Objective:To differentiate human embryonic stem cells ( hESCs ) into keratinocytes ( K-hESCs) and analyse the expression characteristics of biomarkers of K-hESCs.Methods: The hESCs of line H9 were seeded on matrigel in mTeSR1 medium.The hESCs were directly differentiated into kerati-nocytes in epithelial differentiation medium with bone morphogenetic protein 4, retinoic acid and N2 sup-plement.The karyotype of K-hESCs was analyzed, comparing the gene expression differences of K-hESCs with human gingival epithelial cells (HGECs), human immortalized oral epithelial cells (HIOECs) and HaCaT by Real-time PCR.Molecular characteristics of the cell differentiation were observed throughout the process by immunocytochemical techniques.Results:H9-hESCs were successfully differentiated into the cells that exhibited characteristics of keratinocytes in epithelial differentiation medium.The karyotype of K-hESCs was 46, XX; and the keratinocyte gene p63 expression in K-hESCs was significantly lower than that in HaCaT ( P<0.05) , but there was no significant difference of p63 expression in K-hESCs, comparing with that in HGECs and HIOECs ( P >0.05 ) .Conclusion: H9-hESCs could be directly differentiated into K-hESCs.The gene expression of K-hESCs was similar to that of epithelial cells in the early stage of monolayer cells differentiation with high proliferative activity.

To explore the analysis methods for composite traditional Chinese medicine (TCM) constitutions.

Objective:To explore the role and mechanism of the B7-H1 expressed by auto-keratinocytes in the intermingled skin grafting model in vitro(MELC). Methods:The intermingled skin grafting model(MELC) was established in vitro. Flow cytometry was performed to detect the expressions of B7-H1 in keratinocytes. The expressions of PD-1 in lymphocytes were measured at the same time. The levels of IL-10,Foxp3 and GATA-3 mRNA in lymphocytes were detected by Real-time quantitative PCR. Results: Through flow eytometry,in the MELC with auto-keratinocytes,the expression of B7-H1 in auto-keratinocytes and the PD-1 in lymphocytes were rising trend and the rising rate was in time-dependent manners(P<0. 01). RT-PCR assay indicated that the relative levels of IL-10, Foxp3,GATA-3mRNA expression were significant raised and the rising rate was in time-dependent manners (P<0. 01). Conclusion:In the intermingled skin grafting model,the auto-keratinocytes could express B7-H1 to enhance the expression of PD-1 in T cells. When B7-H1 combined with PD-1,the Th2 cells and Foxp3+Tregs were induced and suppressed the immune response in the intermingled skin grafting model.

Objective To explore the genetic mechanism of Yang-deficiency constitution by detecting genomic copy number variations (CNVs). Methods Thirty cases of Yang-deficiency constitution and 30 cases of balanced constitution were included according to the standards of Classification and Determination of Constitution in Traditional Chinese Medicine. DNA was extracted from white blood cells in peripheral blood. A genome-wide association study was conducted by using Affymetrix SNP 6.0 platform. CNVs of each sample were analyzed using PennyCNV software. The Yang-deficiency constitution-specific copy number variation regions (CNVRs) of each autosome were identified. CNVR-related genes and their annotations were searched at online Human Genome Browser. Results The mean number of CNVs in balanced constitution group was 12.63±3.39, ranging from 8 to 20. After stepwise elimination of two Yang-deficiency constitution subjects, the mean number of CNVs in Yang-deficiency constitution group was 15.04±8.95, ranging from 2 to 38. A total of 26 CNVRs were identified from 28 Yang-deficiency constitution subjects, including 19 duplicated CNVRs, 6 deleted CNVRs, and 1 mixed type CNVR. Most CNVRs were shared by a few Yang-deficiency constitution subjects, and only 7 CNVRs were shared by more than 5 Yang-deficiency constitution subjects. The functions of representative genes in Yang-deficiency constitution-specific CNVRs were related with extracellular and intracellular signal transduction, metabolic regulation, and immune response, etc. Conclusion Yang-deficiency constitution subjects have some specific genomic CNVs, which might result in Yang-deficiency constitution phenotypes by influencing the expression of genes associated with extracellular and intracellular signal transduction, material metabolism (energy metabolism), and immune response, etc.

Objective:To investigate the possible role of NKT cells in unexplained recurrent spontaneous abortion by measuring the NKT cell numbers,maturity and cytokine secretion of the placenta of mice with unexplained recurrent spontaneous abortion.Methods:Normal pregnancy model in hybrid by feeding CBA / J and BABL/C in a cage,and the model of unexplained recurrent spontaneous abortion was established by feeding CBA / J and DBA2/J in a cage.The number of NKT and CD3~+T cells was determined by flow-cytometry;Th1/Th2-relative cytokines were assayed by ELISA and T-bet expression was determinded by fluorescence quantitative PCR.Results:There was not significant change of CD3~+ T cells when compared between normal pregnancy and unexplained recurrent spontaneous abortion group (P＞0.05).In the course of normal pregnancy,the IFN-γ secreted by placenta lymphocytes decreased gradually,accompanied by the decline of NKT cell number and the proportion of mature cells;whereas in the course of unexplained recurrent spontaneous abortion,it was on the opposite.There was significant difference of T-bet mRNA expression between the two groups.T-bet mRNA expression was related to the proportion of mature NKT cells or placenta IFN-γ secretion by lymphocytes.Conclusion:Unexplained recurrent spontaneous abortion may be related to NKT cells disorders,NKT cells are of low-mature proportion and inadequate secretion of IFN-γ during early pregnancy,whereas are shown high-mature proportion and excessive secretion of IFN-γ during latter pregnancy;the anomaly of T-bet mRNA expression may be one of the factors leading to NKT cells disorder.

Objective:To apply the best evidence for the prevention of radial artery occlusion after transradial coronary angiography or intervention to clinical practice and evaluate its effect.Methods:This was a quasi-experimental study. Based on the evidence continuous quality improvement model, evidence-based practice method was used to obtain the best evidence, formulated review indicators, analyzed the obstacles in the practice process and took action strategies. The 88 patients who underwent transradial coronary angiography or intervention in the Cardiology Department of Qilu Hospital of Shandong University (Qingdao) from June 1 to 30, 2020 were selected as the baseline review group by convenience sampling. The 94 patients who underwent this treatment from September 1 to 30, 2020 were selected as the evidence application group. The baseline review group used the original perioperative management plan, and the evidence application group used the perioperative management plan based on the best evidence. The implementation rate of each review indicator, the incidence of radial artery occlusion, the degree of compression pain, and the comfort level of patients were compared between the two groups.Results:The implementation rates of review indicators 1, 2, 3, 5, 6, 7, 8 in the evidence application group were 100.0% (94/94), 100.0% (94/94), 11.7(11/94), 88.3% (83/94), 100.0% (94/94), 100.0%(94/94), 85.1%(80/94), respectively, which were higher than those in the baseline review group(all 0), except for the review indicator 4, the differences were statistically significant ( χ2 values were 9.00-178.02, all P<0.05). The incidence of radial artery occlusion and the incidence of pain Numerical Rating Scale>3 points in the evidence application group were 2.1% (2/94) and 3.2% (3/94), respectively, which were lower than 14.8% (13/88) and 23.9% (21/88) in the baseline review group; the comfort level of patients in the evidence application group was 96.8% (91/94), which was higher than 63.6% (56/88) in the baseline review group. The differences were statistically significant ( χ2 = 8.01, 15.21, 30.10, all P<0.05). Conclusions:The best evidence for the prevention of radial artery occlusion after transradial coronary angiography or intervention can be applied to clinical practice, which can standardize the behavior of medical staff, reduce the incidence of postoperative radial artery occlusion, reduce the degree of compression pain, and improve the comfort of patients.

SUMMARY Thehumanembryonicstemcells(hESCs)serveasaself-renewable,genetically-healthy, pluripotent and single source of all body cells,tissues and organs.Therefore,it is considered as the good standard for all human stem cells by US,Europe and international authorities.In this study,the standard and healthy human mesenchymal progenitors,ligament tissues,cardiomyocytes,keratinocytes,primary neurons,fibroblasts,and salivary serous cells were differentiated from hESCs.The human cellular health-safety of NaF,retinoic acid,5-fluorouracil,dexamethasone,penicillin G,adriamycin,lead ace-tate PbAc,bisphenol A-biglycidyl methacrylate (Bis-GMA)were evaluated selectively on the standar-dized platforms of hESCs,hESCs-derived cardiomyocytes,keratinocytes,primary neurons,and fibro-blasts.The evaluations were compared with those on the currently most adopted cellular platforms.Parti-cularly,the sensitivity difference of PM2.5 toxicity on standardized and healthy hESCs derived fibroblasts, currently adopted immortalized human bronchial epithelial cells Beas-2B and human umbilical vein endo-thelial cells (HUVECs)were evaluated.The results showed that the standardized hESCs cellular plat-forms provided more sensitivity and accuracy for human cellular health-safety evaluation.

Embryonic stem cells have unlimited proliferative capacity, which may provide a source of tendon stem/progenitor cells for tissue engineering. Experts of International Science and Technology Collaborative Program of Ministry of Science and Technology have developed a protocol consensus on differentiation of human embryonic stem cells into the tendon cells. The consensus recommends a protocol of two-step generation of human embryonic stem cells into tendon cells: the human embryonic stem cells are first differentiated into mesenchymal stem cells on different material surfaces; then with the scaffold-free tissue engineering tendon formed by high-density planting, the mesenchymal stem cells are induced into tendon cells under static or dynamic mechanical stimulation in vivo and in vitro. Tissue engineering tendon established in vitro by the protocol can be used as a model in toxicological analysis and safety evaluation of tendon-relevant small molecule compounds, medical materials and drugs.
Cell Differentiation ; Consensus ; Human Embryonic Stem Cells ; cytology ; Humans ; Mesenchymal Stromal Cells ; cytology ; Tendons ; cytology ; Tissue Engineering