OBJECTIVETo investigate the effects of oral cancer-associated fibroblasts (CAFs) on lymphangiogenesis in oral squamous cell carcinoma (OSCC).

METHODSCAFs and normal fibroblasts (NFs) were obtained from the tissues of patients with OSCC who did not receive radio-chemotherapy before operation. And the CAFs and NFs were isolated by method of tissue block and identified by immunohistochemical staining. The effects of CAFs (group A) and NFs (group B) to human lymphatic endothelial cells (HLEC) were detected by using a 24-multiwell transwell cell culture chamber. DMEM sugar medium was as blank control group. The number of proliferative, migratory, invasive and tubes of HLEC were counted under inverted phase contrast microscope.

RESULTSThe proliferative number of HLEC of group A for 96, 144, 196 h was significantly higher than that of group B and blank control group, group B higher than blank control group (P<0.01). The migratory and invasive number of HLEC of group A for 96 h was significantly higher than that of group B and blank control group, group B higher than blank control group (P<0.01). The number of tube formation of HLEC of group A for 24 h was significantly higher than that of group B and blank control group, group B higher than blank control group (P<0.01).

CONCLUSIONCAFs promote HLEC's proliferation, migration, invasion, tube formation, and these effects are stronger than NFs.

Carcinoma, Squamous Cell ; Cell Culture Techniques ; Cell Movement ; Cell Proliferation ; Endothelial Cells ; Fibroblasts ; physiology ; Humans ; Mouth Neoplasms ; pathology

Objective:This study aimed to investigate the possible mechanism of 32P colloid induced apoptosis of craniopharyngi-oma (CP) cells in vitro and the relationship between dose effect and time effect. Methods:This study established a primary cell culture of CP limited subculture cell line. Methyl thiazolyl tetrazolium (MTT) assay was performed to plot the cell survival curve after the CP cells were treated with 32P colloid at different concentrations and time. Apoptotic rate was detected by flow cytometry(FCM). Apoptosis related DNA was investigated by TUNEL fluorescent staining. The morphological characteristics of apoptotic cells were determined by Hoechst33342 fluorescence staining. The ultrastructure of apoptotic cells was investigated by transmission electron microscopy (TEM). Results:Hoechst33342 fluorescence staining, TUNEL fluorescence staining, and TEM revealed that 32P colloid induced the apoptosis of CP cells. 32P colloid reduced the survival rate and increased the apoptotic rate of CP cells as concentration (0 MBq/mL to 14.80 MBq/mL) and time (1 d to 14 d) were increased. Conclusion: 32P colloid could effectively inhibit the growth of CP cells and induce apoptosis in vitro. High concentrations and prolonged time could induce a remarkable effect.

Objective To investigate the application of statins in secondary prevention of ischemic stroke and transient ischemic attacks (TIA) in different risk groups,and to identify the factors influencing the compliance of statins. Methods Data were prospectively collected on consecutively encountered ischemic stroke or TIA patients admitted to the First Affiliated Hospital of Zhengzhou University from April 2009 to January 2010.All clinical characteristics and possible factors influencing the compliance of statins were collected; the application of statins was investigated at 3-month follow-up.The multivariate Logistic regression analysis was used for the analysis of influence factors of the compliance of statins.Results All 369 patients were collected,52.8％ of cases were prescribed statins for therapy during hospitalization.The application rate of statins in accordance with guidelines in high-risk group,extremely high-risk Ⅱ group and extremely high-risk Ⅰ group was 25.0％ (16/64),44.1％ (30/68) and 71.4％ (135/189),respectively. Logistic regression analysis showed that the statins application during hospitalization was associated with diabetes history ( P =0.032,OR =1.789,95％ CI 1.052-3.043 ) and the presence of carotid vulnerable plaques(P =0.000,OR =5.308,95％ CI 3.340-8.434).The general application rate of statins was 22.3％ (81/363),which was significantly lower than that during hospitalization. The application rate of statins in accordance with guidelines in high-risk group,extremely high-risk Ⅱ group and extremely high-risk Ⅰ group was 9.7％ ( 6/62 ),25.8％ (17/66) and 29.4％ (55/187) respectively.Logistic regression analysis showed that good compliance of statins was associated with discharge instructions on statins application ( P =0.000,OR =34.852,95％ CI 14.673-175.452 ). Conclusions The compliance of statins in secondary prevention of ischemic stroke and TIA is poor,and there is still a large gap between clinical practice and guidelines; Discharge instructions on statins application increase the compliance of statins.

Objective To investigate the predictive value of the modified National Institutes of Health Stroke Scale ( mNIHSS),measured within 9 days after stroke,for the outcome in terms of modified Rankin Scale (mRS) at 6 months after stroke.Methods AII 161 patients with acute anterior circulation ischemic stroke were recruited consecutively from July 2010 until November 2010.The mNIHSS score was assessed within 3 days,4 to 6 days and 7 to 9 days after stroke,and the mRS score at 6 months after stroke was assessed by a neurology resident.Spearman rank correlation and ROC curve were used for statistic analysis.Results One hundred and sixty-one patients were assessed within 3 days,at 4 to 6 days and 7 to 9 days after stroke.Significant Spearman rank correlation coefficients were found between mRS at 6 months and mNIHSS scores within 3 days (rs =0.592,P＜0.01 ),4 to 6 days (rs =0.597,P＜0.01 ) and 7 to 9days (rs =0.595,P＜0.01).At 6 months after stroke,the AUC ranged from 0.789 (95％ CI 0.708-0.856) for measurements within 3 days to 0.792 (95％ CI 0.712-0.857 ) and 0.799 ( 95％ CI 0.721-0.864) for 4 to 6 days and 7 to 9 days,respectively. There was no significant difference for the AUCbetween them.The optimal cut-off values for the prediction of prognosis at 6 months were 8,7 and 6 points of mNIHSS score within 3 days,4 to 6 days and 7 to 9 days,respectively.The negative predictive value gradually increased from 0.847 for assessment within 3 days to 0.867 ( 95％ CI 0.578-0.765 ) for 7 to 9 days,whereas positive predictive value declined from 0.601 for assessment within 3 days to 0.533 for 7 to 9 days.The overall accuracy of predtictions decreased from 0.753 for assessment within 3 days to 0.709 for 7 to 9 days.Conclusions When measured within 9 days,the mNIHSS has a good predictive value for final outcome in terms of mRS at 6 months after stroke.However,the optimal cut-off values for the prediction of prognosis at 6 months are different when early mNIHSS scores were assessed at different periods.

To investigate the RNA interference (RNAi) effect induced by vector-derived small interfering RNA (siRNA) targeting the three gatekeeper genes (Rad52, Ku70, Ku80) and screen the more effective target sites from candidates for further research, by using siRNA design tools online, we selected 2 candidate sequences directed to every gatekeeper gene. According to the sequences, six vector-derived siRNAs (denoted psiRNA1-6) and one mocking psiRNA7 were constructed. Among them, psiRNA1 and psiRNA2 targeted Rad52, psiRNA3 and psiRNA4 to Ku70, psiRNA5 and psiRNA6 to Ku80. The mocking psiRNA7 was used as control. After sequence identification, the seven plasmids were transfected into HepG2 cell line. siRNA-induced silencing of gatekeeper genes was determined by using RT-PCR at RNA level and Western Blot at protein level. The results showed that the six plasmids specifically targeting the coding region of gatekeeper genes were successfully designed and constructed. To some extent, the six plasmids could reduce the expression of target gene. Comparatively, the plasmid-derived siRNA psiRNA1, psiRNA4 and psiRNA5 were more effective than their counterparts. The results suggest that the gene silencing efficiency of siRNA is different, depending on their targeted region, and siRNA may provide us with practical tools for further study on the three gatekeeper genes, i.e. Rad52, Ku70, Ku80.

Objective:To explore the application value of bedside lung ultrasound score(LUS)in evaluating patients with primary graft dysfunction(PGD)after lung transplantation.Methods:A total of 35 PGD patients after lung transplantation in surgical intensive care unit from June 2018 to May 2020 were selected as research objects. After lung transplantation, one physician collected the clinical data, including age, gender, preoperative 24 h APACHE II score, postoperative hemodynamic parameters, lactate and respiratory parameters; another physician was responsible for LUS examination and LUS at 12/24/48/72 h post-operation. The correlation between LUS and oxygenation index was analyzed by bivariate correlation analysis. Receiver operating characteristic curve(ROC)was utilized for calculating the predictive value, sensitivity and specificity of LUS score for severe PGD.Results:A negative correlation existed between LUS and oxygenation index in single lung transplant( r=-0.536, P<0.01)and a negative correlation between LUS and oxygenation index ( r=-0.518, P<0.01)in double lung transplant. The area under ROC curve of LUS showed that the predictive value of LUS of severe PGD patients with single lung transplant was 7.0 with a sensitivity of 86.7% and a specificity of 72.1%, the predicted value of LUS of severe PGD in double lung transplant was 13.0 with a sensitivity of 83.3% and a specificity of 60.0%. Conclusions:Lung ultrasound is simple and easy to operate and LUS can evaluate the severity of PGD patients after lung transplantation. It has a high potency of guiding clinical diagnosis and treatment.

Objective:To study the efficacy of different surgical methods in treatment of hilar cholangiocarcinoma (HCCA), and to analyze the factors affecting long-term prognosis of HCCA patients after surgical treatment.Methods:The clinical data of 105 patients who underwent surgical treatment for HCCA at the First Affiliated Hospital of Soochow University from April 2010 to October 2021 were retrospectively analysed. There were 58 males and 47 females, with age (64.2±10.6) years old. Data analysed included surgical treatments, postoperative pathological data, perioperative complications and survival on follow-up. The Kaplan Meier survival curve was plotted, and the log-rank test and Cox proportional hazard model were performed to analyze the key factors affecting long-term prognosis.Results:Of 105 patients, 4 (3.8%) patients died during the perioperative period, and 58 patients (55.2%) developed complications with included 32 (30.5%) patients with pleural effusion and 12 (11.4%) patients with biliary leakage. The follow-up data was available in 85 patients with the overall median survival time of 19 months, and the 1-, 3-, 5-year cumulative survival rates of 58.1%, 27.0% and 24.8% respectively. The 1-, 3-, and 5-year cumulative survival rates for the R 0 resection patients ( n=59) were 69.4%, 36.2%, 27.4%, respectively, which were significantly better than 49.4%, 12.3%, 0% for the R 1/2 resection patients ( n=20), and 0% for the palliative drainage patients ( n=6) (all P<0.05). Univariate analysis and Cox multivariate analysis showed that age ≥70 years ( HR=2.158, 95% CI: 1.175-3.965), preoperative CA19-9 level ≥1 015 U/ml ( HR=1.981, 95% CI: 1.009-3.894), resection margin ( HR=2.587, 95% CI: 1.371-4.881), and lymph node metastasis ( HR=2.308, 95% CI: 1.167-4.567) were independent risk factors for long-term prognosis of HCCA patients (all P<0.05). Conclusions:R 0 resection was an effective way to prolong survival of patients with HCCA. Age, preoperative CA19-9 level, resection margin and lymph node metastasis were related to long-term survival of HCCA patients.

To investigate the RNA interference (RNAi) effect induced by vector-derived small interfering RNA (siRNA) targeting the three gatekeeper genes (Rad52, Ku70, Ku80) and screen the more effective target sites from candidates for further research, by using siRNA design tools online,we selected 2 candidate sequences directed to every gatekeeper gene. According to the sequences, six vector-derived siRNAs (denoted psiRNA1-6) and one mocking psiRNA7 were constructed. Among them, psiRNA1 and psiRNA2 targeted Rad52, psiRNA3 and psiRNA4 to Ku70, psiRNA5 and psiRNA6 to Ku80. The mocking psiRNA7 was used as control. After sequence identification, the seven plasmids were transfected into HepG2 cell line. siRNA-induced silencing of gatekeeper genes was determined by using RT-PCR at RNA level and Western Blot at protein level. The results showed that the six plasmids specifically targeting the coding region of gatekeeper genes were successfully designed and constructed. To some extent, the six plasmids could reduce the expression of target gene.Comparatively, the plasmid-derived siRNA psiRNA1, psiRNA4 and psiRNA5 were more effective than their counterparts. The results suggest that the gene silencing efficiency of siRNA is different,depending on their targeted region, and siRNA may provide us with practical tools for further study on the three gatekeeper genes, i.e. Rad52, Ku70, Ku80.

In order to screen potential mRNA locations of P gene in which targeting siRNAs can effectively inhibit HBV expression, 5 recombinant plasmids containing 4 targeting-specific siRNA fragments and a control were prepared and transfected into 2.2.15 cells respectively. The expression levels of HBx mRNA, HBs mRNA and HBc mRNA were detected by RT-PCR. The concentrations of the hepatitis B virus antigens, including HBsAg and HBeAg harvested from the culture supernatant of transfected 2.2.15 cells, were measured by ELISA. X protein was tested by Western blot. The results showed that four siRNAs against distinct mRNA locations of HBV polymerse gene had different inhibitory effects on their targeted mRNA. The plasmid-derived psiRNAl and psiRNA2 could effectively inhibit the transcription and translation of HBs gene, whereas the inhibitory efficiency of psiRNA3, psiRNA4 for HBe gene was much higher than that of psiRNA1 and psiRNA2. In comparison to the rest of psiRNAs in this study, psiRNA4 was the most effective to suppress the transcription and translation of HBx. It is suggested that siRNA can be considered as a powerful therapeutic agent for reducing HBV expression. The siRNAs against HBV polymerase are effective largely depending on the location of targeted sites. To enhance inhibitory efficiency, hunting for high effective target in polymerase gene is necessary and feasible.

ObjectiveTo analyze the expression of molecular marker affecting the prognosis of acute myeloid leukemia （AML） patients from bioinformatics database， thus providing an experimental basis for further exploration of a novel molecular marker for the prognosis of AML. MethodsThe prognostic data of 179 AML patients from The Cancer Genome Atlas （TCGA） database were examined for differential gene analysis and survival analysis. The bone marrow samples of 74 healthy individuals （HI） and 542 de novo AML patients in the dataset GSE13159 downloaded from the Gene Expression Omnibus （GEO） database were analyzed to detect the difference in the expression levels of differential target genes. Peripheral blood and bone marrow samples were collected from 18 de novo AML patients and 20 age- and gender-matched healthy controls， and real-time fluorescent quantitative PCR was used to validate the expression levels of the differential genes in the AML patients. ResultsBioinformatics data analysis showed that the optimal cut-off value of Homo sapiens NK2 homeobox 3 （NKX2-3） calculated by R language was 0.051. Survival analysis revealed a statistically poorer overall survival in de novo AML patients with high NKX2-3 expression than in those with low NKX2-3 expression （P = 0.0036）. NKX2-3 was highly expressed in patients with de novo AML than in HI and the difference was statistically significant （P < 0.001）. Real-time fluorescence quantitative PCR verified the expression levels of the NKX2-3 gene in AML patients and confirmed that compared with those in HI， in the de novo AML patients， NKX2-3-1 and NKX2-3-2 were highly expressed and were significantly correlated （P = 0.000， P = 0.000）. ConclusionNKX2-3 is highly expressed in de novo AML patients， and the AML patients with high NKX2-3 expression have poor overal survival. NKX2-3 may be closely related to the clinical outcome and prognosis of AML.