發(fā)布時(shí)間：2018-04-29 04:21

  本文選題：順鉑 + 腎毒性��； 參考：《江蘇大學(xué)》2017年博士論文

【摘要】：目的:順鉑是一種常見的臨床化療藥物,但腎毒性限制了其應(yīng)用。課題組前期研究顯示人臍帶間質(zhì)干細(xì)胞(human umbilical cord mesenchymal stem cells,hucMSC)來(lái)源的外泌體(exosome)(簡(jiǎn)稱hucMSC-ex)在體內(nèi)外模型中可以降低順鉑誘導(dǎo)的腎臟過(guò)氧化損傷及細(xì)胞凋亡,但hucMSC-ex預(yù)處理在順鉑誘導(dǎo)的腎損傷中的作用以及對(duì)順鉑抗腫瘤效果的影響研究甚少;本研究旨在揭示體內(nèi)外模型中hucMSC-ex在順鉑誘導(dǎo)的腎毒性中的預(yù)防作用及機(jī)制,為腎損傷的預(yù)防提供新的策略。方法:(1)貼壁法分離培養(yǎng)hucMSC,并用試劑盒法提取exosomes,通過(guò)Nanosight納米顆粒分析儀對(duì)exosome的大小及濃度進(jìn)行檢測(cè)分析;采用成像流式觀察hucMSC-ex表面標(biāo)記CD9和CD63的表達(dá)。(2)使用CM-Dil染料對(duì)hucMSC-ex染色后與NRK-52E細(xì)胞共孵育,超分辨顯微鏡觀察NRK-52E細(xì)胞對(duì)hucMSC-ex的攝取。建立體外hucMSC-ex預(yù)防順鉑誘導(dǎo)腎小管上皮細(xì)胞(NRK-52E)損傷模型,分為3組:Control組,NRK-52E細(xì)胞不加任何處理;PBS組,與hucMSC-ex相同體積的PBS作用于NRK-52E細(xì)胞24h,順鉑損傷16h;hucMSC-ex組,hucMSC-ex作用于NRK-52E細(xì)胞24h后,順鉑處理16h。流式細(xì)胞儀Annexin V/PI雙染法分析NRK-52E細(xì)胞的凋亡情況;Western blot方法分析bax、Cyclin D3蛋白的表達(dá);免疫熒光技術(shù)檢測(cè)核增殖抗原(PCNA)的表達(dá)情況。NRK-52E細(xì)胞轉(zhuǎn)染mRFP-GFP-LC3雙標(biāo)腺病毒,超分辨顯微鏡觀察不同組別中自噬小體的形成;免疫熒光技術(shù)及Western blot分析自噬相關(guān)蛋白LC3B的表達(dá);Western blot檢測(cè)14-3-3ζ蛋白的表達(dá)。構(gòu)建體內(nèi)hucMSC-ex預(yù)防順鉑誘導(dǎo)的SD大鼠急性腎損傷模型,分為3組:Control組,SD大鼠背部切開不加處理再縫合;PBS組,SD大鼠雙側(cè)腎臟腎被膜注射PBS 24h后腹腔注射順鉑作用3d;hucMSC-ex組,SD大鼠雙側(cè)腎臟腎被膜注射hucMSC-ex 24h后腹腔注射順鉑作用3d。每天收集血液樣本,檢測(cè)血清尿素氮(BUN)和肌酐(Crea)的水平。順鉑作用3d后將大鼠處死,留取腎組織。HE染色觀察腎組織的損傷情況;免疫組織化學(xué)染色檢測(cè)PCNA的表達(dá);TUNEL染色分析腎小管上皮細(xì)胞的凋亡情況;westernblot檢測(cè)大鼠腎組織凋亡相關(guān)蛋白bax的表達(dá)情況,綜合評(píng)價(jià)hucmsc-ex對(duì)急性腎損傷的預(yù)防作用。westernblot分析大鼠腎組織lc3b的表達(dá);免疫組織化學(xué)染色及westernblot檢測(cè)腎組織中14-3-3ζ的表達(dá)。(3)體外順鉑作用于胃癌細(xì)胞之前,用hucmsc-ex預(yù)處理,通過(guò)流式細(xì)胞技術(shù)分析胃癌細(xì)胞的凋亡情況及細(xì)胞周期改變;transwell實(shí)驗(yàn)分析胃癌細(xì)胞的遷移能力;平板克隆形成實(shí)驗(yàn)檢測(cè)胃癌細(xì)胞的增殖能力。(4)通過(guò)14-3-3ζ腺病毒過(guò)表達(dá)或慢病毒shrna干擾hucmsc中的14-3-3ζ并收集上清提取exosomes,分別為ad-gfp-ex、ad-14-3-3ζ-ex、shgfp-ex、sh14-3-3ζ-ex。熒光顯微鏡檢測(cè)hucmsc細(xì)胞中綠色熒光強(qiáng)度;westernblot分析hucmsc及hucmsc-ex中14-3-3ζ的表達(dá);超分辨顯微鏡觀察nrk-52e細(xì)胞對(duì)ad-14-3-3ζ-ex(exosomes以cm-dil標(biāo)記,14-3-3ζ蛋白表達(dá)gfp)的內(nèi)化情況。分別用pbs、ad-gfp-ex、ad-14-3-3ζ-ex、shgfp-ex、sh14-3-3ζ-ex預(yù)處理nrk-52e細(xì)胞24h后順鉑損傷16h,超分辨顯微鏡觀察細(xì)胞中自噬小體的形成情況;westernblot檢測(cè)細(xì)胞中14-3-3ζ和lc3b的表達(dá);免疫熒光技術(shù)觀察細(xì)胞中pcna的表達(dá);tunel或流式細(xì)胞術(shù)分析細(xì)胞的凋亡情況。在體內(nèi)control組、pbs組、ad-gfp-ex組、ad-14-3-3ζ-ex組中,免疫組織化學(xué)技術(shù)檢測(cè)腎組織中14-3-3ζ的表達(dá);westernblot分析lc3b的表達(dá);血清學(xué)檢測(cè)bun、crea的水平;he染色觀察腎組織的病理形態(tài);tunel染色分析腎小管上皮細(xì)胞的凋亡情況;westernblot檢測(cè)大鼠腎組織凋亡相關(guān)蛋白caspase3的表達(dá)情況;免疫組織化學(xué)染色檢測(cè)pcna的表達(dá)。(5)通過(guò)免疫共沉淀技術(shù)和超分辨顯微鏡檢測(cè)14-3-3ζ與自噬相關(guān)蛋白atg16l之間的相互作用。結(jié)果:(1)人臍帶間質(zhì)干細(xì)胞來(lái)源的exosome,nanosight分析發(fā)現(xiàn)其是直徑為97nm左右的囊泡;成像流式檢測(cè)發(fā)現(xiàn)hucmsc-ex表達(dá)cd9和cd63,陽(yáng)性率約為80%。(2)hucmsc-ex預(yù)處理減少順鉑誘導(dǎo)的nrk-52e細(xì)胞凋亡數(shù)量,降低bax的表達(dá),增加cyclind3和pcna的表達(dá)。在體內(nèi)hucmsc-ex降低腎小管細(xì)胞tunel陽(yáng)性細(xì)胞數(shù)量及bax的表達(dá),增加pcna陽(yáng)性細(xì)胞數(shù)。在體外hucmsc-ex預(yù)防模型中,發(fā)現(xiàn)hucMSC-ex可以被轉(zhuǎn)運(yùn)到靶細(xì)胞中。在NRK-52E細(xì)胞中hucMSC-ex預(yù)處理增加自噬小體的數(shù)量及LC3B蛋白的表達(dá)。在體內(nèi)hucMSCex預(yù)處理具有相同的作用。(3)在順鉑處理的胃癌細(xì)胞模型中,hucMSC-ex預(yù)處理不會(huì)改變順鉑誘導(dǎo)的細(xì)胞凋亡及周期阻滯,胃癌細(xì)胞遷移和增殖能力與單獨(dú)順鉑處理組相比無(wú)明顯差異。(4)液相質(zhì)譜分析(LC-MS/MS)發(fā)現(xiàn)hucMSC-ex攜帶14-3-3ζ蛋白,并且將其轉(zhuǎn)運(yùn)至NRK-52E細(xì)胞中。在體內(nèi)外模型中hucMSC-ex預(yù)處理促進(jìn)14-3-3ζ蛋白表達(dá)升高。HucMSC-ex中14-3-3ζ過(guò)表達(dá)明顯增加NRK-52E細(xì)胞中自噬小體的形成和LC3B的表達(dá),同時(shí)降低順鉑誘導(dǎo)的NRK-52E細(xì)胞凋亡并促進(jìn)細(xì)胞增殖。在體內(nèi)AD-14-3-3ζ-ex降低血清BUN、Crea的水平以及腎臟TUNEL陽(yáng)性細(xì)胞數(shù)量。敲減hucMSC-ex中14-3-3ζ減弱自噬并加重順鉑誘導(dǎo)的NRK-52E細(xì)胞凋亡,抑制細(xì)胞增殖。(5)在體外模型中HucMSC-ex預(yù)處理促進(jìn)NRK-52E細(xì)胞ATG16L蛋白的表達(dá)。超分辨顯微鏡結(jié)果顯示14-3-3ζ促進(jìn)ATG16L蛋白的表達(dá)和聚集。免疫共沉淀結(jié)果顯示14-3-3ζ可以與ATG16L相互作用。結(jié)論:HucMSC-ex可以通過(guò)轉(zhuǎn)運(yùn)14-3-3ζ蛋白增強(qiáng)自噬從而預(yù)防順鉑誘導(dǎo)的急性腎損傷,同時(shí)不影響順鉑的抗腫瘤效果;14-3-3ζ與ATG16L相互作用,可以促進(jìn)自噬小體的形成。本研究為預(yù)防順鉑誘導(dǎo)的急性腎損傷及擴(kuò)展順鉑臨床應(yīng)用提供了新的思路和依據(jù)。
[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.

【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R730.5

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