ABSTRACT
Objective:To investigate the effect of KRT5 knockdown in keratinocytes on melanin content in co-cultured melanocytes, and to explain mechanisms underlying formation of hyperpigmented lesions in reticulate pigmented anomaly of the flexures (Dowling-Degos disease, DDD) .Methods:HaCaT cells with heterozygous mutations in the KRT5 gene were obtained by using clustered regularly interspaced short palindromic repeats (CRISPR) -CRISPR-associated protein 9 (Cas9) technology (experimental group) , and HaCaT cells transfected with non-targeting single guide RNA:Cas9 protein complex served as control group, both of which were in vitro co-cultured with primary human melanocyte cells (HEMn) separately. Immunofluorescence study was conducted to determine the expression of cytokeratin and melanosomes in co-cultured cells; melanin content was detected in melanocytes in different co-culture groups, which were obtained by differential trypsinization. Immunohistochemical study was performed to determine the expression of melanocyte-specific premelanosome protein 17 (Pmel17) in skin lesions in a patient with DDD carrying a KRT5 mutation and normal skin tissues in a healthy control. Results:Sanger sequencing showed a heterozygous mutation (c.1delA) at the initiation codon of exon 1 of the KRT5 gene in HaCaT cells in the experimental group, but no mutation in the KRT5 gene in the control group. Western blot analysis showed that the KRT5 protein expression was significantly lower in the experimental group (0.60 ± 0.05) than in the control group (1.00 ± 0.00, t = 32.38, P = 0.001) . Compared with the co-culture system in the control group, the number of Pmel17-labeled melanosomes markedly increased with the melanin content elevated by 52.5% ( t = -3.48, P = 0.025) in the HEMn cells co-cultured with HaCaT cells in the experimental group. Immunohistochemical study showed that the Pmel17 expression increased in the skin lesions in the DDD patient with KRT5 mutation compared with the normal skin tissues in the healthy control. Conclusion:The effect of HaCaT cells with CRISPR-Cas9-induced KRT5 mutation on the co-cultured HEMn melanocytes was verified by the successfully established in vitro co-culture system, which provides a primary cell model for further studies on interaction mechanisms between keratinocytes and melanocytes, and on pathogenesis of skin pigmentation abnormalities.
ABSTRACT
【Objective】 To investigate the airway parameters of adult-onset eosinophilic asthma （EA） and analyze the correlation between airway remodeling and lung function by quantitative CT. 【Methods】 From March 2015 to November 2016， totally 94 subjects from the “FACT-Digital Lung” Multi-research Center were divided into three groups： 30 normal subjects， 33 EA patients and 31 non-eosinophilic asthma （NEA） patients. We measured and recorded the bronchial parameters of RB1， LB1+2， RB10， and LB10， and small airway disease parameters. The indicators for quantitative evaluation of bronchial parameters include lumen area （LA）， wall thickness （WT）， wall area （WA）， and wall area percentage （WA%）. The parameters for the quantitative assessment of small airway disease included the percentage of inspiratory voxels below -950HU （IN-950）， the mean lung density （MLDin）， and the whole volume of the lung in inspiration （Vin）. Pearson or Spearman rank correlation analysis was used to evaluate the correlation between these parameters and lung function. 【Results】 The differences in LA/BSA， WT/√BSA， and WA/BSA between the three groups were statistically significant （P<0.05）. FEV1% had a significant correlation with IN-950 and MLDin （P<0.01）. FEV1/FVC had a significant correlation with Vin， IN-950， and MLDin （P<0.01）. EOS counts were positively related to IN-950 （r=0.343， P=0.011）， while EOS had a negative correlation with FEV1% （r=－0.343， P=0.015）. 【Conclusion】 With the increase of eosinophils counts in peripheral blood， the airway's stenosis in asthma patients gradually increased， and the extent of airflow limitation steadily increased. The IN-950 may be a sensitive imaging biomarker for evaluating the small airway disease in adult-onset eosinophilic asthma patients.
ABSTRACT
Objective To evaluate the feasibility and effects of endoscopic thyroidectomy(ETE) for papillary thyroid carcinoma(PTC).Methods A total of 137 patients with PTC were enrolled from January 2013 to January 2016.The patients were divided into two groups depended on different surgical approaches.The patients'' characteristics,such as age,gender,pain,intraoperative blood loss,hospitalization,central lymph node metastasis etc were retrospectively analyzed in the two groups.Results In the study,78 patients performed ETE,and 58 performed COT.There was one case converted to open surgery from ETE due to intraoperative bleeding.The clinicopathological characteristics and operative complications between the two groups had no significant differences,such as gender,time of drainage,postoperative hospitalized days,positive lymph node metastasis,injury of recurrent laryngeal nerve,permanent injury of parathyroid glands.The age of patients was younger in ETE(t=-4.299,P=0.000).The drainage content(t=9.539,P=0.000),intraoperative blood loss(t=2.862,P=0.005) and subcutaneous ecchymosis (χ2=13.482,P=0.000) were found more in the ETE.ETE required a longer operative time(t=8.162,P=0.000).However,ETE provided better cosmetic outcomes than COT [(9.4±0.5)points vs.(5.4±1.0)points,t=30.142,P=0.000].No clinical recurrence and metastasis occurred for at least one-year follow-up.Conclusion With excellent cosmetic results,ETE is a feasible and safe operation for patients with PTC without metastasis to lateral cervical lymph nodes.