一起云南不明原因猝死案件中4种野生菌的细胞毒性  

Cytotoxicity of 4 Wild Mushrooms in a Case of Yunnan Sudden Unexplained Death

作　　者：龙武 瞿鹏飞 马琳[3] 王蕊 习严梅 李玉华 聂胜洁 段婷 杜进良 唐雪[3] 赵静峰[4] 雷普平 王跃兵[3] LONG Wu;QU Peng-fei;MA Lin;WANG Rui;XI Yan-mei;LI Yu-hua;NIE Sheng-jie;DUANTing;DU Jin-liang;TANG Xue;ZHAO Jing-feng;LEI Pu-ping;WANG Yue-bing(College of Forensic Medicine,Kunming Medical University,Kunming 650500,China;Department of Pathology,School of Basic Medicine,Peking University Health Science Center,Beijing 100191,China;Yun-nan Institute of Endemic Diseases Control and Prevention,Dali 671000,Yunnan Province,China;Key Laboratory of Medicinal Chemistry for Natural Resource,Ministry of Education,Yunnan Research and De-velopment Center for Natural Products,School of Chemical Science and Technology,Yunnan University,Kunming 650500,China)

机构地区：[1]昆明医科大学法医学院,云南昆明650500 [2]北京大学医学部基础医学院病理学系,北京100191 [3]云南省地方病防治所,云南大理671000 [4]云南大学化学科学与工程学院教育部自然资源药物化学重点实验室云南省天然产物转化与应用重点实验室,云南昆明650500

出　　处：《法医学杂志》2023年第2期121-128,共8页Journal of Forensic Medicine

基　　金：昆医联合专项-重点项目(202101AY070001-008)。

摘　　要：目的探究一起云南不明原因猝死(Yunnan sudden unexplained death,YNSUD)案件中涉及的4种野生菌的细胞毒性,为YNSUD的防治提供实验依据。方法采集事件发生家庭食用过的4种野生菌,通过专家辨认和基因测序鉴定种属。运用超声波萃取法提取4种野生菌的生品浸膏干预HEK293细胞,然后用细胞计数试剂盒-8(Cell Counting Kit-8,CCK-8)筛选出有明显细胞毒性的野生菌。将筛选出的野生菌再分别制成生品、熬煮和熬煮后酶解3种浸膏,所得3种浸膏以不同浓度干预HEK293细胞,用CCK-8与乳酸脱氢酶(lactate dehydrogenase,LDH)检测法联合检测细胞毒性,并用倒置相差显微镜观察细胞形态。结果4种野生菌分别为粉黄黄肉牛肝菌(Butyriboletus roseoflavus)、美味牛肝菌(Boletus edulis)、变绿红菇(Rus⁃sula virescens)和隐花青鹅膏(Amanita manginiana)。仅在隐花青鹅膏中发现细胞毒性,其生品浸膏在质量浓度为0.1 mg/mL时显示出细胞毒性,熬煮浸膏和熬煮后酶解浸膏分别在质量浓度为0.4 mg/mL和0.7 mg/mL时有明显细胞毒性。除数量明显减少外,隐花青鹅膏提取物干预后的HEK293细胞还表现出突触增多及折光性差等改变。结论该起YNSUD案件涉及的野生菌中,隐花青鹅膏的提取物具有明显细胞毒性,通过熬煮和酶解两种工艺可以降低其部分毒性,但不能完全灭毒,食用该菌具有一定的安全隐患,隐花青鹅膏可能是导致该起YNSUD案件的原因之一。Objective To explore the cytotoxicity of four wild mushrooms involved in a case of Yunnan sudden unexplained death(YNSUD),to provide the experimental basis for prevention and treat‐ment of YNSUD.Methods Four kinds of wild mushrooms that were eaten by family members in this YNSUD incident were collected and identified by expert identification and gene sequencing.Raw extracts from four wild mushrooms were extracted by ultrasonic extraction to intervene HEK293 cells,and the mushrooms with obvious cytotoxicity were screened by Cell Counting Kit-8(CCK-8).The selected wild mushrooms were prepared into three kinds of extracts,which were raw,boiled,and boiled followed by enzymolysis.HEK293 cells were intervened with these three extracts at different concentrations.The cytotoxicity was detected by CCK-8 combined with lactate dehydrogenase(LDH)Assay Kit,and the morphological changes of HEK293 cells were observed under an inverted phase contrast microscope.Results Species identification indicated that the four wild mushrooms were Butyriboletus roseoflavus,Boletus edulis,Russula virescens and Amanita manginiana.Cytotoxicity was found only in Amanita manginiana.The raw extracts showed cytotoxicity at the mass concentration of 0.1 mg/mL,while the boiled extracts and the boiled followed by enzymolysis extracts showed obvious cytotoxicity at the mass concentration of 0.4 mg/mL and 0.7 mg/mL,respectively.In addition to the obvious decrease in the number of HEK293 cells,the number of synapses increased and the refraction of HEK293 cells was poor after the intervention of Amanita manginiana extracts.Conclusion The extracts of Amanita manginiana involved in this YNSUD case has obvious cytotoxicity,and some of its toxicity can be reduced by boiled and enzymolysis,but cannot be completely detoxicated.Therefore,the consumption of Amanita manginiana is potentially dangerous,and it may be one of the causes of the YNSUD.

关 键 词：法医病理学 法医毒理学 云南不明原因猝死 野生菌 细胞毒性 隐花青鹅膏 

分 类 号：DF795.1[医药卫生—法医学] D919.1[政治法律—诉讼法学] R89[政治法律—法学]