本文選題：腎癌 + exosomes��； 參考：《重慶醫(yī)科大學》2014年博士論文

【摘要】：目的 提取腎癌細胞株786-0來源的exosomes，探討腎癌細胞株786-0來源的exosomes對腎癌細胞株786-0侵襲轉(zhuǎn)移的影響；建立腎癌患者和健康志愿者血清中exosomes的提取及提純方法，飛行質(zhì)譜技術(shù)檢測腎癌患者血清的exosomes與健康志愿者血清exosomes的差異蛋白，發(fā)現(xiàn)與腎癌侵襲轉(zhuǎn)移相關(guān)蛋白；利用siRNA技術(shù)選擇性阻斷與腎癌侵襲轉(zhuǎn)移相關(guān)蛋白血小板反應蛋白-1，進一步研究腎癌細胞分泌exosomes的血小板反應蛋白1在腎癌細胞侵襲轉(zhuǎn)移中的作用及其相關(guān)機制。為腎癌的侵襲轉(zhuǎn)移提供理論補充以及為轉(zhuǎn)移性腎癌的生物治療提供新思路。 方法 1.786-0細胞培養(yǎng)上清液中exosomes的提取與鑒定：采用蔗糖/重水密度梯度超速離心法提取并純化細胞培養(yǎng)上清液中的exosomes。利用透射電鏡、western-blot技術(shù)對exosomes的形態(tài)學以及蛋白分子進行。Bradford法檢測exosomes的蛋白濃度。 2. Exosomes對腎癌細胞株786-0的侵襲轉(zhuǎn)移的影響，，將exosomes（100μg/mL）、exosomes特異性抑制劑二甲基阿米洛利與786-0細胞共培養(yǎng)，劃痕實驗，Matrigel成膠Transwell小室實驗以及粘附實驗檢測786-0細胞的侵襲、轉(zhuǎn)移能力的變化。 3. western-blot技術(shù)檢測共培養(yǎng)前后786-0的CXCR4以及MMP-9的表達。 4.血清中exosomes的提取，搜集腎癌病人18例及健康志愿者6例血清，蔗糖/重水密度梯度超速離心法提取并純化exosomes。 5.差異蛋白分析，利用Itraq技術(shù)分析腎癌病人血清來源的exosomes與健康志愿者來源的exosomes的差異蛋白組學 6.分析差異蛋白結(jié)果，KEGG搜庫以及PUBMED搜索，選擇侵襲轉(zhuǎn)移相關(guān)蛋白，利用免疫組織化學技術(shù)檢測35例癌組織以及24例癌旁組織中血小板反應蛋白1（TSP-1）的表達； western-blott技術(shù)檢測腎癌病人血清來源的exosomes與健康志愿者血清來源的exosomes中TSP-1的表達。 7.Exosomes中TSP-1的敲除。化學合成TSP-1干擾片段， siRNA技術(shù)沉默786-0細胞TSP-1的表達，流式細胞技術(shù)檢測轉(zhuǎn)染的效率。western-blot技術(shù)檢測沉默前后TSP-1在786-0來源的exosomes中的表達。 8. Exosomes影響腎癌細胞株786-0侵襲轉(zhuǎn)移機制的探討。將TSP-1敲除的exosomes與786-0細胞共培養(yǎng)，劃痕實驗，Matrigel成膠Transwell小室實驗以及粘附實驗檢測786-0細胞的侵襲、轉(zhuǎn)移能力的變化；western-blott技術(shù)檢測CXCR4以及MMP-9的表達。 結(jié)果 1.利用蔗糖/重水密度梯度超速離心法成功從病人血清以及細胞培養(yǎng)上清液中提取并純化exosomes，透射電鏡下exosomes為類球體。Western blot檢測：腎癌細胞株786-0源性exosomes表達細胞間粘附分子（ICAM-1）、熱休克蛋白70（HSP70）和G250。Bradford蛋白法檢測發(fā)現(xiàn)exosomes的總蛋白濃度為1.5mg/ml～2.0mg/ml。為后續(xù)試驗奠定基礎。 2.正常786-0來源的exosomes處理786-0細胞后，786-0細胞運動能力明顯強于exosomes抑制組（二甲基阿米洛利處理組）以及PBS對照組，單位面積細胞數(shù)量分別為（55.84±7.60VS16.06±4.08VS29.17±1.72）；細胞侵襲轉(zhuǎn)移能力明顯增強，在侵襲轉(zhuǎn)移實驗中在24小時細胞穿過Matrigel成膠的Transwell小室基底膜的數(shù)量分別為（87.5±7.8VS29.3±11.7VS57.6±5.4）;細胞粘附能力降低，三組實驗的粘附細胞數(shù)分別為（42.5±6.5VS71.5±7.5VS51.5±8.5）. Western-blott檢測三組CXCR4以及MMP-9的表達，exosomes處理組786-0的CXCR4以及MMP-9的表達明顯增高。 3.Itraq分析顯示腎癌患者血清來源的exosomes含有蛋白393種，其中腎癌病人來源的exosomes高表達蛋白51種，低表達5種。在高表達蛋白中，與腫瘤轉(zhuǎn)移相關(guān)的功能蛋白3種：血小板反應蛋白1（TSP1）、細胞外基質(zhì)蛋白1（ECM1）、色素上皮衍生因子（PEDF）。其中TSP-1在腎癌患者來源的exosomes中表達量最高（114：113=4.092）。低表達蛋白中胰島素生長因子受體結(jié)合蛋白3（IBP3）與腫瘤轉(zhuǎn)移相關(guān)。 4.免疫組化結(jié)果顯示TSP-1在腎癌以及癌旁組織中的表達沒有顯著區(qū)別，而Western-blot結(jié)果顯示腎癌患者血清來源的exosomes中TSP-1的表達明顯高于健康志愿者來源的exosomes。 5.利用siRNA技術(shù)成功轉(zhuǎn)染786-0細胞株，流式細胞結(jié)果顯示在細胞轉(zhuǎn)染72h轉(zhuǎn)染效率為：46.21%。轉(zhuǎn)染后786-0細胞株來源的exosomes中TSP-1表達量明顯降低。 6.沉默TSP-1蛋白的exosomes處理786-0細胞后，786-0細胞的遷徙能力明顯弱于正常exosomes，單位面積細胞數(shù)量分別為（16.44±6.08VS53.84±6.70）；細胞侵襲轉(zhuǎn)移能力明顯減弱，在侵襲轉(zhuǎn)移實驗中在24小時細胞穿過Matrigel成膠的Transwell小室基底膜的數(shù)量分別為（22.5±12.8VS89.5±7.2）;細胞粘附能力增強，兩組實驗的粘附細胞數(shù)分別為（67.5±6.5VS38.5±6.5）. Western-blott檢測三組CXCR4以及MMP-9的表達，TSP-1敲除exosomes處理組786-0的CXCR4以及MMP-9的表達明顯降低。 結(jié)論 1.利用蔗糖/重水密度梯度超速離心法可以成功從病人血清以及細胞培養(yǎng)上清液中提取并純化exosomes。提取的exosomes含有ICAM-1、HSP70以及G250等exosomes的結(jié)構(gòu)蛋白以及來源細胞蛋白標記物。Exosomes的成功提取為后續(xù)試驗奠定了堅實的實驗基礎。 2.786-0來源的exosomes可以促進腫瘤細胞的侵襲轉(zhuǎn)移能力，該促進作用可以被exosomes特異性抑制劑二甲基阿米洛利阻斷。正常786-0細胞來源的exosomes可以促進786-0細胞的CXCR4以及MMP-9蛋白的表達。該研究結(jié)果為后續(xù)試驗提供實驗依據(jù)。 3.Itraq技術(shù)可用于分析腎癌患者血清來源的exosomes的差異蛋白組學。差異蛋白分析結(jié)果顯示TSP-1在腎癌病人血清來源的exosomes中高表達，且與腫瘤的轉(zhuǎn)移密切相關(guān)。該研究結(jié)果可能為exosomes誘導腎癌細胞株786-0侵襲轉(zhuǎn)移提供研究方向。 4.TSP-1在腎癌組織以及癌旁組織中表達無顯著差異，而在腎癌患者血清來源的exosomes中TSP-1的表達量明顯高于健康志愿者血清來源的exosomes。提示腫瘤分泌的exosomes中的TSP-1可能參與了腫瘤的侵襲轉(zhuǎn)移，為本實驗提供了理論依據(jù)以及實驗基礎。 5.利用TSP-1-shRNA技術(shù)能夠成功的轉(zhuǎn)染786-0細胞，并成功的沉默786-0細胞的TSP-1的表達。TSP-1敲除的exosomes對腎癌的侵襲轉(zhuǎn)移能力的促進作用明顯減弱，下游CXCR4以及MMP-9的表達量明顯下調(diào)。提示exosomes促進腫瘤細胞侵襲轉(zhuǎn)移的可能機制為exosomes利用自身的TSP-1蛋白，促進786-0細胞的CXCR4以及MMP-9的表達。
[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 鹵 7.60V16.06 鹵 4.08VS29.17 鹵 1.72 ) , and the number of cells per unit area was ( 55.84 鹵 7.60V16.06 鹵 4.08VS29.17 鹵 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 鹵 7.8VS29 . 3 鹵 11.7VS57.6 鹵 5.4 ) , and cell adhesion decreased , and the number of adherent cells in three groups were ( 42.5 鹵 6.5 VS71.5 鹵 7.5 V51.5 鹵 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 鹵 6.08VS53.84 鹵 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 鹵 12.8VS89.5 鹵 7.2 ) , and cell adhesion increased . The number of adherent cells in both groups were ( 67.5 鹵 6.5VS38.5 鹵 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 .
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R737.11

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相關(guān)期刊論文 前3條

1 劉安全;吳小候;羅春麗;;HepaCAM蛋白通過exosomes途徑分泌到細胞外并抑制腫瘤細胞增殖[J];第三軍醫(yī)大學學報;2012年02期

2 張俊;吳小候;陳剛;羅春麗;;腎細胞癌源性exosomes體外誘導單核細胞分化為PD-L1髓源性抑制細胞[J];第三軍醫(yī)大學學報;2012年02期

3 郭剛;張帆;杜青山;馮丹;張旭;;舒尼替尼在晚期腎細胞癌二線序貫治療中的臨床應用研究[J];臨床泌尿外科雜志;2014年01期

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