本文選題：氯硝柳胺 + 腎上腺皮質(zhì)癌��； 參考：《第二軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：研究背景:腎上腺皮質(zhì)癌(Adrenocortical carcinoma,ACC)是一種來(lái)源于腎上腺皮質(zhì)的上皮性惡性腫瘤,臨床罕見(jiàn),每年的發(fā)病率約為1-2/100萬(wàn)人,發(fā)病多見(jiàn)于兩個(gè)年齡高峰,5歲的兒童和50歲左右的成人,平均發(fā)病年齡45歲,女性發(fā)病率略高于男性。ACC的病理生理機(jī)制仍未能明確,目前認(rèn)為ACC的病理是一個(gè)多因素參與的復(fù)雜過(guò)程,染色體畸變、基因突變、DNA的甲基化,胰島素樣生長(zhǎng)因子(IGF)和Wnt/β-catenin信號(hào)通路的功能異常都對(duì)ACC的發(fā)生發(fā)展起重要作用。ACC惡性程度高,多數(shù)腫瘤直徑5cm,平均10cm,瘤體內(nèi)多伴有出血及壞死,病程進(jìn)展迅速且隱匿,大多數(shù)患者就診時(shí)已屬癌癥晚期,伴發(fā)有淋巴或遠(yuǎn)處器官轉(zhuǎn)移,最常見(jiàn)于肺、肝、腹膜后淋巴結(jié)和骨轉(zhuǎn)移。ACC的臨床表現(xiàn)由腫瘤的功能和體積決定,大多數(shù)成人ACC具有內(nèi)分泌功能,分泌皮質(zhì)醇和性激素的庫(kù)欣綜合癥(Cushing'syndrome,CS)以及男性化在臨床中最為常見(jiàn),約占ACC的40%,單純CS和男性化表現(xiàn)分別占30%和20%,女性化約占10%,分泌醛固酮的ACC臨床中較為少見(jiàn),約2%。兒童ACC90%以上具有內(nèi)分泌功能,臨床表現(xiàn)多為男性化或假青春期表現(xiàn),其中以分泌雄激素的腫瘤最為多見(jiàn),約占兒童ACC的55%以上,混合分泌皮質(zhì)醇和雄激素的占30%,單純分泌皮質(zhì)醇的腫瘤較少見(jiàn),不足5%。非功能性的ACC因起病隱匿,一般臨床表現(xiàn)多以腫瘤進(jìn)展后引起的腹部癥狀為主。影像學(xué)和內(nèi)分泌檢查是臨床診斷ACC的主要手段。目前ACC的治療由于缺乏特效的化療和靶向治療藥物,手術(shù)仍是其主要的治療手段,但30%-85%患者就診時(shí)腫瘤已處于進(jìn)展期,伴發(fā)有淋巴或其它臟器轉(zhuǎn)移,無(wú)法進(jìn)行有效的手術(shù)切除或根治性手術(shù)切除,平均生存期僅為3～9個(gè)月,大部分生存時(shí)間不超過(guò)1年,5年生存率不到15%,即使手術(shù)治療后的患者5年生存率也不足30%。所以對(duì)于無(wú)法進(jìn)行手術(shù)或無(wú)法進(jìn)行根治性手術(shù)的患者仍須考慮藥物治療,或是術(shù)后輔以藥物治療來(lái)延長(zhǎng)期生存時(shí)間。ACC藥物治療國(guó)外推薦使用米托坦,但其有效性及安全性仍存在爭(zhēng)議。EDPM(順鉑、依托泊甙、多柔比星、米托坦)和Sz/m(鏈霉素、米托擔(dān))是米托坦聯(lián)合細(xì)胞毒性藥物作為進(jìn)展期ACC的一線治療方案,但總的療效也非常有限。放療、射頻消融、介入栓塞只能使少數(shù)患者受益,因此為了有效提高ACC患者的生存率,手術(shù)聯(lián)合藥物治療仍是一項(xiàng)重要的研究課題。由于ACC對(duì)傳統(tǒng)的化療藥物不敏感,所以尋找有效的、安全的、高選擇性藥物也是當(dāng)今研究ACC治療的重點(diǎn)。氯硝柳胺(Niclosamide),又名滅絳靈,化學(xué)名:N-(2'-氯-4'-硝基苯)-5-氯水楊酰胺,分子式C13H8C12N2O4,臨床中用來(lái)治療人腸道寄生蟲(chóng)病,是美國(guó)食品藥品監(jiān)督管理局(FDA)推薦治療蠕蟲(chóng)的藥物。作用機(jī)制通過(guò)抑制蟲(chóng)體細(xì)胞內(nèi)氧化磷酸化過(guò)程和線粒體功能、阻斷NADPH向NADP+的轉(zhuǎn)化、抑制ATP的產(chǎn)生、改變細(xì)胞能量代謝發(fā)揮治療作用。近年來(lái),在抗腫瘤藥物的的研發(fā)過(guò)程中,通過(guò)大量的藥物篩選實(shí)驗(yàn)發(fā)現(xiàn)氯硝柳胺還具有潛在的抗腫瘤作用,相對(duì)于新研究合成的抗腫瘤藥物,氯硝柳胺具有細(xì)胞毒性強(qiáng),人體毒副作用小的優(yōu)勢(shì)。WANG于2009報(bào)道氯硝柳胺可以抑制白血病細(xì)胞K562靶基因蛋白的表達(dá),隨后其他學(xué)者在研究中發(fā)現(xiàn)氯硝柳胺可以通過(guò)影響 Wnt/β-catenin、mTORC1、STAT3、NF-κB、Notch、Hedgehog 等信號(hào)通路和腫瘤細(xì)胞內(nèi)線粒體、溶酶體來(lái)產(chǎn)生抑制腫瘤細(xì)胞增殖和誘導(dǎo)凋亡的作用,進(jìn)一步研究發(fā)現(xiàn)氯硝柳胺不僅作用于腫瘤細(xì)胞,還可以作用于擁有自我更新能力并能產(chǎn)生異質(zhì)性細(xì)胞的腫瘤干細(xì)胞。除此之外,在另一些研究中還發(fā)現(xiàn),氯硝柳胺能夠增加腫瘤細(xì)胞對(duì)化療和放療的敏感性,甚至可以逆轉(zhuǎn)腫瘤細(xì)胞對(duì)化療和放療的耐受性。氯硝柳胺對(duì)正常細(xì)胞的能量代謝和信號(hào)傳導(dǎo)影響甚微,作為潛在的抗腫瘤藥物展現(xiàn)出良好的應(yīng)用前景。研究目的:1.研究氯硝柳胺對(duì)人腎上腺皮質(zhì)癌SW-13細(xì)胞株細(xì)胞增殖、周期、凋亡和遷移力、侵襲力的影響并初步探討其機(jī)制。2.研究氯硝柳胺對(duì)腎上腺皮質(zhì)癌SW-13細(xì)胞裸鼠皮下移植瘤生長(zhǎng)的抑制作用和機(jī)制。研究方法:1.通過(guò)體外培養(yǎng)SW-13細(xì)胞,采用CCK-8法和iCELLigence實(shí)時(shí)無(wú)標(biāo)記細(xì)胞增殖監(jiān)測(cè)儀檢測(cè)不同濃度氯硝柳胺對(duì)SW-13細(xì)胞增殖的影響。2.采用流式細(xì)胞技術(shù)檢測(cè)氯硝柳胺作用SW-13細(xì)胞后對(duì)細(xì)胞周期分布率的影響。3.采用AO/EB染色法和Annexin V/PI染色流式細(xì)胞技術(shù)檢測(cè)氯硝柳胺作用SW-13細(xì)胞后對(duì)細(xì)胞凋亡率的影響。4.采用Transwell實(shí)驗(yàn)和細(xì)胞劃痕實(shí)驗(yàn)檢測(cè)氯硝柳胺作用SW-13細(xì)胞后對(duì)細(xì)胞侵襲力、遷移能力的影響。5.采用Western Blotting檢測(cè)氯硝柳胺作用SW-13細(xì)胞后對(duì)β-catenin、LRP6、vimentin、E-cadherin 表達(dá)的影響。6.建立SW-13細(xì)胞裸鼠移植瘤模型,用隨機(jī)數(shù)字表法設(shè)對(duì)照組、DMSO組,氯硝柳胺低濃度組、氯硝柳胺高濃度組。經(jīng)腹腔注藥,動(dòng)態(tài)監(jiān)測(cè)瘤體直徑、瘤重,觀察氯硝柳胺作用裸鼠后對(duì)腫瘤體積和瘤重的影響,并計(jì)算生長(zhǎng)抑制率。部分瘤體行HE染色,鏡下觀察組織形態(tài)。7.設(shè)順鉑組為陽(yáng)性對(duì)照,各組裸鼠處死前通過(guò)眼眶內(nèi)眥靜脈采血,對(duì)血液樣本進(jìn)行血細(xì)胞、生化分析。8.采用TUNEL法檢測(cè)氯硝柳胺作用裸鼠后對(duì)移植瘤組織細(xì)胞凋亡率的影響。9.采用免疫組織化學(xué)染色法檢測(cè)氯硝柳胺作用裸鼠后對(duì)移植瘤組織中β-catenin、LRP6、vimentin、E-cadherin、Bc 1-2,caspase-3 表達(dá)的影響。結(jié)果:1.不同濃度的氯硝柳胺分別作用SW-13細(xì)胞24、48、72、96、120h后,呈時(shí)間和濃度依賴性抑制SW-13細(xì)胞增殖(F=157.93,P0.001)。2.流式細(xì)胞儀細(xì)胞周期檢測(cè)結(jié)果顯示24、48、72h后,與對(duì)照組相比氯硝柳胺組將SW-13 細(xì)胞周期阻滯在 G0/G1 期(P=0.001、0.005、0.008)。3.AO/EB染色法和Annexin V/PI染色法結(jié)果均顯示氯硝柳胺組的凋亡率高于照組(均 P0.001)。4.Transwell實(shí)驗(yàn)結(jié)果示氯硝柳胺組SW-13細(xì)胞的侵襲力低于對(duì)照組(P0.001),細(xì)胞劃痕實(shí)驗(yàn)結(jié)果顯示氯硝柳胺組SW-13細(xì)胞的遷移能力低于對(duì)照組(P=0.002)。5.Western Blotting 結(jié)果示氯硝柳胺組 SW-13 細(xì)胞 β-catenin、LRP6、vimentin 的表達(dá)低于對(duì)照組(均P0.001),而E-cadherin的表達(dá)高于對(duì)照組(P=0.004)。6.HE染色組織切片證實(shí)成功建立SW-13細(xì)胞裸鼠移植瘤模型,氯硝柳胺低濃度組、氯硝柳胺高濃度組的終末腫瘤體積和瘤重,低于與對(duì)照組(均P0.001),對(duì)照組與DMSO組差異無(wú)統(tǒng)計(jì)學(xué)意義(均P0.05)。7.順鉑組的白細(xì)胞低于氯硝柳胺組、對(duì)照組(均P0.001),氯硝柳胺組與對(duì)照組比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。氯硝柳胺組、順鉑組的紅細(xì)胞和血小板與對(duì)照組之間比較差異無(wú)統(tǒng)計(jì)學(xué)意義(均P0.05)。順鉑組的尿素氮、肌酐、總膽紅素、谷丙轉(zhuǎn)氨酶均高于氯硝柳胺組、對(duì)照組(均P0.001),氯硝柳胺組與對(duì)照之間比較差異無(wú)統(tǒng)計(jì)學(xué)意義(均P0.05)。8.移植瘤組織TUNEL檢測(cè)結(jié)果示氯硝柳胺低濃度組、氯硝柳胺高濃度組的細(xì)胞凋亡率高于對(duì)照組(P=0.004,P=0.005)。9.移植瘤組織免疫組織化學(xué)染色結(jié)果顯示氯硝柳胺低濃度組、氯硝柳胺高濃度組的β-catenin、LRP6、vimentin、Bcl-2 的表達(dá)低于對(duì)照組(均 P0.001),E-cadherin、caspase-3的表達(dá)高于對(duì)照組(均P0.001)。結(jié)論:1.氯硝柳胺呈時(shí)間-濃度依賴性抑制人腎上腺皮質(zhì)癌SW-13細(xì)胞增殖,阻滯細(xì)胞周期,抑制侵襲力和遷移能力,促進(jìn)細(xì)胞凋亡;抑制SW-13細(xì)胞裸鼠皮下移植瘤生長(zhǎng),其機(jī)制可能涉及Wnt/β-catenin信號(hào)通路,上皮間質(zhì)轉(zhuǎn)化(EMT)和凋亡相關(guān)蛋白表達(dá)異常。2.氯硝柳胺對(duì)血細(xì)胞、肝腎功能的影響小于順鉑。
[Abstract]:Background: Adrenocortical carcinoma (ACC) is an epithelial malignant tumor of the adrenal cortex. It is rare in the adrenal cortex. The incidence of the adrenal cortical carcinoma is about 1-2/100 million per year. The incidence of adrenal cortical cancer is about two age peaks, 5 years old and 50 years old, with an average age of 45 years. The incidence of women is slightly higher than that of male.ACC. The pathophysiological mechanism is still not clear. At present, the pathology of ACC is a complex process involving multiple factors. Chromosomal aberration, gene mutation, DNA methylation, insulin like growth factor (IGF) and Wnt/ beta -catenin signaling pathway are important for the development of ACC, and the malignant degree of.ACC is high, and most of the tumor diameter 5cm is 5cm. The average 10cm, with more bleeding and necrosis in the tumor, the course of the disease progresses rapidly and occult. Most patients have advanced cancer, with lymphatic or distant organ metastasis. The most common clinical manifestations of the lung, liver, retroperitoneal lymph nodes and bone metastases are determined by the ability and volume of the tumor, and most of the adult ACC has endocrine function. Cushing'syndrome (Cushing'syndrome, CS), which secretes cortisol and sex hormones, is the most common in the clinic, accounting for about 40% of ACC, 30% and 20% for simple CS and masculine, 10% in feminization, and rare in the ACC secreting aldosterone. About 2%. children have endocrine function above ACC90%, and the clinical manifestations are mostly male. It is the most common manifestation of the androgenic hormone, accounting for more than 55% of the children's ACC, 30% of the mixed secretion of cortisol and androgens, and a rare tumor that secretes cortisol only, and the 5%. nonfunctional ACC is concealed because of the onset of the disease, and the general clinical manifestations are mainly abdominal symptoms after the tumor progresses. Study and endocrine examination are the main means of clinical diagnosis of ACC. Currently, the treatment of ACC is still the main treatment because of the lack of effective chemotherapy and targeted therapy, but the tumor in 30%-85% patients is in progress, with lymph or other organ metastasis, and no effective surgical resection or radical operation can be carried out. The average survival time is only 3~9 months, most of the survival time is not more than 1 years, and the 5 year survival rate is less than 15%. Even if the 5 year survival rate of the patients after the operation is less than 30%., the patients who are unable to perform the operation or do not have a radical operation still have to consider the drug treatment or the postoperative adjuvant treatment to prolong the long-term survival. Inter.ACC drug therapy is recommended for the use of mitoxan, but its effectiveness and safety are still controversial.EDPM (cisplatin, etoposide, doxorubicin, mitoxan) and Sz/m (streptomycin, mitoxin) are the frontline treatments for the combined cytotoxic drugs of mitoxan as advanced ACC, but the total efficacy is very limited. Radiotherapy, radiofrequency ablation, intervention Embolization can only benefit a small number of patients, so in order to effectively improve the survival rate of ACC patients, operation combined with drug therapy is still an important research topic. Because ACC is insensitive to traditional chemotherapeutic drugs, the search for effective, safe and high selective drugs is also the focus of the study of ACC today. Niclosamide, N- (2'- chlorine -4'- nitrobenzene) -5- chlorosalicamide, molecular C13H8C12N2O4, used in the clinical treatment of human intestinal parasitic diseases, is the American food and Drug Administration (FDA) recommended for the treatment of worms. The mechanism of action blocks NADPH to NADP+ by inhibiting the phosphorylation and mitochondrial function of the insect body cells. Transformation, inhibiting the production of ATP and changing cell energy metabolism play a therapeutic role. In recent years, in the process of research and development of antitumor drugs, a large number of drug screening experiments have found that niclosamide also has potential antitumor effects. Compared with the newly synthesized antitumor drugs, niclosamide has a strong cytotoxicity and human toxicity. Using small advantage.WANG in 2009 reports that niclosamide inhibits the expression of K562 target gene protein in leukemia cells, other scholars have found that niclosamide can inhibit tumor cells by affecting Wnt/ beta -catenin, mTORC1, STAT3, NF- kappa B, Notch, Hedgehog and other signaling pathways and tumor cell mitochondria, lysosomes Further studies have shown that niclosamide not only acts on tumor cells but also acts on tumor stem cells that have self renewal capacity and can produce heterogeneous cells. In addition, in other studies, niclosamide can increase the sensitivity of tumor cells to chemotherapy and radiotherapy, even in some other studies. It can reverse the tolerance of tumor cells to chemotherapy and radiotherapy. The effect of niclosamide on the energy metabolism and signal transduction of normal cells is very little. As a potential antitumor drug, it can be used as a potential antitumor drug. Objective: 1. to study the proliferation, cycle, apoptosis and migration of niclosamide on human adrenocortical carcinoma SW-13 cell lines. The effect of attack and preliminary study on its mechanism.2. to study the inhibitory effect and mechanism of niclosamide on the growth of subcutaneous xenografts in nude mice of adrenocortical cancer SW-13 cells. 1. through the culture of SW-13 cells in vitro, CCK-8 and iCELLigence real-time unmarked cell proliferation monitoring apparatus were used to detect the effect of different concentrations of niclosamide on SW-13 cells Effects of proliferation on.2. using flow cytometry to detect the cell cycle distribution of niclosamide using SW-13 cells.3. AO/EB staining and Annexin V/PI staining flow cytometry were used to detect the effect of niclosamide on the apoptosis rate of SW-13 cells..4. using Transwell test and cell scratch test to detect clonite Effect of salamine on SW-13 cells on cell invasiveness and mobility,.5. using Western Blotting to detect the effects of nitramine on SW-13 cells on the expression of beta -catenin, LRP6, vimentin and E-cadherin.6. to establish a SW-13 cell xenografts in nude mice. The random number table method was used to set up the control group, DMSO group, nitramine low concentration group and clonite. In the Liu Angao concentration group, the diameter of the tumor and the weight of the tumor were monitored dynamically by intraperitoneal injection. The effects of nude mice on tumor volume and weight were observed and the growth inhibition rate was calculated. Part of the tumor body was stained with HE, and the tissue morphology.7. was observed in the group of cisplatin as positive control. Blood cells, biochemical analysis of.8., TUNEL method was used to detect the effect of niclosamide on nude mice. The effect of.9. on the expression of beta -catenin, LRP6, vimentin, E-cadherin, Bc 1-2 and Caspase-3 in transplanted tumor tissues by immunohistochemical staining. Results: 1. After the same concentration of niclosamide acted on SW-13 cell 24,48,72,96120h respectively, the time and concentration dependent inhibition of SW-13 cell proliferation (F=157.93, P0.001).2. flow cytometry showed 24,48,72h, and the nitramine group blocked the SW-13 cell cycle in G0/G1 phase (P=0.001,0.005,0.008).3.AO/EB, compared with the control group. The results of staining and Annexin V/PI staining showed that the apoptosis rate of the niclosamide group was higher than that of the irradiated group (P0.001).4.Transwell experimental results showed that the invasiveness of SW-13 cells in the niclosamide group was lower than that of the control group (P0.001). The results of cell scratch test showed that the migration ability of SW-13 cells in the niclosamide group was lower than that of the control group (P=0.002).5.Western Blott. The results of ing showed that the expression of beta -catenin, LRP6 and vimentin in the SW-13 cells of the niclosamide group was lower than that of the control group (P0.001), while the expression of E-cadherin was higher than that of the control group (P=0.004).6.HE staining tissue section confirmed the successful establishment of the tumor model of nude mice in SW-13 cells, the low concentration group of niclosamide, the final tumor volume and tumor of the high concentration group of niclosamide. Weight, lower than the control group (all P0.001), the control group and the DMSO group had no statistically significant difference (all P0.05) the white cells in the.7. cisplatin group were lower than the niclosamide group, the control group (P0.001), the niclosamide group and the control group had no significant difference (P0.05). The difference between the red cells and the platelets in the niclosamide group and the cisplatin group was different from the control group. No statistical significance (all P0.05). The urea nitrogen, creatinine, total bilirubin and alanine transaminase in the cisplatin group were higher than the niclosamide group and the control group (P0.001). There was no significant difference between the niclosamide group and the control group (all P0.05) the TUNEL test results of.8. transplanted tumor showed the low concentration group of niclosamide, the cells of the high concentration group of niclosamide The apoptosis rate was higher than that of the control group (P=0.004, P=0.005).9. transplantation tumor tissue immunohistochemical staining results showed the low concentration group of niclosamide, the expression of beta -catenin, LRP6, vimentin, Bcl-2 in the high concentration group of niclosamide was lower than that of the control group (P0.001), E-cadherin, caspase-3 expression was higher than that of the control group (P0.001). Conclusion: 1. niclosamide was present. The proliferation of SW-13 cells in human adrenocortical cancer cells, inhibition of cell cycle, inhibition of invasion and migration, and apoptosis, and the inhibition of the growth of subcutaneous xenografts in nude mice of SW-13 cells may involve the Wnt/ beta -catenin signaling pathway, epithelial mesenchymal transition (EMT) and apoptosis related protein expression of.2. niclosamide The effect on blood cells, liver and kidney function is less than cisplatin.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R736.6

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