【作者】 季鑫；

【导师】 高春芳；

【作者基本信息】 第二军医大学 ， 临床检验诊断学， 2010， 硕士

【摘要】 目的：肝细胞癌(hepatocellular carcinoma, HCC)是世界上最常见的恶性肿瘤之一,在恶性肿瘤致死原因中位列第三。肝炎病毒、黄曲霉毒素、酒精、饮水污染和遗传因素都是肝细胞癌的主要病因,其中最重要的一个病因是乙肝病毒(hepatitis B virus, HBV)或丙肝病毒(hepatitis C virus, HCV)的慢性感染。慢性乙型肝炎(chronic hepatitis B, CHB)是一种最常见的高发病率和高死亡率的传染性肝病。全球乙肝病毒感染者约有20亿,其中乙肝病毒携带者约为3.5亿,每年大约有一百万人死于HBV感染继发的肝衰竭、肝硬化和肝癌。我国是乙肝的高发地,据流行病学调查资料显示,HBsAg阳性者占总人口的7.18%,约1.2亿人。其中慢性乙型肝炎患者约2000-3000万人。作为乙肝主要不良结局的肝纤维化/硬化和原发性肝癌同样具有较高的发病率,以细胞外基质(extracellular matrix, ECM)过度沉积为特征的肝纤维化是急、慢性肝病的主要病理转归,且早期具有可逆性,但如果得不到及时诊断和治疗,就可进一步发展成为肝硬化,肝功能可从代偿走向失代偿,患者最终因肝功能衰竭或并发肝癌而影响生存。肝星状细胞(hepatic stellate cell, HSC)是肝脏的间质细胞,在慢性肝脏疾病的发生发展过程中具有重要的作用。最新研究表明肝星状细胞可以作为抗原提呈细胞,表达多种抗原提呈相关的重要分子,在肝脏相关的免疫功能中也发挥重要的作用。转化生长因子β1(transforming growth factorβ1, TGFβ1)是强效致纤维化因子,介导细胞对组织的损伤,在纤维硬化性疾病中能激活纤维形成细胞,促进细胞外基质产生,改变基质金属蛋白酶(matrix metalloproteinase, MMP)及其抑制剂的活性,增强靶细胞对细胞因子的反应性,对肝炎、肝损伤后肝纤维化、肝硬化具有重要起始促进作用。TGFB1至少存在10个以上的单核苷酸多态性(single nucleotide polymorphism, SNP)位点,-509位点位于TGFβ1启动子区域,codon10 (Leu>Pro)存在于TGFβ1前体分子的信号肽中,而信号肽中氨基酸序列的改变理论上可影响相应蛋白质的分泌。我们设计相应实验检测八种肝脏细胞系的TGFβ1基因-509位点的基因型及针对codon10 (Leu>Pro)的SNP对肝脏相关细胞功能的影响进行研究。方法：用PCR-RFLP方法检测八种肝脏细胞系的TGFβ1基因-509位点的基因型。构建含codon10(Leu>Pro)的TGFβ1(LAP+成熟态单体)真核表达重组体CMV-Leu、CMV-Pro。在L02、HepG2、SMMC-7721、LX-2细胞中转染pcDNA3.1空载体、CMV-Leu、CMV-Pro,培养24小时后,ELISA检测细胞培养上清TGFβ1量。LO2和HepG2细胞转染三种质粒后进行培养,在培养的第12、24、36、48小时,分别进行MTT实验,以检测转染质粒对细胞增殖情况的影响；L02和HepG2细胞转染质粒后48小时,用AnnexinⅤ和PI进行双染色,流式细胞仪检测细胞凋亡情况。HepG2细胞转染三种质粒后培养24小时,用CD105-FITC抗体对细胞进行标记,用流式细胞仪检测CD105分子的表达情况。LX-2细胞转染三种质粒后培养24小时,用CD80-FITC、CD83-PC5、CD1α-PE荧光标记抗体进行避光孵育,流式细胞仪检测三种CD分子的表达情况。实验数据采用SPSS11.0 (Statistical Package for the Social Science 11.0)统计软件用两独立样本t检验进行统计分析。结果：确定了八种肝脏细胞系的TGFβ1基因-509位点和codon10的基因型。构建的含codon10(Leu>Pro)的TGFβ1(LAP+成熟态单体)真核表达重组体CMV-Leu、CMV-Pro通过测序确认序列正确。在四种细胞系中,分别转染CMV-Leu、CMV-Pro组的TGFβ1分泌量比pcDNA3.1组高,证明CMV-Leu、CMV-Pro能成功转染进入细胞并能对其产生作用,提高其TGFβ1分泌量。转染CMV-Pro组细胞的TGFβ1分泌量比转染CMV-Leu组高,且有统计学意义(P<0.05)。在LO2细胞凋亡实验中,转染CMV-Leu、CMV-Pro的凋亡率比转染pcDNA3.1的凋亡率低,其中转染CMV-Pro的凋亡率又低于转染CMV-Leu的凋亡率,且有统计学意义(P<0.01)。在HepG2细胞的凋亡实验中可以发现,转染CMV-Leu组和CMV-Pro组的细胞凋亡率显著高于转染pcDNA3.1组的凋亡率(P<0.01),其中转染CMV-Leu组的细胞凋亡率又显著高于转染CMV-Pro组的凋亡率(P<0.01)。在LO2和HepG2细胞的细胞增殖实验中,两种细胞转染CMV-Pro组的细胞增殖活性都高于相应的CMV-Leu转染组。在HepG2细胞转染pcDNA3.1、CMV-Leu、CMV-Pro后,转染CMV-Pro组CD105表达率比转染CMV-Leu组高,且有统计学意义(P<0.05)。在LX-2实验中,转染CMV-Leu、CMV-Pro细胞的CDla、CD80表达率并无明显差异,转染CMV-Pro组的CD83表达率明显低于转染CMV-Leu组,有统计学差异(P<0.01)。结论：TGFβ1基因codon10为Pro时比为Leu时更能促进细胞TGFβ1的分泌活性,并能增加HepG2细胞的CD105表达。TGFβ1 codon10为Pro时比为Leu时更能促进L02、HepG2细胞的增殖。CMV-Leu、CMV-Pro对LO2细胞有一定的保护作用,其中TGFβ1基因codon10 Pro型较Leu型对L02细胞凋亡的保护作用更强。而CMV-Leu、CMV-Pro则能促进HepG2细胞的凋亡,其中codon10为Leu型时促进作用更强。TGFβ1基因codon10 Leu型能显著增加LX-2细胞CD83的表达,但对CD80、CD1α的表达并无明显影响。更多还原

【Abstract】 Objective:Hepatocellular carcinoma (HCC) is one of the most common neoplasms worldwide and the third cause of death among those patients with malignant tumors. One of the important risk factors for HCC is chronic infection by hepatitis B or C virus, although other risk factors, such as Aflatoxin and alcoholism, have been implicated in its pathogenesis. Chronic hepatitis B is one of the most common infectious liver diseases which had high morbidity and mortality because of the development of liver fibrosis, cirrhosis and subsequently hepatocellular carcinoma. About 2 billion people are affected and more than 10% of these populations are persistent into chronic status all over the world. The mortality rate during HBV infection caused by liver failure, liver cirrhosis and HCC is approximately 1 million each year. About half a million Chinese die from HCC caused by HBV and from end stage cirrhosis each year. The patients with chronic HBV are at increased risk of progression to cirrhosis and development of HCC. Liver cirrhosis in its early stage is able to reverse, so it is important for diagnosis and treatment of liver diseases to early diagnose of liver fibrosis.Accumulating evidences indicated that Hepatic stellate cells (HSCs) play an important role in the progression of chronic liver diseases. HSCs could express the molecules necessary for antigen presentation and modulate lymphocyte proliferation, so HSCs also could play a role in the immune function of the liver.TGFβ1 acts as an important profibrogenic cytokine in liver diseases.It plays an important role in the fibroproliferative changes that follow liver tissue damage, for it can initially recruit and stimulate fibroblasts to produce ECM, enhance ECM synthesis and inhibit ECM degradation by downregulating the expression of matrix-degrading enzymes, promoting expression of matrix metalloproteinase (MMP) inhibitors and inducing tissue inhibitor of metalloproteinases-1 (TIMP-1).Until now at least 10 single nucleotide polymorphisms (SNPs) in TGFβ1 gene have been recognized. LeulOPro is located in the signal peptide of TGFβ1 precursor; leucine is encoded when the codon is CTG while proline is encoded when the codon is CCG. Base substitution causing amino acid replacement in signal peptide might influence the secretion of some related proteins. Since TGFβ1 plays such a pivotal role in liver fibrosis and liver malignancy, serial experiments were designed in this study to analyze TGFβ1 gene-509 polymorphisms of eight cell lines, to determine whether the leucine/proline substitution influences TGFβ1 secretion and thus changes the functions of liver cells.Method:TGFβ1 gene polymorphisms at positions-509 of Huh-7, HepG2, BEL-7402, SMMC-7721, PLC/PRF/5, Hep3B, QGY, L02 cells were analyzed. Two TGFβ1 plasmid CMV-Leu and CMV-Pro were constructed by cloning TGFβ1 DNA fragment (including LAP and active portion), which encoding either the Leu or Pro forms of TGFβ1, into pcDNA3.1 vectors. L02, HepG2, SMMC-7721 and LX-2 cell lines were chosen to be transfected with CMV-Leu、CMV-Pro or pcDNA3.1 empty vectors. The cell medium was collected and assayed by ELISA kit specific for TGFβ1 separately at 24 hours after the transfection. The transfected L02 and HepG2 cells were collected at 48 hours after the transfection, stained by Annexin V and PI, then analyzed by flow cytometry. All data was calculated by Student’s t test. Cell proliferation assays (MTT test) of L02 and HepG2 cells were carried out at the time point of 12,24,36,48 hours after initial transfection. HepG2 cells were collected at 24 hours after the transfection, stained by CD105-FITC and analyzed by flow cytometry. LX-2 cells were collected at 24 hours after the transfection, stained by CD80-FITC, CD83-PC5, CD1α-PE and analyzed by flow cytometry. The data were analyzed with SPSS 11.0 by Student’s t test.Results:The genotype of eight cell lines’TGFβ1 gene-509 and codon10 were confirmed successfully. CMV-Leu and CMV-Pro vectors were proved to be exactly correct by DNA sequencing. The amounts of TGFβ1 secreted from both CMV-Leu and CMV-Pro transfected cells were higher than pcDNA3.1-transfected cells in four cell lines. CMV-Pro-transfected cells secreted much more TGFβ1 than CMV-Leu-transfected cells, and the difference was statistically significant (P<0.05). The apoptotic rates of CMV-Leu or CMV-Pro transfected cells were greater than pcDNA3.1-transfected cells in HepG2 cells. While in L02 cells the apoptotic rates of CMV-Leu or CMV-Pro transfected cells were much lower than pcDNA3.1-transfected cells. The apoptotic rate of CMV-Pro-transfected cells was also lower than that of CMV-Leu-transfected cells in two cells. And the differences between all transfected cells were statistically significant (P<0.05). Both TGFβ1 codon 10 genotypes could cause an advancing proliferation of liver cells and the genotype of Pro 10 is more effective than Leu 10. CMV-Pro-transfected HepG2 cells expressed a higher level of CD105 than CMV-Leu-transfected cells did (P<0.05). CMV-Pro-transfected LX-2 cells expressed lower level of CD83 than CMV-Leu-transfected cells did (P<0.01), while CD 80 and CD1αexpression pattern were not affected by the genotypes.Conclusion:TGFβ1 gene Pro10 could enhance the secretion of TGFβ1 than LeulO in liver cells. Both TGFβ1 Pro10 and Leu10 could accelerate the proliferation activity of L02 cells and HepG2 cells, and Pro10 had a better acceleration effect than Leu10. LeulOPro encoding either Pro10 or Leu10 could promote the apoptosis of hepatoma cells, but prevent the apoptosis of normal liver cells. And TGFβ1 Pro10 had stronger protective ability than Leu10. TGFβ1 Pro10 also could increase the CD105 expression than Leu10 in HepG2 cells, but Leu10 increase the CD83 expression than Pro in LX-2 cells.更多还原