肾癌源性exosomes对肾癌细胞株786-0侵袭转移的影响及相关机制研究

发布时间：2018-05-19 02:39

本文选题：肾癌 + exosomes　；参考：《重庆医科大学》2014年博士论文

【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。
[Abstract]:Purpose

The effect of exosomes derived from renal cancer cell line 786 - 0 on invasion and metastasis of renal cancer cell line 786 - 0 was investigated .
To establish the extraction and purification methods of exosomes from serum of renal cancer patients and healthy volunteers , and the difference protein of exosomes and serum exosomes from healthy volunteers was detected by flight mass spectrometry .
Using siRNA technique to selectively block the expression of platelet - reactive protein - 1 associated with invasion and metastasis of renal cell carcinoma , we further study the role of exosomes - secreting exosomes in the invasion and metastasis of renal cancer cells and related mechanisms . It provides theoretical supplement for invasion and metastasis of renal cancer and provides a new idea for the biological treatment of metastatic renal cancer .

method

1 . Extraction and identification of exosomes from culture supernatant of 786 - 0 cell culture : The exosomes were extracted and purified from cell culture supernatant by means of sucrose / heavy water density gradient method . The morphology of exosomes and protein molecules were determined by means of transmission electron microscope and western - blot .

2 . The effects of Exosomes on invasion and metastasis of renal cancer cell line 786 - 0 , exosomes ( 100 渭g / mL ) , exosomes - specific inhibitor dimethyl amiloride and 786 - 0 cell co - culture , scratch test , Matrigel gel - forming Transwell chamber experiment and adhesion experiment were used to detect the invasion and metastasis ability of 786 - 0 cells .

3 . The expression of CXCR4 and MMP - 9 was detected by western - blot .

4 . The exosomes were extracted from serum , 18 patients with renal cancer and 6 healthy volunteers were collected , and the exosomes were extracted and purified by sucrose / heavy water density gradient centrifugation .

5 . Differential protein analysis , using Itraq technique to analyze the difference of exosomes and exosomes from healthy volunteers from patients with renal cancer .

6 . Analysis of the results of variance protein , the search of the cell search and PUBMED search , the selection of invasion and metastasis related proteins , the detection of 35 cancer tissues by immunohistochemistry and the expression of thrombospondin 1 ( TSP - 1 ) in 24 cases of adjacent tissues .
The expression of TSP - 1 in exosomes derived from serum derived exosomes and healthy volunteers was detected by western - blott technique .

7 . The expression of TSP - 1 in Exosomes was determined by chemical synthesis of TSP - 1 interfering fragment , siRNA technology silencing 786 - 0 cell TSP - 1 and flow cytometry . Western - blot was used to detect the expression of TSP - 1 in exosomes from 786 - 0 .

8 . Effects of Exosomes on the invasion and metastasis mechanism of 786 - 0 cell line 786 - 0 in renal cell carcinoma cell line 786 - 0 . The exosomes were co - cultured with 786 - 0 cells , scratch test , Matrigel gel - gel Transwell chamber experiment and adhesion experiment were used to detect the invasion and metastasis ability of 786 - 0 cells .
The expression of CXCR4 and MMP - 9 was detected by western - blott technique .

Results

1 . exosomes were extracted and purified from serum and cell culture supernatant by sucrose / heavy water density gradient method . The exosomes of exosomes were detected by Western blot . The total protein concentration of exosomes was 1.5mg / ml ~ 2.0mg / ml .

2 . After 786 - 0 cells were treated with exosomes from 786 - 0 source , 786 - 0 cells were significantly stronger than exosomes ( 55.84 卤 7.60V16.06 卤 4.08VS29.17 卤 1.72 ) , and the number of cells per unit area was ( 55.84 卤 7.60V16.06 卤 4.08VS29.17 卤 1.72 ) .
The invasion and metastasis ability of cells was significantly increased , and the number of basement membrane of Transwell cells passing through Matrigel in invasion and metastasis was ( 87.5 卤 7.8VS29 . 3 卤 11.7VS57.6 卤 5.4 ) , and cell adhesion decreased , and the number of adherent cells in three groups were ( 42.5 卤 6.5 VS71.5 卤 7.5 V51.5 卤 8.5 ) . The expression of CXCR4 and MMP - 9 was detected by Western - blott . The expression of CXCR4 and MMP - 9 in the exosomes treated group was significantly increased .

3 . Itraq analysis showed that exosomes derived from patients with renal cell carcinoma contain 393 kinds of protein , including 51 kinds of exosomes derived from renal cell carcinoma and 5 kinds of low expression . In high expression protein , three kinds of functional protein related to tumor metastasis : thrombospondin 1 ( TSP1 ) , extracellular matrix protein 1 ( ECM1 ) and pigment epithelium derived factor ( PEDF ) . Among them , TSP - 1 is the highest in exosomes derived from renal cancer patients ( 114 : 113 = 4.092 ) . Insulin growth factor receptor binding protein 3 ( IBP3 ) in a low expression protein is associated with tumor metastasis .

4 . Immunohistochemical results showed that TSP - 1 had no significant difference in the expression of TSP - 1 in renal cell carcinoma and adjacent tissues . Western - blot showed that the expression of TSP - 1 in exosomes of renal cancer patients was significantly higher than that of exosomes derived from healthy volunteers .

5 . Using siRNA technology successfully transfected the 786 - 0 cell line , the results of flow cytometry showed that the transfection efficiency was 46.21 % after transfection , and the expression of TSP - 1 in exosomes derived from 786 - 0 cell line was significantly decreased after transfection .

6 . After the treatment of 786 - 0 cells by exosomes of TSP - 1 protein , the migration ability of 786 - 0 cells was significantly weaker than that of normal exosomes , and the number of cells per unit area was ( 16.44 卤 6.08VS53.84 卤 6.70 ) .
The cell invasion and metastasis ability was significantly decreased , and the number of basement membrane of Transwell cells passing through Matrigel in invasion and metastasis was ( 22.5 卤 12.8VS89.5 卤 7.2 ) , and cell adhesion increased . The number of adherent cells in both groups were ( 67.5 卤 6.5VS38.5 卤 6.5 ) . The expression of CXCR4 and MMP - 9 was detected by Western - blott . The expression of CXCR4 and MMP - 9 in the exosomes treated by TSP - 1 was significantly decreased .

Conclusion

1 . exosomes were extracted and purified from serum and cell culture supernatant by sucrose / heavy water density gradient method . exosomes extracted from exosomes contain the structural proteins of exosomes such as ICAM - 1 , HSP70 and G250 , as well as source cell protein markers . The successful extraction of Exosomes provides a solid experimental basis for subsequent experiments .

2.786 - 0 - derived exosomes can promote the invasion and metastasis of tumor cells , which can be blocked by exosomes - specific inhibitor dimethyl amiloride . exosomes derived from normal 786 - 0 cells can promote CXCR4 expression and MMP - 9 protein expression in 786 - 0 cells .

3 . Itraq technique can be used to analyze the differential protein group of exosomes from patients with renal cancer . The results of differential protein analysis indicate that TSP - 1 is highly expressed in exosomes derived from serum from patients with renal cancer , and is closely related to metastasis of tumor . The results may provide a research direction for exosomes to induce invasion and metastasis of renal cancer cell line 786 - 0 .

4 . There was no significant difference in the expression of TSP - 1 in the tissues of renal cancer and adjacent tissues , but the expression of TSP - 1 in exosomes from patients with renal cancer was significantly higher than that from healthy volunteers .

5 . The expression of TSP - 1 in 786 - 0 cells was successfully transfected with TSP - 1 - shRNA technology . The effect of exosomes on invasion and metastasis of renal cell carcinoma was significantly reduced . The possible mechanism of exosomes to promote invasion and metastasis of tumor cells was exosomes , which promoted the expression of CXCR4 and MMP - 9 in 786 - 0 cells .
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.11

【参考文献】