PDTC通過減少肌肉萎縮及脂肪降解緩解腫瘤惡病質(zhì)
發(fā)布時間：2018-07-13 17:12
【摘要】：背景：腫瘤惡病質(zhì)(cancer cachexia)是一種進(jìn)行性骨骼肌消耗(伴隨或不伴隨脂肪消耗),并且不能通過營養(yǎng)支持完全逆轉(zhuǎn)的功能性損傷疾病。其發(fā)生機(jī)制復(fù)雜,目前的治療效果有限。研究抗腫瘤惡病質(zhì)藥物尚無統(tǒng)一標(biāo)準(zhǔn)和系統(tǒng)的體內(nèi)外生物篩選評價模型,昂貴費(fèi)時的體內(nèi)篩選延緩了抗腫瘤惡病質(zhì)藥物的發(fā)現(xiàn)與研究,建立并優(yōu)化具有代表性的體內(nèi)外抗腫瘤惡病質(zhì)生物評價模型,將為抗腫瘤惡病質(zhì)的研究帶來極大便利。NF-κB信號通路與腫瘤惡病質(zhì)中肌肉萎縮和脂肪消耗密切相關(guān),吡咯烷二硫代氨基甲酸鹽(PDTC)能通過抑制腫瘤組織中NF-κB的表達(dá)緩解惡病質(zhì)小鼠體重下降,但其對脂肪細(xì)胞和骨骼肌細(xì)胞的直接作用,緩解腫瘤惡病質(zhì)的分子作用機(jī)制仍不明確,有待進(jìn)一步的研究。目的：建立具有代表性的穩(wěn)定可行的體內(nèi)外腫瘤惡病質(zhì)模型,用于抗腫瘤惡病質(zhì)新藥的研究與開發(fā)。利用建立的體內(nèi)外模型評價PDTC對腫瘤惡病質(zhì)的緩解作用,探究其對脂肪和骨骼肌的直接作用及可能的分子作用機(jī)制。方法：(1)采用MTT法、甘油檢測試劑盒、免疫熒光法及Western Blot檢測TNF-α對成熟脂肪細(xì)胞及肌管的影響,構(gòu)建TNF-α誘導(dǎo)的肌萎縮及脂解模型；(2)C26細(xì)胞培液直接作用成熟脂肪細(xì)胞,或C26細(xì)胞培液與2% HS分化液1：1稀釋后作用于肌管,分別采用油紅O染色、甘油三脂GPO-POD酶法檢測試劑盒、免疫熒光法及Western Blot檢測C26細(xì)胞培液對成熟脂肪細(xì)胞及肌管的影響,構(gòu)建C26細(xì)胞培液誘導(dǎo)的肌萎縮及脂解模型；(3)以C26細(xì)胞懸液接種SPF級BALB/c雄鼠腋下,監(jiān)測小鼠體重、瘤體積及攝食量等,構(gòu)建C26腫瘤惡病質(zhì)小鼠模型；(4)MTT法或CCK8法檢測PDTC對成熟脂肪細(xì)胞或成熟肌管細(xì)胞的增殖活力影響；(5)油紅O染色法、甘油含量檢測試劑盒及甘油三脂GPO-POD酶法檢測試劑盒測定PDTC對TNF-α及C26細(xì)胞培液誘導(dǎo)的脂解的緩解作用,Western Blot檢測NF-κB的活化、Perilipin變化及相關(guān)蛋白表達(dá)；(6)免疫熒光進(jìn)行染色后,]mage J軟件統(tǒng)計分化肌管直徑檢測：PDTC對TNF-α及C26細(xì)胞培液誘導(dǎo)的肌管萎縮的緩解作用,Western Blot檢測NF-κB的活化狀況、萎縮相關(guān)蛋白MuRF-1和Atrogin-1以及肌生成相關(guān)蛋白MyoD的表達(dá)情況；(7)監(jiān)測C26荷瘤小鼠體重,腓腸肌及附睪脂肪組織的重量,生化檢測儀測定血生化指標(biāo),HE染色觀察腓腸肌及附睪脂肪組織的顯微結(jié)構(gòu)變化,評價PDTC對C26荷瘤小鼠腫瘤惡病質(zhì)的緩解作用。結(jié)果：(1)甘油檢測及MTT法顯示TNF-α 50 ng/mL時甘油釋放量最大且對脂肪細(xì)胞活性無影響,并且p-p65表達(dá)增加,Perilipin蛋白表達(dá)減少；Image J及免疫熒光實(shí)驗(yàn)發(fā)現(xiàn)TNF-α 100 ng/mL時肌管直徑明顯小于對照組,且p-p65表達(dá)上調(diào),MHC表達(dá)下調(diào)；(2)甘油三脂檢測及免疫印跡實(shí)驗(yàn)表明在C26細(xì)胞培液刺激下,脂肪細(xì)胞中甘油三脂含量顯著下降,p-p65表達(dá)增加,Perilipin表達(dá)下調(diào)；Image J測量發(fā)現(xiàn)肌管直徑減小,且p-p65表達(dá)增加,MHC表達(dá)減少；(3)C26荷瘤模型組在實(shí)驗(yàn)第12天左右出現(xiàn)惡病質(zhì)表型,瘤體積持續(xù)增大,體重及腓腸肌重量下降：(4)MTT法測得PDTC (0-100 pM)對成熟脂肪細(xì)胞作用24 h及48 h,對細(xì)胞活性無影響；CCK8法顯示當(dāng)PDTC濃度大于50州時對成熟肌管作用48 h,對肌管細(xì)胞活性有一定影響：(5)脂解模型中,PDTC 5μM時即可降低TNF-α誘導(dǎo)的甘油釋放,100μM時極大的緩解了脂解情況,Perilipin的表達(dá)隨PDTC濃度的增加逐漸增加；PDTC 10 μM時脂肪細(xì)胞內(nèi)甘油三脂含量顯著高于C26細(xì)胞培液組,且隨PDTC濃度的增大,脂肪細(xì)胞甘油三脂量逐漸增多,p-p65及p-HSL表達(dá)隨PDTC濃度的增加逐漸減少,脂滴圍蛋白(Perilipin)的表達(dá)則隨PDTC濃度的增加逐漸增加；(6)肌萎縮模型中,PDTC在25μM及50μM都能顯著改善TNF-α及C26細(xì)胞培液引起的肌管萎縮,p-p65、E3泛素鏈接酶MuRF-1及Atrogin-1表達(dá)隨PDTC濃度的增加逐漸減少,MyoD及MI-IC表達(dá)呈劑量依賴性增加；(7)小鼠腫瘤惡病質(zhì)模型中,PDTC劑量依賴性的緩解惡病質(zhì)小鼠體重、腓腸肌及附睪脂肪組織重量的下降,改善血中TG、GLU、ALB含量及甘油釋放量。結(jié)論：成功建立了由TNF-α及C26細(xì)胞培液誘導(dǎo)的體外脂解模型、肌萎縮模型及體內(nèi)C26小鼠惡病質(zhì)模型,為腫瘤惡病質(zhì)藥物研究提供了一個便捷系統(tǒng)的平臺；PDTC能通過降低NF-κB活性,抑制酯酶及泛素蛋白酶體系統(tǒng)的激活,改善肌生成途徑,從而很好緩解由TNF-α及C26培液誘導(dǎo)的成熟脂肪細(xì)胞的脂解及成熟肌管的萎縮程度,也能較好的延緩腫瘤惡病質(zhì)小鼠體重、腓腸肌及附睪脂肪重量的下降,表現(xiàn)出顯著的緩解腫瘤惡病質(zhì)的效果,是一個非常有潛力的惡病質(zhì)治療藥物。
[Abstract]:Background: tumor cachexia (cancer cachexia) is a functional lesion of progressive skeletal muscle consumption (with or without fat consumption), and can not be completely reversed through nutritional support. Its mechanism is complex and current therapeutic effects are limited. There is no unified standard and systemic in vivo and in vivo biology for the study of antitumor drugs. The screening and evaluation model, expensive and time-consuming screening delayed the discovery and study of antitumor cachexia drugs, and the establishment and optimization of a representative biological evaluation model of cachexia in vivo and in vitro, will bring great convenience to the.NF- kappa B signaling pathway and tumor cachexia for muscle atrophy and fat consumption of tumor cachexia. Cut correlation, pyrrolidine two thiocarbamate (PDTC) can alleviate the weight decline of cachexia mice by inhibiting the expression of NF- kappa B in tumor tissue, but its direct effect on adipocytes and skeletal muscle cells and the molecular mechanism of cancerous cachexia are still unclear. In vivo and in vivo tumor cachexia model, used for the research and development of new antitumor cachexia drugs. Use the established model in vivo and in vitro to evaluate the remission of PDTC on cachexia, explore its direct effect on fat and skeletal muscle and possible molecular mechanism. Methods: (1) MTT, glycerol detection kit, immunofluorescence The effects of TNF- alpha on mature adipocytes and myotubes were detected by light method and Western Blot, and the model of Myoatrophy and lipolysis induced by TNF- alpha was constructed. (2) C26 cell culture directly acted on mature adipocytes, or C26 cell culture and 2% HS differentiation liquid 1:1 diluted to myotubes, using oil red O staining and glycerol three fat GPO-POD enzyme assay reagent, respectively. The effects of C26 cell culture on mature adipocyte and myotube were detected by immunofluorescence and Western Blot, and the model of Myoatrophy and adipization induced by C26 cell culture was constructed. (3) C26 cell suspension was used to inoculate SPF grade BALB/c male rat axillary, to monitor mouse weight, tumor volume and intake, and to construct the mouse model of C26 cachexia; (4) MTT method or The effect of PDTC on the proliferation of mature adipocytes or mature myoductus cells was detected by CCK8. (5) oil red O staining, glycerol content detection kit and glycerol three fat GPO-POD enzyme detection kit were used to determine the alleviating effect of PDTC on TNF- alpha and C26 cells induced by culture, Western Blot detection of NF- kappa B activation, Perilipin changes and correlation Protein expression; (6) after immunofluorescence staining,]mage J software statistical differentiation myotube diameter detection: PDTC to TNF- alpha and C26 cells induced myotube atrophy, Western Blot detection of NF- kappa B activation status, atrophy associated protein MuRF-1 and Atrogin-1, and myogenerative protein MyoD expression; (7) monitoring The weight of the tumor bearing mice, the weight of the gastrocnemius and epididymal adipose tissue, biochemical indicator and HE staining to observe the microstructural changes of the gastrocnemius and epididymal adipose tissue, and evaluate the remission of PDTC on the cachexia of C26 tumor bearing mice. Results: (1) glycerol test and MTT method showed the maximum glycerol release of TNF- alpha 50 ng/mL There was no effect on the activity of adipocyte, and the expression of p-p65 increased, and the expression of Perilipin protein decreased. The Image J and immunofluorescence test found that the diameter of the myotube was obviously smaller than the control group when TNF- alpha 100 ng/mL was less than the control group, and the expression of p-p65 was up and the expression of MHC was down regulated. (2) the test of glycerol three fat and immunoblot test showed that the fat was fine under the stimulation of C26 cell culture. The content of glycerol three in the cell decreased significantly, the expression of p-p65 increased, and the expression of Perilipin was down. The Image J measurements found that the myotube diameter decreased and the p-p65 expression increased, and the MHC expression decreased. (3) the C26 bearing tumor model group had the cachexic phenotype around twelfth days of experiment, the tumor volume continued to increase, and the weight and the gastrocnemius weight decreased: (4) PDTC (0) was measured by MTT method (0 The effect of -100 pM on mature adipocytes was 24 h and 48 h, which had no effect on cell activity. CCK8 method showed that when PDTC concentration was more than 50 state, the effect of 48 h on mature myotubes and the activity of myotube cells had a certain effect. (5) the release of glycerol induced by TNF- a could be reduced when PDTC 5 micron was in the lipid solution, and the lipid release was greatly relieved when 100 um M. With the increase of PDTC concentration, the content of glycerol three fat in the adipocytes was significantly higher than that of the C26 cell culture group. With the increase of PDTC concentration, the content of glycerol three fat increased gradually, and the expression of p-p65 and p-HSL decreased with the increase of PDTC concentration, and the expression of lipid peri protein (Perilipin) was followed by PDTC concentration. Increase gradually; (6) in the Myoatrophy model, PDTC can significantly improve the myotube atrophy caused by TNF- alpha and C26 cell culture, p-p65, E3 ubiquitin linked enzyme MuRF-1 and Atrogin-1 expression gradually decreased with the increase of PDTC concentration, MyoD and MI-IC expression increased in a dose-dependent manner; (7) the dose of the tumor cachexia model in mice. Reducing the weight of cachexia mice, the decrease of the weight of the gastrocnemius and epididymis, and the improvement of TG, GLU, ALB and glycerol release in the blood. Conclusion: a successful establishment of an in vitro adipose model, a model of muscle atrophy and a cachexia model in the body of C26 mice induced by TNF- alpha and C26 cells, and a model of cachexia in the body of C26 mice, are provided for the research of cachexia drugs. A convenient system platform; PDTC can reduce the activation of NF- kappa B and inhibit the activation of esterase and ubiquitin proteasome system, improve the myogenesis pathway, thus alleviating the lilipsis of mature adipocytes induced by TNF- alpha and C26 culture and the atrophy of mature myotubes, and also better retarding the weight of tumor cachexia mice, gastrocnemius The decrease of body weight and epididymal fat weight has shown a significant effect on relieving cancer cachexia, and is a potential therapeutic agent for cachexia.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R96
[Abstract]:Background: tumor cachexia (cancer cachexia) is a functional lesion of progressive skeletal muscle consumption (with or without fat consumption), and can not be completely reversed through nutritional support. Its mechanism is complex and current therapeutic effects are limited. There is no unified standard and systemic in vivo and in vivo biology for the study of antitumor drugs. The screening and evaluation model, expensive and time-consuming screening delayed the discovery and study of antitumor cachexia drugs, and the establishment and optimization of a representative biological evaluation model of cachexia in vivo and in vitro, will bring great convenience to the.NF- kappa B signaling pathway and tumor cachexia for muscle atrophy and fat consumption of tumor cachexia. Cut correlation, pyrrolidine two thiocarbamate (PDTC) can alleviate the weight decline of cachexia mice by inhibiting the expression of NF- kappa B in tumor tissue, but its direct effect on adipocytes and skeletal muscle cells and the molecular mechanism of cancerous cachexia are still unclear. In vivo and in vivo tumor cachexia model, used for the research and development of new antitumor cachexia drugs. Use the established model in vivo and in vitro to evaluate the remission of PDTC on cachexia, explore its direct effect on fat and skeletal muscle and possible molecular mechanism. Methods: (1) MTT, glycerol detection kit, immunofluorescence The effects of TNF- alpha on mature adipocytes and myotubes were detected by light method and Western Blot, and the model of Myoatrophy and lipolysis induced by TNF- alpha was constructed. (2) C26 cell culture directly acted on mature adipocytes, or C26 cell culture and 2% HS differentiation liquid 1:1 diluted to myotubes, using oil red O staining and glycerol three fat GPO-POD enzyme assay reagent, respectively. The effects of C26 cell culture on mature adipocyte and myotube were detected by immunofluorescence and Western Blot, and the model of Myoatrophy and adipization induced by C26 cell culture was constructed. (3) C26 cell suspension was used to inoculate SPF grade BALB/c male rat axillary, to monitor mouse weight, tumor volume and intake, and to construct the mouse model of C26 cachexia; (4) MTT method or The effect of PDTC on the proliferation of mature adipocytes or mature myoductus cells was detected by CCK8. (5) oil red O staining, glycerol content detection kit and glycerol three fat GPO-POD enzyme detection kit were used to determine the alleviating effect of PDTC on TNF- alpha and C26 cells induced by culture, Western Blot detection of NF- kappa B activation, Perilipin changes and correlation Protein expression; (6) after immunofluorescence staining,]mage J software statistical differentiation myotube diameter detection: PDTC to TNF- alpha and C26 cells induced myotube atrophy, Western Blot detection of NF- kappa B activation status, atrophy associated protein MuRF-1 and Atrogin-1, and myogenerative protein MyoD expression; (7) monitoring The weight of the tumor bearing mice, the weight of the gastrocnemius and epididymal adipose tissue, biochemical indicator and HE staining to observe the microstructural changes of the gastrocnemius and epididymal adipose tissue, and evaluate the remission of PDTC on the cachexia of C26 tumor bearing mice. Results: (1) glycerol test and MTT method showed the maximum glycerol release of TNF- alpha 50 ng/mL There was no effect on the activity of adipocyte, and the expression of p-p65 increased, and the expression of Perilipin protein decreased. The Image J and immunofluorescence test found that the diameter of the myotube was obviously smaller than the control group when TNF- alpha 100 ng/mL was less than the control group, and the expression of p-p65 was up and the expression of MHC was down regulated. (2) the test of glycerol three fat and immunoblot test showed that the fat was fine under the stimulation of C26 cell culture. The content of glycerol three in the cell decreased significantly, the expression of p-p65 increased, and the expression of Perilipin was down. The Image J measurements found that the myotube diameter decreased and the p-p65 expression increased, and the MHC expression decreased. (3) the C26 bearing tumor model group had the cachexic phenotype around twelfth days of experiment, the tumor volume continued to increase, and the weight and the gastrocnemius weight decreased: (4) PDTC (0) was measured by MTT method (0 The effect of -100 pM on mature adipocytes was 24 h and 48 h, which had no effect on cell activity. CCK8 method showed that when PDTC concentration was more than 50 state, the effect of 48 h on mature myotubes and the activity of myotube cells had a certain effect. (5) the release of glycerol induced by TNF- a could be reduced when PDTC 5 micron was in the lipid solution, and the lipid release was greatly relieved when 100 um M. With the increase of PDTC concentration, the content of glycerol three fat in the adipocytes was significantly higher than that of the C26 cell culture group. With the increase of PDTC concentration, the content of glycerol three fat increased gradually, and the expression of p-p65 and p-HSL decreased with the increase of PDTC concentration, and the expression of lipid peri protein (Perilipin) was followed by PDTC concentration. Increase gradually; (6) in the Myoatrophy model, PDTC can significantly improve the myotube atrophy caused by TNF- alpha and C26 cell culture, p-p65, E3 ubiquitin linked enzyme MuRF-1 and Atrogin-1 expression gradually decreased with the increase of PDTC concentration, MyoD and MI-IC expression increased in a dose-dependent manner; (7) the dose of the tumor cachexia model in mice. Reducing the weight of cachexia mice, the decrease of the weight of the gastrocnemius and epididymis, and the improvement of TG, GLU, ALB and glycerol release in the blood. Conclusion: a successful establishment of an in vitro adipose model, a model of muscle atrophy and a cachexia model in the body of C26 mice induced by TNF- alpha and C26 cells, and a model of cachexia in the body of C26 mice, are provided for the research of cachexia drugs. A convenient system platform; PDTC can reduce the activation of NF- kappa B and inhibit the activation of esterase and ubiquitin proteasome system, improve the myogenesis pathway, thus alleviating the lilipsis of mature adipocytes induced by TNF- alpha and C26 culture and the atrophy of mature myotubes, and also better retarding the weight of tumor cachexia mice, gastrocnemius The decrease of body weight and epididymal fat weight has shown a significant effect on relieving cancer cachexia, and is a potential therapeutic agent for cachexia.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R96
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