初治结核病患者自噬相关基因表达水平的研究

A Study on Expression of Autophagy Related Gene in Tuberculosis Patients Undergoing Initial Treatment

目的研究初治结核病患者自噬相关基因表达水平。方法选取2013年12月至2016年3月在首都医科大学附属北京胸科医院就诊的12例初治肺结核病患者作为病例组；选取首都医科大学附属北京胸科医院体检健康的新入职职工和在读学生12名作为对照组。收集研究对象外周血，提取RNA，将RNA逆转录为cDNA，进行实时定量PCR。使用H37Rv标准株感染人外周血单核细胞（THP-1细胞）作为感染组，而未感染H37Rv标准株的THP-1细胞作为未感染组，分别在感染4h和6h使用Trizol法提取RNA，将RNA逆转录为cDNA，进行实时定量PCR检测。实时定量PCR检测使用β-肌动蛋白（β—actin）作为内参基因，测量各个基因的循环阈值（cycle threshold，Ct），并计算各个基因的△Ct值。不同样品同一基因的检测通过计算两者之间的△Ct值差值，使用2^-△△△比较基因之间的表达水平。结果病例组Ras蛋白脑组织同源类似物（Rheb）基因△Ct值为8．71±0．58，对照组△Ct值为7．83±0．58，两组相比差异有统计学意义（t=3．74，P=0．001）；病例组Rheb基因表达下调。病例组哺乳动物雷帕霉素靶蛋白（mTOR）基因△Ct值为11．88±0．97，对照组△Ct值为10.81±1．04，两组相比差异有统计学意义（t=2．60，P=0．016）；病例组历mTOR基因表达下调。使用2^-△△△法比较两组间自噬相关基因表达倍数关系，对照组Rheb基因表达水平是病例组的（185±0．91）倍；对照组mTOR基因表达水平是病例组的（2．76±1．56）倍。H37Rv标准株感染THP-1细胞6h后，感染组自噬相关蛋白LC3B基因△Ct值为6．41±0．15，未感染组△Ct值为7．04±0．05，两组相比差异有统计学意义（t=-4．50，P=0．046）；感染组表达上调。感染组mTOR基因△Ct值为8．73±0．16，对照组△Ct值为10.10±0．20，两组相比差异有统计学意义（t=4．79，P=0．041），感染组表达上调。结论初治结核病患者患病后Rheb和mTOR基因表达下调，调节细胞的自噬；而H37Rv标准株感染THP-1细胞6h后LC3B和mTOR基因表达上调，调节细胞自噬水平。

Objective To explore expression of autophagy related gene in tuberculosis patients undergoing initial treatment. Methods Twelve first- treated tuberculosis patients from Beijing Chest Hospital were selected as case group and 12 newly recruited employees and students of Beijing Chest Hospital were in control group. The RNA was extracted from the peripheral blood and reversed to cDNA and then tested using qPCR. THP-1 cells infected by H37Rv were defined as infectious group and those without H37Rv were defined as the control group, then, using Trizol, RNA was extracted 4 and 6 hours after being infected with THP- 1, and then reversed to cDNA and tested using qPCR. The gene of β-actin was defined as reference gene. After cycle threshold （Ct） values being tested by qPCR, we calculated the △Ct by subtracted Ct values of reference gene from autophagy genes. In addition, we used 2 ^△△△, method to compare levels of the same genes from difference groups. Results The △Ct values of Rheb gene in case group and control group were 8.71 ± 0.58 versus 7.83 ± 0.58 （t=3.74, P=0.001）. The △Ct values ofmTOR gene were 11.88 ± 0.97 versus 10.81 ±. 1.04 （t=-2.60, P=0.016） in case group and control group. Both Rheb gene and mTOR gene were lowly expressed in case group. However, after infected for 6 hours, △Ct values of LC3B gene in infected group and control group were 6.41 ± 0.15 versus 7.04 ± 0.05 （t=-4.50, P=0.046）; △Cts of m TOR were 8.73 ± 0.16 versus 10.10 ± 0.20 （t=-4.79, P=0.041）. The LC3B gene and the mTOR gene were highly expressed in infectious group. All the differences were statistically significant. Conclusion The Rheb and mTOR genes were lowly expressed in first-treated tuberculosis patients, which could regulate and increase the autophagy in defending Mycobacterium tuberculosis infection. In contrast, the gene expressions of LC3B and mTOR were upgrading after infected by H37Rv for 6 hours.