OBJECTIVE To establish a method for determining the contents of clopidogrel （CLP）， clopidogrel carboxylate （CLP-C）， clopidogrel acyl-β-D-glucuronide （CLP-G） and contents of clopidogrel active metabolite （CAM） in rat plasma， and to investigate their in vivo pharmacokinetic characteristics. METHODS The Shisedo CAPCELL ADME column was used with a mobile phase consisting of water and acetonitrile （both containing 0.1% formic acid） in a gradient elution. The flow rate was 0.4 mL/min， and the column temperature was maintained at 20 ℃. The injection volume was 2 μL. The analysis was performed in positive ion mode using electrospray ionization with multiple reaction monitoring. The ion pairs for quantitative analysis were m/z 322.1→211.9 （for CLP）， m/z 308.1→197.9 （for CLP-C）， m/z 322.1→154.8 （for CLP-G）， m/z 504.1→154.9 [for racemic CAM derivative （CAMD）]. Six rats were administered a single intragastric dose of CLP （10 mg/kg）. Blood samples were collected before medication and at 0.08， 0.33， 0.66， 1， 2， 4， 6， 10， 23 and 35 hours after medication. The established method was used to detect the serum contents of various components in rats. Pharmacokinetic parameters were then calculated using WinNonlin 6.1 software. RESULTS The linear ranges for CLP， CLP-C and CAMD were 0.08-20.00， 205.00-8 000.00， and 0.04-25.00 ng/mL， respectively （r≥0.990）. The relative standard deviations for both intra-day and inter-day precision tests were all less than 15%， and the relative errors for accuracy ranged from －11.68% to 14.40%. The coefficients of variation for the matrix factors were all less than 15%， meeting the requirements for bioanalytical method validation. The results of the pharmacokinetic study revealed that， following a single intagastric administration of CLP in rats， the exposure to the parent CLP in plasma was extremely low. Both the area under the drug concentration-time curve （AUC0-35 h） and the peak concentration of the parent CLP were lower than those of its metabolites. The AUC0-35 h of the active metabolite CAM was approximately 43 times that of CLP， though it had a shorter half-life （2.53 h）. The inactive metabolite CLP-C exhibited the highest exposure level， but it reached its peak concentration the latest and was eliminated slowly. The AUC0-35 h of CLP-G was about four times that of CAM， and its half-life was similar to that of CLP-C. CONCLUSIONS This study successfully established an liquid chromatography-tandem mass spectrometry method for the determination of CLP and its three metabolites， and revealed their pharmacokinetic characteristics in rats. Specifically， the parent drug CLP was rapidly eliminated， while the inactive metabolites CLP-C and CLP-G exhibited long half-lives， and active metabolite CAM displayed a transient exposure pattern.

Endovascular treatment has become the standard treatment method for acute ischemic stroke caused by large vessel occlusion. The application of neuroimaging techniques for appropriate patient selection and prognosis prediction is of great significance for successful endovascular treatment. This article reviews the application progress of fluid-attenuated inversion recovery sequence vascular hyperintensity in patients with acute ischemic stroke underwent endovascular treatment.

[摘 要] 目的：设计和构建表达PD-1 shRNA的靶向CD19 CAR-T细胞并验证其体外肿瘤细胞杀伤能力。方法：设计并构建表达PD-1 shRNA的CD19 CAR分子基因，将其包装成逆转录病毒载体，通过qPCR法检测病毒载体拷贝数。将慢病毒转导人原代T细胞，获得三种CAR-T细胞，分别为RNAU6-CD19 CAR-T、PD-1 shRNA1-CD19 CAR-T、PD-1 shRNA2-CD19 CAR-T细胞。采用qPCR法检测三种CAR-T细胞中PD-1 mRNA的表达水平，流式细胞术检测三种CAR-T细胞中PD-1表达水平，萤光素酶报告基因实验、流式细胞术检测在不同效靶比时CAR-T细胞对CD19阳性靶细胞（人淋巴瘤daudi细胞）的杀伤功能。结果：RNAU6-CD19 CAR、PD-1 shRNA1-CD19 CAR、PD-1 shRNA2-CD19 CAR三种CAR分子成功包装成逆转录病毒载体，病毒载体拷贝数均高于1×107拷贝/mL，转导人原代T细胞获得CAR-T细胞，RNAU6-CD19 CAR-T、PD-1 shRNA1-CD19 CAR-T、PD-1 shRNA2-CD19 CAR-T细胞转导效率分别为43.1%、55.1%、41.7%。与RNAU6-CD19 CAR-T细胞相比，PD-1 shRNA1-CD19 CAR-T、PD-1 shRNA2-CD19 CAR-T细胞中PD-1 mRNA表达水平均显著降低（均P<0.01）、细胞表面PD-1表达水平更低（均P<0.01）、体外对daudi细胞的杀伤率更高（P<0.05或P<0.01）。结论：成功构建表达PD-1 shRNA的靶向CD19 CAR-T细胞，其对CD19阳性靶细胞的杀伤率显著提高，PD-1 mRNA及其翻译产物PD-1的表达减少，CAR-T细胞的耗竭减缓。

Phlegm-dampness syndrome is a common syndrome type of rheumatoid arthritis (RA), which is closely related to spleen dysfunction. Combined with the understanding of modern medicine on the pathogenesis of phlegm dampness and spleen dysfunction, it is believed that the important factors to promote the development of RA include inflammation related to lipid metabolism disorders, abnormal glucose regulation, and intestinal flora imbalance. Improper diet or external factors lead to glucose and lipid metabolism disorders and affect the imbalance of intestinal microbiota, causing damage to the intestinal mucosal barrier. The metabolites produced promote endogenous glucose and lipid metabolism imbalance and inflammatory response, thus forming the biological basis of RA with phlegm dampness syndrome. The method of invigorating spleen, removing dampness and resolving phlegm can correct the disorder of glucose and lipid metabolism in the body. It can also adjust endogenous glucose and lipid metabolism and inhibit RA inflammation by improving the composition of intestinal flora in RA patients, thus playing a therapeutic role.

Post-stroke cognitive impairment (PSCI), one of the important complications of stroke, seriously affects the quality of life of these patients. PSCI is an important cause of disease burden of stroke. In recent years, more and more evidences show that blood biomarkers are of great significance in PSCI diagnosis, and the detection of blood biomarkers is relatively simple and more suitable for clinical application. Therefore, this paper sorts out the values of 5 blood biomarkers, nerve injury marker, metabolic biomarker, inflammatory biomarker, oxidative stress marker and other biomarker, in diagnosing PSCI, to provide references for early diagnosis and intervention of PSCI.

OBJECTIVE To establish a method for simultaneous determination of atorvastatin （ATV） and its active metabolites 2-hydroxy atorvastatin acid （2-HAT）， 4-hydroxy atorvastatin acid （4-HAT） and toxic metabolite atorvastatin lactone （ALT） in rat plasma and apply it for pharmacokinetic study. METHODS LC-MS/MS method was adopted for analysis. The one-step precipitation method was used for processing plasma samples （plasma samples were pretreated by acidification to adjust pH value so as to prevent inversion of configuration）， gradient elution was used to analyze the samples， and the analysis time was 5 min. Electrospray positive ionization was adopted， and positive ion scanning was performed in multi-reaction monitoring. The m/z of quantified ion pairs of ATV and its metabolites such as 2-HAT， 4-HAT and ATL， and internal standard pitavastatin were 559.3→ 440.2， 575.2→440.3， 575.0→440.2， 540.9→448.2 and 422.2→290.0， respectively. After conducting a comprehensive methodological investigation of the analytical method， the concentrations of ATV and its metabolites 2-HAT， 4-HAT，and ATL were determined， and the pharmacokinetic parameters of ATV and its metabolites were calculated using the non- compartment model of WinNonlin 6.1. RESULTS The results of methodological validation showed that endogenous substances in blank plasma did not interfere with the determination of the components to be tested， and the standard curve had a good linear relationship； the lower limits of quantification for ATV， 2-HAT， 4-HAT and ATL were 0.5， 0.5， 0.25 and 0.063 nmol/L， respectively. The precision， accuracy， recovery， matrix effect and stability investigation were all in line with the requirements of biological analysis. Pharmacokinetic analysis showed that after intragastric administration in rats， ATV calcium metabolized rapidly， and was mainly exposed to blood circulation in the form of ATV and 2-HAT， with the lowest concentration of lactone-type metabolites. CONCLUSIONS The established method is precise， rapid and accurate for plasma concentration analysis of ATV and its active/toxic metabolites. The application of the method could help to fully elucidate the pharmacokinetic characteristics of atorvastatin calcium in rats.

In the online teaching of Biochemistry course, a variety of network resource platforms (such as Zhihuishu learning network, teleconference, WeChat, QQ, etc) were used to establish a learning community. The teaching content and teaching plan were carefully designed and implemented, enriching the knowledge system of the learning community. And then blending teaching was performed through the combination of live broadcasting and online interaction. In addition to teaching students the basic knowledge of biochemistry, it is also combined with clinical cases and life examples to interact and discuss with students in various forms, giving full play to the advantages of learning community and improving the quality and effect of online learning.

Compared with routine imaging examination, magnetic resonance vascular wall imaging (MR-VWI) has the advantages of non-invasive and accurate in the diagnosis and differentiation of moyamoya disease and atherosclerotic moyamoya syndrome, and is of great significance in guiding clinical treatment and predicting outcomes. This article reviews the features of MR-VWI in moyamoya disease and atherosclerotic moyamoya syndrome.

Objective:To explore the value of enhancement rate of aneurysm wall by high-resolution magnetic resonance vessel wall imaging (HR-VWI) in assessing the risk of intracranial aneurysm rupture.Methods:Seventy patients with 80 intracranial aneurysms, admitted to our hospital from January 2016 to December 2019, were chosen in our study; morphological parameters (shape, neck width, height, diameter) of the intracranial aneurysms, and signal intensity and enhancement rate of aneurysm wall were collected from HR-VWI. These patients were divided into a high-risk rupture group (PHASES scores>3) and a low-risk rupture group (PHASES scores≤3) according to PHASES scores. Univariate analysis was used to compare the differences of clinical data (age, gender, blood pressure, blood lipids, and smoking history) and aneurysm imaging data between the two groups of patients. Multivariable Logistic regression was used to determine the independent influencing factors for aneurysm rupture, and receiver-operating characteristic (ROC) curve of enhancement rate of aneurysm wall in predicting aneurysm rupture was drew to determine the best diagnostic value.Results:Univariate analysis showed that the percentages of patients with hypertension, multiple aneurysms and irregular aneurysm morphology, aneurysm height, aneurysm diameter and aneurysm wall enhancement rate were significantly different between the high-risk rupture group and low-risk rupture group ( P<0.05). Multivariate Logistic regression analysis showed that aneurysm diameter ( OR=1.647, 95%CI: 1.177-2.306, P=0.004) and aneurysm wall enhancement rate ( OR=5.317, 95%CI: 1.527-18.512, P=0.009) were independent influencing factors for rupture of intracranial aneurysms. The area of ROC curve was 0.735, the optimal cut-off value was 0.583; the sensitivity of predicting rupture of intracranial aneurysms was 72.7% and the specificity was 70.2%. Conclusion:The aneurysm wall enhancement rate is significantly correlated with risk of aneurysm rupture; when the PHASES scores>3 and aneurysm wall enhancement rate>58.3%, clinical intervention should be vigilant.

With the development of imaging technology, high-resolution magnetic resonance vascular wall imaging technology has been gradually applied to the risk assessment of intracranial aneurysms rupture. It has provided more comprehensive information for clinicians to assess the stability of intracranial aneurysms. This article summarizes the indicators used for assessing the risk of intracranial aneurysms rupture in recent years with high-resolution magnetic resonance vascular wall imaging.