OATP1B1基因多态性对他莫昔芬及其代谢产物的摄取研究

发布时间：2018-09-04 08:21

【摘要】：目的:构建OATP1B1(Organic anion transporting polypeptide1B1)野生型和突变型过表达慢病毒载体基因平台,并在此平台上进行OATP1B1388和521位点的突变对他莫昔芬及其代谢产物的摄取差异的比较研究。方法:从人肝脏组织提取总RNA并逆转录成c DNA,利用PCR技术钓取OATP1B1基因片段,同时对载体GV358进行酶切,扩增产物的两末端序列与线性化克隆载体两末端序列完全一致。以线性化载体和目的基因扩增产物配制反应体系,进行重组反应,实现线性化载体和目的基因片段的体外环化。重组产物转化到DH5α感受态细胞进行扩增,抽提、纯化后进行测序比对,测序比对正确的进行定点突变得到OATP1B1388G(OATP1B1*1b)和521C(OATP1B1*5)的突变体质粒。在脂质体Lip2000介导下,把重组质粒和慢病毒包装质粒p Helper1.0和p Helper2.0共同转染293T细胞,包装生产慢病毒,然后收获病毒液,对病毒液进行浓缩纯化。符合实验要求且滴度合格的病毒液感染目的293T细胞,荧光定量PCR和蛋白免疫印迹法检测293T细胞中OATP1B1野生型(OATP1B1*1a)及其突变型质粒在基因和蛋白水平的表达情况。同时,建立Z-他莫昔芬(Z-tamoxifen,缩写为tamoxifen)及其活性代谢产物4-羟基-N-去甲基他莫昔芬(endoxifen)的高效液相串联质谱(HPLC-MS/MS)方法学。实验组分别为HEK293T细胞组、HEK293T细胞加药组、阴性对照病毒-HEK293T组(HEK293T细胞转染载体质粒组)、OATP1B1*1a-HEK293T组、OATP1B1*1b-HEK293T组和OATP1B1*5-HEK293T组。六组细胞分别加入不同浓度梯度的tamoxifen和endoxifen作用24小时和48小时后用清水裂解细胞,运用HPLC-MS/MS方法学比较OATP1B1基因多态性对tamoxifen和endoxifen的摄取差异。结果:1)加药24小时检测细胞裂解液中tamoxifen和endoxifen的含量,HEK293T细胞加药组与阴性对照病毒-HEK293T加药组,细胞裂解液中药物含量对比,差异不具有统计学意义(P0.05)。OATP1B1*1a-HEK293T组、OATP1B1*1b-HEK293T组和OATP1B1*5-HEK293T组,三组细胞裂解液中药物含量明显高于HEK293T细胞加药组且差异具有统计学意义(P0.01),说明tamoxifen和endoxifen可以通过转运体OATP1B1摄取进入细胞内。2)加药24小时细胞裂解液中tamoxifen和endoxifen的含量,OATP1B1*1b-HEK293T组细胞裂解液中药物含量对比OATP1B1*1a-HEK293T组差异不具有统计学意义(P0.05);说明OATP1B1388位点碱基由A突变为G时,对转运体蛋白摄取能力没有影响。OATP1B1*5-HEK293T组细胞裂解液中药物含量对比OATP1B1*1a-HEK293T组含量偏低,且差异具有统计学意义(P0.05);说明OATP1B1521位点碱基由T突变为C时会抑制转运体蛋白的摄取转运功能。3)加药48小时检测细胞裂解液中tamoxifen和endoxifen的含量,HEK293T细胞加药组与阴性对照病毒-HEK293T加药组药物含量对比,差异不具有统计学意义(P0.05)。OATP1B1*1a-HEK293T组、OATP1B1*1b-HEK293T组和OATP1B1*5-HEK293T组,三组细胞裂解液中药物含量明显高于HEK293T细胞加药组且差异具有统计学意义(P0.01)。4)加药48小时,OATP1B1*1b-HEK293T组细胞裂解液中药物含量对比OATP1B1*1a-HEK293T组差异不具有统计学意义(P0.05);OATP1B1*5-HEK293T组细胞裂解液中药物浓度对比OATP1B1*1a-HEK293T组含量偏低,且差异具有统计学意义(P0.05);48小时各组细胞裂解液中药物含量对比同一条件24小时细胞裂解液中药物浓度略高,但是差异不具有统计学意义(P0.05)说明24小时内转运体OATP1B1对tamoxifen和endoxifen的摄取基本达到饱和。结论:1)过表达慢病毒质粒细胞模型构建成功,包括OATP1B1*1a-HEK29T、OATP1B1*1b-HEK293T和OATP1B1*5-HEK293T细胞模型,且感染效率达到80%以上,OATP1B1基因在m RNA、蛋白水平可以高度表达。2)tamoxifen及其主要代谢产物endoxifen可以通过OATP1B1摄取进入细胞,OATP1B1521位点碱基T突变为C时,可以抑制转运体蛋白的摄取转运功能,导致细胞裂解液中药物浓度对比野生组偏低且差异具有统计学意义(P0.05);OATP1B1388位点碱基由A突变为G时,细胞裂解液药物的含量对比野生组差异不具有统计学意义(P0.05)
[Abstract]:AIM: To construct a wild-type and mutant overexpression lentiviral gene platform of OATP1B1 (Organic anion transporting polypeptide 1B1) and to compare the uptake of tamoxifen and its metabolites by mutations at sites 1388 and 521. Methods: Total RNA was extracted from human liver tissues and reverse transcribed into C DNA. OATP1B1 gene fragments were harvested by PCR, and GV358 was digested by enzyme. The two terminal sequences of the amplified product were identical with those of the linearized cloning vector. The recombinant reaction system was prepared with the linearized vector and the target gene amplified product to realize in vitro cyclization of the linearized vector and the target gene fragment. The recombinant product was transformed into DH5a competent cells for amplification, extraction, purification and sequencing. The mutant plasmids of OATP1B1388G (OATP1B1*1b) and 521C (OATP1B1*5) were obtained by site-directed mutagenesis. The recombinant plasmids and lentiviral packaging plasmids P Helper 1.0 and P Helper 2.0 were co-transfected by liposome Lip2000. 293T cells were packaged to produce lentiviruses, and then harvested to concentrate and purify the virus solution. The expression of OATP1B1 wild type (OATP1B1 * 1a) and its mutant plasmids at gene and protein levels in 293T cells were detected by fluorescence quantitative PCR and Western blot. Meanwhile, a high performance liquid chromatography-mass spectrometry (HPLC-MS/MS) method was established for the determination of Z-tamoxifen and its active metabolite 4-hydroxy-N-demethyltamoxifen. The experimental groups were HEK293T cell group, HEK293T cell dosage group, negative control virus-HEK293T cell transfection vector plasmid. Group A, OATP1B1*1a-HEK293T group, OATP1B1*1b-HEK293T group and OATP1B1*5-HEK293T group. Six groups of cells were treated with different concentration gradient of tamoxifen and endoxifen for 24 hours and 48 hours respectively. The uptake of tamoxifen and endoxifen was compared by HPLC-MS/MS. The contents of tamoxifen and endoxifen in cell lysate were detected by hourly method. There was no significant difference in the contents of tamoxifen and endoxifen between the HEK293T cell lysate group and the negative control virus-HEK293T cell lysate group (P 0.05). OATP1B1*1a-HEK293T group, OATP1B1*1b-HEK293T group and OATP1B1*5-HEK293T cell lysate group contained drugs. The amount of tamoxifen and endoxifen in the 24-hour cell lysate was significantly higher than that in the HEK293T group (P 0.01), indicating that tamoxifen and endoxifen could be uptaken into the cells by transporter OATP1B1. The content of tamoxifen and endoxifen in the 24-hour cell lysate was lower in the OATP1B1 * 1b-HEK293T group than in the OATP1B1 * 1a-HEK293T group. There was no significant difference between the two groups (P 0.05), indicating that there was no effect on transporter protein uptake when the base of OATP1B1388 changed from A to G. The drug content in cell lysate of OATP1B1*5-HEK293T group was lower than that of OATP1B1*1a-HEK293T group, and the difference was statistically significant (P 0.05); indicating that the base of OATP1B1521 mutated from T to C. It can inhibit the transporter protein uptake and transport function. 3) The content of tamoxifen and endoxifen in cell lysate was detected 48 hours after treatment. The content of tamoxifen and endoxifen in HEK293T cell treatment group was not significantly different from that in negative control virus-HEK293T treatment group (P 0.05). OATP1B1*1a-HEK293T group, OATP1B1*1b-HEK293T group and OATP1B1*5-HEK293T group were not significantly different (P 0.05). The drug content in cell lysate of OATP1B1*1b-HEK293T group was not significantly different from that of OATP1B1*1a-HEK293T group (P 0.05); the drug concentration in cell lysate of OATP1B1*5-HEK293T group was significantly higher than that of HEK293T group (P 0.01). Compared with OATP1B1*1a-HEK293T group, the content of drug in cell lysate was lower, and the difference was statistically significant (P 0.05); 48 hours after treatment, the drug concentration in cell lysate was slightly higher than that in 24 hours under the same condition, but the difference was not statistically significant (P 0.05). CONCLUSIONS: 1) Overexpression of lentivirus plasmid cell model was successfully constructed, including OATP1B1*1a-HEK29T, OATP1B1*1b-HEK293T and OATP1B1*5-HEK293T cell models, and the infection efficiency was above 80%. OATP1B1 gene was highly expressed in M RNA and protein level. 2) tamoxifen and its main metabolite endoxifen could be uptaken by OATP1B1. When OATP1B1521 base T mutated into C, the uptake and transport of transporter proteins were inhibited, resulting in a low and statistically significant difference in the concentration of drugs in cell lysate compared with the wild group (P 0.05); when OATP1B1388 base mutated from A to G, there was no uniform difference in the content of drugs in cell lysate compared with the wild group. Academic significance (P0.05)
【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R96

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