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.

【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.

Objective:To explore the selection of surgical methods for different sites of symptomatic Rathke's cleft cyst (RCC) and the clinical efficacies of these patients.Methods:Forty-seven patients with symptomatic RCC, admitted to our hospital from January 2016 to December 2019, were chosen in our study; 21 patients with intrasellar symptomatic RCC accepted surgery via unilateral nasal approach at the right side, 19 patients with intra-suprasellar symptomatic RCC accepted surgery via bilateral nasal approach, 3 patients with suprasellar symptomatic RCC accepted endonasal transsphenoidal surgery under endoscope, and 4 patients with suprasellar symptomatic RCC accepted craniotomy via pterion approach. The clinical efficacies and complications of patients accepted different surgical methods were compared. All patients were followed up for 3-36 months to observe the recurrence.Results:The postoperative symptoms of the patients were effectively improved, including headache relief ratio of 27/31, vision loss improvement ratio of 5/5, high prolactin relief ratio of 11/13, pituitary function improvement ratio of 9/18. Complications occurred in 6 patients, presenting as diabetes insipidus. Four patients recurred during follow-up.Conclusion:Intrasellar and intra-suprasellar symptomatic RCC accepted surgery via endoscopic transnasal transsphenoidal approach are safe and effective; selection of surgical methods for suprasellar symptomatic RCC should be determined according to the sizes and growth directions of cysts.

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.

Objective To investigate the changes and clinical significance of neck-shaft angle in the treatment of proximal humerus fractures with locking plate.Methods A retrospective case-control study was done on 214 cases of proximal humerus fractures treated with locking plate in our hospital from January 2008 to June 2016.According to the presence of medial bone defects,the cases were divided into medial bone defect group (85 cases) and non-medial bone defect group (129 cases).In medial bone defect group,there were 35 males and 50 females,with an average age of 63.3 years old.In this group,the cases of Neer three-part fractures and Neer four-part fractures were 47 and 38,respectively.In non-medial bone defect group,there were 51 males and 78 females,with an average age of 65.9 years old.In this group,the cases of Neer three-part fractures and Neer four-part fractures were 57 and 72,respectively.The preoperative humeral neck-shaft angle of contralateral humerus,and the humeral neck-shaft angle with its changes on the second postoperative day as well as 1,3,6 and 12 months after surgery were evaluated by the X-ray film.The Constant scores of the shoulder function were also assessed.Results The follow-up time for the patients ranged from 12 to 42 months (mean 24.2 months).In medial bone defect group,the preoperative neck-shaft angle of contralateral humerus,as well as neck-shaft angles of fractured side at five follow-up visits were (133.4±4.0) °,(134.7 ±11.5) °,(120.8±4.0) °,(118.5 ±3.5) °,(117.9 ±10.4) °and (114.7 ±4.4)°,respectively.While in the other group,the corresponding parameters were (134.0±2.4)°,(133.8± 17.1)°,(135.6± 2.9)°,(132.7± 2.8) °,(131.5 ± 13.5) °,(135.2 ± 2.8) °,respectively.There were significant differences between the two groups of the neck-shaft angle at 1,3,6 and 12 months (P ＜ 0.05).In the medial bone defect group,there were no significant differences between neck-shaft angle of contralateral humerus and the second postoperative day (P ＞0.05),but both of them are significantly greater than others(P ＜0.05).In the other group,except for the neck-shaft angles of postoperative 1 month and 6 months (P ＜ 0.05),there was no statistically significant difference between the angles in different time points(P ＞ 0.05).The mean Constant scores of medial bone defect group and no medial bone defect group were (69.3 ± 14.6) and (75.2 ± 12.6),respectively.Conclusions After the recovery of neck shaft angle,there is still a need of attention to the losses of neck shaft angle after surgery.Neck-shaft angle could be a reference for the treatment strategy of proximal humerus fractures with locking plate,which could guide the anatomical reduction as well as be used to evaluate the effects after surgery.It could also be a reference for function exercise.Moreover,the losses of neck shaft angle will be influenced by supporting from internal side,which could have effects on the function recovery after surgery.

Objective To investigate the association between different number of inferomedial screws (0-3 screws) and their efficacy in locking plate of proximal humerus fractures.Methods Data of 90 patients who had undergone locking plate for proximal humerus fracture between January 2007 and December 2012 were retrospectively analyzed.They were divided into four groups according to the number of inferomedial screws.In the NS (no-screw support) group,36 patients did not have inferomedial screws for mechanical support of the inferomedial region of the proximal humerus; in the OS (one-screw support) group,23 patients had the reconstruction by insertion of only one inferomedial screw; there were 19 patients in the TS (two-screw support) group and 12 in the ThS (three-screw support) group.There were no significant differences among 4 groups in sex,fracture type and age.The Constant scores of the shoulder function,changes in humeral head height at the latest follow-up,incidence of complications and time for fracture healing among the 4 groups were collected.Results The follow-ups for the patients ranged from 12 to 56 months (mean,21.4 months).At the latest follow-up,the ThS,TS,OS and NS groups had a mean Constant score of 76.7 (SD,11.6),74.1 (SD,7.4),66.8 (SD,10.7),65.8 (SD,10.2),respectively; the mean change in humeral head height were 1.4 (SD,1.0) mm,2.9 (SD,1.2) mm,5.3(SD,3.2) mm and 6.7(SD,3.3) mm,respectively.There were no significant differences between the ThS and TS groups in Constant score,and Constant scores in ThS and TS groups were significantly higher than those in OS and NS groups.However,the loss in humeral head height in the ThS group was significantly less than those in the TS,OS and NS groups,and the loss in humeral head height in TS group was significantly less than those in the OS and NS groups.There were no significant differences among 4 groups in time for fracture healing and incidence of complications.Conclusion Locking plate in general do not appear to be a panacea for proximal humerus fractures especially in cases of medial comminution or bone defects because of the malreduction of the medial cortical bone.However,medial support with two or three inferomedial screws being meticulously placed in the inferomedial region may enhance the mechanical stability of medial column support and allow for better maintenance of reduction.

Background and purpose:Colorectal serrated adenoma (SA) was ofifcially named in 2000 by the WHO as a separate disease, with unique properties compared with traditional adenoma (TA), and its relationship with colorectal cancer (CRC) is very concerned. This study was to analyze and compare the telomerase, p53 and Ki-67 immunohistochemical expression on the tissues of SA, TA and CRC. Methods:Immunohistochemistry was adopted to analyze the expression of telomerase, p53, and Ki-67 in 37 cases of SA, 36 cases of TA and 34 cases of CRC. Results:The p53-positive percentage of SA was signiifcantly lower than that of TA (P<0.01), and the p53-positive percentage of TA was signiifcantly lower than that of CRC (P<0.01). No signiifcant difference of Ki-67 expression was found between SA and TA, and the Ki-67-positive percentage of SA and TA was lower than that of CRC (P<0.01). The telomerase-positive percentage of TA was signiifcantly lower than that of SA (P<0.01), and the telomerase-positive percentage of SA was signiifcantly lower than that of CRC (P<0.05). Conclusion:Telomerase, P53, and Ki-67 immunohisto chemical analysis indicated that SA is a kind of proliferative adenoma, and telomerase activation may play a role in the cancer process.

Objective To evaluate correlation of radial head fracture with forearm interosseous membrane (IOM) injury.Methods Twenty-six patients with radial head fractures were studied prospectively between September 2007 and June 2010.There were 15 men and 11 women,with an average age of 37.6years (range,21-53).According to the Mason classification,there were 7 cases of type Ⅰ,9 cases of type Ⅱ,10 cases of type ⅢL All patients were subjected to forearm X-ray,CT scans and the MR within a week.Clinical and radiographic data of all the patients were collected.Spearman rank correlation statistical analysis was used to analyze the correlation between the radial head fracture and the IOM injury.Results The radial head fractures and IOM injury were directly related.The IOM injury was noted in all type of radial head fracture.The more severity radial head fracture had,the more IOM injury happened.In Mason Ⅰ-Ⅲ fractures,IOM injury was found in 2,4 and 7 cases respectively.The different degree of radial head fracture caused different effects on IOM injury.The severity of radial head fracture was correlated with damage degree of IOM.In Mason type Ⅰ and type Ⅱ fractures,the IOM injury were just partial disruption with distal part of the IOM and did not reach the biomechanically essential central band.In type Ⅲ fractures,central band disruption was found in 3 cases.Conclusion Mason Ⅰ-Ⅲ radial head fractures are associated with forearm IOM injury.There was a positive correlation between radial head fractures and IOM injury.If IOM lesions are suspected,magnetic resonance imaging should be performed,especially Mason Ⅲ fractures.

Objective To evaluate the effect of pronator teres tendon reconstructing the forearm interosseous membrane(IOM)central band on restoring the longitudinal stability of the forearm after Essex-Lopresti injury.Methods A total of ten fresh-frozen adult cadaveric forearms were loaded axially through the humeral and the distal radius.Each forearm was tested as following six steps:radial head excision,radial head plus excision of triangular fibrocartilage complex(TFCC),radial head plus TFCC plus excision of IOM central band,reconstructing central band with pronator teres tendon,metallic radial head prosthesis replacement,central band reconstruction plus radial head prosthesis replacement.The difference of the load exerted to displace the radius to the proximal for 5 mm was compared at different steps by using single-factor analysis of variance.Then,the effect of each step on restoring the forearm longitudinal stability of the forearmwas evaluated.Results There was statistical difference upon load for the radial displacement to the proximal for 5 mm in all six steps,ie,(74.51±15.17)N,(49.23±6.14)N,(17.83±4.73)N,(27.40±3.56)N,(140.25±25.39)Nand(164.21±28.26)N,respectively(P＜0.01).With migration of the radius to proximal for 5 mm,the load for pronator teres reconstruction was 55.66% of radial head plus TFCC,and the load for pronator teres reconstruction plus radial head prosthesis replacement was 559.31% of IOM reconstruction step and 333.56% of radial head excision.Conclusions Reconstruction of the central band with pronator teres tendon is insufficient to restore the longitudinal stability of the forearm after Essex-Lopresti injury.Reconstruction of the central band in combination with metallic radial head prosthesis replacement are beneficial to restoring the longitudinal stability of the forearm.