BACKGROUND:In the research and application of neural stem cells,cell culture and passage are key links,which directly affect the quality of cells and experimental results.It is of great significance to find the most suitable digestive enzymes that can maintain the biological characteristics of embryonic mouse spinal cord neural stem cells and enhance their passage efficiency.OBJECTIVE:To explore the most suitable digestive enzyme for passage of neural stem cells from the spinal cord of embryonic mice.METHODS:Microscopic dissection was used to isolate and extract spinal cord tissue from E14 d embryonic mice,which was cultured in DMEM/F12 serum-free medium containing epidermal growth factor,basic fibroblast growth factor,and B27.After spherulation,Nestin and Sox2 immunofluorescence identification was performed.During neural stem cell passage and culture,single-cell suspensions were prepared using trypsin,papain,and TrypLETM Express enzyme digestion combined with blow molding.The cell dispersion and spheroidization were observed,and passage 3 cells were stained with propidium iodide to detect cell death.Cell proliferation was detected by counting the total number of cells.Immunofluorescence staining,western blot assay and RT-PCR were used to detect the expression of Olig2,Tuj1,GFAP,and NeuN at the protein and mRNA levels and to identify cell differentiation.RESULTS AND CONCLUSION:After 72 hours of culture,E14 d embryonic mouse spinal cord tissue cells could form suspended neurospheres,which could be passaged after 5-7 days.Compared with trypsin and papain,TrypLETM Express enzyme combined with blow beating method was used for passage.The cell dispersion rate was high,the activity was good,and more NeuN-and Tuj1-positive neurons differentiated.This study optimized the culture and passaging process of neural stem cells,laying a foundation for further research on stem cell transplantation therapy for spinal cord diseases.

BACKGROUND:In the research and application of neural stem cells,cell culture and passage are key links,which directly affect the quality of cells and experimental results.It is of great significance to find the most suitable digestive enzymes that can maintain the biological characteristics of embryonic mouse spinal cord neural stem cells and enhance their passage efficiency.OBJECTIVE:To explore the most suitable digestive enzyme for passage of neural stem cells from the spinal cord of embryonic mice.METHODS:Microscopic dissection was used to isolate and extract spinal cord tissue from E14 d embryonic mice,which was cultured in DMEM/F12 serum-free medium containing epidermal growth factor,basic fibroblast growth factor,and B27.After spherulation,Nestin and Sox2 immunofluorescence identification was performed.During neural stem cell passage and culture,single-cell suspensions were prepared using trypsin,papain,and TrypLETM Express enzyme digestion combined with blow molding.The cell dispersion and spheroidization were observed,and passage 3 cells were stained with propidium iodide to detect cell death.Cell proliferation was detected by counting the total number of cells.Immunofluorescence staining,western blot assay and RT-PCR were used to detect the expression of Olig2,Tuj1,GFAP,and NeuN at the protein and mRNA levels and to identify cell differentiation.RESULTS AND CONCLUSION:After 72 hours of culture,E14 d embryonic mouse spinal cord tissue cells could form suspended neurospheres,which could be passaged after 5-7 days.Compared with trypsin and papain,TrypLETM Express enzyme combined with blow beating method was used for passage.The cell dispersion rate was high,the activity was good,and more NeuN-and Tuj1-positive neurons differentiated.This study optimized the culture and passaging process of neural stem cells,laying a foundation for further research on stem cell transplantation therapy for spinal cord diseases.

Objective:To evaluate the application value of amplicon sequencing in identification of drug-contami-nating microbes in Chinese patent drugs.Methods:Both culturing method and Amplicon Sequencing technology were used to identify bacteria and fungi in 16 batches of Chinese patent drugs.The microbial colonies isolated by culturing medium were identified by 16srDNA/ITS sequencing and MALID-TOF-MS.The amplicon sequencing reads were clustered to OTU(Operational Taxonomic Units)by 97％similarity and BLAST in NCBI to get species taxonomy.Alpha and beta diversity index were used to analyze the microbial community composition in and between batches of drugs.Results:Fifteen species of bacteria and 3 species of fungi were isolated by culturing method,while 75 species of bacteria and 78 species of fungi were identified by amplicon sequencing.Between pre-cultured and non-cultured drug groups,the α diversity index and abundance of most dominant microbial genus were not changed significantly(P＞0.05).Beta diversity analysis showed that microbial diversity between different drugs are more than the same drugs between different pre-treated groups.Conclusion:This study revealed that amplicon sequencing method can provide more comprehensive information on the microbial community composition than tradi-tional culture method.It contributes great value to quick inspection and monitor for microbial contamination in chines patent drugs.

Objective:To evaluate the application value of amplicon sequencing in identification of drug-contami-nating microbes in Chinese patent drugs.Methods:Both culturing method and Amplicon Sequencing technology were used to identify bacteria and fungi in 16 batches of Chinese patent drugs.The microbial colonies isolated by culturing medium were identified by 16srDNA/ITS sequencing and MALID-TOF-MS.The amplicon sequencing reads were clustered to OTU(Operational Taxonomic Units)by 97％similarity and BLAST in NCBI to get species taxonomy.Alpha and beta diversity index were used to analyze the microbial community composition in and between batches of drugs.Results:Fifteen species of bacteria and 3 species of fungi were isolated by culturing method,while 75 species of bacteria and 78 species of fungi were identified by amplicon sequencing.Between pre-cultured and non-cultured drug groups,the α diversity index and abundance of most dominant microbial genus were not changed significantly(P＞0.05).Beta diversity analysis showed that microbial diversity between different drugs are more than the same drugs between different pre-treated groups.Conclusion:This study revealed that amplicon sequencing method can provide more comprehensive information on the microbial community composition than tradi-tional culture method.It contributes great value to quick inspection and monitor for microbial contamination in chines patent drugs.

Objective:To investigate risk factors for decompressive craniectomy (DC) after endovascular therapy (EVT) in patients with acute anterior circulation ischemic stroke.Methods:Patients underwent EVT due to acute anterior circulation large vessel occlusion in Liaocheng Brain Hospital from January 2018 to January 2020 were retrospectively included. They were divided into DC group and non-DC group. Univariate and multivariate logistic regression analyses were used to determine risk factors for DC after EVT. Results:A total of 207 patients were enrolled, 126 were male (60.87%), and their age was 66.22±11.24 years old. The baseline National Institutes of Health Stroke Scale (NIHSS) score was 19.84±9.20, and the Alberta Stroke Program Early CT Score (ASPECTS) was 7.98±0.66. The immediate postoperative modified Treatment In Cerebral Ischemia (mTICI) blood flow grade in seven patients (5.80%) was ≤2a, 30 (14.49%) experienced hemorrhagic transformation (HT) after procedure, and 28 (13.5%) received DC. There were statistically significant differences between the DC group and the non-DC group in terms of past stroke history, preoperative NIHSS score and ASPECTS, vascular occlusion site, EVT time, immediate postoperative mTICI ≤2a, and HT (all P<0.05). Multivariate logistic regression analysis showed that atrial fibrillation (odds ratio [ OR] 3.202, 95% confidence interval [ CI] 1.335-9.796; P=0.011), previous stroke history ( OR 2.655, 95% CI 1.016-6.938; P=0.046), high preoperative NIHSS score ( OR 1.074, 95% CI 1.026-1.124; P=0.002), internal carotid artery occlusion ( OR 4.268, 95% CI 1.399-13.024; P=0.011), longer EVT time ( OR 1.010, 95% CI 1.003-1.016; P=0.003), mTICI grade ≤2a ( OR 5.342, 95% CI 1.565-18.227; P=0.007) and postoperative HT ( OR 3.036, 95% CI 1.024-9.004; P=0.045) were independent risk factors for DC. Conclusions:It is not uncommon for patients with acute anterior circulation ischemic stroke to need DC after EVT. Previous stroke history, atrial fibrillation, high baseline NIHSS score, internal carotid artery occlusion, prolonged blood EVT time, mTICI grade ≤2a and postoperative HT are independent predictors of needing DC after EVT.

This study aimed to compare the safety and effectiveness between unilateral biportal endoscopy (UBE) technique and microscopic decompression (MD) technique in lumbar spinal stenosis treatment. PubMed, Cochrane Library, Embase, Web of Science, China National Knowledge Infrastructure, and other databases were used to conduct extensive literature searches. RevMan ver. 5.3 software was used for the statistical analysis. Eleven studies were included with 930 patients, including 449 patients in the UBE group and 521 in the MD group. Both techniques revealed similar operative times at −1.77 minutes (95% confidence interval [CI], −7.59 to 4.05 minutes; p =0.55), the postoperative dural expansion area at −1.27 (95% CI, −19.30 to 16.77; p =0.89), the postoperative complications at 0.76 (95% CI, 0.47 to 1.22; p =0.26), the preoperative Visual Analog Scale (VAS) for leg pain, and the last follow-up (>12 months) VAS for leg pain at −0.04 (95% CI, −0.14 to 0.06; p =0.47), the preoperative Oswestry Disability Index (ODI), and the last follow-up (>12 months) ODI scores at −0.18 (95% CI, −0.76 to 0.40; p =0.54), and patient satisfaction (the modified MacNab score) at 1.15 (95% CI, 0.54 to 2.42; p =0.72). However, intraoperative bleeding was lower following the UBE technique at −52.78 mL (95% CI, −93.47 to −12.08 mL; p =0.01) and was shorter following the UBE technique at −3.06 (95% CI, −3.84 to −2.28; p <0.01). UBE and MD technology have no significant differences in efficacy or safety in the treatment of patients with lumbar spinal stenosis based on this meta-analysis. However, the UBE technique has less intraoperative bleeding and a shorter hospital stay. It has a slight advantage and is a better surgical option than the MD technique. It can be an alternative minimally invasive spinal surgery method.

Objective:To explores the clinical efficacy and safety of carotid endarterectomy in patients with high bifurcation carotid artery stenosis.Methods:A total of 169 patients with carotid artery stenosis (147 patients with non-high bifurcation carotid artery stenosis and 22 patients with high bifurcation carotid artery stenosis), underwent carotid endarterectomy under surgical microscope in our hospital from January 2017 to January 2020, were included in the study. Patients with high bifurcation carotid artery stenosis were operated by cutting off the posterior abdomen of the digastric muscle to assist in exposing the distal end of the internal carotid artery plaque. Cervical CTA/DSA examination was performed within one week of surgery to confirm whether carotid artery stenosis was relieved; ultrasound examination of cervical vessels was performed 6 months and 1 year after surgery to determine whether restenosis was appeared in the carotid artery. The surgical efficacy, perioperative complications and re-examination results were compared between the two groups.Results:Postoperative carotid artery stenosis was relieved in both groups. The proportions of new cerebral infarction and temporary neurological impairment between the two groups (4.5% vs. 2.0%; 9.1% vs. 3.4%) showed no significant differences ( P>0.05). Carotid artery restenosis was not found in both groups. There was no obvious functional abnormality in the digastric muscles of patients with high bifurcation carotid stenosis. Conclusion:Cutting off the posterior abdomen of the digastric muscle during carotid endarterectomy can better expose the distal end of the internal carotid plaque in patients with high bifurcation carotid stenosis, and provide convenience for effective relief of carotid artery stenosis with high safety.

Objective To investigate the feasibility and safety of hybrid surgery in treatment of chronic symptomatic internal carotid artery occlusion.MethodsFrom April 2016 to December 2018, 9 patients with chronic symptomatic internal carotid artery occlusion confirmed by digital subtraction angiography were treated with carotid endarterectomy (CEA)+double chamber Fogarty balloon (3F) embolectomy or stent implantation when necessary. The clinical data of these patients were retrospectively analyzed. Vascular recanalization and complications during perioperative period and follow-up of the patients were analyzed.ResultsCEA+double chamber Fogarty balloon (3F) thrombectomy was performed in 5 patients; and stent placement was performed in 4 patients after Fogarty balloon thrombectomy. The internal carotid arteries of all 9 patients were unobstructed immediately during the operation. Postoperative hyperperfusion syndrome occurred in 3 patients; blood pressure was strictly controlled and the symptoms gradually relieved after proper dehydration. No serious complications such as cerebral hemorrhage, cerebral infarction, or nerve injury occurred. The duration of follow-up was 4-36 months; no patient developed a new stroke or death; no re-occlusion appeared; but one mild restenosis (50％) was detected. one patient suffered from a TIA attack 6 months after operation. ConclusionHybrid surgery is an effective method for treatment of chronic symptomatic internal carotid artery occlusion; however, preoperative evaluation and selection of patients should be carefully; individualized treatment plans should be developed.

Coronary artery disease (CAD) is a complex human disease, involving multiple genes and their nonlinear interactions, which often act in a modular fashion. Genome-wide single nucleotide polymorphism (SNP) profiling provides an effective technique to unravel these underlying genetic interplays or their functional involvements for CAD. This study aimed to identify the susceptible pathways and modules for CAD based on SNP omics. First, the Wellcome Trust Case Control Consortium (WTCCC) SNP datasets of CAD and control samples were used to assess the joint effect of multiple genetic variants at the pathway level, using logistic kernel machine regression model. Then, an expanded genetic network was constructed by integrating statistical gene–gene interactions involved in these susceptible pathways with their protein–protein interaction (PPI) knowledge. Finally, risk functional modules were identified by decomposition of the network. Of 276 KEGG pathways analyzed, 6 pathways were found to have a significant effect on CAD. Other than glycerolipid metabolism, glycosaminoglycan biosynthesis, and cardiac muscle contraction pathways, three pathways related to other diseases were also revealed, including Alzheimer’s disease, non-alcoholic fatty liver disease, and Huntington’s disease. A genetic epistatic network of 95 genes was further constructed using the abovementioned integrative approach. Of 10 functional modules derived from the network, 6 have been annotated to phospholipase C activity and cell adhesion molecule binding, which also have known functional involvement in Alzheimer’s disease. These findings indicate an overlap of the underlying molecular mechanisms between CAD and Alzheimer’s disease, thus providing new insights into the molecular basis for CAD and its molecular relationships with other diseases.

Objective Colour ultrasound was used to detect the hemodynamic changes in patients with internal carotid artery kingking,in order to investigate the relationship between the carotid distortion angle and blood flow changes and to explore the assessment of severe internal carotid artery twist operation indications.Methods Forty-five patients with carotid artery kingking hospitalized in the Brain Hospital of Liaocheng were performed colour ultrasound to detect systolic blood flow velocity (PSV), end-diastolic velocity EDV) and to measure the angle of carotid artery kingking.Results According Metz classification, of the 45 patients, Ⅰ level 17 cases, Ⅱ level 17 cases, Ⅲ level 11 cases.With the decrease of carotid distortion angle,the influence on hemodynamics was more and more obvious, especially while the angle less than 30 degrees, the carotid artery blood flow was severely affected.The difference of PSV before and after Kinking was statistically significant in patientes of Metz Ⅲ level (Z=-2.934,P=0.003) and Metz Ⅱ level (Z=-3.053,P=0.002), but was statistically no significant in patientes of Metz Ⅰ level (Z=-0.382, P=0.702).There was a negative correlation between the ratio of the twist angle and PSV before kinking/PSV after kinking (rz =-0.842, P ＜0.05),that was, with the decrease of the twist angle, PSV before kinking/ PSV after kinking increase accordingly.Conclusion Kinking seriously affect the carotid artery blood flow dynamics.Ultrasound can accurately detect distortions arterial hemodynamics