摘要
胃癌是严重威胁人类生命健康的主要疾病之一，当前已经成为全球重大的公共卫生问题。在我国胃癌发病率和死亡率均占消化系统恶性肿瘤的第一位。由于胃癌的早期症状不典型，当患者出现明显不适症状时，病情往往已经发展的较晚，失去了行根治性手术的机会，对于这部分患者，化疗成为其治疗的主要手段。虽然近年来不断研究出新的化疗药物及改进的联合化疗方案，但胃癌患者的化疗效果仍然不尽如人意。胃癌的多药耐药（multidrug resistance，MDR）往往是导致化疗治疗效果欠佳的主要原因之一。MDR发生的机制较为复杂，而且受到多种因素影响，研究逆转MDR的有效方法已经成为提高化疗疗效的关键。因此，如何逆转胃癌的MDR，提高胃癌化疗疗效，成为当前胃癌研究中的热点，也是临床胃癌治疗中急待解决的问题。
蛋白质组学（Proteomics）是以基因编码的所有蛋白质组为研究对象，从整体水平上分析细胞内动态变化的蛋白质组成成份、表达水平与修饰状态，了解蛋白质之间的相互作用与联系，进而揭示蛋白质功能及其与细胞生命活动规律的关系。蛋白质组学的技术体系是以双相凝胶电泳（two-dimensional polyacrylamide gel electrophoresis，2-DE）为主的蛋白质分离技术和以质谱（mass spectrometry，MS）、生物信息学（Bioinformatics）为主的蛋白质鉴定技术。
本实验共分为四部分，第一部分应用siRNA抑制人胃癌SGC7901细胞中ZNF139表达及并研究其对化疗药物敏感性影响；第二部分应用siRNA抑制人胃癌裸鼠原位移植瘤中ZNF139表达及并研究其对化疗药物敏感性影响。通过第一、二部分的研究，得出应用siRNA能够抑制人胃癌SGC7901细胞及其裸鼠原位移植瘤中ZNF139的表达，抑制ZNF139的表达后能够提高胃癌细胞对于化疗药物的敏感性。第三部分应用蛋白质组学技术对siRNA抑制人胃癌SGC7901细胞及其裸鼠原位移植瘤中前后的差异蛋白进行分离鉴定，寻找与ZNF139参与胃癌发生、发展及多药耐药有可能相关的蛋白。通过第三部分的研究，得出ZNF139有可能通过PDXK、ANXA2及Fascin而参与胃癌的发生、发展及多药耐药。第四部分应用qRT-PCR及Western blot法检测各实验组人胃癌SGC7901细胞及其裸鼠原位移植瘤中PDXK、ANXA2及Fascin的表达情况。通过第四部分的研究，得出ZNF139有可能通过促进ANXA2、Fascin的表达及抑制PDXK的表达来参与胃癌的发生、发展及多药耐药等过程。
本课题四部分的具体内容如下：
第一部分：siRNA抑制人胃癌SGC7901细胞中ZNF139表达及其对化疗药物敏感性影响的研究
目的：以人胃癌SGC7901细胞为研究对象，检测siRNA抑制人胃癌SGC7901细胞前后ZNF139的表达情况，并研究其对化疗药物敏感性的影响。
方法：
1设计合成针对ZNF139的小干扰RNA，构建siRNA-ZNF139表达质粒。培养人胃癌SGC7901细胞，应用siRNA-ZNF139质粒转染人胃癌SGC7901细胞。
2qRT-PCR检测各实验组人胃癌SGC7901细胞中ZNF139mRNA表达水平的变化。
3Western blot法检测各实验组人胃癌SGC7901细胞中ZNF139蛋白表达水平的变化。
4MTT法检测各实验组人胃癌SGC7901细胞对于化疗药物敏感性的变化。
结果：
1各实验组人胃癌SGC7901细胞中ZNF139mRNA表达水平的变化。分别用3个阳性质粒及阴性对照质粒以浓度为0.8μg/μl转染人胃癌SGC7901细胞48小时，依据结果可以发现空白对照组与阴性对照组中ZNF139mRNA的相对表达量最高，且二者之间表达差异无统计学意义（P>0.05）；1号阳性质粒组中ZNF139mRNA的相对表达量为最低，且其与空白对照组ZNF139mRNA的相对表达量比较差异有统计学意义（P<0.05）。各实验组中以1号阳性质粒组ZNF139的抑制效率最高，为84.41%。
选用1号阳性质粒，以0.4μg/μl、0.6μg/μl、0.8μg/μl、1.0μg/μl的浓度梯度转染人胃癌SGC7901细胞48小时，依据结果发现空白对照组ZNF139mRNA的相对表达量最高；浓度为0.8μg/μl组的ZNF139mRNA的相对表达量最低，且其与空白对照组ZNF139mRNA的相对表达量比较差异有统计学意义（P <0.05）。各实验组中以浓度0.8μg/μl组ZNF139的抑制效率最高，为84.42%。
选用浓度为0.8μg/μl的1号阳性质粒，分别转染胃癌细胞24小时、48小时、72小时，依据结果发现空白对照组ZNF139mRNA的相对表达量最高；转染48小时实验组的ZNF139mRNA表达量最低，且其与空白对照组ZNF139mRNA的相对表达量比较差异有统计学意义（P <0.05）。各实验组中以转染48小时实验组ZNF139的抑制效率最高，为84.36%。
2各实验组人胃癌SGC7901细胞中ZNF139蛋白表达水平的变化。
应用Western blot法检测各实验组ZNF139蛋白表达水平的变化。实验结果与qRT-PCR结果一致。
3各实验组人胃癌SGC7901细胞对于化疗药物敏感性的变化依据前面实验部分得到结果，选用0.8μg/μl浓度的1号阳性质粒及阴性对照质粒，转染人胃癌SGC7901细胞48小时后，应用MTT法检测各实验组人胃癌SGC7901细胞对于5-FU、ADR、CDDP、L-OHP、MMC、MTX、VP-16这7种化疗药物敏感性的变化。依据结果发现空白对照组与阴性对照组中相互之间各自比较细胞平均抑制率，差异无统计学意义（P>0.05）。siRNA-ZNF139组中各自较空白对照组细胞平均抑制率明显升高，且二者之间比较差异有统计学意义（P <0.05）。
结论：
1siRNA能够抑制人胃癌SGC7901细胞中ZNF139mRNA的表达。
2siRNA能够抑制人胃癌SGC7901细胞中ZNF139蛋白的表达。
3siRNA抑制人胃癌SGC7901细胞中ZNF139的表达能够提高胃癌细胞对于5-FU、ADR、CDDP、L-OHP、MMC、MTX、VP-16这7种化疗药物的敏感性。
第二部分：siRNA抑制人胃癌裸鼠原位移植瘤中ZNF139表达及其对化疗药物敏感性影响的研究
目的：以人胃癌SGC7901细胞裸鼠原位移植瘤为研究对象，检测siRNA抑制人胃癌裸鼠原位移植瘤前后ZNF139的表达情况，并研究其对化疗药物敏感性的影响。
方法：
1人胃癌裸鼠原位移植瘤模型的建立及干预
收集体外培养的人胃癌SGC7901细胞，建立裸鼠皮下移植瘤模型，传代5次，获取生长旺盛的第6代皮下移植瘤，将第6代皮下移植瘤应用OB生物胶粘贴法进行原位移植。术后每日观察裸鼠腹部切口、进食及腹腔原位移植瘤生长情况。待原位移植瘤长至直径约10mm左右时，分别随机按照空白对照组、siRNA-ZNF139组、siRNA-ZNF139+奥沙利铂组、奥沙利铂组及阴性对照组进行干预。干预期间，隔日测量一次瘤体长、短径，并按照V=ab2/2（a为瘤体长径，b为瘤体短径）计算原位移植瘤体积。干预处理两周后，取出原位移植瘤。
2qRT-PCR检测各实验组人胃癌裸鼠原位移植瘤中ZNF139mRNA表达水平的变化。
3Western blot法检测各实验组人胃癌裸鼠原位移植瘤中ZNF139蛋白表达水平的变化。
4MTT法检测各实验组人胃癌裸鼠原位移植瘤对于化疗药物敏感性的变化。
结果：
1人胃癌裸鼠原位移植瘤模型干预后的形态观察干预处理两周后，统计各实验组原位移植瘤体积（V=ab2/2，a为瘤体长径，b为瘤体短径）。依据结果可以发现空白对照组与阴性对照组中原位移植瘤的体积最大，且二者之间差异无统计学意义（P>0.05）；siRNA-ZNF139+奥沙利铂组中原位移植瘤的体积最小，且其与空白对照组比较差异有统计学意义（P<0.05）；siRNA-ZNF139组与空白对照组原位移植瘤的体积比较差异有统计学意义（P<0.05）；siRNA-ZNF139组与siRNA-ZNF139+奥沙利铂组原位移植瘤的体积比较差异有统计学意义（P<0.05）；奥沙利铂组与空白对照组原位移植瘤的体积比较差异有统计学意义（P<0.05）；奥沙利铂组与siRNA-ZNF139+奥沙利铂组原位移植瘤的体积比较差异有统计学意义（P<0.05）；siRNA-ZNF139组与奥沙利铂组原位移植瘤的体积比较差异无统计学意义（P>0.05）。
2各实验组人胃癌裸鼠原位移植瘤中ZNF139mRNA表达水平的变化
依据结果可以发现空白对照组与阴性对照组中ZNF139mRNA的相对表达量最高，且二者之间表达差异无统计学意义（P>0.05）；siRNA-ZNF139组中ZNF139mRNA的相对表达量为最低，且其与空白对照组ZNF139mRNA的相对表达量比较差异有统计学意义（P<0.05）。siRNA-ZNF139组ZNF139的抑制效率为81.05%。
3各实验组人胃癌裸鼠原位移植瘤中ZNF139蛋白表达水平的变化应用Western blot法检测各实验组ZNF139蛋白表达水平的变化。实验结果与qRT-PCR结果一致。
4各实验组人胃癌裸鼠原位移植瘤组织对于化疗药物敏感性的变化应用MTT法检测各实验组人胃癌裸鼠原位移植瘤组织细胞对于5-FU、ADR、 CDDP、L-OHP、MMC、MTX、VP-16这7种化疗药物敏感性的变化。依据结果发现空白对照组与阴性对照组中相互之间各自比较细胞平均抑制率，差异无统计学意义（P>0.05）。siRNA-ZNF139组中各自较空白对照组细胞平均抑制率明显升高，且二者之间比较差异有统计学意义（P <0.05）。
结论：
1siRNA-ZNF139能够抑制人胃癌裸鼠原位移植瘤的生长。
2siRNA能够抑制人胃癌裸鼠原位移植瘤中ZNF139mRNA的表达。
3siRNA能够抑制人胃癌裸鼠原位移植瘤中ZNF139蛋白的表达。
4siRNA抑制人胃癌裸鼠原位移植瘤中ZNF139的表达能够提高原位移植瘤组织对于5-FU、ADR、 CDDP、L-OHP、MMC、MTX、VP-16这7种化疗药物的敏感性。
第三部分：siRNA抑制人胃癌SGC7901细胞及其裸鼠原位移植瘤中ZNF139表达的蛋白质组学研究
目的：以人胃癌SGC7901细胞及其裸鼠原位移植瘤为研究对象，通过双向荧光差异凝胶电泳（2D-DIGE）和液相色谱-质谱联用（LC-MS）等蛋白质组学技术研究各实验组人胃癌SGC7901细胞及其裸鼠原位移植瘤中差异蛋白的表达情况。
方法：应用双向荧光差异凝胶电泳（2D-DIGE）技术分别分离各实验组人胃癌SGC7901细胞及其裸鼠原位移植瘤蛋白质，选择差异点，并用Ettan spot picker挖取，挖取点行胶内酶解，应用液相色谱-质谱联用（LC-MS）鉴定技术对挖取的蛋白质点鉴定，并对鉴定出的蛋白质进行分析。
结果：人胃癌SGC7901细胞双向荧光差异凝胶电泳（2D-DIGE）图谱上蛋白质点为1958±67个，匹配率为90.5%，人胃癌裸鼠原位移植瘤2D-DIGE图谱上蛋白质点为5227±59，匹配率为88.7%。
在人胃癌SGC7901细胞中选取8个差异明显的蛋白点，经由LC-MS鉴定出6种蛋白质：吡哆醛激酶（pyridoxal kinase，PDXK）、结蛋白（Desmin）、核磷蛋白（nucleophosmin，NPM）、热休克蛋白70（heat shockprotein70，HSP70）、膜联蛋白A2（annexin A2，ANXA2）、和Fascin。在siRNA-ZNF139转染后的人胃癌SGC7901细胞中PDXK、Desmin、NPM表达上调，HSP70、ANXA2、Fascin表达下调。
在人胃癌裸鼠原位移植瘤中选取6个差异明显的蛋白点，经由LC-MS鉴定出5种蛋白质：ANXA1、PDXK、Fascin、ANXA2、不均一核糖核蛋白（heterogeneous nuclear ribonucleoprotein，hnRNP）。在siRNA-ZNF139转染后的人胃癌裸鼠原位移植瘤中ANXA1、PDXK表达上调，Fascin、ANXA2、hnRNP表达下调。
结论：
1在siRNA-ZNF139转染后的人胃癌SGC7901细胞中PDXK、Desmin、NPM表达上调，HSP70、ANXA2、Fascin表达下调。
2在siRNA-ZNF139转染后的人胃癌裸鼠原位移植瘤中ANXA1、PDXK表达上调，Fascin、ANXA2、hnRNP表达下调。
3ZNF139可能通过调节PDXK、ANXA2及Fascin而参与胃癌的发生、发展及多药耐药。
第四部分：siRNA-ZNF139对人胃癌SGC7901细胞及其裸鼠原位移植瘤中PDXK、ANXA2及Fascin表达影响的研究
目的：以人胃癌SGC7901细胞及其裸鼠原位移植瘤为研究对象，检测各实验组人胃癌SGC7901细胞及其裸鼠原位移植瘤中PDXK、ANXA2及Fascin的表达情况。
方法：
1培养人胃癌SGC7901细胞，应用siRNA-ZNF139质粒转染人胃癌SGC7901细胞。
2人胃癌裸鼠原位移植瘤模型的建立及干预
3qRT-PCR检测各实验组人胃癌SGC7901细胞及其裸鼠原位移植瘤中PDXK、ANXA2及Fascin mRNA表达水平的变化。
4Western blot法检测各实验组人胃癌SGC7901细胞及其裸鼠原位移植瘤中PDXK、ANXA2及Fascin蛋白表达水平的变化。
结果：
1各实验组人胃癌SGC7901细胞及其裸鼠原位移植瘤中PDXK、ANXA2及Fascin mRNA表达水平的变化
应用qRT-PCR检测空白对照组、siRNA-ZNF139组及阴性对照组中人胃癌SGC7901细胞中PDXK mRNA表达水平的变化。依据结果可以发现空白对照组与阴性对照组中PDXK mRNA的相对表达量较低，且二者之间表达差异无统计学意义（P>0.05）；siRNA-ZNF139组中PDXK mRNA的相对表达量为最高，且其与空白对照组PDXK mRNA的相对表达量比较差异有统计学意义（P<0.05）。
应用qRT-PCR检测空白对照组、siRNA-ZNF139组及阴性对照组中人胃癌SGC7901细胞中ANXA2mRNA表达水平的变化。依据结果可以发现空白对照组与阴性对照组中ANXA2mRNA的相对表达量最高，且二者之间表达差异无统计学意义（P>0.05）；siRNA-ZNF139组中ANXA2mRNA的相对表达量为最低，且其与空白对照组ANXA2mRNA的相对表达量比较差异有统计学意义（P<0.05）。
应用qRT-PCR检测空白对照组、siRNA-ZNF139组及阴性对照组中人胃癌SGC7901细胞中Fascin mRNA表达水平的变化。依据结果可以发现空白对照组与阴性对照组中Fascin mRNA的相对表达量最高，且二者之间表达差异无统计学意义（P>0.05）；siRNA-ZNF139组中Fascin mRNA的相对表达量为最低，且其与空白对照组Fascin mRNA的相对表达量比较差异有统计学意义（P<0.05）。
应用qRT-PCR检测空白对照组、siRNA-ZNF139组及阴性对照组中人胃癌裸鼠原位移植瘤中PDXK mRNA表达水平的变化。依据结果可以发现空白对照组与阴性对照组中PDXK mRNA的相对表达量较低，且二者之间表达差异无统计学意义（P>0.05）；siRNA-ZNF139组中PDXKmRNA的相对表达量为最高，且其与空白对照组PDXK mRNA的相对表达量比较差异有统计学意义（P<0.05）。
应用qRT-PCR检测空白对照组、siRNA-ZNF139组及阴性对照组中人胃癌裸鼠原位移植瘤中ANXA2mRNA表达水平的变化。依据结果可以发现空白对照组与阴性对照组中ANXA2mRNA的相对表达量最高，且二者之间表达差异无统计学意义（P>0.05）；siRNA-ZNF139组中ANXA2mRNA的相对表达量为最低，且其与空白对照组ANXA2mRNA的相对表达量比较差异有统计学意义（P<0.05）。
应用qRT-PCR检测空白对照组、siRNA-ZNF139组及阴性对照组中人胃癌裸鼠原位移植瘤中Fascin mRNA表达水平的变化。依据结果可以发现空白对照组与阴性对照组中Fascin mRNA的相对表达量最高，且二者之间表达差异无统计学意义（P>0.05）；siRNA-ZNF139组中FascinmRNA的相对表达量为最低，且其与空白对照组Fascin mRNA的相对表达量比较差异有统计学意义（P<0.05）。
2各实验组人胃癌SGC7901细胞及其裸鼠原位移植瘤中PDXK、ANXA2及Fascin蛋白表达水平的变化。
应用Western blot法检测各实验组PDXK、ANXA2及Fascin蛋白表达水平的变化。实验结果与qRT-PCR结果一致。
结论：
1siRNA-ZNF139转染人胃癌SGC7901细胞及其裸鼠原位移植瘤后，PDXK mRNA的表达水平上升，ANXA2mRNA及Fascin mRNA的表达水平降低。
2siRNA-ZNF139转染人胃癌SGC7901细胞及其裸鼠原位移植瘤后，PDXK蛋白的表达水平上升，ANXA2蛋白及Fascin蛋白的表达水平降低。
3ZNF139有可能通过促进ANXA2、Fascin的表达及抑制PDXK的表达来参与胃癌的发生、发展及多药耐药等过程。
综合上述四部分实验内容，本研究小结如下：
1siRNA能够抑制人胃癌SGC7901细胞及其裸鼠原位移植瘤中ZNF139mRNA的表达。
2siRNA能够抑制人胃癌SGC7901细胞及其裸鼠原位移植瘤中ZNF139蛋白的表达
3siRNA抑制人胃癌SGC7901细胞及其裸鼠原位移植瘤中ZNF139的表达能够提高胃癌细胞对于化疗药物的敏感性。
4siRNA-ZNF139能够抑制人胃癌裸鼠原位移植瘤的生长。
5在siRNA-ZNF139转染后的人胃癌SGC7901细胞中PDXK、Desmin、 NPM表达上调， HSP70、 ANXA2、 Fascin表达下调。在siRNA-ZNF139转染后的人胃癌裸鼠原位移植瘤中ANXA1、PDXK表达上调，Fascin、ANXA2、hnRNP表达下调。
6siRNA-ZNF139转染人胃癌SGC7901细胞及其裸鼠原位移植瘤后，PDXK mRNA的表达水平上升，ANXA2mRNA及Fascin mRNA的表达水平降低。
7siRNA-ZNF139转染人胃癌SGC7901细胞及其裸鼠原位移植瘤后，PDXK蛋白的表达水平上升，ANXA2蛋白及Fascin蛋白的表达水平降低。
8ZNF139有可能通过促进ANXA2、Fascin的表达及抑制PDXK的表达来参与胃癌的发生、发展及多药耐药等过程。
Gastric cancer is one of the major diseases considered a serious threat tohuman life and health, and it has become a major global public healthproblem.In China gastric carcinoma is the first place of the incidence andmortality rate in malignant tumors of the digestive system. The earlysymptoms of stomach cancer are atypical; therefore, when patients exhibit theapparent symptoms, the disease has already developed into the late stage.Therefore, the opportunity for radical surgery for these patients is lost, andchemotherapy becomes the primary means of their treatment. In recent years,new chemotherapy drugs and improved combined chemotherapy have beendeveloped. However, the effects of chemotherapy on gastric cancer patientsare still not satisfactory. The multidrug resistance (MDR) of gastric cancer isone of the main causes that result in the ill effects of chemotherapy treatment.Mechanisms of MDR are more complex, and it also influence by multiplefactors. Researching the effective ways of reversing MDR has become the keyto improving the efficacy of chemotherapy. Therefore, how to reverse MDR ingastric carcinoma and improve chemotherapy of gastric carcinoma become thecurrent hot spots in the study of gastric carcinoma and the pressing problemsin the clinical treatment of gastric carcinoma.
Proteomics focuses on studies of the all proteins encoded by genes, andmakes complete analysis of the protein composition, expression levels andmodification state of the intracellular proteins with dynamic changes, andunderstands the interactions and associations among proteins, thus revealingthe protein function and its relationship with cell life activity Proteomicstechnological system is protein separation techniques based ontwo-dimensional polyacrylamide gel electrophoresis (2-DE) and proteinidentification techniques based on mass spectrometry (MS) and bioinformatics.
The experiment was divided into four parts. In the first part, it was usedsiRNA to inhibit the expression of ZNF139in human gastric carcinoma cellSGC7901and researched its effect on chemotherapeutic sensitivity. In thesecond part, it was used siRNA to inhibit the expression of ZNF139inorthotopic transplantation tumor of human gastric cancer in nude mice andresearched its effect on chemotherapeutic sensitivity. From the research of thefirst and second part, it concluded that using siRNA can inhibit the expressionof ZNF139in human gastric carcinoma cell SGC7901and orthotopictransplantation tumor of human gastric cancer in nude mice, and sensitivity ofgastric carcinoma cell to chemotherapy drugs can be improve after inhibitingthe expression of ZNF139. In the third part, it was used the proteomicstechnology to separate and identify difference protein from human gastriccarcinoma cells SGC7901and orthotopic transplantation tumor of humangastric cancer in nude mice, and it was purpose to search the proteins whichrelated with ZNF139participation occurrence, development and multidrugresistance of gastric carcinoma. From the research of the third part, itconcluded that ZNF139possible participate occurrence, development andmultidrug resistance of gastric carcinoma through regulating the expression ofPDXK, ANXA2and Fascin. In the fourth part, it was used the qRT-PCR andWestern blot to detect the expression of PDXK, ANXA2and Fascin in humangastric carcinoma cell SGC7901and orthotopic transplantation tumor ofhuman gastric cancer in nude mice which ZNF139inhibited by siRNA. Fromthe research of the fourth part, it concluded that ZNF139possible participateoccurrence, development and multidrug resistance of gastric carcinomathrough increasing the expression of ANXA2, Fascin and reducing theexpression of PDXK.
The specific contents of the four parts in this experiment are as follows:
Part I: Inhibiting ZNF139expression by siRNA in human gastriccarcinoma cell SGC7901and researching its effect onchemotherapeutic sensitivity.
Objective: to object human gastric carcinoma cell SGC7901, to detectthe expression of ZNF139in human gastric carcinoma cell SGC7901beforeand after using siRNA, to research its effect on chemotherapeutic sensitivity.
Methods:
1The siRNA was designed and synthesized according to the ZNF139.The siRNA-ZNF139plasmids were constructed. Human gastric carcinomacell SGC7901was cultured. The siRNA-ZNF139plasmid was transfected intohuman gastric carcinoma cell SGC7901.
2The qRT-PCR method was applied to detect the expression of ZNF139on the mRNA level in human gastric carcinoma cell SGC7901in eachexperimental group.
3The Western Blot method was applied to detect the expression ofZNF139on the protein level in human gastric carcinoma cell SGC7901ineach experimental group.
4The MTT method was applied to detect sensitivity to chemotherapydrugs in human gastric carcinoma cell SGC7901in each experimental group.
Results:
1The expression of ZNF139on the mRNA level in human gastriccarcinoma cell SGC7901in each experimental group.
It was used the3positive plasmids and the negative control plasmid withconcentration for0.8μg/μl that transfected human gastric carcinoma cellSGC7901for48hours. From the results, it can be found that the relativeexpression quantity of ZNF139mRNA were highest in both blank controlgroup and negative control group, and there was no statistically significantdifference within both group (P>0.05). The relative expression quantity ofZNF139mRNA was lowest in no.1plasmid group, and there was statisticallysignificant difference within blank control group and no.1plasmidgroup(P<0.05). The inhibition rate of ZNF139was lowest in no.1plasmidgroup,84.41%.
It was used the no.1plasmids concentration for0.4μg/μl,0.6μg/μl,0.8μg/μl and1.0μg/μl that transfected human gastric carcinoma cell SGC7901 for48hours. From the results, it can be found that the relative expressionquantity of ZNF139mRNA were highest in blank control group. The relativeexpression quantity of ZNF139mRNA was lowest in0.8μg/μl group, andthere was statistically significant difference within blank control group and0.8μg/μl group (P<0.05). The inhibition rate of ZNF139was lowest in0.8μg/μl group,84.42%.
It was used the no.1plasmids concentration for0.8μg/μl that transfectedhuman gastric carcinoma cell SGC7901for24hours,48hours and72hours.From the results, it can be found that the relative expression quantity ofZNF139mRNA were highest in blank control group. The relative expressionquantity of ZNF139mRNA was lowest in48hours group, and there wasstatistically significant difference within blank control group and48hoursgroup (P<0.05). The inhibition rate of ZNF139was lowest in0.8μg/μl group,84.36%.
2The expression of ZNF139on the protein level in human gastriccarcinoma cell SGC7901in each experimental group.
The Western Blot method was applied to detect the expression ofZNF139on the protein level. Western blot results were consistent withqRT-PCR results.
3The sensitivity to chemotherapy drugs in human gastric carcinoma cellSGC7901in each experimental group.
According to the previous results, it was used the no.1plasmidsconcentration for0.8μg/μl that transfected human gastric carcinoma cellSGC7901for48hours. The MTT method was applied to detect sensitivity to5-FU, and ADR, CDDP, L-OHP, MMC, MTX, VP-16in human gastriccarcinoma cell SGC7901in each experimental group. From the results, it canbe found that the cell inhibition rate between blank control group and negativecontrol group was no statistically significant difference within both group(P>0.05). The cell inhibition rate was obviously higher compared with blankcontrol group, and there was statistically significant difference within bothgroup (P<0.05).
Conclusions:
1The expression of ZNF139on the mRNA level can be inhibited inhuman gastric carcinoma cell SGC7901by siRNA.
2The expression of ZNF139on the protein level can be inhibited inhuman gastric carcinoma cell SGC7901by siRNA.
3The sensitivity of gastric carcinoma cell to chemotherapy drugs5-FU,ADR, CDDP, L-OHP, MMC, MTX, VP-16can be improve after inhibiting theexpression of ZNF139in human gastric carcinoma cell SGC7901by siRNA.Part II: Inhibiting ZNF139expression by siRNA in orthotopic
transplantation tumor of human gastric cancer in nude mice
and researching its effect on chemotherapeutic sensitivity.
Objective: to object orthotopic transplantation tumor of human gastriccancer in nude mice, to detect the expression of ZNF139in orthotopictransplantation tumor of human gastric cancer in nude mice before and afterusing siRNA, to research its effect on chemotherapeutic sensitivity.
Methods:
1Established orthotopic transplantation nude mice model of humangastric cancer and intervention
Human gastric carcinoma cell SGC7901cultured in vitro were collected.It was established subcutaneous transplantation tumor model in nude mice,and passage5times. It was get the vigorous growth of the6th generation ofsubcutaneous transplantation tumors. It was used the OB biological glue pastemethod transplanted the6th generation subcutaneously to orthotopictransplantation. It was observed the nude mice abdominal incision eating andceliac orthotopic tumor growth after surgery everyday. When diameter of theorthotopic transplantation tumor was10mm or so, it was begin to intervention.According to the blank control group, siRNA-ZNF139group, siRNA-ZNF139+oxaliplatin group, oxaliplatin and negative control groups intervene nudemice in random. During intervention every other day, it was measured the sizeof tumors, and calculated the volume of orthotopic transplantation tumoraccording to V=ab2/2(a, length diameter of tumor; b, short diameter of tumor). It was take out orthotopic transplantation tumor after two weeks.
2The qRT-PCR method was applied to detect the expression of ZNF139on the mRNA level in orthotopic transplantation tumor of human gastriccancer in nude mice in each experimental group.
3The Western Blot method was applied to detect the expression ofZNF139on the protein level in orthotopic transplantation tumor of humangastric cancer in nude mice in each experimental group.
4The MTT method was applied to detect sensitivity to chemotherapydrugs in orthotopic transplantation tumor of human gastric cancer in nudemice in each experimental group.
Results:
1Morphological observation of orthotopic transplantation nude micemodel of human gastric cancer after intervention
After two weeks during intervention, it was statistical volume oforthotopic transplantation tumor (V=ab2/2; a, length diameter of tumor; b,short diameter of tumor). From the results, it can be found that the volume oforthotopic transplantation tumor were biggest in both blank control group andnegative control group, and there was no statistically significant differencewithin both group (P>0.05). The volume of orthotopic transplantation tumorwas smallest in siRNA-ZNF139+oxaliplatin group, and there was statisticallysignificant difference within blank control group and siRNA-ZNF139+oxaliplatin group (P<0.05). There was statistically significant differencewithin siRNA-ZNF139group and blank control group (P<0.05). There wasstatistically significant difference within siRNA-ZNF139group andsiRNA-ZNF139+oxaliplatin group (P<0.05).There was statistically significantdifference within oxaliplatin group and blank control group (P<0.05). Therewas statistically significant difference within oxaliplatin group andsiRNA-ZNF139+oxaliplatin group (P<0.05). There was no statisticallysignificant difference within siRNA-ZNF139group and oxaliplatin group(P>0.05).
2The expression of ZNF139on the mRNA level in orthotopic transplantation tumor of human gastric cancer in nude mice in eachexperimental group.
From the results, it can be found that the relative expression quantity ofZNF139mRNA were highest in both blank control group and negative controlgroup, and there was no statistically significant difference within both group(P>0.05). The relative expression quantity of ZNF139mRNA was lowest insiRNA-ZNF139group, and there was statistically significant difference withinblank control group and siRNA-ZNF139group(P<0.05). The inhibition rate ofZNF139was81.05%in siRNA-ZNF139group.
3The expression of ZNF139on the protein level in orthotopictransplantation tumor of human gastric cancer in nude mice in eachexperimental group.
The Western Blot method was applied to detect the expression ofZNF139on the protein level. Western blot results were consistent withqRT-PCR results.
4The sensitivity to chemotherapy drugs in orthotopic transplantationtumor of human gastric cancer in nude mice in each experimental group.
The MTT method was applied to detect sensitivity to5-FU, and ADR,CDDP, L-OHP, MMC, MTX, VP-16in orthotopic transplantation tumor ofhuman gastric cancer in nude mice in each experimental group. From theresults, it can be found that the cell inhibition rate between blank controlgroup and negative control group was no statistically significant differencewithin both group (P>0.05). The cell inhibition rate was obviously highercompared with blank control group, and there was statistically significantdifference within both group (P<0.05).
Conclusions:
1The growth of orthotopic transplantation tumor of human gastric cancerin nude mice by siRNA-ZNF139.
2The expression of ZNF139on the mRNA level can be inhibited inorthotopic transplantation tumor of human gastric cancer in nude mice bysiRNA.
3The expression of ZNF139on the protein level can be inhibited inorthotopic transplantation tumor of human gastric cancer in nude mice bysiRNA.
4The sensitivity of gastric carcinoma cell to chemotherapy drugs5-FU,ADR, CDDP, L-OHP, MMC, MTX, VP-16can be improve after inhibiting theexpression of ZNF139in orthotopic transplantation tumor of human gastriccancer in nude mice by siRNA.
Part III: Proteomics study on the expression of ZNF139in human gastriccarcinoma cell SGC7901and orthotopic transplantation tumorof human gastric cancer in nude mice inhibited by siRNA
Objective: to object human gastric carcinoma cell SGC7901andorthotopic transplantation tumor of human gastric cancer in nude mice, todetect the differences proteins in human gastric carcinoma cell SGC7901andorthotopic transplantation tumor of human gastric cancer in nude mice in eachexperimental group by2D-DIGE and LC-MS.
Methods:
The differences proteins in human gastric carcinoma cell SGC7901andorthotopic transplantation tumor of human gastric cancer in nude mice in eachexperimental group were separated by2D-DIGE.Application DeCyderDifferential Analysis Software selected difference obvious proteins and usedEttan spot picker to dig the points. The difference proteins were identified byLC-MS after in-gel digest. The SEQUEST computing method in BioWorkssoftware was applied for database retrieval.
Results:
About1958±67proteins are separated by2D-DIGE and the matchingrate is90.5%in human gastric carcinoma cell SGC7901. About5227±59proteins are separated by2D-DIGE and the matching rate is88.7%inorthotopic transplantation tumor of human gastric cancer in nude mice.
It was select eight differences proteins in human gastric carcinoma cellSGC7901. Six kinds of proteins are identified by LC-MS: Pyridoxal kinase(PDXK)，Desmin, Nucleophosmin (NPM), Heat shock protein70(HSP70), Annexin A2(ANXA2) and Fascin. PDXK, Desmin and NPM wereup-regulating while HSP70, ANXA2and Fascin were down-regulate insiRNA-ZNF139group.
It was select six differences proteins in orthotopic transplantation tumorof human gastric cancer in nude mice. Five kinds of proteins are identified byLC-MS: ANXA1, PDXK, Fascin, ANXA2and Heterogeneous nuclearribonucleoprotein (hnRNP). ANXA1, PDXK were up-regulating while Fascin,ANXA2and hnRNP were down-regulate in siRNA-ZNF139group.
Conclusions:
1PDXK, Desmin and NPM were up-regulating while HSP70, ANXA2and Fascin were down-regulate in human gastric carcinoma cell SGC7901after using siRNA-ZNF139.
2ANXA1, PDXK were up-regulating while Fascin, ANXA2and hnRNPwere down-regulate in siRNA-ZNF139group in orthotopic transplantationtumor of human gastric cancer in nude mice after using siRNA-ZNF139.
3ZNF139possible participate occurrence, development and multidrugresistance of gastric carcinoma through regulating the expression of PDXK,ANXA2and Fascin.
Part IV: Research on effects of siRNA-ZNF139on expression of PDXK,ANXA2and Fascin in human gastric carcinoma cell SGC7901and orthotopic transplantation tumor of human gastric cancerin nude mice
Objective: to object human gastric carcinoma cell SGC7901andorthotopic transplantation tumor of human gastric cancer in nude mice, todetect the expression of PDXK, ANXA2and Fascin in human gastriccarcinoma cell SGC7901and orthotopic transplantation tumor of humangastric cancer in nude mice before and after using siRNA,
Methods:
1Human gastric carcinoma cell SGC7901was cultured. ThesiRNA-ZNF139plasmid was transfected into human gastric carcinoma cellSGC7901
2Established orthotopic transplantation nude mice model of humangastric cancer and intervention
3The qRT-PCR method was applied to detect the expression of PDXK,ANXA2and Fascin on the mRNA level in human gastric carcinoma cellSGC7901and orthotopic transplantation tumor of human gastric cancer innude mice in each experimental group.
4The Western Blot method was applied to detect the expression ofPDXK, ANXA2and Fascin on the protein level in human gastric carcinomacell SGC7901and orthotopic transplantation tumor of human gastric cancer innude mice in each experimental group.
Results:
1The expression of PDXK, ANXA2and Fascin on the mRNA level inhuman gastric carcinoma cell SGC7901and orthotopic transplantation tumorof human gastric cancer in nude mice in each experimental group.
The qRT-PCR method was applied to detect the expression of PDXK onthe mRNA level in human gastric carcinoma cell SGC7901in eachexperimental group. From the results, it can be found that the relativeexpression quantity of PDXK mRNA were lowest in both blank control groupand negative control group, and there was no statistically significant differencewithin both group (P>0.05). The relative expression quantity of PDXK mRNAwas highest in siRNA-ZNF139group, and there was statistically significantdifference within blank control group and siRNA-ZNF139group (P<0.05).
The qRT-PCR method was applied to detect the expression of ANXA2onthe mRNA level in human gastric carcinoma cell SGC7901in eachexperimental group. From the results, it can be found that the relativeexpression quantity of ANXA2mRNA were highest in both blank controlgroup and negative control group, and there was no statistically significantdifference within both group (P>0.05). The relative expression quantity ofANXA2mRNA was lowest in siRNA-ZNF139group, and there wasstatistically significant difference within blank control group andsiRNA-ZNF139group (P<0.05).
The qRT-PCR method was applied to detect the expression of Fascin onthe mRNA level in human gastric carcinoma cell SGC7901in eachexperimental group. From the results, it can be found that the relativeexpression quantity of Fascin mRNA were highest in both blank control groupand negative control group, and there was no statistically significant differencewithin both group (P>0.05). The relative expression quantity of Fascin mRNAwas lowest in siRNA-ZNF139group, and there was statistically significantdifference within blank control group and siRNA-ZNF139group (P<0.05).
The qRT-PCR method was applied to detect the expression of PDXK onthe mRNA level in orthotopic transplantation tumor of human gastric cancer innude mice in each experimental group. From the results, it can be found thatthe relative expression quantity of PDXK mRNA were lowest in both blankcontrol group and negative control group, and there was no statisticallysignificant difference within both group (P>0.05). The relative expressionquantity of PDXK mRNA was highest in siRNA-ZNF139group, and therewas statistically significant difference within blank control group andsiRNA-ZNF139group (P<0.05).
The qRT-PCR method was applied to detect the expression of ANXA2onthe mRNA level in orthotopic transplantation tumor of human gastric cancer innude mice in each experimental group. From the results, it can be found thatthe relative expression quantity of ANXA2mRNA were highest in both blankcontrol group and negative control group, and there was no statisticallysignificant difference within both group (P>0.05). The relative expressionquantity of ANXA2mRNA was lowest in siRNA-ZNF139group, and therewas statistically significant difference within blank control group andsiRNA-ZNF139group (P<0.05).
The qRT-PCR method was applied to detect the expression of Fascin onthe mRNA level in orthotopic transplantation tumor of human gastric cancer innude mice in each experimental group. From the results, it can be found thatthe relative expression quantity of Fascin mRNA were highest in both blankcontrol group and negative control group, and there was no statistically significant difference within both group (P>0.05). The relative expressionquantity of Fascin mRNA was lowest in siRNA-ZNF139group, and there wasstatistically significant difference within blank control group andsiRNA-ZNF139group (P<0.05).
2The expression of PDXK, ANXA2and Fascin on the protein level inhuman gastric carcinoma cell SGC7901and orthotopic transplantation tumorof human gastric cancer in nude mice in each experimental group.
The Western Blot method was applied to detect the expression of PDXK,ANXA2and Fascin on the protein level. Western blot results were consistentwith qRT-PCR results.
Conclusions:
1The expression of PDXK on the mRNA level increased and theexpression ANXA2, Fascin of on the mRNA level reduced in human gastriccarcinoma cell SGC7901and orthotopic transplantation tumor of humangastric cancer in nude mice after using siRNA-ZNF139.
2The expression of PDXK on the protein level increased and theexpression of ANXA2, Fascin on the protein level reduced in human gastriccarcinoma cell SGC7901and orthotopic transplantation tumor of humangastric cancer in nude mice after using siRNA-ZNF139.
3ZNF139possible participate occurrence, development and multidrugresistance of gastric carcinoma through increasing the expression of ANXA2,Fascin and reducing the expression of PDXK.
Based on the four parts of this research, we could draw conclusionsas follows:
1The expression of ZNF139on the mRNA levelcan be inhibited inhuman gastric carcinoma cell SGC7901and orthotopic transplantation tumorof human gastric cancer in nude mice by siRNA.
2The expression of ZNF139on the protein levelcan be inhibited inhuman gastric carcinoma cell SGC7901and orthotopic transplantation tumorof human gastric cancer in nude mice by siRNA.
3The sensitivity of gastric carcinoma cell to chemotherapy drugs can be improve after inhibiting the expression of ZNF139in human gastriccarcinoma cell SGC7901and orthotopic transplantation tumor of humangastric cancer in nude mice by siRNA.
4The growth of orthotopic transplantation tumor of human gastric cancerin nude mice by siRNA-ZNF139.
5PDXK, Desmin and NPM were up-regulating while HSP70, ANXA2and Fascin were down-regulate in human gastric carcinoma cell SGC7901after using siRNA-ZNF139. ANXA1, PDXK were up-regulating while Fascin,ANXA2and hnRNP were down-regulate in siRNA-ZNF139group inorthotopic transplantation tumor of human gastric cancer in nude mice afterusing siRNA-ZNF139.
6The expression of PDXK on the mRNA level increased and theexpression ANXA2, Fascin of on the mRNA level reduced in human gastriccarcinoma cell SGC7901and orthotopic transplantation tumor of humangastric cancer in nude mice after using siRNA-ZNF139.
7The expression of PDXK on the protein level increased and theexpression of ANXA2, Fascin on the protein level reduced in human gastriccarcinoma cell SGC7901and orthotopic transplantation tumor of humangastric cancer in nude mice after using siRNA-ZNF139.
8ZNF139possible participate occurrence, development and multidrugresistance of gastric carcinoma through increasing the expression of ANXA2,Fascin and reducing the expression of PDXK.
引文
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