本文選題：巴西蘇木素 + 膀胱癌�。� 參考：《山西大學》2017年碩士論文

【摘要】：膀胱癌是泌尿系統(tǒng)中的主要癌癥,具有發(fā)病率高和易復(fù)發(fā)等特點。手術(shù)后進行膀胱灌注是術(shù)后防復(fù)發(fā)的重要手段,課題組合作單位研制的膀胱灌注藥物“蘇復(fù)寧洗液”能有效的預(yù)防膀胱癌術(shù)后復(fù)發(fā)。課題組研究發(fā)現(xiàn),蘇復(fù)寧洗液的主效成分巴西蘇木素(Brazilin)能有效的致死膀胱癌T24細胞。但目前尚缺乏對巴西蘇木素作為膀胱癌治療藥物的廣譜性、安全性及其致死膀胱癌細胞的死亡機制的系統(tǒng)研究。在本文中我們以不同來源膀胱細胞為對象,檢測巴西蘇木素作為膀胱癌治療藥物的廣譜性和安全性;通過檢測巴西蘇木素對膀胱癌T24細胞中的疊加增殖性質(zhì)、乳酸脫氫酶、鈣離子和線粒體膜電位等生理生化性質(zhì)的影響,進一步明確巴西蘇木素對膀胱癌細胞的毒性;檢測不同死亡方式抑制劑對巴西蘇木素致死T24細胞效應(yīng)的影響,結(jié)合半胱氨酸蛋白酶(Caspase)酶活性測定及透射電鏡形態(tài)學分析,證明巴西蘇木素通過程序性壞死致死膀胱癌T24細胞;進一步利用巴西蘇木素作用T24細胞6 h后的數(shù)字基因表達譜數(shù)據(jù)分析及RT-qPCR(Real-time qPCR),提出巴西蘇木素是通過RIP1/RIP3/MLKL介導(dǎo)的細胞程序性壞死途徑致死T24細胞。具體研究有以下四部分內(nèi)容:1.利用低血清實驗檢測巴西蘇木素對不同類型膀胱癌細胞系的影響,發(fā)現(xiàn)巴西蘇木素能有效致死T24、5637和J82細胞,證明巴西蘇木素作為膀胱癌治療藥物具備一定的廣譜性。比較巴西蘇木素對正常膀胱上皮細胞系SV-HUC-1及膀胱上皮細胞癌細胞系T24、5637的細胞毒性,發(fā)現(xiàn)巴西蘇木素在濃度效應(yīng)和時間效應(yīng)方面對膀胱上皮細胞SV-HUC-1的致死效應(yīng)小于膀胱癌T24細胞和5637細胞。說明巴西蘇木素作為膀胱癌藥物具有一定的藥物安全性。2.檢測巴西蘇木素對膀胱癌T24細胞一些生理生化性質(zhì)的影響,發(fā)現(xiàn)巴西蘇木素能夠抑制T24細胞增殖、阻礙其不斷重疊生長;引起細胞膜通透性改變、促進乳酸脫氫酶釋放;引起細胞內(nèi)參與眾多信號傳導(dǎo)的鈣離子濃度顯著升高;導(dǎo)致參與細胞代謝的線粒體膜電位降低。進一步明確了巴西蘇木素對膀胱癌細胞的毒性效應(yīng),為巴西蘇木素的藥物開發(fā)提供了實驗依據(jù)。3.通過不同死亡方式抑制劑處理,半胱氨酸蛋白酶活性測定及形態(tài)學觀察探究巴西蘇木素致死T24細胞的死亡方式。研究發(fā)現(xiàn)凋亡抑制劑z-VAD-fmk不能抑制巴西蘇木素的藥性,結(jié)合半胱氨酸蛋白酶(Caspase)家族酶活測定,證明巴西蘇木素不通過Caspase誘導(dǎo)的凋亡信號致死T24細胞。而自噬小體抑制劑3-MA和程序性壞死抑制劑Nec-1均能在一定程度上抑制巴西蘇木素對T24細胞的致死效應(yīng)。透射性電鏡觀察發(fā)現(xiàn)巴西蘇木素引起T24細胞的細胞膜破裂、線粒體崩裂、細胞核彌散等程序性壞死形態(tài)學特征,這些數(shù)據(jù)表明,巴西蘇木素通過誘導(dǎo)程序性壞死致死膀胱癌T24細胞。4.半致死濃度的巴西蘇木素作用T24細胞6 h后的數(shù)字基因表達譜數(shù)據(jù)顯示程序性壞死關(guān)鍵基因RIP1/MLKL超表達,RT-qPCR實驗證明巴西蘇木可誘導(dǎo)T24細胞中程序性壞死關(guān)鍵基因RIP1/RIP3/MLKL表達顯著上調(diào),初步探明巴西蘇木素是通過RIP1/RIP3/MLKL介導(dǎo)的細胞程序性壞死途徑致死T24細胞。
[Abstract]:Bladder cancer is the major cancer in the urinary system, and has a high incidence of recurrence and characteristics. After the operation of bladder is an important means of postoperative recurrence, intravesical drug development research group unit of Funing lotion Su "can effectively prevent bladder cancer after relapse. The study group found the main topic. The effective component of Funing lotion Brazil Su hematoxylin (Brazilin) can effectively kill T24 cells of bladder cancer. But there is still a lack of Brazil hematoxylin as broad-spectrum drugs for the treatment of bladder cancer, study mechanism of death safety and mortality of bladder cancer cells. In this paper we use different sources of bladder cells as the object. Brazil hematoxylin as broad-spectrum detection and safety of drugs for the treatment of bladder cancer through the detection of Brazil; hematoxylin is added to the proliferation properties of bladder cancer T24 cells in lactate dehydrogenase, and mitochondrial calcium Effect of membrane potential and other physiological and biochemical properties, to further clarify the toxicity of Brazil hematoxylin on bladder cancer cell death detection; effects of different inhibitors on Brazil hematoxylin lethal effect of T24 cells combined with cysteine protease (Caspase) analysis of enzyme activity assay and transmission electron microscope morphology, proved Brazil hematoxylin by programmed necrosis of bladder cancer death T24 cells; the further use of Brazil hematoxylin T24 cells after 6 h the number of gene expression data analysis and RT-qPCR (Real-time qPCR), the Brazil hematoxylin is programmed cell necrosis pathway mediated by RIP1/RIP3/MLKL cells. The specific lethal T24 study has the following four parts: 1. the effect of low serum test of Brazil sun the different types of bladder cancer cell lines and found that Brazil hematoxylin can effectively lethal T245637 and J82 cells, hematoxylin as evidence of Brazil Drugs for the treatment of bladder cancer with a broad-spectrum. Brazil hematoxylin of normal bladder epithelial cell line SV-HUC-1 and bladder epithelial cell carcinoma cell line T245637 cells, found in Brazil hematoxylin in concentration and time effects on bladder epithelial cells SV-HUC-1 induced death effect is smaller than the bladder cancer T24 cells and 5637 cells in Brazil. Hematoxylin as has an impact on drug safety.2. detection of certain Brazil hematoxylin on some physiological and biochemical properties of bladder cancer T24 cells of bladder cancer drugs found in Brazil, hematoxylin can inhibit the proliferation of T24 cells, which hinder the continuous growth caused by overlapping; cell membrane permeability changes, promote the release of lactate dehydrogenase; intracellular calcium concentration in many signal transduction was significantly increased; lead to mitochondrial membrane potential in cell metabolism decreased. To further clarify the Brazil hematoxylin of bladder The toxic effect of bladder cancer cells, provides the experimental basis for.3. through different forms of death inhibitors for drug development in Brazil hematoxylin, cysteine protease activity was observed on Brazil hematoxylin lethal T24 cell death and apoptosis morphology. The study found that the inhibitor z-VAD-fmk can inhibit the growth of Brazil hematoxylin resistance, combined with the cysteine protease (Caspase) determination of enzyme family live, Brazil hematoxylin not through the apoptotic signal induced by Caspase and T24 cells. Lethal autophagosome inhibitor 3-MA and programmed necrosis inhibitor Nec-1 could inhibit the Brazil sum to a certain extent on T24 cells were observed by transmission electron microscope. The lethal effect of Brazil hematoxylin caused T24 cell membrane rupture. Mitochondrial crack, nucleus dispersion necroptosis morphological characteristics, these data suggest that Brazil hematoxylin induced through Guide necroptosis lethal T24 bladder cancer cell.4. semi lethal concentration Brazil hematoxylin T24 cells after 6 h the number of gene expression data showed programmed necrosis of key genes over expression of RIP1/MLKL, RT-qPCR proved that Brazil hematoxylin can induce programmed necrosis of T24 cells in the key gene RIP1/RIP3/MLKL expression was significantly up-regulated, preliminary study of Brazil wood in the programmed cell necrosis pathway mediated by RIP1/RIP3/MLKL cell death T24.

【學位授予單位】：山西大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.14

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