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Objective:To construct a serine protease inhibitor Kazal type-5 (Spink5) conditional knockout mouse model, and to identify its phenotype.Methods:B cell-specific Spink5 conditional knockout mice of genotype Mb1 cre/+Spink5 floxp/floxp were constructed by using clustered regularly interspaced short palindromic repeats (CRISPR) /CRISPR-associated protein 9 (Cas9) technology, and served as the knockout group. Mice of genotype Mb1 +/+Spink5 floxp/floxp served as the control group. The mice of genotype Mb1 cre/+Spink5 floxp/floxp or Mb1 +/+Spink5 floxp/floxp were sacrificed when they were 4 to 6 weeks old, splenic mononuclear cells were isolated, and B lymphocytes and non-B lymphocytes were sorted by flow cytometry and fluorescence-activated cell sorting. Genotype identification was performed by PCR, and protein expression of lymphoepithelial Kazal-type-related inhibitor (LEKTI) was determined by Western blot analysis. Skin tissues were resected from the mice, and subjected to hematoxylin-eosin staining for measuring the epidermal thickness. Immunofluorescence staining was performed to determine fluorescence intensity of LEKTI protein in the mouse skin tissues. Paired t test or two-independent-sample t test was used for comparisons between groups. Results:Genotype identification results demonstrated that the stable B lymphocyte-specific Spink5 conditional knockout mouse model was successfully constructed. Western blot analysis revealed that the relative protein expression of LEKTI in the B lymphocytes in the knockout group was 0.01 ± 0.02, which was significantly lower than that in the non-B lymphocytes in the knockout group (0.66 ± 0.11, t = 9.99, P < 0.001) , and that in the B lymphocytes in the control group (1.08 ± 0.13, t = 13.78, P < 0.001) . Among 39 mice in the knockout group, 4 presented with dry skin and scattered scaly hypertrophic maculopapules. The epidermal thickness of the lesional skin tissues in the knockout group was 90.42 ± 21.31 μm, significantly higher than that of the non-lesional skin tissues in the knockout group (29.71 ± 3.63 μm, t = 5.05, P = 0.002) and that of normal skin tissues in the control group (12.42 ± 2.21 μm, t = 6.74, P < 0.001) . Immunofluorescence staining showed no significant difference in the fluorescence intensity of LEKTI protein among the lesional skin tissues (46.21 ± 1.21) , non-lesional skin tissues (46.62 ± 2.13) in the knockout group and normal skin tissues in the control group (47.69 ± 1.71, P > 0.05) . Conclusion:The B lymphocyte-specific Spink5 conditional knockout mouse model was successfully constructed, which provides a basis for further exploring mechanisms underlying skin barrier defects and immune dysfunction in Netherton syndrome.
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Objective To analyze the regulatory of miRNA (miR)-149-5p for the expression of Aurora-B in esophageal squamous cell carcinoma (ESCC). Methods The pathologic histology paraffin blocks of 61 patients with ESCC in Shanxi Provincial People's Hospital from January 2010 to December 2017 were collected. The immunohistochemical staining and tissue section in situ hybridization method were used to observe the expressions of Aurora-B and miR-149-5p in the tumor tissues and adjacent mucosas of the patients, and their relationship with clinicopathological parameters was analyzed. miRNA was predicted by using software TargetScan and miR walk. The relationship between Aurora-B and miR-149-5p were verified by using Western blot in ESCC TE-1 cells. Results The result of immunohistochemical staining showed that in 61 patients, 37 (61％) tumor tissues showed higher expression of Aurora-B compared with adjacent mucosas, the Aurora-B expression in 15 (26％) tumor tissues were in line with benign tissues, the Aurora-B expression in 9 (13％) tumor tissues were inferior to benign tissues. The expression of Aurora-B were not correlated with age (χ2=0.008, P=0.929), gender (χ2=0.088, P=0.767), grade of differentiation (χ2=2.632, P=0.268), but correlated with TNM staging (χ2=15.153, P<0.01) and lymph node metastasis (χ2=5.979, P=0.014). The miR-149-5p was predicted to combine with Aurora-B 3'untraslated region (UTR) by using TargetScan and miRwalk software. The result of in situ hybridization showed that the miR-149-5p showed low expression in 22 (36％) tumor tissues. The expression of miR-149-5p was correlated with Aurora-B expression (χ2 = 5.622,P= 0.018), and not correlated with age (χ2= 2.617, P= 0.106), gender (χ2= 1.529, P= 0.216), grade of differentiation (χ2 = 2.854, P= 0.240), TNM staging (χ2 = 0.870, P= 0.351) and lymph node metastasis (χ2= 0.128, P= 0.351). The Western blot results of TE-1 cells showed that the expression of Aurora-B in simultaneous over-expression of miR-149-5p and Aurora-B group was higher than that in over-expression of miR-149-5p group, and lower than that in over-expression of Aurora-B group. Conclusion The miR-149-5p can be involved in ESCC progression through regulating the expressions of Aurora-B.
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Mast cells (MC),a major participant in inflammation and allergy,can initiate an early defense response to foreign invaders.Besides expressing high-affinity IgE receptor,MC also expresse a large number of G protein-coupled receptors (GPCRs).Various studies have shown that cationic micromolecules represented by substance P and many peptidergic drugs can induce MC degranulation through a novel receptor,Mas-related G protein-coupled receptor X2 (MrgprX2).MrgprX2 plays an important role in allergy,itching and pseudo-allergic drug reactions.It will help explain some clinical difficulties which can not be explained by classical IgE-dependent activation of MC,and provides potential therapeutic targets for MC-mediated diseases.
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Objective@#To investigate the clinical and immunological laboratory features and gene mutation in a female patient who carried a germline gain-of-function mutation in STAT3.@*Method@#A patient with lymphadenopathy and pancytopenia, visited the Department of Rheumatology and Immunology of Children′s Hospital of Chongqing Medical University in May 2016. The clinical and laboratory characteristics, results of immunophenotyping and exome sequencing were analyzed retrospectively and related literature was reviewed.@*Result@#The patient was a four years old girl. The clinical manifestation consisted of autoimmune pancytopenia, lymphadenopathy and recurrent infections. Multiple exams showed that peripheral blood leukocyte count was (2.2-4.9)×109/L, red blood cell count was (2.09-5.75)×109/L, hemoglobin level was 64-165 g/L, platelet count was (52-138) ×109/L. Percentages of lymphocyte subsets showed that CD3+ T lymphocyte was 0.716 0 (CD4+ T lymphocyte was 0.326 0, CD8+ T lymphocyte was 0.323 0 and CD4- CD8-T TCRαβ+ lymphocyte was 0.029 0), CD19+ B lymphocyte was 0.235 0 (transitional B was 0.004 3), NK was 0.032 0. Percentages of CD4+ T lymphocyte release IL-4, IFN-γ, IL-17 and IL-21 were 0.014 9, 0.213, 0.024 0 and 0.021 0, respectively. Lymphocyte proliferation function and TCRVβ diversity were normal. The serum immunoglobulin levels were 16.4 g/L (IgG), 1.53 g/L (IgA), 3.99 g/L (IgM) and 3.20 kU/L (IgE). The patient carried a missense variant in the 21st exon of STAT3, c. 1974G>C, p.K658N, which was previously described as a gain-of-function mutation. The patient was treated with methylprednisolone and prednisone intermittently. There were significant improvements of hepatosplenomegaly, lymphadenopathy and pancytopenia. We searched internal database and literature for cases with gain-of-function mutations in STAT3. A total of 19 cases were identified, all were non-Chinese. Among 16 cases who had clinical data, age of onset of 11 patients was less than 5 years. 14 cases had autoimmune hemolytic anemia, autoimmune thrombocytopenia or autoimmune neutropenia. Twelve patients had lymphadenopathy while 11 had infections and 5 had endocrine abnormalities.@*Conclusion@#The patient with Primary immunodeficiency disease (PID) due to gain-of-function mutation in STAT3 gene often has early-onset autoimmune disorders, lymphadenopathy and recurrent infections. Since the routine immunological examination may be normal or slightly abnormal, comprehensive evaluation of immune function should be done. Genetic testing ultimately helps to confirm the diagnosis.
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Objective:To explore the effect of prostaglandin E2 (PGE2) on the expression of high mobility group box-1 protein (HMGB1) in peritoneal macrophages of septic mice and its possible mechanisms.Methods:Ihe mouse peritoneal macrophages were isolated and cultured by conventional methods.The model of inflammation was established by using lipopolysaccharide (LPS) to incubate with mouse peritoneal macrophages.The PGE2,prostaglandin E receptor (EP) 4 agonist,EP4 RNAi,and DN.CREB inhibitory plasmid were used to interfere with the LPS-treated mouse peritoneal macrophage.The levels of HMGB 1 was determined by Western blot.Results:Compared with LPS alone treatment,the expression of HMGB 1 in peritoneal macrophages was increased obviously after 24 h by treatment with PGE2 and LPS,and it was also increased after the combined treatment of EP4 receptor agonist with LPS for 24 h (both P0.05);compared with LPS alone treatment,the combined treatment of EP4 receptor agonist with LPS for 24 h could up-regulate the phosphorylation of epidermal growth factor receptor (EGFR) and protein kinase B (Akt) thr308 (P<0.05),which were blocked by EGFR inhibitor.Once Akt specific inhibitor was used before EP4 and LPS treatment,the expression of HMGB1 was declined (P<0.05).Conclusion:PGE2 can up-regulate the expression of HMGB1 in sepsis of peritoneal macrophages through EP4 receptor,which may be related to the activation of EGFR/PI3K/Akt signaling pathway.
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Objective To investigate the effect of video -assisted thoracoscopic surgery (VATS)and conventional thoracotomy in treatment of thoracic esophagus cancer,to provide the reference for clinical.Methods 90 cases of thoracic esophageal cancer in our hospital from January 2012 to January 2015 were chosen as the research subjects.They were randomly divided into observation group(application of VATS treatment)and the control group (application of traditional open chest surgery).The quantity difference,chest drainage,hospitalization time,complica-tions,recovery conditions and other indicators of bleeding were compared in the two groups.Results The operation time of the observation group[(267.6 ±76.5)min]was shorter than that of the control group[(324.4 ±87.6)min]. The amount of intraoperative bleeding[(235.3 ±79.5)mL],drainage volume[(327.5 ±95.2)mL]of the observation group were less than the control group[(398.2 ±98.3)mL and (752.6 ±156.4)mL].Postoperative hospitalization time[(12.2 ±3.2)d]of the observation group was shorter than the control group[(15.8 ±4.4)d].The differences were statistically significant (t =3.276,8.644,15.575,4.439,all P 0.05).The complication rate of the observation group was lower than that of the control group,the difference was statistically significant (χ2 =4.865,P <0.05).Conclusion In the treatment of thoracic esophageal carcinoma,VATS and conventional thoracotomy surgery has good effect,and VATS has small injury,less complication.
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Chromosome 22q11.2 deletion syndrome,also called DiGeorge syndrome or Velo-CardiacFacial syndrome,has all expansive phenotype involving essentially every organ and system,such as cardiovascular abnormalities,abnormal face,immunodeficiency,even psychiatric illnesses,and etc.Fluorescence in situ hybridization analysis test for the microdeletion from chromosome 22 at the q11.2 band is the comqrmed diagnostic method So far,it has not been known thoroughly in China and there has not been a normative screening system yet.Close relations between the microdeletion and congenital cardiovascular abnormalities especially conotruncal cardiac defects and arcus aortae abnormalities have been shown in reported cases.This review will describe the 22q11 DS and how to screen it in the congenital cardiovascular abnormalities so that it Can be diagnosed early and managed properly.which will benefit the patients and their later generations.
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Objective To investigate the expression of Ezrin protein and c-met in esophageal squamous cell carcinoma, their significance in the progress of esophageal squamous cell carcinoma development and metastasis. Methods The expression of Ezrin protein and c-met in 50 patients with esophageal squamous cell carcinoma was studied using immunohistochemical (SP) method. The correlation of each score according to the intensity and percentage of labeled cells or intercellular substance with relevant clinical data was statistically analyzed. Results In normal esophageal squamous epithelium, the positive expression of Ezrin protein was 34 %, the positive expression of c-met was 8.0 %. In esophageal squamous cell carcinoma, the positive expression of Ezrin protein was 76.0 %, the positive expression of c-met was 58.0 %. The expression of Ezrin protein had close relation to depth of invasion and lymph node metastasis(P < 0.05). The positive rate of c-met protein expression in lymph node metastasis was obviously higher than those without lymph node metasis (P <0.05). The expressinn of Ezrin protein in c-met positive eases was obviously higher than those of c-met negative cases(P <0.05). Conclusion The test of Ezrin protein and c-met may have significant prognostic value for assessing the degree of malignancy and potential lymph node metastasis probability of esophageal squamous cell carcinoma. Ezrin protein may be a target to evaluate the tumor prognosis.