Objective : To investigate the brain microvascular tissue block culture method for extracting primary rat brain microvascular endothelial cells and identify its effect. Methods : Brain tissue from 4-week-old Sprague Dawley rats was screened, pre-digested and solidified to obtain brain microvascular segments. These segments were subsequently placed in a CO2incubator for primary culture. The target cells were identified by cell morphology and immunocytochemical staining for factor Ⅷ-related antigen. Results : After a 48-hour culture periodin vitro, the short spindle cells crawled out from around the brain microvascular segments. After 72 hours, island-like cell culsters formed. After 96 hours the clusters fused and the cells formed a typical monolayer, cobble stone-like, and mosaic arrangement. Factor Ⅷ-related antigen immunocytochemical staining showed that the cytoplasm of the cells appeared brown-red, indicating positive expression; DAB stained the nucleus, showing blue-dark. Conclusion The brain microvascular tissue block culture method can isolate and culture primary rat brain microvascular endothelial cells.

On the premise of respecting the objective law of the occurrence and development of traditional Chinese medicine（TCM） dispensing granules， relevant national departments have gradually formed the research and formulation ideas of national drug standards for dispensing granules based on the experiences and lessons learned in the development process of quality standards， as well as the formation mechanism of national standards for dispensing granules. This has certain reference significance for the formulation path of TCM quality standards. Combined with the general situation of the published standards and specific cases， the research concepts of the national standards for dispensing granules were analyzed and summarized in this paper， and the analysis of the technical characteristics of the issued national standards was focused， including the introduction of standard decoction， the overall quality control of TCM， the whole process quality control and other research ideas. At the same time， it summarized the industry common problems in the research and development process of national standards for dispensing granules， such as the source and process control of medicinal materials， and strived to solve them together， encouraging the demonstration and application of new technological means in the field of TCM dispensing granules. Finally， based on the literature analysis， the shortcomings of the current national standards were discussed， and relevant suggestions were put forward to further improve the national standards for dispensing granules. Through the overall analysis， it is helpful to comprehensively understand the technical characteristics of the national standards for TCM dispensing granules， and provide reference for the scientific exploration and practice of quality control methods for TCM.

Objective To investigate the effect and mechanism of microRNA-10b(miR-10b)on idiopathic short stature(ISS).Methods A total of 54 children with ISS and 54 healthy children were collected.The serum expression of miR-10b was detected by RT-qPCR,and the relationship between serum miR-10b expression and clinical data of children with ISS was analyzed.miR-10b inhibitor,si-TGFBR1 and their negative control transfection C28/I2 cells were used.CCK-8 experimental detection was used to detect C28/I2 cell proliferation.Western blot assay was used to detect gnome related transcription factor 2(RUNX2),collagen type X alpha 1 chain(COL10A1),transforming growth factor beta receptor 1(TGFBR1),SMAD3 and pSMAD3 protein expression.The target of miR-10b was screened in StarBase database,and the targeting relationship between miR-10b and TGFBR1 was verified by dual luciferase reporter gene assay.Results The serum expression of miR-10b was higher in the ISS group than that of the healthy control group,and the higher the miR-10b expression,the more obvious the decrease of child height,IGF-1 and alkaline phosphatase(P＜0.05).Compared with the NC group,the cell proliferation ability and RUNX2,COL10A1,TGFBR1,and pSMAD3 protein expression were up-regulated in the miR-10b inhibitor group(P＜0.05).StarBase database suggested that miR-10b had a binding site of TGFBR1,and dual luciferase reporter gene assay confirmed that TGFBR1 interacted with miR-10b(P＜0.05).Compared with the si-NC group,the expression of TGFBR1 was down-regulated and the cell proliferation ability was decreased in the si-TGFBR1 group(P＜0.05).Conclusion miR-10b inhibits chondrocyte proliferation and hypertrophy in idiopathic short stature by targeting TGFBR1/SMAD3 pathway.

OBJECTIVE To establish the characteristic chromatogram of the volatile oil in the standard decoction of Qingshang juantong decoction， and preliminarily infer the main active components of volatile oil that affect the clinical therapeutic effect. METHODS The volatile oil in the standard decoction of Qingshang juantong decoction was extracted by steam distillation. The ultra-high performance liquid chromatography （UPLC） characteristic chromatograms of 15 batches of samples were established by the Similarity Evaluation System of TCM Chromatographic Fingerprint （2012 edition）， and the similarity evaluation was carried out. The volatile oil of standard decoction was identified by UPLC coupled with quadrupole time-of-flight mass spectrometry （UPLC-Q-TOF/MS）. Then the volatile oil components were analyzed by GC-MS. RESULTS The similarities of UPLC characteristic chromatograms for volatile oil of 15 batches of Qingshang juantong decoction were between 0.949 and 0.997. A total of 12 common peaks were obtained. According to the UPLC-Q-TOF/MS， the main components were methyl eugenol， E-ligustilide， E-butylidenephthalide and so on. A total of 23 components were identified by GC-MS， which were mainly 3，4，5-trimethoxy- methylbenzene， patchouli alcohol， Z-ligustilide and so on. CONCLUSIONS The characteristic chromatograms of the volatile oil in the standard decoction of Qingshang juantong decoction is established， and it is inferred that methyl eugenol， ligustilide， E- butylidenephthalide， patchouli alcohol， 3，4，5-trimethoxy-methylbenzene might be the main active components affecting the clinical therapeutic effect of the volatile oil of Qingshang juantong decoction.

Objective To optimize the formula of Dahuang Xiaoshi decoction components based on its hepatoprotective activity and evaluate their efficacy.Methods The Wistar rats were randomly divided into blank group,model group,ursodeoxycholic acid group(positive group),and orthogonal groups of Dahuang Xiaoshi decoction components.The acute liver injury model induced by alpha-napthyl-i sothiocyanate(ANIT)was used to optimize the allocation ratio of Dahuang Xiaoshi decoction components by taking liver function indicators as an index and combining with multiple statistical methods.The additional Wistar rats were taken to induce liver injury and optimize the compatible dosage of Natrii Sulfas(0,1,2,4 g)in Dahuang Xiaoshi decoction components based on the biological signs and liver function biochemical indicators.On this basis,the Wistar rats were randomly divided into blank group,model group,ursodeoxycholic acid group and different dosages(low,medium and high)of the Zhibaihuang components group.The liver protective effect of Zhibaihuang components was systematically evaluated through the general biological signs,liver function biochemical indicators,lipid peroxide indicators,liver pathological examination and bile transport-related indicators.Results Dahuang Xiaoshi decoction components optimized by orthogonal and multiple statistics could improve the biological signs and ameliorate the biochemical abnormalities in rats with liver injury.Dahuang Xiaoshi decoction components combined with different dosages of Natrii Sulfas could slow down the mass loss of ANIT-induced acute liver injury rats(P＜0.01 or P＜0.05)and recall the abnormally elevated serum liver function enzyme activities(P＜0.01 or P＜0.05).Except for alkaline phosphatase(ALP),there was no statistical difference in regulating other liver function enzyme activity between different allocations of Natrii Sulfas.After comprehensive consideration,its composition was optimized to the Zhibaihuang components without Natrii Sulfas.Further pharmacodynamic evaluation results showed that the optimized Zhibaihuang components could improve their abnormal biological signs(P＜0.01 or P＜0.05),decrease the serum liver function enzyme activity(P＜0.01 or P＜0.05)and the levels of T-BiL and TG(P＜0.05),and restore the levels of hepatic lipid peroxide(P＜0.01 or P＜0.05),repair liver pathological injury,adjust the expression of bile transport proteins(P＜0.05),and thus exert good liver protective activity.Conclusion The optimized Dahuang Xiaoshi decoction components,Zhibaihuang components,was obtained through orthogonal,multiple statistics and univariate investigation.It could improve the abnormal biological signs of animals,protect the liver and reduce enzymes,resist lipid peroxidation,restore abnormal metabolic indicators,and repair liver pathological injury,which provides a reference for its further clinical application and development.

Objective To analyze the epidemiological characteristics of population-based cohort of patients with the comorbidity of cardiovascular and cerebrovascular diseases and type 2 diabetes in Lingnan area,and to study the related influencing factors in the onset and progression of the disease. Methods A retrospective cohort study was used to collect data from people who underwent physical examination in the Eleventh People's Hospital of Guangzhou from May 2022 to December 2023. Data mainly included questionnaire surveys,physical examinations,and laboratory testing indicators. The 2022 was defined as the baseline to statistically analyze the occurrence and development of the comorbidity of cardiovascular and cerebrovascular diseases and type 2 diabetes in this population,and to analyze the related influencing factors of comorbidity and distribution of traditional Chinese medicine constitution in comorbidity population. Results Finally,a total of 26498 subjects were included,from which there were 359 patients with the comorbidity of cardiovascular and cerebrovascular diseases and type 2 diabetes (comorbidity group),accounting for 1.4％ of the total. Among them,290 were male,accounting for 80.8％,which is much higher than female. The mean age was(61.6±9.5)years old,which was significantly higher than that of the non-comorbidity group. The cases of comorbidity group were mainly concentrated in the age group of 45-75 years old,and no cases were found in people under 35 years old. There were 293 patients with the comorbidity of ischemic cardiovascular disease and type 2 diabetes,whose proportion (81.6％) is much higher than that of other types. Significant differences between comorbidity group and non-comorbidity group were found in terms of gender,age,age distribution,height,body mass,body mass index (BMI),smoking,alcohol consumption,marital status,exercise,and dampness syndrome (P<0.05). About 1.0％ of population at the baselined converted from non-comorbidities or single disease to comorbidities. The proportion of newly diagnosed patients with the comorbidity of ischemic cardiovascular disease and type 2 diabetes is the highest,up to 68.9％. BMI overweight or obesity,large waist circumference,smoking,dampness syndrome and exercise were the risk factors affecting the comorbidity of cardiovascular and cerebrovascular diseases and type 2 diabetes. A total of 264 cases of comorbidity group had finished evaluation of traditional Chinese medicine body constitutions. The proportion of balanced constitution was the highest (31.1％),followed by dampness-heat constitution (18.2％),yang-deficiency constitution (13.3％) and phlegm-dampness constitution (11.7％). Conclusion The incidence of the comorbidity of cardiovascular and cerebrovascular diseases and type 2 diabetes is high in Lingnan area,which may be related to dampness constitution,BMI overweight or obesity,large waist circumference,smoking,dampness syndrome and lack of exercise.

Objective:To conduct stakeholder identification and problem analysis of inclusive health insurance participation in rare disease coverage based on the stakeholder perspective.Methods:A literature review and a Mitchell scale were used to identify,categorize,and analyze the relationship among the stakeholders,and qualitative interviews were conducted with identified stakeholders to explore their demands,positioning,and barriers to participation in inclusive health insurance.Results:The Healthcare Security Ad-ministration,Banking and Insurance Regulatory Commission,and the primary insurer were the most influential"identified stakehold-ers"in the development of inclusive health insurance.A total of 63 concepts were coded,and the stakeholders were analyzed and de-scribed in categories of claims,roles,strengths,difficulties,and behavioral strategies.Conclusion:There were differences in the de-mands of the stakeholders,lack of clarity of roles,and lack of effective synergy.

Objective:To extract rat brain microvascular endothelial cells(BMECs)using thin-layer cell culture method.Methods:The brain cortex of four-week-old SD rats was obtained,which was chopped,sieved,and digested with type II collagenase to obtain microvascular segments.The amount of culture medium was strictly controlled,and it was inoculated into culture flasks for primary culture.The target cells were morphologically observed by using an invert-ed phase contrast microscope,and the factor Ⅷ related antigen(FⅧRag)was identified by using immunocytochemi-cal staining.Results:The target cells cultured in the inverted phase contrast microscope showed typical endothelial-like monolayer cobble stone-like mosaic growth;FⅧRag positive cells accounted for more than 99％of the total cells.Conclusion:Thin-layer cell culture method can successfully isolate and cultivate high-purity rat BMECs.

Objective:To assess the imaging characteristics of muscle FDG metabolism, tumor incidence, and pulmonary interstitial changes in patients with anti-melanoma differentiation-associated gene 5 (MDA5) antibody positivity in 18F-FDG PET/CT imaging, and the value of 18F-FDG PET/CT in differentiating anti-MDA5 antibody positive dermatomyositis. Methods:From June 2016 to July 2019, the PET/CT images of 75 patients with dermatomyositis (21 males, 54 females, age (52.3±14.3) years; 34 anti-MDA5 antibody positive and 41 anti-MDA5 antibody negative) and 30 healthy controls (10 males, 20 females; age (53.5±11.8) years) were retrospectively analyzed in Renji Hospital, School of Medicine, Shanghai Jiao Tong University. The SUV max of muscle was measured and the mean of SUV max (mSUV max) was calculated. Statistics of patients with dermatomyositis complicated with neoplastic lesions and the SUV max of pneumonia lesions in patients with dermatomyositis complicated with interstitial pneumonia was determined. Independent sample t test, one-way analysis of variance, Student-Newman-Keuls (SNK) test and χ2 test were used to analyze data. The ROC curve analysis was used to analyze the diagnostic efficacy of mSUV max for the differential diagnosis of anti-MDA5 antibody positive dermatomyositis. Results:The muscle mSUV max of the control group, anti-MDA5 antibody positive and negative groups were 0.39±0.05, 0.66±0.21 and 0.87±0.29 ( F=39.93, P<0.001), respectively. The muscle mSUV max of dermatomyositis patients was increased compared with healthy controls ( q values: 6.76, 12.63, both P<0.001), and the muscle mSUV max of anti-MDA5 antibody negative was higher than positive ( q=5.79, P<0.001). The AUC was 0.74, and the cut-off value of muscle mSUV max was 0.75 with the accuracy of 74.7%(56/75). Of 41 patients with negative anti-MDA5 antibody, there were 6 (14.6%) had malignant tumor, while there was no malignant tumor in patients with positive anti-MDA5 antibody (0/34; χ2=5.41, P=0.020). There were 11 patients (26.8%, 11/41) with anti-MDA5 antibody negative dermatomyositis complicated with interstitial pneumonia and 33 patients (97.1%, 33/34) with anti-MDA5 antibody positive dermatomyositis complicated with interstitial pneumonia ( χ2=37.81, P<0.001). FDG metabolism in anti-MDA5 antibody positive patients was higher than that in anti-MDA5 antibody negative patients (lesion SUV max: 3.65±1.83 and 2.38±1.27; t=2.13, P=0.039). Conclusions:The muscle FDG metabolism of anti-MDA5 antibody positive dermatomyositis patients is higher than that of healthy controls, but lower than that of anti-MDA5 antibody negative patients. The incidence of neoplastic lesions in patients with positive anti-MDA5 antibody is lower than that in patients with negative anti-MDA5 antibody. The proportion and severity of interstitial pneumonia are higher in patients with positive anti-MDA5 antibody than in those with negative anti-MDA5 antibody. 18F-FDG PET/CT has certain value on identifying anti-MDA5 antibody positive dermatomyositis.

At present, the diagnosis, treatment, and social security of rare diseases have attracts social attention. As an important part of the multi-level health insurance system in China, commercial health insurance plays an increasingly important role in the supplement to basic medical insurance. Moreover, recently, the benefits of new healthcare reform policies have provided much room and great opportunities for the application of commercial health insurance for rare disease.This paper presents current medical insurance for patients with rare diseases and discuss the necessity and feasibility of bringing rare diseases into the coverage of commercial health insurance, and analyzes the current application and the potential problems of rare disease insurance in the commercial health insurance. This article also gives suggestions to the policies to make full use of the positive role of commercial insurance for the completion of multi-level health insurance system for rare disease.