本文選題：順鉑 + 腎毒性��； 參考：《江蘇大學》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用。課題組前期研究顯示人臍帶間質(zhì)干細胞(human umbilical cord mesenchymal stem cells,hucMSC)來源的外泌體(exosome)(簡稱hucMSC-ex)在體內(nèi)外模型中可以降低順鉑誘導的腎臟過氧化損傷及細胞凋亡,但hucMSC-ex預處理在順鉑誘導的腎損傷中的作用以及對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在順鉑誘導的腎毒性中的預防作用及機制,為腎損傷的預防提供新的策略。方法:(1)貼壁法分離培養(yǎng)hucMSC,并用試劑盒法提取exosomes,通過Nanosight納米顆粒分析儀對exosome的大小及濃度進行檢測分析;采用成像流式觀察hucMSC-ex表面標記CD9和CD63的表達。(2)使用CM-Dil染料對hucMSC-ex染色后與NRK-52E細胞共孵育,超分辨顯微鏡觀察NRK-52E細胞對hucMSC-ex的攝取。建立體外hucMSC-ex預防順鉑誘導腎小管上皮細胞(NRK-52E)損傷模型,分為3組:Control組,NRK-52E細胞不加任何處理;PBS組,與hucMSC-ex相同體積的PBS作用于NRK-52E細胞24h,順鉑損傷16h;hucMSC-ex組,hucMSC-ex作用于NRK-52E細胞24h后,順鉑處理16h。流式細胞儀Annexin V/PI雙染法分析NRK-52E細胞的凋亡情況;Western blot方法分析bax、Cyclin D3蛋白的表達;免疫熒光技術檢測核增殖抗原(PCNA)的表達情況。NRK-52E細胞轉(zhuǎn)染mRFP-GFP-LC3雙標腺病毒,超分辨顯微鏡觀察不同組別中自噬小體的形成;免疫熒光技術及Western blot分析自噬相關蛋白LC3B的表達;Western blot檢測14-3-3ζ蛋白的表達。構建體內(nèi)hucMSC-ex預防順鉑誘導的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;免疫組織化學染色檢測PCNA的表達;TUNEL染色分析腎小管上皮細胞的凋亡情況;westernblot檢測大鼠腎組織凋亡相關蛋白bax的表達情況,綜合評價hucmsc-ex對急性腎損傷的預防作用。westernblot分析大鼠腎組織lc3b的表達;免疫組織化學染色及westernblot檢測腎組織中14-3-3ζ的表達。(3)體外順鉑作用于胃癌細胞之前,用hucmsc-ex預處理,通過流式細胞技術分析胃癌細胞的凋亡情況及細胞周期改變;transwell實驗分析胃癌細胞的遷移能力;平板克隆形成實驗檢測胃癌細胞的增殖能力。(4)通過14-3-3ζ腺病毒過表達或慢病毒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hucmsc細胞中綠色熒光強度;westernblot分析hucmsc及hucmsc-ex中14-3-3ζ的表達;超分辨顯微鏡觀察nrk-52e細胞對ad-14-3-3ζ-ex(exosomes以cm-dil標記,14-3-3ζ蛋白表達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預處理nrk-52e細胞24h后順鉑損傷16h,超分辨顯微鏡觀察細胞中自噬小體的形成情況;westernblot檢測細胞中14-3-3ζ和lc3b的表達;免疫熒光技術觀察細胞中pcna的表達;tunel或流式細胞術分析細胞的凋亡情況。在體內(nèi)control組、pbs組、ad-gfp-ex組、ad-14-3-3ζ-ex組中,免疫組織化學技術檢測腎組織中14-3-3ζ的表達;westernblot分析lc3b的表達;血清學檢測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胞的凋亡情況;westernblot檢測大鼠腎組織凋亡相關蛋白caspase3的表達情況;免疫組織化學染色檢測pcna的表達。(5)通過免疫共沉淀技術和超分辨顯微鏡檢測14-3-3ζ與自噬相關蛋白atg16l之間的相互作用。結(jié)果:(1)人臍帶間質(zhì)干細胞來源的exosome,nanosight分析發(fā)現(xiàn)其是直徑為97nm左右的囊泡;成像流式檢測發(fā)現(xiàn)hucmsc-ex表達cd9和cd63,陽性率約為80%。(2)hucmsc-ex預處理減少順鉑誘導的nrk-52e細胞凋亡數(shù)量,降低bax的表達,增加cyclind3和pcna的表達。在體內(nèi)hucmsc-ex降低腎小管細胞tunel陽性細胞數(shù)量及bax的表達,增加pcna陽性細胞數(shù)。在體外hucmsc-ex預防模型中,發(fā)現(xiàn)hucMSC-ex可以被轉(zhuǎn)運到靶細胞中。在NRK-52E細胞中hucMSC-ex預處理增加自噬小體的數(shù)量及LC3B蛋白的表達。在體內(nèi)hucMSCex預處理具有相同的作用。(3)在順鉑處理的胃癌細胞模型中,hucMSC-ex預處理不會改變順鉑誘導的細胞凋亡及周期阻滯,胃癌細胞遷移和增殖能力與單獨順鉑處理組相比無明顯差異。(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)運至NRK-52E細胞中。在體內(nèi)外模型中hucMSC-ex預處理促進14-3-3ζ蛋白表達升高。HucMSC-ex中14-3-3ζ過表達明顯增加NRK-52E細胞中自噬小體的形成和LC3B的表達,同時降低順鉑誘導的NRK-52E細胞凋亡并促進細胞增殖。在體內(nèi)AD-14-3-3ζ-ex降低血清BUN、Crea的水平以及腎臟TUNEL陽性細胞數(shù)量。敲減hucMSC-ex中14-3-3ζ減弱自噬并加重順鉑誘導的NRK-52E細胞凋亡,抑制細胞增殖。(5)在體外模型中HucMSC-ex預處理促進NRK-52E細胞ATG16L蛋白的表達。超分辨顯微鏡結(jié)果顯示14-3-3ζ促進ATG16L蛋白的表達和聚集。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可以通過轉(zhuǎn)運14-3-3ζ蛋白增強自噬從而預防順鉑誘導的急性腎損傷,同時不影響順鉑的抗腫瘤效果;14-3-3ζ與ATG16L相互作用,可以促進自噬小體的形成。本研究為預防順鉑誘導的急性腎損傷及擴展順鉑臨床應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.

【學位授予單位】：江蘇大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R730.5

【相似文獻】

相關博士學位論文 前1條

1 賈浩源;hucMSC-exosome來源的14-3-3ζ活化自噬在預防順鉑毒中的作用及機制[D];江蘇大學;2017年

，

本文編號：1818365