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核苷类似物对小鼠肝细胞线粒体DNA ND1和ND4的影响

2018-11-15张维乔录新丁渭陈德喜张玉林

中国医药导报 2018年21期

张维 乔录新 丁渭 陈德喜 张玉林

[摘要] 目的 探討长期使用核苷类似物(NAs)是否导致小鼠肝脏线粒体DNA(mtDNA)ND1和ND4区损伤。 方法 7周龄雌性Balb/c小鼠25只,采用简单随机分组法分为5组,对照组和4种核苷类似物组,每组各5只。实验组司他夫定(D4T)50 mg/kg,齐多夫定(AZT)100 mg/kg,拉米夫定(3TC)50 mg/kg和去羟肌苷(DDI)50 mg/kg,对照组为双蒸水,分别腹腔内注射,每周5次,连续3个月。留取各组肝组织,应用激光捕获显微技术获取肝细胞,对mtDNA ND1和ND4区克隆和测序。 结果 其余各组肝细胞的mtDNA ND4序列距离与参考序列的平均距离与对照组比较,差异均有高度统计学意义(P < 0.01);AZT组肝细胞的mtDNA ND4平均dN与对照组比较,差异有统计学意义(P < 0.05);肝细胞mtDNA ND1的序列距离与参考序列的平均距离,AZT组和3TC组序列距离与对照组比较,差异均有统计学意义(P < 0.01);DDI组肝组织mtDNA ND1的平均dS与对照组比较,差异有统计学意义(P < 0.05);AZT组和3TC组肝细胞mtDNA ND1的平均dS与对照组比较,差异均有统计学意义(P < 0.05)。 结论 长期暴露于NAs可导致小鼠肝细胞mtDNA ND1和ND4区病变。

[关键词] mtDNA ND1;mtDNA ND4;激光捕获显微切割;线粒体毒性;核苷类似物

[中图分类号] R978.7;R512.91 [文献标识码] A [文章编号] 1673-7210(2018)07(c)-0008-06

Mitochondrial DNA ND1 and ND4 mutation in liver and captured hepatocytes of mice after long-term nucleoside analogues exposure

ZHANG Wei1* QIAO Luxin1,2* DING Wei1 CHEN Dexi1,2 ZHANG Yulin1▲

1.Beijing You′an Hospital, Capital Medical University, Beijing 100069, China; 2.Beijing Institute of Hepatology, Beijing 100069, China

[Abstract] Objective To investigate whether long-term use of nucleoside analogues (NAs) can lead to damage of DNA (mtDNA) ND1 and ND4 regions in liver mitochondria of mice. Methods According to the simple random method, twenty-five 7-weeks-old female Balb/c mice were divided into 5 groups, with 5 cases in each group. The control group received double-distilled water by intraperitoneal injection 5 days per week for 3 months. The each test group was respectively given D4T 50 mg/kg, AZT 100 mg/kg,3TC 50 mg/kg,or DDI 50 mg/kg by intraperitoneal injection per day,5 days per week for 3 months. Single liver cell was captured from mouse liver tissues by laser capture microdissection. The products of mtDNA ND1 and ND4 region PCR amplicons were cloned and sequenced in mice liver cells. Results The mean distance to the reference sequence of mtDNA ND4 in the captured hepatocytes of AZT, D4T and 3TC groups were longer than that in the control group, the differences were statistically significant (P < 0.01). The mean mtDNA ND4 dN of the captured hepatocytes in the AZT group was higher than that in the control group, the difference was statistically significant (P < 0.05). The mean distance to the reference sequence of mtDNA ND1 in the captured hepatocytes of AZT and 3TC groups were longer than that in the control group, the differences were statistically significant(P < 0.01). The mean mtDNA ND1 dS of the liver tissue in the DDI group was higher than that in the control group, the difference was statistically significant (P < 0.05). The mean mtDNA ND1 dS of AZT and 3TC groups were higher than that in the control group, the differences were statistically significant(P < 0.05). Conclusion Long-term exposure to nucleoside analogue can result in mtDNA ND1 and ND4 regions lesion in mouse hepatocytes.

[Key words] mtDNA ND1; mtDNA ND4; Laser capture microdissection; Mitochondrial toxicity; Nucleoside analogue

核苷类似物(NAs)通过宿主细胞酶磷酸化为三磷酸形式,插入病毒DNA链,作为链终止剂和/或抑制人类免疫缺陷病毒(HIV)/乙型肝炎病毒(HBV)逆转录,用于抗HIV和HBV治疗[1-2]。NAs也能抑制人细胞DNA聚合酶?捃(pol?捃),干扰线粒体DNA的合成和修复,导致线粒体功能障碍[3],长期应用导致相关系统疾病,如乳酸酸中毒、脂肪变性、胰腺炎、神经肌肉病变等[4]。但NAs的肝毒性机制尚不清楚。前期研究表明长期暴露于NAs的小鼠存在肝细胞mtDNA D-loop区序列变化[5],但NAs对于mtDNA编码基因的损伤尚不清楚。本研究拟通过分析NAs对位于mtDNA主环和小环上的编码基因NADH脱氢酶亚单位4(ND4)和亚单位1(ND1)序列的影响,并通过激光捕获显微切割技术更精确研究NAs对组织获取肝细胞的mtDNA损伤,探讨NAs小鼠肝脏线粒体毒性。

1 材料与方法

1.1 实验动物与分组

7周龄,体质量28~30 g Balb/C雌性小鼠25只,采用简单随机分组法分为司他夫定(D4T)组、齐多夫定(AZT)组、拉米夫定(3TC)组、去羟肌苷(DDI)组和对照组,每组各5只小鼠。D4T、AZT、3TC、DDI均由东北制药集团有限责任公司惠赠。Balb/C小鼠购自军事医学科学院,合格证号:2012-0004。每组小鼠每天分别经腹腔注射D4T 50 mg/kg、AZT 100 mg/kg、3TC 50 mg/kg和DDI 50 mg/kg,每周5 d,连续12周。对照组腹腔内注射双蒸水。5组小鼠给予相同光照、室温、饲料等饲养条件。严格按照《首都医科大学实验室实验动物使用管理规定》使用实验动物。

1.2 小鼠肝组织的激光捕获显微切割(LCM)

每组小鼠均在12周给药结束时,颈椎脱臼处死小鼠,迅速分离小鼠肝组织,于液氮中速冻。实验时应用最佳切片温度(OCT)复合物包埋冰冻肝组织,-20℃将组织切为6 μm薄片,置于聚乙烯包被的玻片上。继而进行HE染色。风干5 min后,染色的载玻片在2 h内按照厂家说明通过P.A.L.M.自动微光束系统(Oberkochen公司,德国)对HE染色的小鼠肝组织切片进行激光捕获显微切割,获取组织肝细胞。

1.3 mtDNA ND1和ND4基因的克隆和测序

通过QIAGEN公司生产的DNA提取试剂盒,按说明书提取小鼠肝组织和肝细胞基因组DNA。PCR扩增肝组织和肝细胞mtDNA ND1和ND4基因。每个反应约10 ng基因组DNA作为模板。ND1引物对F1:5′-GAAGCAACCTTAATCCCAACAC-3′,R1:5′-GA-AGGATGAAGGGGTATGCTAT-3′;ND4引物对F1:5′-GAAGCAACCTTAATCCCAACAC-3′;R1:5′-GAAGGATGAAGGGGTATGCTAT-3′。引物由上海英峻公司合成。PCR反应体系:双蒸水33.5 μL+10×buffer 5 μL+dNTP Mix(2 mmol/L)4 μL+双向引物各1 μL+TaqDNA聚合酶0.5 μL+DNA模板5 μL。双蒸水作为阴性对照。PCR反应条件:94°C变性3 min,继而30个循环扩增(94°C变性30 s,56℃退火30 s,72℃延伸1 min),最后72℃ 5 min。PCR产物克隆于pMD-18T载体,具体步骤参照分子生物学方法进行[6]。运用ABI 3730基因分析仪器对每个样本随机选择的10个克隆测序,通过BioEdit软件对正反双向测序核苷酸序列进行连接,纠错分析。

1.4 统计学方法

运用Mega 5.0软件,通过Clustal W多序列比对程序将测序序列与参考序列(NC_005089.1,GenBank)进行比对。运用Mega 5.0软件分析实验序列与参考序列之间的核苷酸距离、同义替换率(dS)和错义替换率(dN)。采用PASW 18.0统计学软件进行数据分析,计量资料数据用均数±标准差(x±s)表示,组间比较采用Mann–Whitney非参数检验,以P < 0.05为差异有统计学意义。

2 结果

2.1 显微切割捕获肝细胞前后图片对比

应用LCM技术每组每个鼠肝样本获取200个肝细胞,显微切割前后具有代表性的图片见图1。经HE染色后,肝细胞核染清楚,肝细胞核形态各异,从大圆形到卵圆形。其他细胞显示小和黑染椭圆形和长圆形核。各NAs组肝小叶存在轻中度的脂肪变性。

2.2 NAs组mtDNA ND1序列的变异

肝组织mtDNA ND1序列距离与参考序列的平均距离,NAs组分别与对照组比较,差异无统计学意义(P > 0.05)。肝细胞mtDNA ND1序列距离与参考序列的平均距离,AZT组和3TC组长于对照组(P < 0.01)。肝组织与对照组的平均dS比较,DDI组较之对照组有较高的dS值(P < 0.05)。肝组织与对照组的平均dN比较,差异均無统计学意义(P > 0.05)。肝细胞平均dS比较,AZT和3TC组较之对照组有较高的dS值(P < 0.05)。肝细胞平均dN比较,NAs组与对照组比较,差异无统计学意义(P > 0.05)。见图2。

2.3 NAs组mtDNA ND4序列的变异

肝组织mtDNA ND4与参考序列的平均距离,D4T组与对照组相比存在更远的距离(P < 0.05)。肝细胞的mtDNA ND4序列与参考序列的平均距离,除DDI组,余NAs组较之对照组差异均有统计学意义(P < 0.01)。与对照组比较,4种NAs组肝组织dS和dN差异均无统计学意义(P > 0.05)。与对照组比较,肝细胞的mtDNA ND4NAs组平均dS差异均无统计学意义(P > 0.05)。肝细胞的mtDNA ND4平均dN,与对照组比较,仅AZT组存在相对高的dN值,差异有统计学意义(P < 0.05)。见图3。

2.4 NAs组肝组织和肝细胞mtDNA ND4基因突变

NAs组肝组织ND4区序列单碱基的转换率分别与对照组比较,差异无统计学意义(P > 0.05)。各NAs组肝组织ND4的过渡亚型分别与对照组比较,差异无统计学意义(P > 0.05)。捕获肝细胞的ND4序列单碱基的转换率AZT组和D4T组均高于对照组(P < 0.05)。仅AZT组T→A转化率高于对照组(P < 0.05)。D4T组T→A转化率与对照组比较,差异无统计学意义(P > 0.05)。D4T组T→C转化率高于对照组(P < 0.05)。见图4。

2.5 NAs组肝组织和肝细胞mtDNA ND1基因突变

NAs组肝组织ND1区序列单碱基的转换率分别与对照组比较,差异无统计学意义(P > 0.05)。3TC组肝组织ND4的过渡亚型高于对照组,差异有统计学意义(P < 0.05)。捕获肝细胞的ND1序列单碱基的转换率AZT组和3TC组均高于对照组,差异有统计学意义(P < 0.05);捕获肝细胞的ND1序列单碱基的转换率AZT组和3TC组均高于对照组,差异有统计学意义(P < 0.05)。3TC组和DDI组A→G转化率均高于对照组(P < 0.05);AZT组和3TC组C→T转化率数值均高于对照组(P < 0.05);AZT组和3TC组T→C转化率数值均高于对照组(P < 0.05);3TC组A→T颠换率、3TC组T→G颠换率以及AZT组C→G颠换率数值与对照组比较,差异无统计学意义(P > 0.05)。见图5。

3 讨论

NAs易致肌细胞、神经元细胞和脂肪细胞病变,导致肌病、周围神经病、胰腺炎、脂肪萎缩、肝脂肪变性和乳酸酸中毒等线粒体功能障碍相关的疾病,但机制尚不清楚[7-9]。一项临床研究中,约20%接受抗逆转录病毒治疗的患者表现出肝损伤[109]。另一研究12%接受干扰素联合利巴韦林治疗HIV/HCV合并感染患者,表现出无症状的线粒体毒性[11]。NAs的肝毒性可能促进HIV/HCV合并感染患者的肝纤维化。此外,与CART相关的免疫重建似乎可减轻患者HCV相关肝损伤[12]。NAs可以导致肝脏相关的线粒体毒性[13],具体机制需要深入研究。此前研究表明长期暴露于NAs的小鼠存在肝细胞mt DNA D-loop区的缺失[5]。除D-loop区,ND4和ND1是NADH脱氢酶的亚基,位于线粒体内膜,由线粒体基因MT-ND4和MT-ND1编码。MT-ND4基因的变异可导致遗传性和年龄相关性疾病[14-16]。而MT-ND1基因变异相关疾病的出现类似于NAs诱导的线粒体功能障碍,包括线粒体脑肌病和乳酸酸中毒[15-17]。体外研究中,以10倍的药物最大血浆浓度将NAs作用于HepG2细胞可导致mt DNA ND4的水平下降[18]。本研究表明,小鼠暴露于NAs 3个月后,小鼠肝细胞mtDNA编码基因ND4和ND1出现变化,与本课题组此前小鼠暴露于NAs 4个月后,导致小鼠神经元细胞mtDNA ND4和ND1的拷贝缺失的结果基本一致[19]。因此,推测NA导致的肝脏相关的线粒体毒性可通过影响基因水平的ND1和ND4,进而引起mtDNA的损伤,对线粒体功能的影响仍需要深入研究。

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(收稿日期:2018-03-05 本文編辑:李岳泽)