Objective:To establish an UHPLC-MS/MS method to determine the contents of baicalin,scutellarin,wogonin,caffeic acid,ephedrine,belamcandin,irisflorentin and baicalein in Shegan mixtures. Methods:The chromatographic separation was performed on a ZORBAX SB-C18 column(2. 1 mm × 50 mm,1. 8 μm)with the mobile phase consisting of acetonitrile and water(0. 1% formic acid),the flow rate was 0.3 ml·min-1,and the column temperature was 35℃. Electrospray ionization source(ESI)was used with multiple reaction monitoring( MRM)combined with positive and negative scanning switch. üegative ion mode detection was used for caf-feic acid,belamcandin and scutellarin ,while positive ion mode detection was used for baicalin,wogonin,irisflorentin,baicalein and e-phedrine. Results:The quantification limit of baicalin,scutellarin,wogonin,caffeic acid,ephedrine,belamcandin,irisflorentin and ba-icalein was 1. 44 × 10-4 ,4. 20 × 10-3 ,2. 95 × 10-4 ,7. 80,4. 90 × 10-3 ,4. 6 × 10-2 ,3. 18 × 10-4 ng·ml-1 and 4. 85 ng·ml-1 . The detection limit was 4.32 ×10-5,1.3 ×10-3,8.84 ×10-5,0.77,2.90 ×10-4,3.33 ×10-4,9.5 ×10-5 ng·ml-1 and 1.46 ng· ml-1 ,respectively. The correlation coefficients( R2 )were all higher than 0. 992 3 within the linear ranges. Both intra- and inter-day RSD were less than 5%. The average recoveries of the eight components were within the range of 80%-120%. Conclusion:The method is simple,rapid,sensitive and accurate. It can be used to determine the contents of baicalin,scutellarin,wogonin,caffeic acid,ephed-rine,belamcandin,irisflorentin and baicalein in Shegan mixtures for the quality control.

Objective:To investigate the expression of microRNA-324-5p (miR-324-5p) and transcription factor forkhead box C1 (FOXC1) in glioma and their relationship with the prognosis of patients.Methods:From March 2012 to March 2015, a total of 72 cases of glioma tissues were collected from glioma patients who were admitted to Chongqing Hygeia Tumor Hospital and the People's Hospital of Nanchuan in Chongqing, and 28 cases of normal human brain tissues resected in craniocerebral surgery were also collected. The expressions of miR-324-5p and FOXC1 mRNA were detected by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR), and the expression of FOXC1 protein was detected by immunohistochemistry. Pearson method was used to analyze the correlation between the expressions of miR-324-5p and FOXC1 in glioma tissues; Kaplan-Meier method was used to analyze the survival of patients with glioma; Cox regression analysis was used to analyze the risk factors affecting the prognosis of patients with glioma.Results:FOXC1 protein was mainly located in the cytoplasm of glioma, and its positive expression rate in glioma tissues was 81.94% (59/72), which was significantly higher than that in normal brain tissues [17.86% (5/28)], and the difference was statistically significant ( χ2 = 35.938, P<0.01). Compared with normal brain tissues, the expression of miR-324-5p was down-regulated in glioma tissues (0.62±0.19 vs. 0.98±0.02, t = 9.974, P < 0.05), and the expression of FOXC1 mRNA was up-regulated (1.41±0.29 vs. 0.99±0.02, t = 7.633, P < 0.05). The expressions of miR-324-5p and FOXC1 protein were correlated with the number of primary lesions, differentiation degree, TNM stage and lymph node metastasis of glioma (all P<0.05). Pearson analysis showed that the expressions of miR-324-5p and FOXC1 mRNA were negatively correlated ( r = -0.550, P<0.01). The 5-year overall survival rate of patients in miR-324-5p high-expression group was significantly higher than that of patients in miR-324-5p low-expression group (45.71% vs. 24.33%, χ2 = 6.531, P = 0.011), and the 5-year overall survival rate of patients in FOXC1 protein high-expression group was significantly lower than that of patients in FOXC1 protein low-expression group (30.41% vs. 42.34%, χ2 = 3.631, P = 0.047). Multivariate Cox regression analysis showed that low differentiation, TNM stage Ⅲ-Ⅳ, lymph node metastasis, low expression of miR-324-5p and high expression of FOXC1 protein were independent risk factors for prognosis of glioma patients (all P < 0.05). Conclusions:The expression of miR-324-5p is low and the expression of FOXC1 is high in glioma. They may be involved in the regulation of tumor differentiation and metastasis, and related to the poor prognosis of patients. They may be potential therapeutic targets for glioma.

Objectiye To study the levels of cartilage oligomeric matrixprotein (COMP) and matrix metalloproteinase-3 (MMP-3) in the serum fluid of osteoarthritic rabbit models and their relationships with the severity of pathological changes, so as to investigate their correlation with osteoarthritis(OA). Methods The osteoarthritic animal models were get from immobilizing the right knees of 18 rabbits in full extension using plaster cast. Knee joint pathological changes of 2,6 weeks were examined for pathological severity of OA; ELISA sandwich method was used to measure the levels of COMP and MMP-3 in serum before and after modeling( at 2, 6 weeks respectively); X ray of model keens was also obtained in different period.Correlation analysis was performed to demonstrate the relationship between the levels of COMP, MMP-3 in the serum and the pathological severity of OA. Results ( 1 ) Morphological observations: immobilizing the right knees of rabbits in full extension using plaster cast was a reliable methed for osteoarthritic animal models and the typical histopathologic character was seen; the severity of osteoarthritisgradually increased with time extended. (2) The levels of COMP[(3.64 ±0. 18)μg/L], MMP-3 [(1.99 ±0. 81 ) μg/L]in the serum of 2 weeks osteoarthritic animal models were higher than those before immobilizing with plaster cast [COMP(3.35 ±0. 20) μg/L,MMP-3( 1.61 ±0. 71 ) μg/L]. The levels of COMP[(3.96 ±0. 44) μg/L],MMP-3[(3.44 ±0. 91) μg/L] of 6 weeks were much higher,with a significant difference(P ＜0.05). The levels of COMP, MMP-3 in serum had a linear correlation with the pathological severity of OA (r ＞0. 710,and P ＜ 0. 05 ). Conclusion The levels of COMP and MMP-3 in serum can help to predict and evaluate the progression of OA.

Objective To study the diagnostic value of cartilage oligomeric matrix protein for early cartilage destruction in osteoarthritis and assess its value in the prediction of the disease progression.Methods The osteoarthritis animal models were developed by immobilizing the right knees of 18 rabbits in full extension position using plaster East.Knee joint pathological changes at week 2 and 6 were examined for pathological severity evaluation of osteoarthritis.ELISA sandwich method was used to measure the levels of cartilage oligomeric matrix protein(COMP) in serum before and after modeling(at week 2 and 6 respectively) and immunohistolgy method was used to examine the levels of COMP in knee articular cartilage of osteoarthritis animal models.Correlation analysis was performed to demonstrate the relationship between the levels of COMP in the serum and the pathological severity of osteoarthritis.Pearson's test and t-test were used for correlation analysis.Results ①) Osteoarthritis animal models could be successfully developed by immobilizing the right knees of rabbits in full extension position using plaster east for 2 weeks.Early histopathological changes in the articular cartilage could be observed,At week 6,the typical histopathological characteristics could be seen.②With the extension of modeling time,serum COMP levels persistently increased.The serum COMP levels before modeling,at modeling week 2,week 6 were (3.35±0.20),(3.64±0.18),(3.96±0.44) μg/L respectively,the difference was significant (P＜0.05).③ The level of COMP in the articular cartilage of non-osteoarthritis animal models,models at week 2,week 6 were (2.7±1.8 )％ ,(5.7±0.7)％,(7.6±0.7)％ respectively (P＜0.05 for all).④ The level of COMP in the serum was linearily correlated with the pathological severity of osteoarthritis(r＞0.770 for all,and P＜0.05 for all).Conclusion Levels of COMP in the serum can help to make early diagnosis of osteoarthritis,and elevated COMP level can predict the progression of osteoarthritis.

According to the specific construction of the pSUPER. retro. puro vector, RNA interfering with the hypoxia-inducible factor 1alpha (HIF-1alpha) plasmid was constructed in this research programme. The efficiency was (87.15 +/- 2.36)% and green fluorescent protein was observed after 36 hours when the constructed plasmid co-transformed into the prostatic carcinoma cell line PC-3; compared with the control groups, this constructed plasmid could reduce the expression of total RNA and protein in PC-3 cells significantly after 48 hours. The cells were checked out by the plasmid resistance against the puromycin biotin, the clones of which were selected and enlarged for two weeks, then the cell strain of pSUPER-siHIF-1alpha/PC-3 was collected. The HIF-1alpha protein expression of the pSUPER-siHIF-1alpha/ PC-3 cells of also decreased significantly. The results showed that the RNA interference could be used in the construction of expression vector of constructed siRNA inhibiting the expression of HIF-1alpha with PC-3 cells, which is the basis of researching the pathology, multiplication, and metastasis of HIF-1alpha in the prostatic carcinoma and other cancers.
Base Sequence ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Male ; Molecular Sequence Data ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; RNA Interference ; RNA, Messenger ; genetics ; metabolism ; Transfection

The progress of research on the the anterior cruciate ligament (ACL) wound healing demonstrates that the synovial tissue in the knee joint plays a very important role in the healing process of injured ACL. Therefore, the molecular response mechanisms of lysyl oxidase (LOX) and matrix metalloproteina (MMP) in normal/injured ACL fibroblast cells could be considered to perform the major analysis function of injured ACL healing mechanism. The mRNA expressions of LOXs and MMPs and the activity expressions of MMP-2 in ACL fibroblasts co-cultured with synovial cells were analyzed by quantitative real-time PCR and zymography. The results showed that co-culture could regulate the mRNA expressions of LOXs and MMPs in the ACL fibroblasts cells. These results suggest that the differential expressions of LOXs and MMP-1, 2, 3 in co-cultured ACL indicate that interaction crosstalk do exist between ACL cells and synovial cells and provide a theoretical basis for subsequent exploration of the mechanisms and treatment of ACL injury and repair.
Anterior Cruciate Ligament ; cytology ; Anterior Cruciate Ligament Injuries ; Coculture Techniques ; Fibroblasts ; cytology ; metabolism ; Humans ; Knee Injuries ; physiopathology ; Knee Joint ; cytology ; Matrix Metalloproteinases ; genetics ; metabolism ; Protein-Lysine 6-Oxidase ; genetics ; metabolism ; Synovial Membrane ; cytology ; Wound Healing ; physiology

AIM:To investigate the effect of CYP2C19 gene polymorphism combined with plate-let function test guiding antiplatelet drug selection on gastrointestinal bleeding in elderly patients with acute coronary syndrome(ACS)after percutaneous coronary intervention(PCI).METHODS:A total of 216 elderly patients with ACS who were admitted to our hospital from April 2021 to December 2022 and successfully completed PCI were selected and divided into study group and control group accord-ing to the random number table method,with 108 cases in each group.All patients received 4 days the same dose aspirin combined with clopidogrel antiplatelet therapy before surgery.After surgery,CYP2C19 genotype detection was performed on the study group to obtain genotype grouping to dis-tinguish different metabolites.Thromboelastogram(TEG)detection was performed on the patients with intermediate metabolites.According to the re-sults,patients with platelet inhibition rate less than 50％(clopidogrel resistance type)were selected and their medication was changed from aspirin and clopidogrel to aspirin and ticagrelor.The control group was treated with aspirin and clopidogrel.Se-rum levels of cardiac troponin(cTnl),creatine ki-nase isoenzyme(CK-MB)and hypersensitive C-reac-tive protein(hs-CRP)were observed 5 weeks after PCI,and major adverse cardiovascular events(MACE)were observed 1 and 6 months after PCI.The expression of CD62P and fibrinogen receptor 1(PAC-1)in platelets of the two groups were com-pared before and after 7 and 14 days of treatment.The platelet aggregation rate of the two groups was compared.Gastrointestinal bleeding events and cardiac emergencies were recorded after treat-ment.RESULTS:Genetic tests showed that there were 21 cases(19.44％)of ultrafast metabolites and 25 cases(23.15％)of fast metabolites,42 cases of abnormal metabolites including intermediate metabolites(genotypes accounting for 10.19％,13.89％,9.26％and 5.56％,respectively)and 20 cas-es of slow metabolites(genotypes accounting for 11,respectively).8.33％,7.41％and 2.78％).5 weeks after surgery,the serum levels of cTnl,hs-CRP and CK-MB in the study group were lower than those in the control group(P＜0.05,P＜0.01).One month after operation,the total incidence of MACE between the observation group and the control group was similar(P＞0.05).Six months after opera-tion,the total incidence of MACE in the observa-tion group was significantly lower than that in the control group(P＜0.05).The expression levels of CD62P and PAC-1 in the study group were lower than those in the control group on day 7 and day 14 after treatment(P＜0.01).The maximum platelet aggregation rate in the study group was lower than that in the control group(P＜0.01).The occurrence of cardiac emergencies between the two groups was similar(P＞0.05).The number of postoperative gastrointestinal bleeding in the study group was lower than that in the control group(x2=9.479,P＜0.01).CONCLUSION:Aspirin combined with ticagre-lor antiplatelet therapy can reduce platelet activity,aggregation rate and gastrointestinal bleeding events in patients with low response to chlorpyrro-lore who are co-screened by CYP2C19 genotype and platelet function test.

Objective:To investigate the diagnosis and clinical characteristics of five elderly patients with Chlamydia psittaci-caused severe pneumonia.Methods:Through retrospective analysis, diagnosis and treatment process and clinical characteristics of five elderly inpatients with severe pneumonia caused by Chlamydia psittaci were summarized in the Department of Critical Care Medicine, Quanzhou First Hospital East Street Branch Area, affiliated to Fujian Medical University between January to June 2021.Results:Five patients with severe pneumonia caused by Chlamydia psittaci were aged from 64 to 74 years, with various underlying diseases such as coronary heart disease, chronic heart failure, chronic obstructive pulmonary disease, etc.All patients had an established history of poultry exposure.These cases had high fever, cough, spitting, and dyspnea as the main clinical manifestations.Some of them also had systemic symptoms or weakness of the limbs as the prodromal symptoms.The disease progressed rapidly, with severe respiratory failure, acute kidney injury, and shock appearing soon, accompanied by different degrees of muscle injury, and damage to the heart, liver, blood coagulation, and immune systems at the same time.Laboratory examination showed that levels of inflammatory indicators were increased: at 3, 5, 7 d after admission, the level of C reactive protein was 214.6(153.9-256.3)mg/L, 199.2(115.8-333.8)mg/L, 151.0(11.19-173.7)mg/L, respectively; and interleukin 6 level was 1 241.0(912.1-6822.0)ng/L, 779.1(451.2-7122.0)ng/L, 631.2(7.0-4 321.0)ng/L, respectively.And monitoring results of nutritional index indicated a high metabolic state.The imaging examinations showed that consolidation and ground-glass shadows spreaded to both lungs, may accompany the miliary and nodular shadows, and may also involve pleural which caused pleural effusion.After the clinical use of metagenomic next-generation sequencing(mNGS), mNGS has been confirmed as an important method for the diagnosis of Chlamydia psittaci infection.The disease course and prognosis were related to the severity of the disease, advanced age, underlying diseases, and timely diagnosis and effective treatment.Conclusions:The progression of Chlamydia psittaci pneumonia to severe disease may be related to advanced age, more basic diseases such as chronic cardiopulmonary disease, smoking, and timely diagnosis and treatment.Generally, laboratory and imaging examinations have no diagnostic specificity, but mNGS is of great significance for early diagnosis, transition to target treatment and improvement of prognosis.

Objective To investigate whether quality assurance and quality control using multimodal image guidance and radiation dosimetry optimization could ensure and improve the targeting of 125I seeds brachytherapy.Methods A total of 287 patients (184 males,103 females,average age 61.9 years) with non-small-cell lung cancer (NSCLC) and 122 patients (average age 65.5 years) with prostate cancer who were diagnosed by pathology or imaging methods (coincidence imaging,CT,flexible fiberoptic bronchoscopy,ultrasonography) from October 2002 to October 2016 were retrospectively enrolled.All imaging methods were used to locate the target area.Optimization of radiation dosimetry was made according to treatment planning system (TPS) and evaluated by dose-volume histogram (DVH).125I seeds implantation was performed under the imaging guidance,followed by real-time location verification,dosimetric verification.Therapeutic efficacy was evaluated.Paired t test was used to analyze the data.Results The prescribed dose (PD) of planning target volume (PTV) was above 140 Gy.Immediate dosimetric verification was performed in 59 patients with NSCLC and 31 patients with pulmonary metastases.The radiation dose before and after the implantation was coincident in 93.2％ (55/59) of NSCLC and 93.5％ (29/31) of pulmonary metastases.The coincident rate of patients with dose delivered to 90％ GTV (D90)＞matched peripheral dose (MPD) was 93.2％ (55/59) and 87.1％ (27/31) respectively in NSCLC and pulmonary metastases.There was no significant difference in MPD,D90 and conformity index (CI) before and after implantation (t values:2.11-9.71,all P＞0.05).The average dose of the risk organs was significantly lower than the tolerance dose of the normal tissue.The incidence of radiation pneumonitis was 1.05％ (3/287).No other serious complications were found.Conclusion Targeted diagnosis and therapy of multimodal image guidance could be optimized to improve the effect of targeting and therapeutic gain ratio of 125I seeds brachytherapy.

This paper is aimed to investigate the effect of titanium (Ti) particles and tumor necrosis factor alpha (TNF-alpha) on the expressions of MMP-1, 2, 3 in human synovial cells, so as to explore the possible mechanism of osteolysis post-operation of metal-on-metal total joint arthroplasty in human synovial cells induced by Ti particles. In vitro cell cultures, human synovial cells were treated by Ti particles and/or TNF-alpha. The total RNA was isolated at 2 hours after the treatment. The gene expression of MMP-1, 2, 3 was analyzed by Semi-quantitative Reverse-transcriptional PCR and quantitative real-time PCR. Cell supernatant was collected at 12, 24, 48 hours after the treatment and Gelatin zymography was performed to detect the activity of MMP-2. Compared to those in the control group (untreated), Ti particles and TNF-alpha increased the gene expression of MMP-1, 2, 3 respectively (P < 0.05), and the effect of combination of the two was even more significant (P < 0.01). The trend of activities of MMP-2 is similar with gene expression. Ti particles and TNF-alpha increased MMP-2 activities by 1.3 times and 1.5 times respectively (P < 0.05), and the combination of the two increased by 1.7 times (P < 0.01). Ti particles and TNF-alpha-induced the stimulation of MMP-1, 2, 3 expressions and MMP-2 activities in human knee joint synovial cells may be involved in aseptic loosening after metal-on-metal arthroplasty through increasing the degradation of bone matrix and declining of osseous support structure mechanics.
Cells, Cultured ; Humans ; Joint Prosthesis ; Knee Joint ; cytology ; Matrix Metalloproteinase 1 ; genetics ; metabolism ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Matrix Metalloproteinase 3 ; genetics ; metabolism ; Matrix Metalloproteinases ; genetics ; metabolism ; Particle Size ; Prosthesis Failure ; adverse effects ; RNA ; genetics ; metabolism ; Synovial Membrane ; cytology ; enzymology ; Titanium ; pharmacology ; Tumor Necrosis Factor-alpha ; pharmacology