VKORC1-3673G>A、CYP2C9*3、CYP4F2 rs2108622和CYP2C19*2基因多态性对中国汉族房颤患者华法林维持剂量的影响

目的 探讨VKORC1-3673G>A、CYP2C9^*3、CYP4F2 rs2108622和CYP2C19^*2位点基因多态性对中国汉族房颤患者华法林维持剂量的影响。方法 收集107例服用华法林达维持剂量的汉族房颤患者的血样和临床相关资料,应用PCR-RFLP法检测VKORC1-3673G>A、CYP2C9^*3、CYP4F2 rs2108622和CYP2C19^*2基因型,采用独立样本t检验分析基因型与华法林维持剂量的相关性。多元线性回归建立给药模型,探讨基因多态性对华法林维持剂量的影响。结果 VKORC1-3673G>A、CYP2C9^*3、CYP4F2 rs2108622基因多态性和患者年龄、体质量能解释45.2%的华法林维持剂量差异。CYP2C19^*2基因多态性对本研究人群华法林维持剂量无影响。结论 VKORC1-3673G>A、CYP2C9^*3、CYP4F2 rs2108622基因多态性显著影响中国汉族房颤患者的华法林维持剂量。     

HLA-B*1502基因多态性与肾移植受者环孢素所致肝功能异常的相关性研究

摘 要 目的： 研究肾移植术后患者HLA-B^*     

肾移植患者SLCO1B3基因多态性与他克莫司血药浓度相关性研究

目的 探究肾移植患者SLCO1B3基因多态性与术后早期他克莫司血药浓度的相关性。方法 选取昆明市第一人民医院68例肾移植患者为研究对象,运用化学免疫发光法监测他克莫司血药浓度,同时采用聚合酶链反应法检测CYP3A5^*3、SLCO1B3 T334G和G699A基因多态性,并进行基因分型,分析各基因型与他克莫司血药浓度的相关性。结果 CYP3A5^*3不同基因型对术后他克莫司血药浓度和标准化血药浓度有影响,差异具有统计学意义(P＜0.05),SLCO1B3 T334G和G699A基因位点不同基因型对术后他克莫司血药浓度和标准化血药浓度无影响,差异均无统计学意义。结论 与CYP3A5^*3/^*3基因型相比,CYP3A5^*1等位基因携带者达到相同的他克莫司浓度需要增加他克莫司给药剂量。SLCO1B3 T334G和G699A基因多态性对肾移植术后早期他克莫司血药浓度无影响。     

肇庆地区人群华法林相关基因CYP2C9*3和VKORC1的多态性分布研究及指导价值

目的 探讨广东省肇庆地区人群华法林相关基因细胞色素P450复合物亚基2C9（CYP2C9）和维生素K环氧还原酶复合物亚基1（VKORC1）多态性分布,并比较性别和中国西双版纳傣族、北京汉族、南方汉族间差异性的分布,为临床医生精准使用华法林进行抗凝治疗提供理论基础。方法 选取2019年5月-2022年1月于肇庆市第一人民医院进行华法林相关基因检测的患者122例,所有患者均采用数字荧光分子杂交技术对CY92C9^*3和VKORC1进行基因多态性检测,比较患者性别间和中国西双版纳傣族、北京地区汉族、南方地区汉族间的基因多态性分布情况,并对比基于药物基因组学指导下的华法林使用剂量与常规剂量使用华法林治疗后2~3 d后国际标准化比值（INR）达标率。结果 122例检测样本中,CY92C9^*3基因位点AA、AC、CC基因型所占的比例分别为95.90%、4.10%、0,C等位基因和T等位基因频率分别为97.95%和2.05%;VKORC1基因位点GG、GA、AA基因型分别为0.82%、19.67%、79.51%,A等位基因和C等位基因频率分别为10.66%和89.34%。不同性别间CY92C9^*3与VKORC1的基因型分布和等位基因分布差异均无统计学意义（P>0.05）。通过已有的数据库进行对比,肇庆地区的CY92C9^*3基因型、等位基因与1000 Genomes Project （1000 GP）西双版纳傣族、北京汉族与南方汉族对比无统计学差异（P>0.05）;但与1000 GP北京汉族对比,VKORC1的基因型和等位基因频率有统计学差异（P<0.05）;与1000GP西双版纳傣族对比,VKORC1的等位基因频率有统计学差异（P<0.05）;华法林在基因组学指导下的剂量与常规剂量治疗后INR达标率差异有统计学意义（P<0.01）。结论 肇庆地区人群存在CY92C9^*3和VKORC1基因多态性,其中VKORC1基因可能存在地域的差异,进行华法林相关基因检测可以为临床制定个体化华法林抗凝方案提供重要的参考价值。     

NUDT15c.415C>T和TPMT*3C基因多态性检测方法的比较与临床应用

目的 建立准确、快速、经济的方法,检测NUDT15 c.415C>T和TPMT*3C基因多态性,探讨临床应用价值。方法 收集2017年5月-2018年5月期间福建汉族患者服用硫唑嘌呤2周以上的血清样本,提取DNA或白细胞后分别采用PCR-RFLP法、PCR-Sanger测序法和荧光定量PCR法对NUDT15 c.415C>T和TPMT*3C进行基因多态性分型,比较这3种方法的准确性、简便性及经济性。根据白细胞值分组,结合临床资料,探讨基因多态性等因素与硫唑嘌呤致白细胞减少的相关性。结果 共纳入129例患者,其中硫唑嘌呤致白细胞减少15例（11.6%）。3种方法的基因多态性检测结果一致,TPMT*3C未发现突变纯合子。携带NUDT15c.415C>T突变等位基因者服用硫唑嘌呤致白细胞减少的风险高于携带野生等位基因者（OR=6.2,95%CI：2.5~15.4,P=0.000 054）,而携带TPMT*3C突变等位基因者与野生等位基因者出现白细胞减少比例并无显著性差异（P=0.393）。NUDT15c.415C>T基因多态性预测白细胞减少敏感度为53.3%,特异度为85.1%,ROC曲线AUC为0.69。结论 3种方法都可用于临床检测NUDT15 c.415C>T和TPMT*3C基因多态性。PCR-RFLP法不需要专用试剂盒,也不需要昂贵的仪器设备,成本较低,过程简单,易于操作,特别适合条件有限的单位开展工作。福建汉族患者在服用硫唑嘌呤前进行NUDT15c.415C>T基因多态性检测比TPMT*3C更具临床价值。     

13C核磁共振技术测定鱼肝油中ω-3脂肪酸α(1,3)-酰基和β(2)-酰基的位次分布

目的 应用¹³C核磁共振（¹³C-NMR）光谱测定鱼肝油中ω-3脂肪酸[十八碳四烯酸（C18：4 n-3,moroctic acid,MA）、二十碳五烯酸（C20：5 n-3,eicosapentaenoic acid,EPA）、二十二碳六烯酸（C22：6 n-3,docosahexaenoic acid,DHA）]形成的甘油三脂中α（1,3）-酰基、β（2）-酰基的位次分布。方法 用CDCl₃溶解样品,利用高分辨核磁共振光谱直接测定。结果 鱼肝油中MA、EPA和DHA α（1,3）-酰基、β（2）-酰基在δ 171.5~173.5 ppm的化学位移与文献报道基本一致。2批次鱼肝油未发现上述ω-3脂肪酸特征峰,4批次鱼肝油有特征峰检出,但是β（2）-酰基的位次分布不同。按照β（2）-酰基的比例,4批鱼肝油来源于家养鱼类提取的可能性较大。结论 应用该¹³C核磁共振技术可以鉴别鱼肝油的优劣,方法简单、快捷。     

丸剂中非法添加环丙沙星的快速检测方法研究

目的 建立一种丸剂中非法添加环丙沙星的快速检测方法。方法 通过¹⁹F-核磁共振定量（¹⁹F-qNMR）法对添加入当归苦参丸中的环丙沙星进行检测,以4,4’-二氟二苯甲酮为内标,以氘代DMSO为溶剂,采集δ-120.3处环丙沙星信号和δ-104.8处内标信号面积,计算环丙沙星的含量。结果 以环丙沙星和内标信号面积比为纵坐标,环丙沙星和内标质量比为横坐标,得到线性回归方程为：Y=3.384 1X+0.114 8,R²=0.992,环丙沙星在5～30 mmol/L具有良好的线性,检出限为7.798×10^-2 mg/mL,定量限为0.260 mg/mL。建立的¹⁹F-qNMR法精密度、重复性、稳定性、加样回收率均符合检测要求。结论 所建立的¹⁹F-qNMR法样品制备简单,检测时间短,灵敏度高,适合对丸剂中非法添加的环丙沙星进行含量测定。     

14C尿素呼气试验固体闪烁法检测幽门螺杆菌感染的安全性及有效性

目的 评价¹⁴C尿素呼气试验（¹⁴C-UBT）新型固体闪烁方法检测幽门螺杆菌（HP）感染的安全性和有效性。方法 连续纳入2017年11月1~31日因上消化道症状就诊安徽医科大学第一附属医院和安徽医科大学第二附属医院内镜中心的门诊患者100例,对每位患者先后采用¹⁴C-UBT液体闪烁检测方法和YH10型固体闪烁法检测HP,以¹⁴C-UBT液闪检测的结果为金标准,观察比较YH10型固体闪烁法检测HP感染的安全性及诊断符合率。结果 ①在采用YH10型固体闪烁采样瓶对患者进行HP检测的过程中,均未发生机械、化学、电击、火灾等伤害,没有闪烁液倒吸入口和泼洒的风险。②YH10型固体闪烁采样瓶临床样本以100每分钟衰变率为判断分界值,固闪样本诊断HP为阳性的病例数为59例,液闪样本诊断HP为阳性的病例数为59例,两种检测方法检测HP感染阳性的符合率为100%。结论 与¹⁴C-UBT液闪检测法相比,新型¹⁴C-UBT固体闪烁法检测HP感染,安全有效,值得临床推广应用。     

1,2,3,4,6-O-五没食子酰基葡萄糖对MPP+诱导PC12细胞凋亡的保护作用

目的 考察1,2,3,4,6-O-五没食子酰基葡萄糖(1,2,3,4,6-penta-O-galloyl-β-D-glucose,β-PGG)对MPP⁺诱导的帕金森病细胞模型中PC12细胞凋亡的保护作用及其机制研究。方法 PC12细胞孵育于高糖DMEM培养基中,在药物处理前1周,将神经生长因子(NGF)加入培养基中,使培养基中NGF的终质量浓度为50 ng/mL。将细胞分为对照组、MPP⁺组以及50 μmol/L β-PGG预处理7、12、20、30 h组,观察预处理不同时间对MPP⁺中PC12细胞存活影响。采用台盼蓝染色法检测细胞死亡情况,MTT法检测细胞活力,免疫印迹法检测Bcl-2、Bax、Fas、FasL、procaspase-3、procaspase-8、procaspase-9蛋白表达情况,并检测caspase-3、caspase-8、caspase-9活力。结果 对照组PC12细胞死亡率最低,MPP⁺组PC12细胞死亡率最高,从β-PGG预处理12 h起PC12细胞死亡率较MPP⁺组均明显降低(P< 0.01)。MPP⁺组PC12细胞活力最低,50 μmol/L β-PGG预处理12 h时PC12细胞活力进一步增高,预处理20 h时细胞活力最高。β-PGG预处理5 h后即可见Bcl-2、procaspase-3、procaspase-8、procaspase-9蛋白含量增加,至15 h时增加达到高峰;与之相反的是,β-PGG预处理5 h后即可见Bax、Fas、FasL蛋白含量减少,至30 h时达最少。50 μmol/L β-PGG预处理PC12细胞15 h后caspase-3、caspase-8、caspase-9的活力分别为MPP⁺组的36.5%、40.2%、42.2%。结论 β-PGG对MPP⁺诱导PC12细胞凋亡具有保护作用,其机制是通过增强Bcl-2的表达、抑制Bax、Fas、FasL的表达以及降低caspase-3、caspase-8、caspase-9的活力实现了抑制MPP⁺引起的PC12细胞凋亡,促进PC12细胞的存活。     

石菖蒲中β-细辛醚的提取工艺优化及其体外抗氧化活性研究

目的 采用乙醇加热回流法研究石菖蒲β-细辛醚的最佳提取工艺,并考察其不同部位抗氧化活性。方法 以石菖蒲β-细辛醚提取量为评价指标,优化提取方法,在单因素试验的基础上,采用正交设计与响应面法考察乙醇浓度、料液比、提取时间3个因素对石菖蒲β-细辛醚提取量的影响。在确定最佳提取工艺后,对其不同部位抗氧化活性进行研究。结果 石菖蒲β-细辛醚最佳提取工艺：乙醇浓度为95%,料液比为1∶20 g·mL^–1,提取时间为2.5 h。在此条件下,石菖蒲β-细辛醚提取量为0.918 7 mg·g^–1。体外抗氧化活性结果表明抗氧化能力强弱顺序依次为乙酸乙酯>石油醚>乙醇>正丁醇。其中,乙酸乙酯部位抗氧化活性最强,具有较好清除DPPH、ABTS自由基的能力,同时具备一定的还原能力。结论 优选工艺方法简单、稳定可靠,可用于石菖蒲β-细辛醚的提取和抗氧化活性测定,为石菖蒲的进一步开发提供依据。     

Association between the HLA-B*15:02 allele and carbamazepine-induced Stevens-Johnson syndrome/toxic epidermal necrolysis in Han individuals of northeastern China

BackgroundThis study examined the significant association between carbamazepine (CBZ)-induced Stevens–Johnson Syndrome (SJS)/toxic epidermal necrolysis (TEN) and HLA-B*15:02 in epilepsy patients of Han ethnicity living in northeastern China.MethodsCBZ–SJS/TEN patients and CBZ-tolerant control patients were genotyped for HLA-B*15:02 by PCR amplification using sequence-specific primers. Patients then were evaluated for HLA genotypes using PCR with sequence-based typing.ResultsEight of 35 CBZ–SJS/TEN patients carried HLA-B*15:02 (22.9%) versus 2 of 125 in CBZ-tolerant control patients (OR = 18.222, 95% CI = 3.662–90.662, p = 0.000). Our results suggest that HLA-B*15:02 is necessary but is not sufficient to produce SJS/TEN following CBZ treatment among Han individuals from northeastern China. Other HLA alleles, including A*33:03, B*58:01, C*03:02, DQB1*03:03, and DRB1*07:01 may be associated weakly with CBZ–SJS/TENConclusionOur results are not consistent with previous studies reporting a strong association between HLA-B*15:02 and CBZ–SJS/TEN among individuals from southern, southwestern, and central China. Other genes may be more tightly associated with CBZ–SJS/TEN. Screening for HLA-B*15:02 still may be recommended for patients in northeastern China before starting CBZ.     

别嘌呤醇用药调查及其药疹患者HLA-B*5801基因型分析

目的:调查别嘌呤醇用药情况并分析可能引起别嘌呤醇皮疹的HIA-B^*5801基因型。方法:收集我院应用别嘌呤醇患者的人口流行病学特征,分析发生皮疹患者的HIA-B^*5801基因型。结果:监测服用别嘌呤醇患者421名,别嘌呤醇平均剂量145.8mg·d^-1。4例患者分别出现单纯斑丘疹、结节性药疹、慢性荨麻疹和大疱型类天疱疮等药疹,其中仅1例患者检测出HLA-B^*5801。结论:我院患者别嘌呤醇用药剂量小于成人最大量,严重药疹发生率低,HIA-B^*5801与别嘌呤醇致严重皮疹的相关性有待进一步确证。     

Immunomodulatory effect and quantitation of a cytotoxic glycosphingolipid from Murdannia loriformis

NMR signal reassignments for a cytotoxic glycosphingolipid compound, 2, -O-D-glucopyranosyl-2-(2-hydroxy-Z-6-enecosamide)sphingosine, isolated from an ethanolic extract of the herb Murdannia loriformis, have been achieved by use of FAB-MS, and 1D and 2D ¹H and ¹³C NMR. The amount of 2 in the herb juice was quantitatively determined by use of a validated HPLC method (RP-18, MeOH–H₂O, UV detection at 210 nm). The immunomodulatory effect of the herb juice and of 2 was proved by means of in vitro cellular immunological assays. Compound 2 at a concentration of 13 nmol L^–1 stimulated PBMC proliferation and increased the CD 3,4:CD 3,8 ratio in T lymphocytes.     

A study on the genotoxic activities of some new benzoxazoles

The Bacillus subtilis rec assay has been specially developed to detect DNA-damaging potential in chemicals, with the rationale based on the relative difference of survival of a DNA repair combination proficient strains and its deficient strain, which is interpreted as genotoxicity. The genotoxic activities of newly (1–6) and previously (7–18) synthesized various benzoxazoles and benzimidazoles were analyzed via the B. subtilis rec assay. Newly obtained benzoxazole and benzimidazole derivatives (1–6) were synthesized in the presence of polyphosphoric acid (PPA) and 6 N HCl, respectively to detect their DNA-damaging activities. Among the tested compounds, 6-methyl-2-(o-chlorophenyl)benzoxazole (9), 5-amino-2-(p-methylbenzyl)benzoxazole (4), 5-(p-fluorobenzamido)-2-phenylbenzoxazole (13), and 2-(p-methylaminophenyl)benzoxazole (18) showed genotoxic activities having Rec₅₀ values of 1.85, 1.74, 1.60, and 1.50 or S-probit values of 0.534, 0.482, 0.460, and 0.357, respectively. On the other hand, 2-(p-bromobenzyl)-5-methylbenzimidazole (6) and 2-benzyl-5-(p-fluorophenylacetamido)-benzoxazole (15) were exhibited a reverse effect that displayed a bacterial growth in the rec ^- strains while there was no any bacterial growth in rec ⁺ strains at the same concentration.     

光泽汀体内遗传毒性风险评价

目的 评价光泽汀小鼠体内的遗传毒性。方法 C57BL/6J小鼠分为溶剂对照（0.5% CMC-Na）组、茜草素（200 mg·kg^-1，结构对照）组、乙酰基亚硝基脲（ENU，40 mg·kg^-1，阳性对照）组、甲基磺酸乙酯（EMS，200 mg·kg^-1，阳性对照）组和光泽汀低、中、高剂量（100、200、300 mg·kg^-1）组，溶剂、光泽汀和茜草素连续7 d ig给予，给药第1天记为D1，阳性对照ENU和EMS分别连续3 d给予，均每天给药1次。于D7、D56采集约0.5 mL外周血用于血清生化检测；于D14、D28、D42、D56采集外周血开展Pig-a基因突变试验；末次给药后采集肝、肾细胞开展彗星试验，分析每只动物至少100个细胞的尾DNA百分含量；末次给药后制备骨髓细胞样本，计算嗜多染红细胞的微核发生率。解剖后取心、肝、脾、肺以及肾脏进行组织病理学检查。结果 试验期间所有动物一般症状未见明显异常，各组动物体质量未见明显差异，未见与给予受试物有关的组织病理学改变。光泽汀低、中、高剂量组及EMS组肾脏尾DNA百分率均显著高于溶剂对照组（P<0.05、0.001），光泽汀高剂量组及EMS组肝脏尾DNA百分率与溶剂对照组比较显著增加（P<0.05、0.001）。光泽汀与茜草素的小鼠骨髓微核试验、Pig-a基因突变试验均为阴性。结论 100～300 mg·kg^-1光泽汀未见对小鼠整体产生明显毒性。光泽汀可导致小鼠肝、肾细胞DNA损伤，肾细胞DNA损伤程度更为严重。     

Radioligand-Binding Assay Employing P-Glycoprotein-Overexpressing Cells: Testing Drug Affinities to the Secretory Intestinal Multidrug Transporter

Purpose. To develop a rapid and reliable system for affinity determination of conventional as well as newly synthesized compounds to P-gp. Methods. The principles of radioligand-binding assay were adapted to the human intestinal P-gp. Acceptor protein was obtained from the human carcinoma cell line Caco-2, where overexpression of P-gp was induced by growing cells in the presence of the cytostatic drug vinblastine. ³H-Verapamil was chosen as radioligand. Results. The saturability and specificity of ³H-verapamil as the radioligand for the binding to P-gp was demonstrated. From concentration dependence of displacement of the radioligand by various non-labeled ligands for P-gp, affinity constants to P-gp binding sites were calculated. The binding results obtained were in agreement with those published earlier where influx and efflux experiments with cell monolayers had been conducted in order to functionally characterize the P-gp -drug interaction. Conclusions. A radioligand-binding assay on the basis of P-gp overexpressing Caco-2 cells has been developed. The method might be suitable for high-throughput screening of drug interaction with human P-gp. It will allow modeling of the interaction of drugs with the human multi-drug transporter and has also the potential to serve as a high-throughput screening tool to detect compounds prone to P-gp mediated intestinal secretion and potential P-gp related drug/drug interactions in drug discovery and early development.     

Performance of a miniaturized algal bioassay in phytotoxicity screening

A miniaturized and low-cost algal growth-inhibition assay, with Pseudokirchneriella subcapitata, based on the standard ISO 8692 and using 96-well microplates, was tested and optimized in this work, to be used as a useful tool for pollutant phytotoxicity screening. For validation, the performance of the microplate algal growth-inhibition assay was first compared with the standard flask assay for the toxicity testing of five reference toxicants (copper(II) sulfate, zinc sulfate, potassium permanganate, potassium dichromate and 3,5-Dichlorophenol) and six wastewater samples. Statistical evaluation of EC₅₀ results from both methods demonstrated a good agreement between microplate and flask assays either in testing chemicals (r ² = 0.975, p < 0.0017) or environmental samples toxicity (r ² = 0.984, p < 0.0001). In addition, the performance of this algal microplate bioassay was also evaluated in comparison with Lemna test, ISO 20079, for phytotoxicity assessment of 27 wastewater samples from industries and treatment plants. The results showed that the algal test was more sensitive for most of the samples, but a significant agreement between both tests was observed (r ² = 0.644, p < 0.0001). In conclusion, this miniaturized test can be a good tool to include in a battery of tests for phytotoxicity screening of a wide range of chemicals and environmental samples, with the advantage of requiring low sample volumes for the test, allowing large numbers of samples to be tested, and generating low volumes of waste.     

WBSCR22基因调控人恶性肿瘤细胞铁死亡的研究

目的 研究WBSCR22是否参与调控细胞铁死亡。方法 设计合成靶向人WBSCR22基因的siRNA (siWBSCR22),瞬时转染敲低WBSCR22 mRNA;RT-qPCR、Western blotting分别检测WBSCR22 mRNA、蛋白表达水平;铁死亡诱导剂Erastin、RSL3单独或联合抗氧化剂NAC、坏死抑制剂NSA、凋亡抑制剂ZVAD-FMK、自噬抑制剂3-MA作用于人结直肠癌细胞系HCT116和RKO、人肺癌细胞系H460、人肝癌细胞系SMMC7721,CCK8法检测细胞死亡率;丙二醛法、菲罗嗪法分别检测细胞脂质过氧化水平、Fe²⁺浓度。结果 siWBSCR22瞬时转染显著降低WBSCR22 mRNA及蛋白水平;WBSCR22敲低显著促进Erastin、RSL3诱导的HCT116、RKO、H460、SMMC7721细胞铁死亡;加入NAC后显著降低Erastin、RSL3诱导的WBSCR22敲低细胞铁死亡,而加入NSA、ZVAD-FMK、3-MA对Erastin、RSL3诱导的WBSCR22敲低细胞铁死亡无影响;Erastin、RSL3诱导后细...