【作者】 刘艳；

【导师】 刘良；

【作者基本信息】 华中科技大学 ， 法医学， 2009， 博士

【摘要】 1研究背景有毒动植物中毒是具有中国特色法医毒理学的重要组成部分，也是我国法医毒理学研究的重点内容之一。乌头与乌头属植物是我国临床常用的重要中药，是药用有毒植物的典型代表，也为我国最早有记载的有毒植物，其主要毒性成分为乌头类生物碱，其中以含有双酯基化学结构的乌头碱毒性最强。乌头碱的主要毒性作用的靶器官，主要为心脏与神经系统。有大量研究表明，乌头碱的毒性成份，也是其药性成份，具有强心、镇痛、抗炎、抗肿瘤、降低血压、降低血管通透性等作用，广泛地被应用于临床治疗。由于乌头碱的毒性剧烈，其有效治疗剂量与中毒剂量或致死量极为接近，用药稍有不慎，如因炮制不当，或误服等，即可引起中毒甚至死亡。而利用乌头属植物及乌头碱自杀、投毒他杀的案件时有发生，可见乌头碱中毒在有毒动植物中毒中占有重要位置。为了进一步提高乌头碱中毒的诊断、治疗，规范乌头属有毒中药在疾病中的使用，同时，也为乌头碱中毒的法医学鉴定提供相关的理论依据，对乌头碱中毒机制研究，特别是乌头碱对心肌细胞的毒性作用机制研究，成为了中药研究与应用、临床急救医学和法医毒理学研究的重点问题。近年来，国内外对乌头碱心肌细胞毒性作用机制的研究，已进入到细胞质膜动态变化的分子作用机理水平。但大多仍局限于心肌细胞损伤及单个心肌细胞离子通道检测等方面，对毒物靶器官心室肌细胞群毒性作用的分子毒理学机制，特别是乌头碱对心肌细胞之间信息传递的影响机制、基因表达调控机制尚不明确。2研究目的2．1系统观察不同浓度乌头碱染毒后心肌细胞的毒理病理学变化，研究不同中毒剂量、中毒时间与病变间的效应关系，优化、规范乌头碱靶器官的细胞培养及分子毒理学研究的基础方法；2．2研究乌头碱中毒与心肌细胞DNA损伤的关系，为从分子毒理学角度研究DNA损伤与乌头碱中毒机制的关系提供基础；2．3研究乌头碱染毒对心肌细胞Ca²⁺依赖性蛋白激酶Cα亚型（PKCα）的表达、PKCα磷酸化水平，以及PKCα磷酸化对Cx43磷酸化表达的影响，明确其内在级联效应关系，及参与乌头碱心肌细胞毒性作用分子机制的途径；2．4研究乌头碱对心肌细胞内Ca²⁺调控蛋白表达的影响，观察Ca²⁺调控蛋白参与乌头碱心肌细胞毒性作用机制的方式；2．5研究乌头碱中毒特征及法医学鉴定注意事项。3研究方法3．1新生大鼠心肌细胞原代培养及其方法优化研究选择1～2d的SD新生大白鼠，雄雌不限，消毒后直接剪取心室肌，用胰蛋白酶等消化液消化，制作心肌细胞悬液；于37℃、5％CO₂培养箱，以差速贴壁法培养原代心肌细胞，优化心肌细胞原代培养方法，提高心室肌细胞纯度、成活率和心肌细胞群整体性。选用台盼蓝染色法进行培养心肌细胞活性鉴定；采用抗心肌特异性单克隆抗体，结合间接免疫荧光法检测，对心肌细胞纯度进行鉴定。规范、优化新生大鼠心肌细胞体外原代培养的方法，为后续研究建立良好地基础。3．2乌头碱染毒模型的建立及毒性效应研究于心肌细胞原代培养第6天，更换无血清培养液培养16～18h，以去除血清干扰，再加入不同浓度乌头碱，构建心肌细胞乌头碱染毒模型，根据实验目的不同，设定不同的阳性和阴性对照。实时动态监测乌头碱染毒心肌细胞的形态、功能的变化，研究乌头碱的毒性对心肌细胞损伤的量效作用。3．3乌头碱对大鼠心肌培养细胞DNA损伤的研究新生SD大鼠24只，随机分为6组；取心室肌以差速贴壁法原代培养心室肌细胞。培养第6天，更换无血清培养液培养后，制成密度为2×10⁵个细胞／mL的细胞悬液，分别加入浓度为0.1、0.5、1.0、2.0μmol／L的乌头碱混合液，染毒30 min；并以PBS溶液作阴性对照。采用彗星电泳技术及CASP分析软件，检测不同浓度乌头碱染毒后心室肌细胞DNA损伤程度。实验重复6次。3．4乌头碱对新生大鼠心肌培养细胞内PKCα表达的影响取12孔原代心肌培养细胞组，分成对照组（Normal）、乌头碱染毒组（ACO）、碱性磷酸酶处理组（A．P．）、AAP干预组（AAP）、AAP干预后乌头碱染毒组（AAP+ACO）、PKCα抑制剂G（o|¨） 6976干预组（G（o|¨） 6976），及AAP与G（o|¨） 6976联合干预组（AAP+G（o|¨） 6976），共7组。提取各实验组及对照组心肌细胞总蛋白，以BCA法标准蛋白曲线定量，采用Western-blotting技术，选用P-Cx43（Ser-368）蛋白抗体、NP-Cx43（Ser-368）蛋白抗体、抗PKCα蛋白抗体，及P-PKCα（Ser-657）蛋白抗体，定量检测各组在培养心肌细胞内P-Cx43（Ser-368）蛋白、NP-Cx43（Ser-368）蛋白、总PKCα蛋白，及P-PKCα（Ser-657）蛋白表达含量变化。实验重复6次。3．5乌头碱对心肌细胞PKCα及P-PKCα（Ser-657）位点磷酸化状态的影响选择兔抗鼠总PKCα多克隆抗体（稀释度为1：200）、兔抗鼠磷酸化PKCα（Ser-657）（稀释度为1：200）功能性抗体，用Fluorescein标记山羊抗兔IgG和Rhodamine标记山羊抗兔IgG（稀释度为1：100），应用激光共聚焦扫描显微镜，结合细胞图像荧光定分析技术，检测乌头碱染毒前后，心肌细胞激酶Cα亚型及其第657位丝氨酸残基（Ser657）位点，特异性磷酸化状态的改变。3．6乌头碱对大鼠心肌培养细胞Ca²⁺调控蛋白表达的影响取12孔原代心肌培养细胞组，运用荧光标记技术及RT-PCR技术，建立钠钙交换体（NCX）、肌浆网钙泵Ca²⁺-ATP酶（SERCA₂）、磷酸受钠蛋白（PLB）、兰尼碱受体（RyR₂），及管家基因β-actin共5个基因座的多重荧光复合RT-PCR反应体系及扩增方法，在3100 DNA测序仪中进行毛细管电泳，检测乌头碱染毒组及正常对照组心肌细胞内NCX、SERCA₂、RyR₂、PLB四种基因mRNA表达的差异与变化。实验重复6次。3．7乌头碱中毒10例法医学尸检资料分析收集10例乌头碱中毒死亡的法医学鉴定资料，包括系统的法医学解剖资料及组织病理学检查结果，部分案例还有案情调查、抢救病历资料等；并按年龄、性别、中毒原因、中毒类型、病理变化及特征等分类整理。结乌头碱中毒的毒理病理组织学变化，探讨乌头碱中毒法医学鉴定的注意事项。3．8实验数据的统计学分析本实验研究采用SSPS统计软件（version 10.0，USA）分析，数据以（?）±s表示。3．8．1彗星电泳结果采用CASP软件分析HDNA％、TDNA％、TL、TM、OTM，5个检测指标。计算各组平均值和标准差，以（?）±s表示，应用SSPS在方差齐性条件下做单因素方差分析，对各剂量组组间差异进行比较分析。3．8．2正常对照组和各实验组中P-Cx43（Ser-368）蛋白表达和NP-Cx43（Ser-368）蛋白表达差异、总PKCα蛋白表达和P-PKCα（Ser-657）蛋白表达相对含量分析，应用SSPS做单变量两因素方差分析（ANOVA），对组间均数的多重比较选用Games-Howell检验。3．8．3对ca²⁺调控蛋白NCX、SERCA₂、RyR₂、PLB基因mRNA表达的差异与变化，应用SSPS做t检验。4研究结果4．1原代培养心肌细胞及其方法优化在倒置相差显微镜下观察，刚接种时的心肌细胞悬浮于培养液中，为均一、分散的圆形折光颗粒；12 h后细胞已开始贴壁生长，偶尔可见个别细胞自发搏动；培养24 h后，贴壁细胞互相连接呈稀疏网状，并出现缓慢的同步化搏动；48 h后，心肌细胞在培养孔底部进一步伸展，呈现快速同步化搏动的细胞单层，搏动频率约80～120次／min；培养第6天，心肌细胞在培养孔底部充分伸展，形成稳定的同步化搏动细胞单层，频率约120次／min。采用台盼蓝染色法测定，活细胞的存活率平均达到97.33％；选用抗心肌肌钙蛋白Ⅰ（cTnⅠ）特异性单克隆抗体，结合免疫荧光法检测，心肌培养细胞平均细胞纯度为97.1％。4．2乌头碱对心肌培养细胞染毒模型的建立及毒性效应观察建立乌头碱染毒模型，与正常对照组比较，不同剂量乌头碱染毒后，心肌细胞出现非同步性搏动，搏动减慢，甚至停博。心肌细胞乌头碱染毒后，毒理病理变化与中毒剂量、中毒时间呈现一定的相关性。4．3乌头碱对大鼠心肌培养细胞DNA损伤的观察心肌培养细胞被不同浓度乌头碱染毒后，尾部DNA含量、彗尾长度、尾矩、Olive-尾矩均随乌头碱浓度增加而升高，头部DNA含量则逐渐降低，与对照组相比，均有极显著性差异（P＜0.01）；结果显示，乌头碱染毒剂量越大，心肌细胞DNA损伤越严重。4．4乌头碱对心肌培养细胞内PKCα蛋白表达的影响4．4．1对心肌细胞内P-Cx43（Ser-368）表达的观察各实验组均以Normal为参照。不同干预组对P-Cx43（Ser-368）蛋白表达均有所不同。与正常对照组比较，ACO、A．P．、G（o|¨） 6976组心肌细细胞P-Cx43（Ser-368）蛋白表达均下降；AAP、AAP+ACO、AAP+G（o|¨）9676组P-Cx43（Ser-368）蛋白表达均上升。选用Games-Howell统计方法，检验多组间均数的多重比较，显示ACO、A．P．、G（o|¨） 6976三组间，仅ACO染毒组具有统计学差异（P＜0.05）；而AAP、AAP+ACO、AAP+G（o|¨）9676三个干预组之间未见明显差异。从Western Blotting检测结果显示：ACO、A．P．、G（o|¨） 6976干预后，Cx43（Ser-368）位点磷酸化蛋白表达减少；经APP预处理后，磷酸化表达增强，且效果明显（P＜0.01）。4．4．2对心肌细胞内NP-Cx43（Ser-368）表达的观察各组NP-Cx43（Ser-368）位点磷酸化状态蛋白定量分析结果显示：与正常对照比较，A．P．组、ACO染毒组、G（o|¨） 6976组表达增强（P＜0.01），其中A．P．组表达最强。而AAP组、AAP+ACO组、AAP+G（o|¨） 6976联用组则表达降低（P＜0.01）。4．4．3对心肌细胞中总PKCα表达的观察各组总PKCα蛋白定量分析结果显示：与正常对照比较，A．P．组、ACO染毒组、G（o|¨） 6976组、AAP组、AAP+ACO组、AAP+G（o|¨） 6976联用组心肌细胞内总PKCα表达水平无显著差异（P＞0.05）。4．4．4对心肌细胞中总P-PKCα（Ser-657）表达的观察各实验组均以Normal为参照。不同实验干预组P-PKCα（Ser-657）蛋白均有不同程度的表达。正常组和ACO组心肌细胞内均有P-PKCα（Ser-657）蛋白表达，而A．P．组心肌细胞内无P-PKCα（Ser-657）蛋白表达。与正常对照组相比较，ACO组、G（o|¨） 6976组心肌细胞中P—PKCα（Ser-657）磷酸化蛋白表达显著降低（P＜0.01）。而AAP组、AAP+ACO组、AAP+G（o|¨） 6976联合处理组，磷酸化表达增强，心肌细胞P-PKCα（Ser-657）蛋白表达显著增高（P＜0.01）。4．4．5乌头碱染毒前后心肌细胞PKCα及PKCα（Ser-657）位点磷酸化状态的观察应用激光扫描共聚焦显微镜，观察免疫荧光检测结果显示：标记总PKCα的Fluorescein呈绿色荧光信号，弥散性分布于心肌细胞胞浆中。与正常对照组相比较，ACO染毒组、G（o|¨） 6976组、AAP组、AAP+ACO组、AAP+G（o|¨） 6976联合处理组总PKCα绿色荧光信号差异无显著性。标记P-PKCα（Ser-657）的Rhodamine呈红色荧光信号，弥散性分布于心肌细胞胞浆中。与正常对照组相比较，ACO染毒组、G（o|¨） 6976组，红色荧光信号显著减弱，有极显著性差异（P＜0.01）；而AAP组、AAP+ACO组、AAP+G（o|¨） 6976联合处理组红色荧光信号较正常对照组，也有极显著性差异（P＜0.01）。4．5乌头碱对大鼠心肌培养细胞Ca²⁺调控蛋白表达的影响多重荧光复合RT-PCR检测发现，与正常对照组比较，乌头碱染毒组RyR₂、NCX基因mRNA表达增加，而PLB、SERCA₂基因mRNA表达减少。4．6乌头碱中毒尸检资料分析病理变化特征主要为：①心肌灶性出血，细胞横纹不清，肌浆凝聚，细胞间质淤血；②肝可见肝细胞灶性或点状坏死，可见肝细胞脂肪变性或水样变性；③肺脏可有灶性或点状出血，灶性肺水肿；④可见部分胃粘膜有散在性点状出血；⑤偶见肾组织点状出血和散在性坏死。5研究结论5．1优化、规范心肌细胞培养方法，建立稳定的体外新生大鼠培养细胞的乌头碱染毒模式，为法医毒理学研究提供了方法学上的参考。5．2乌头碱染毒可引起心肌细胞DNA损伤，并呈明显的剂量—效应关系，推测细胞DNA损伤参与乌头碱毒性作用机制。5．3乌头碱染毒可影响PKCα本身的磷酸化状态，同时乌头碱染毒所致的PKCα（Ser657）位点蛋白磷酸化表达下降，可进一步导致心肌细胞Cx43磷酸化状态减弱。推测PKCα磷酸化、Cx43磷酸化状态的改变是乌头碱心肌细胞毒性作用机制之一。5．4乌头碱可影响心肌细胞Ca²⁺调控蛋白的表达，使RyR₂、NCX基因mRNA表达增加，PLB、SERCA₂基因mRNA表达减少，推测Ca²⁺调控蛋白参与了乌头碱毒性作用机制。其毒性作用机制的方式仍有待进一步研究。。Ca²⁺调控蛋白参与毒性作用机制有待进一步研究。5．5对10例乌头碱中毒尸检资料进行分析，总结其毒理病理变化特点，并提出乌头碱中毒法医学鉴定的注意事项。更多还原

【Abstract】 1 BackgroundToxic animals and plants poisoning are one of important componentelements of forensic toxicology with Chinese characteristic, and a part ofimportant content of forensic toxicology research in China. Aconite andAconitium plants, which are important Chinese medicine widely used inclinic, are typical representative for herb poisonous plants, and they arethe poisonous plant recorded earliest. Aconitium alkaloid is their chiefingredient; and Aconitine, chemical structure of which containsdiesterditerpene, is the most poisonous. The target organs of aconitinetoxic effect are mainly heart and nervous system. It has been confirmedthat toxic ingredient of aconitine, which plays roles of drug, have widespread pharmaceutical properties including cardio-tonic, analgesic,anti-inflammatory, tumor-suppressant, lowering blood pressure, reducinghemal wall and so on. As aconitine are of severe toxicity and therapy doseis so close to poisonous dose or lethal dose that use immodestly, such asimproper process, misuse and so on, would lead to poisoning, even death.Meanwhile, related homicide or veneficium suicide using Aconite andAconitium plants are matters of common occurrences, which indicate thatAconitine intoxication lies important position of toxic animals and plantspoisoning.In order to further improve the diagnosis and healing of aconitinepoisoning, specify the therapeutic action of Aconitium poisonous Chinesetraditional medicines for diseases and offer relevant theoretical foundationfor forensic evaluation of aconitine intoxication, the investigation of the mechanism of aconitine poisoning, especially the mechanism of toxiceffect on cardiomyocytes, turn into the important issue for research andapplication of Chinese traditional medicines, clinical emergency medicineand forensic toxicology study.At present, mechanism studies of toxic effect on aconitine poisoningare mainly focused on molecular action of cytoplasma membrane dynamicchange, most of which are limited to cardiomyocytes injury, detection ofion channel of single cardiac myocyte and so on. It is uncertain thatmolecular toxic mechanism of toxic effect on groups of ventricularmyocytes as target organ, especially influence on information transferbetween cardiomyocytes and mechanism of gene expression regulatory.2 Objectives●To systematic observe toxicological pathology change of cardiomyocytesafter incubation with different concentrations of aconitine,investigate the effect relationship between different poisonous dose,poisonous period and pathological change, optimize and specify cell ofaconitine target organ culture and basic method of moleculartoxicologic study.●To study the DNA damage of aconitine cultured cardiomyocytes andoffer theoretical principle for the relationship between DNA damageand aconitine intoxication mechanism from molecular toxicologicpoint of view.●To investigate the expression of protein kinase C （PKCα） andphosphorylation level in aconitine cultured cardiomyocytes and howPKCαphosphorylation affect Cx43 （Ser368） phosphorylation; affirmwhether PKCαand P-Cx43 （Ser368） phosphorylation be of cascadeeffect relation after aconitine incubation and play role in molecularmechanism of aconitine cardiomyocytes. ●To explore the influence of aconitine on calmodulin in cardiomyocytes.Observe calmodulinin involved in the way of toxicitymechanism of aconitine cardiomyocytes.●To analyze the character of aconitine poisoning and attentions inforensic identification.3 Methods3.1 Optimizing neonatal rats’ cardomyocytes primary culturecondition.1-2 day-old Spague-Dawley rats, regardless of the gender, weredisinfected, and ventricles were minced and digested in solutioncontaining trypsinase to be myocardial cell suspension. After incubation at37℃in 5% CO₂, selective attachment of cardiomyocytes was induced forprimary culture cell to optimize primary cardomyocytes culture conditionand increase the purity, survival rates of ventricular myocytes and integrityof cardomyocytes group. Identify the activity of cultured cardiomyocytesby trypan blue staining method, and evaluate the purity of cardiomyocytesby anti-myocardium specific monoclonal antibody combining withindirect immunofluorescence. Specify and optimize in vitro neonatal rats’cardomyocytes primary culture condition and set up preferred basis forfollowing study.3.2 Setting up aconitine cultured model and studying toxic effect ofaconitine.On 6^th day of primary cardiomyocytes culture, after replenishmentwith serum-free medium for 16～18h incubation to wipe off interference ofserum, cardiomyocytes was incubated with different doses of aconitine tobe cultured models. Meanwhile, set up various intoxicated and controlgroups according to different objectives. Real-time monitoringmorphologic and functional change of aconitine cultured cardiomyocytes, and analyze the dose-effect manner of aconitine toxicity tocardiomyocytes injury.3.3 Studying the DNA damage of aconitine cultured cardiomyoeytes.Ventricular myocytes of 24 neonatal rats （randomly divided into 6groups） were incubated by differential adherent culture. On 6^th day ofcardiomyocytes primary culture, after replenishment with serum-freemedium for 16～18h incubation, unattached cells were adjusted to cellsuspension at a density of 2×10⁵ cell/ml. Add aconitine solution in cellsuspensions to aconitine mixture at different concentrations of 0.1μM/L,0.5μM/L, 1μM/L and 2μM/L for 30 min incubation. Set PBS to benegative control group. Detect DNA damage level of ventricular myocytesafter aconitine incubation by comet assay and acridine orange dye andanalyze the damage by CASP analysis software. All the steps repeated 6times.3.4 Investigating the expression of PKCαin aconitine culturedneonatal rats’ cardiomyocytes.12 wells for primary cultured cardiomyocytes group were dividedinto 7groups, including normal group （Normal）, aconitine incubated group（ACO）, alkaline phosphatase treated group （A.P.）, AAP treated group（AAP）, aconitine incubated after AAP treated group （AAP+ACO）, G（o|¨）6976 group （PKCαdepressant, G（o|¨） 6976） and AAP and G（o|¨） 6976 co-treatedgroup （AAP+G（o|¨） 6976）. Extract total protein of cardiomyocytes ofexperimental groups and control group, and quantify the total protein byBCA standard protein regression curve. Quantify the change of expressionamounts of P-Cx43 （Ser-368）, NP-Cx43 （Ser-368） and total PKCαandP-PKCα（Ser-657） in groups of cultured cardiomyocytes byWestern-bloting with anti-P-Cx43 （Ser-368） antibody, anti-NP-Cx43（Ser-368） antibody and anti-PKCαantibody and anti-P-PKCα（Ser-657）antibody. All the steps repeated 6 times. 3.5 Influence of the PKCαand P-PKCα（Ser-657） locusphosphorylation state in cardiomyocytes after aconitine incubation.Anti-rabbit-anti-rat PKCαpolyclonal antibody （dilution at 1:200）,rabbit anti-mouse phosphorylation PKCα（Ser-657） functional antibody（dilution at 1:200）, Fluorescein mark goat via rabbit IgG and Rhodaminemark goat via rabbit IgG （dilution at 1:100） were selected to detect thespecific phosphorylation state of P-PKCα（Ser-657） locus with laserscanning confocal microscope and cellular image fluorescent locationanalysis technique before and after aconitine incubation.3.6 Influence of the expression of calmodulin in aconitine culturedrats’ cardiomyocytes.Multiplex fluorescence reaction system and amplification methodwere set up with Na⁺-Ca²⁺ crossover （NCX）, phosphoric receptor sodiumalbumen （PLB）, sarcoplasmic reticulum calcium pump Ca²⁺-ATP ase（SERCA₂）, Ryanodine receptor （RyR₂） and housekeeping gene （β-actin）by fluorescence labeling technique and RT-PCR in 12 wells of primarycultured cardiomyocytes groups. Capillary electrophoresis was used by3100 DNA Sequencer to detect the difference and change of transcriptmRNA of NCX, SERCA₂, RyR₂ and PLB in aconitine cultured groups andcontrol group. All the steps repeated 6 times.3.7 Analyzing forensic postmortem examination documents of 10aconitine poisoning cases.Collect forensic postmortem examination documents of 10 aconitinepoisoning cases, including systematic forensic anatomical data andhistopathological outcomes, investigation documents, rescue record and soon for some of the cases. Sort the data by age, gender, poisoning cause,poisoning type, and pathological change, characteristic and so on.Summarize the toxicological pathologic change of aconitine poisoning and investigate attention of forensic identification for aconitine poisoning.3.8 Statistical analysis of experimental data.Data from the study analyzed by SSPS statistical software （version10.0, USA） were expressed as （?）±s.●Outcomes of comet assay were processed by CASP analysis software.Experimental data, such as head DNA （HDNA %）, tail DNA（TDNA %）, tail length （TL）, tail moment （TM） and Olive tail moment（OTM） were measured and average value and standard deviation werecalculated and expressed by （?）±s. Each dosage group was comparedby one-way analysis of variance （ANOVA） with homogeneity ofvariance, and differences between groups were analyzed.●The differences between expressions of P-Cx43 （Ser-368） andNP-Cx43 （Ser-368）, relative expression amounts of total PKCαandP-PKCα（Ser-657） from control group and each experimental groupwere analyzed with univariate two-way （ANOVA） by SSPS statisticalsoftware; and multiple comparisons were made using Games-Howellpost hoc test.●The difference and change of transcript mRNA of NCX, SERCA₂,RyR₂ and PLB in aconitine cultured groups and control group wereanalyzed by t-test with SPSS statistical software.4 Results4.1 Primary cardiomyocytes culture and condition optimization.When plated in culture well, the cardiomyocytes were bright pellets inshape and suspended in medium. After 12 hours, they began to crawl onthe bottom of the culture well and spontaneous beating could be found infew cells. After 24 hours, attached myocytes connected each other, looked like a net; and slow synchronous beating could be found in all cultures.After 48 hours, the cardiomyocytes stretched on the bottom of culturewells, and high-speed （80～120 times per minute） synchronous beatingmonolayers of syncytium were formed. On 6th day of culture, meanlivability of cardiomyocytes were 97.33 % using trypan blue stainingmethod. Applying immunofluorescent microscopy with specificmonoclonal anti-cTnI antibody, we estimated that the mean purity ofcultured cardiomyocytes were 97.1%.4.2 Setting-up models of cultured cardiomyocytes treated by aconitineand observing the toxic effect.Aconitine incubated models were set up and it was verified by H.E.staining that the morphological structure of cardiomyocytes were hardlychanged after incubation with different dosages of aconitine compared tothe control cultures. But cardiomyocytes showed asynchronic beat,slowing beat and even arrest. There were certain correlation amongtoxicological pathologic change, toxic dose and poisoning period.4.3 Observation of DNA damage of cultured rats’ cardiomyocytesafter aconitine incubation.After different concentrations of aconitine incubation, tail DNA, taillength, tail moment ^TM and Olive tail moment of cultured neonatal rats’cardiomyocytes increased, and head DNA decreased while theconcentration of aconitine increased. And there were all extremelysignificant differences compared with the control group （P＜0.01）. Theoutcomes indicated that higher the dose of aconitine, severer the DNAdamage of cardiomyocytes.4.4 The influence of expression of PKCαin cultured cardiomyocytes.4.4.1 Observation of expression of P-Cx43 （Ser-368） in cardimyocytes.Every experimental group was compared with the normal group. Andthe expressions of P-Cx43 （Ser-368） in cardiomyocytes with different treated patterns were not the same. The expression amounts of P-Cx43（Ser-368） in cardiomyocytes of ACO incubated group, A.R group and G（o|¨）6976 group decreased, and all increased in AAP group, AAP+ACO groupand AAP+G（o|¨） 6976 group. Games-Howell statistical method was selectedfor multiple comparisons of means between groups. Among ACOincubated group, A.P. group and G（o|¨） 6976 group, there was significantstatistical difference in ACO incubated group （P＜0.05）, while there werehardly obvious difference among AAP group, AAP+ACO group andAAP+G（o|¨） 6976 group. The outcomes by Western Blotting presented thatafter ACO, A.R and G（o|¨） 6976 treated, the expression protein of Cx43（Ser-368） locus decreased, while after APP pre-treated increased obviously（P＜0.01）.4.4.2 Observation of expression of NP-Cx43（Ser-368） in cardiomyocytes.The quantitative analytic outcomes of expression protein of NP-Cx43（Ser-368） locus phosphorylation state presented that the expression in A.P.group, ACO incubated group and G（o|¨） 6976 group reinforce （P＜0.01）,especially A.P. group the most, while AAP group, AAP+ACO group andAAP+G（o|¨） 6976 group reduce （P＜0.01）.4.4.3 Observation of expression of total PKCαin cardiomyocytes.The quantitative analytic result of total PKCαshowed that there wereno significant difference of expression of total PKCαin A.R group, ACOincubated group, G（o|¨） 6976 group, AAP group, AAP+ACO group andAAP+G（o|¨） 6976 group, compared with the control group （P＞0.05）.4.4.4 Observation of expression of total P- PKCα（Ser-657） incardiomyocytes.Every experimental group was compared with the normal group. Andthe expressions of P-PKCα（Ser-657） in cardiomyocytes with differenttreated patterns were different. The P-PKCα（Ser-657） expressed incardiomyocytes of the normal group and ACO group, but none of A.Rgroup.Compared with the normal group, the expressions of P-PKCα （Ser-657） phosphorylation protein in cardiomyocytes of ACO group andG（o|¨） 6976 group decreased obviously （P＜0.01）. And the expressions ofphosphorylation increased in AAP group, AAP+ACO group and AAP+G（o|¨）6976 group （P＜0.01）.4.4.5 Observation of PKCαand PKCα（Ser-657） locus phosphorylationstate before and after aconitine incubationApplying laser scanning confocal microscope, the immumofluorescenedetection results presented:Total PKCαlabeled by Fluorescein, as green fluorescene signals, weredispersed distributed in cytoplasm of cardiomyocytes. There were nosignificant differences in ACO incubated group, G（o|¨） 6976 group, AAPgroup, AAP+ACO group and AAP+G（o|¨） 6976 co-treated group comparedwith the normal group.Total P-PKCα（Ser-657） labeled by Rhodamine red fluorescene signalslabeling, as red fluorescene signals, were dispersed distributed incytoplasm of cardiomyocytes. The red fluorescene signals weakenedobviously and there were extremely significant differences in ACOincubated group and G（o|¨） 6976 group compared with the normal group（P＜0.01）. There were also extremely significant differences of redfluorescene signals in AAP group, AAP+ACO group and AAP+G6 6976co-treated group （P＜0.01）.4.5 Influence of expression of calmodulin in aconitine cultured rats’cardiomyocytes.It was detected that mRNA of RyR₂ and NCX expressed more, andPLB and SERCA₂ less by RT-PCR combined with multiplex fluorescence.4.6 Analysis of forensic postmortem examination documents ofaconitine poisoning cases.Characteristic of pathological changes were mainly that:①myocardium focal hemorrhage, transverse striation of cardiomyocytes wasunclear, sarcoplasm condensed and intercellular congestion;②focal or spotty necrosis of hepatocytes and fatty degeneration or hydropicdegeneration;③focal or globular hemorrhage of lung, focalpneumonedema;④scattered globular hemorrhage in gastric mucosa ofsome cases;⑤globular hemorrhage or scattered necrosis in kidney of afew cases.5 Conclusions●5.1 Optimization and specify cardiomyocytes culture method,establishment of aconitine incubated in vitro neonatal rats’ cell modeland reference for forensic toxicologic study.●5.2 DNA damage of aconitine incubated cardiomyocytes was ofobvious dose-effect relation, which would indicate that DNA damageplayed a role in toxic effect mechanism.●5.3 Aconitine incubation would influence phosphorylation state ofPKCαand phosphorylation expression of PKCα（Ser657） decreased,which further induced that phosphorylated Cx43 of cardiomyocytesweakened. It inferred that the change of phosphorylation state ofPKCαand Cx43 was one of the paths of the aconitine poisoning.●5.4 Aconitine influenced the expression of calmodulin incardiomyocytes, and mRNA of RyR₂ and NCX expressed more andPLB and SERCA₂ less. That calmodulin take part in toxic effectmechanism deserves further research.●5.5 Announcements of forensic identification for aconitine poisoningwere raised after collection of forensic postmortem examinationdocuments of 10 aconitine poisoning cases and summarize thecharacters of toxicological pathologic change.更多还原