hucMSC-exosome来源的14-3-3ζ活化自噬在预防顺铂毒中的作用及机制

发布时间：2018-04-29 04:21

本文选题：顺铂 + 肾毒性　；参考：《江苏大学》2017年博士论文

【摘要】：目的:顺铂是一种常见的临床化疗药物,但肾毒性限制了其应用。课题组前期研究显示人脐带间质干细胞(human umbilical cord mesenchymal stem cells,hucMSC)来源的外泌体(exosome)(简称hucMSC-ex)在体内外模型中可以降低顺铂诱导的肾脏过氧化损伤及细胞凋亡,但hucMSC-ex预处理在顺铂诱导的肾损伤中的作用以及对顺铂抗肿瘤效果的影响研究甚少;本研究旨在揭示体内外模型中hucMSC-ex在顺铂诱导的肾毒性中的预防作用及机制,为肾损伤的预防提供新的策略。方法:(1)贴壁法分离培养hucMSC,并用试剂盒法提取exosomes,通过Nanosight纳米颗粒分析仪对exosome的大小及浓度进行检测分析;采用成像流式观察hucMSC-ex表面标记CD9和CD63的表达。(2)使用CM-Dil染料对hucMSC-ex染色后与NRK-52E细胞共孵育,超分辨显微镜观察NRK-52E细胞对hucMSC-ex的摄取。建立体外hucMSC-ex预防顺铂诱导肾小管上皮细胞(NRK-52E)损伤模型,分为3组:Control组,NRK-52E细胞不加任何处理;PBS组,与hucMSC-ex相同体积的PBS作用于NRK-52E细胞24h,顺铂损伤16h;hucMSC-ex组,hucMSC-ex作用于NRK-52E细胞24h后,顺铂处理16h。流式细胞仪Annexin V/PI双染法分析NRK-52E细胞的凋亡情况;Western blot方法分析bax、Cyclin D3蛋白的表达;免疫荧光技术检测核增殖抗原(PCNA)的表达情况。NRK-52E细胞转染mRFP-GFP-LC3双标腺病毒,超分辨显微镜观察不同组别中自噬小体的形成;免疫荧光技术及Western blot分析自噬相关蛋白LC3B的表达;Western blot检测14-3-3ζ蛋白的表达。构建体内hucMSC-ex预防顺铂诱导的SD大鼠急性肾损伤模型,分为3组:Control组,SD大鼠背部切开不加处理再缝合;PBS组,SD大鼠双侧肾脏肾被膜注射PBS 24h后腹腔注射顺铂作用3d;hucMSC-ex组,SD大鼠双侧肾脏肾被膜注射hucMSC-ex 24h后腹腔注射顺铂作用3d。每天收集血液样本,检测血清尿素氮(BUN)和肌酐(Crea)的水平。顺铂作用3d后将大鼠处死,留取肾组织。HE染色观察肾组织的损伤情况;免疫组织化学染色检测PCNA的表达;TUNEL染色分析肾小管上皮细胞的凋亡情况;westernblot检测大鼠肾组织凋亡相关蛋白bax的表达情况,综合评价hucmsc-ex对急性肾损伤的预防作用。westernblot分析大鼠肾组织lc3b的表达;免疫组织化学染色及westernblot检测肾组织中14-3-3ζ的表达。(3)体外顺铂作用于胃癌细胞之前,用hucmsc-ex预处理,通过流式细胞技术分析胃癌细胞的凋亡情况及细胞周期改变;transwell实验分析胃癌细胞的迁移能力;平板克隆形成实验检测胃癌细胞的增殖能力。(4)通过14-3-3ζ腺病毒过表达或慢病毒shrna干扰hucmsc中的14-3-3ζ并收集上清提取exosomes,分别为ad-gfp-ex、ad-14-3-3ζ-ex、shgfp-ex、sh14-3-3ζ-ex。荧光显微镜检测hucmsc细胞中绿色荧光强度;westernblot分析hucmsc及hucmsc-ex中14-3-3ζ的表达;超分辨显微镜观察nrk-52e细胞对ad-14-3-3ζ-ex(exosomes以cm-dil标记,14-3-3ζ蛋白表达gfp)的内化情况。分别用pbs、ad-gfp-ex、ad-14-3-3ζ-ex、shgfp-ex、sh14-3-3ζ-ex预处理nrk-52e细胞24h后顺铂损伤16h,超分辨显微镜观察细胞中自噬小体的形成情况;westernblot检测细胞中14-3-3ζ和lc3b的表达;免疫荧光技术观察细胞中pcna的表达;tunel或流式细胞术分析细胞的凋亡情况。在体内control组、pbs组、ad-gfp-ex组、ad-14-3-3ζ-ex组中,免疫组织化学技术检测肾组织中14-3-3ζ的表达;westernblot分析lc3b的表达;血清学检测bun、crea的水平;he染色观察肾组织的病理形态;tunel染色分析肾小管上皮细胞的凋亡情况;westernblot检测大鼠肾组织凋亡相关蛋白caspase3的表达情况;免疫组织化学染色检测pcna的表达。(5)通过免疫共沉淀技术和超分辨显微镜检测14-3-3ζ与自噬相关蛋白atg16l之间的相互作用。结果:(1)人脐带间质干细胞来源的exosome,nanosight分析发现其是直径为97nm左右的囊泡;成像流式检测发现hucmsc-ex表达cd9和cd63,阳性率约为80%。(2)hucmsc-ex预处理减少顺铂诱导的nrk-52e细胞凋亡数量,降低bax的表达,增加cyclind3和pcna的表达。在体内hucmsc-ex降低肾小管细胞tunel阳性细胞数量及bax的表达,增加pcna阳性细胞数。在体外hucmsc-ex预防模型中,发现hucMSC-ex可以被转运到靶细胞中。在NRK-52E细胞中hucMSC-ex预处理增加自噬小体的数量及LC3B蛋白的表达。在体内hucMSCex预处理具有相同的作用。(3)在顺铂处理的胃癌细胞模型中,hucMSC-ex预处理不会改变顺铂诱导的细胞凋亡及周期阻滞,胃癌细胞迁移和增殖能力与单独顺铂处理组相比无明显差异。(4)液相质谱分析(LC-MS/MS)发现hucMSC-ex携带14-3-3ζ蛋白,并且将其转运至NRK-52E细胞中。在体内外模型中hucMSC-ex预处理促进14-3-3ζ蛋白表达升高。HucMSC-ex中14-3-3ζ过表达明显增加NRK-52E细胞中自噬小体的形成和LC3B的表达,同时降低顺铂诱导的NRK-52E细胞凋亡并促进细胞增殖。在体内AD-14-3-3ζ-ex降低血清BUN、Crea的水平以及肾脏TUNEL阳性细胞数量。敲减hucMSC-ex中14-3-3ζ减弱自噬并加重顺铂诱导的NRK-52E细胞凋亡,抑制细胞增殖。(5)在体外模型中HucMSC-ex预处理促进NRK-52E细胞ATG16L蛋白的表达。超分辨显微镜结果显示14-3-3ζ促进ATG16L蛋白的表达和聚集。免疫共沉淀结果显示14-3-3ζ可以与ATG16L相互作用。结论:HucMSC-ex可以通过转运14-3-3ζ蛋白增强自噬从而预防顺铂诱导的急性肾损伤,同时不影响顺铂的抗肿瘤效果;14-3-3ζ与ATG16L相互作用,可以促进自噬小体的形成。本研究为预防顺铂诱导的急性肾损伤及扩展顺铂临床应用提供了新的思路和依据。
[Abstract]:Objective: cisplatin (cisplatin) is a common clinical chemotherapeutic drug, but renal toxicity restricts its application. Earlier studies have shown that the external secretory (hucMSC-ex) derived from human umbilical cord mesenchymal stem cells (human umbilical cord mesenchymal stem cells, hucMSC) can reduce the renal peroxidation injury induced by cisplatin in the model in vivo and in vitro And apoptosis, but the role of hucMSC-ex preconditioning in cisplatin induced renal injury and the effect of cisplatin on the anti-tumor effect of cisplatin are seldom studied. This study aims to reveal the preventive effect and mechanism of hucMSC-ex in the renal toxicity induced by cisplatin in vitro and in vivo, and provide a new strategy for the prevention of renal injury. Method: (1) isolation and isolation of the renal injury. HucMSC was cultured and exosomes was extracted with kit method. The size and concentration of exosome were detected and analyzed by Nanosight nanoparticle analyzer. The expression of CD9 and CD63 on hucMSC-ex surface was observed by imaging flow pattern. (2) CM-Dil dyestuff was used to reincubate NRK-52E cells with hucMSC-ex staining, and NRK-52E cells were observed by superresolution microscope. HucMSC-ex intake. Establish a hucMSC-ex prevention model of cisplatin induced renal tubular epithelial cell (NRK-52E) injury in vitro, divided into 3 groups: group Control, NRK-52E cells without any treatment; PBS group, PBS in the same volume as hucMSC-ex acts on NRK-52E cell 24h, cisplatin damage 16h; hucMSC-ex group, after the action of cisplatin treatment 16 H. flow cytometry Annexin V/PI double staining method was used to analyze the apoptosis of NRK-52E cells; Western blot method was used to analyze the expression of Bax, Cyclin D3 protein; immunofluorescence technique was used to detect the expression of nuclear proliferation antigen (PCNA),.NRK-52E cells were transfected into the mRFP-GFP-LC3 double standard adenovirus, and the formation of autophagic bodies in different groups was observed by superresolution microscope. The expression of autophagy related protein LC3B was analyzed by immunofluorescence technique and Western blot, and Western blot was used to detect the expression of 14-3-3 zeta protein. The model of acute renal injury in SD rats induced by cisplatin induced by hucMSC-ex was divided into 3 groups: Control group, SD rats' back incision without treatment and re suture. Intraperitoneal injection of cisplatin effect 3D; group hucMSC-ex, SD rats were injected with hucMSC-ex 24h after hucMSC-ex 24h injection of cisplatin to collect blood samples every day to detect the level of serum urea nitrogen (BUN) and creatinine (Crea). After the action of cisplatin, the rats were killed and renal tissue.HE staining was left to observe the damage of renal tissue; immunohistochemical staining was used. The expression of PCNA, TUNEL staining analysis of renal tubular epithelial cell apoptosis, Westernblot detection of apoptosis related protein Bax expression in renal tissue of rats, comprehensive evaluation of the preventive effect of hucmsc-ex on acute renal injury by.Westernblot analysis of lc3b in renal tissue of rats; immunohistochemical staining and Westernblot detection Expression of 14-3-3 zeta in renal tissue. (3) prior to cisplatin in vitro, hucmsc-ex pretreatment was used to analyze the apoptosis and cell cycle changes of gastric cancer cells by flow cytometry; Transwell test was used to analyze the migration ability of gastric cancer cells; the proliferation ability of gastric cancer cells was detected by flat clones. (4) through the 14-3-3 Zeta Adenovirus overexpression or lentivirus shRNA interfered with the 14-3-3 zeta in hucmsc and collected the supernatant to extract exosomes, which were ad-gfp-ex, ad-14-3-3 zeta -ex, shgfp-ex, and sh14-3-3 zeta -ex. fluorescence microscope to detect the green fluorescence intensity in hucmsc cells, Westernblot analysis and the expression of zeta; super-resolution microscopy observation of cell pairs The internalization of ad-14-3-3 zeta -ex (exosomes with cm-dil, 14-3-3 zeta protein expressed by 14-3-3 zeta protein). The formation of autophagic bodies in the cells was observed with PBS, ad-gfp-ex, ad-14-3-3 zeta -ex, shgfp-ex, and sh14-3-3 zeta, respectively, and the formation of autophagic bodies in the cells was observed by superresolution microscopy. The expression of PCNA in cells was observed by immunofluorescence; TUNEL or flow cytometry was used to analyze the apoptosis of cells. In group control, PBS, ad-gfp-ex, and ad-14-3-3 zeta -ex, immunohistochemical technique was used to detect the expression of 14-3-3 zeta in renal tissue; Westernblot analysis of lc3b, bun, level of crea Pathological morphology of tissue; TUNEL staining analysis of apoptosis of renal tubular epithelial cells; Westernblot detection of expression of apoptosis related protein Caspase3 in renal tissue of rats; immunohistochemical staining to detect the expression of PCNA. (5) detection of the phase between 14-3-3 zeta and autophagy related protein atg16l through immunoprecipitation and superresolution microscopy Results: (1) the exosome of human umbilical cord stromal stem cells was found by nanosight analysis. The imaging flow detection found that hucmsc-ex expressed CD9 and CD63, and the positive rate was about 80%. (2) hucmsc-ex pretreatment reduced the number of NRK-52E fine cell apoptosis induced by cisplatin, reduced the expression of Bax, and increased cyclind3 and PCNA. In vivo hucmsc-ex reduced the number of TUNEL positive cells in renal tubular cells and the expression of Bax, and increased the number of PCNA positive cells. In the hucmsc-ex prevention model in vitro, it was found that hucMSC-ex could be transported to the target cells. In NRK-52E cells, the number of autophagic bodies and the expression of LC3B protein were increased by hucMSC-ex preconditioning in NRK-52E cells. In vivo hucMSCex The treatment had the same effect. (3) in cisplatin treated gastric cancer cell model, hucMSC-ex pretreatment did not change cisplatin induced apoptosis and cycle arrest. There was no significant difference in the migration and proliferation ability of gastric cancer cells compared with that of the single cisplatin treatment group. (4) liquid phase mass spectrometry (LC-MS/MS) found that hucMSC-ex carried 14-3-3 zeta protein, and In vitro and in vivo models, hucMSC-ex pretreatment promoted 14-3-3 zeta protein expression to increase the expression of 14-3-3 zeta in.HucMSC-ex, which significantly increased the formation of autophagic bodies and LC3B expression in NRK-52E cells, and reduced the apoptosis of cisplatin induced NRK-52E cells and promoted cell proliferation. In vivo AD-14-3-3 zeta -ex reduced serum. The level of BUN, Crea and the number of TUNEL positive cells in the kidney. 14-3-3 zeta in hucMSC-ex reduced autophagy and increased cisplatin induced NRK-52E cell apoptosis and inhibited cell proliferation. (5) HucMSC-ex preconditioning promotes the expression of ATG16L protein in NRK-52E cells in an in vitro model. The results of ultra resolution microscope show that 14-3-3 Zeta promotes the expression of ATG16L protein. The results of immunoprecipitation showed that 14-3-3 zeta could interact with ATG16L. Conclusion: HucMSC-ex can enhance autophagy by transferring 14-3-3 zeta protein to prevent cisplatin induced acute renal injury, without affecting the antitumor effect of cisplatin, and the interaction of 14-3-3 zeta with ATG16L can promote the formation of autophagic bodies. This study is to prevent the formation of autophagic corpuscles. Cisplatin induced acute kidney injury and the clinical application of extended cisplatin provide a new idea and basis.

【学位授予单位】：江苏大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R730.5

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