结核杆菌inhA基因突变与肺痨康治疗耐异烟肼肺结核分子机制关系初探

发布时间：2018-03-26 16:57

本文选题：肺痨康　切入点：耐异烟肼肺结核　出处：《成都中医药大学》2016年硕士论文

【摘要】：目的：研究中药复方肺痨康治疗耐异烟肼肺结核小鼠的分子机制,从分子生物学角度,探讨其治疗作用与inhA基因突变之间的关系,为中医药治疗耐药肺结核提供一些研究思路。方法：本研究先取80只昆明种小鼠,尾静脉注射法建立耐异烟肼肺结核动物模型,随机分为模型组、异烟肼组、肺痨康组及异烟肼联合肺痨康组,分别给予生理盐水、异烟肼、肺痨康、异烟肼联合肺痨康灌胃治疗56天；选取20只同等小鼠,尾静脉注射H37RV标准结核分枝杆菌菌悬液,建立H37RV标准菌小鼠模型,以生理盐水灌胃56天；选取20只同等小鼠,尾静脉注射生理盐水作为空白组,以生理盐水灌胃56天；比较各组小鼠一般体征及治疗前后体重变化,56天后取小鼠肺脏组织进行细菌培养：肉眼及光镜下观察各组小鼠肺脏组织病理变化；利用全基因组重测序技术对治疗后小鼠肺组织中结核杆菌基因组DNA进行重测序,以标准菌株(H37RV)为参考序列,筛选对比分析；采用SPSS21.0统计软件对实验中的计量、计数资料进行统计学分析,采用bwa(version 0.7.8)、samtools、Picard等软件处理全基因测序后的数据并进行碱基与基因组表达结果的比对。结果：通过对比治疗前后的的小鼠体重变化,各组小鼠治疗前体重组间差异无统计学意义(P0.05),治疗后各自小鼠体重组间差异有统计学意义(P0.05),标准菌株组与模型组比较,差异无统计学意义(P0.05),异烟肼组、肺痨康组、联合用药组与模型组比较,差异有统计学意义(P0.05)；肉眼观察小鼠肺部形态发现标准菌株组和模型组肺部形态有明显改变,每只小鼠肺组织均有大小不等结节,异烟肼组、肺痨康组、联合用药组仅仅有部分小鼠肺脏现有结节,比较各组小鼠结节数差异性,标准菌株与模型组差异无统计学意义(P0.05)；异烟肼组与模型组比较差异有统计学意义(P0.05)；肺痨康组与模型组比较差异有统计学意义(P0.05)；联合用药组与模型组比较差异有统计学意义(P0.05),肺痨康、模型组、联合用药组之间差异性无统计学意义(P0.05)；光镜下观察治疗组肺脏炎症反应较模型组明显减轻；研究发现与结核杆菌耐药基因inhA基因突变相关的可能的突变碱基位点有三个,分别位于基因组的第1673425、1676290、1678706碱基位置,对应碱基C、C、A突变为T、A、C。该位置碱基突变分别位于编码序列上游、下游、下游,基因组编码序列的改变分别在777、1279、3695。对比各组碱基位点,标准菌株这三个位置碱基与参考碱基一致,模型组、肺痨康组、异烟肼组、联合组耐异烟肼结核杆菌与inhA对应碱基均有突变,且突变位置、基因组编码序列、基因一致。结论：1.肺痨康治疗耐异烟肼肺结核模型小鼠,能显著改善小鼠的一般体征及组织病变,减轻肺部炎症反应,为临床应用治疗耐药肺结核提供更多依据；2.通过全基因测序发现一些新的与inhA基因突变相关位点,分别位于编码序列上游、下游、下游,基因组编码序列的改变分别在777、1279、3695；3.初步研究发现肺痨康抗耐异烟肼肺结核的分子机制与诱发inhA基因回复突变关系不明显,这为中药复方抗结核的进一步研究提供参考。
[Abstract]:Objective : To study the molecular mechanism of traditional Chinese medicine compound pulmonary tuberculosis in the treatment of resistant pulmonary tuberculosis in mice , and to explore the relationship between the therapeutic action and inhA gene mutation , and to provide some research thinking for the treatment of drug - resistant pulmonary tuberculosis in Chinese medicine .
Twenty - one equivalent mice were selected and H37RV standard mycobacterium suspension was injected intravenously to establish a model of H37RV standard strain mice .
Twenty - five mice were treated with normal saline ( normal saline ) as blank group , and saline was used for 56 days .
After 56 days , the lung tissues of mice were cultured for bacterial culture : the pathological changes of lung tissues were observed with naked eyes and under light microscope .
The genomic DNA of mycobacterium tuberculosis in lung tissues of mice after treatment was re - sequenced by full - genome re - sequencing technology , and the reference sequence of standard strain ( H37RV ) was used as reference sequence , and the comparative analysis was screened ;
The results showed that the difference between the two groups was not statistically significant ( P0.05 ) , and the difference was significant ( P0.05 ) .
The lung morphology of mice was observed to be significantly different from the standard strain group and the model group , and the lung tissues of each mouse had a large number of nodules , and the difference between the number of nodules and the standard strain and the model group was not statistically significant ( P0.05 ) .
Compared with the model group , the difference was significant ( P0.05 ) .
There was significant difference between the tuberculosis group and the model group ( P0.05 ) .
There was no significant difference between the combination therapy group and the model group ( P0.05 ) , but there was no significant difference between the two groups ( P0.05 ) .
Under light microscope , the inflammatory response of lung in the treatment group was significantly reduced compared with the model group .
The results showed that 1 . tuberculosis model mice could significantly improve the general physical signs and tissue pathological changes of mice , reduce the inflammatory response of the lung , and provide more basis for the clinical application of drug - resistant pulmonary tuberculosis .
2 . Some new genes related to inhA gene mutation were identified by full - gene sequencing , which were located in the upstream , downstream and downstream of the coding sequence , respectively , and the changes of genomic coding sequence were 777 , 1279 , 3695 , respectively .
3 . It was found that the relationship between the molecular mechanism of pulmonary tuberculosis and the reversion of inhA gene was not obvious , which provided a reference for the further study of the compound anti - tuberculosis of Chinese medicine .

【学位授予单位】：成都中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R285.5

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