蛋白质Z对肺腺癌生长与转移作用研究

发布时间：2018-04-22 06:09

本文选题：肺腺癌 + PZ蛋白　；参考：《广东药科大学》2017年硕士论文

【摘要】：研究背景:肺癌是一种以肺部组织内肿瘤细胞生长失控为特征的恶性肿瘤。如果不接受有效治疗,肿瘤细胞能够迅速转移到人体其他组织与器官。目前,肺癌的发病率位居所有恶性肿瘤疾病中第二位,死亡率位居第一位。近年来,随着研究者们对肺癌的细胞遗传学特点、分子生物学机制等领域进行深入的研究,一些新型的分子靶点治疗(EGFR-TKIs药物:Osmertinib)、化学免疫治疗(CRLX101、TAS-102化疗药物)为肺癌患者获得更好的疗效与预后效果。尽管如此,肺癌的转移与侵袭现象仍然是临床上所要面临的难题。肺腺癌,恶性程度高,是最容易出现早期转移的非小细胞型肺癌。在首诊时,疾病通常已达晚期。目前,对肺腺癌转移与复发的治疗效果不理想,导致患者死亡率高,这对人类生命健康造成巨大威胁。因此,探讨肺癌的转移与侵袭机制具有十分重要的意义。蛋白质Z(Protein Z,PZ)是一种主要由肝脏产生具有抗凝血作用的血浆糖蛋白因子。生理状态下,它与蛋白质Z依赖的蛋白酶抑制剂(Protein Z dependent protease inhibitor,ZPI)形成复合物在血液中循环。PZ作为ZPI的辅助因子,在ZPI抑制FXa的过程中发挥抗凝作用并能够使抗凝作用提升1000倍。在PZ缺乏的情况下,ZPI抑制FXa的作用减弱,凝血活性增强,容易增加体内血栓形成风险。已有研究报道,在胃癌、结肠癌、肺癌等恶性肿瘤组织中发现PZ及其m RNA表达。我们前期的实验研究发现,PZ的血浆水平随着恶性实体瘤病程的进展而明显下降,这提示PZ可能是肿瘤患者预后不良的因素之一。而我们最新研究发现PZ在肺腺癌组织及细胞中表达,这与之前报道的文献结果一致,这意味着PZ与恶性肿瘤之间可能存在着更深层的关系。然而,PZ在恶性肿瘤中表达是否对肿瘤本身的生长及转移或者其他生物学发面发挥作用,目前尚不清楚。本实验拟在前期探索性研究的基础上,主要通过细胞分子生物学实验,探讨PZ对肺腺癌转移及生长的影响,并初步探讨PZ对肺腺癌转移的作用机制,为临床上肺腺癌的疾病诊治提供一定理论依据。目的:1.了解PZ在肺腺癌中的表达情况。2.探讨PZ对肺腺癌细胞的迁移、侵袭、增殖与凋亡的影响。3.初步探讨PZ在肺腺癌转移机制中的作用。研究方法:一免疫组织化学、蛋白质印迹方法分别检测肺腺癌组织、肺腺癌细胞中PZ的表达1、免疫组织化学方法检测肺腺癌组织中PZ的表达:完成收集肺腺癌组织标本,标本收集来自未曾经过放疗及化疗的患者。用免疫组化方法将30例肺腺癌和16例癌旁病理组织标本染色,观察染色情况检测肺腺癌及癌旁组织中PZ的表达。收集数据并完成统计分析,比较PZ在肺腺癌与癌旁组织中的表达情况。2、蛋白质印迹方法检测肺腺癌细胞中PZ的表达:实验细胞分别采用ACC212102、A549、SPCA-1三种人源肺腺癌细胞系进行培养。收集对数生长期细胞,冰冻PBS轻柔冲洗细胞代谢产物,加入RIPA裂解液,放置冰上充分裂解,低温超速离心提取细胞蛋白质。利用BCA方法检测蛋白浓度,严格按照Western blot操作流程完成整个实验过程。收集数据并完成统计分析,了解PZ在不同肺腺癌细胞系上的表达情况,并筛选出在强表达PZ的肺腺癌细胞系。二合成靶向沉默目的基因PZ的si RNA,并转染入A549肺腺癌细胞系,方法检测沉默效果根据第一部分中Western blot方法检测肺腺癌细胞中的PZ表达结果,筛选出PZ表达效果最强的A549肺腺癌细胞系培养并进行下一步实验。收集对数生长期细胞,并将其分为5组,分别是:空白对照Mock组、无效序列阴性对照si RNA-NC组,实验组si RNA-PZ001、002、003(三组)。严格按照si RNA试剂盒操作说明书完成实验操作。将靶向目的基因si RNA脂质载体转染入A549肺腺癌细胞中,置于37℃、5%CO2培养箱中培养24-96小时,收集目的蛋白。BCA方法检测蛋白浓度,Western blot方法检测转染效果。收集数据并完成统计分析,对比转染前后PZ在肺腺癌细胞中的表达情况,筛选最有效沉默PZ的靶向序列,为下一步细胞实验:划痕实验、细胞迁移、侵袭实验、细胞增殖及凋亡实验准备。三划痕实验、Transwell方法分别检测si RNA转染前后A549细胞迁移及侵袭能力变化1、划痕实验检测沉默PZ前后的A549细胞迁移能力:收集对数生长期细胞,并分为3组,分别是:空白对照Mock组、阴性对照si RNA-NC组,实验组si RNAPZ组。选取6孔板并在底部标记横线,将3组细胞分别接种适量细胞到孔中,过夜长满单层细胞,用小枪头垂直背后划痕,PBS轻柔冲洗去除划下的细胞,加入无血清培养液,置于37℃、5%CO2恒温细胞培养箱中培养,0小时、24小时、48小时取样拍照。收集数据并完成统计分析,对比对照组与实验组结果,了解PZ对肺腺癌细胞迁移能力的影响。2、Transwell方法检测沉默目PZ前后的A549细胞迁移及侵袭能力:收集对数生长期细胞,并分为3组,分别是:Mock组、si RNA-NC组,si RNA-PZ组。根据A459细胞的直径,选择孔径为0.8um的Transwell小室。将3组细胞分别接种适量细胞到上层小室中并加入无双抗无胎牛血清的培养液,下层为含有20%胎牛血清的培养液(若实验为侵袭实验,则在接种细胞前需要先在小室的上室中按比例铺一层基质胶),置于37℃、5%CO2恒温细胞培养箱中培养24小时后取出,甲醇固定,0.1%结晶紫染色,棉签轻轻擦掉上层未迁移细胞,显微镜下观察细胞。收集数据并完成统计分析,对比对照组与实验组结果,了解PZ对肺腺癌细胞迁移、侵袭转移能力的影响。四蛋白印迹方法检测PZ对细胞间质化肿瘤转移机制作用收集对数生长期细胞,分为3组:Mock组、si RNA-NC组与si RNA-PZ组。对三组肺腺癌细胞同时检测Slug、Vimentin、N-cadherin三种蛋白。收集数据并进行统计分析,对比三组之间Slug、Vimentin、N-cadherin三种蛋白表达差异,了解PZ对肺腺癌细胞间质化(epithelial-mesenchymal transition,EMT)转移机制的作用。五CCK-8方法检测si RNA转染前后A549细胞的增殖变化情况收集对数生长期细胞,分为3组:Mock组、si RNA-NC组与si RNA-PZ组。选择96孔板,将不同组细胞分别接种到小孔中,每次实验每组细胞均接种至5个孔中,并放置于37℃、5%CO2恒温细胞培养箱中培养24小时后,每个孔加入10ul CCK溶液,并于细胞培养箱中培养12小时、24小时、48小时、72小时,并分别检测对应时间点的吸光度值。收集数据并进行统计分析,对比不同时间点三组之间细胞增殖情况,了解PZ对肺腺癌细胞增殖能力的影响。六流式细胞方法检测si RNA转染前后的A549细胞凋亡坏死变化情况分别选取已被si RNA-PZ转染及没有被转染的A549细胞。收集对数生长期细胞,分为2组:对照Control组与实验si RNA-PZ组。将各组细胞消化、离心收集5×10^5个细胞,取500ul 1×Binding Buffer重悬细胞,然后每管加入5ul Annexin V-FITC和10ul PI,轻柔涡旋之后,室温避光孵育5分钟,放置在流式仪上分析。收集数据并进行统计分析,对比两组细胞之间的结果,了解PZ对肺腺癌细胞凋亡的影响。结果:一PZ在肺腺癌组织及肺腺癌细胞中高表达1.免疫组化实验结果显示:PZ在肺腺癌组织中表达明显高于癌旁组织,差异有统计学意义(P0.05)。2.Western blot实验结果显示:PZ分别在ACC212102、A549、SPCA-1三种不同的肺腺癌细胞中均有表达。PZ蛋白在A549细胞中表达尤为突出。A549细胞与SPCA-1、ACC212102两种肺腺癌细胞相比,PZ的表达量差异均有统计学意义(P0.05)。二si RNA有效沉默PZ并筛选效果最佳效序列经过si RNA脂质体转染后,si RNA-PZ001组与Mock组及si RNA-NC组相比,si RNA-PZ001中的PZ在表达水平明显下降。差异有统计学意义(P0.001),提示si RNA脂质体靶向沉默PZ基因有效,可以进行下一步实验;而Mock组与si RNA-NC组细胞中PZ的表达,差异无统计学意义(P0.05)。三si RNA-PZ组细胞迁移及侵袭能力较靶向沉默前明显下降1.划痕实验结果发现:si RNA-PZ组肺腺癌细胞与Mock组及si RNA-NC组细胞迁移速度比较,无论是在24小时还是48小时后,si RNA-PZ组细胞向中线运动迁移速度明显缓慢,差异均有统计学意义(P0.05)。抑制PZ表达能够抑制A549细胞的迁移,这提示PZ表达在A549细胞中可能参与并且有助于肺腺癌细胞的迁移运动。而Mock组与si RNA-NC组细胞迁移速度比较,差异无统计学意义(P0.05)。2.Transwell迁移及侵袭实验结果发现:无论在迁移实验还是侵袭实验中,si RNA-PZ组肺腺癌细胞与Mock组及si RNA-NC组比较,穿过Transwell小室膜的细胞数目明显减少,差异均有统计学意义(P0.05)。抑制PZ表达能够抑制A549细胞迁移及侵袭能力,这提示PZ在肺腺癌细胞中表达,能够促进肺腺癌细胞的迁移能力,有助肺腺癌细胞的转移。而Mock组与si RNA-NC组比较,穿过小室膜的细胞数目差异无统计学意义(P0.05)。四PZ可能通过EMT途径影响A549细胞的迁移及侵袭利用Western blot检测与细胞间质化转移途径相关蛋白Slug、Vimentin及Ncadherin三种蛋白结果发现:si RNA-PZ组与Mock组、si RNA-NC组相比较,Slug、Vimentin及N-cadherin三种蛋白表达均下降,差异均有统计学意义(P0.05),这提示PZ的表达能够影响Slug、Vimentin及N-cadherin蛋白的表达,初步提示了PZ可能通过作用EMT通路影响肺腺癌细胞的转移。五PZ可能不参与A549细胞增殖CCK-8实验结果发现:si RNA-PZ组、Mock组、si RNA组三组细胞增殖情况无明显差异,差异无统计学意义(P0.05),这提示PZ蛋白的表达可能不参与肺腺癌细胞的增殖过程。六PZ可能不影响A549细胞的凋亡流式细胞术检测细胞凋亡实验结果发现:si RNA-PZ组、Mock组、si RNA组三组细胞凋亡与坏死情况均无明显差异,差异均无统计学意义(P0.05),这提示PZ蛋白的表达不参与肺腺癌细胞的凋亡过程,对肺腺癌细胞的凋亡与坏死无影响。结论:1.PZ蛋白在肺腺癌组织与肺腺癌细胞中均出现高表达,临床标本检测提示PZ表达可能与肺腺癌发生、发展相关。2.通过细胞生物学实验与分子实验证实PZ与肺腺癌的迁移与侵袭呈正相关。3.PZ蛋白可能通过EMT影响肿瘤转移。4.PZ蛋白的表达可能不参与肺腺癌细胞的增殖过程。5.PZ蛋白的表达不参与肺腺癌细胞的凋亡过程,对肺腺癌细胞的凋亡与坏死无影响。
[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.

【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

【参考文献】