Objective To explore the application value of anhydrous ethanol as an alternative to methanol in the preparation of chromosomal specimens from peripheral blood lymphocytes, and to establish a set of quantitative analytical methods for objectively evaluating the effectiveness of specimen preparation. Methods Residual blood samples from routine laboratory slide preparation were used for lymphocyte culture. The standard slide preparation method was employed. The fixative in the control group was methanol and glacial acetic acid (3∶1). Four experimental groups were set up based on the ratio of anhydrous ethanol to glacial acetic acid in the fixative (volume ratios of 3∶1, 5∶1, 7∶1, and 9∶1 for experimental groups 1, 2, 3, and 4, respectively). A chromosomal analysis was conducted using an automated chromosome scanning/image analysis system to evaluate the morphology and dispersion of metaphase chromosomes in both control and experimental groups. Comparisons were made between the control and experimental groups regarding the dic + r aberration rate, ace aberration rate, chromosomal aberration rate, chromosome dispersion index, chromosome overlapping ratio, and dispersion index/overlapping ratio. Results Microscopic evaluation revealed that the preparation quality of experimental groups 1 and 2 was comparable to the control group. No statistically significant differences were observed in dic + r aberration rate between each of the experimental groups and the control (P > 0.05). All experimental groups except group 4 showed no significant differences in ace aberration rate and chromosome aberration rate compared with the control group (P > 0.05). Experimental groups 1 and 2 showed no significant differences in chromosome dispersion index, overlapping ratio, and dispersion index/overlapping ratio compared with the control group (P > 0.05). Conclusion A mixture of anhydrous ethanol and glacial acetic acid at a 5∶1 ratio is recommended for use as a fixative in the preparation of chromosomal specimens from peripheral blood lymphocytes. A quantitative index system for assessing the quality of chromosomal specimens was established, enabling objective evaluation of slide preparation effectiveness.

ObjectiveTo establish an allele-specific polymerase chain reaction （PCR） method for identifying Scolopendra dispensing granules， so as to ensure the quality and therapeutic effects of Scolopendra and its preparations. MethodThe primer interval suitable for the PCR was selected based on the cytochrome c oxidase subunit 3（COX-3） gene sequence of Scolopendra， and the single nucleotide polymorphism （SNP） loci of Scolopendra and its adulterants were mined from the interval for the design of specific primers. The samples of Scolopendra and its adulterants were collected. The PCR system was established and optimized regarding the annealing temperature， cycles， Taq enzymes， DNA template amount， PCR instruments， and primer concentrations， and the specificity and applicability of this method were evaluated. ResultThe PCR system was composed of 12.5 μL 2×M5 PCR Mix， 0.4 μL forward primer （10 μmol·L^-1）， 0.4 μL reverse primer （10 μmol·L^-1）， 2.5 μL DNA template， and 9.2 μL sterile double distilled water. PCR parameters： Pre-denaturation at 94 ℃ for 3 min， 30 cycles （94 ℃ for 20 s， 62 ℃ for 20 s， 72 ℃ for 45 s）， and extension at 72 ℃ for 5 min. After PCR amplification with the system and parameters above， the electrophoresis revealed a bright band at about 135 bp for Scolopendra and no band for the adulterants. ConclusionThe established allele-specific PCR method can accurately identify the medicinal materials， decoction pieces， and standard decoction freeze-dried powder of Scolopendra， as well as the intermediates and final products of Scolopendra dispensing granules， which is of great significance for ensuring the quality and clinical efficacy of Scolopendra and its preparations.

Objective:To explore the impact of health self-focused attention on knowledge, attitude and practice of health management in patients with essential hypertension.Methods:This was a cross-sectional study. A total of 190 patients with essential hypertension were randomly selected from Shanghai Dapuqiao Community Health Service Center to participate in a questionnaire survey from October to December, 2021. The contents of the questionnaire included the general information of the patients, the degree of influence of hypertension on individuals, and the patients′ knowledge and personal cognition of hypertension, self-management behaviors, health self-focused attention, and blood pressure control. SPSS 25.0 and AMOS 24.0 were used to develop a structural equation model for analysis.Results:The recovery rate of questionnaires was 100.0% (190/190). Of the 190 patients, there were 82 males and 108 females with a mean age of (71.2±8.5) years. According to blood pressure, 190 hypertensive patients were divided into blood pressure controlled group ( n=119) and blood pressure non-controlled group ( n=71). There were significant differences in the total scores of hypertension self-management behavior and health self-focused attention between the two groups ( P<0.05). Correlation analysis showed that the degree of influence of hypertension on individuals was negatively correlated with the disease knowledge and personal cognition, and the performance of hypertension self-management behavior ( r=-0.39 and -0.28, P<0.01), while the disease knowledge and personal cognition were positively correlated with the performance of hypertension self-management behavior and health self-focused attention ( r=0.29 and 0.27, P<0.01). Hypertension self-management behavior was positively correlated with health self-focused attention and blood pressure control ( r=0.28 and 0.48, P<0.01), and health self-focused attention was positively correlated with blood pressure control ( r=0.21, P<0.01). The equation model analysis showed that health self-focused attention had a driving effect on patients′ disease knowledge and cognition and self-management behavior. Health self-focused attention significantly affected patients′ mastery of disease knowledge and formation of correct cognition (path coefficient=0.28). In addition, hypertension patients′ disease knowledge storage and internal recognition of prevention and treatment strategies determined their self-management behavior (path coefficient=0.20). On the other hand, the impact of hypertension on patients life, work and economic burden played a negative role in their acceptance of disease management knowledge, formation of correct concepts, and adoption of self-management behaviors with adjustment coefficients of -0.48 and -0.22, respectively. Conclusion:Health self-focused attention may play an initial role in the process of changing the knowledge, attitude and behavior of hypertension patients.

With the optimization of surgical technologies and postoperative management regimens, the number of lung transplantation has been significantly increased, which has become an important treatment for patients with end-stage lung disease. However, due to the impact of comprehensive factors, such as bronchial ischemia and immunosuppression, the incidence of airway stenosis after lung transplantation is relatively high, which severely affects postoperative survival and quality of life of lung transplant recipients. In recent years, with the improvement of perioperative management, organ preservation and surgical technologies, the incidence of airway stenosis after lung transplantation has been declined, but it remains at a high level. Early diagnosis and timely intervention play a significant role in enhancing clinical prognosis of patients with airway stenosis. In this article, the general conditions, diagnosis, treatment and prevention of airway stenosis after lung transplantation were reviewed, aiming to provide reference for comprehensive management of airway stenosis after lung transplantation and improving clinical prognosis of lung transplant recipients.

ObjectiveTo determine the serum antibody level and risk factors in the adolescent population in a county in Zhejiang Province， following the immunization with inactivated COVID-19 vaccine， and to construct a prediction model for antibody concentration. MethodsWe conducted the study in a county in Zhejiang Province， employing a stratified cluster random sampling strategy in school children who had received the inactivated COVID-19 vaccine. Data on gender， age， type of vaccine， and time of vaccination was collected. Serum samples were also collected to test for anti-S and N IgG antibody against the SARS-CoV-2 by using chemiluminescent immunoassay （CLIA）. Risk factors were determined to construct a prediction model for antibody concentration. ResultsThe IgG antibody concentration was significantly higher in girls， those who received two doses， and those who had simply received the KX vaccine . It decreased with age and time interval between the sampling and last vaccination. The prediction model constructed by random forest regression in the study had a better model fit and predictive ability than that by the multivariable linear stepwise regression. ConclusionGender， age， vaccination dose， type of vaccine， and time of vaccination are associated with vaccination effectiveness of inactivated COVID-19 vaccines in adolescents. Prediction model could predict the antibody level in the vaccinated population， which can provide a new tool for better evaluation of vaccination effectiveness against emerging infectious diseases in future.

ObjectiveTo understand the acceptance of sex-related behaviors， sexual safety awareness， and refusal skills among junior high school students and conduct association analysis， and to provide a reference for the implementation of sex and reproductive health education for adolescents. MethodsA questionnaire survey related to knowledge of sexual and reproductive health was conducted among 478 students from two junior high schools in Jiading District， Shanghai. ResultsThe pass rate for sex and reproductive health knowledge among junior high school students was only 36.6%， with acceptance rates for dating， watching pornography， engaging in sexual behavior， pregnancy， and artificial abortion at 24.5%， 11.1%， 5.6%， 4.8%， and 5.9%， respectively. Binary logistic regression analysis showed that students who passed the sexual and reproductive health assessment had a significantly lower acceptance rate of sex-related behaviors than those who did not （aOR=1.666， 95%CI： 1.124‒2.470， P=0.011）. Adolescents with a passing knowledge of sexual and reproductive health （aOR=1.607， 95%CI： 1.060‒2.438， P=0.026） or a lower acceptance of sex-related behaviors （aOR=4.199， 95%CI： 2.804‒6.289， P<0.001） had higher awareness of sexual safety. Adolescents with a passing knowledge of sexual and reproductive health （aOR=1.691， 95%CI： 1.066‒2.684， P=0.026）， a lower acceptance of sex-related behaviors （aOR=1.756， 95%CI： 1.130‒2.729， P=0.012）， or higher awareness of sexual safety （aOR=2.872， 95%CI： 1.826‒4.516， P<0.001） had stronger skills to refuse sex-related behaviors. ConclusionSexual and reproductive attitudes， sexual safety awareness， and refusal skills for sex-related behaviors among junior high school students in Jiading District need to be improved. Schools should focus on cultivating adolescents’ sexual and reproductive knowledge and related skills， and advocate for relevant institutions to provide appropriate sexual healthcare services to meet their needs for reproductive healthcare.

ObjectiveTo explore the correlation between blood stasis constitution （BSC） and diseases based on constitution literature involving “constitution-diseases correlation”. MethodsA comprehensive search was conducted on six Chinese and English electronic databases including CNKI， Wanfang， VIP， SinoMed， PubMed and Embase to find all clinical researches on the correlation between constitution and diseases using the Classification and Identification of Chinese Medicine Constitution standard from April 1st， 2009 to December 31st， 2022， and the participants of the research were BSC related. By analyzing the characteristics of the literature， such as authors， publication institutes， participants， and results， the disease with the highest proportion of BSC distribution or BSC as their risk factors or protective factors were summarized to explore the correlation between BSC and diseases. ResultsTotally 135 clinical studies on diseases highly related to BSC were included， with a total sample size of 71 172 cases.There were 27 keywords in the articles appeared more than 3 times， including the elderly， lumbar disc herniation， coronary heart disease， cardiovascular disease， and endometriosis. In the author's clustering， included studis were mainly from Shenzhen Hospital of Beijing University of Chinese Medicine， Second Affiliated Hospital of Guangzhou University of Chinese Medicine， and Wenzhou Central Hospital. In terms of blood stasis related diseases， 81 studies showed that BSC was the most common type of constitution in the study population， involving 48 disease or morbid states. The diseases and median proportions of BSC with reported literature ≥3 included coronary heart disease （28.8%）， endometriosis （31.3%）， neurocognitive impairment （26.4%）， lumbar disc herniation （26.0%）， ischemic stroke （25.0%）， adenomyosis （34.7%）， and endometrial polyps （25.0%）. Fifty-eight studies found that BSC was a risk factor for disease occurrence，and these diseases reported more than 3 times included hypertension （median OR = 2.956）， type 2 diabetes （median OR = 3.436），osteoporosis （median OR = 5.171）， sudden deafness （median OR = 3.827） and endometriosis （median OR = 5.412）. One study indicated BSC as the protective factor of lateral growth tumor of large intestine （median OR = 0.161）. ConclusionBSC is closely related to circulatory system diseases， urogenital system diseases， and musculoskeletal system diseases.

ObjectiveTo understand the usage of antibiotics in outpatients in a county of Zhejiang Province from 2015 to 2020， so as to provide a basis for further standardizing the clinical application of antibiotics in this region. MethodsOutpatient records and prescription records of 146 public medical institutions from 2015 to 2020 were extracted from the electronic medical record data sharing platform system of medical institutions in a county of Zhejiang Province. The utilization rates and the number of types of antibiotics used in outpatient patients were described. According to the drug anatomy， therapeutic and chemical classification system （ATC）， clinical application classification and dosage form， the use status of different types of antibiotics was observed. The annual percentage change （APC） was used to analyze the time trend of antibiotic use. Among the antibiotic prescriptions in each year， the proportions of prescriptions aimed for different use reasons and patients were compared to analyze the changes in the structures of antibiotic prescriptions. ResultsDuring 2015‒2020， a total of 2 861 438 prescriptions were issued in146 primary public medical institutions in this county， including 314 642 prescriptions for antibiotics， and the overall utilization rate of antibiotics was 11%. The utilization rate of antibiotics showed a decreased trend across the years （from 14.23% in 2015 to 7.59% in 2020，APC=-11.51%， 95%CI=-0.01%‒-21.7%， t=-12.05， P<0.001） and an obvious seasonal variation character （higher in winter and spring， lower in summer and autumn）. Cefoxitin sodium for injection was the most used antibiotic in 2015， and cefuroxime was the most used antibiotic from 2015 to 2020， respectively. The utilization rates of cephalosporins （APC=-11.06%， 95%CI=0‒-20.89%， t=-22.233， P<0.001）， quinolones （APC=-9.74%， 95%CI=0‒-18.53%， t=-7.652， P=0.002）， macrolides （APC=-17.52%， 95%CI=0‒-17.52%， t=-3.626， P=0.022） and lincoamides （APC=-49.01%， 95%CI=0‒-74%， t=-3.35， P=0.029） showed a decreasing trend across the years. There were differences in the distribution of drug use reasons （χ²=9 458.427， P<0.001）， as well as the age （χ²=5 421.709， P<0.001） and sex （χ²=245.322， P<0.001） of patients. During the observation period， 50.69% of antibiotic prescriptions were used to treat respiratory diseases， with the highest proportion of 57.67% in 2015 and the lowest proportion of 34.93% in 2020. 58.37% of patients were aged 50‒70 years， with a decreasing proportion of patients under 50 years （17.83% in 2015， 6.64% in 2020） and an increasing proportion of patients over 70 years （24.1% in 2015， 38.41% in 2020）. ConclusionThe utilization rate of antibiotics used in outpatients from primary public medical institutions in a county of Zhejiang Province shows a decreasing trend across the years， but higher than that of secondary and above hospitals in China， and also higher than that of primary medical institutions in economically-developed areas. The management and monitoring of the clinical use of antibiotics in the county should be actively strengthened.

BACKGROUND:Compared with traditional two-dimensional culture,three-dimensional microtissue culture can show greater advantages.However,more favorable cultivation methods in three-dimensional culture still need to be further explored. OBJECTIVE:To evaluate the cell behavior of microtissue and its ability to promote cartilage formation under two three-dimensional culture methods. METHODS:Cartilage-derived microcarriers were prepared by chemical decellularization and tissue crushing.DNA quantification and nuclear staining were used to verify the success of decellularization,and histological staining was used to observe the matrix retention before and after decellularization.The microcarriers were characterized by scanning electron microscopy and CCK-8 assay.Cartilage-derived microtissues were constructed by combining cartilage-derived microcarriers with human adipose mesenchymal stem cells through three-dimensional static culture and three-dimensional dynamic culture methods.The cell viability and chondrogenic ability of the two groups of microtissues were detected by scanning electron microscopy,live and dead staining,and RT-qPCR. RESULTS AND CONCLUSION:(1)Cartilage-derived microcarriers were successfully prepared.Compared with before decellularization,the DNA content significantly decreased after decellularization(P<0.001).Scanning electron microscope observation showed that the surface of the microcarrier was surrounded by collagen,maintaining the characteristics of the natural extracellular matrix of cartilage cells.CCK-8 assay indicated that microcarriers had no cytotoxicity and could promote cell proliferation.(2)Scanning electron microscopy and live and dead staining results showed that compared with the three-dimensional static group,the three-dimensional dynamic group had a more extended morphology of microtissue cells,and extensive connections between cells and cells,between cells and matrix,and between matrix.(3)The results of RT-qPCR showed that the expressions of SOX9,proteoglycan,and type Ⅱ collagen in microtissues of both groups were increased at 7 or 14 days.The relative expression levels of each gene in the three-dimensional dynamic group were significantly higher than those in the three-dimensional static group at 14 days(P<0.05).At 21 days,the three-dimensional static group had significantly higher gene expression compared with the three-diomensional dynamic group(P<0.001).(4)The results showed that compared with three-dimensional static culture microtissue,three-dimensional dynamic culture microtissue could achieve higher expression of chondrogen-related genes in a shorter time,showing better cell viability and chondrogenic ability.

Objective:To study the in vitro construction of functional and self-renewing cartilage organoids based on cartilage acellular extracellular matrix (ECM) microcarriers.Methods:Fresh porcine articular cartilage was taken. The merely crushed cartilage particles were set as natural cartilage group and ECM microcarriers of appropriate particle size, which were prepared by the acellular method of combining physical centrifugation and chemical extraction, were set as microcarrier group. Cartilage organoids were constructed by loading human umbilical cord mesenchymal stem cells (hUCMSCs) and human chondrocytes (hCho) with a ratio of 3∶1 with microcarriers through a rotating bioreactor. The organoids with different induction times were divided into 0-, 7-, 14-, and 21-day induction groups. The cell residues of the microcarrier group and natural cartilage group were evaluated by 4′, 6-diaminidine 2-phenylindole (DAPI) fluorescence staining and DNA quantitative analysis. The retention of microcarrier components was observed by Safranin O and toluidine blue stainnings, and the collagen and glycosaminoglycan (GAGs) levels in the microcarrier group and the natural cartilage group were determined by colorimetric method and dimethyl-methylene blue (DMMB) method. The microcarriers were further characterized by scanning electron microscopy and energy dispersive spectroscopy. The hUCMSCs cultured with Dulbecco′s Modified Eagle′s Medium (DMEM) supplemented with fetal bovine serum (FBS) in a volume fraction of 10% was used as the control group and the hUCMSCs cultured with the microcarrier extract was used as the experimental group. Subgroups of hUCMSCs cultured at 3 time points: 1, 3 and 5 days were set up in the two groups separately. Cell Counting Kit 8 (CCK-8) was used to detect the biocompatibility of the two groups. The cellular activity of the organoids of the 0-, 7-, 14-, and 21-day induction groups was detected by live/dead staining and the self-renewal ability of the cartilage organoids of the 14-day induced group was identified by Ki67 fluorescence staining. The organoids of the 7-, 14-, and 21-day induction groups were detected by RT-PCR in terms of the expression levels of chondrogenesis-related marker aggrecan (ACAN), type II collagen (COL2A1), SRY-related high mobility group-box gene-9 (SOX9), cartilage hypertrophy-and mineralization-related marker type I collagen (COL1A1), Runt-related transcription factor-2 (RUNX2), and osteocalcin (OCN). Colorimetric and DMMB assays were performed to determine the ability of organoids in the 0-, 7-, 14-, and 21-day induction groups to secrete collagen and GAGs.Results:The results of DAPI fluorescent staining showed that the natural cartilage group had a large number of nuclei while the microcarrier group hardly had any nuclei. The DNA content of the microcarrier group was (7.8±1.8)ng/mg, which was significantly lower than that of the natural cartilage group [(526.7±14.7)ng/mg] ( P<0.01). Saffranin O and toluidine blue staining showed that the microcarrier was dark- and uniform-colored and it kept a lot of cartilage ECM components. The collagen and GAGs contents of the microcarrier group were (252.9±1.4)μg/mg and (173.4±0.8)μg/mg, which were significantly lower than those of the natural cartilage group [(311.9±2.2)μg/mg and (241.3±0.7)μg/mg] ( P<0.01). Scanning electron microscopy showed that the surface of the microcarriers had uneven and interleaved collagen fiber network. The results of energy spectrum analysis showed that elements C, O and N were evenly distributed in the microcarriers, indicating that the composition of the microcarrier was uniform. The microcarrier had good biocompatibility and there was no statistical significance in the results of CCK-8 test between the control group and the experimental group after 1 and 3 days of culture ( P>0.05). After 5 days of culture, the A value of the experimental group was 0.53±0.02, which was better than that of the control group (0.44±0.03) ( P<0.05). In the 0-, 7-, 14-, and 21-day induction groups, hUCMSCs and hCho were attached to the surface of the microcarriers, with good cellular activity, and the live/death rates were (70.6±1.1)%, (80.5±0.6)%, (94.5±0.9)%, and (90.8±0.5)% respectively ( P<0.01). There were a large number of Ki67 positive cells in cartilage organoids. RT-PCR showed that the expression levels of ACAN, COL2A1, SOX9, COL1A1, RUNX2 and OCN were 1.00±0.09, 1.00±0.24, 1.00±0.18, 1.00±0.03, 1.00±0.06 and 1.00±0.13 respectively in the 7-day induction group; 4.16±0.28, 5.09±1.25, 5.65±1.05, 0.47±0.01, 1.68±0.02 and 0.21±0.06 respectively in the 14-day induction group; 13.42±0.92, 3.07±0.21, 1.84±1.08, 2.72±0.17, 2.91±0.18 and 3.32±1.20 respectively in the 21-day induction group. Compared with the 7-day induction group, the expression levels of ACAN, COL2A1, SOX9 and RUNX2 in the 14-day group were increased ( P<0.05), but COL1A1 expression level was decreased ( P<0.05), with no significant difference in OCN expression level ( P>0.05). Compared with the 7-day induction group, the expression levels of ACAN, COL1A1 and RUNX2 in the 21-day induction group were significantly increased ( P<0.01), with no significant differences in the expression levels of COL2A1, SOX9 and OCN ( P>0.05). Compared with the 14-day induction group, the expression levels of ACAN, COL1A1, RUNX2 and OCN in the 21-day group were increased ( P<0.05 or 0.01), with no significant difference in the expression level of COL2A1 ( P>0.05), but the expression level of SOX9 was decreased ( P<0.05). The contents of collagen in 0-, 7-, 14-and 21-day induction groups were (219.15±0.48)μg/mg, (264.07±1.58)μg/mg, (270.83±0.84)μg/mg and (280.01±0.48)μg/mg respectively. The GAGs contents were (171.18±1.09)μg/mg, (184.06±1.37)μg/mg, (241.08±0.84)μg/mg and (201.14±0.17)μg/mg respectively. Compared with the 0-day induction group, the contents of collagen and GAGs in 7-, 14-, and 21-day induction groups were significantly increased ( P<0.01), among which the content of collagen was the lowest in 7-day induction group ( P<0.01) but the highest in the 21-day induced group ( P<0.01); the content of GAGs was the lowest in the 7-day induced group ( P<0.01) but the highest in the 14-day induction group ( P<0.01). Conclusions:The microcarriers prepared by combining physical and chemical methods are decellularized successfully, with more matrix retention, uniform composition and on cytotoxicity. By loading microcarriers with hUCMSCs and hCho, cartilage organoids are successfully constructed in vitro, which are characterized by good cell activity, self-renewal ability, strong expression of genes related to chondrogenesis and secretion of collagen and GAGs. The cartilage organoids constructed at 14 days of induction have the best chondrogenic activity.