磷脂混雜酶1和中期因子在原發(fā)性肝細(xì)胞癌發(fā)生發(fā)展中作用的研究
發(fā)布時間：2018-08-02 19:24
【摘要】：目的:通過檢測PLSCR1與MK在肝癌組織及癌旁肝正常組織中的基因表達(dá)差異并分析PLSCR1與MK和肝癌病理特征的相關(guān)性及兩者之間的相關(guān)性,并運用Cox回歸模型分析臨床基礎(chǔ)資料對患者總生存期的影響;構(gòu)建重組質(zhì)粒,在細(xì)胞水平探討PLSCR1與MK相互作用在肝癌發(fā)生發(fā)展中可能的作用機制。方法:1)收集我院2009年1月至2013年6月期間收治的原發(fā)性肝細(xì)胞癌手術(shù)切除患者肝癌組織和對應(yīng)癌旁肝正常組織標(biāo)本各40例,所有病例確診為原發(fā)性肝細(xì)胞癌,所有病例均為初診,術(shù)前均未接受過放化療、中醫(yī)及免疫治療,臨床資料完整。每例標(biāo)本均留取腫癌組織和癌旁正常肝組織。手術(shù)標(biāo)本切除后于30min內(nèi)取樣本并液氮速凍,保存于-80℃冰箱備用。采用Real-Time PCR技術(shù)(Q-PCR)檢測40例肝癌組織與癌旁正常組織中PLSCR1與MK表達(dá)量,統(tǒng)計分析其與患者臨床病理特征關(guān)系,采用Cox回歸模型分析臨床基礎(chǔ)資料對患者總預(yù)后生存期的影響以及分析PLSCR1和MK表達(dá)水平之間的相關(guān)性。2)根據(jù)質(zhì)粒pET28a(+)-PLSCR1、pGEX-4T1-MK、pcDNA3.1/myc-His(-)A-PLSCR1 和 pcDNA3.1-MK 設(shè)計引物,構(gòu)建質(zhì)粒;然后將測序確認(rèn)的重組質(zhì)粒pET28a(+)-PLSCR1和pGEX-4T1-MK重組表達(dá)質(zhì)粒分別轉(zhuǎn)入BL21菌株中,分別將上述重組表達(dá)質(zhì)粒轉(zhuǎn)化菌株中加入IPTG進(jìn)行誘導(dǎo)表達(dá),利用GST-pull down技術(shù)和免疫共沉淀(CO-IP)技術(shù)驗證PLSCR1與相互作用蛋白MK之間的相互作用。3)研究PLSCR1和MK相互作用對肝癌細(xì)胞增殖的影響。分別構(gòu)建PLSCR1和MK的siRNA干擾質(zhì)粒,分組轉(zhuǎn)染 HepG2 細(xì)胞:MK-siRNA 組,PLSCR1-siRNA 組,MK+PLSCR1-siRNA 共轉(zhuǎn)組和未加任何處理的對照組,借助酶聯(lián)免疫檢測儀測定并記錄各孔在450nm波長下對應(yīng)的吸收值。4)檢測PLSCR1和MK相互作用對肝癌細(xì)胞遷移的影響:將MK-si-RNA-HepG2,PLSCR1-siRNA-HepG2 細(xì)胞、MK+PLSCR1-siRNA-HepG2 細(xì)胞、Control-siRNA-HepG2細(xì)胞及HepG2細(xì)胞接種于小室內(nèi),培養(yǎng)48h后進(jìn)行細(xì)胞染色,通過普通倒置顯微鏡下對細(xì)胞的遷移情況進(jìn)行觀察,在560nm下測定細(xì)胞裂解液的吸收值,并定量計算其含量。結(jié)果:1)肝癌組織中PLSCR1和MK基因的表達(dá)量明顯高于癌旁組織,PLSCR1的 A△Ct 值分別為-2.36+1.49,2-△△Ct值(RQ 值)均值為 8.55±7.67;MK 的 △△Ct值為-2.97±0.86,RQ值為9.05±3.97,具有差異有統(tǒng)計學(xué)意義(P0.05)。依據(jù)40例患者RQ值均值將PLSCR1和MK進(jìn)行分組,RQ值≥10為高表達(dá)組,RQ值10為低表達(dá)組進(jìn)行分析,結(jié)果表明PLSCR1與MK的表達(dá)與患者的年齡、是否伴隨肝硬化、血液AFP值、腫瘤大小及是否包膜侵犯無關(guān)(P0.05),而與患者的性別、腫瘤分化程度、腫瘤數(shù)目、血管浸潤及臨床分期相關(guān)性顯著(P0.05)。2)我們通過Kaplan-meier生存分析PLSCR1和MK的癌/癌旁組織的倍數(shù)變化(RQ值)對肝癌術(shù)后總生存期的影響,發(fā)現(xiàn)PLSCR1和MK高表達(dá)組的生存率明顯低于低表達(dá)組,Breslow法檢驗水平間的兩兩比較具有顯著性差異(P0.05)。運用Cox回歸模型分析患者臨床基礎(chǔ)資料及患者PLSCR1和MK基因的表達(dá)量與肝癌術(shù)后生存期相關(guān)風(fēng)險因素,發(fā)現(xiàn)包膜侵犯、血管浸潤、Ki-67值、性別、肝硬化及PLSCR1和MK高表達(dá)是評估總生存期的風(fēng)險因素(P0.05),包膜侵犯風(fēng)險系數(shù)最高為8.04(P=0.001);肝癌組織中PLSCR1和MK表達(dá)水平Spearman相關(guān)性分析結(jié)果顯示r=0.30(P=0.03),兩者存在弱正相關(guān)性。3)對成功構(gòu)建的質(zhì)粒進(jìn)行酶切鑒定,測序結(jié)果表明,構(gòu)建四個質(zhì)粒的外源片段按正確的閱讀框架連入pET28a(+)、pGEX-4T1、pcDNA3.1/myc-His(-)A和pcDNA3.1載體,陽性克隆經(jīng)測序無誤的抽提質(zhì)粒;融合蛋白、蛋白沉降分析及CO-IP試驗結(jié)果顯示,PLSCR1和MK蛋白可以發(fā)生相互作用。4)成功構(gòu)建MK和PLSCR1干擾細(xì)胞株,通過轉(zhuǎn)染細(xì)胞干擾PLSCR1和MK的表達(dá)后,用CCK-8檢測HepG2細(xì)胞的增殖情況,HepG2細(xì)胞的增殖效率顯著降低,并同時干擾PLSCR1和MK對HepG2細(xì)胞增殖抑制作用更為顯著(P0.01);PLSCR1和MK相互作用同樣對HepG2細(xì)胞遷移具有抑制作用,侵移實驗表明干擾PLSCR1可以明顯影響HepG2細(xì)胞的遷移作用,并且同時干擾PLSCR1和MK對HepG2細(xì)胞的遷移抑制作用更顯著(P0.01)。結(jié)論:1)PLSCR1與MK在肝癌組織中的表達(dá)明顯高于癌旁組織,其表達(dá)水平與患者臨床病理特征相關(guān)性表明與患者的性別、腫瘤分化程度、腫瘤數(shù)目、血管浸潤及臨床分期相關(guān)性顯著(P0.05)。2)Cox回歸模型分析患者臨床基礎(chǔ)資料及患者PLSCR1和MK基因的表達(dá)量與肝癌術(shù)后生存期相關(guān)風(fēng)險因素,發(fā)現(xiàn)包膜侵犯、血管浸潤、Ki-67值、性別、肝硬化及PLSCR1和MK高表達(dá)是評估總生存期的風(fēng)險因素(P0.05);肝癌組織中PLSCR1和MK表達(dá)水平Spearman相關(guān)性分析兩者存在正相關(guān)性(P0.05)。3)融合蛋白、蛋白沉降分析、CO-IP實驗及GST-Pulldown實驗結(jié)果表明,PLSCR1和MK蛋白可以發(fā)生相互作用。4)PLSCR1與MK蛋白相互作用影響HepG2細(xì)胞增殖和遷移,干擾PLSCR1可抑制HepG2細(xì)胞的增殖和遷移效果,同時干擾PLSCR1和MK對HepG2細(xì)胞的增殖和遷移的抑制作用更為顯著(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).
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.7
本文編號：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).
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.7
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