恶性高热实验室诊断方法的初步建立

目的 初步建立恶性高热(MH)的实验室诊断方法-咖啡因-氟烷骨骼肌收缩试验(CHCT).方法 选取10头实验小型猪和1头皮特兰猪,肌肉注射氯胺酮和地西泮麻醉后,静脉注射硫喷妥钠5mg/kg和琥珀胆碱2mg/kg麻醉诱导后气管插管,机械通气,吸入2%～3%氟烷4 h,新鲜气流量为2 L/min,纯氧和空气各1 L/min.记录麻醉过程中及麻醉结束后48 h内MH的发生情况.气管插管后即刻取耳组织,采用常规酚氯仿方法提取基因组DNA,检测蓝尼定受体-1(RYR1)基因的突变情况.根据北美MH研究组的标准建立CHCT的实验装置,气管插管后即刻取股四头肌近肌腱部位的肌纤维,进行CHCT.结果 实验小型猪均未见MH发作,而皮特兰猪在麻醉过程中出现MH的特征性表现.RYR1基因检测显示,实验小型猪均为氟烷抗性基因型(HALNN);皮特兰猪为氟烷易感基因型(HALnn).CHCT结果表明,实验小型猪诊断为非MH易感猪,皮特兰猪诊断为MH易感猪.结论 .实验小型猪和皮特兰猪的CHCT诊断结果与特征性表现及RYR1基因检测结果均一致,初步验证本实验室CHCT设备的稳定性及测定结果的可靠性.

Preliminary establishment of the laboratory diagnostic test for malignant hyperthermia

Objective To preliminarily establish laboratory diagnostic test for malignant hyperthermia (MH) -- caffeine and halothane contracture test (CHCT). Methods Ten Chinese experimental miniature pigs (CEMP) and one pietrain swine were selected in this study. The animals were anesthetized with ketamine and diasepam. Anesthesia was induced with thiopental sodium 5 mg/kg and tracheal intubation was facilitated with suecinylcholine 2 mg/kg intravenously. The animals were mechanically ventilated and anesthesia was maintained with inhalation of 2%-3% halothane for4 h. Frash gas flow was set at 2 L/min and oxygen and air flow at 1 L/ min. All the aninals were observed whether the MH episodes were initiated or not during the process of anesthesia and 48 h after anesthesia. Ear tissues were removed immediately after tracheal intuhation and the genome DNA was extracted by classic SDS-proteinase K-hydroxybenzene-chloroform method to detect mutations in the RYR1 gene. The laboratory devices of CHCT were established according to the North America MH Group protocol and muscle fibers of femoral quadrieeps near tendon were taken to perform CHCT. Results Characteristic manifestations of MH were not found in the CEMPs during and after anesthesia but in the Pietrain swine during anesthesia. The RYR1 genotype of the CEMPs was HALNN and the Pietrain swine HALnn. The CEMPs were diagnosed as MH non-susceptibility and the Pietrain swine as MH susceptibility according to CHCT. Conclusion The CHCT results of CEMPs and Pietrain swine are consistent with those of characteristic manifestations and genetic detection, demonstrating that the devices of CHCT are overall steady-going and the results reliable.