AIM: To investigate the effect of interleukin-8(IL-8)on the regulation of monocyte chemotactic protein-1(MCP-1)secreted by lens epithelial cells(LEC)during cell migration in the development of posterior capsule opacification(PCO).METHODS: A rat lens capsule model was established and cultured in medium supplemented with 10% fetal bovine serum. Upon migration of LEC to 30%-50% of the posterior capsule, serum was removed. The capsule was subsequently divided into two groups: a control group and an IL-8(15 ng/mL)treatment group. LEC migration was captured at multiple time points. The secretion and mRNA expression of MCP-1 were quantified using ELISA and RT-qPCR, respectively. Immunofluorescence was used to assess MCP-1 expression in the different experimental groups. SRA01/04 cells were divided into three groups: control, IL-8(15 ng/mL), and IL-8(15 ng/mL)+200 μmol/L Bindarit(BND)groups, with migration measured by the Transwell assay. Additionally, SRA01/04 cells were divided into negative control(NC), NC+15 ng/mL IL-8, and 15 ng/mL IL-8+p65 siRNA groups, and MCP-1 secretion and mRNA expression were further analyzed by ELISA and RT-qPCR.RESULTS:LEC migration in the rat lens capsule cultured in vitro showed that the cells migration of the 15 ng/mL IL-8 group significantly increased at 48, 72 and 96 h(all P<0.05). ELISA results revealed that MCP-1 levels in SRA01/04 cells from the 15 ng/mL IL-8-treated group were markedly higher than those in the control group at both 12 and 24 h(all P<0.05). RT-qPCR analysis also demonstrated a significant increase in MCP-1 mRNA expression in the 15 ng/mL IL-8 group at both time points(all P<0.05). Immunofluorescence staining indicated greater MCP-1 expression in capsular epithelial cells of the 15 ng/mL IL-8 group at 24 h(P=0.007). Transwell assays further confirmed increased cell migration in the 15 ng/mL IL-8 group compared to the control group(P=0.001), while the migration reduced in the 15 ng/mL IL-8+200 μmol/L BND group compared to the 15 ng/mL IL-8 group(P=0.003). Moreover, ELISA and RT-qPCR results demonstrated a significant increase in MCP-1 secretion and mRNA expression in the NC+15 ng/mL IL-8 group at both 12 and 24 h compared to the NC group(all P<0.01). In contrast, MCP-1 secretion and mRNA expression were reduced in the 15 ng/mL IL-8+p65 siRNA group compared to the NC+15 ng/mL IL-8 group at both time points(all P<0.01).CONCLUSION: IL-8 promotes the migration of residual epithelial cells and regulates the secretion and expression of MCP-1 in LEC. The mechanism underlying IL-8's effects appears to be mediated through the activation of the NF-κB/p65 signaling pathway.

Objective:To investigate the anti-aging and antioxidant effect of the heat shock transcription factor 1 (HSF1) on human retinal pigment epithelial cells.Methods:Two HSF1-deficient ARPE cells (ARPE/Hsf1 -/-) were constructed by using the clustered regularly interspaced short palindromic repeat and associated protein 9 (CRISPR/Cas9) gene editing system and named H8, H9 konckout cell strains.Experiments were operated on the 3 cell strains: wild-type, H8 and H9 cells.The content of reactive oxygen species in ARPE-19 cell was measured by DHE probe staining combined with flow cytometry technology, and the cell cycle was measured by flow cytometry technology.The cell viability at different time points was measured using cell counting kit-8 (CCK-8).Crystal violet staining assay was used to measure the relative ratio of cell survival.SA-β-gal staining assay was used to detect the ratio of ARPE-19 senescent cells.The expressions of HSP70, HSP27, clusterin (CLU), p53, p21 and interleukin (IL)-1β proteins were measured by Western blot technology.The expressions of p53, p21, IL-6, IL-8, IL-1β and monocyte chemoattractant protein 1 (MCP1) mRNA were measured by quantitative real-time PCR technology.Relative expression of heat shock response protein under different heat shock treatment conditions and HSP90 inhibitor IPI504, relative survival with different concentrations of H 2O 2, relative expression of p21 protein after treatment with or without ROS scavenger N-acetylcysteine (NAC) were compared in each cell strain. Results:Gene sequencing showed that H8 and H9 cell strains successfully carried mutated genes.Western blot experiment results showed that H8 and H9 cell strains did not express HSF1 protein, and HSF1 was successfully knocked out in ARPE-19 cells.Compared with wild-type cell, the expression levels of HSP70, HSP27 and CLU proteins in H8 and H9 cell strains significantly decreased, with statistically significant differences (all at P<0.05), and no significant difference was found in the relative HSP90 protein expression level ( F=0.29, P>0.05).Under different heat shock stimulation and IPI504 induction, the HSP70, HSP27, and CLU protein levels significantly increased in wild-type cells compared with before treatment, and the HSP70, HSP27, and CLU protein levels were significantly lower in H8 and H9 cell strains than in corresponding treated wild-type cells (all at P<0.05).Compared with wild-type cell strains, cell viability significantly decreased in H8 and H9 cell strains at 24, 48, 72, and 96 hours (all at P<0.05).Compared with wild-type cell strains, the percentage of cells in G1 phase was significantly higher and the mRNA and protein levels of the cell cycle inhibitors p53 and p21 significantly increased in H8 and H9 strains, showing statistically significant differences (all at P<0.05), and the ratio of positive cells for SA-β-gal staining significantly increased, showing statistically significant differences (all at P<0.001).The relative expression of aging-related inflammatory factors IL-6, IL-8, IL-1β, and MCP1 mRNA decreased, and the differences were statistically significant (all at P<0.001).In addition, compared with wild-type cell strains, the content of reactive oxygen species (ROS) was higher in H8 and H9 cell strains, and the differences were statistically significant (all at P<0.001).The expression of p21 protein in H8 and H9 cell strains wtih NAC treatment decreased significantly compared with non-NAC treatment cells (both at P<0.05).Compared with wild-type cell strains, H8 and H9 cell viability decreased at 200, 400, 600, and 800 μmol/L H 2O 2 treatment conditions, and the differences were statistically significant (all at P<0.05). Conclusions:Knockdown of HSF1 can downregulate the expression of heat shock proteins, activate the ROS/P53/P21 pathway, induce senescence in RPE cells, and increase the sensitivity of RPE to oxidative stress stimuli.HSF1 may have anti-senescence and anti-oxidant regulatory effects in RPE cells.

Objective:To investigate the expression of nicotinamide-N-methyltransferase (NNMT) in various tumor tissues and its prognostic value in papillary thyroid carcinoma.Methods:The paraffin samples of surgically resected tissues from 168 cases of colorectal cancer, 75 cases of gastric cancer, 178 cases of lung cancer, 15 cases of liver cancer, 60 cases of thyroid cancer, 7 cases of prostate cancer, 74 cases of breast cancer, and 14 cases of renal cancer were collected from Sir Run Run Show Hospital, Zhejiang University School of Medicine and the Third Affiliated Hospital of Zhejiang University of Traditional Chinese Medicine between January 2016 and December 2021; tissue samples of 58 cases of papillary thyroid cancer and another 19 cases of thyroiditis were collected between January 2016 and December 2016. Immunohistochemistry kits were prepared and performance tests were performed. Normal specimens (>5 cm from the margin of paracancerous tissues) and the samples of gastric cancer, colorectal cancer, lung cancer, thyroid cancer, prostate cancer, breast cancer, and kidney cancer tissues as well as their paracancerous tissues (3 cm from the tumor edge) were selected. Immunohistochemistry kits were used to detect the expression of NNMT protein in normal tissue samples, different tumor tissues and their paracancerous tissues. X-tile software combined with the receiver operating characteristics curve of NNMT in the diagnosis of tumor tissues and paracancerous tissues in papillary throid carcinoma were used to determine the optimal cut-off value (41.5); < 41.5 was treated as the NNMT protein low expression group and ≥ 41.5 was treated as the NNMT protein high expression group. The expression of NNMT protein in patients with papillary thyroid carcinoma with different clinicopathological characteristics was compared; Kaplan-Meier method was used to analyze the overall survival of the patients with papillary thyroid carcinoma; Cox proportional risk model was used to conduct multivariate analysis on the influencing factors of overall survival.Results:The prepared immunohistochemistry kits were valid for at least 12 months, with good intra-batch and batch-to-batch repeatability, good stability and specificity. NNMT protein was not or occasionally lowly expressed in colorectal, lung, thyroid, prostate, breast, kidney, and gastric tissues. NNMT protein was highly expressed in colorectal cancer, gastric cancer, breast cancer, kidney cancer, thyroid cancer, lung cancer, prostate cancer tissues, while lowly expressed in colorectal cancer, gastric cancer, breast cancer, kidney cancer, thyroid cancer, lung cancer, prostate cancer adjacent tissues. The high expression rates of NNMT protein in thyroiditis tissue, papillary thyroid cancer tumor tissue and paracancerous tissues were 15.79% (3/19), 68.97% (40/58) and 31.03% (18/58), respectively, and the high expression rate of NNMT protein in papillary thyroid carcinoma tissue was higher than that in thyroiditis tissue and paracancerous tissue. All patients with papillary thyroid cancer were divided into the NNMT protein high expression group (40 cases) and the low expression group (18 cases). There were no statistically significant differences in NNMT protein expression among patients with different age, gender, degree of differentiation, lump diameter, TNM stage, lymph node metastasis, and serum anti-thyroglobulin antibody (TgAb) level (all P > 0.05). The median overall survival time of 58 patients was 18.5 months, and the 5-year overall survival rate was 90.0%. The overall survival of patients with a lump diameter of ≥2 cm was worse than that of those with a lump diameter of < 2 cm ( P < 0.001), and the overall survival of patients with lymph node metastasis was worse than that of those without lymph node metastasis ( P = 0.041). The overall survival of patients in the NNMT protein high expression group was worse than that of those in the NNMT protein low expression group, and the overall survival of patients with high serum TgAb level was worse than that of those with low serum TgAb level, while the differences were not statistically significant (all P >0.05). Lump diameter ( HR = 35.56, 95% CI 2.64-478.25, P = 0.007), NNMT protein expression ( HR = 308.12, 95% CI 2.21-42 958.20, P = 0.023), serum TgAb level ( HR = 142.85, 95% CI 1.88-10 854.25, P = 0.025) were independent influencing factors for the OS of patients with papillary thyroid carcinoma. Conclusions:NNMT is highly expressed in various tumor tissues. NNMT expression is related to the prognosis of patients with papillary thyroid carcinoma;the patients with high expression of NNMT have worse prognosis compared with those with low expression of NNMT.

Objective:To observe the inhibition of SARS-CoV-2 spike protein (S-protein) on the proliferation of human retinal pigment epithelium (RPE) cells.Methods:SARS-CoV-2 S-protein gene fragment expression plasmid (p3xflag-S) was constructed and transfected into human RPE, HEK293 cells. DNA sequencing was used for identification, and the expression of Flag-S was detected by Western blot. HEK293 cells were divided into the cells 1, 2, 3 and 4 and transfected with GFP11 plasmid and vector, GFP1-10 plasmid and vector, transfected with GFP11 and pCMV-HA-ACE2 plasmid, GFP1-10 and p3xflag-S plasmid. Cell 1 was co-cultured with cell 2 (control group 1), cell 2 with cell 3 (control group 2), cell 3 with cell 4 (observation group), and cell 1 mixed with cells 2, 3 and 4 (control group 3). Bright-field microscopy and fluorescence microscopy were used to observe cell fusion. RPE cells were divided into control group and overexpression S-protein group. The cell cycle was detected by flow cytometry; the cell proliferation level was detected by Counting Kit 8 (CCK-8); and the S-protein expression level in RPE cells was detected by Western blot. The Student’s t-test was performed for comparison between groups. Results:DNA sequence assay showed that S-protein cDNA was fused with flag-tagged protein. Western blot assay showed that S-protein-related expression was elevated in transfected HEK293 cells compared with untransfected p3xflag-S cells. Large, multinucleated fused cell clusters were visible under bright-field microscopy; multiple nuclear with distinct green fluorescence were visible in the fused cells under fluorescence microscopy. Western blot assay showed elevated S-protein-related expression in transfected p3xflag-S plasmid RPE cells compared to untransfected p3xflag-S plasmid RPE cells. CCK-8 results showed that the proliferative capacity of RPE cells in the S-protein overexpression group was significantly reduced compared with the control group, with statistically significant differences ( t=22.70, 16.75, 23.38; P<0.000 1). The results of flow cytometry showed that the G1 phase cells in the control and overexpression S-protein groups were 41.1 % and 67.0%, respectively; compared with the control group, the G1 phase cells in the overexpression S-protein group were significantly higher, and the difference was statistically significant ( t=4.76, P=0.018). The apoptosis rate was significantly increased in the S-protein overexpression group compared with the control group, and the difference was statistically significant ( t=4.91, P=0.008). Conclusion:Overexpression of the SARS-CoV-2 spike protein reduced the proliferation of human RPE cells.

Objective:To discuss the diagnostic methods, clinical features and treatment options of bronchogenic cysts.Methods:A total of 86 patients with bronchogenic cysts and 5 patients with esophageal cysts and esophageal cysts were selected from January 2011 to May 2020 in the Affiliated Tumor Hospital of Harbin Medical University. There were 37 males and 49 females with bronchogenic cysts, aged 23 to 70(49.27±10.70)years old. According to the location of the disease, the patients were divided into mediastinal type(65 cases, 75.6%); intrapulmonary type(21 cases, 24.4%); bronchogenic cyst originating from the esophagus(9 cases, 10.5%).Results:The preoperative diagnosis coincidence rate was 9.3% in 8 cases. The rate of thoracoscopic surgery(59.3% in 51 cases), compared with the indwelling time of thoracic drainage tube after thoracotomy[(3.80±1.25) days vs.(4.97±1.54)days, P<0.001] and hospital stay[(7.08±1.75) days vs.(9.60±2.58)days, P<0.001] significantly shortened. 65 cases(71.4%, 65/91) were successfully followed up, with a median follow-up time of 34(2-111) months, and no recurrence was found. Conclusion:Bronchial cysts have no characteristic clinical manifestations, and it is difficult to make a clear diagnosis before surgery. Chest MRI has a great advantage in the diagnosis of cysts. For most cases, thoracoscopic surgery can achieve better clinical treatment results and has minimally invasive advantages. It is difficult to distinguish between bronchogenic cysts that originated in the esophagus and esophageal cysts, and there is no significant difference in clinical characteristics.

Objective:To explore the correlation between preoperative serum hyaluronic acid (HA) level and prognosis of breast cancer patients.Methods:The 98 patients with breast cancer who underwent surgical treatment in the Oncology Department of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine from January 2004 to November 2014 in a historical cohort were included, aged (52.5±9.4) years.The preoperative serum HA contents of the patients were detected. According to the median of 53.7 μg/L, the patients were divided into high and low groups with 49 patients in each group.The χ 2 test was used to analyze the correlation between the serum HA content and the general clinical data of the patients, and the Kaplan-Meier method, Log-rank test and multivariate Cox regression model wereusedto analyze the correlation between HA content and patients′ survival. Results:The percentages of patients with high HA levels in menopause and non-menopause patientswere 55.7% and 40.5%, respectively; in progesterone receptor (PR) positive and negative patients were 54.1% and 43.2%, respectively; in estrogen receptor (ER) positive and negative patients were 45.7% and 60.7%, respectively; in Ki-67 positive and negative patients were 55.6% and 43.2%, respectively; in the tumor size stage TⅠ, TⅡ, TⅢ, and TⅣ patients were 50.0%, 41.7%, 72.7%, and 1/1, respectively; in lymph node metastasis and non-metastasispatients were 45.7% and 53.8%, respectively. There was no significant correlation between the level of HA and the menopausal status, the expressions of PR, ER and Ki-67, tumor size, and lymph node metastasis in breast cancer patients (χ2=2.128, 1.086, 1.800, 1.485, 4.273, 0.656, P>0.05). Patients with high HA levels accounted for 30.9% of patients aged 52 years or less and 74.4% of patients older than 52 years (χ2=18.274, P=0.000); 43.4% of patients with early TNM and 72.7% of patients with advanced TNM (χ2=5.861, P=0.015); 45.2% of patients without distant metastasis and 78.6% of patients with distant metastasis (χ2=5.333, P=0.023); 38.1% of Her-2 negative patients and 58.9% of Her-2 positive patients(χ2=4.167, P=0.041); and the median survival of patients with high HA levels was 70 months, which was shorter than 83 months for patients with low HA levels (χ2=6.799, P=0.007). Therefore, ahigh HA content predicts an older age, a later tumor stage, higher risk of distant metastasis, positive expression of Her-2 and shorter survival. Multivariate Cox regression model analysis suggested that high levels of serum HA may be a risk factor for patients′ survival, with HR (95% CI) value of 9.98 (1.16-85.88) and P value of 0.036. Conclusion:The high level of preoperative serum HA has a certain correlation with the poor prognosis of breast cancer patients.

Objective:To explore the correlation between preoperative serum hyaluronic acid (HA) level and prognosis of breast cancer patients.Methods:The 98 patients with breast cancer who underwent surgical treatment in the Oncology Department of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine from January 2004 to November 2014 in a historical cohort were included, aged (52.5±9.4) years.The preoperative serum HA contents of the patients were detected. According to the median of 53.7 μg/L, the patients were divided into high and low groups with 49 patients in each group.The χ 2 test was used to analyze the correlation between the serum HA content and the general clinical data of the patients, and the Kaplan-Meier method, Log-rank test and multivariate Cox regression model wereusedto analyze the correlation between HA content and patients′ survival. Results:The percentages of patients with high HA levels in menopause and non-menopause patientswere 55.7% and 40.5%, respectively; in progesterone receptor (PR) positive and negative patients were 54.1% and 43.2%, respectively; in estrogen receptor (ER) positive and negative patients were 45.7% and 60.7%, respectively; in Ki-67 positive and negative patients were 55.6% and 43.2%, respectively; in the tumor size stage TⅠ, TⅡ, TⅢ, and TⅣ patients were 50.0%, 41.7%, 72.7%, and 1/1, respectively; in lymph node metastasis and non-metastasispatients were 45.7% and 53.8%, respectively. There was no significant correlation between the level of HA and the menopausal status, the expressions of PR, ER and Ki-67, tumor size, and lymph node metastasis in breast cancer patients (χ2=2.128, 1.086, 1.800, 1.485, 4.273, 0.656, P>0.05). Patients with high HA levels accounted for 30.9% of patients aged 52 years or less and 74.4% of patients older than 52 years (χ2=18.274, P=0.000); 43.4% of patients with early TNM and 72.7% of patients with advanced TNM (χ2=5.861, P=0.015); 45.2% of patients without distant metastasis and 78.6% of patients with distant metastasis (χ2=5.333, P=0.023); 38.1% of Her-2 negative patients and 58.9% of Her-2 positive patients(χ2=4.167, P=0.041); and the median survival of patients with high HA levels was 70 months, which was shorter than 83 months for patients with low HA levels (χ2=6.799, P=0.007). Therefore, ahigh HA content predicts an older age, a later tumor stage, higher risk of distant metastasis, positive expression of Her-2 and shorter survival. Multivariate Cox regression model analysis suggested that high levels of serum HA may be a risk factor for patients′ survival, with HR (95% CI) value of 9.98 (1.16-85.88) and P value of 0.036. Conclusion:The high level of preoperative serum HA has a certain correlation with the poor prognosis of breast cancer patients.

Objective: To investigate the diagnostic value of MRI and molybdenum target in ductal carcinoma in situ (DCIS). Methods: From June 2014 to June 2018, 58 patients diagnosed as DCIS in Heji Hospital Affiliated to Changzhi Medical College were chosen.MRI and mammography were performed, and the diagnostic potency of the two methods was observed. Results: The coincidence degree of molybdenum target X-ray and MRI diagnosis: the coincidence coefficient kappa=0.452 of molybdenum target X ray and MRI showed that the two diagnostic methods were in general anastomosis.The diagnostic consistency of different examination methods: the diagnostic consistency of molybdenum target combined with MRI (96.6%) was significantly higher than that of molybdenum target X(82.8%), the difference was statistically significant (χ²=5.95, P<0.05), and there was no statistically significant difference compared with MRI(89.7%) (χ²=2.43, P>0.05). Conclusion Molybdenum target and MRI in diagnosis of DCIS have their own characteristics.Combined application can improve the diagnostic effect.

Objective To investigate the clinical characteristics of DCD donor-derived CRKP infection and bleeding in kidney transplantation,and to summarize the experience of diagnosis,treatment and prevention.Methods A retrospective analysis was carried out from July 2016 to December 2017 in hospital,containing clinical data of 4 cases of CRKP-infected DCD donors and 7 cases of kidney transplantation recipients.Results In the CRKP culture of 4 cases of DCD donors,1 case was positive for blood culture,1 case was positive for urine culture,1 case was positive for sputum culture,and 1 case was negative for blood,urine and sputum culture.The corresponding 7 recipients were all positive for blood culture after renal transplantation,4 cases were positive for urine culture,3 cases were positive for sputum culture,and 5 cases were positive for perirenal drainage.Of the 7 patients,4 cases had renal artery hemorrhage,1 of them was died.The average bleeding time was 17.75 days after operation (14-19 days).In 7 patients with renal transplantation,CRP increasd.And in 3 cases of deaths,CRP was stably higher than normal.Meanwhile,CRP in 4 surviving patients gradually decreased to the normal range after effective anti-infection treatment.All 7 patients were treated with carbapenems;2 patients were dead without avibactam therapy;and 5 cases were treated with avibactam and carbapenems and survived,1 case died and 1 case had good renal function recovery.Conclusion Positive CRKP in blood,urine and sputum of DCD donors can lead to CRKP infection in kidney transplant recipients.Even if the body fluids of donors are all negative,the false negative results could not be excluded.Persistent or increased high-level CRP after operation is an early warning on CRKP infection.And CRP can be used as an indicator for evaluating the effectiveness of anti CRKP therapy.The combination of avibactam and carbapenem antibiotics is an effective regimen in the treatment of DCD donor-derived CRKP.

Objective To investigate the magnetic resonance imaging (MRI) features and differential diagnosis of axillary schwannomas, and analyze the causes of misdiagnosis. Methods A retrospective study from October 2014 to October 2017 was performed in 5 patients with axillary schwannomas, confirmed by surgery and pathology, in whom clinically suspected axillary metastases have been diagnosed. All the patients underwent breast MRI to summarize the key points of diagnosis and differential diagnosis. Results Five tumors located in neurovascular bundles extending along the brachial plexus nerve distribution. The tumors presented as single, spindle or ovoid masses, well-circumscribed margins in 4 cases, entering and exiting nerve signs in 5 cases, the split fat sign in 4 cases, vascular compressed sign in 5 cases, and target sign in 1 case. The tumors were isointense or slight hypointense on T1WI compared to the adjacent muscle. Three tumors manifested heterogeneous slight hyperintense, 1 tumor was slight hyperintense, and the target sign was seen in 1 tumor on T2WI. Diffusion-weighted image was heterogeneous hyper or slightly hyper-intense. When b＝500 and 1 000 s/mm2, the apparent diffusion coefficient (ADC) was (1.40 - 2.23) × 10 -3mm2/s and (1.31 -2.94) × 10-3mm2/s respectively. All the 5 tumors manifested persistent enhancement on dynamic contrast-enhanced MRI (DCE-MRI). Three tumors were heterogeneously enhanced, and 2 tumors were circularly enhanced. Conclusions Axillary schwannomas has certain characteristics which can be helpful for the identification of metastatic lymph nodes.