuǎn)DATS對PI3K/Akt通路蛋白的下調(diào),說明DATS誘導(dǎo)的骨肉瘤細胞MG63和MNNG/HOS凋亡是依賴于ROS增加導(dǎo)致的PI3K/Akt信號通路下調(diào)起作用的。結(jié)論1. DATS具有明顯的抑制骨肉瘤細胞生長的作用。2. DATS通過增加ROS的產(chǎn)生及下調(diào)cyclin D1、上調(diào)p21和p27蛋白表達而誘導(dǎo)骨肉瘤細胞G0/G1周期阻滯。3. DATS通過誘導(dǎo)ROS產(chǎn)生導(dǎo)致骨肉瘤細胞線粒體膜電位水平下降。4. DATS通過增加ROS增加誘導(dǎo)骨肉瘤細胞凋亡,其分子機制可能與ROS依賴的PI3K/Akt信號通路下調(diào)及線粒體凋亡通路活化有關(guān)。第二部分辛伐他汀對骨肉瘤細胞的作用及機制研究研究背景骨肉瘤是主要發(fā)生于青少年人群的最常見的原發(fā)性惡性骨腫瘤,是青少年腫瘤致死的主要原因之一。即使采用了廣泛的外科手術(shù)切除及新輔助化療方案,目前骨肉瘤的治療效果仍然較差,5年生存率在過去二十年來并未得到明顯的提高。因此,研究或開發(fā)新的治療骨肉瘤的有效藥物迫在眉睫。他汀類藥物是羥甲基戊二酸單酰輔酶A (3-hydroxy-3-methylglutaryl coenzyme A, HMG-CoA)還原酶的競爭性抑制劑,而HMG-CoA還原酶是人體內(nèi)內(nèi)源性膽固醇合成的限速酶。他汀類藥物通過抑制HMG-CoA還原酶的活性,能夠有效的減少細胞內(nèi)膽固醇的合成,從而降低血液中的膽固醇水平。近階段以來,他汀類藥物的抗腫瘤作用獲得了越來越多研究者的注意,并且被證實能夠抑制多種腫瘤的生長,促進其凋亡。在諸多的他汀類藥物中,辛伐他汀在體內(nèi)、體外和動物實驗中顯示出了相較其他同類藥物更強的抗腫瘤特性。之前的體外研究證實,辛伐他汀能有效的抑制卵巢癌、膽管癌、腎癌、前列腺癌和肝癌的生長,其主要的抗腫瘤機制是誘導(dǎo)凋亡及周期阻滯。然而,辛伐他汀對骨肉瘤的作用目前研究較少,其抗骨肉瘤效果及機制文獻報道不多。本研究將通過一系列方法檢測辛伐他汀對骨肉瘤的作用,并探尋可能涉及的分子機制,為辛伐他汀治療骨肉瘤的可能前景提供理論依據(jù)。研究目的1.觀察辛伐他汀對骨肉瘤細胞的作用。2.探尋辛伐他汀抑制骨肉瘤生長的可能信號通路及分子機制。研究方法1.應(yīng)用不同濃度的辛伐他汀處理骨肉瘤細胞MNNG/HOS 24h,48h,72h后,用CCK-8法檢測骨肉瘤細胞細胞活性,分析辛伐他汀對骨肉瘤細胞增殖的影響。2.應(yīng)用細胞劃痕實驗(愈傷實驗)檢測辛伐他汀處理骨肉瘤細胞MNNG/HOS后的細胞遷移距離,觀察辛伐他汀對骨肉瘤細胞遷移能力影響。3.應(yīng)用底面鋪有Matrigel基質(zhì)膠的Transwell小室檢測辛伐他汀對骨肉瘤細胞侵襲能力的影響。應(yīng)用Western Blot檢測基質(zhì)金屬蛋白酶(MMPs) MMP-2、MMP-9的蛋白表達,分析辛伐他汀抑制抑制骨肉瘤細胞遷移侵襲的可能分子機制。4.應(yīng)用細胞周期檢測試劑盒檢測辛伐他汀處理骨肉瘤細胞MNNG/HOS后的細胞周期分布變化,觀察辛伐他汀對骨肉瘤細胞周期分布的影響。應(yīng)用Western Blot檢測細胞周期相關(guān)蛋白cyclin D1、CDK2、CDK4、p21、p27的表達,分析辛伐他汀誘導(dǎo)骨肉瘤細胞GO/G1周期阻滯的可能分子機制。5.應(yīng)用細胞凋亡檢測試劑盒檢測辛伐他汀對骨肉瘤細胞凋亡的影響。應(yīng)用Western Blot檢測PI3K/Akt信號通路中PI3K、Akt、p-Akt的表達及下游的Bax、Bcl-2、cleaved PARP蛋白表達變化。分析辛伐他汀誘導(dǎo)骨肉瘤細胞凋亡的可能分子機制。6.應(yīng)用PI3K/Akt信號通路激活劑IGF-1和特異性抑制劑LY294002及不同濃度的辛伐他汀分別或者共同作用MNNG/HOS細胞后,提取總蛋白,檢測PI3K、Akt、p-Akt的表達及下游的Bax、Bcl-2、cleaved PARP蛋白表達變化,分析PI3K/Akt信號通路在辛伐他汀誘導(dǎo)骨肉瘤細胞凋亡中的作用。7.建立骨肉瘤MNNG/HOS細胞的動物模型,給予腹腔注射辛伐他汀或生理鹽水,定期稱量裸鼠體重,測量計算移植瘤的體積。觀察辛伐他汀對動物模型中骨肉瘤的生長抑制情況。實驗結(jié)果1.辛伐他汀以時間和濃度依賴方式抑制骨肉瘤細胞MNNG/HOS的增殖。2.辛伐他汀抑制骨肉瘤細胞MNNG/HOS的遷移,并呈劑量依賴性。3.辛伐他汀抑制骨肉瘤細胞MNNG/HOS的侵襲,并呈劑量依賴性。同時,MNNG/HOS細胞中的MMP-2和MMP-9蛋白的表達明顯下降,說明辛伐他汀抑制骨肉瘤細胞侵襲的分子機制可能與下調(diào)MMP-2和MMP-9蛋白的表達有關(guān)。4.辛伐他汀誘導(dǎo)MNNG/HOS細胞G0/G1周期阻滯。Western Blot結(jié)果顯示,細胞內(nèi)cyclin D1、CDK2、CDK4蛋白的表達明顯下調(diào),而p21和p27蛋白的表達則被明顯上調(diào),這個結(jié)果解釋了辛伐他汀誘導(dǎo)骨肉瘤細胞G0/G1周期阻滯的可能分子機制。5.辛伐他汀作用后,MNNG/HOS細胞凋亡比率明顯增加并與劑量正相關(guān)。Western blot結(jié)果顯示,辛伐他汀作用后PI3K和p-Akt (Ser 473)蛋白的表達明顯下調(diào),而總Akt的表達沒有顯著變化。同時,Bax的表達增加,而Bcl-2的表達下降,導(dǎo)致Bax/Bcl-2的表達比率明顯上升,下游cleaved PARP的表達增加。6.IGF-1能通過上調(diào)PI3K和p-Akt蛋白的表達而活化PI3K/Akt信號通路,而辛伐他汀與之作用相反,起到了類似于LY294002的通路抑制作用,其下游的Bax/Bcl-2的表達比率也發(fā)生了對應(yīng)的變化。IGF-1與辛伐他汀共同作用MNNG/HOS細胞后辛伐他汀對PI3K/Akt信號通路的抑制作用被逆轉(zhuǎn),證明辛伐他汀誘導(dǎo)凋亡的主要分子機制涉及PI3K/Akt信號通路的失活。7.成功利用裸鼠建立骨肉瘤細胞的動物模型。辛伐他汀對荷瘤裸鼠的體重及日�；顒記]有明顯影響,具有較好的耐受性,辛伐他汀組的移植瘤體積要明顯小于對照組腫瘤體積,證實了辛伐他汀抗骨肉瘤作用的有效性。結(jié)論1.辛伐他汀能夠有效的抑制體外骨肉瘤細胞系的增殖并明顯抑制動物模型中骨肉瘤的生長。2. 辛伐他汀抑制骨肉瘤細胞遷移和侵襲,可能與下調(diào)MMP-2和-9的表達有關(guān)。3.辛伐他汀通過下調(diào)細胞周期調(diào)節(jié)蛋白cyclin D1, CDK2和CDK4表達而上調(diào)p21和p27蛋白表達誘導(dǎo)G0/G1周期阻滯。4.辛伐他汀誘導(dǎo)骨肉瘤細胞凋亡,可能與PI3K/Akt信號通路失活有關(guān)。
[Abstract]:Research background osteosarcoma is the most common primary malignant bone tumor originating in the skeletal system, mainly in children and adolescents. Lung metastasis is the most common early metastasis site of osteosarcoma and is also an important cause of death. The standard treatment of osteosarcoma at present is the standard treatment for osteosarcoma. Neoadjuvant chemotherapy, surgical resection, and postoperative adjuvant chemotherapy. However, even with extensive surgical excision and high intensity adjuvant chemotherapy, tumor recurrence or metastasis will occur in approximately 35-55% of first diagnosed tumor patients. The presence of chemotherapy resistance and the side effects of chemotherapy further affect the treatment of osteosarcoma Therefore, it is necessary to continue to develop new therapeutic drugs to further improve the effect of osteosarcoma treatment and improve the prognosis. The allicin is a natural organic sulfide (organosulfur compounds, OSCs) extracted from the bulbs of garlic, a mixture of many volatile allyl sulfides, the main components including the diallyl sulphide vulcanization. Substances (diallyl sulfide, DAS), diallyl two sulfides (diallyl disulfide, DADS), diallyl three sulfides (diallyl trisulfide, DATS) and allicene (Ajoene). In recent years, epidemiological studies have found that the intake of garlic is negatively related to the occurrence of some tumors, and many in vivo and in vitro experiments have also confirmed that allicin is obvious. Among the various active ingredients of allicin, DATS shows a potential anti-cancer drug that is stronger than several other components. Previous studies have shown that DATS has an obvious antitumor effect on gastric cancer, colon cancer, lung cancer, breast cancer, and prostate cancer. However, the role and mechanism of DATS on osteosarcoma In this study, two cells of osteosarcoma cell line MG63 and MNNG/HOS were used as research objects to study the effect of DATS on two kinds of cells and the possible molecular mechanisms involved, in order to provide a theoretical basis for the use of DATS for the treatment of osteosarcoma. Objective 1. to observe the effect of DATS on osteosarcoma cells by.2. to explore the inhibitory bone of the bone. The possible mechanism of action of sarcoma growth. 1. the use of different concentrations of DATS to treat osteosarcoma cells MG63 and MNNG/HOS 24h, 48h, 72h, CCK-8 method was used to detect the cell activity of osteosarcoma cells, and the effect of DATS on the proliferation of osteosarcoma cells was analyzed. The application of DATS incubating MG63 and MNNG/HOS cells was observed under the inverted microscope. Morphological changes of cells, the morphological changes of osteosarcoma cells were observed by DATS..3. cell cycle detection kit was used to detect the cell cycle distribution changes after DATS treatment of osteosarcoma cells MG63 and MNNG/HOS. The effect of DATS on the cell cycle distribution of osteosarcoma cells was observed. Western Blot was used to detect the cell cycle related proteins cyclin D1, p21, p27. Expression and analysis of the possible molecular mechanism of DATS induced osteosarcoma cell G0/G1 cycle arrest.4. application cell apoptosis detection kit to detect the cell apoptosis ratio after DATS treatment of osteosarcoma cells MG63 and MNNG/HOS, observe the effect of DATS on osteosarcoma cell apoptosis,.5. application fluorescence probe DCFH-DA through fluorescence microscopy and flow cytometry Detection of DATS induced MG63 and MNNG/HOS intracellular reactive oxygen (ROS) change level.6. application fluorescence probe JC-1 to detect the mitochondrial membrane potential changes of MG63 and MNNG/HOS after DATS treatment.7. DATS treatment of MG63 and purified cells. Expression of osoho-Akt and the expression of downstream molecules Bad, Bax, Bcl-2, Bcl-XL, to detect the changes in the expression of cytochrome c, caspase-9, Caspase-3, cleaved PARP protein in the mitochondrial apoptosis pathway. The possible molecular mechanism of apoptosis of osteosarcoma induced osteosarcoma cells was analyzed. Experimental results 1. The increase of concentration and treatment time increased the inhibitory effect of.DATS on the activity of MG63 cells. After.2. DATS processing cell 48h, the osteosarcoma cells had obvious morphological changes..3. DATS induced MG63 and MNNG/HOS cells to induce MG63 and MNNG/HOS cell cycle arrest, and antioxidant NAC could reverse DATS induced periodic block to treat bone meat. After the tumor cells, the expression of cyclin D1 protein was downregulated, while the expression of p21 and p27 protein was obviously up regulated by.4. DATS, which could induce apoptotic.NAC pretreated cells in osteosarcoma cells in a dose and time dependent manner, and could reverse DATS induced apoptotic.5. DATS action MG63 and MNNG/HOS. Prolonging the action time and obviously increasing the intracellular ROS level of osteosarcoma cells.NAC completely eliminated the increase of ROS and the effect of.6. DATS on osteosarcoma cells, the mitochondrial membrane potential decreased significantly, NAC against the mitochondrial membrane potential damage effect of DATS,.7. DATS after.7. DATS, PI3Kp11o beta, PI3Kp85a, Akt, and protein tables The expression of Bad and Bax was up-regulated, while the expression of Bad and Bax was up, while the expression of Bcl-2 and Bcl-XL was down, the level of caspase-9 and caspase-3 activated in the cells and the expression of cleaved PARP protein increased obviously. I3K/Akt signaling pathway played a similar role in the PI3K/Akt signaling pathway inhibitor LY294002, while LY294002 and DATS played a significant synergistic inhibitory effect when.NAC completely reversed the DATS down regulation of the PI3K/Akt pathway protein, indicating that the DATS induced osteosarcoma cell MG63 and MNNG/HOS apoptosis is dependent on ROS increases. Conclusion 1. DATS can inhibit the growth of osteosarcoma cells obviously..2. DATS can induce ROS production and downregulation of cyclin D1, up regulation of p21 and p27 protein expression and induce G0/G1 cycle block of osteosarcoma cells,.3. DATS is induced to lead to the decrease of mitochondrial membrane potential in osteosarcoma cells. DATS induced osteosarcoma cell apoptosis by increasing ROS, its molecular mechanism may be related to the downregulation of ROS dependent PI3K/Akt signaling pathway and the activation of mitochondrial apoptosis pathway. Part second the role and mechanism of simvastatin on osteosarcoma cells Malignant bone tumors are one of the main causes of death in juvenile tumors. Even with extensive surgical resection and neoadjuvant chemotherapy, the treatment effect of osteosarcoma is still poor and the 5 year survival rate has not been significantly improved over the past twenty years. Therefore, the effective drugs for the study or development of new osteosarcoma are forced to The statin is a competitive inhibitor of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and the HMG-CoA reductase is a speed limiting enzyme in the endogenous cholesterol synthesis in the human body. Statins can effectively reduce intracellular cholesterol by inhibiting the activity of HMG-CoA reductase. In recent years, the antitumor effects of statins have gained more and more attention and have been shown to inhibit the growth of various tumors and promote their apoptosis. In many statins, simvastatin has shown more in vivo, in vitro and in animal experiments. Previous in vitro studies have proved that simvastatin can effectively inhibit the growth of ovarian, cholangiocarcinoma, kidney, prostate and liver cancer. The main anti-tumor mechanism is to induce apoptosis and cycle arrest. However, the effect of simvastatin on osteosarcoma is less, and its anti osteosarcoma effect and the effect of simvastatin are less. This study will detect the role of simvastatin on osteosarcoma by a series of methods and explore the possible molecular mechanism to provide a theoretical basis for the possible prospect of simvastatin in the treatment of osteosarcoma. 1. the effect of simvastatin on osteosarcoma cells was observed by.2. exploration of simvastatin for the inhibition of osteosarcoma. Long possible signaling pathways and molecular mechanisms. Method 1. the effects of different concentrations of simvastatin on osteosarcoma cells MNNG/HOS 24h, 48h, 72h were used to detect the cell activity of osteosarcoma cells by CCK-8, and the effects of simvastatin on the proliferation of osteosarcoma cells were analyzed by.2. application cell scratch test (callus test) to detect simvastatin treatment of bone and meat Cell migration distance after tumor cell MNNG/HOS, the effect of simvastatin on osteosarcoma cell migration ability was observed. The effect of simvastatin on the invasion ability of osteosarcoma cells was detected by the Transwell chamber with Matrigel matrix glue at the bottom of.3. application. Western Blot was used to detect the matrix metalloproteinase (MMPs) MMP-2, the expression of MMP-9 protein and the analysis of symplectic protein. The possible molecular mechanism of lovastatin inhibition of osteosarcoma cell migration and invasion.4. application cell cycle detection kit to detect the cell cycle distribution changes after simvastatin treatment of osteosarcoma cell MNNG/HOS, and observe the effect of simvastatin on the cell cycle distribution of osteosarcoma cells. Western Blot was used to detect cell cycle related protein cyclin D1, Expression of CDK2, CDK4, p21, p27, the possible molecular mechanism of GO/G1 cycle arrest induced by simvastatin in osteosarcoma cells.5. application cell apoptosis detection kit to detect the effect of simvastatin on the apoptosis of osteosarcoma cells. Western Blot was used to detect PI3K, Akt, p-Akt, and downstream. Changes in white expression. Analysis of the possible molecular mechanism of simvastatin induced osteosarcoma cell apoptosis.6. use PI3K/Akt signaling pathway activator IGF-1 and specific inhibitor LY294002 and different concentrations of simvastatin, respectively, to extract total protein, and detect the expression of PI3K, Akt, p-Akt, and Bax, Bcl-2, cleave downstream of the MNNG/HOS cells. Changes in the expression of D PARP protein, analysis of the role of PI3K/Akt signaling pathway in the apoptosis of osteosarcoma induced by simvastatin.7. to establish an animal model of osteosarcoma MNNG/HOS cells, give intraperitoneal injection of simvastatin or physiological saline, weigh the body weight of nude mice regularly and measure the volume of the transplanted tumor. Observe the bone meat of simvastatin in animal model Experimental results 1. simvastatin inhibits the proliferation of osteosarcoma cell MNNG/HOS in time and concentration dependent manner,.2. simvastatin inhibits the migration of MNNG/HOS in osteosarcoma cells, and dose dependent.3. simvastatin inhibits the invasion of osteosarcoma cells MNNG/HOS, and is dose-dependent. Meanwhile, MM in MNNG/HOS cells The expression of P-2 and MMP-9 protein decreased obviously. The molecular mechanism of inhibition of osteosarcoma cell invasion by simvastatin may be related to the down-regulation of MMP-2 and MMP-9 protein expression related to.4. simvastatin induced MNNG/HOS cell G0/G1 cycle block.Western Blot results, and the expression of cyclin D1, CDK2, protein expression in cell is obviously down. The expression of white was obviously up-regulated, which explained the possible molecular mechanism of G0/G1 cycle block in osteosarcoma induced by simvastatin. After the action of.5. simvastatin, the apoptosis ratio of MNNG/HOS cells increased significantly and the dose positive correlation.Western blot results showed that the expression of PI3K and p-Akt (Ser 473) protein was obvious after the use of simvastatin. At the same time, the expression of total Akt did not change significantly. At the same time, the expression of Bax increased, while the expression of Bcl-2 decreased, resulting in a significant increase in the expression ratio of Bax/Bcl-2. The expression of.6.IGF-1 in the downstream cleaved PARP can activate the PI3K/Akt signaling pathway by up regulation of the expression of PI3K and p-Akt protein, while simvastatin is the opposite of the action of simvastatin. LY294002 pathway inhibition, the downstream Bax/Bcl-2 expression ratio also has a corresponding change in the inhibitory effect of simvastatin on the PI3K/Akt signaling pathway after the co action of simvastatin and simvastatin. It is proved that the main molecular mechanism of simvastatin induced apoptosis involves the inactivation of the PI3K/Akt signaling pathway in.7. formation. Animal models of osteosarcoma cells were established in nude mice. Simvastatin did not show significant weight and daily activities in nude mice bearing tumor.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R738
[Abstract]:Research background osteosarcoma is the most common primary malignant bone tumor originating in the skeletal system, mainly in children and adolescents. Lung metastasis is the most common early metastasis site of osteosarcoma and is also an important cause of death. The standard treatment of osteosarcoma at present is the standard treatment for osteosarcoma. Neoadjuvant chemotherapy, surgical resection, and postoperative adjuvant chemotherapy. However, even with extensive surgical excision and high intensity adjuvant chemotherapy, tumor recurrence or metastasis will occur in approximately 35-55% of first diagnosed tumor patients. The presence of chemotherapy resistance and the side effects of chemotherapy further affect the treatment of osteosarcoma Therefore, it is necessary to continue to develop new therapeutic drugs to further improve the effect of osteosarcoma treatment and improve the prognosis. The allicin is a natural organic sulfide (organosulfur compounds, OSCs) extracted from the bulbs of garlic, a mixture of many volatile allyl sulfides, the main components including the diallyl sulphide vulcanization. Substances (diallyl sulfide, DAS), diallyl two sulfides (diallyl disulfide, DADS), diallyl three sulfides (diallyl trisulfide, DATS) and allicene (Ajoene). In recent years, epidemiological studies have found that the intake of garlic is negatively related to the occurrence of some tumors, and many in vivo and in vitro experiments have also confirmed that allicin is obvious. Among the various active ingredients of allicin, DATS shows a potential anti-cancer drug that is stronger than several other components. Previous studies have shown that DATS has an obvious antitumor effect on gastric cancer, colon cancer, lung cancer, breast cancer, and prostate cancer. However, the role and mechanism of DATS on osteosarcoma In this study, two cells of osteosarcoma cell line MG63 and MNNG/HOS were used as research objects to study the effect of DATS on two kinds of cells and the possible molecular mechanisms involved, in order to provide a theoretical basis for the use of DATS for the treatment of osteosarcoma. Objective 1. to observe the effect of DATS on osteosarcoma cells by.2. to explore the inhibitory bone of the bone. The possible mechanism of action of sarcoma growth. 1. the use of different concentrations of DATS to treat osteosarcoma cells MG63 and MNNG/HOS 24h, 48h, 72h, CCK-8 method was used to detect the cell activity of osteosarcoma cells, and the effect of DATS on the proliferation of osteosarcoma cells was analyzed. The application of DATS incubating MG63 and MNNG/HOS cells was observed under the inverted microscope. Morphological changes of cells, the morphological changes of osteosarcoma cells were observed by DATS..3. cell cycle detection kit was used to detect the cell cycle distribution changes after DATS treatment of osteosarcoma cells MG63 and MNNG/HOS. The effect of DATS on the cell cycle distribution of osteosarcoma cells was observed. Western Blot was used to detect the cell cycle related proteins cyclin D1, p21, p27. Expression and analysis of the possible molecular mechanism of DATS induced osteosarcoma cell G0/G1 cycle arrest.4. application cell apoptosis detection kit to detect the cell apoptosis ratio after DATS treatment of osteosarcoma cells MG63 and MNNG/HOS, observe the effect of DATS on osteosarcoma cell apoptosis,.5. application fluorescence probe DCFH-DA through fluorescence microscopy and flow cytometry Detection of DATS induced MG63 and MNNG/HOS intracellular reactive oxygen (ROS) change level.6. application fluorescence probe JC-1 to detect the mitochondrial membrane potential changes of MG63 and MNNG/HOS after DATS treatment.7. DATS treatment of MG63 and purified cells. Expression of osoho-Akt and the expression of downstream molecules Bad, Bax, Bcl-2, Bcl-XL, to detect the changes in the expression of cytochrome c, caspase-9, Caspase-3, cleaved PARP protein in the mitochondrial apoptosis pathway. The possible molecular mechanism of apoptosis of osteosarcoma induced osteosarcoma cells was analyzed. Experimental results 1. The increase of concentration and treatment time increased the inhibitory effect of.DATS on the activity of MG63 cells. After.2. DATS processing cell 48h, the osteosarcoma cells had obvious morphological changes..3. DATS induced MG63 and MNNG/HOS cells to induce MG63 and MNNG/HOS cell cycle arrest, and antioxidant NAC could reverse DATS induced periodic block to treat bone meat. After the tumor cells, the expression of cyclin D1 protein was downregulated, while the expression of p21 and p27 protein was obviously up regulated by.4. DATS, which could induce apoptotic.NAC pretreated cells in osteosarcoma cells in a dose and time dependent manner, and could reverse DATS induced apoptotic.5. DATS action MG63 and MNNG/HOS. Prolonging the action time and obviously increasing the intracellular ROS level of osteosarcoma cells.NAC completely eliminated the increase of ROS and the effect of.6. DATS on osteosarcoma cells, the mitochondrial membrane potential decreased significantly, NAC against the mitochondrial membrane potential damage effect of DATS,.7. DATS after.7. DATS, PI3Kp11o beta, PI3Kp85a, Akt, and protein tables The expression of Bad and Bax was up-regulated, while the expression of Bad and Bax was up, while the expression of Bcl-2 and Bcl-XL was down, the level of caspase-9 and caspase-3 activated in the cells and the expression of cleaved PARP protein increased obviously. I3K/Akt signaling pathway played a similar role in the PI3K/Akt signaling pathway inhibitor LY294002, while LY294002 and DATS played a significant synergistic inhibitory effect when.NAC completely reversed the DATS down regulation of the PI3K/Akt pathway protein, indicating that the DATS induced osteosarcoma cell MG63 and MNNG/HOS apoptosis is dependent on ROS increases. Conclusion 1. DATS can inhibit the growth of osteosarcoma cells obviously..2. DATS can induce ROS production and downregulation of cyclin D1, up regulation of p21 and p27 protein expression and induce G0/G1 cycle block of osteosarcoma cells,.3. DATS is induced to lead to the decrease of mitochondrial membrane potential in osteosarcoma cells. DATS induced osteosarcoma cell apoptosis by increasing ROS, its molecular mechanism may be related to the downregulation of ROS dependent PI3K/Akt signaling pathway and the activation of mitochondrial apoptosis pathway. Part second the role and mechanism of simvastatin on osteosarcoma cells Malignant bone tumors are one of the main causes of death in juvenile tumors. Even with extensive surgical resection and neoadjuvant chemotherapy, the treatment effect of osteosarcoma is still poor and the 5 year survival rate has not been significantly improved over the past twenty years. Therefore, the effective drugs for the study or development of new osteosarcoma are forced to The statin is a competitive inhibitor of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and the HMG-CoA reductase is a speed limiting enzyme in the endogenous cholesterol synthesis in the human body. Statins can effectively reduce intracellular cholesterol by inhibiting the activity of HMG-CoA reductase. In recent years, the antitumor effects of statins have gained more and more attention and have been shown to inhibit the growth of various tumors and promote their apoptosis. In many statins, simvastatin has shown more in vivo, in vitro and in animal experiments. Previous in vitro studies have proved that simvastatin can effectively inhibit the growth of ovarian, cholangiocarcinoma, kidney, prostate and liver cancer. The main anti-tumor mechanism is to induce apoptosis and cycle arrest. However, the effect of simvastatin on osteosarcoma is less, and its anti osteosarcoma effect and the effect of simvastatin are less. This study will detect the role of simvastatin on osteosarcoma by a series of methods and explore the possible molecular mechanism to provide a theoretical basis for the possible prospect of simvastatin in the treatment of osteosarcoma. 1. the effect of simvastatin on osteosarcoma cells was observed by.2. exploration of simvastatin for the inhibition of osteosarcoma. Long possible signaling pathways and molecular mechanisms. Method 1. the effects of different concentrations of simvastatin on osteosarcoma cells MNNG/HOS 24h, 48h, 72h were used to detect the cell activity of osteosarcoma cells by CCK-8, and the effects of simvastatin on the proliferation of osteosarcoma cells were analyzed by.2. application cell scratch test (callus test) to detect simvastatin treatment of bone and meat Cell migration distance after tumor cell MNNG/HOS, the effect of simvastatin on osteosarcoma cell migration ability was observed. The effect of simvastatin on the invasion ability of osteosarcoma cells was detected by the Transwell chamber with Matrigel matrix glue at the bottom of.3. application. Western Blot was used to detect the matrix metalloproteinase (MMPs) MMP-2, the expression of MMP-9 protein and the analysis of symplectic protein. The possible molecular mechanism of lovastatin inhibition of osteosarcoma cell migration and invasion.4. application cell cycle detection kit to detect the cell cycle distribution changes after simvastatin treatment of osteosarcoma cell MNNG/HOS, and observe the effect of simvastatin on the cell cycle distribution of osteosarcoma cells. Western Blot was used to detect cell cycle related protein cyclin D1, Expression of CDK2, CDK4, p21, p27, the possible molecular mechanism of GO/G1 cycle arrest induced by simvastatin in osteosarcoma cells.5. application cell apoptosis detection kit to detect the effect of simvastatin on the apoptosis of osteosarcoma cells. Western Blot was used to detect PI3K, Akt, p-Akt, and downstream. Changes in white expression. Analysis of the possible molecular mechanism of simvastatin induced osteosarcoma cell apoptosis.6. use PI3K/Akt signaling pathway activator IGF-1 and specific inhibitor LY294002 and different concentrations of simvastatin, respectively, to extract total protein, and detect the expression of PI3K, Akt, p-Akt, and Bax, Bcl-2, cleave downstream of the MNNG/HOS cells. Changes in the expression of D PARP protein, analysis of the role of PI3K/Akt signaling pathway in the apoptosis of osteosarcoma induced by simvastatin.7. to establish an animal model of osteosarcoma MNNG/HOS cells, give intraperitoneal injection of simvastatin or physiological saline, weigh the body weight of nude mice regularly and measure the volume of the transplanted tumor. Observe the bone meat of simvastatin in animal model Experimental results 1. simvastatin inhibits the proliferation of osteosarcoma cell MNNG/HOS in time and concentration dependent manner,.2. simvastatin inhibits the migration of MNNG/HOS in osteosarcoma cells, and dose dependent.3. simvastatin inhibits the invasion of osteosarcoma cells MNNG/HOS, and is dose-dependent. Meanwhile, MM in MNNG/HOS cells The expression of P-2 and MMP-9 protein decreased obviously. The molecular mechanism of inhibition of osteosarcoma cell invasion by simvastatin may be related to the down-regulation of MMP-2 and MMP-9 protein expression related to.4. simvastatin induced MNNG/HOS cell G0/G1 cycle block.Western Blot results, and the expression of cyclin D1, CDK2, protein expression in cell is obviously down. The expression of white was obviously up-regulated, which explained the possible molecular mechanism of G0/G1 cycle block in osteosarcoma induced by simvastatin. After the action of.5. simvastatin, the apoptosis ratio of MNNG/HOS cells increased significantly and the dose positive correlation.Western blot results showed that the expression of PI3K and p-Akt (Ser 473) protein was obvious after the use of simvastatin. At the same time, the expression of total Akt did not change significantly. At the same time, the expression of Bax increased, while the expression of Bcl-2 decreased, resulting in a significant increase in the expression ratio of Bax/Bcl-2. The expression of.6.IGF-1 in the downstream cleaved PARP can activate the PI3K/Akt signaling pathway by up regulation of the expression of PI3K and p-Akt protein, while simvastatin is the opposite of the action of simvastatin. LY294002 pathway inhibition, the downstream Bax/Bcl-2 expression ratio also has a corresponding change in the inhibitory effect of simvastatin on the PI3K/Akt signaling pathway after the co action of simvastatin and simvastatin. It is proved that the main molecular mechanism of simvastatin induced apoptosis involves the inactivation of the PI3K/Akt signaling pathway in.7. formation. Animal models of osteosarcoma cells were established in nude mice. Simvastatin did not show significant weight and daily activities in nude mice bearing tumor.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R738
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