發(fā)布時(shí)間：2018-01-05 06:36

  本文關(guān)鍵詞：食管癌微環(huán)境中PKM2參與細(xì)胞惡性表型的調(diào)控機(jī)制研究　出處：《新疆醫(yī)科大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 食管癌 M2型丙酮酸激酶(PKM2) 惡性表型 調(diào)控機(jī)制

【摘要】：目的:探討M2型丙酮酸激酶(Pyruvate Kinase M2,PKM2)與食管鱗狀細(xì)胞癌(Esophageal Squamous Cell Carcinoma,ESCC)臨床惡性表型的相關(guān)性;驗(yàn)證微環(huán)境中PKM2對(duì)食管癌細(xì)胞惡性表型的功能影響;初步探討食管癌微環(huán)境中PKM2表達(dá)參與的細(xì)胞調(diào)控通路。方法:(1)在組織水平,通過免疫組化方法檢測(cè)PKM2在食管鱗癌癌組織及癌旁正常組織中的表達(dá),分析PKM2與食管癌臨床病理參數(shù)(患者年齡、性別,腫瘤浸潤(rùn)深度、淋巴結(jié)轉(zhuǎn)移等)和預(yù)后的相關(guān)性。此外,運(yùn)用酶聯(lián)免疫吸附法(Enzyme Linked Immunosorbent Assay,ELISA)驗(yàn)證PKM2在食管癌患者血清中的表達(dá)。(2)通過實(shí)時(shí)熒光定量PCR(Quantitative Real Time PCR,qRT-PCR)檢測(cè)不同食管癌細(xì)胞系中PKM2的本底表達(dá)量差異。設(shè)計(jì)合成三條不同的PKM2干擾序列,通過siRNA干擾技術(shù)敲低食管癌Eca109細(xì)胞中PKM2的表達(dá),qRT-PCR技術(shù)檢測(cè)siRNA瞬時(shí)轉(zhuǎn)染后PKM2 mRNA表達(dá)水平的變化;Westem blot技術(shù)檢測(cè)蛋白水平PKM2的表達(dá)改變;采用四甲基偶氮唑鹽(MTT)比色法檢測(cè)細(xì)胞增殖變化;流式細(xì)胞術(shù)(Flow Cytometry,FCM)檢測(cè)細(xì)胞凋亡及周期改變;細(xì)胞劃痕實(shí)驗(yàn)檢測(cè)對(duì)食管癌細(xì)胞遷移能力的影響;Transwell實(shí)驗(yàn)檢測(cè)對(duì)食管癌細(xì)胞侵襲能力的影響。(3)進(jìn)一步選擇干擾效率最高的siRNA序列設(shè)計(jì)合成攜帶GFP的干擾PKM2表達(dá)的慢病毒包裝載體,同時(shí)設(shè)計(jì)合成攜帶GFP的PKM2過表達(dá)慢病毒包裝載體,通過轉(zhuǎn)染食管癌Eca109細(xì)胞、FCM分選得到穩(wěn)定轉(zhuǎn)染的干擾以及過表達(dá)PKM2的細(xì)胞株,MTT法驗(yàn)證細(xì)胞增殖變化,細(xì)胞劃痕實(shí)驗(yàn)驗(yàn)證PKM2表達(dá)量改變對(duì)食管癌細(xì)胞遷移能力的影響,Transwell實(shí)驗(yàn)驗(yàn)證PKM2表達(dá)量改變對(duì)食管癌細(xì)胞侵襲能力的影響。(4)選取4~5周齡14~17g的雌性BALB/c Nude鼠,構(gòu)建裸鼠異位移植瘤模型。采用PKM2干擾及過表達(dá)慢病毒包裝載體穩(wěn)定轉(zhuǎn)染的食管癌Eca109細(xì)胞系,細(xì)胞計(jì)數(shù)后按2×107個(gè)細(xì)胞/0.2m L/鼠的劑量接種于裸鼠右側(cè)后肢部皮下,接種后每周測(cè)量裸鼠體重并觀察瘤塊的生長(zhǎng)情況。瘤塊長(zhǎng)出后,用游標(biāo)卡尺測(cè)量裸鼠瘤塊長(zhǎng)徑(A)及垂直橫徑(B),按公式V=A×B2/2計(jì)算裸鼠腫瘤體積。組織樣本,福爾馬林固定后制成石蠟包埋的組織樣本,切片后進(jìn)行HE染色及免疫組化染色,觀察移植瘤組織形態(tài)并檢測(cè)移植瘤組織中PKM2的表達(dá)。(5)為探討PKM2調(diào)控食管癌惡性表型的機(jī)制,將PKM2 siRNA轉(zhuǎn)染食管癌eca109細(xì)胞及kyse150細(xì)胞,正常培養(yǎng)的eca109細(xì)胞及kyse150細(xì)胞設(shè)為對(duì)照組,轉(zhuǎn)染后48h收集細(xì)胞并提取總rna,明確pkm2sirna干擾效果后通過mrna表達(dá)譜芯片檢測(cè),分析基因的改變和信號(hào)通路變化。結(jié)果:(1)食管癌組織中pkm2陽性表達(dá)為在細(xì)胞胞漿中呈棕褐色著色,pkm2在食管癌組織中的表達(dá)(69/75,92.0%)明顯高于其在配對(duì)癌旁正常組織中的表達(dá)(9/75,12.0%),差異有統(tǒng)計(jì)學(xué)意義(p0.05)。kaplan-meier生存分析發(fā)現(xiàn),與pkm2低表達(dá)的食管癌患者相比較,pkm2高表達(dá)的患者顯示出生存時(shí)間較短的趨勢(shì)(log-rank檢驗(yàn),p0.05)。此外,檢測(cè)哈族食管癌臨床樣本中pkm2的表達(dá),發(fā)現(xiàn):pkm2在食管癌組織中的表達(dá)(37/54,68.5%)明顯高于其在配對(duì)癌旁正常組織中的表達(dá)(8/54,14.8%),差異有統(tǒng)計(jì)學(xué)意義(p0.05)。通過kaplan-meier生存分析,顯示:pkm2高表達(dá)的哈族食管癌患者其生存期明顯短于pkm2低表達(dá)的哈族食管癌患者(log-rank檢驗(yàn),p0.05),即pkm2高表達(dá)的哈族食管癌患者預(yù)后較差。食管癌患者血清中pkm2的表達(dá)(78.84ng/ml)顯著高于正常對(duì)照人群血清中pkm2的表達(dá)(13.55ng/ml,p0.05)。(2)在細(xì)胞水平檢測(cè)不同食管癌細(xì)胞系中pkm2的本底表達(dá)量差異,結(jié)果發(fā)現(xiàn)在eca109、ec9706、kyse30、kyse150、kyse450及t46種細(xì)胞系中,kyse450細(xì)胞中pkm2表達(dá)量最高,其次是t4、ec9706、eca109、kyse150,kyse30細(xì)胞中pkm2表達(dá)量最低。為驗(yàn)證微環(huán)境中pkm2表達(dá)對(duì)食管癌細(xì)胞惡性表型的功能影響,通過設(shè)計(jì)、合成并瞬時(shí)轉(zhuǎn)染pkm2sirna,結(jié)果發(fā)現(xiàn)三條pkm2sirna在mrna水平和蛋白水平均能顯著敲低食管癌eca109細(xì)胞中pkm2的表達(dá),其表達(dá)量改變與隨機(jī)序列對(duì)照組(sirna-scramble)以及正常培養(yǎng)對(duì)照組(normalcontrol)比較均顯著降低,差異有統(tǒng)計(jì)學(xué)意義(p0.05)。通過pkm2sirna瞬時(shí)轉(zhuǎn)染干擾pkm2表達(dá)后進(jìn)行細(xì)胞功能學(xué)實(shí)驗(yàn),mtt檢測(cè)發(fā)現(xiàn)敲低pkm2的表達(dá)后食管癌eca109細(xì)胞增殖受到顯著抑制,劃痕實(shí)驗(yàn)和transwell侵襲實(shí)驗(yàn)發(fā)現(xiàn)eca109遷移能力和侵襲能力均顯著降低,fcm檢測(cè)發(fā)現(xiàn)敲低pkm2的表達(dá)后細(xì)胞凋亡增多,細(xì)胞周期被阻滯在g0/g1期。檢測(cè)細(xì)胞功能變化,發(fā)現(xiàn):sirna瞬時(shí)轉(zhuǎn)染成功敲低pkm2的表達(dá)后,食管癌eca109細(xì)胞的增殖受到抑制,細(xì)胞凋亡增加,細(xì)胞侵襲、遷移能力顯著下降,同時(shí)細(xì)胞周期被阻滯在g0/g1期。(3)為進(jìn)一步明確pkm2表達(dá)對(duì)食管癌細(xì)胞功能的影響,采用慢病毒穩(wěn)定轉(zhuǎn)染pkm2基因并在體內(nèi)驗(yàn)證該結(jié)果,本研究選擇干擾效果最顯著的sirna序列進(jìn)一步設(shè)計(jì)合成干擾pkm2穩(wěn)定表達(dá)的慢病毒包裝載體,以及pkm2過表達(dá)慢病毒包裝載體,根據(jù)細(xì)胞轉(zhuǎn)染的慢病毒種類將實(shí)驗(yàn)分5組:干擾pkm2表達(dá)實(shí)驗(yàn)組(轉(zhuǎn)染sirna-pkm2慢病毒包裝載體)、干擾pkm2表達(dá)對(duì)照組(轉(zhuǎn)染sirna-scramble慢病毒包裝載體)、pkm2過表達(dá)實(shí)驗(yàn)組(轉(zhuǎn)染pkm2過表達(dá)慢病毒包裝載體)、pkm2過表達(dá)對(duì)照組(轉(zhuǎn)染pkm2-scramble慢病毒包裝載體)、陰性對(duì)照組(正常培養(yǎng)的eca109細(xì)胞)。通過轉(zhuǎn)染干擾以及過表達(dá)pkm2的慢病毒載體改變pkm2的表達(dá)后,檢測(cè)細(xì)胞功能變化,細(xì)胞增殖實(shí)驗(yàn)結(jié)果顯示,干擾PKM2表達(dá)后食管癌Eca109細(xì)胞的增殖受到顯著抑制,過表達(dá)PKM2促進(jìn)了Eca109細(xì)胞的增殖;劃痕實(shí)驗(yàn)結(jié)果發(fā)現(xiàn),干擾PKM2表達(dá)后Eca109細(xì)胞的相對(duì)遷移距離減小,過表達(dá)PKM2后Eca109細(xì)胞的相對(duì)遷移距離增加;Transwell侵襲實(shí)驗(yàn)發(fā)現(xiàn),干擾PKM2表達(dá)后侵襲細(xì)胞數(shù)顯著減少,過表達(dá)PKM2后侵襲細(xì)胞數(shù)顯著增多,即干擾PKM2表達(dá)抑制了食管癌Eca109細(xì)胞的增殖、遷移和侵襲,過表達(dá)PKM2促進(jìn)食管癌Eca109細(xì)胞的增殖、遷移和侵襲。(4)在裸鼠皮下分別注射干擾、過表達(dá)PKM2的Eca109穩(wěn)定轉(zhuǎn)染細(xì)胞株一周后成瘤,注射干擾PKM2表達(dá)的Eca109細(xì)胞成瘤后組織低表達(dá)PKM2,注射過表達(dá)PKM2的Eca109細(xì)胞成瘤后組織高表達(dá)PKM2,此外,PKM2過表達(dá)后裸鼠皮下移植瘤體積大于對(duì)照組,統(tǒng)計(jì)分析結(jié)果顯示兩組之間平均瘤體體積差異有統(tǒng)計(jì)學(xué)意義(P0.05),即過表達(dá)PKM2促進(jìn)了裸鼠皮下移植瘤的生長(zhǎng)。以上結(jié)果說明在體內(nèi)PKM2的表達(dá)影響食管癌細(xì)胞的增殖。(5)mRNA表達(dá)譜芯片差異基因篩選常規(guī)標(biāo)準(zhǔn)為FC(abs)在2.0倍以上,按此標(biāo)準(zhǔn),干擾PKM2表達(dá)后Eca109細(xì)胞中有4592個(gè)上調(diào)基因,2497個(gè)下調(diào)基因;干擾PKM2表達(dá)后KYSE150細(xì)胞中有1796個(gè)上調(diào)基因,1936個(gè)下調(diào)基因。干擾PKM2表達(dá)后Eca109細(xì)胞和KYSE150細(xì)胞中mRNA水平表達(dá)均上調(diào)的基因有298個(gè),其中差異倍數(shù)大于5倍的基因共有16個(gè);表達(dá)均下調(diào)的基因有277個(gè),其中差異倍數(shù)大于4倍的基因共有8個(gè)。此外,Eca109細(xì)胞中干擾PKM2表達(dá)后受到影響的細(xì)胞功能和信號(hào)通路包括p53信號(hào)通路、自噬、蛋白質(zhì)消化和吸收、補(bǔ)體系統(tǒng)以及化學(xué)致癌作用;KYSE150細(xì)胞中干擾PKM2表達(dá)后受到影響的功能和信號(hào)通路變化包括核糖體起源、N-聚糖生物合成、凋亡、TNF信號(hào)通路、Notch信號(hào)通路、DNA復(fù)制、細(xì)胞周期以及對(duì)轉(zhuǎn)錄的異常調(diào)控。結(jié)論:PKM2在食管癌中呈高表達(dá),哈族食管癌患者高表達(dá)PKM2其預(yù)后差。PKM2高表達(dá)能促進(jìn)食管鱗癌細(xì)胞的增殖、遷移以及侵襲,抑制細(xì)胞凋亡,并影響細(xì)胞的周期分布。PKM2對(duì)食管癌細(xì)胞惡性表型的影響可能受p53信號(hào)通路、TNF信號(hào)通路和Notch信號(hào)通路等多種因素的調(diào)控。
[Abstract]:Objective: To investigate the effect of pyruvate kinase M2 (Pyruvate Kinase M2, PKM2) and esophageal squamous cell carcinoma (Esophageal Squamous Cell Carcinoma, ESCC) the clinical relevance of the malignant phenotype; influence of PKM2 verification in the microenvironment of the malignant phenotype of esophageal cancer cell function; preliminary study of cell regulation pathways involved in the expression of PKM2 esophageal cancer microenvironment. Methods: (1) at the organizational level, expression was detected by immunohistochemistry method PKM2 in esophageal squamous cell carcinoma and normal tissues, analysis of PKM2 and the clinical pathological parameters of esophageal cancer (age, gender, tumor invasion depth, lymph node metastasis) correlation and prognosis. In addition, by ELISA immunoassay (Enzyme Linked Immunosorbent Assay, ELISA) to confirm the expression of PKM2 in esophageal cancer. (2) by real-time fluorescence quantitative PCR (Quantitative Real Time PCR, qRT-PCR) detection of esophageal cancer cells In the end the difference in the expression of PKM2. The design and synthesis of three different PKM2 interference sequence expression by siRNA interference on PKM2 in esophageal cancer Eca109 cells, qRT-PCR detection of siRNA after transient transfection of PKM2 mRNA expression; to detect protein expression level changes PKM2 Westem blot technology; using four methyl thiazolyl tetrazolium (MTT) assay in cell proliferation; flow cytometry (Flow Cytometry, FCM) changes of apoptosis and cell cycle detection; effects of cell scratch assay and migration of esophageal carcinoma cells; effect of Transwell assay on invasion of esophageal carcinoma cells. (3) lentiviral vector packaging further interference PKM2 interference siRNA sequence design of the highest efficiency of synthesis carrying GFP expression, at the same time the design and synthesis of GFP carrying PKM2 overexpression lentivirus packaging carrier by transfection of human esophageal carcinoma cells Eca109 and FCM Isolated interference stable transfection and overexpression of PKM2 cell line, cell proliferation was determined by MTT method, the expression of cell scratch experiments to verify the PKM2 volume change effect on esophageal cancer cell migration and expression of Transwell experiment PKM2 effects on invasion of esophageal carcinoma cells. (4) female BALB/c Nude rats from 4~5 week at the age of 14~17g, construct the heterotopic nude mouse model. Using PKM2 interference and overexpression of Eca109 in esophageal carcinoma cell lines lentiviral vector was successfully transfected into the packaging cell, after counting according to the dose inoculation of 2 * 107 cells in /0.2m L/ rats in nude rat right hindlimb subcutaneous growth of mice weight was measured weekly, and observe the tumor after inoculation of tumor. After the long, measured with vernier caliper tumor block length (A) and vertical diameter (B), calculate the tumor volume according to the formula of V=A * B2/2. Tissue samples were formalin fixed after made of stone Paraffin embedded tissue samples, sections were stained with HE and immunohistochemistry to observe the expression of PKM2 in transplanted tumor tissue morphology were detected in transplanted tumor tissue. (5) to explore the mechanism of regulation of malignant phenotype of PKM2 esophageal carcinoma, PKM2 siRNA transfected esophageal cancer Eca109 cells and kyse150 cells, the normal cultured Eca109 and kyse150 cells as control group, transfected 48h cells were collected and extracted the total RNA, clear pkm2sirna jamming effect by mRNA microarray detection, change and signal pathway of gene. Results: (1) the positive expression of pkm2 in esophageal cancer tissue was brown coloration in the cell cytoplasm, the expression of pkm2 in esophageal carcinoma (69/75,92.0%) was significantly higher than the expression in adjacent normal tissues (9/75,12.0%), the difference was statistically significant (P0.05) and pkm2.Kaplan-meier survival analysis showed that low expression of esophageal cancer patients In comparison, patients with high expression of pkm2 showed a survival in a relatively short time trend (log-rank test, P0.05). In addition, detected expression of Kazakh's esophageal cancer in clinical samples of pkm2: the expression of pkm2 in esophageal carcinoma (37/54,68.5%) was significantly higher than the expression in adjacent normal tissues (8/54,14.8%), the difference was statistically significant (P0.05). The Kaplan-Meier survival analysis showed that the high expression of pkm2 in Kazakh esophageal cancer patients whose survival was significantly shorter than the low expression of pkm2 in Kazakh esophageal cancer patients (log-rank test, P0.05), the high expression of pkm2 in Kazakh esophageal cancer patients with poor prognosis. The expression of pkm2 in serum of patients with esophageal cancer in (78.84ng/ml) pkm2 expression was significantly higher than that of normal control group in serum (13.55ng/ml, P0.05). (2) pkm2 at the cellular level detection of esophageal carcinoma cell line in the bottom of the expression differences were found in the Eca109, EC9706, kyse30, Kyse150, KYSE450 and T46 cell lines, the highest expression of pkm2 in KYSE450 cells, followed by T4, EC9706, Eca109, kyse150, pkm2 was the lowest in kyse30 cells. Pkm2 verification in the micro environment affect the expression, the malignant phenotype of esophageal cancer cell function through the design, synthesis and transient transfection of pkm2sirna results found that the expression of three pkm2sirna were significantly lower on pkm2 in esophageal cancer Eca109 cells at mRNA and protein level, the expression changes and random sequence control group (sirna-scramble) and normal control group (normalcontrol) training is significantly reduced, the difference was statistically significant (P0.05). Experimental study by cell function the expression of pkm2sirna after transient transfection of pkm2 interference, MTT detected that the expression knockdown of pkm2 after esophageal cancer Eca109 cells proliferation was significantly inhibited, cell scratch test and Transwell test showed that Eca109 migration and The invasion ability decreased significantly, the apoptosis that knockdown of pkm2 expression after FCM detection increased, cell cycle arrest in g0/g1 phase. It is found that changes in detection of cell function: siRNA transient transfection knockdown pkm2 protein after Eca109 esophageal cancer cell proliferation inhibition, cell apoptosis, cell invasion and migration ability at the same time significantly decreased, cell cycle arrest in g0/g1 phase. (3) to further clarify the effect of pkm2 expression on esophageal cancer cell function, using lentivirus transfected with pkm2 gene and verified the results in vivo, siRNA sequences in this study disturbance is the most significant effect of the further design package lentivirus vectors for the synthesis of stable expression of pkm2 interference the expression of pkm2 and lentiviral packaging carrier, according to the types of cells transfected with lentivirus were divided into 5 groups: interference of pkm2 expression in the experimental group (sirna-pkm2 transfected with lentiviral packaging carrier), PK interference The expression of M2 in the control group (transfected with sirna-scramble lentiviral vector packaging), over expression of pkm2 in experimental group (transfected with pkm2 overexpression lentivirus vector, overexpression of pkm2 packaging) and control group (transfected with pkm2-scramble lentiviral vector packaging), negative control group (normal Eca109 cells). By transfection of interference and overexpression of Lentivirus Expression Vector the change of pkm2 after pkm2, to detect the changes of cell function, cell proliferation experiments showed that PKM2 knockdown esophageal carcinoma Eca109 cells significantly inhibited the proliferation, overexpression of PKM2 promotes the proliferation of Eca109 cells; scratch experiment results showed that the relative migration in PKM2 knockdown Eca109 cells after overexpression of PKM2 distance decreases. The relative migration distance of Eca109 cells increased; Transwell invasion experiments showed that PKM2 knockdown significantly reduce the number of invasive cells, overexpression of PKM2 cells increased significantly after the invasion, dry PKM2 interference inhibited the expression of Eca109 esophageal cancer cell proliferation, migration and invasion, overexpression of PKM2 promotes Eca109 esophageal cancer cell proliferation, migration and invasion. (4) in nude mice were injected with interference and overexpression of Eca109 stable transfected cell line PKM2 carrying tumor one week, tumor tissue with low expression of PKM2 rejection PKM2 the expression of Eca109 stem cell injection, injection of PKM2 overexpressing Eca109 cells into tumor tissue after high expression of PKM2, in addition, over expression of PKM2 volume after subcutaneous transplantation tumor in nude mice than in the control group, statistical analysis showed that there were statistically significant differences in average tumor volume between the two groups (P0.05), which promotes the over expression of PKM2 in nude mice the growth of xenografts. The above results showed that the effect of the proliferation of esophageal carcinoma cells PKM2 expression in vivo. (5) the difference of gene expression microarray screening standard for FC mRNA (ABS) in more than 2 times, according to this standard, the expression of PKM2 after interference There are 4592 up-regulated genes in Eca109 cells, 2497 down regulated genes; interference PKM2 expression of KYSE150 cells after 1796 up-regulated, 1936 down regulated genes. PKM2 knockdown Eca109 cells and KYSE150 cells in the expression of mRNA were up-regulated and 298 genes, of which more than 5 times the number of times the difference between the total gene 16; the expression of 277 genes were down regulated, which showed over 4 times for a total of 8 genes. In addition, cellular functions and signaling pathways affected by PKM2 expression including p53 signaling pathway, Eca109 interference cell autophagy, protein digestion and absorption, complement system and chemical carcinogenesis; function and signal affected by the expression of PKM2 pathway changes including the ribosome origin interference in KYSE150 cells, N- glycan biosynthesis, apoptosis, TNF signaling pathway, Notch signaling pathway, DNA replication, cell cycle and abnormal regulation of transcription. Conclusion: the high expression of PKM2 in esophageal carcinoma was high expression in patients with esophageal carcinoma PKM2 Kazakh poor prognosis of esophageal squamous cell carcinoma with high expression of.PKM2 can promote cell proliferation, migration and invasion, apoptosis, and cell cycle distribution of.PKM2 on the malignant phenotype of esophageal carcinoma cells may be affected by the impact of the p53 signaling pathway, regulate a variety of the factors of TNF signaling pathway and Notch signaling pathway.

【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.1

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