本文選題：肺腺癌 + PZ蛋白��； 參考：《廣東藥科大學(xué)》2017年碩士論文

【摘要】：研究背景:肺癌是一種以肺部組織內(nèi)腫瘤細(xì)胞生長失控為特征的惡性腫瘤。如果不接受有效治療,腫瘤細(xì)胞能夠迅速轉(zhuǎn)移到人體其他組織與器官。目前,肺癌的發(fā)病率位居所有惡性腫瘤疾病中第二位,死亡率位居第一位。近年來,隨著研究者們對(duì)肺癌的細(xì)胞遺傳學(xué)特點(diǎn)、分子生物學(xué)機(jī)制等領(lǐng)域進(jìn)行深入的研究,一些新型的分子靶點(diǎn)治療(EGFR-TKIs藥物:Osmertinib)、化學(xué)免疫治療(CRLX101、TAS-102化療藥物)為肺癌患者獲得更好的療效與預(yù)后效果。盡管如此,肺癌的轉(zhuǎn)移與侵襲現(xiàn)象仍然是臨床上所要面臨的難題。肺腺癌,惡性程度高,是最容易出現(xiàn)早期轉(zhuǎn)移的非小細(xì)胞型肺癌。在首診時(shí),疾病通常已達(dá)晚期。目前,對(duì)肺腺癌轉(zhuǎn)移與復(fù)發(fā)的治療效果不理想,導(dǎo)致患者死亡率高,這對(duì)人類生命健康造成巨大威脅。因此,探討肺癌的轉(zhuǎn)移與侵襲機(jī)制具有十分重要的意義。蛋白質(zhì)Z(Protein Z,PZ)是一種主要由肝臟產(chǎn)生具有抗凝血作用的血漿糖蛋白因子。生理狀態(tài)下,它與蛋白質(zhì)Z依賴的蛋白酶抑制劑(Protein Z dependent protease inhibitor,ZPI)形成復(fù)合物在血液中循環(huán)。PZ作為ZPI的輔助因子,在ZPI抑制FXa的過程中發(fā)揮抗凝作用并能夠使抗凝作用提升1000倍。在PZ缺乏的情況下,ZPI抑制FXa的作用減弱,凝血活性增強(qiáng),容易增加體內(nèi)血栓形成風(fēng)險(xiǎn)。已有研究報(bào)道,在胃癌、結(jié)腸癌、肺癌等惡性腫瘤組織中發(fā)現(xiàn)PZ及其m RNA表達(dá)。我們前期的實(shí)驗(yàn)研究發(fā)現(xiàn),PZ的血漿水平隨著惡性實(shí)體瘤病程的進(jìn)展而明顯下降,這提示PZ可能是腫瘤患者預(yù)后不良的因素之一。而我們最新研究發(fā)現(xiàn)PZ在肺腺癌組織及細(xì)胞中表達(dá),這與之前報(bào)道的文獻(xiàn)結(jié)果一致,這意味著PZ與惡性腫瘤之間可能存在著更深層的關(guān)系。然而,PZ在惡性腫瘤中表達(dá)是否對(duì)腫瘤本身的生長及轉(zhuǎn)移或者其他生物學(xué)發(fā)面發(fā)揮作用,目前尚不清楚。本實(shí)驗(yàn)擬在前期探索性研究的基礎(chǔ)上,主要通過細(xì)胞分子生物學(xué)實(shí)驗(yàn),探討PZ對(duì)肺腺癌轉(zhuǎn)移及生長的影響,并初步探討PZ對(duì)肺腺癌轉(zhuǎn)移的作用機(jī)制,為臨床上肺腺癌的疾病診治提供一定理論依據(jù)。目的:1.了解PZ在肺腺癌中的表達(dá)情況。2.探討PZ對(duì)肺腺癌細(xì)胞的遷移、侵襲、增殖與凋亡的影響。3.初步探討PZ在肺腺癌轉(zhuǎn)移機(jī)制中的作用。研究方法:一免疫組織化學(xué)、蛋白質(zhì)印跡方法分別檢測(cè)肺腺癌組織、肺腺癌細(xì)胞中PZ的表達(dá)1、免疫組織化學(xué)方法檢測(cè)肺腺癌組織中PZ的表達(dá):完成收集肺腺癌組織標(biāo)本,標(biāo)本收集來自未曾經(jīng)過放療及化療的患者。用免疫組化方法將30例肺腺癌和16例癌旁病理組織標(biāo)本染色,觀察染色情況檢測(cè)肺腺癌及癌旁組織中PZ的表達(dá)。收集數(shù)據(jù)并完成統(tǒng)計(jì)分析,比較PZ在肺腺癌與癌旁組織中的表達(dá)情況。2、蛋白質(zhì)印跡方法檢測(cè)肺腺癌細(xì)胞中PZ的表達(dá):實(shí)驗(yàn)細(xì)胞分別采用ACC212102、A549、SPCA-1三種人源肺腺癌細(xì)胞系進(jìn)行培養(yǎng)。收集對(duì)數(shù)生長期細(xì)胞,冰凍PBS輕柔沖洗細(xì)胞代謝產(chǎn)物,加入RIPA裂解液,放置冰上充分裂解,低溫超速離心提取細(xì)胞蛋白質(zhì)。利用BCA方法檢測(cè)蛋白濃度,嚴(yán)格按照Western blot操作流程完成整個(gè)實(shí)驗(yàn)過程。收集數(shù)據(jù)并完成統(tǒng)計(jì)分析,了解PZ在不同肺腺癌細(xì)胞系上的表達(dá)情況,并篩選出在強(qiáng)表達(dá)PZ的肺腺癌細(xì)胞系。二合成靶向沉默目的基因PZ的si RNA,并轉(zhuǎn)染入A549肺腺癌細(xì)胞系,方法檢測(cè)沉默效果根據(jù)第一部分中Western blot方法檢測(cè)肺腺癌細(xì)胞中的PZ表達(dá)結(jié)果,篩選出PZ表達(dá)效果最強(qiáng)的A549肺腺癌細(xì)胞系培養(yǎng)并進(jìn)行下一步實(shí)驗(yàn)。收集對(duì)數(shù)生長期細(xì)胞,并將其分為5組,分別是:空白對(duì)照Mock組、無效序列陰性對(duì)照si RNA-NC組,實(shí)驗(yàn)組si RNA-PZ001、002、003(三組)。嚴(yán)格按照si RNA試劑盒操作說明書完成實(shí)驗(yàn)操作。將靶向目的基因si RNA脂質(zhì)載體轉(zhuǎn)染入A549肺腺癌細(xì)胞中,置于37℃、5%CO2培養(yǎng)箱中培養(yǎng)24-96小時(shí),收集目的蛋白。BCA方法檢測(cè)蛋白濃度,Western blot方法檢測(cè)轉(zhuǎn)染效果。收集數(shù)據(jù)并完成統(tǒng)計(jì)分析,對(duì)比轉(zhuǎn)染前后PZ在肺腺癌細(xì)胞中的表達(dá)情況,篩選最有效沉默PZ的靶向序列,為下一步細(xì)胞實(shí)驗(yàn):劃痕實(shí)驗(yàn)、細(xì)胞遷移、侵襲實(shí)驗(yàn)、細(xì)胞增殖及凋亡實(shí)驗(yàn)準(zhǔn)備。三劃痕實(shí)驗(yàn)、Transwell方法分別檢測(cè)si RNA轉(zhuǎn)染前后A549細(xì)胞遷移及侵襲能力變化1、劃痕實(shí)驗(yàn)檢測(cè)沉默PZ前后的A549細(xì)胞遷移能力:收集對(duì)數(shù)生長期細(xì)胞,并分為3組,分別是:空白對(duì)照Mock組、陰性對(duì)照si RNA-NC組,實(shí)驗(yàn)組si RNAPZ組。選取6孔板并在底部標(biāo)記橫線,將3組細(xì)胞分別接種適量細(xì)胞到孔中,過夜長滿單層細(xì)胞,用小槍頭垂直背后劃痕,PBS輕柔沖洗去除劃下的細(xì)胞,加入無血清培養(yǎng)液,置于37℃、5%CO2恒溫細(xì)胞培養(yǎng)箱中培養(yǎng),0小時(shí)、24小時(shí)、48小時(shí)取樣拍照。收集數(shù)據(jù)并完成統(tǒng)計(jì)分析,對(duì)比對(duì)照組與實(shí)驗(yàn)組結(jié)果,了解PZ對(duì)肺腺癌細(xì)胞遷移能力的影響。2、Transwell方法檢測(cè)沉默目PZ前后的A549細(xì)胞遷移及侵襲能力:收集對(duì)數(shù)生長期細(xì)胞,并分為3組,分別是:Mock組、si RNA-NC組,si RNA-PZ組。根據(jù)A459細(xì)胞的直徑,選擇孔徑為0.8um的Transwell小室。將3組細(xì)胞分別接種適量細(xì)胞到上層小室中并加入無雙抗無胎牛血清的培養(yǎng)液,下層為含有20%胎牛血清的培養(yǎng)液(若實(shí)驗(yàn)為侵襲實(shí)驗(yàn),則在接種細(xì)胞前需要先在小室的上室中按比例鋪一層基質(zhì)膠),置于37℃、5%CO2恒溫細(xì)胞培養(yǎng)箱中培養(yǎng)24小時(shí)后取出,甲醇固定,0.1%結(jié)晶紫染色,棉簽輕輕擦掉上層未遷移細(xì)胞,顯微鏡下觀察細(xì)胞。收集數(shù)據(jù)并完成統(tǒng)計(jì)分析,對(duì)比對(duì)照組與實(shí)驗(yàn)組結(jié)果,了解PZ對(duì)肺腺癌細(xì)胞遷移、侵襲轉(zhuǎn)移能力的影響。四蛋白印跡方法檢測(cè)PZ對(duì)細(xì)胞間質(zhì)化腫瘤轉(zhuǎn)移機(jī)制作用收集對(duì)數(shù)生長期細(xì)胞,分為3組:Mock組、si RNA-NC組與si RNA-PZ組。對(duì)三組肺腺癌細(xì)胞同時(shí)檢測(cè)Slug、Vimentin、N-cadherin三種蛋白。收集數(shù)據(jù)并進(jìn)行統(tǒng)計(jì)分析,對(duì)比三組之間Slug、Vimentin、N-cadherin三種蛋白表達(dá)差異,了解PZ對(duì)肺腺癌細(xì)胞間質(zhì)化(epithelial-mesenchymal transition,EMT)轉(zhuǎn)移機(jī)制的作用。五CCK-8方法檢測(cè)si RNA轉(zhuǎn)染前后A549細(xì)胞的增殖變化情況收集對(duì)數(shù)生長期細(xì)胞,分為3組:Mock組、si RNA-NC組與si RNA-PZ組。選擇96孔板,將不同組細(xì)胞分別接種到小孔中,每次實(shí)驗(yàn)每組細(xì)胞均接種至5個(gè)孔中,并放置于37℃、5%CO2恒溫細(xì)胞培養(yǎng)箱中培養(yǎng)24小時(shí)后,每個(gè)孔加入10ul CCK溶液,并于細(xì)胞培養(yǎng)箱中培養(yǎng)12小時(shí)、24小時(shí)、48小時(shí)、72小時(shí),并分別檢測(cè)對(duì)應(yīng)時(shí)間點(diǎn)的吸光度值。收集數(shù)據(jù)并進(jìn)行統(tǒng)計(jì)分析,對(duì)比不同時(shí)間點(diǎn)三組之間細(xì)胞增殖情況,了解PZ對(duì)肺腺癌細(xì)胞增殖能力的影響。六流式細(xì)胞方法檢測(cè)si RNA轉(zhuǎn)染前后的A549細(xì)胞凋亡壞死變化情況分別選取已被si RNA-PZ轉(zhuǎn)染及沒有被轉(zhuǎn)染的A549細(xì)胞。收集對(duì)數(shù)生長期細(xì)胞,分為2組:對(duì)照Control組與實(shí)驗(yàn)si RNA-PZ組。將各組細(xì)胞消化、離心收集5×10^5個(gè)細(xì)胞,取500ul 1×Binding Buffer重懸細(xì)胞,然后每管加入5ul Annexin V-FITC和10ul PI,輕柔渦旋之后,室溫避光孵育5分鐘,放置在流式儀上分析。收集數(shù)據(jù)并進(jìn)行統(tǒng)計(jì)分析,對(duì)比兩組細(xì)胞之間的結(jié)果,了解PZ對(duì)肺腺癌細(xì)胞凋亡的影響。結(jié)果:一PZ在肺腺癌組織及肺腺癌細(xì)胞中高表達(dá)1.免疫組化實(shí)驗(yàn)結(jié)果顯示:PZ在肺腺癌組織中表達(dá)明顯高于癌旁組織,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。2.Western blot實(shí)驗(yàn)結(jié)果顯示:PZ分別在ACC212102、A549、SPCA-1三種不同的肺腺癌細(xì)胞中均有表達(dá)。PZ蛋白在A549細(xì)胞中表達(dá)尤為突出。A549細(xì)胞與SPCA-1、ACC212102兩種肺腺癌細(xì)胞相比,PZ的表達(dá)量差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。二si RNA有效沉默PZ并篩選效果最佳效序列經(jīng)過si RNA脂質(zhì)體轉(zhuǎn)染后,si RNA-PZ001組與Mock組及si RNA-NC組相比,si RNA-PZ001中的PZ在表達(dá)水平明顯下降。差異有統(tǒng)計(jì)學(xué)意義(P0.001),提示si RNA脂質(zhì)體靶向沉默PZ基因有效,可以進(jìn)行下一步實(shí)驗(yàn);而Mock組與si RNA-NC組細(xì)胞中PZ的表達(dá),差異無統(tǒng)計(jì)學(xué)意義(P0.05)。三si RNA-PZ組細(xì)胞遷移及侵襲能力較靶向沉默前明顯下降1.劃痕實(shí)驗(yàn)結(jié)果發(fā)現(xiàn):si RNA-PZ組肺腺癌細(xì)胞與Mock組及si RNA-NC組細(xì)胞遷移速度比較,無論是在24小時(shí)還是48小時(shí)后,si RNA-PZ組細(xì)胞向中線運(yùn)動(dòng)遷移速度明顯緩慢,差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。抑制PZ表達(dá)能夠抑制A549細(xì)胞的遷移,這提示PZ表達(dá)在A549細(xì)胞中可能參與并且有助于肺腺癌細(xì)胞的遷移運(yùn)動(dòng)。而Mock組與si RNA-NC組細(xì)胞遷移速度比較,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。2.Transwell遷移及侵襲實(shí)驗(yàn)結(jié)果發(fā)現(xiàn):無論在遷移實(shí)驗(yàn)還是侵襲實(shí)驗(yàn)中,si RNA-PZ組肺腺癌細(xì)胞與Mock組及si RNA-NC組比較,穿過Transwell小室膜的細(xì)胞數(shù)目明顯減少,差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。抑制PZ表達(dá)能夠抑制A549細(xì)胞遷移及侵襲能力,這提示PZ在肺腺癌細(xì)胞中表達(dá),能夠促進(jìn)肺腺癌細(xì)胞的遷移能力,有助肺腺癌細(xì)胞的轉(zhuǎn)移。而Mock組與si RNA-NC組比較,穿過小室膜的細(xì)胞數(shù)目差異無統(tǒng)計(jì)學(xué)意義(P0.05)。四PZ可能通過EMT途徑影響A549細(xì)胞的遷移及侵襲利用Western blot檢測(cè)與細(xì)胞間質(zhì)化轉(zhuǎn)移途徑相關(guān)蛋白Slug、Vimentin及Ncadherin三種蛋白結(jié)果發(fā)現(xiàn):si RNA-PZ組與Mock組、si RNA-NC組相比較,Slug、Vimentin及N-cadherin三種蛋白表達(dá)均下降,差異均有統(tǒng)計(jì)學(xué)意義(P0.05),這提示PZ的表達(dá)能夠影響Slug、Vimentin及N-cadherin蛋白的表達(dá),初步提示了PZ可能通過作用EMT通路影響肺腺癌細(xì)胞的轉(zhuǎn)移。五PZ可能不參與A549細(xì)胞增殖CCK-8實(shí)驗(yàn)結(jié)果發(fā)現(xiàn):si RNA-PZ組、Mock組、si RNA組三組細(xì)胞增殖情況無明顯差異,差異無統(tǒng)計(jì)學(xué)意義(P0.05),這提示PZ蛋白的表達(dá)可能不參與肺腺癌細(xì)胞的增殖過程。六PZ可能不影響A549細(xì)胞的凋亡流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡實(shí)驗(yàn)結(jié)果發(fā)現(xiàn):si RNA-PZ組、Mock組、si RNA組三組細(xì)胞凋亡與壞死情況均無明顯差異,差異均無統(tǒng)計(jì)學(xué)意義(P0.05),這提示PZ蛋白的表達(dá)不參與肺腺癌細(xì)胞的凋亡過程,對(duì)肺腺癌細(xì)胞的凋亡與壞死無影響。結(jié)論:1.PZ蛋白在肺腺癌組織與肺腺癌細(xì)胞中均出現(xiàn)高表達(dá),臨床標(biāo)本檢測(cè)提示PZ表達(dá)可能與肺腺癌發(fā)生、發(fā)展相關(guān)。2.通過細(xì)胞生物學(xué)實(shí)驗(yàn)與分子實(shí)驗(yàn)證實(shí)PZ與肺腺癌的遷移與侵襲呈正相關(guān)。3.PZ蛋白可能通過EMT影響腫瘤轉(zhuǎn)移。4.PZ蛋白的表達(dá)可能不參與肺腺癌細(xì)胞的增殖過程。5.PZ蛋白的表達(dá)不參與肺腺癌細(xì)胞的凋亡過程,對(duì)肺腺癌細(xì)胞的凋亡與壞死無影響。
[Abstract]:Background: lung cancer is a malignant tumor characterized by the uncontrollable growth of tumor cells in the lung tissue. If it is not treated effectively, the tumor cells can quickly transfer to other tissues and organs of the human body. At present, the incidence of lung cancer ranks the second in all of the malignant tumor diseases, and the death rate is the first in recent years. The cytogenetic and molecular biological mechanisms of lung cancer are studied in depth. Some new molecular target therapy (EGFR-TKIs drug: Osmertinib), chemical immunotherapy (CRLX101, TAS-102 chemotherapy) can achieve better curative effect and prognosis for lung cancer patients. However, the metastasis and invasion of lung cancer still remain. It is a difficult problem to face in clinical. Lung adenocarcinoma, high degree of malignancy, is the most prone to early metastasis of non small cell lung cancer. In the first diagnosis, the disease is usually advanced. At present, the treatment effect on the metastasis and recurrence of lung adenocarcinoma is not ideal, which leads to the high mortality of the patients, which is a great threat to human life and health. Therefore, the lung is discussed. The mechanism of metastasis and invasion of cancer is of great significance. The protein Z (Protein Z, PZ) is a plasma glycoprotein factor, which mainly produces anticoagulant action by the liver. In physiological state, it forms complex with the protein Z dependent protease inhibitor (Protein Z dependent protease inhibitor, ZPI) in the blood circulation.PZ in the blood. As a cofactor for ZPI, anticoagulant action and anticoagulant action can be enhanced by 1000 times in the process of ZPI inhibition of FXa. In the absence of PZ, the effect of ZPI to inhibit FXa is weakened, the activity of coagulation is enhanced, and the risk of thrombus formation is increased easily. Studies have reported that PZ and its m R are found in cancer tissues such as gastric cancer, colon cancer and lung cancer. NA expression. Our previous experimental study found that the plasma level of PZ decreased significantly along with the progression of malignant solid tumor, suggesting that PZ may be one of the factors of poor prognosis in cancer patients. Our latest study found that PZ is expressed in lung adenocarcinoma tissue and cells, which is consistent with the previous reports, which means that PZ and evil are evil. There may be a deeper relationship between sexual tumors. However, it is not clear whether the expression of PZ may play a role in the growth and metastasis of the tumor or other biological surfaces in the malignant tumor. This experiment is based on a preliminary exploratory study of the cell molecular biology experiment to explore the transfer of PZ to lung adenocarcinoma. The effect of PZ on the metastasis of lung adenocarcinoma is preliminarily discussed in order to provide a certain theoretical basis for the diagnosis and treatment of lung adenocarcinoma. Objective: 1. to understand the expression of PZ in lung adenocarcinoma.2. to explore the effect of PZ on the migration, invasion, proliferation and apoptosis of lung adenocarcinoma cells..3. preliminarily discussed the role of PZ in the mechanism of metastatic adenocarcinoma of the lung. Methods: an immunohistochemical method was used to detect the expression of PZ in the lung adenocarcinoma and lung adenocarcinoma cells 1. The expression of PZ in the lung adenocarcinoma tissue was detected by immunohistochemistry. The specimens were collected from the lung adenocarcinoma tissue, and the specimens were collected from patients who had not been treated with chemotherapy and chemotherapy. 30 cases of lung adenocarcinoma were treated by immunohistochemical method. The expression of PZ in lung adenocarcinoma and para cancerous tissue was detected by staining of cancer and 16 paracancerous pathological tissue specimens. The data were collected and the expression of PZ in lung adenocarcinoma and para cancer tissue was compared. The expression of PZ in lung adenocarcinoma cells was detected by the method of Western blot: the experimental cells used ACC212102, A549, SPCA-1 respectively. Three human lung adenocarcinoma cell lines were cultured. The logarithmic growth period cells were collected. Frozen PBS flushed the metabolites of cells gently and added RIPA lysate. The cells were fully cracked on the ice, and the cell protein was extracted by the low temperature overspeed centrifugation. The protein concentration was detected by the BCA method, and the whole experiment process was completed strictly according to the Western blot operation process. The data and statistical analysis were performed to understand the expression of PZ in different lung adenocarcinoma cell lines, and to screen out the lung adenocarcinoma cell lines strongly expressing PZ. Two Si RNA, which was targeted to the target gene PZ, was synthesized and transfected into the A549 lung adenocarcinoma cell line. The effect of the silence was detected in the first part of the Western blot method in the detection of lung adenocarcinoma cells. The PZ expression results were screened out and the A549 lung adenocarcinoma cell lines with the strongest expression of PZ were cultured and the next experiment was carried out. The logarithmic growth phase cells were collected and divided into 5 groups: blank control group Mock, null sequence negative control Si RNA-NC group and experimental group Si RNA-PZ001002003 (three groups). The operation instructions of Si RNA kit were strictly followed. The target gene Si RNA lipid carrier was transfected into A549 lung adenocarcinoma cells, placed in 37 C and cultured in 5%CO2 incubator for 24-96 hours. The target protein.BCA method was collected to detect protein concentration and Western blot method was used to detect the transfection effect. The data were collected and the system analysis was completed, and the PZ in the lung adenocarcinoma cells before and after transfection was compared. The target sequence of the most effective silent PZ was screened. The next step cell experiment: scratch test, cell migration, invasion experiment, cell proliferation and apoptosis experiment preparation. Three scratch test, Transwell method to detect the change of A549 cell migration and invasion ability of A549 cells before and after Si RNA transfection, and the scratch test to detect the A549 cell migration before and after the silent PZ Migration ability: collect the logarithmic growth phase cells and divide them into 3 groups: blank control Mock group, negative control Si RNA-NC group and Si RNAPZ group in experimental group. Select 6 orifice plates and mark transverse lines at the bottom. The 3 groups of cells are inoculated with proper amount of cells into the pores, the single cells are covered with single cell in the night, and the scratch in the head of the gun is vertical, and the PBS is gently rinsed and removed. The cells were added to the serum-free culture medium and placed in the incubator of 5%CO2 constant temperature cell at 37 degrees C, 0 hours, 24 hours and 48 hours for sampling and taking pictures. The data were collected and the statistical analysis was completed. The effects of the control group and the experimental group on the migration ability of lung adenocarcinoma cells were compared with the control group.2, and the Transwell method was used to detect the migration and invasion of A549 cells before and after the silence of PZ. Attack ability: collect the logarithmic growth period cells and divide into 3 groups, which are group Mock, Si RNA-NC group, Si RNA-PZ group. According to the diameter of A459 cells, the Transwell small room with the pore size of 0.8um is selected. The 3 groups of cells are inoculated to the upper chamber and added to the culture medium with no double anti fetal bovine serum, and the lower layer is the culture containing 20% fetal bovine serum. If the experiment was the invasion experiment, we need to spread a layer of matrix glue in the upper chamber of the small room before inoculation, and put it at 37 C, and then take out the incubator in the 5%CO2 incubator for 24 hours, the methanol is fixed, the crystal violet is stained, the cotton swabs are gently erased on the upper layer of the non migrating cells, and the cells are observed under the microscope. The data are collected and completed. In comparison with the results of the control group and the experimental group, the effect of PZ on the migration of lung adenocarcinoma cells and the ability of invasion and metastasis was understood. Four the Western blot method was used to detect the mechanism of PZ for the transfer mechanism of interstitial tumor to collect logarithmic growth phase cells, which were divided into 3 groups: Mock group, Si RNA-NC group and Si RNA-PZ group. Three groups of lung adenocarcinoma cells were simultaneously detected Slug, Vimenti N, N-cadherin three proteins. Collect data and carry out statistical analysis, compare the differences in the expression of three kinds of protein, Slug, Vimentin, N-cadherin between the three groups, to understand the effect of PZ on the mechanism of interstitial lung adenocarcinoma (epithelial-mesenchymal transition, EMT) metastasis. Five CCK-8 method was used to detect the proliferation of the cells before and after the Si RNA. The logarithmic growth stage cells were divided into 3 groups: Mock group, Si RNA-NC group and Si RNA-PZ group. Select 96 orifice plates and inoculate different groups of cells into small pores. Each cell was inoculated into 5 holes each time, and placed at 37, 5%CO2 constant temperature cell culture incubator for 24 hours, each hole was added to 10ul CCK solution, and cultured in cell culture box. 12 hours, 24 hours, 48 hours and 72 hours, and measured the absorbance of the corresponding time points. Collect data and analyze the proliferation of cells between three groups at different time points, and understand the effect of PZ on the proliferation of lung adenocarcinoma cells. Six flow cytometry was used to detect the changes of apoptosis and necrosis of A549 cells before and after the transfection of RNA. Si RNA-PZ transfected and not transfected A549 cells were selected to collect the logarithmic growth phase cells and divided into 2 groups: control group Control and experimental Si RNA-PZ group. The cells were digested and centrifuged to collect 5 x 10^5 cells, and 500ul 1 x Binding Buffer heavy suspension cells were taken, then each tube was added to 5ul Annexin, followed by gentle vortex, The effect of PZ on the apoptosis of lung adenocarcinoma cells was compared between the two groups of cells. Results: a high expression of a PZ in the lung adenocarcinoma and lung adenocarcinoma cells and 1. immunohistochemical results showed that the expression of PZ in the lung adenocarcinoma tissue was significantly higher than that in the lung adenocarcinoma tissue. The difference was statistically significant (P0.05).2.Western blot experimental results showed that PZ expressed.PZ protein in A549 cells in three different lung adenocarcinoma cells, ACC212102, A549, SPCA-1, especially.A549 cells and SPCA-1, and there were significant differences in the expression of the two types of lung adenocarcinoma cells. 0.05). Two Si RNA effectively silenced PZ and the best effect sequence was transfected by Si RNA liposome. The expression level of Si RNA-PZ001 group was significantly lower than that in Mock group and Si RNA-NC group. The difference was statistically significant. There was no significant difference in the expression of PZ in the cells of group CK and Si RNA-NC group (P0.05). The cell migration and invasion ability of the three Si RNA-PZ group was significantly lower than that before the target silencing. The results showed that the migration velocity of the lung adenocarcinoma cells in the Si RNA-PZ group and the Mock group and Si RNA-NC group was compared, whether at 24 hours or 48 hours later. The migration velocity of the cells to the middle line was obviously slow, and the difference was statistically significant (P0.05). The inhibition of PZ expression could inhibit the migration of A549 cells. This suggested that the expression of PZ may be involved in the A549 cells and may contribute to the migration of lung adenocarcinoma cells, but there is no significant difference between the Mock group and the Si RNA-NC group (P0.05). The results of.2.Transwell migration and invasion experiments showed that the number of cells passing through the Transwell compartment was significantly reduced in both the Si RNA-PZ group and the Mock group and the Si RNA-NC group in both the Migration Experiment and the invasion experiment, and the difference was statistically significant (P0.05). The inhibition of PZ expression could inhibit the migration and invasion of A549 cells. This suggested that the proliferation and invasion ability of A549 cells could be inhibited. The expression of PZ in lung adenocarcinoma cells can promote the migration ability of lung adenocarcinoma cells and help the metastasis of lung adenocarcinoma cells. Compared with the Si RNA-NC group, there is no significant difference in the number of cells passing through the cell membrane in the Mock group (P0.05). Four PZ may affect the migration and invasion of A549 cells through EMT pathway and the detection and intercellular use of Western blot. The results of three proteins, Slug, Vimentin and Ncadherin, were found in Si RNA-PZ group and Mock group and Si RNA-NC group. The expression of Slug, Vimentin and N-cadherin three proteins decreased, and the difference was statistically significant (P0.05). PZ may affect the metastasis of lung adenocarcinoma cells through the action of EMT pathway. Five PZ may not participate in the CCK-8 experiment of A549 cell proliferation. The proliferation of Si RNA-PZ group, Mock group and Si RNA group has no significant difference, the difference is not statistically significant (P0.05), which suggests that the expression of the PZ protein may not participate in the proliferation process of lung adenocarcinoma cells. Six PZ may not affect apoptotic cell apoptosis in A549 cells. The apoptosis and necrosis of Si RNA-PZ group, Mock group and Si RNA group have no significant difference (P0.05), which suggests that the expression of PZ protein is not involved in the apoptosis process of lung adenocarcinoma cells and the apoptosis of lung adenocarcinoma cells. Conclusion: 1.PZ protein is highly expressed in lung adenocarcinoma and lung adenocarcinoma cells.

【學(xué)位授予單位】：廣東藥科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R734.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 黃方;潘學(xué)誼;王宏;劉亞楠;關(guān)則兵;郭煜;李銘杰;曾文彬;徐之龍;;蛋白質(zhì)Z在肺腺癌的表達(dá)及其臨床意義[J];重慶醫(yī)學(xué);2015年36期

2 殷濤;王春友;熊炯p，

本文編號(hào)：1785930