Objective:To establish a presenilin enhancer-2 (PSENEN) gene-silenced human immortalized keratinocyte (HaCaT) cell model, and to evaluate the effect of PSENEN gene silencing on the proliferation of and γ-secretase expression in HaCaT cells.Methods:Three shRNAs targeting the PSENEN gene were constructed, and inserted into the linearized LV3-pGLV-h1-GFP-puro vector to establish a recombinant lentiviral expression plasmid. After restriction enzyme digestion and sequencing, lentiviral packaging and purification were performed, and lentiviral titer was determined. Cultured HaCaT cells were divided into 5 groups: shRNA1, shRNA2 and shRNA3 groups treated with the lentivirus solutions containing PSENEN gene-targeted shRNA1, shRNA2 and shRNA3 respectively, NC group treated with the lentivirus solution containing a negative control shRNA (shNC) , and blank group treated without lentivirus solution. After transfection, inverted fluorescence microscopy was performed, and transfection efficiency was determined by flow cytometry. Cell counting kit-8 (CCK8) assay was performed to evaluate the effect of PSENEN gene silencing on the proliferation of HaCaT cells, and real-time fluorescence-based quantitative PCR (qPCR) and Western blot analysis were conducted to determine the mRNA and protein expression of PSENEN, nicastrin (NCT) , presenilin-1 (PS1) and anterior pharynx defective 1a (APH1a) genes respectively. Statistical analysis was carried out by using repeated measures analysis of variance, one-way analysis of variance, and least significant difference t test for multiple comparisons. Results:Inverted fluorescence microscopy showed that fluorescence was observed in the shRNA1 group, shRNA2 group, shRNA3 group and NC group, and flow cytometry showed that the transfection efficiency was over 98% in the above 4 groups. qPCR and Western blot analysis revealed that the mRNA and protein expression of PSENEN gene significantly decreased in the shRNA1 (0.187 ± 0.010, 0.219 ± 0.097, respectively) , shRNA2 (0.163 ± 0.022, 0.208 ± 0.014, respectively) and shRNA3 (0.174 ± 0.009, 0.185 ± 0.062, respectively) groups compared with the NC group (1.054 ± 0.272, 1.076 ± 0.075, respectively, all P < 0.001) . CCK8 assay showed that the cellular proliferative activity significantly increased in the shRNA1 group compared with the NC group at 0, 12, 36 and 48 hours (all P < 0.05) , and there was no significant difference between the 2 groups at 24 or 60 hours (both P > 0.05) ; the cellular proliferative activity was significantly higher in the shRNA2 and shRNA3 groups than in the NC group at 0, 12, 24, 36, 48 and 60 hours (all P < 0.05) . There was no significant difference in the mRNA expression of NCT, PS1 and APH1a genes among the shRNA1 group, shRNA2 group, shRNA3 group, NC group, and blank group ( F= 8.168, 4.644, 1.981, respectively, all P > 0.05) , while the relative protein expression level of mature NCT (mNCT) , immature NCT (imNCT) , carboxyl-terminal fragment of PS1 (PS1-CTF) and APH1a significantly differed among the above 5 groups ( F= 39.268, 5.929, 27.842, 20.663, respectively, all P ≤ 0.01) . Compared with the NC group, the shRNA1, shRNA2 and shRNA3 groups all showed significantly decreased protein expression of mNCT, PS1-CTF and APH1a (all P < 0.01) , but insignificant changes in imNCT protein expression (all P > 0.05) . Conclusion:The PSENEN gene-silenced HaCaT cell model was successfully constructed, and the PSENEN gene silencing could lead to an increase in the cellular proliferative activity of HaCaT cells and a decrease in the protein expression of γ-secretase subunits mNCT, PS1-CTF and APH1a.

Objective To measure expressions of nicastrin and its downstream Notch-HES signaling pathwayassociated proteins in skin lesions of patients with acne inversa harbouring nicastrin gene mutations.Methods An immunohistochemical study was performed to measure the expressions of nicastrin and Notch-HES signaling pathwayassociated proteins in paraffin-embeded skin samples from lesions of 4 patients with acne inversa and confirmed nicastrin mutations and from normal skin of 6 human controls.Spearman correlation analysis was carried out to assess the relationship between the expressions of nicastrin and Notch-HES signaling pathway-associated proteins.Results In normal control skin samples,nicastrin was widely distributed in the full-thickness epidermis and skin appendages such as pilosebaceous units,apocrine glands and eccrine glands.However,the expressions of nicastrin and Notch-HES signaling pathway-associated proteins were markedly decreased in the epidermis and hair follicle infundibulum in lesions of patients harbouring nicastrin gene mutations compared with normal control skin.Furthermore,nicastrin expression was positively correlated with Notchl,Notch3 and HES-1 expressions (r =0.831,0.748 and 0.807,P ＜ 0.01,0.05 and 0.01 respectively),but not significantly correlated with Notch2 or HES-5 expressions (r =0.597,0.591 respectively,both P ＞0.05).Conclusion Nicastrin expression markedly decreases in lesions of patients with acne inversa harbouring nicastrin gene mutations,and is positively correlated with the expressions of several Notch-HES signaling pathway-associated proteins,suggesting that the decrease in nicastrin expression may take part in the pathogenesis of acne inversa by influencing the expression of the downstream Notch-HES signaling pathway.

Objective To investigate the efficacy of enteral nutrition containing glutamine on improving blood lipids and immune parameters in elderly patients with gastrointestinal cancer.Methods 98 cases of elderly patients with gastrointestinal cancer were randomly divided into observation group(n=5 1)and control group (n=47).The control group was additionally given conventional enteral nutrition,and the observation group was additionally given enteral nutrition containing glutamine.The levels of albumin (ALB),tumor necrosis factor (TNF),indicators of cellular immunity,indicators of humoral immune function and blood lipid,before and after operation,were observed in the two groups.Results 9 days after operation,the levels of ALB and total cholesterol,per-centage of CD8 + cells were lower than those in the control group,and levels of TFN,IgA,triacylglycerol,low density lipoprotein cholesterin,percentage of CD4 + cells and CD4/CD8 ratio were higher than those in the control group,and have significant differ-ences (P <0.05).While,there was no statistically significant difference of the percentage of CD3 cells between the groups(P >0.05).Conclusion Enteral nutrition containing glutamine can significantly improve blood lipids and immune function in elderly pa-tients with gastrointestinal cancer,which may have important clinical research value and be worthy of further application.

Epithelioid Hemangioendotheliomais a rare, low-grade malignant vascular tumour. It’scalled hepatic epithelioid hemangioendothelioma(HEHE), when it occurs in liver. It can be metastatic and postoperative recurrence. There are few cases have been reported in the literature at home and abroad because of its rarity. The treatment of HEHE is also controversial. With the continuous improvement of surgical techniques of liver transplantation, it is increasingly applied to treat liver failure patients caused by HEHE. Our paper reviews the literature on disease characteristics of HEHE, and liver transplantation for HEHE indications, immunotherapy and prognosis, to illustrate the status and progress of liver transplantation for HEHE.

Objective To construct a lentiviral vector delivering the Nicastrin (NCT) gene-targeted short hairpin RNA (shRNA) and determine gene-silencing efficiency of the vector in the human immortalized keratinocyte cell line HaCaT,and to construct a NCT gene-silenced HaCaT cell model to lay an experimental foundation for subsequently studying effects of NCT gene silencing on biological behavior of keratinocytes.Methods Three NCT gene-targeted shRNAs were designed and inserted into the pGLV3/ H1/GFP + Puro vector to construct three recombinant plasmids,which were then confirmed by sequencing.Recombinant plasmids combined with lentivirus packaging plasmids were co-transfected into 293T cells to obtain lentivirus particles,and the virus titer was determined.Cultured HaCaT cells were divided into 3 groups:blank group receiving no treatment,negative control group infected with the empty vector LV3-shNC,interference groups infected with lentivirus NCT-shRNA1,-shRNA2,-shRNA3,respectively.Flow cytometry was performed to determine transfection efficiency,and real-time fluorescence-based quantitative PCR (qRT-PCR) and Western blot analysis were conducted to determine efficiency of target gene silencing in HaCaT cells,so as to select the most efficient interference sequence.Results Sequencing analysis indicated that recombinant lentiviral vector NCT-shRNA was constructed successfully.After co-transfection of recombinant plasmids and lentivirus packaging plasmids into 293T cells,the titer of recombinant lentivirus particles was about 109 TU/ml.Flow cytometry showed that the transfection efficiency was greater than 95％.qRT-PCR revealed that the NCT mRNA expression was obviously down-regulated in the interference group compared with the negative control group,and NCT-shRNA1 was the most efficient sequence with interference efficiency being 75％.Western blot analysis showed that the inhibition rate of NCT protein expression was 71.7％ in the shRNA1 group compared with the negative control group.Conclusion The most efficient NCT-shRNA interference sequence is screened out,and the recombinant lentiviral vector NCT-shRNA and an NCT gene-silenced HaCaT cell model are both constructed successfully.

Objective:To investigate changes of nicastrin (NCSTN) downstream molecules in signaling pathways related to cell proliferation and differentiation after silencing the expression of the NCSTN gene in the human immortalized keratinocyte cell line HaCaT.Methods:HaCaT cells were divided into 3 groups: interference group transfected with a specific small interfering RNA (siRNA) targeting NCSTN (NCSTN-siRNA) , negative control group transfected with a negative control siRNA, and blank control group transfected with the equal amount of transfection reagent. Real-time PCR and Western blot analysis were performed to measure the NCSTN mRNA and protein expression in groups, in order to verify the transfection efficiency. Differences in gene expression profiles in HaCaT cells were detected between the interference group and negative control group by using Agilent whole-genome microarray, and differentially expressed genes were identified based on a fold change ≥ 2.0 with a P value ≤ 0.05. Gene ontology (GO) enrichment analysis was employed to identify the roles of the differentially expressed genes, and then to screen out significantly differentially expressed genes associated with proliferation and differentiation of keratinocytes, some of which were verified by real-time PCR. Results:The interference group showed significantly decreased mRNA and protein expression of NCSTN (0.287 ± 0.090, 0.443 ± 0.085, respectively) compared with the negative control group (0.969 ± 0.127, 1.047 ± 0.114, respectively) and blank control group (1.000 ± 0.151, 1.000 ± 0.111, F = 30.787, 31.139, respectively, both P = 0.001) . Whole genome-expression analysis using an Agilent microarray platform revealed 605 downregulated genes and 444 upregulated genes in HaCaT cells in the interference group compared with the negative control group. GO analysis showed that differentially expressed genes were enriched into 4 biological processes, including epithelial development, epithelial cell differentiation, keratinocyte differentiation and keratinization. The significantly differentially expressed genes associated with proliferation and differentiation of keratinocytes, including the Sprouty-related protein with EVH1 domain 2, fibroblast growth factor 7, insulin-like growth factor-binding protein 5, Rho-associated coiled-coil kinase 2 and bone morphogenetic protein 6 genes, were verified by real-time PCR, and the verification results were consistent with the difference trend shown by the microarray results. Conclusion:The loss of NCSTN gene function may affect the normal proliferation and differentiation of keratinocytes by regulating the expression of its downstream molecules in signaling pathways associated with cell proliferation and differentiation.

OBJECTIVETo detect mutation of adenosine deaminase acting on RNA1 (ADAR1) gene in a pedigree affected with dyschromatosis symmetrical hereditaria (DSH).

METHODSClinical data and peripheral blood samples of the patients from the pedigree were collected. Potential mutations of the ADAR1 gene were screened among 2 patients, 2 unaffected individual from the pedigree as well as 50 unrelated healthy controls by PCR amplification and direct sequencing.

RESULTSA c.3463C>T (p.R1155W) missense mutation of the ADAR gene was identified in the 2 patients, which was absent in the 2 healthy relatives and 50 unrelated controls. The mutation has been previously identified among 5 Chinese families and was the most common mutation site.

CONCLUSIONThe c.3463C>T missense mutation of the ADAR gene probably underlies the disease in this pedigree.

Hidradenitis suppurativa (HS) /acne inversa (AI) is a chronic, recurrent and inflammatory skin disease caused by follicular atresia with varying clinical manifestations, and there is no unified classification criteria for it in China and other countries. Based on its clinical characteristics, gene mutations and their correlations, researchers have enriched the subtype classification of HS/AI. This review summarizes recent advances in clinical phenotypes and genotypes of HS/AI as well as their relationships, in order to help clinicians better understand the disease, and provide a theoretical basis for correct diagnosis and individualized treatment strategies.

Objective:To evaluate the proliferative activity of and changes in the expression of related differentiation proteins in a stably NCSTN gene-silenced human immortalized keratinocyte cell line HaCaT, and to preliminarily explore the possible mechanism underlying the occurrence of acne inversa.Methods:By lentivirus-mediated short hairpin RNA (shRNA) , a NCSTN gene-silenced HaCaT cell model was established (shRNA group) , and other HaCaT cells transfected with empty lentivirus served as a negative control group. Real-time quantitative PCR and Western blot analysis were performed to determine the NCSTN gene-silencing efficiency. Cell counting kit-8 (CCK8) assay was conducted to evaluate the proliferative activity of HaCaT cells, and real-time quantitative PCR and Western blot analysis were performed to determine the mRNA and protein expression of cytokeratins (CK1, CK5, CK7, CK10, CK14, CK16, CK17, CK18, CK19 and CK20) and other differentiation molecules (involucrin and loricrin) respectively in HaCaT cells. Two-independent-sample t test was used to compare the measurement data between two groups. Results:NCSTN mRNA and protein expression were significantly lower in the shRNA group (0.42 ± 0.19, 0.30 ± 0.07 respectively) than in the negative control group (1.00 ± 0.34, 1.00 ± 0.26; t = 5.196, 2.637, P < 0.001, < 0.05, respectively) , and the gene-silencing efficiency was 70%. Compared with the negative control group, the shRNA group showed higher cellular proliferative activity, but decreased protein expression of CK16, CK19 and terminal differentiation molecule involucrin ( t = 3.787, 3.817, 2.904, P < 0.01, < 0.05, < 0.05, respectively) . Conclusion:Stable silencing of NCSTN gene can lead to abnormal proliferation and differentiation of HaCaT cells, which provides new ideas for subsequent exploration of acne inversa caused by NCSTN gene mutation.

Objective:To investigate the safety and feasibility of the CHESS endoscpic ruler (CHESS ruler), and the consistency between the measured values and the interpretation values by endoscopic physician experience.Methods:From January 2021 to January 2022, a total of 105 liver cirrhosis patients with portal hypertension were prospectively enrolled from General Hospital, Xixia Branch Hospital, Ningnan Hospital of People′s Hospital of Ningxia Hui Autonomous Region (29 cases), and the First People′s Hospital of Yinchuan (25 cases), General Hospital of Ningxia Medical University (18 cases), Wuzhong People′s Hospital (10 cases), the Fifth People′s Hospital of Ningxia Hui Autonomous Region (10 cases), Shizuishan Second People′s Hospital (6 cases), Yinchuan Second People′s Hospital (5 cases), and Zhongwei People′s Hospital (2 cases) 8 hospitals. The clinical characteristics of all the patients, including gender, age, nationality, etiolog of liver cirrhosis, and Child-Pugh classification of liver function were recorded. A big gastroesophageal varices was defined as diameter of varices ≥5 mm. Endoscopist (associated chief physician) performed gastroscopy according to the routine gastroscopy procedures, and the diameter of the biggest esophageal varices was measured by experience and images were collected, and then objective measurement was with the CHESS ruler and images were collected. The diameter of esophageal varices of 10 randomly selected patients (random number table method) was determined by 6 endoscopists (attending physician or associated chief physician) with experience or measured by CHESS ruler. Kappa test was used to test the consistency in the diameter of esophageal varices between measured values by CHESS ruler and the interpretation values by endoscopic physician experience.Results:Among 105 liver cirrhosis patients with portal hypertension, male 65 cases and female 40 cases, aged (54.8±12.2) years old, Han nationality 82 cases, Hui nationality 21 cases and Mongolian nationality 2 cases. The etiology of liver cirrhosis included chronic hepatitis B (79 cases), alcoholic liver disease (7 cases), autoimmune hepatitis (7 cases), chronic hepatitis C (2 cases), and other etiology (10 cases). Liver function of 32 cases was Child-Pugh A, Child-Pugh B 57 cases, and Child-Pugh C 16 cases. All 105 liver cirrhosis patients with cirrhotic portal hypertension were successfully measured the diameter of gastroesophageal varices by CHESS ruler, and the success rate of application of CHESS ruler was 100.0% (105/105). The procedure time from the CHESS ruler into the body to the exit of the body after measurement was (3.50±2.55) min. No complications happened in all the patients during measurement. Among 105 liver cirrhosis patients with cirrhotic portal hypertension, 96 cases (91.4%) were recognized as big gastroesophageal varices by the endoscopists. Totally 93 cases (88.6%) were considered as big gastroesophageal varices by CHESS ruler. Eight cases were recognized as big gastroesophageal varices by the endoscopist, however not by the CHESS ruler; 5 cases were recognized as big gastroesophageal varices by the CHESS ruler, but not by the endoscopists; 4 cases were not recognized as big gastroesophageal varices both by the endoscopists and CHESS ruler; 88 cases were recognized as big gastroesophageal varices both by the endoscopists and CHESS ruler. The missed diagnostic rate of big gastroesophageal varices by the endoscopists experience was 5.4% (5/93), and the Kappa value of consistency coefficient between the measurement by the CHESS ruler and the interpretation by endoscopists experience was 0.31 (95% confidence interval 0.03 to 0.60). The overall Kappa value of consistency coefficient by 6 endoscopists measured by CHESS ruler in big gastroesophageal varices diagnosis was 0.77 (95% confidence interval 0.61 to 0.93).Conclusion:As an objective measurement tool, CHESS ruler can make up for the deficiency of subjective judgment by endoscopists, accurately measure the diameter of gastroesophageal varices, and is highly feasible and safe.