磷脂混杂酶1和中期因子在原发性肝细胞癌发生发展中作用的研究
发布时间：2018-08-02 19:24
【摘要】：目的:通过检测PLSCR1与MK在肝癌组织及癌旁肝正常组织中的基因表达差异并分析PLSCR1与MK和肝癌病理特征的相关性及两者之间的相关性,并运用Cox回归模型分析临床基础资料对患者总生存期的影响;构建重组质粒,在细胞水平探讨PLSCR1与MK相互作用在肝癌发生发展中可能的作用机制。方法:1)收集我院2009年1月至2013年6月期间收治的原发性肝细胞癌手术切除患者肝癌组织和对应癌旁肝正常组织标本各40例,所有病例确诊为原发性肝细胞癌,所有病例均为初诊,术前均未接受过放化疗、中医及免疫治疗,临床资料完整。每例标本均留取肿癌组织和癌旁正常肝组织。手术标本切除后于30min内取样本并液氮速冻,保存于-80℃冰箱备用。采用Real-Time PCR技术(Q-PCR)检测40例肝癌组织与癌旁正常组织中PLSCR1与MK表达量,统计分析其与患者临床病理特征关系,采用Cox回归模型分析临床基础资料对患者总预后生存期的影响以及分析PLSCR1和MK表达水平之间的相关性。2)根据质粒pET28a(+)-PLSCR1、pGEX-4T1-MK、pcDNA3.1/myc-His(-)A-PLSCR1 和 pcDNA3.1-MK 设计引物,构建质粒;然后将测序确认的重组质粒pET28a(+)-PLSCR1和pGEX-4T1-MK重组表达质粒分别转入BL21菌株中,分别将上述重组表达质粒转化菌株中加入IPTG进行诱导表达,利用GST-pull down技术和免疫共沉淀(CO-IP)技术验证PLSCR1与相互作用蛋白MK之间的相互作用。3)研究PLSCR1和MK相互作用对肝癌细胞增殖的影响。分别构建PLSCR1和MK的siRNA干扰质粒,分组转染 HepG2 细胞:MK-siRNA 组,PLSCR1-siRNA 组,MK+PLSCR1-siRNA 共转组和未加任何处理的对照组,借助酶联免疫检测仪测定并记录各孔在450nm波长下对应的吸收值。4)检测PLSCR1和MK相互作用对肝癌细胞迁移的影响:将MK-si-RNA-HepG2,PLSCR1-siRNA-HepG2 细胞、MK+PLSCR1-siRNA-HepG2 细胞、Control-siRNA-HepG2细胞及HepG2细胞接种于小室内,培养48h后进行细胞染色,通过普通倒置显微镜下对细胞的迁移情况进行观察,在560nm下测定细胞裂解液的吸收值,并定量计算其含量。结果:1)肝癌组织中PLSCR1和MK基因的表达量明显高于癌旁组织,PLSCR1的 A△Ct 值分别为-2.36+1.49,2-△△Ct值(RQ 值)均值为 8.55±7.67;MK 的 △△Ct值为-2.97±0.86,RQ值为9.05±3.97,具有差异有统计学意义(P0.05)。依据40例患者RQ值均值将PLSCR1和MK进行分组,RQ值≥10为高表达组,RQ值10为低表达组进行分析,结果表明PLSCR1与MK的表达与患者的年龄、是否伴随肝硬化、血液AFP值、肿瘤大小及是否包膜侵犯无关(P0.05),而与患者的性别、肿瘤分化程度、肿瘤数目、血管浸润及临床分期相关性显著(P0.05)。2)我们通过Kaplan-meier生存分析PLSCR1和MK的癌/癌旁组织的倍数变化(RQ值)对肝癌术后总生存期的影响,发现PLSCR1和MK高表达组的生存率明显低于低表达组,Breslow法检验水平间的两两比较具有显著性差异(P0.05)。运用Cox回归模型分析患者临床基础资料及患者PLSCR1和MK基因的表达量与肝癌术后生存期相关风险因素,发现包膜侵犯、血管浸润、Ki-67值、性别、肝硬化及PLSCR1和MK高表达是评估总生存期的风险因素(P0.05),包膜侵犯风险系数最高为8.04(P=0.001);肝癌组织中PLSCR1和MK表达水平Spearman相关性分析结果显示r=0.30(P=0.03),两者存在弱正相关性。3)对成功构建的质粒进行酶切鉴定,测序结果表明,构建四个质粒的外源片段按正确的阅读框架连入pET28a(+)、pGEX-4T1、pcDNA3.1/myc-His(-)A和pcDNA3.1载体,阳性克隆经测序无误的抽提质粒;融合蛋白、蛋白沉降分析及CO-IP试验结果显示,PLSCR1和MK蛋白可以发生相互作用。4)成功构建MK和PLSCR1干扰细胞株,通过转染细胞干扰PLSCR1和MK的表达后,用CCK-8检测HepG2细胞的增殖情况,HepG2细胞的增殖效率显著降低,并同时干扰PLSCR1和MK对HepG2细胞增殖抑制作用更为显著(P0.01);PLSCR1和MK相互作用同样对HepG2细胞迁移具有抑制作用,侵移实验表明干扰PLSCR1可以明显影响HepG2细胞的迁移作用,并且同时干扰PLSCR1和MK对HepG2细胞的迁移抑制作用更显著(P0.01)。结论:1)PLSCR1与MK在肝癌组织中的表达明显高于癌旁组织,其表达水平与患者临床病理特征相关性表明与患者的性别、肿瘤分化程度、肿瘤数目、血管浸润及临床分期相关性显著(P0.05)。2)Cox回归模型分析患者临床基础资料及患者PLSCR1和MK基因的表达量与肝癌术后生存期相关风险因素,发现包膜侵犯、血管浸润、Ki-67值、性别、肝硬化及PLSCR1和MK高表达是评估总生存期的风险因素(P0.05);肝癌组织中PLSCR1和MK表达水平Spearman相关性分析两者存在正相关性(P0.05)。3)融合蛋白、蛋白沉降分析、CO-IP实验及GST-Pulldown实验结果表明,PLSCR1和MK蛋白可以发生相互作用。4)PLSCR1与MK蛋白相互作用影响HepG2细胞增殖和迁移,干扰PLSCR1可抑制HepG2细胞的增殖和迁移效果,同时干扰PLSCR1和MK对HepG2细胞的增殖和迁移的抑制作用更为显著(P0.01)。
[Abstract]:Objective: to detect the difference of gene expression between PLSCR1 and MK in liver cancer tissues and normal liver tissues, and to analyze the correlation between PLSCR1 and the pathological features of MK and liver cancer and the correlation between them, and analyze the effect of the clinical basic data on the total survival time of the patients with the Cox regression model, and to construct the recombinant plasmid and discuss the PLSCR1 at the cell level. The possible mechanism of interaction with MK in the development and development of liver cancer. Methods: 1) 40 cases of primary hepatocellular carcinoma (HCC) and 40 normal tissues adjacent to the paracancerous liver were collected from January 2009 to June 2013 in our hospital. All cases were diagnosed as primary hepatocellular carcinoma. All cases were first diagnosed before operation. There was no chemotherapy, traditional Chinese medicine and immunotherapy, and the clinical data were complete. Each specimen was left with tumor tissue and normal liver tissue near cancer. The surgical specimens were removed in 30min and frozen in liquid nitrogen and stored at -80 centigrade refrigerators. Real-Time PCR technique (Q-PCR) was used to detect the PLSCR1 and MK in 40 cases of liver cancer tissues and normal tissues adjacent to the cancer. The relationship between the clinical and pathological features of the patients was statistically analyzed, and the Cox regression model was used to analyze the effect of the clinical basic data on the patient's total prognosis and the correlation between the expression level of PLSCR1 and MK. The design of the primers based on the plasmid pET28a (+) -PLSCR1, pGEX-4T1-MK, pcDNA3.1/myc-His (-) A-PLSCR1 and pcDNA3.1-MK was designed. The recombinant plasmid pET28a (+) -PLSCR1 and pGEX-4T1-MK recombinant plasmid, confirmed by sequencing, were then transferred into the BL21 strain respectively, and the recombinant plasmid was transformed into IPTG for induction expression respectively. GST-pull down technology and immunoprecipitation (CO-IP) technology were used to verify the phase between PLSCR1 and interacting protein MK. Interaction.3) study the effect of PLSCR1 and MK interaction on the proliferation of hepatoma cells. Construct siRNA interference plasmids of PLSCR1 and MK respectively, and transfect HepG2 cells into group MK-siRNA, PLSCR1-siRNA group, MK+PLSCR1-siRNA co rotation group and no treated control group, and determine and record the pores at 450nm wavelength with the aid of enzyme immunoassay detector. The effect of the absorption value.4) to detect the effect of PLSCR1 and MK interaction on the migration of hepatoma cells: MK-si-RNA-HepG2, PLSCR1-siRNA-HepG2 cells, MK+PLSCR1-siRNA-HepG2 cells, Control-siRNA-HepG2 cells and HepG2 cells were inoculated into the chamber, and the cells were stained after cultivating 48h, and the cell migration under the common inverted microscope was carried out. The absorption value of the cell lysate was measured under 560nm and the content was calculated. Results: 1) the expression of PLSCR1 and MK genes in the liver cancer tissues was significantly higher than that in the paracancerous tissues, and the A Delta Ct values of PLSCR1 were respectively -2.36+1.49,2- Delta and Ct (RQ) value of 8.55 + 7.67, and MK Delta Ct was 0.86 and 9.05 + 3.97. The difference was statistically significant (P0.05). PLSCR1 and MK were grouped according to the mean value of RQ in 40 patients. The RQ value was more than 10 in the high expression group and the RQ value 10 was analyzed in the low expression group. The results showed that the expression of PLSCR1 and MK was not associated with the patient's age, whether the liver cirrhosis, the blood AFP, the size of the tumor and the invasion of the capsule (P0.05), and the sex with the patient's sex. Difference, tumor differentiation, tumor number, vascular invasion and clinical staging correlation significant (P0.05).2) we analyzed the effects of multiple changes (RQ values) on the total survival of liver cancer after Kaplan-meier survival analysis of PLSCR1 and MK, and found that the survival rate of the PLSCR1 and MK high table group was significantly lower than that of the low expression group, and the Breslow method was used to test the water. The 22 comparison of the 22 in the flat was significant (P0.05). The Cox regression model was used to analyze the patient's clinical basis and the risk factors associated with the expression of PLSCR1 and MK genes and the survival period of the postoperative liver cancer. The risk factors for the total survival were found by the invasion of the capsule, the vascular invasion, the Ki-67 value, the sex, the cirrhosis and the high expression of PLSCR1 and MK. P0.05), the highest risk factor of envelope invasion was 8.04 (P=0.001), and the Spearman correlation analysis of PLSCR1 and MK expression levels in liver cancer tissue showed r=0.30 (P=0.03), and there was a weak positive correlation between the two.3) and the plasmid was successfully constructed by enzyme digestion. The result of sequencing showed that the external fragments of the construction of the plasmid were linked to the correct reading frame into pE. T28a (+), pGEX-4T1, pcDNA3.1/myc-His (-) A and pcDNA3.1 vectors, positive clones were sequenced by unmistakable plasmids; fusion protein, protein sedimentation analysis and CO-IP test results showed that PLSCR1 and MK protein could interact.4) to construct MK and PLSCR1 interfering fine cell lines. The proliferation of HepG2 cells, the proliferation efficiency of HepG2 cells decreased significantly, and the inhibitory effect of PLSCR1 and MK on the proliferation of HepG2 cells was more significant (P0.01). The interaction of PLSCR1 and MK also inhibited the migration of HepG2 cells. The invasion experiment showed that the interference of PLSCR1 could significantly affect the migration of HepG2 cells. The inhibitory effect of PLSCR1 and MK on the migration of HepG2 cells was more significant (P0.01). Conclusion: 1) the expression of PLSCR1 and MK in the liver cancer tissues was significantly higher than that of the para cancerous tissue. The correlation between the expression level and the clinicopathological features of the patients showed that the correlation was significant with the sex, the degree of tumor differentiation, the number of tumor, the invasion of blood vessels and the clinical stage (P0.0 5).2) Cox regression model analysis of patients' clinical basic data and the risk factors associated with the expression of PLSCR1 and MK gene in patients with the survival period of liver cancer. It was found that the invasion of the capsule, the vascular invasion, the Ki-67 value, the sex, the liver cirrhosis and the high expression of PLSCR1 and MK were the risk factors for evaluating the total survival time (P0.05), and the expression level of PLSCR1 and MK was Spearm in the liver cancer tissue Spearm An correlation analysis has positive correlation (P0.05).3) fusion protein, protein sedimentation analysis, CO-IP experiment and GST-Pulldown experiment results show that PLSCR1 and MK protein can interact.4) the interaction of PLSCR1 and MK proteins affects the proliferation and migration of HepG2 cells, and interference PLSCR1 can inhibit the proliferation and migration effect of the cells. The inhibition of PLSCR1 and MK on proliferation and migration of HepG2 cells was more significant (P0.01).
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7
本文编号：2160481
[Abstract]:Objective: to detect the difference of gene expression between PLSCR1 and MK in liver cancer tissues and normal liver tissues, and to analyze the correlation between PLSCR1 and the pathological features of MK and liver cancer and the correlation between them, and analyze the effect of the clinical basic data on the total survival time of the patients with the Cox regression model, and to construct the recombinant plasmid and discuss the PLSCR1 at the cell level. The possible mechanism of interaction with MK in the development and development of liver cancer. Methods: 1) 40 cases of primary hepatocellular carcinoma (HCC) and 40 normal tissues adjacent to the paracancerous liver were collected from January 2009 to June 2013 in our hospital. All cases were diagnosed as primary hepatocellular carcinoma. All cases were first diagnosed before operation. There was no chemotherapy, traditional Chinese medicine and immunotherapy, and the clinical data were complete. Each specimen was left with tumor tissue and normal liver tissue near cancer. The surgical specimens were removed in 30min and frozen in liquid nitrogen and stored at -80 centigrade refrigerators. Real-Time PCR technique (Q-PCR) was used to detect the PLSCR1 and MK in 40 cases of liver cancer tissues and normal tissues adjacent to the cancer. The relationship between the clinical and pathological features of the patients was statistically analyzed, and the Cox regression model was used to analyze the effect of the clinical basic data on the patient's total prognosis and the correlation between the expression level of PLSCR1 and MK. The design of the primers based on the plasmid pET28a (+) -PLSCR1, pGEX-4T1-MK, pcDNA3.1/myc-His (-) A-PLSCR1 and pcDNA3.1-MK was designed. The recombinant plasmid pET28a (+) -PLSCR1 and pGEX-4T1-MK recombinant plasmid, confirmed by sequencing, were then transferred into the BL21 strain respectively, and the recombinant plasmid was transformed into IPTG for induction expression respectively. GST-pull down technology and immunoprecipitation (CO-IP) technology were used to verify the phase between PLSCR1 and interacting protein MK. Interaction.3) study the effect of PLSCR1 and MK interaction on the proliferation of hepatoma cells. Construct siRNA interference plasmids of PLSCR1 and MK respectively, and transfect HepG2 cells into group MK-siRNA, PLSCR1-siRNA group, MK+PLSCR1-siRNA co rotation group and no treated control group, and determine and record the pores at 450nm wavelength with the aid of enzyme immunoassay detector. The effect of the absorption value.4) to detect the effect of PLSCR1 and MK interaction on the migration of hepatoma cells: MK-si-RNA-HepG2, PLSCR1-siRNA-HepG2 cells, MK+PLSCR1-siRNA-HepG2 cells, Control-siRNA-HepG2 cells and HepG2 cells were inoculated into the chamber, and the cells were stained after cultivating 48h, and the cell migration under the common inverted microscope was carried out. The absorption value of the cell lysate was measured under 560nm and the content was calculated. Results: 1) the expression of PLSCR1 and MK genes in the liver cancer tissues was significantly higher than that in the paracancerous tissues, and the A Delta Ct values of PLSCR1 were respectively -2.36+1.49,2- Delta and Ct (RQ) value of 8.55 + 7.67, and MK Delta Ct was 0.86 and 9.05 + 3.97. The difference was statistically significant (P0.05). PLSCR1 and MK were grouped according to the mean value of RQ in 40 patients. The RQ value was more than 10 in the high expression group and the RQ value 10 was analyzed in the low expression group. The results showed that the expression of PLSCR1 and MK was not associated with the patient's age, whether the liver cirrhosis, the blood AFP, the size of the tumor and the invasion of the capsule (P0.05), and the sex with the patient's sex. Difference, tumor differentiation, tumor number, vascular invasion and clinical staging correlation significant (P0.05).2) we analyzed the effects of multiple changes (RQ values) on the total survival of liver cancer after Kaplan-meier survival analysis of PLSCR1 and MK, and found that the survival rate of the PLSCR1 and MK high table group was significantly lower than that of the low expression group, and the Breslow method was used to test the water. The 22 comparison of the 22 in the flat was significant (P0.05). The Cox regression model was used to analyze the patient's clinical basis and the risk factors associated with the expression of PLSCR1 and MK genes and the survival period of the postoperative liver cancer. The risk factors for the total survival were found by the invasion of the capsule, the vascular invasion, the Ki-67 value, the sex, the cirrhosis and the high expression of PLSCR1 and MK. P0.05), the highest risk factor of envelope invasion was 8.04 (P=0.001), and the Spearman correlation analysis of PLSCR1 and MK expression levels in liver cancer tissue showed r=0.30 (P=0.03), and there was a weak positive correlation between the two.3) and the plasmid was successfully constructed by enzyme digestion. The result of sequencing showed that the external fragments of the construction of the plasmid were linked to the correct reading frame into pE. T28a (+), pGEX-4T1, pcDNA3.1/myc-His (-) A and pcDNA3.1 vectors, positive clones were sequenced by unmistakable plasmids; fusion protein, protein sedimentation analysis and CO-IP test results showed that PLSCR1 and MK protein could interact.4) to construct MK and PLSCR1 interfering fine cell lines. The proliferation of HepG2 cells, the proliferation efficiency of HepG2 cells decreased significantly, and the inhibitory effect of PLSCR1 and MK on the proliferation of HepG2 cells was more significant (P0.01). The interaction of PLSCR1 and MK also inhibited the migration of HepG2 cells. The invasion experiment showed that the interference of PLSCR1 could significantly affect the migration of HepG2 cells. The inhibitory effect of PLSCR1 and MK on the migration of HepG2 cells was more significant (P0.01). Conclusion: 1) the expression of PLSCR1 and MK in the liver cancer tissues was significantly higher than that of the para cancerous tissue. The correlation between the expression level and the clinicopathological features of the patients showed that the correlation was significant with the sex, the degree of tumor differentiation, the number of tumor, the invasion of blood vessels and the clinical stage (P0.0 5).2) Cox regression model analysis of patients' clinical basic data and the risk factors associated with the expression of PLSCR1 and MK gene in patients with the survival period of liver cancer. It was found that the invasion of the capsule, the vascular invasion, the Ki-67 value, the sex, the liver cirrhosis and the high expression of PLSCR1 and MK were the risk factors for evaluating the total survival time (P0.05), and the expression level of PLSCR1 and MK was Spearm in the liver cancer tissue Spearm An correlation analysis has positive correlation (P0.05).3) fusion protein, protein sedimentation analysis, CO-IP experiment and GST-Pulldown experiment results show that PLSCR1 and MK protein can interact.4) the interaction of PLSCR1 and MK proteins affects the proliferation and migration of HepG2 cells, and interference PLSCR1 can inhibit the proliferation and migration effect of the cells. The inhibition of PLSCR1 and MK on proliferation and migration of HepG2 cells was more significant (P0.01).
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7
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