利福平致小鼠肝损伤特点的研究及ABCC2基因过表达细胞株的构建

发布时间：2018-05-13 23:39

本文选题：利福平 + 肝损伤　；参考：《第三军医大学》2017年硕士论文

【摘要】：研究背景及目的最近几年,结核病的发病率较往年有了显著升高的趋势,因结核造成的死亡率也有了明显的提高,居各类传染病之首。在世界范围来说,中国是结核病最多的国家之一[1-2]。在抗结核病的治疗中,抗结核药所致的肝损害的发病率约为2%-28%[3-5],在抗结核药所致的不良反应中,肝损害占首位,而重度肝损伤的发病率约为1%-2%[6-7],但引起肝毒性的机制还未明确。因此,我们需要更多的研究,以找到抗结核药造成肝损害的机制。尽量减少抗结核药在使用过程中导致的肝损害,或者寻找新的抗结核药。这也是临床上一直想解决的问题。利福平(rifampicin,RIF)是属于利福霉素类的半合成抗生素,它能够抑制细菌RNA的合成,可用于肺结核、肠结核、结核性腹膜炎、关节结核等各种结核杆菌感染,以及肠球菌属等感染的治疗。是常用的抗结核药物之一,在临床上广泛运用。因而,它也成了我国药物引起的急性肝损伤的常见的主要原因。利福平有许多副反应,肝脏毒性是它最为常见的不良反应,长期使用利福平可引起药物性肝损伤,因而严重影响了它在临床上的使用[8-11]。目前对利福平的肝损伤机制是不清楚的。在抗结核过程中没有证据证明利福平在肝脏代谢当中会出现毒性产物[12]。本文通过给予小鼠不同剂量的利福平,观察利福平是否致小鼠肝损伤,以及给予不同剂量的利福平后小鼠肝损伤的特点,为进一步探讨利福平致肝损伤的机制奠定基础。并构建ABCC2基因过表达细胞株,为今后研究ABCC2基因,MRP2蛋白的表达和定位与利福平导致的胆汁淤积的关系奠定基础。方法1.利福平致小鼠肝损伤特点的研究方法:将24只健康雌性ICR小鼠用随机数字法分成4组,每组各6只,分别为对照组、低、中、高剂量组。每天早上8点半定时灌胃1次,连续2周。低剂量组每只小鼠按每天100mg/kg给予利福平;中剂量组每只小鼠按每天200mg/kg给予利福平;高剂量组每只小鼠按每天400 mg/kg给予利福平;对照组:予以等容积的羧甲基纤维素钠的水溶液,以作对照。末次给药后禁食6 h取材,收集小鼠血液和肝组织样品进行血清学检测并观察小鼠肝脏病理改变。2.ABCC2基因过表达细胞株的构建:(1)ABCC2-eGFP定点整合载体构建,(2)电转HEK-293细胞,筛选阳性克隆。结果1.各组小鼠间所有肝生化指标无明显统计学差异。2.各实验组小鼠肝脏均发生明显病理改变。3.肝HE染色示:各实验组小鼠肝细胞排列紊乱,部分肝细胞肿胀、见空泡状的细胞,考虑为不同程度的脂肪变性细胞,并且在汇管区周围出现大量炎性细胞浸润,可见散在的胆汁淤积。其中高剂量组表现最为明显,出现广泛的炎性细胞浸润及肝细胞脂肪变性。4.肝电镜照片示:各实验组肝脏组织电镜照片可见肝细胞包浆中充满大量的脂肪滴,可见部分细胞核萎缩,部分毛细胆管轻微扩张、其内可见胆汁淤积,部分线粒体、内质网肿胀断裂。也是在高剂量组肝脏病理改变最明显。5.构建了ABCC2基因过表达细胞株。结论连续2周给予不同剂量的利福平均可引起小鼠肝损伤,主要表现为肝细胞脂肪变性、炎性细胞浸润、部分细胞核核萎缩,部分线粒体肿胀,内质网肿胀以及散在的胆汁淤积,利福平对小鼠肝脏的损伤呈浓度依赖性的,但其机制不明确有待进一步研究。成功构建ABCC2基因过表达细胞株。目的这个meta分析的目的是要阐明SLCO1B1 T521C多态性对他汀类药物血液浓度的影响。方法通过计算机检索pubmed、Cochrane Library、EMBASE、中国生物医学文献数据库、相关期刊论文、维普、万方等数据库,共检索到的25篇相关研究并提取数据。提取AUC0-6h、AUC0-12h、AUC0-24h、AUC0-inf、Cmax和CL/F等药代动力学参数。用标准均数差(SMD)和95%的可信区间(95%CI)比较野生型(TT)基因型和突变基因型(TC、CC、TC+CC)。亚组分析根据种族人群、他汀类药物的类型进行。用敏感性分析对各项研究进行异质性检验。从纳入研究提取资料然后用rev Man5.3进行Meta分析。结果共纳入25篇文献,包含903例病例,Meta分析结果显示:全部研究的AUC0-inf标准均数差TT-TC为0.51(95%CI:0.29,0.72),TT-CC为1.80(95%CI:1.31,2.29),TT-(TC+CC)为0.78(95%CI:0.57,0.99);总体而言,突变基因型的AUC0-inf的标准均数差较高。在白种人和亚洲人的亚组分析得出同样的结果。全部研究的Cmax标准均数差TT-TC为0.48(95%CI:0.18,0.77),TT-CC为1.24(95%CI:0.77,1.70),TT-(TC+CC)为0.65(95%CI:0.43,0.87);突变基因型的Cmax的标准均数差也较高。在白种人和亚洲人的亚组分析也得出同样的结果。全部研究的CL/F标准均数差TT-TC为-1.01(95%CI:-1.91,-0.11),TT-CC为-1.51(95%CI:-2.08,-0.94),TT-(TC+CC)为-1.07(95%CI:-1.55,-0.59),突变基因型的CL/F的标准均数差较低。结论SLCO1B1 T521C等位基因可能对羟甲基戊二酰辅酶还原酶抑制剂药代动力学起着重要影响。
[Abstract]:In recent years, the incidence of tuberculosis has been significantly higher than in previous years, and the death rate caused by tuberculosis has also increased significantly. In the world, China is one of the most tuberculosis countries, [1-2]., in the treatment of anti nodule disease, the liver damage caused by anti tuberculosis drugs. The incidence of the disease is about 2%-28%[3-5]. In the adverse reactions caused by anti tuberculous drugs, liver damage is the first, and the incidence of severe liver injury is about 1%-2%[6-7], but the mechanism of hepatotoxicity is not clear. Therefore, we need more research to find the mechanism of anti tuberculosis drug causing liver damage. Rifampicin (rifampicin, RIF) is a semi synthetic antibiotic of rifamycin, which inhibits the synthesis of bacterial RNA and can be used in tuberculosis, intestinal tuberculosis, tuberculosis peritonitis, tuberculosis of joint, and Enterococcus, and Enterococcus. It is one of the commonly used antituberculous drugs and is widely used in clinical practice. Therefore, it has also become a common cause of acute liver injury caused by drugs in our country. Rifampin has many side effects. Liver toxicity is the most common adverse reaction. The long-term use of rifampin can cause drug induced liver damage and therefore serious shadow. The mechanism of the clinical use of [8-11]. is not clear. There is no evidence that rifampin may have a toxic product in the liver metabolism during the anti tuberculosis process, [12]., by giving rifampicin at different doses in mice, to observe whether rifampin causes liver damage in mice and to give different doses. The characteristics of liver injury in Li Fuping's post Li Fuping mice lay the foundation for further study of the mechanism of Li Fuping induced liver injury, and constructed the overexpressed cell line of the ABCC2 gene to lay the foundation for the future study of the relationship between the expression and location of the ABCC2 gene, the expression of MRP2 protein and the cholestasis caused by Li Fuping. Methods: 24 healthy female ICR mice were divided into 4 groups by random number method, 6 rats in each group, respectively, the control group, low, middle, high dose group. Every day at 8:30 every morning, the stomach was given 1 times for 2 weeks. Each mouse in the low dose group was given rifampin per day at 100mg/kg; each mouse in the middle dose group was given rifampin at 200mg/kg per day; the high dose group per mouse was given every day. The mice were given rifampicin at 400 mg/kg per day; the control group was treated with the same volume of sodium carboxymethyl cellulose water as the control. After the last dose of 6 h, the samples of blood and liver tissues of mice were collected for serological examination and the construction of.2.ABCC2 gene overexpressed cell lines in the liver pathological changes of mice was observed: (1) ABCC2-eGFP determination. Point integration carrier construction, (2) electric transfer of HEK-293 cells and screening positive clones. Results there was no significant difference in all liver biochemical indexes between each group of 1. groups. The liver of all mice in each group had obvious pathological changes in.3. liver HE staining: the liver cells in each experimental group were in disorder, some of the hepatocytes were swollen, and the vacuolated cells were considered. A large number of inflammatory cells were infiltrated around the manifold and scattered cholestasis was seen around the pipe area. The high dose group showed the most obvious, extensive inflammatory cell infiltration and hepatocyte fatty degeneration.4. liver electron microscopy. A large number of fat drops, visible part of the nucleus atrophy, partial capillary bile duct dilated slightly, it can be seen in the cholestasis, some mitochondria, the endoplasmic reticulum swelling and fracture. Also in the high dose group, the most obvious liver pathological changes.5. constructed the ABCC2 gene overexpressed cell line. Conclusion the 2 weeks of continuous dose of rifampin can cause the liver of mice. The main manifestations were hepatocyte fatty degeneration, inflammatory cell infiltration, partial nucleus atrophy, partial mitochondria swelling, endoplasmic reticulum swelling and scattered cholestasis. Rifampin was dependent on the damage of liver in mice, but the mechanism was not clear. The ABCC2 gene overexpressed cell line was successfully constructed. The purpose of this meta analysis is to clarify the effect of SLCO1B1 T521C polymorphism on the blood concentration of statins. Methods 25 related studies and extraction data were retrieved by computer retrieval of PubMed, Cochrane Library, EMBASE, Chinese biomedical literature database, Chinese journal full text database, VIP and Wanfang Data base. Pharmacokinetic parameters, such as AUC0-6h, AUC0-12h, AUC0-24h, AUC0-inf, Cmax and CL/F, compared wild type (TT) genotypes and mutant genotypes (TC, CC, TC+CC) with standard mean number difference (SMD) and 95% confidence interval (95%CI). Subgroups analyzed the types of statins based on racial populations. Sensitivity analysis was used to test the heterogeneity of various studies by sensitivity analysis. A total of 25 articles were included in 25 articles and 903 cases were included. The results of Meta analysis showed that the average number difference TT-TC of the AUC0-inf standard was 0.51 (95%CI:0.29,0.72), TT-CC was 1.80 (95%CI:1.31,2.29), TT- (TC+CC) was 0.78 (95%CI:0.57,0.99); in general, the mutant genotype The standard mean difference of 0-inf was high. The same results were obtained in the subgroup of white people and Asians. The average number difference TT-TC of the Cmax standard was 0.48 (95%CI:0.18,0.77), TT-CC was 1.24 (95%CI:0.77,1.70), TT- (TC+CC) was 0.65 (95%CI:0.43,0.87); the standard mean mean difference of the mutant genotype was also higher. The subgroup analysis also obtained the same results. The CL/F standard mean difference TT-TC of all studies is -1.01 (95%CI:-1.91, -0.11), TT-CC is -1.51 (95%CI:-2.08, -0.94), TT- (TC+CC) is -1.07. Pharmacokinetics of preparation plays an important role.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R52;R575

【参考文献】