Objective: To construct, reproduce and identify the global microRNA-34a (miR-34a) gene knockout DBA1/J mice. Methods: According to the principle of CRISPR/cas9, miR-34a-/+DBA1/J mice were constructed as F0 generation mice; they were hybridized with DBA1/J wild type mice (WT), and miR-34a-/+F1 mice with genotype were screened out by PCR identification; miR-34a-/-F2 generation mice were screened out, and their growth characteristics and reproductive ability were analyzed; the proportion of T and B lymphocytes in immune organs and peripheral blood of mice was detected by flow cytometry. Results: Two F0 mice, four F1 mice and five F2 mice were obtained. The proportions of CD4+T, CD8+T lymphocyte subpopulations had no significant changes(P>0.05), but increased in the propotion of B lymphocytes in immune organs and peripheral blood of miR-34a-/- knockout mice(P<0.05). There were no significant differences in weights and reproductive capacities between the miR-34a knockout mice and WT DBA1/J mice(P>0.05). Conclusion: The global miR-34a knockout mice were constructed successfully via CRISPR/Cas9 technique.

［Key words］CRISPR/Cas9; miR-34a; gene knockout; reproduction; identification

Objective: To investigate the effect and mechanism of mammalian target of rapamycin complex 1（mTORC1）on the stability of microtubules in Hela cells. Methods: Hela cells was pretreated with rapamycin（50 nmol/L）for 24 hours, then treated with Nocodazole（5 μmol）for 30, 60 or 120 minutes, the stability of microtubule protein was detected by immunofluorescence. Hela cells was treated with rapamycin(50 nmol/L) for 24 hours, the expression of microtubule depolymerizing protein stathmin, KIF2A, microtubule polymerizing protein CLIP170, microtubule cutting protein Katanin and Spastin were detected by Western blotting. The stability of microtubule protein was detected by immunofluorescence after autophagy related gene 5 (ATG5) was inhibited by ATG5-shRNA. Hela cells was pretreated with rapamycin(50 nmol/L) for 24 hours, the expression of Ras homologous gene family member A (RhoA) was detected by Western blotting; the stability of microtubules was examined by immunofluorescence after transfection with GFP RhoA-Q63l or GFP RhoA-N19, P190RhoGAP-siRNA plasmids. Results: Inhibiting the activity of mTORC1 with rapamycin significantly enhanced the stability of microtubules through resisting depolymerization while the stathmin, KIF2A, CLIP170, Katanin and Spastin expression were not changed.The stability of microtubules mediated by mTORC1 was independent on the autophagy. After the activity of mTORC1 was inhibited by rapamycin, the activation of RhoA GTP was down-regulated and the stability of microtubule was attenuated along with decreased activation of P190 RhoGAP. Conclusion: mTORC1 regulated the stability of microtubules through  RhoA pathway in Hela cells.

［Key words］mTORC1; microtubules; RhoA; autophagy； Hela cells

Objective: We aim to develop a rapid and efficient method of myocardial ischemia reperfusion (MIR) without the requirement of ventilation and chest opening in mice. Methods: Forty male Kunming mice (6-8 weeks old and weighing 18-25 g) were randomly divided into sham operation group, ischemia 15 min+reperfusion 24 h group, ischemia 30 min + reperfusion 24 h group and ischemia 45 min+reperfusion 24 h group(10 mice in each group). Mice were anesthetized with 2% isoflurane gas, the heart were fully exposed through a small incision of the forth intercostal space on the left side. After rapidly locating and ligating the left anterior descending coronary artery (LAD) for 15, 30 and 45 minutes, the myocardial ischemia-reperfusion model was established by loosening the ligature for 24 hours. At the same time, electrocardiogram was performed before, 5 min and 24 h after surgery. The ligation site was re-ligated 24 h after surgery, and Evan′s Blue-TTC double staining was performed to detect myocardial ischemic area and infarct area. Results: By 24 hours after surgery, the survival rate of mice was 90%, and the success rate of MIR modeling was 86.7%. Electrocardiogram showed that the ST segment and the T wave was elevated after LAD ligation, and the QRS wave was broadened, followed by the gradually decrease of the ST segment. After ligation of LAD, the ventricular wall below the ligature line showed grayish white, and the pale infarct area was observed 24 h after operation. The frequency and amplitude of heart beat were significantly weaker than before ligation. Cardiac sections were stained with Evan′s Blued-TTC to present a clearly defined ischemic, infarcted, and normal myocardial tissue. In ischemia 45 min+reperfusion 24 h group, the ratio of the ischemia area + infarction area to the total myocardial area of the left ventricle was（63.8±6.36）% and the ratio of the area of the infarct area to the area of the ischemic area + infarct area was （46.7±5.77）%. Conclusion: This new rapid and convenient method of I/R in mice may be a more efficient model of MIR. It reduces the time required for the use of the ventilator, and the success rate of modeling is more than 85%.

［Key words］mouse； coronary artery ligation； ischemia reperfusion； animal model

Objective: To establish a high performance liquid chromatography (HPLC) method for simultaneous determination of the contents of gallic acid, propyl gallate and naringenin in Paeoniae Rubrathe Radix. Methods: The analysis of three compounds was performed on a 30 ℃ thermostatic ODS-C18 HPLC column (250 mm×4.6 mm, 5μm), with the mobile phase comprising of methanol-0.1% acetic acid flowing at 1.0 mL/min in a gradient elution manner. And the detection wavelength was set at 270 nm. Results: For three compounds, gallic acid, propyl gallate, and naringenin, good linear relationship could be obtained within the concentration ranges of 5-250 μg/mL (R²>0.999), respectively. Results also indicated the good precision of the analytical method and good accuracy within the optimal recovery range. It was shown that 1 g of the raw material of Paeoniae Rubrathe Radix contained 1.867 mg of gallic acid, 0.561 mg of propyl gallate, and 0.093 mg of naringenin, respectively. Conclusion: This method was able to accurately determine the contents of three active compounds in Paeoniae Rubrathe Radix, and could be used for the quality control of Paeoniae Rubrathe Radix.

［Key words］Paeoniae Rubrathe Radix； gallic acid； propyl gallate； naringenin； high performance liquid chromatography (HPLC)

Objective: To investigate the effect of COTE1 gene on hepatocarcinoma cell line PLC/PRF/5 apoptosis induced by doxorubicin(DOX). Methods: The expression level of COTE1 mRNA and protein were detected by qPCR or Western blotting after transfected with COTE1 shRNA lentivirus.CCK-8 assay was used to detect the inhibition rate of DOX on the proliferation of HCC cells.The apoptosis rate of HCC cells treated with DOX was detected by flow cytometry. Results: qPCR and Western Blotting results showed the expression of COTE1 was down-regulated after transfection with lentivirus and CCK-8 results showed the inhibitory effect of DOX was increased. Flow cytometry results showed that DOX could promote the apoptosis rate, while inhibition of the expression of COTE1 can enhance the apoptosis rate.Conclusion: Inhibition of COTE1 could enhance the sensitivity of PLC/PRF/5 cells to doxorubicin by enhancing apoptosis.

［Key words］COTE1; hepatoacrcinoma cell; doxorubicin; apoptosis

Objective: To investigate the cytokines and master regulator transcription factors of CD4⁺ subsets in pancreatic cancer patients. Methods: Specimens of peripheral blood were collected from 27 pancreatic cancer patients and 30 healthy controls. The serum levels of IFN-γ, IL-2, IL-4, IL-17, TGF-β and IL-10 were determined by ELISA. The mRNA expression level of T-bet, Gata-3, RORγt and Foxp3 were detected by real time-PCR. Results: Compared with healthy controls, the level of IFN-γ, IL-2 and T-bet mRNA were markedly decreased in pancreatic cancer patients(P<0.05), but the level of IL-4, IL-17, TGF-β, IL-10 and mRNA expression level of Gata-3, RORγt and Foxp3 were significantly increased in pancreatic cancer patients(all P<0.05).Conclusion: Pancreatic cancer patients showed lower levels of Th1 cytokines and master regulator transcription factors, higher levels of Th2, Th17 and Tregs cytokines and master regulator transcription factors; Th2, Th17 and Treg cells may synergistically promote the development of pancreatic cancer disease.

［Key words］pancreatic cancer; Th1； Th2； Th17; Tregs

Objective: To evaluate the effect of obesity on Lp-PLA2 in peripheral blood of adolescents and analyze the effect of Lp-PLA2 inhibition on the secretion of oxidized low density lipoprotein (oxLDL) by adipocytes in vitro. Methods: Peripheral blood was collected from 36 obese adolescents (obese group) and 36 healthy weight adolescents (control group), and the concentration of serum Lp-PLA2 was detected by chemiluminescence method. The adipocytes were cultured in vitro, and the culture supernatant was collected after stimulation with 67 pmol/L lipoprotein cholesterol (LDL-C) and 20 μmol/L Lp-PLA2 inhibitor for 48 and 72 hours. The concentration of Lp-PLA2 and oxLDL in the supernatant were detected by chemiluminescence and enzyme-linked immunosorbent assay (ELISA), respectively. The expression of Lp-PLA2 mRNA in adipocytes was measured by RT-PCR. Results: Compared with the control group\[（123.5±27.3）ng/mL], the level of Lp-PLA2 in peripheral blood of obese adolescents[（353.4±30.4）ng/mL]was significantly increased (t=33.76，P<0.05). The level of peripheral blood Lp-PLA2 in obese adolescents was positively correlated with body mass index (r=0.414, P=0.012) and waist circumference (r=0.460, P=0.004). Univariate regression analysis showed that total cholesterol, LDL-C, apolipoprotein A1 and apolipoprotein B were associated with elevated Lp-PLA2. Compared to the control group, the concentration of Lp-PLA2 and the level of Lp-PLA2 mRNA in adipocytes were significantly increased after stimulation with LDL-C for 48 hours（P<0.01）. Furthermore, the production of oxLDL by adipocytes was significantly decreased after treating with Lp-PLA2 inhibitors（P<0.01）.Conclusion: Lp-PLA2 may be an important factor in the increase of cardiovascular risk in adulthood of obese adolescents.

［Key words］adolescen; Lp-PLA2; cardiovascular risk; obesity

Objective: To observe the uptake and metabolism of 18F-Fluorodeoxyglucose (¹⁸F-FDG) in various brain regions of the emotional regulationloop in generalized anxiety disorder (GAD) based on the PET,and the mechanism of brain dysfunction in GAD was studied. Methods: Dynamic PET-CT scanning was performed on 20 GAD patients and 20 healthy subjects. 18F-FDG whole-brain uptake and metabolism data were collected. SUV_max, dynamic uptake and metabolism changes of each brain structure at 11 time points in 150 mins were measured based on image fusion and semi-quantitative analysis. Results: Compared to the control group, there were less and slower uptake of ¹⁸F-FDG, faster metabolism in the bilateral prefrontal cortex in GAD group（P<0.05）.There were less, slower uptake of ¹⁸F-FDG and slower metabolism in the left striatum and left hippocampus in GAD group（P<0.05）. There were more, faster uptake of 18F-FDG and faster metabolism in the left thalamus in GAD group（P<0.05）.There were more and faster uptake of ¹⁸F-FDG in the right thalamus（P<0.05）, but no significant metabolism change in GAD group. The 3 indicators in right striatum and right hippocampus had no significant change in GAD group.Conclusion: There are ¹⁸F-FDG uptake and metabolism disorder of multiple brain structures of this loop in GAD, and its abnormal uptake peak, rate of uptake and rate of metabolism may be involved in the GAD.

［Key words］generalized anxiety disorder; emotional regulationloop; PET-CT; maximum standardized uptake value

Objective: To summarize the clinical experience of diagnosis and treatment of acute myocardial infarction caused by coronary artery spasm. Methods: A retrospective analysis of 12 patients with acute myocardial infarction who underwent coronary artery spasm from March 2014 to June 2019 was performed. Results: Through coronary angiography and intravascular ultrasound (IVUS) or optical coherence tomography(OCT), the diagnosis of different coronary artery spasm was confirmed. Eight patients (66.7%) of acute inferior myocardial infarction caused by right coronary artery spasm, 2 patients (16.7%) of acute anterior myocardial infarction caused by the left anterior descending coronary artery spasm, 2 patients (16.6%) of acute anterior and inferior myocardial infarction caused by three coronary arteryies spasm. The coronary artery spasm was disappeared after intracoronary administration of nitroglycerin. After treatment with nitrates, calcium antagonists and statins, the clinical symptoms of chest tightness and chest pain were relieved. Conclusion: Coronary angiography, IVUS and OCT can be used to diagnose acute myocardial infarction caused by coronary artery spasm and guide drug treatment effectively. The clinical effect of nitrate drugs and calcium antagonist drugs is significant.

［Key words］coronary artery spasm； coronary angiography； acute myocardial infarction

Objective: To investigate the expression of Beta-lactamase-like protein (LACTB) in endometrial cancer tissues and its clinical significance. Methods: The protein expression of LACTB, etrogen receptor and progesterone receptor in 89 cases of endometrial cancer and 21 cases of normal endometrial tissues were assessed by immunohistochemistry. Meanwhile, the expression of estrogen receptor and progesterone receptor in endometrial cancer tissues was assessed by immunohistochemistry．The known Kaplan-Meier Plotter database was used to analyze the relationship between the expression changes of LACTB mRNA transcriptome sequencing and the prognosis of endometrial cancer in endometrial cancer tissues. Results: The position rate of LACTB protein expression in endometrial carcinoma tissues was significantly lower than that in normal endometrial tissues(P<0.05). The expression of LACTB protein in endometrial carcinoma tissues was not associated with age grouping(P>0.05). Compared with clinical stage Ⅰ-Ⅱ, the position rate of LACTB protein expression was significantly decreased in clinical stage Ⅲ-Ⅳ(P<0.05). The positive expression rate of LACTB protein was lower in type Ⅰ endometrial cancer than in type Ⅱ endometrial cancer, while the difference was not statistically significant. The position rate of LACTB protein expression was correlated with estrogen receptor and progesterone receptor(P<0.05). The overall survival and progression-free survival of the LACTB positive expression group were higher than the negative expression group, while there was no stastical difference. Conclusion: Low expression of LACTB in endometrial cancer tissues may be an important molecule involved in the progression of endometrial cancer, which is associated with etrogen receptor and progesterone receptor expression.

［Key words］endometrial cancer; Beta-lactamase-like protein(LACTB); etrogen receptor; progesterone receptor; immunohistochemistry; prognostic analysis

Objective: To explore pregnancy management through risk factors analysis of adverse pregnancy outcomes. Methods: A cohort study was conducted on 872 patients excluded prepregnancy complications from August 2016 to May 2017. The following data on pregnancy and adverse pregnancy outcomes were collected, i.e. pre-pregnancy related information, pregnancy examination data, gestational week, newborn birth weight and common complications during pregnancy. The risk factors of premature birth, postpartum hemorrhage, macrosomia, low birth weight and cesarean section were analyzed by multivariate regression analysis, and the management plan was proposed according to the risk factors. Results: The risk factors of gestational hypertension were advanced age, high BMI before pregnancy, excessive weight gain and increased triglyceride level in late stage. Risk factors for gestational diabetes were advanced age, high pre-pregnancy BMI and insufficient weight gain. The main risk factors of premature birth were gestational hypertension and premature rupture of membranes. The main risk factors of postpartum hemorrhage are high pre-pregnancy weight and large fetal weight. The main risks of giving birth to giant baby at full term were excessive weight gain during pregnancy, high BMI before pregnancy and prolonged gestational period.Conclusion: The management of peri-pregnancy was suggested as that pregnancy should be conducted at an appropriate age, pre-pregnancy weight should be controlled, microelements should be added appropriately, weight growth rate should be controlled in an appropriate range during pregnancy, and reproductive tract infection should be actively screened and controlled.

［Key words］adverse pregnancy outcomes; risk factors; multivariate analysis

Objective: To detect the changes of serum uric acid in patients with anterior and post circulation acute ischemic stroke (AIS), and to explore its prognosis significance. Methods: The clinical data of 141 patients with AIS were analyzed retrospectively. They were divided into the anterior circulation group and the posterior circulation group according to the injury site. The levels of serum uric acid, Barthel index (BI), and neurological deficit (NIS) were measured, and the correlation between serum uric acid level and BI index or NIS score was analyzed by Pearson correlation analysis. The patients were divided into good prognosis group and bad prognosis group according to their prognosis. Risk factors for poor prognosis in patients with anterior and posterior circulation AIS were determined by multivariate logistic regression analysis. Results: According to Pearson correlation analysis, there was a significant negative correlation between serum uric acid level and BI index in the anterior and posterior circulation groups (r=-0.832, -0.801, P=0.002, 0.005), while there was a significant positive correlation between serum uric acid level and NIS score in the anterior and posterior circulation groups (r=0.874, 0.885, P=0.000, 0.000). There were no significant differences in the proportion of sex, smoking history, past transient ischemic attack history, hypertension, coronary heart disease, hyperlipidemia, atrial fibrillation, diffuse intravascular coagulation branch number and the proportion of patients receiving cranial valve decompression between the good and bad prognosis groups (P>0.05), while the proportion of age over or equal to 60 years old, duration from onset to thrombolysis more than 4.5 hours, admission hyperuricemia, BI index below 60 points, NIS score higher than 12 points,were higher than those with good prognosis (P<0.05). Logistic regression analysis showed that age over or equal to 60 years old, duration from onset to thrombolysis more than 4.5 hours, admission hyperuricemia, BI index below 60 points, NIS score higher than 12 points, were independent risk factors for poor prognosis in patients with anterior and posterior circulation AIS (P<0.05). Conclusion: The levels of serum uric acid in patients with anterior and post circulation AIS are closely related to the patient′s self-care ability and neurological deficit, and it can be used as reference indexes for prognosis.

［Key words］anterior and posterior circulation; acute ischemic stroke; blood uric acid; Barthel index; neurological deficit score

Objective: To study the relationship between the magnitude platelet decline and the prognosis in patients with early septic shock. Methods: Data of 327 patients with septic shock were collected. The percentage of platelet decline that was less than 30% or more than reference value within 48 hours was considered as control group, and that was more than 30% was enrolled in the study group. In addition, by comparing the lactate level, SOFA score, infection sites, and platelet decline ratio in the two groups, we determined to analyze the factors affecting the cumulative survival rate. Results: The 28-day cumulative survival rate was 75.5% (control group) and 9.8% (study group), respectively. Besides, the results in study group was significantly lower than that in control group (χ²=170.1, P<0.01). Both SOFA score within 24 hours and decline rate of platelet (more than 30%) were correlated with patient death by Binary logistic regression (P<0.01). Moreover, compared with the platelet count (less than 100×10⁹/L), the ROC curve also suggested that decline rate of platelet (more than 30%) was a more specific predictive indicator. Conclusion: Patients with decline rate of platelet (more than 30%) in the early stage of septic shock may have worse prognosis.

［Key words］sepsis； septic shock； platelet decline ratio； SOFA； prognosis

Objective: To compare the effects of ultrasound-guided thoracic paravertebral nerve block(TPVB) and serratus plane block(SPB) on thoracoscopic surgery under general anesthesia with spontaneous breathing. Methods: Sixty patients aged 18~65 years, with BMI between 18~24 kg/m2 and ASA physical status Ior II, undergoing thoracoscopic surgery were selected. They were divided randomly into two groups: TPVB group and SPB group; both groups underwent ultrasound guided TPVB or SPB with 0.375% ropivacaine 20 mL before induction of anesthesia; the laryngeal mask was placed after intravenous anesthesia. Blood pressure, heart rate, arterial blood oxygen saturation (SaO2), end-tidal carbon dioxide partial pressure (PetCO₂) were recorded before induction of anesthesia (T0), when spontaneous breathing was recoveried after anesthesia induction(T1), 30(T2) and 60(T3) minutes after onset of surgery, at the end of surgery(T4), and after recovery(T5). Visual analog scale(VAS) scores at rest and cough were recorded at different time-points: 2,4,6,12,24 and 48 h after surgery. The number of effective compressions for patient -controlled analgesia and the cumulative dosage of opioids drugs, duration of block and complications were recorded. Results: There were no significant differences in blood pressure, heart rate, SaO₂ and PetCO₂ among T0, T1, T2, T3, T4 and T5 between TPVB group and SPB group. The time of the sensory block was significantly longer in the SPB group than that of the TPVB group(P<0.05). The VAS at cough was lower than the TPVB group at 6 h, 12 h after surgery(P<0.05). The number of effective compressions for patient -controlled analgesia and the cumulative dosage of opioids drugs was lower in the SPB group than that of the TPVB group(P<0.05). Conclusion: Ultrasound-guided TPVB and SPB could be safe and effective for the thoracoscopic surgery under general anesthesia with spontaneous breathing. However, SPB could significantly prolong postoperative analgesia, reduce the total amount of opioids administration.

［Key words］ultrasound; thoracic paravertebral nerve block; serratus plane block; thoracoscopic surgery

Objective: To explore the related factors of early neurological deterioration (END) in patients with acute isolated pontine infarction(AIPI). Methods: A retrospective analysis of the patients with AIPI was conducted, these patients were treated in the Department of Neurology, Affiliated Hospital of Jiangsu University from January 2016 to January 2019. According to the occurrence of END, the patients were divided into END group and non-END group. The data of patients were collected and the independent risk factors of END in AIPI patients were analyzed by multivariate Logistic regression. Results: There were significant differences in NIHSS score, fasting blood sugar, basilar artery perforator disease and small artery disease between the two groups at admission (all P＜0.05). Basilar artery perforator disease was an independent risk factor for END (OR=5.614，95%CI 1.165~27.055).Conclusion: Basilar artery perforator disease may be attributed to END in patients with AIPI.

［Key words］pontine; cerebral infarction; deterioration of disease; risk factors