本文選題：結(jié)直腸癌　切入點(diǎn)：CD95　出處：《吉林大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：目的 本研究探討介導(dǎo)炎癥反應(yīng)的JAK2和介導(dǎo)免疫反應(yīng)的CD95與結(jié)直腸癌預(yù)后的相關(guān)性；探討JAK2和CD95受體在結(jié)直腸癌組織及細(xì)胞株中的表達(dá)水平，分析二者的相關(guān)性；通過抑制或增強(qiáng)JAK2的活化，探討大腸癌細(xì)胞在增殖能力、細(xì)胞表型（上皮型/間質(zhì)型）以及CD95活性方面的變化，從細(xì)胞和分子水平證實(shí)JAK2介導(dǎo)的炎癥信號(hào)通路與大腸癌細(xì)胞的免疫功能之間的關(guān)系及其作用機(jī)制。 方法 1.利用生物信息學(xué)分析的方法：借助荷蘭醫(yī)學(xué)生物數(shù)據(jù)平臺(tái)，分析來源于荷蘭8家醫(yī)院的大腸癌數(shù)據(jù)庫中CD95和JAK2的表達(dá)水平、CD95和JAK2的表達(dá)與生存期的Kaplan-Meier曲線，借助平臺(tái)中的基因熱圖（geneheatmap），將與CD95關(guān)系密切的基因呈現(xiàn)出來，以形象地呈現(xiàn)出CD95表達(dá)相關(guān)的功能基因；利用STRING在線軟件分析預(yù)測CD95相關(guān)蛋白及其功能；利用基因文氏圖（Gvenn）分析CD95相關(guān)基因與JAK2相關(guān)基因的重疊程度，并預(yù)測數(shù)據(jù)庫中CD95與JAK2的相關(guān)性。利用在線meta軟件計(jì)算3個(gè)數(shù)據(jù)庫中CD95與JAK2總體相關(guān)性系數(shù)（r值、p值）。 2.結(jié)直腸癌組織切片和細(xì)胞的獲取：2011年10月-2012年5月在荷蘭Utrecht大學(xué)醫(yī)學(xué)中心胃腸外科經(jīng)手術(shù)切除的結(jié)直腸癌組織，經(jīng)福爾馬林固定、石蠟包埋后制成結(jié)直腸癌組織切片，從中選取10例石蠟包埋組織切片；取同期手術(shù)切除的結(jié)直腸癌新鮮組織進(jìn)行細(xì)胞建株并穩(wěn)定傳代獲得結(jié)直腸癌（CRC）細(xì)胞，本實(shí)驗(yàn)選取其中9株用于研究。 3. CD95和JAK2相關(guān)性分析：通過免疫組化染色檢測CD95和JAK2在結(jié)直腸癌組織切片中的表達(dá)水平，通過免疫印跡法檢測二者在CRC細(xì)胞中的表達(dá)水平，并分析二者的相關(guān)性。 4.結(jié)直腸癌細(xì)胞中JAK2/STAT3的阻斷：在細(xì)胞培養(yǎng)體系中加入JAK2特異性抑制劑AZD1480和CEP33779，通過western blot檢測AZD1480和CEP33779在不同濃度（0.5μM~5μM）和不同作用時(shí)間（2h~24h）下JAK2、STAT3和pSTAT3水平，對(duì)照組為同一細(xì)胞株在相應(yīng)濃度DMSO和作用時(shí)間下JAK2、STAT3和pSTAT3水平，陽性對(duì)照采用同一細(xì)胞株在IL-6（10ng/ml，30min）刺激后JAK2、STAT3和pSTAT3的水平。 5.結(jié)直腸癌細(xì)胞的克隆形成能力檢測：結(jié)直腸癌細(xì)胞團(tuán)被解離成單個(gè)細(xì)胞后，以1000個(gè)細(xì)胞/100μl基質(zhì)膠的濃度接種到12孔板中，待基質(zhì)膠凝固后每孔添加2ml培養(yǎng)基，實(shí)驗(yàn)組及對(duì)照組試劑添加到培養(yǎng)基中。2天換1次液，12天后觀察細(xì)胞團(tuán)形成數(shù)量。 6.流式細(xì)胞技術(shù)檢測細(xì)胞凋亡：結(jié)直腸癌細(xì)胞分別經(jīng)過一定濃度的JAK2特異性抑制劑AZD1480、CEP33779作用2天后，以等劑量的DMSO處理CRC細(xì)胞2天作為對(duì)照，采用熒光染料碘化吡啶（PI）染色（4℃）固定，4h后流式細(xì)胞儀檢測細(xì)胞凋亡的百分率。 7.免疫熒光和實(shí)時(shí)熒光定量PCR檢測：JAK2特異性抑制劑CEP33779抑制JAK2/STAT3通路活化，對(duì)參與上皮-間質(zhì)轉(zhuǎn)化（EMT）的相關(guān)蛋白進(jìn)行免疫熒光檢測，對(duì)EMT標(biāo)志物HIF-1、ZEB-1、ZEB-2、FABP、Snail-1、Snail-2、vimentin等轉(zhuǎn)錄因子的mRNA進(jìn)行逆轉(zhuǎn)錄后熒光定量PCR檢測。 8. FasL刺激下細(xì)胞分泌細(xì)胞因子的檢測：實(shí)驗(yàn)組培養(yǎng)體系FasL終濃度20ng/ml，對(duì)照組為同株無FasL的細(xì)胞，12h后收集上清液，經(jīng)層析、濃縮后，通過細(xì)胞因子芯片檢測結(jié)直腸癌細(xì)胞分泌細(xì)胞因子的變化。 9. CD95-黃色熒光蛋白報(bào)告基因（YFP-CD95）的轉(zhuǎn)染及CRC細(xì)胞的共聚焦顯微鏡檢查：分別構(gòu)建pWPT-CD95-YFP，同時(shí)構(gòu)建pWPT-YFP作為對(duì)照。采用磷酸鈣法轉(zhuǎn)染，，使用FuGene轉(zhuǎn)染試劑將慢病毒包裝載體和pWPT-CD95-YFP或pWPT-YFP轉(zhuǎn)染進(jìn)293T細(xì)胞。細(xì)胞培養(yǎng)24-48h后收集上清液獲得病毒。CRC29和L145細(xì)胞培養(yǎng)6-16h后進(jìn)行解離，用聚凝胺進(jìn)行慢病毒上清轉(zhuǎn)染24h。轉(zhuǎn)染后的細(xì)胞根據(jù)YFP-CD95表達(dá)高低經(jīng)流式細(xì)胞分選，從而獲得純度較高的CD95高表達(dá)細(xì)胞株。利用FasL和/或CEP33779作用CRC細(xì)胞1h、2h后，在共聚焦熒光顯微鏡下觀察細(xì)胞表面YFP-CD95熒光信號(hào)的變化。 結(jié)果 1. CD95與JAK2在結(jié)直腸癌中的相關(guān)性 （1）生物信息學(xué)分析CD95和JAK2與結(jié)直腸癌患者預(yù)后呈正相關(guān)CD95和JAK2在8個(gè)結(jié)直腸癌數(shù)據(jù)庫共1293例結(jié)直腸癌病人組織中每組內(nèi)部表達(dá)水平差異較大，而各數(shù)據(jù)庫間無顯著性差異；CD95和JAK2高表達(dá)，病人生存期較短。 （2）CD95與JAK2在結(jié)直腸癌中的表達(dá)及功能相關(guān)性 STRING分析顯示CD95與結(jié)直腸癌的免疫反應(yīng)、炎癥反應(yīng)與炎癥因子關(guān)系密切。Gvenn分析顯示CD95相關(guān)基因與JAK2高度相關(guān)。在線meta分析發(fā)現(xiàn)CD95相關(guān)基因中JAK2與其相關(guān)性最好。與CD95同時(shí)表達(dá)的基因，能夠調(diào)控炎癥反應(yīng)、JAK2/STAT3信號(hào)通路、上皮-間質(zhì)轉(zhuǎn)化（EMT）。對(duì)10例結(jié)直腸癌石蠟切片的免疫組化檢查發(fā)現(xiàn)CD95高表達(dá)的腫瘤組織同時(shí)存在JAK2高表達(dá)，反之亦然，二者呈正相關(guān)。 （3）CD95和JAK2在結(jié)直腸癌細(xì)胞中表達(dá)的相關(guān)性 經(jīng)細(xì)胞建株并穩(wěn)定傳代的結(jié)直腸癌細(xì)胞株，包括來源于結(jié)直腸癌原發(fā)腫瘤的細(xì)胞株CRC26、CRC29、CR9、CR16、CRC48、CRC47，來源于肝轉(zhuǎn)移灶的細(xì)胞株為L145、L167、L169。JAK2表達(dá)水平CRC29、CR9、CR16最高，其余依次為L169、CRC47、CRC48、L167、L145、CRC26，CD95表達(dá)水平由高到低依次為CRC29、L145、CRC47、CR16、CRC48、CRC26、L169、CR9、L167。9株CRC細(xì)胞株中CD95與JAK2的表達(dá)具有一定的相關(guān)性（r2=0.5087，p=0.0470），而來源結(jié)直腸的原發(fā)腫瘤細(xì)胞CD95與JAK2的表達(dá)相關(guān)性較好（r2=0.7360，p=0.0289）。 2. JAK2對(duì)結(jié)直腸癌細(xì)胞增殖、凋亡、EMT的影響 （1）JAK2活化抑制后結(jié)直腸癌細(xì)胞的克隆增殖能力變化結(jié)直腸癌細(xì)胞在基質(zhì)膠中培養(yǎng)，對(duì)照組用等濃度的DMSO處理，實(shí)驗(yàn)組利用JAK2抑制劑阻斷JAK2活化，其克隆增殖能力明顯降低。 （2）JAK2活化后結(jié)直腸癌細(xì)胞的克隆增殖能力變化結(jié)直腸癌細(xì)胞在基質(zhì)膠中培養(yǎng)，實(shí)驗(yàn)組利用合適濃度的IL-6活化JAK2，其克隆增殖能力較對(duì)照組明顯增強(qiáng)。不同IL-6濃度刺激下結(jié)直腸癌細(xì)胞的克隆增殖無明顯差異。 （3）JAK2阻斷后CRC細(xì)胞凋亡率變化 對(duì)于懸浮培養(yǎng)的結(jié)直腸癌細(xì)胞CRC29，用不同作用濃度的JAK2特異性抑制劑AZD1480和CEP33779分別阻斷JAK2活化，抑制劑作用2天后，采用碘化吡啶（Propidium Iodide，PI）染色法檢測CRC細(xì)胞凋亡。與對(duì)照組相比，JAK2特異性抑制后CRC細(xì)胞凋亡無明顯增加（p0.05）。 （4）JAK2阻斷下FasL誘導(dǎo)細(xì)胞凋亡率變化 對(duì)于懸浮培養(yǎng)的結(jié)直腸癌細(xì)胞CRC29，JAK2特異性抑制AZD1480和CEP33779阻斷JAK2活化，2h后在培養(yǎng)體系中加入殺傷劑量（20ng/ml）的FasL，作用2天后檢測CRC細(xì)胞凋亡。AZD1480和CEP33779兩組中CRC29細(xì)胞凋亡率分別為33.52%、38.76%，AZD1480預(yù)先抑制JAK2活化后FasL誘導(dǎo)CRC29細(xì)胞凋亡增至41.20%、45.19%，CEP33779預(yù)先抑制JAK2活化后FasL誘導(dǎo)CRC29細(xì)胞凋亡增至62.34%、62.65%。 （5）JAK2抑制后與EMT相關(guān)的蛋白表達(dá)及轉(zhuǎn)錄因子水平發(fā)生變化 ①CRC29細(xì)胞系在懸浮培養(yǎng)條件下，與IL-6刺激的CRC29細(xì)胞系相比，受到JAK2抑制的CRC29細(xì)胞系Caveolin-1表達(dá)減低，E-cadherin表達(dá)升高，共聚焦顯微鏡下可見到實(shí)驗(yàn)組細(xì)胞－細(xì)胞連接處Caveolin-1熒光信號(hào)減弱，E-cadherin熒光信號(hào)明顯增強(qiáng)。②CRC29細(xì)胞系在懸浮培養(yǎng)條件下，與IL-6刺激的CRC29細(xì)胞系相比，受到JAK2抑制的CRC29細(xì)胞系HIF-1、ZEB-1、FABP、Snail-1、等基因mRNA表達(dá)降低，其中ZEB-2、Snail-2和HIF-2變化最為明顯，vimentin表達(dá)增加。 3.特異性阻斷JAK2對(duì)CRC細(xì)胞CD95表型的影響 （1）FasL能夠刺激CRC細(xì)胞分泌細(xì)胞因子變化 FasL作用24h后CRC細(xì)胞分泌IL-8、IL-15R alpha水平升高，而預(yù)先加入CEP33779抑制JAK2活化的CRC細(xì)胞分泌IL-8水平進(jìn)一步升高。 （2）轉(zhuǎn)染CD95-YFP細(xì)胞株對(duì)FasL及JAK2阻斷的反應(yīng) 對(duì)成功轉(zhuǎn)染了CD95-YFP質(zhì)粒并穩(wěn)定表達(dá)CD95-YFP的結(jié)直腸癌細(xì)胞系CRC29，分別用FasL、CEP33779、CEP33779預(yù)處理1h而后FasL（CEP33779→FasL），作用1h后，給予固定，隨后通過熒光顯微鏡觀察細(xì)胞膜上帶有黃色熒光信號(hào)的CD95的表型變化，即是否聚集成為活性形式，對(duì)照組以等濃度的DMSO作用于培養(yǎng)體系，結(jié)果發(fā)現(xiàn)在JAK2阻斷1h后，細(xì)胞膜表面熒光強(qiáng)度明顯高于對(duì)照組，而CEP33779→FasL處理組CRC細(xì)胞表面CD95表達(dá)明顯耗減。 結(jié)論 1.對(duì)多個(gè)結(jié)直腸癌數(shù)據(jù)庫的生存曲線薈萃分析得知，結(jié)直腸癌組織中CD95和JAK2高表達(dá)，患者的生存期較短。 2.結(jié)直腸癌數(shù)據(jù)庫中CD95與JAK2在表達(dá)、功能相關(guān)通路等方面的相關(guān)性分析表明，CD95與JAK2在結(jié)直腸癌組織中的高度相關(guān)性，主要體現(xiàn)在腫瘤的免疫反應(yīng)、炎癥反應(yīng)、JAK2/STAT3信號(hào)通路、上皮-間質(zhì)轉(zhuǎn)化（EMT）幾個(gè)方面。 3.體外培養(yǎng)的結(jié)直腸癌細(xì)胞在增殖過程中，JAK2/STAT3處于較低水平的活化狀態(tài)，特異性阻斷JAK2/STAT3通路能夠降低或完全抑制腫瘤細(xì)胞的克隆形成能力，提示JAK2在腫瘤細(xì)胞增殖方面具有關(guān)鍵作用；特異性阻斷JAK2/STAT3通路在一定程度上抑制了腫瘤EMT，從而對(duì)腫瘤細(xì)胞的遷移可能產(chǎn)生一定的抑制作用；而JAK2/STAT3的過度活化則促進(jìn)腫瘤細(xì)胞增殖。JAK2抑制不能誘導(dǎo)CRC細(xì)胞凋亡，而FasL可誘導(dǎo)腫瘤細(xì)胞凋亡增多。 4.通過轉(zhuǎn)染CD95-YFP報(bào)告基因并使其穩(wěn)定表達(dá)CD95的結(jié)直腸癌細(xì)胞系CRC29（CRC29CD95-YFP），抑制JAK2/STAT3通路有助于腫瘤細(xì)胞表面CD95單體寡聚化形成轉(zhuǎn)變成活性形式，為介導(dǎo)細(xì)胞凋亡提供細(xì)胞表面結(jié)構(gòu)條件，從而證明JAK2對(duì)CRC細(xì)胞膜上CD95具有一定的穩(wěn)定作用。
[Abstract]:objective
This study was to investigate the relationship between CD95 and prognosis of colorectal cancer mediated inflammation and JAK2 mediated immune response; to investigate the expression of JAK2 and CD95 receptor in colorectal cancer tissues and cell lines, analysis of the relationship between the two; by inhibiting or enhancing the activation of JAK2 on colorectal cancer cell in proliferation, cell phenotype (epithelial / mesenchymal type) changes and the activity of CD95, confirmed the relationship between immune function and inflammatory signaling pathways in colorectal cancer cells mediated by JAK2 and the mechanism of the cellular and molecular level.
Method
1. by bioinformatics analysis: with the Holland medical and biological data platform, the expression level of CD95 and JAK2 analysis from 8 hospitals in Holland of colorectal cancer database, CD95 and JAK2 expression and survival curve of Kaplan-Meier, with the help of gene thermography in the platform (geneheatmap), the close relationship with CD95 gene to show, vividly showing the expression of CD95 related functional genes; analysis and prediction of CD95 protein and its function using STRING online software; using gene Venn diagram (Gvenn) analysis of overlap CD95 related genes associated with JAK2 gene, and to predict the relationship between CD95 and JAK2 in the database. Using the online software meta 3 the database CD95 and JAK2 general correlation coefficient (r value, P value).
To obtain 2. colorectal cancer tissues and cells: October 2011 -2012 year in May in gastrointestinal surgery, Utrecht University Medical Center in Holland after surgical resection of colorectal cancer, formalin fixed, paraffin embedded tissue sections made of colorectal cancer, 10 cases of paraffin embedded tissue sections from; take over the same period surgical resection of colorectal cancer tissues cell lines stably and obtain colorectal cancer (CRC) cells, 9 of them were selected for this experiment.
3. CD95 and JAK2 correlation analysis: immunohistochemical staining was used to detect the expression level of CD95 and JAK2 in colorectal cancer tissue slice. The expression level of two in CRC cells was detected by Western blotting, and the correlation between the two was analyzed.
Blockade of JAK2/STAT3 4. in colorectal cancer cell: the JAK2 specific inhibitor AZD1480 and CEP33779 were added into the culture system, through the Western blot detection of AZD1480 and CEP33779 in different concentration (0.5 M~5 M) and different time (2h~24h) of JAK2, STAT3 and pSTAT3, the control group for the same cell lines in the corresponding the concentration of DMSO and reaction time JAK2, STAT3 and pSTAT3, positive control using the same cell line in IL-6 (10ng/ml, 30min) after the stimulation of JAK2, STAT3 and pSTAT3 levels.
The ability to detect the formation of clone 5. colorectal cancer cells: colorectal cancer cells were dissociated into single cells after inoculation of 1000 cells in a concentration of /100 l matrix to 12 Kong Banzhong, the matrix Plastic solidified per hole adding 2ml medium, the experimental group and the control group reagent was added to the medium.2 for 1 days liquid, 12 days after the observation of cell colony number.
6. flow cytometry: apoptosis of colorectal cancer cells respectively by JAK2 specific inhibitor AZD1480 in certain concentration, CEP33779 for 2 days, DMSO cells were treated with low-dose CRC for 2 days as control, using the fluorescent dye propidium iodide (PI) staining (4 C) fixed, the percentage of 4H cell apoptosis detection cell apparatus.
7. immunofluorescence and real-time fluorescence quantitative PCR detection of JAK2 specific inhibitor CEP33779 inhibits the activation of JAK2/STAT3 pathway, involved in epithelial mesenchymal transition (EMT) related proteins were detected by immunofluorescence, EMT markers HIF-1, ZEB-1, ZEB-2, FABP, Snail-1, Snail-2, vimentin and other transcription factors mRNA reverse transcription fluorescence quantitative detection of PCR.
8. FasL stimulated the secretion of cytokines detection cell culture system: experimental group FasL final concentration of 20ng/ml, the control group without monoecious FasL cells, 12h cell culture supernatants were collected by chromatography, concentration, the changes of cytokine microarray for detection of colorectal cancer cells to secrete cytokines.
9. CD95- yellow fluorescent protein (YFP-CD95) and confocal microscopy and transfection of CRC cells: pWPT-CD95-YFP were constructed, pWPT-YFP was constructed as control. Using calcium phosphate transfection method, using FuGene transfection reagent package lentivirus vector and pWPT-CD95-YFP or pWPT-YFP and transfected into 293T cells. The cell culture supernatant was collected after dissociation of the 24-48h were obtained.CRC29 virus and L145 cell culture after 6-16h was transfected into 24h. by lentivirus supernatant of transfected cells after Polybrene according to the expression level of YFP-CD95 by flow cytometry, so as to obtain a high degree of purity of CD95 high expression cell line. Using FasL and / or CEP33779 CRC cells 1H, 2h, observe the changes of cell surface YFP-CD95 the fluorescence signal in the confocal fluorescence microscope.
Result
Correlation between 1. CD95 and JAK2 in colorectal cancer
(1) bioinformatics analysis of CD95 and JAK2 in colorectal cancer and the prognosis of the patients was positively related to CD95 and JAK2 within each in 8 colorectal cancer database of 1293 cases of colorectal cancer patients the expression level differences, but no significant difference between each database; high expression of CD95 and JAK2, the survival time is short.
(2) expression and function correlation of CD95 and JAK2 in colorectal cancer
STRING analysis showed that the immune response of CD95 and colorectal cancer, the relationship between inflammatory response and inflammatory factors in close.Gvenn analysis showed that CD95 gene is highly correlated with JAK2. The online meta analysis showed that CD95 related gene JAK2 and its expression in the best correlation. At the same time with the CD95 gene, can control inflammation, JAK2/STAT3 signaling pathway, epithelial mesenchymal transition (EMT). Immunohistochemistry in 10 cases of colorectal cancer paraffin section examination found high expression of CD95 in tumor tissue and high expression of JAK2, and vice versa, two were positively correlated.
(3) the correlation between CD95 and JAK2 expression in colorectal cancer cells
The cell lines and stable passage of the colorectal cancer cell lines, including from the primary colorectal tumor cell lines CRC26, CRC29, CR9, CR16, CRC48, CRC47, derived from liver metastasis cell lines L145, L167, L169.JAK2 expression level of CRC29, CR9, CR16 is the highest, followed by the remaining L169 CRC47, CRC48, L167, L145, CRC26, CD95 expression level from high to low was CRC29, L145, CRC47, CR16, CRC48, CRC26, L169, CR9, CD95 and JAK2 expression of CRC cell lines in L167.9 has a certain correlation (r2=0.5087, p=0.0470), and the primary source of colorectal CD95 and the good correlation between the expression of JAK2 tumor cells (r2=0.7360, p=0.0289).
Effect of 2. JAK2 on the proliferation, apoptosis and EMT of colorectal cancer cells
(1) the clonal proliferation ability of colorectal cancer cells was inhibited after JAK2 activation inhibition. The colorectal cancer cells were cultured in matrix glue. The control group was treated with equal concentration of DMSO. The experimental group used JAK2 inhibitor to block JAK2 activation, and the clonal proliferation ability of colorectal cancer cells was significantly reduced.
(2) the proliferation changes of JAK2 activation after cloning of colorectal cancer cells of colorectal cancer cells cultured on Matrigel, the experimental group using the appropriate concentration of IL-6 activated JAK2, the clone proliferation ability were markedly increased compared to control group. Cloning stimulation under different IL-6 concentration in colorectal cancer cell proliferation had no significant difference.
(3) changes in the apoptosis rate of CRC cells after JAK2 blockage
瀵逛簬鎮(zhèn)誕鍩瑰吇鐨勭粨鐩磋偁鐧岀粏鑳?yōu)CRC29,鐢ㄤ笉鍚屼綔鐢ㄦ祿搴︾殑JAK2鐗瑰紓鎬ф姂鍒跺墏AZD1480鍜孋EP33779鍒嗗埆闃繪柇JAK2媧誨寲,鎶戝埗鍓備綔鐢

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