【作者】 陈洁；

【导师】 蒋俊俊；

【作者基本信息】 广西医科大学 ， 再生医学， 2022， 硕士

【摘要】 目的和意义:本研究旨在探讨复方新诺明(Cotrimoxazole,CTX)对巨噬细胞内外马尔尼菲篮状菌(Talaromyces marneffei,TM)生长的影响,并从二氢蝶酸合成酶(Dihydropteroated synthetase,DHPS),二氢叶酸合成酶(Dihydrofolate synthetase,DHFS)和二氢叶酸还原酶(Dihydrofolate reductase,DHFR)及巨噬细胞Dectin-1信号通路等角度深入研究CTX在体外对TM的作用机制,为马尔尼菲篮状菌病患者提供关于复方新诺明的药物治疗策略和参考。方法:临床上复方新诺明(CTX)又称复方磺胺甲恶唑(Trimethoprim/Sulfamethoxazole,TMP/SMX),它是由甲氧苄啶(Trimethoprim,TMP)和磺胺甲恶唑(Sulfamethoxazole,SMX)按照1:5比例组成的复方药物。通过菌落计数(Colony forming units,CFU)检测TM被TMP/SMX,SMX,TMP直接抑制的程度。用酶联免疫吸附试验(Enzyme-Linked Immunosorbent Assay,ELISA)检测TMP/SMX对TM的DHPS,DHFS,DHFR的作用;SMX对DHPS,DHFS的作用;TMP对DHFR的作用。将THP-1悬浮细胞经乙酸肉豆蔻佛波酯(phorbol myristate acetate,PMA)刺激成巨噬细胞作为细胞模型,检测TMP/SMX,SMX,TMP对巨噬细胞的活性影响确定安全浓度范围,通过CFU检测TM在巨噬细胞内被TMP/SMX,SMX,TMP间接抑制的程度。通过ELISA检测TMP/SMX对巨噬细胞感染TM后分泌IL-6、IL-10、NO、ROS的影响。通过实时荧光定量PCR(Quantitative Realtime PCR,q PCR)检测TMP/SMX对巨噬细胞感染TM后Dectin-1信号通路关键因子的m RNA表达水平。结果:1.TMP/SMX在安全浓度范围内可以抑制TM的生长1:5的TMP/SMX在68/340μg/ml以下,SMX在360μg/ml以下,TMP在400μg/ml以下是安全浓度范围,对细胞造成的毒性作用较低。TMP/SMX,SMX和TMP均可在安全范围内直接抑制TM生长。TMP/SMX在14/70μg/ml到68/340μg/ml,SMX在100μg/ml到360μg/ml,TMP在240μg/ml到400μg/ml,TM被抑制的效果随浓度的增大而越来越明显,差异具有统计学意义(均P<0.05)。2.TMP/SMX可以增强THP-1巨噬细胞杀伤进入细胞内TM的作用TMP/SMX,SMX和TMP均可在安全浓度范围内增强巨噬细胞在细胞内杀伤TM的作用。TMP/SMX从36/180μg/ml到68/340μg/ml,SMX从340μg/ml到360μg/ml,TMP从320μg/ml到400μg/ml,均可以发挥随浓度增大越来越明显的抑制TM的作用,差异具有统计学意义(均P<0.05)3.TMP/SMX通过抑制DHPS,DHFS,DHFR发挥杀伤TM的作用安全有效浓度的TMP/SMX和TM共培养,TM的DHPS,DHFS和DHFR均可被抑制。安全有效浓度的SMX和TM共培养,TM的DHPS和DHFS均可被抑制。TM的DHPS,DHFS和DHFR被抑制的效果随着TMP/SMX浓度的增大而越来越显著(均P<0.05)。没有抑菌效果的TMP不能单独抑制TM的DHFR(P>0.05)。4.TMP/SMX通过调控巨噬细胞Dectin-1信号通路,减少巨噬细胞分泌促TM炎症因子,增加抗TM炎症因子的释放安全有效浓度的TMP/SMX和TM感染的巨噬细胞共培养,巨噬细胞表面Dectin-1受体和下游炎症因子IL-6,IL-10,IL-23A的m RNA水平在TM感染组上调,加TMP/SMX后下调;TNF-α和CXCL8的m RNA水平在TM感染组下调,加TMP/SMX后上调(均P<0.05)。结论:本研究发现CTX对巨噬细胞内外的TM均有抑制效果,可能通过抑制TM的DHPS,DHFS,DHFR等关键酶,并调控巨噬细胞Dectin-1受体及下游炎症因子的表达发挥作用。本研究初步揭示CTX在体外抗TM的作用机制,为临床马尔尼菲篮状菌病提供关于CTX的治疗策略和参考。更多还原

【Abstract】 Objective and Significance: The purpose of this research was to explore the effects of Cotrimoxazole(CTX)to the growth of Talaromyces marneffei(TM)fungus inside and outside of macrophages.We conducted further research on the mechanism of CTX to TM fungus in vitro from the perspectives of Dihydropteroated synthetase(DHPS),Dihydrofolate synthetase(DHFS)and Dihydrofolate reductase(DHFR)and the Dectin-1 signaling pathway of macrophages,providing Talaromycosis patients with drug treatment strategies and reference concerned with CTX.Methods: Cotrimoxazole(CTX)was also called Trimethoprim/Sulfamethoxazole(TMP/SMX)in the clinical,it is a compound drug which consists of two drug compositions named Trimethoprim(TMP)and Sulfamethoxazole(SMX)with the proportion of 1:5.Colony forming units(CFU)was used to detect the direct inhibition degree of TM by TMP/SMX,SMX,TMP.Enzyme-Linked Immunosorbent Assay(ELISA)was used to detect the effect of TMP/SMX to the DHPS,DHFS,DHFR of TM,to detect the effect of SMX to the DHPS,DHFS and to detect the effect of TMP to the DHFR.Phorbol myristate acetate(PMA)was used to stimulate THP-1 suspension cells to macrophages as the cell model.Cell activity experiment was conducted to detect the effects of TMP/SMX,SMX,TMP to macrophages to determine the safe concentration range.CFU was conducted to detect the indirect inhibition degree of TM inside macrophages by TMP/SMX,SMX,TMP.ELISA was used to detect the secretion levels of IL-6,IL-10,NO,ROS by TM-infected macrophages after adding TMP/SMX.Quantitative Realtime PCR(q PCR)was used to detect the m RNA expression levels of the Dectin-1 signaling pathway key factors in TM-infected macrophages after adding TMP/SMX.Results: 1.TMP/SMX inhibit TM growth within a safe concentration range The safe concentration range of 1:5 TMP/SMX was below 68/340μg/ml,SMX was below 360μg/ml,and TMP was below 400μg/ml,which caused little toxicity to cells.TMP/SMX,SMX and TMP can directly inhibit TM growth within a safe concentration range.When the concentration of TMP/SMX ranges from 14/70μg/ml to 68/340μg/ml,SMX ranges from 100μg/ml to 360μg/ml and TMP ranges from 240μg/ml to 400μg/ml,the inhibition trend of TM was more and more obvious with the increasing concentration of TMP/SMX,SMX and TMP,and the difference was statistically significant(all P<0.05).2.TMP/SMX can enhance the killing effect of THP-1 macrophages on TM fungus infected into macrophages TMP/SMX,SMX and TMP can enhance the intracellular killing effect of macrophages within a safe concentration range.When the concentration of TMP /SMX ranges from 36/180μg/ml to 68/340μg/ml,SMX ranges from 340μg/ml to 360μg/ml and TMP ranges from 320μg/ml to 400μg/ml,it showed more and more obvious inhibiting TM effect with the increasing concentration of TMP/SMX,SMX and TMP,and the difference was statistically significant(all P<0.05).3.TMP/SMX can kill TM fungus by inhibiting DHPS,DHFS and DHFR The safe and effective concentration of TMP/SMX co-cultured with TM,the DHPS,DHFS and DHFR of TM can be inhibited.The safe and effective concentration of SMX co-cultured with TM,the DHPS and DHFS of TM can be inhibited.The DHPS,DHFS and DHFR of TM can be more and more obviously inhibited with the increasing concentration of TMP/SMX(all P < 0.05).The ineffective concentration of TMP cannot inhibit the DHFR of TM separately(P>0.05).4.TMP/SMX can reduce the secretion of pro-TM inflammatory factors and increase the release of anti-TM inflammatory factors by regulating the Dectin-1 signaling pathway of macrophages The safe and effective concentration of TMP/SMX co-cultured with TMinfected macrophages,the m RNA expression levels of Dectin-1 receptor on the surface of macrophages and downstream inflammatory factors IL-6,IL-10,IL-23 A were upregulated in TM-infected macrophages group and downregulated after adding TMP/SMX.The m RNA expression levels of TNF-α and CXCL8 were downregulated in TM-infected macrophages group and upregulated after adding TMP/SMX(all P<0.05).Conclusion: This research suggests that CTX has an inhibitory effect on TM fungus inside and outside of macrophages,perhaps by inhibiting the key enzymes named DHPS,DHFS and DHFR of TM and regulating the expression levels of the Dectin-1 receptor and downstream inflammatory factors of macrophages.This study preliminarily revealed the mechanism of CTX resistance to TM fungus in vitro,providing the treatment strategies and reference concerned with CTX for the clinical Talaromycosis patients.更多还原