【作者】 常彬霞；

【导师】 辛绍杰；

【作者基本信息】 中国人民解放军军医进修学院 ， 内科学， 2010， 博士

【摘要】 严重肝脏疾病时的主要功能障碍之一,是药物在肝脏的代谢功能明显下降,从而导致药效降低,并会进一步加重肝脏损害,这主要与肝脏表达的药物代谢酶含量下降以及活性降低有关。为了解决这个问题,本研究将肝脏中较为重要的与药物代谢有关的Ⅰ相药物代谢酶—细胞色素P450 3A4(CYP 3A4)和Ⅱ相药物代谢酶—谷胱甘肽硫转移酶Al (GST A1)构建成一个双表达载体；同时,为了提高CYP3A4和GST A1的表达量,根据人类优势密码子将其基因进行优化后,构建成另一个双表达载体,分别将其转染肝脏肿瘤细胞系C3A中,以期能够增强C3A细胞的药物代谢的功能,在治疗严重肝脏疾病的病人中得到应用。1.重组质粒的构建及鉴定本实验根据人类优势密码子对CYP 3A4和GST A1序列进行优化,由Invitrogen公司进行全基因合成,并将以上两个基因克隆到以pcDNA3.1 (+)载体为骨架的双CMV启动子载体中,构建质粒pcDNA3.1 (+)-OCYP 3A4-OGST A1,以该质粒为模板扩增优化过的OGSTA1和OCYP3A4基因。从含有GSTA1和CYP 3A4的克隆中扩增GSTA1和CYP3A4基因。将获得的4个片段经酶切、连接、转化等步骤最终构建成两个双表达的重组质粒：pBudCE4.1-CYP 3A4-GST A1和pBudCE4.1-OCYP 3A4-OGST A1,序列测定及分析结果表明所构建质粒符合应用要求。2.转染肝细胞系的建立及功能评价将构建好的重组质粒pBudCE4.1-CYP 3A4-GST A1和pBudCE4.1-OCYP 3A4-OGST A1分别转染肝脏肿瘤细胞系C3A,用浓度为400μg／ml的Zeocin筛选出稳定转染的细胞系,成功构建了转染重组质粒pBudCE4.1-CYP 3A4-GST A1的细胞系,命名为C3A-未优势化；而转染重组质粒PBudCE4.1-OCYP 3A4-OGST A1的细胞虽有存活,但生长极为缓慢,至本论文完成之时仍未生长成单层细胞,目前正在进一步研究中。经MTT试验证明：CYP 3A4和／或GSTA1的表达量增加会导致细胞生长受到抑制。构建好的C3A-未优势化细胞系通过qPCR的方法检测其目的基因CYP 3A4和GST A1的表达量较正常C3A细胞系高；通过色谱法证明C3A-未优势化细胞系有明显的CYP 3A4活性,而正常C3A细胞系中CYP 3A4的表达活性较低,未检测到；用免疫组化实验证实C3A-未优势化细胞系中目的基因GST A1有表达,而正常C3A细胞系中的目的基因GST A1表达较低,未检测到；另外,C3A-未优势化细胞系对利多卡因的代谢能力亦有所增强,代谢率为62.5%,而正常C3A细胞系中的利多卡因代谢率仅为30%。结论：构建的C3A-未优势化细胞系功能有所改善,有望成为生物人工肝系统的细胞材料。更多还原

【Abstract】 The hepatic metabolism of drug decreases obviously in patients with serious liver diseases.This reduced the drug effects and aggravated the liver damage, which is due to the declined expression and activity of drug metabolic enzyme. Therefore, we have targeted gene cytochrome P450 3A4 (CYP 3A4) and glutathione-S-transferase A1 (GST A1), which belonged to PhaseⅠand PhaseⅡdrug metabolic enzyme respectively. At the same time, we optimized the codons of CYP 3A4 and GST A1 to enhance the expression. The two vectors transfected C3A cell line and the function of drug metabolism was evaluated.1. Construction and identification of recombinant plasmids The optimized CYP 3A4 and GST A1 were synthesized and cloned to pcDNA3.1(+) which had two CMV promoters. The recombinant plasmid was named pcDNA3.1(+)-OCYP 3A4-OGST A1. And we have gotten the OCYP 3A4 and OGST A1 gene by polymerase chain reaction(PCR) from this plasmid. By means of digestion, ligation and transformation, we got two recombinant plasmids, named pBudCE4.1-CYP 3A4-GST A1 and pBudCE4.1-OCYP 3A4-OGST A1 respectively, and confirmed by sequencing and blast analysis.2. Establishment and function evaluation of transfected liver cell line C3A cell line were transfected with recombinant plasmids of pBudCE4.1-CYP 3A4-GST Al and pBudCE4.1-OCYP 3A4-OGST A1 respectively, and cultured with MEM containing 400μg/ml Zeocin for 2 weeks. The cell lines, named C3A-Unoptimized, expressed CYP 3A4 and GST A1 successfully and stably, while the cell lines expressed OCYP 3A4 and OGST Al unstably. By MTT assay, it showed that the cell growth would be inhibited by the increased expression of CYP 3A4 and GSTAl. The C3A-Unoptimized cell lines expressed more CYP 3A4 and GSTA1 than that of normal C3A cell line tested by qPCR. By chromatogram assay, it showed that the activity of CYP 3A4 existed in the C3A-Unoptimized cell lines whereas it was undetectable in normal C3A cell line. The immuno-histochemical staining indicated the higher expression of GSTA1 in C3A-Unoptimized cell lines than in normal C3A cell line. The ability of metabolizing lidocarine for the C3A-Unoptimized cell lines was enhanced (the metabolizing rate was 62.5%) comparing with that in normal C3A cell line (the metabolizing rate was 30%). Conclusions:The function of C3A-Unoptimized cell line have been improved, and this cell line might become a new cell material in the bioartificial liver support system.更多还原