1.Interpretation of Reporting Items for Practice Guidelines in Healthcare for Chinese Patent Medicines (RIGHT for CPM)
Liaoyao WANG ; Hejing PAN ; Le ZHANG ; Wenya WANG ; Xing LIAO
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(1):211-218
The clinical practice guidelines for Chinese patent medicines (CPM) provide reference for the selection of national drug catalogs, the formulation of prescription collections in medical institutions, and the clinical use of CPM, constituting an important part of traditional Chinese medicine (TCM) guidelines. As a crucial part of Chinese drug supply guarantee system, CPM plays an important role in the treatment, prevention, and healthcare of many disease categories, whereas the application of CPM has problems of misuse and even abuse. To standardize the application of CPM, a research team at Zhejiang Chinese Medical University developed the Reporting Items for Practice Guidelines in Healthcare for Chinese Patent Medicines (RIGHT for CPM) based on the RIGHT checklist framework. The RIGHT for CPM checklist gathers key information from published CPM guidelines, existing TCM reporting checklists, and the RIGHT checklist and its extensions to form an initial pool of reporting items. Seventeen experts from different disciplines were invited to conduct two rounds of Delphi surveys, and the final checklist was reviewed and approved for publication by 18 leading experts in TCM research and guideline reporting from China and abroad. The RIGHT for CPM checklist adds 16 sub-items and revises 2 sub-items on the basis of the RIGHT checklist, highlighting the characteristics of CPM guideline reporting. It considers CPM selection and inclusion criteria, policy access, indications and symptoms, drug combination instructions, drug use in special populations, precautions, and recommendations of Western medical physicians, among others. This can further improve the quality and transparency of CPM guideline reporting, promote standardized reporting of CPM guidelines, and facilitate the rational clinical use of CPM. This article interprets the development process of the RIGHT for CPM checklist and the items that highlight the characteristics of CPM guidelines, with a view to promoting the application of the RIGHT for CPM checklist.
2.Clinical Evidence Profile of Eleven Expensive Chinese Patent Medicines: A Scoping Analysis
Yajing LI ; Miaomiao LI ; Le ZHANG ; Wenya WANG ; Hui ZHAO ; Xing LIAO
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(2):227-238
ObjectiveThis study employed the scoping review method to systematically retrieve and analyze the basic information and clinical research evidence of expensive Chinese patent medicines (CPMs), aiming to provide a basis for future related research and clinical applications. MethodsEight Chinese and English databases were systematically searched for the clinical research evidence on expensive CPMs. ResultsEleven expensive CPMs (Angong Niuhuang Wan, Jufang Zhibao Wan, Suhexiang Wan, Pien Tze Huang, Niuhuang Qingxin Wan, Qinggong Shoutao Wan, Compound Realgar Natural Indigo Tablets, Xihuang Wan, Dingkun Wan, Babao Wan, and Guilingji Capsules) were selected. A total of 365 related studies were included in this review, comprising 331 clinical studies (of which 291 were randomized controlled trials), 30 systematic reviews and Meta-analyses, 3 expert consensus, and 1 rapid health technology assessment. Among the 11 CPMs, 2(Angong Niuhuang Wan and Jufang Zhibao Wan) had a daily price over 500 yuan. The famous and precious Chinese medicinal materials involved included Moschus (frequency of 7), Bovisc Alculus (7), and Borneol (5). The dosage forms included pills, capsules, oral liquid, tablets, and lozenges. The diseases treated by these CPMs mainly included malignant tumors, cerebrovascular diseases, gynecological diseases, and hepatobiliary system diseases. The sample sizes of the clinical studies were mainly concentrated within the range of 51-100 cases, and the main control form was CPM + basic Western medicine treatment vs. basic Western medicine treatment. The 331 clinical studies reported a total of 44 adverse events occurred, of which 36 were determined to be adverse reactions. ConclusionThe scarcity of raw materials leads to the high prices of expensive CPMs. The difficulty of conducting clinical research and the critical and severe cases treated lead to a lack of clinical research evidence with large sample sizes. The uneven distribution of existing studies, incomplete information on medicine package, and non-standard clinical research designs remain to be addressed in the future.
3.Meta-analysis of the relationship of semaglutide and malignant neoplasms risk in type 2 diabetes mellitus patients
Qingchuan LIAO ; Wei YU ; Quan WANG
China Pharmacy 2025;36(1):117-123
OBJECTIVE To systematically evaluate the relationship of semaglutide with malignant neoplasms in type 2 diabetes mellitus (T2DM) patients. METHODS Retrieved from the Cochrane Library, PubMed, Embase, ClinicalTrials.gov, CNKI, Wanfang data and CBM, randomized controlled trials (RCTs) about semaglutide in the treatment of T2DM patients with outcome measures including malignant tumor events were collected from the establishment of the database to June 2024. Meta- analysis was performed by using RevMan 5.3 software to assess the risk of malignant neoplasms. RESULTS A total of 24 RCTs (26 trials) involving 24 145 patients were included. Results of meta-analysis showed that compared to placebo, there was no statistical significance in the risk of semaglutide in pancreatic cancer [RR=0.39, 95%CI(0.10, 1.50), P=0.17], thyroid cancer [RR=1.29, 95%CI(0.38, 4.36), P=0.68], prostate cancer [RR=1.05, 95%CI(0.36, 3.12), P=0.92], skin cancer [RR=1.27, 95%CI(0.80, 2.02), P=0.31], gastrointestinal cancer [RR=1.00, 95%CI(0.47, 2.14), P=1.00], colorectal cancer [RR=0.96, 95%CI(0.40, 2.26), P=0.92], lung cancer [RR=1.62, 95%CI(0.74, 3.55), P=0.23], breast cancer [RR=1.25, 95%CI(0.45, 3.51), P=0.67] or all malignant neoplasms [RR=0.96, 95%CI(0.76, 1.21), P=0.73]. Compared to other antidiabetic drugs, there was no statistical significance in the risk of semaglutide in pancreatic cancer [RR=0.62, 95%CI(0.18, 2.09), P=0.44], thyroid cancer [RR=1.09, 95%CI(0.25, 4.78), P=0.90], prostate cancer [RR=2.09, 95%CI(0.46, 9.47), P=0.34], skin cancer [RR=1.76, 95%CI(0.65, 4.72), P=0.26], gastrointestinal cancer [RR=0.68, 95%CI(0.19, 2.35), P=0.54], colorectal cancer [RR=0.60, 95%CI(0.20, 1.78), P=0.36], lung cancer [RR=1.00, 95%CI(0.24, 4.11), P=1.00], breast cancer [RR=0.82, 95%CI(0.25, 2.66), P=0.74] or all malignant neoplasms [RR=1.36, 95%CI(0.96, 1.94), P=0.09]. CONCLUSIONS Semaglutide does not increase the risk of any type of malignant neoplasms in T2DM patients.
4.Therapeutic effect and mechanism of umbilical cord mesenchymal stem cells in rats with primary graft dysfunction after lung transplantation
Hailin LIAO ; Xiaohua WANG ; Yi LU ; Chunrong JU
Organ Transplantation 2025;16(1):91-98
Objective To explore the therapeutic effect and mechanism of umbilical cord mesenchymal stem cells (UC-MSC) in rats with primary graft dysfunction after lung transplantation. Methods Twenty-four male Lewis rats were randomly divided into donor and recipient groups, with 12 rats in each group. The recipients were further divided into 3 groups: blank control group, negative control group, and treatment group, with 4 rats in each group. The color, size and texture of the transplanted lungs were observed 72 h after lung transplantation. The ventilation status and progression of consolidation in the transplant lungs of rats in each group were evaluated by micro-CT. Plasma, transplant lung tissue and alveolar lavage fluid samples of recipient rats were collected. The wet/dry ratio of lung tissue was measured to evaluate the degree of pulmonary edema. Hematoxylin-eosin (HE) staining was used to evaluate the degree of lung tissue damage. Terminal deoxyribonucleic acid transferase mediated dUTP nick end labeling (TUNEL) staining was used to evaluate cell apoptosis. Myeloperoxidase (MPO) activity in lung tissue was detected, and enzyme-linked immunosorbent assay (ELISA) was used to detect interleukin (IL)-6, IL-10 and tumor necrosis factor (TNF)-α levels in plasma and alveolar lavage fluid. Results The appearance of the transplant lungs in the negative control group was significantly different from that of the autologous lungs, while the transplant lungs in the treatment group were almost identical in color to the autologous lungs compared to the blank control group. Compared with the negative control group, the treatment group showed reduced alveolar exudate and more intact airway epithelial cell structure. No alveolar exudate was observed in the blank control group, and the structure of the airways and alveoli remained normal. The treatment group had lower apoptosis rate of airway epithelial cells, lung tissue wet/dry ratio, and MPO activity compared to the negative control group (all P < 0.05). The levels of IL-6 and TNF-α in the bronchoalveolar lavage fluid of the treatment group were lower than those in the negative control group, while the level of IL-10 was higher than that in the negative control group and the blank control group (all P < 0.05). There were no statistically significant differences in the levels of cytokines in plasma among each group (all P > 0.05). Conclusions UC-MSC may effectively alleviate the severity of primary graft dysfunction in rats by reducing the apoptosis rate of cells in lung tissue and inhibiting inflammatory responses.
5.Experience of Using Charcoal-Processed Traditional Chinese Medicine in the Treatment of Gynecological Hemorrhagic Disorders
Xiaolan WU ; Zhaoling YOU ; Guiyun WANG ; Kailing WANG ; Xiaojuan YE ; Lingyu LIAO ; Yueheng LI ; Huiping LIU
Journal of Traditional Chinese Medicine 2025;66(3):308-311
Charcoal-processed traditional Chinese herbal medicine has various therapeutic effects, including astringing, hemostasis, anti-diarrhea, clearing heat, tonifying, and warming the interior. This paper summarizes the clinical application features, compatible experiences, dosages, and precautions for over 20 types of charcoal-processed herbal medicine in the treatment of gynecological bleeding disorders caused by dysfunctions such as dysfunctional uterine bleeding, endometriosis, uterine incision pseudocavity, and vaginal bleeding resulting from threatened miscarriage. The charcoal-processed herbal medicine include Huangqin (Scutellaria Baicalensis) Charcoal, Dahuang (Rheum Palmatum) Charcoal, Cebai (Platycladus Orientalis) Charcoal, Diyu (Sanguisorba Officinalis) Charcoal, Daji (Cirsium Setosum) Charcoal, Xiaoji (Cirsium Japonicum) Charcoal, Shengdi (Rehmannia Glutinosa) Charcoal, Aiye (Artemisia Argyi) Charcoal, Paojiang (Zingiber Officinale) Charcoal, Xuduan (Dipsacus Asper) Charcoal, Duzhong (Eucommia Ulmoides) Charcoal, Qiancao (Rubia Cordifolia) Charcoal, Puhuang (Typha Angustifolia) Charcoal, Shanzha (Crataegus Pinnatifida) Charcoal, Jingjie (Schizonepeta Tenuifolia) Charcoal, Xueyu (Carthamus Tinctorius) Charcoal, Zonglyu (Areca Catechu) Charcoal, Wumei (Prunus Mume) Charcoal, Shudahuang (Rheum Officinale) Charcoal, Lianfang (Nymphaea Alba) Charcoal, Mianmaguanzhong (Clematis Armandii) Charcoal, and Oujie (Nelumbo Nucifera) Charcoal.
6.Efficacy and Safety of Qihuang Acupuncture Theory Combined with Opioid Analgesics in the Treatment of Moderate to Severe Cancer Pain in Lung Cancer Patients:a Randomize-Controlled Trial
Yingqi WANG ; Ruifang YU ; Jinpeng HUANG ; Guiya LIAO ; Ziyan GAN ; Zhenhu CHEN ; Xiaobing YANG ; Chunzhi TANG
Journal of Traditional Chinese Medicine 2025;66(4):358-366
ObjectiveTo observe the analgesic efficacy and safety of Qihuang acupuncture theory combined with opioid analgesics in patients with moderate to severe cancer pain due to lung cancer. MethodsPatients with moderate to severe cancer pain from lung cancer were randomly divided into Qihuang acupuncture group and control group, with 33 cases in each group. The control group was treated with long-acting opioid analgesics at maintenance doses and supplementary analgesic medications as needed. In case of breakthrough pain, short-acting opioids were used for rescue. The Qihuang acupuncture group received Qihuang acupuncture treatment in addition to the treatment used in the control group, administered once every other day, with 3 sessions constituting one treatment course. The treatment duration for both groups was 5 days. The primary outcome was the change in pain intensity, measured using the numerical rating scale (NRS) before and after treatment, and the NRS change rate was calculated. Secondary endpoints included the daily NRS change rate, the Eastern Cooperative Oncology Group (ECOG) Performance Status (PS) score, the European Organization for Research and Treatment of Cancer Quality of Life Core Questionnaire (EORTC QLQ-C30) score, and the 24-hour equivalent hydrocodone sustained-release tablet dose. Laboratory tests, including routine blood, urine, stool, liver function, and kidney function, were performed before and after treatment. Adverse events were recorded throughout the trial. ResultsAll patients completed the trial, and both groups showed a decrease in average NRS scores and PS scores after treatment, with the Qihuang acupuncture group showing lower average NRS scores and PS scores than the control group (P<0.05 or P<0.01). After treatment, the NRS change rate in the Qihuang acupuncture group was (0.42±0.17), significantly higher than that in the control group (0.14±0.27, P<0.01). The daily NRS change rate during treatment was also higher in the Qihuang acupuncture group compared to the control group (P<0.01). The Qihuang acupuncture group showed an increase in overall health status and functional scores in the EORTC QLQ-C30, and a decrease in symptom scores for fatigue, nausea and vomiting, pain, dyspnea, insomnia, appetite loss, constipation, and financial difficulties. In contrast, overall health status and constipation scores in the control group increased, while scores of fatigue, nausea and vomiting, pain, and appetite loss decreased (P<0.05 or P<0.01). After treatment, the 24-hour equivalent hydrocodone sustained-release tablet dose did not show significant difference in the Qihuang acupuncture group (P>0.05), while the control group showed a significant increase in the 24-hour dose (P<0.01). No significant abnormalities were observed in laboratory tests before and after treatment in either group. During the study, the incidence of nausea and vomiting as well as constipation in the Qihuang acupuncture group was both 3.03% (1/33), while the incidence in the control group was 27.27% (9/33) and 36.36% (12/33), respectively, with the Qihuang acupuncture group showing significantly lower incidence (P<0.01). No serious adverse reactions were observed in either group. ConclusionQihuang acupuncture therapy combined with opioid analgesics is more effective than using opioids alone in relieving pain in patients with moderate to severe cancer pain due to lung cancer. It can improve the patients' physical condition and quality of life, reduce the dose of opioid analgesics, and has good safety.
7.Exploring Academic Characteristics of Contemporary Experts and Schools in Traditional Chinese Medicine Gynecology in Treating Endometriosis Diseases Based on SrTO
Zhiran LI ; Xiaojun BU ; Xiaodan WANG ; Le ZHANG ; Ruixue LIU ; Jingyu REN ; Xing LIAO ; Weiwei SUN
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(6):249-259
ObjectiveStarting from the etiology, pathogenesis, and treatment strategies of endometriosis and adenomyosis, to integrate and sort out the academic characteristics of contemporary renowned experts and schools in the field of traditional Chinese medicine gynecology. MethodsAccording to the systematic review of text and opinion (SrTO) process developed by the Joanna Briggs Institute (JBI) in Australia, this paper determined literature screening criteria by searching China National Knowledge Infrastructure (CNKI), VIP, Wanfang, and China Biomedical Literature Database. Information was extracted after literature screening, and quality evaluation was conducted using the JBI Narrative, Text, and Opinion Systematic Review Strict Evaluation Checklist. The JBI Narrative, Opinion, Text Evaluation, and Review Tool Summary Table was used for information synthesis, and data analysis and display were conducted in the form of text and charts. ResultsThe 146 articles related to 39 renowned experts and 19 articles related to 10 schools of thought were included. Research has found that contemporary experts and schools in traditional Chinese medicine gynecology consider blood stasis as the core pathogenesis in understanding the etiology and pathogenesis of two diseases and related infertility. Their viewpoints varied from multiple aspects such as clinical symptom characteristics, meridian circulation location, pathological product evolution, disease duration, emotional psychology, lifestyle habits, preference for food and drink, innate endowment, and acquired injury. In terms of treatment, it was advocated to divide the stage, treat according to different types, adapt to the times, integrate nature and humans, and combine multiple methods to treat comprehensively when necessary. It was also recommended to skillfully use insects, make good use of classic formulas and small prescriptions, pay attention to protecting the spleen and stomach and regulating emotions, and make good use of self-formulated empirical formulas for internal or external use. Besides, individualized long-term management of patients was also advocated. ConclusionThis study applies the SrTO process to systematically summarize the academic ideas of contemporary renowned experts and schools in traditional Chinese medicine gynecology regarding the causes, mechanisms, diagnosis, and treatments of endometriosis, providing a scientific and standardized reference for future theoretical exploration.
8.Multi-index quantitative detection and quality difference evaluation of Gleditsia sinensis from different producing areas
Meifeng LIANG ; Xiongfei WAN ; Nian LIAO ; Shanshan ZHU ; Zhijian WANG
China Pharmacy 2025;36(5):568-573
OBJECTIVE To establish a multi-index quantitative detection method, and to evaluate the quality difference of Gleditsia sinensis from different producing areas. METHODS The contents of protocatechuic acid, vanillic acid, isoscopoletin, scoparone, isovitexin, fustin, taxifolin, fisetin, quercetin, kaempferol, echinocystic acid, betulinic acid, β -sitosterol and stigmasterol were detected by high performance liquid chromatography-quantitative analysis of multi-components by single marker (HPLC-QAMS). The chromatographic column was Kromasil C18, the mobile phase was 0.2% phosphoric acid-acetonitrile solution (gradient elution), the detection wavelengths were 254, 360, 210 nm for different index components, the column temperature was 30 ℃ , the flow rate was 1.0 mL/min, and the sample injection volume was 10 μL. The contents of extract and total ash were detected according to the method of Chinese Pharmacopoeia. The quality differences of 30 batches of G. sinensis (No. S1-S30) from different producing areas were evaluated by chemometrics, weighted technique for order preference by similarity to an ideal solution (TOPSIS) analysis and Logistic regression model. RESULTS The linear ranges of 14 components were 1.55-77.50, 0.71- 35.50, 0.28-14.00, 0.96-48.00, 1.77-88.50, 0.09-4.50, 4.65-232.50, 1.49-74.50, 0.37-18.50, 1.18-59.00, 7.35-367.50, 3.58- 179.00, 0.49-24.50 and 0.21-10.50 μg/mL, respectively (all r>0.999). The RSDs of precision, stability (24 h) and repeatability were less than 2.00%; the average recoveries were 96.99%-100.13% (all RSDs<2.00%), and the relative correction factor had good repeatability. The contents of extract and total ash were Δ 基金项目 湖北省中医药科研立项青年人才项目 (No. 4.2%-12.5% and 0.5%-2.3%, respectively. There was no ZY2019Q014) significant difference in the content of 14 components measured by QAMS method and external standard method (P>0.05). The results of chemometrics showed that 30 batches of samples were clustered into 3 categories: S1 to S11 form one category, S12 to S20 form another category, and S21 to S30 constitute the third category. Echinocystic acid, betulinic acid, taxifolin, kaempferol, isovitexin, scoparone and protocatechuic acid may be the differential components affecting the quality of G. sinensis from different producing areas. The analysis results of the weighted TOPSIS method revealed that relative closeness (Jb) for 30 batches of G. sinensis ranged from 0.144 5 to 0.721 8, with S27 achieving the highest value (Jb) of 0.721 8. The analysis results of the Logistic regression model showed that S21-S30 batches of samples were of superior grade, S1-S11 were of intermediate grade, and S12-S20 were of inferior grade. CONCLUSIONS The established HPLC-QAMS method is simple and accurate. The comprehensive evaluation method is objective and comprehensive, and can be used to evaluate the quality difference of G. sinensis from different producing areas.
9.Eucommia ulmoides promotes alveolar bone formation in ovariectomized rats
Lin ZHENG ; Wenjun JIN ; Shanshan LUO ; Rui HUANG ; Jie WANG ; Yuting CHENG ; Zheqing AN ; Yue XIONG ; Zipeng GONG ; Jian LIAO
Chinese Journal of Tissue Engineering Research 2025;29(6):1159-1167
BACKGROUND:Eucommia ulmoides has a certain osteogenic effect,which can promote the proliferation and differentiation of osteoblasts.However,it is unclear whether Eucommia ulmoides has effects on alveolar bone formation and Wnt/β-Catenin signaling pathway. OBJECTIVE:To investigate the mechanism by which Eucommia ulmoides promotes alveolar bone formation in ovariectomized rats based on the Wnt/β-Catenin signaling pathway. METHODS:Sixty female Sprague-Dawley rats were selected and randomly divided into five groups:blank control group,sham-operation group,model group,low-dose group Eucommia ulmoides group,and high-dose Eucommia ulmoides group,with twelve rats in each group.Osteoporosis animal models were constructed by bilateral oophorectomy in the model group and the low-dose and high-dose Eucommia ulmoides groups.The sham-operation group underwent the same method to remove adipose tissue of equal mass around the bilateral ovaries.Three months after surgery,the low-and high-dose Eucommia ulmoides groups were given 2.1 g/kg/d and 4.2 g/kg/d Eucommia ulmoides by gavage,respectively.The sham-operation group and model group were given the same amount of physiological saline by gavage.After 12 weeks of drug intervention,the changes in alveolar bone mass of rats in each group were observed through Micro-CT;hematoxylin-eosin staining was used to observe the pathological structural changes of alveolar bone in rats;enzyme linked immunosorbent assay was used to detect the expression levels of alkaline phosphatase and osteocalcin in the serum of rats;western blot was used to detect the expression levels of β-Catenin and Frizzled9 receptor proteins in the alveolar bone of rats;and real-time fluorescence quantitative PCR was used to detect the expression of osteocalcin,Runt-related transcription factor 2(Runx2),alkaline phosphatase,β-catenin,and frizzled9 mRNAs in alveolar bone tissues of rats. RESULTS AND CONCLUSION:Compared with the blank control group,bone volume fraction,trabecular number,trabecular thickness,and bone mineral density were reduced in the model group(P<0.05),and trabecular separation was elevated(P<0.05).Pathological observation showed that the arrangement of trabeculae was disordered and irregular,the trabeculae were thinned or broken,and the marrow cavity was enlarged in the model group,with a significant reduction in bone volume;the level of alkaline phosphatase in the serum was increased(P<0.05),and the level of osteocalcin was decreased(P<0.05);mRNA expression of alkaline phosphatase,osteocalcin,Runx2,β-catenin,and frizzled9 were decreased(P<0.05);protein expression of β-Catenin and Frizzled9 was decreased(P<0.05).Compared with the model group,the low-and high-dose Eucommia ulmoides groups showed an increase in bone volume fraction,trabecular number,trabecular thickness,and bone mineral density(P<0.05)and a decrease in trabecular separation(P<0.05).In the low-and high-dose Eucommia ulmoides groups,bone trabeculae were slightly aligned and thickened,with a significant increase in bone mass.Compared with the model group,the serum level of alkaline phosphatase was reduced(P<0.05)and the serum level of osteocalcin was elevated(P<0.05)in the low-and high-dose Eucommia ulmoides groups.Compared with the model group,the mRNA expression of alkaline phosphatase,osteocalcin,Runx2,β-catenin,and frizzled9 were increased in the low-and high-dose Eucommia ulmoides groups(P<0.05).Compared with the model group,the protein expression of Frizzled9 was increased in the low-dose Eucommia ulmoides group(P<0.05),while the protein expression of β-Catenin and Frizzled9 was increased in the high-dose Eucommia ulmoides group(P<0.05).Compared with the low-dose Eucommia ulmoides group,the high-dose Eucommia ulmoides group had a more significant improvement in the above indexes.To conclude,Eucommia ulmoides can effectively promote the alveolar bone formation,and its mechanism of action might be related to the activation of the Wnt/β-catenin signaling pathway.
10.An experimental method for simultaneous extraction and culture of primary cortical neurons and microglial cells from SD rats
Longcai HE ; Wenxue SONG ; Jiang MING ; Guangtang CHEN ; Junhao WANG ; Yidong LIAO ; Junshuan CUI ; Kaya XU
Chinese Journal of Tissue Engineering Research 2025;29(7):1395-1400
BACKGROUND:Primary cortical neurons and microglial cells play a crucial role in exploring cell therapies for neurological disorders,and most of the current methods for obtaining the two types of cells are cumbersome and require separate extraction.It is therefore crucial to find a convenient and rapid method to extract both types of cells simultaneously. OBJECTIVE:To explore a novel method for simultaneous extraction of primary cortical neurons and microglial cells. METHODS:Newborn suckling SD rats were taken within 24 hours.The brain was removed and placed in a dish with DMEM,and the pia mater was removed for later use.Primary neurons were extracted from the same brain tissue,and then the remaining brain tissue was used to extract microglial cells.The whole process was performed on ice.Extraction and culture steps of primary cortical neurons:The cerebral cortex was taken 2.0-3.0 mm with forceps,and the tissue was digested with papain for 20 minutes.After aborting digestion,the blown tissue presented an adherent tissue suspension.The supernatant cell suspension was obtained,filtered,and dispensed into 15 mL centrifuge tubes.After centrifugation and re-suspension,the cells were inoculated onto 6-well plate crawls coated with L-polylysine.Neuronal morphology was observed at 1-day intervals,and staining could be performed for identification using immunofluorescence staining of MAP2 and β-Tubulin by day 7.Microglia extraction and culture steps:The remaining brain tissue at 8-10 mm thick was subjected to microglial cell extraction,digested by trypsin for 20 minutes.After digestion was stopped,the tissue was blown to a homogenate,and then the homogenate was transferred to the culture bottle for culture.On day 14,the culture flasks were sealed and subjected to constant temperature horizontal shaking for 2 hours.Microglial cells were shed in the supernatant.Purified microglial cells were taken and continued to be cultured for 3 days for identification by Iba1 immunofluorescence staining. RESULTS AND CONCLUSION:(1)After 24 hours of culture,the neurons were adherent to the wall,the cytosol was enlarged,and some neurons developed synapses.After 3 and 5 days of culture,the cytosol was further enlarged,and most of the neurons were in the form of synapses,and some neurons were growing in clusters.On day 7,neuronal synapses were prolonged and thickened,and they were connected with each other to form a network.The neurons were identified by β-Tubulin and MAP2 immunofluorescence staining.(2)The cells grew close to the wall on day 1 of culture.On days 3,5,and 7,the density of microglial cells was small,and the cell morphology was bright oval or round,but the cells basically grew in clumps on the upper layer of other cells.On day 10,the density of microglial cells increased significantly.On day 14,microglial cells grew in dense clumps on the upper layer of other cells,and then they could be isolated and purified.The isolated and purified cells were taken and re-cultured to day 3 and identified as microglial cells by Iba1 immunofluorescence;their purity was greater than 95%.(3)The results show that primary cortical neurons and microglial cells obtained by this method after extraction and culture are of high purity,good morphology,and high viability.

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