【作者】 陈刚；

【导师】 吴小候；

【作者基本信息】 重庆医科大学 ， 泌尿外科， 2014， 博士

【摘要】 目的提取肾癌细胞株786-0来源的exosomes，探讨肾癌细胞株786-0来源的exosomes对肾癌细胞株786-0侵袭转移的影响；建立肾癌患者和健康志愿者血清中exosomes的提取及提纯方法，飞行质谱技术检测肾癌患者血清的exosomes与健康志愿者血清exosomes的差异蛋白，发现与肾癌侵袭转移相关蛋白；利用siRNA技术选择性阻断与肾癌侵袭转移相关蛋白血小板反应蛋白-1，进一步研究肾癌细胞分泌exosomes的血小板反应蛋白1在肾癌细胞侵袭转移中的作用及其相关机制。为肾癌的侵袭转移提供理论补充以及为转移性肾癌的生物治疗提供新思路。方法1.786-0细胞培养上清液中exosomes的提取与鉴定：采用蔗糖/重水密度梯度超速离心法提取并纯化细胞培养上清液中的exosomes。利用透射电镜、western-blot技术对exosomes的形态学以及蛋白分子进行。Bradford法检测exosomes的蛋白浓度。2. Exosomes对肾癌细胞株786-0的侵袭转移的影响，将exosomes（100μg/mL）、exosomes特异性抑制剂二甲基阿米洛利与786-0细胞共培养，划痕实验，Matrigel成胶Transwell小室实验以及粘附实验检测786-0细胞的侵袭、转移能力的变化。3. western-blot技术检测共培养前后786-0的CXCR4以及MMP-9的表达。4.血清中exosomes的提取，搜集肾癌病人18例及健康志愿者6例血清，蔗糖/重水密度梯度超速离心法提取并纯化exosomes。5.差异蛋白分析，利用Itraq技术分析肾癌病人血清来源的exosomes与健康志愿者来源的exosomes的差异蛋白组学6.分析差异蛋白结果，KEGG搜库以及PUBMED搜索，选择侵袭转移相关蛋白，利用免疫组织化学技术检测35例癌组织以及24例癌旁组织中血小板反应蛋白1（TSP-1）的表达； western-blott技术检测肾癌病人血清来源的exosomes与健康志愿者血清来源的exosomes中TSP-1的表达。7.Exosomes中TSP-1的敲除。化学合成TSP-1干扰片段， siRNA技术沉默786-0细胞TSP-1的表达，流式细胞技术检测转染的效率。western-blot技术检测沉默前后TSP-1在786-0来源的exosomes中的表达。8. Exosomes影响肾癌细胞株786-0侵袭转移机制的探讨。将TSP-1敲除的exosomes与786-0细胞共培养，划痕实验，Matrigel成胶Transwell小室实验以及粘附实验检测786-0细胞的侵袭、转移能力的变化；western-blott技术检测CXCR4以及MMP-9的表达。结果1.利用蔗糖/重水密度梯度超速离心法成功从病人血清以及细胞培养上清液中提取并纯化exosomes，透射电镜下exosomes为类球体。Western blot检测：肾癌细胞株786-0源性exosomes表达细胞间粘附分子（ICAM-1）、热休克蛋白70（HSP70）和G250。Bradford蛋白法检测发现exosomes的总蛋白浓度为1.5mg/ml～2.0mg/ml。为后续试验奠定基础。2.正常786-0来源的exosomes处理786-0细胞后，786-0细胞运动能力明显强于exosomes抑制组（二甲基阿米洛利处理组）以及PBS对照组，单位面积细胞数量分别为（55.84±7.60VS16.06±4.08VS29.17±1.72）；细胞侵袭转移能力明显增强，在侵袭转移实验中在24小时细胞穿过Matrigel成胶的Transwell小室基底膜的数量分别为（87.5±7.8VS29.3±11.7VS57.6±5.4）;细胞粘附能力降低，三组实验的粘附细胞数分别为（42.5±6.5VS71.5±7.5VS51.5±8.5）. Western-blott检测三组CXCR4以及MMP-9的表达，exosomes处理组786-0的CXCR4以及MMP-9的表达明显增高。3.Itraq分析显示肾癌患者血清来源的exosomes含有蛋白393种，其中肾癌病人来源的exosomes高表达蛋白51种，低表达5种。在高表达蛋白中，与肿瘤转移相关的功能蛋白3种：血小板反应蛋白1（TSP1）、细胞外基质蛋白1（ECM1）、色素上皮衍生因子（PEDF）。其中TSP-1在肾癌患者来源的exosomes中表达量最高（114：113=4.092）。低表达蛋白中胰岛素生长因子受体结合蛋白3（IBP3）与肿瘤转移相关。4.免疫组化结果显示TSP-1在肾癌以及癌旁组织中的表达没有显著区别，而Western-blot结果显示肾癌患者血清来源的exosomes中TSP-1的表达明显高于健康志愿者来源的exosomes。5.利用siRNA技术成功转染786-0细胞株，流式细胞结果显示在细胞转染72h转染效率为：46.21%。转染后786-0细胞株来源的exosomes中TSP-1表达量明显降低。6.沉默TSP-1蛋白的exosomes处理786-0细胞后，786-0细胞的迁徙能力明显弱于正常exosomes，单位面积细胞数量分别为（16.44±6.08VS53.84±6.70）；细胞侵袭转移能力明显减弱，在侵袭转移实验中在24小时细胞穿过Matrigel成胶的Transwell小室基底膜的数量分别为（22.5±12.8VS89.5±7.2）;细胞粘附能力增强，两组实验的粘附细胞数分别为（67.5±6.5VS38.5±6.5）. Western-blott检测三组CXCR4以及MMP-9的表达，TSP-1敲除exosomes处理组786-0的CXCR4以及MMP-9的表达明显降低。结论1.利用蔗糖/重水密度梯度超速离心法可以成功从病人血清以及细胞培养上清液中提取并纯化exosomes。提取的exosomes含有ICAM-1、HSP70以及G250等exosomes的结构蛋白以及来源细胞蛋白标记物。Exosomes的成功提取为后续试验奠定了坚实的实验基础。2.786-0来源的exosomes可以促进肿瘤细胞的侵袭转移能力，该促进作用可以被exosomes特异性抑制剂二甲基阿米洛利阻断。正常786-0细胞来源的exosomes可以促进786-0细胞的CXCR4以及MMP-9蛋白的表达。该研究结果为后续试验提供实验依据。3.Itraq技术可用于分析肾癌患者血清来源的exosomes的差异蛋白组学。差异蛋白分析结果显示TSP-1在肾癌病人血清来源的exosomes中高表达，且与肿瘤的转移密切相关。该研究结果可能为exosomes诱导肾癌细胞株786-0侵袭转移提供研究方向。4.TSP-1在肾癌组织以及癌旁组织中表达无显著差异，而在肾癌患者血清来源的exosomes中TSP-1的表达量明显高于健康志愿者血清来源的exosomes。提示肿瘤分泌的exosomes中的TSP-1可能参与了肿瘤的侵袭转移，为本实验提供了理论依据以及实验基础。5.利用TSP-1-shRNA技术能够成功的转染786-0细胞，并成功的沉默786-0细胞的TSP-1的表达。TSP-1敲除的exosomes对肾癌的侵袭转移能力的促进作用明显减弱，下游CXCR4以及MMP-9的表达量明显下调。提示exosomes促进肿瘤细胞侵袭转移的可能机制为exosomes利用自身的TSP-1蛋白，促进786-0细胞的CXCR4以及MMP-9的表达。更多还原

【Abstract】 ObjectiveTo extract exosomes from the renal cancer cell line786-0supernant, toinvestigate the effect of786-0derived exosomes on the invasion andmetastasis of786-0and the possible mechanism; To isolate and purify theexosomes from renal cancer and health volunteers serum, then Itraq tabbedflight mass spectrometry derived differential protemics were processed tofind the invasion related proteins; siRNA were used to selectively preventthe renal caner invasion related protein Thrombospondin-1expression inrenal cancer cell line secreted exosomes, then to further investigate thepossible role of renal caner secreted Thrombospondin-1in the invasion andmetastasis of renal cancer cell line786-0and the possible mechanism. Wehope that the above research will provide a theoretical supplement for renalcancer metastasis and new ideas biological treatment of metastatic renal cellcarcinoma.Methods1.Isolation and identification of exosomes from786-0supernatant:Ultracentrifugation and sucrose/water density gradient ultracentrifugation were used to isolate and purified the786-0derived exosomes, Transmissionelectron microscope(TEM) were used to observe the morphology of isolatedexosomes, Western blot were used to analyze the expression of HSP70,ICAM-1and G250. Bradford method were used to quantitatively Analyzethe total protein content of exosomes.2.The effect of renal cancer cell line786-0derived exosomes on theinvasion and migration of renal carcinoma cell line786-0: exosomes (100μg/mL), a specific inhibitor of exosomes Amiloride,5-(N, N-Dimethyl)-,hydrochloride,(7mmol/l) were co-cultured with786-0cells, after24hours,the786-0cells were collected. The wound healing assay, Matrigel basedinvasive Transwell cell experiment and cell adhesion experiments wereprocessed to check the invasion and migration capability of786-0cells.3.Western-blot were used to detecte the expression changes of CXCR4and matrix MMP-9on786-0cell.4.Isolation of exosomes from serum: the serum of18cases renal cancerpatients and6heaith volunteers were collected, Ultracentrifugation andsucrose/water density gradient ultracentrifugation were used to isolate andpurified the exosomes.5.Protemics analysis: Exosomes proteomic analysis were processed byisobaric tags for relative and absolute quantification (iTRAQ) techniquecombined with LC-MALDI-TOF/TOF.6.Protemics results analysis: Protemics results were analyzed by KEGG and PUBMED. The invasion related proteins were selected.Immunohistochemical technique were used to detect theThrombospondin(TSP-1) expression of26cases of renal cell carcinomaspecimens and26cases of cancer adjacent tissues. Western-blot were usedto detected the TSP-1expression in18cases of renal cell carcinoma patientsand6healthy volunteers.7.TSP-1knockdowm in exosomes. TSP-1interference fragment wereproduced by chemical synthesis, then was transfected into786-0.Flow cytometry were used to detect transfection efficiency. Stabletransfected cell line were collected. Exosomes extracted from ransfected celllines culture supernatant were harvested. Western-blott were used to analyzethe TSP-1expression.8.The pivotal role of secreted TSP-1in renal cancer cell line786-0andthe possible mechanism. The TSP-KO exosomes, and normal exosomeswere co-cultured with normal786-0cells for24hours. wound healing assay,Matrigel based invasive Transwell cell experiment and cell adhesionexperiments were processed to check the invasion and migration capabilityof786-0cells. Western-blot were used to detecte the expression changes ofCXCR4and matrix MMP-9on786-0cell.Results1.exosomes were successfully extracted from786-0cell culturesupernatant and patients or healthy volunteer serum. The morphology of the 786-0derived exosomes under Transmission electron microscopy werespheroid. Western blot found renal cell carcinoma cell line786-0inducedexosomes express intercellular adhesion molecule (ICAM-1), heat shockprotein70(HSP70) and G250. The Bradford methods found that theconcentration of extracted exosomes were1.5mg/ml～2.0mg/ml. Theabove findings lay the foundation for the following experiments.2.the renal cancer cell line786-0cells co-cultured with exosomesshowed higher cell migration capability than that of exosomes inhibitiongroup and PBS group, the number of migrated cells per unit area were55.84±7.60;16.06±4.08;29.17±1.72; respectively; cell invasion capabilitysignificantly increased, the cell number of traveling through the MatrigelTranswell basement membrane after24hours as were87.5±7.8,29.3±11.7,57.6±5.4,respectively; cell adhesion capability were significantlydecreased, the number of adherent cells in three groups of experiments were42.5±6.5,71.5±7.5,51.5±8.5, respectively. Western-blott were used todetect the CXCR4and MMP-9expression in the three groups. The CXCR4and MMP-9expression were significantly increased in exosomes treatmentgroup.3.ITAQ analysis showed that the exosomes containing393proteins, inwhich51proteins were highly expressed in renal cell carcinoma patientderived exosomes and5proteins were lowly expressed in renal cellcarcinoma patient derived exosomes. There exist three invasion related functional proteins among the highly expressed protein:Thrombospondin(TSP-1), Extracellular matrix protein1(ECM-1), Pigmentepithelium derived factor (PEDF). TSP-1were with the highest expressionamong the invasion related proteins(P114:113=4.092). Among the lowlyexpressed proteins, Insulin like growth factor binding protein were relatedwith the renal cancer invasion.4.Immunohistochemistry confirmed that the expression of TSP-1inrenal cancer tissue and in adjacent tissue were with no significantlydifference. However, Western-blot found that TSP-1expression in renalcancer patients serum were significantly higher than that in healthyvolunteers.5.Flow cytometry showed that TSP-1interference fragmentsuccessfully transfected into786-0cells by siRNA technology. Thetransfection rate were46.21%. The expression of TSP-1in786-0derivedexosomes were decreased when786-0cell transfected with TSP-1interference fragment.6.TSP-1protein Silenced exosomes decreased the migration ability of786-0cells compared with normal exosomes, the number of cells per unitarea were16.44±6.08,53.84±6.70, respectively; cell invasion capabilitywere significantly decreased, the cell number of traveling through theMatrigel Transwell basement membrane after24hours as were22.5±12.8,89.5±7.2, respectively; cell adhesion ability were enhanced, the number of adherent cells were67.5±6.5,38.5±6.5, respectively. Western-blott wereused to detect the CXCR4and MMP-9expression in the two groups. TheCXCR4and MMP-9expression were significantly decreased in TSP-1KOtreatment group.Conclusions1.The786-0derived exosomes and renal cancer derived exosomescould be successfully extracted and purified from the supernatant of renalcancer cell line786-0and serums by the use of ultrafiltration and sucrose/water density gradient ultracentrifugation. The exosomes expressed ICAM-1,HSP70, G250could be used for the follow-up co-culture experiments. Theseexperiments laid a solid basis for further research.2.Renal cell carcinoma cell line786-0derived exosomes can promotethe migration and invasion of786-0cells, reduce the cell adhesion ability.The above function could be prevented by exosomes specific inhibitorAmiloride,5-(N, N-Dimethyl)-, hydrochloride. The possible mechanismfor renal carcinoma cell line786-0derived exosomes promoting migrationand invasion of786-0cells might be that786-0derived exosomes couldenhance the786-0cell CXCR4and MMP-9expression. These findings mayprovide experimental evidence for the following experiment.3.Itraq technology could be used to analyse the differential protein ofexosomes in the serum of renal cancer patients and healthy volunteers. Theresults showed that TSP-1were highly expressed in renal cancer patients serum and highly related with renal cancer invasion. High expression ofthrombospondin-1may be a specific molecular target for renal cellcarcinoma invasion. These findings may provide a research area forexosomes derived renal cancer cell invasion.4.Immunohistochemistry confirmed that the expression of TSP-1inrenal cancer tissue and in adjacent tissue were with no significantlydifference. However, Western-blot found that TSP-1expression in renalcancer patients serum were significantly higher than that in healthyvolunteers. These findings suggested that TSP-1in tumor secreted exosomesmy play a pivotal role in renal cancer invasion and may provide a theoryevidence and experimental basis for the following experiment.5.TSP-1interference fragment successfully transfected into786-0cellsby siRNA technology. The expression of TSP-1in786-0derived exosomeswere decreased when786-0cell transfected with TSP-1interferencefragment.TSP-1silenced exosomes derived from786-0can inhibitthe migration and invasion of786-0cells, enhance the adhesion ability. Thepossible mechanism might be TSP-1silenced exosomes can inhibit theexpression of CXCR4and MMP-9.更多还原