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Objective:To examine the expression of programmed cell death 1 (PD-1) and its ligand (PD-L1) in epithelial ovarian cancer (EOC) tissues, and investigate the correlation among their expression, clinicopathological features and prognosis.Methods:The specimens of 180 patients with EOC treated in the First Affiliated Hospital of Dalian Medical University from October 2002 to December 2013 were confirmed by pathological examination. The pathological tissue specimens of subtypes ，included 120 cases of serous carcinoma, 30 cases of mucinous carcinoma, 20 cases of endometrioid carcinoma, and 20 cases of clear cell carcinoma. The normal paracancerous tissues of 50 cases randomly selected from the 180 patients as control group. Immunohistochemical SP method was used to detect the expressions of both PD-1 and PD-L1 in epithelial ovarian cancer tissues, and the relationships among their expressions,the clinicopathological parameters and prognosis were respectively analyzed.Results:（1） PD-1 was expressed in lymphocytes infiltrated in EOC tissues, and PD-L1 was expressed in the cell membranes of cancer tissues. In all EOC cases, 33 cases (18.3%, 33/180) of both PD-1 and PD-L1 were highly expressed, and only 1 (2.0%, 1/50) of control group showed high expression. There was statistically significant difference between two groups ( P<0.01). (2) Among the four subtypes tissue specimens of EOC, the high expression rate of PD-1 was 25.0% (30/120) for serous carcinoma, 3/15 for endometrioid carcinoma, 0 (0/30) for mucinous carcinoma, and 0 (0/15) for clear cell carcinoma. The high expression rate of PD-L1 was 23.3% (28/120) for serous carcinoma, 3.3% (1/30) for mucinous carcinoma, 2/15 for endometrioid carcinoma, and 2/15 for clear cell carcinoma. Both PD-1 and PD-L1 expressions in the four sub-types of tissue specimens were significantly different ( P<0.05). The high expression rate of both PD-1 and PD-L1 was 9.2% (8/87) in the early stage and 26.9% (25/93) in the late stage. There was a statistically significant difference between the two groups ( P<0.01). Similarly, the expression of both PD-1 and PD-L1 were significantly higher in the cases of high-grade EOC （type Ⅱ） than those of low-grade （type Ⅰ） and in the cases of EOC distributed bilaterally than that distributed unilaterally, and there were statistically significant differences ( P<0.05). (3) The Kaplan-Meier survival analysis showed that the survival time were respectively 35 and 36 months in the cases with high expressions of both PD-1 and PD-L1, and the survival time were the same as 61 months in the cases with low expression of both PD-1 and PD-L1, and the comparison was statistically significant ( P<0.05). Conclusions:The expression levels of PD-1 and PD-L1 in EOC tissues are higher than those in adjacent tissues, especially in serous carcinomas. The expression of both PD-1 and PD-L1 is higher in specimens of the patients with advanced stages. The results showed that the high expression of both PD-1 and PD-L1 is an indicator of poor prognosis of patients suffering from EOC.
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Objective To detect phosphorylated-extracellular signal-regulated kinase (p-ERK1/2) protein expression in epithelial ovarian cancer and cell lines,and to examine the effects of mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor AZD6244 on cell proliferation,apoptosis as well as cell cycle of ovarian cancer cells.To explore the function and significance of MAPK/extracellular signal-regulated kinase (ERK) signaling pathway in the development of ovarian cancer.Methods (1) A total of 104 cases of patients with ovarian cancer who accepted the treatment of gynecological surgery and being confirmed by pathological examination in First Affiliated Hospital,Dalian Medical University from January 2004 to December 2013 were selected.The expressions of p-ERK1/2 protein were detected by immunohistochemistry in ovarian cancer specimens,and the relationship between the expressions of p-ERK1/2 and the clinical features of patients was analyzed.(2) p-ERK1/2 and other related proteins were determined by western blot in various ovarian cancer cells,including SKOV3,OV2008,C13,A2780S,A2780CP,OVCAR4,OVCAR5,OVCAR8 and CAOV3 treated with or without MEK inhibitor.The cellular proliferation,apoptosis and cell cycle of ovarian cancer cells after treatment with MEK inhibitor were analyzed by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry,respectively.Results (1) The immunohistochemical method showed that p-ERK1/2 between low grade serous carcinoma and clear cell carcinoma were not significantly higher expressed (P＞0.05).However,a lower level of the p-ERK1/2 expression were observed among high grade serous carcinoma,mucinous carcinoma and endometrioid carcinoma (all P＜0.05).There was no significant correlation between the protein expression of p-ERK1/2 and patients' age,pathological stage of surgery,and preoperative serum CA125 level (P＞0.05).(2) Western blot showed that the protein p-ERK1/2 was widely expressed in various ovarian cancer cell lines such as SKOV3,OV2008,C13,A2780S,A2780CP,OVCAR4,OVCAR5,OVCAR8 and CAOV3.After treatment with AZD6244 (5,10 μmol/L),the level of p-ERK 1/2 in OVCAR5 and OVCAR8 decreased significantly in dose-dependent manner.Additionally,we found a reduction of the expression level of cyclin D 1,caspase-3 and appeared cleaved poly adenosine diphosphate ribose polymerase (PARP) in OVCAR5 and OVCAR8,compared with control groups.MTT assays showed that OVCAR5,OVCAR8 and A2780S were differently inhibited in the dose-dependent manner after being treated with different concentrations of AZD6244 (0,2.5,5,10,25,50 and 100 μmol/L,all P＜0.05).Further tested by flow cytometry,the results showed that AZD6244 (5,10 μmol/L) was able to induce the apoptosis of OVCAR5,OVCAR8 and A2780S,as well as G0/G1 phase arrest,both in a dose-dependent manner (P＜0.05).Conclusions As the main active and functional unit of MAPK/ERK signaling pathway,p-ERK1/2 protein is expressed in both the tissues and various ovarian cancer cell lines.AZD6244 could down-regulated the expression of p-ERK1/2 in ovarian cancer cells,accompanied by the decreased proliferation and increased cell apoptosis of ovarian cancer cells.In conclusion,MAPK/ERK signaling pathway might play a role in the development and progression of ovarian cancer,and may be provide a novel option for molecular targeted therapies of the disease.
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To better analyze the problem of abnormal neuromuscular coupling related to motor dysfunction for stroke patients, the functional coupling of the multichannel electromyography (EMG) were studied and the difference between stroke patients and healthy subjects were further analyzed to explore the pathological mechanism of motor dysfunction after stroke. Firstly, the cross-frequency coherence (CFC) analysis and non-negative matrix factorization (NMF) were combined to construct a CFC-NMF model to study the linear coupling relationship in bands and the nonlinear coupling characteristics in different frequency ratios during elbow flexion and extension movement. Furthermore, the significant coherent area and sum of cross-frequency coherence were respectively calculated to quantitatively describe the intermuscular linear and nonlinear coupling characteristics. The results showed that the linear coupling relationship between multichannel muscles was different in frequency bands and the overall coupling was stronger in low frequency band. The linear coupling strength of the stroke patients was lower than that of the healthy subjects in different frequency bands especially in beta and gamma bands. For the nonlinear coupling, the intermuscular coupling strength of stroke patients in different frequency ratios was significantly lower than that of the healthy subjects, and the coupling strength in the frequency ratio 1∶2 was higher than that in the frequency ratio 1∶3. This method can provide a theoretical basis for exploring the intermuscular coupling mechanism of patients with motor dysfunction.
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Humans , Case-Control Studies , Electromyography , Muscle, Skeletal , Stroke , Stroke RehabilitationABSTRACT
Objective To examine the expressions of IKKε protein in the specimens and cells of epithelial ovarian cancer and investigate the effect of IKKε inhibitor on cell proliferation and apoptosis. Methods (1) A total of 118 cases of patients with the median age of 59 who have accepted surgical treatment due to ovarian cancer in the First Affiliated Hospital of Dalian Medical University from January 2006 to April 2013 were selected. Twenty cases of patients with the median age of 55 who have accepted hysterectomy and salpingo-oophorectomy due to uterine leiomyoma during the same period were selected as the control. The expressions of IKKε protein were detected by immunohistochemistry in normal ovarian tissues and epithelial ovarian cancer specimens,and the relationship between the expressions of IKKε and the clinical features of patients was analyzed. IKKε protein was determined by western blot in various ovarian cancer cells, including SKOV3, OV2008, C13, A2780S, A2780CP, OV4, OV5, OV8, and CAOV3 treated with or without IKKε inhibitor. The cellular proliferation and apoptosis of ovarian cancer cells after 48 hours treatment of IKKε inhibitor were analyzed by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry, respectively. Results (1) The immunohistochemical results showed that IKKε was highly expressed in epithelial ovarian cancer specimens with the expression rate 66.1% (78/118), compared with normal ovarian tissue with the expression rate 35.0% (7/20), which exhibited statistically significant difference (χ2=6.993, P=0.008). The expression of IKKε protein was correlated with International Federation of Gynecology and Obstetrics (FIGO) stage, histological grade, the level of CA125 in preoperative serum and distribution of the tumor (P0.05). (2) IKKε was widely overexpressed in different levels in SKOV3, OV2008, C13, A2780S, A2780CP, OV4, OV5, OV8, and CAOV3 cells, and the expression of IKKε decreased as the increase of the concentration of IKKε inhibitor (0.1 and 0.5 μmol/L) in OV2008, C13, A2780S, and A2780CP cells after 48 hours treatment. Different concentrations of IKKε inhibitor (0.05, 0.1, 0.5, 1, 5, 10, and 25 μmol/L) significantly inhibited the proliferation of OV2008, C13, A2780S, A2780CP, and SKOV3 cells in a concentration-dependent manner (P<0.05), and the half maximal inhibitory concentration (IC50) was 0.43, 0.86, 0.10, 0.19, and 0.24 μmol/L, respectively. The cell apoptotic rate of OV2008, C13, A2780S, A2780CP, and SKOV3 cells was significantly increased after 48 hours treatment of IKKεinhibitor with the concentration of 0.1 and 0.5 μmol/L (P<0.05). Conclusions The IKKε protein in epithelial ovarian cancer specimens and cells is overexpressed. IKKε inhibitor could inhibit cellular proliferation and induce apoptosis in a concentration-dependent manner. Together, the result indicated that IKKε may be a candidate target for the treatment of ovarian cancer in future.
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Objective To detect and explore the expression and clinical significance of dual specificity tyrosine phosphorylation regulated kinase1b (Dyrk1b) in the specimens and cells of cervical lesions. Methods (1)All the data were collected from 75 patients with cervical cancer and 52 cases with squamous intraepithelial lesion(SIL)admitted in the First Affiliated Hospital of Dalian Medical College during Jan. 2011 to Dec. 2013 and confirmed by pathological examination, included 60 cases of stageⅠand 15 cases of stageⅡ, 12 cases with low-grade squamous intraepithelial lesion(LSIL)and 40 cases with high-grade squamous intraepithelial lesion(HSIL). While, 28 cases with chronic cervicitis were chosen as the control group. The protein expression of Dyrk1b was detected by immunohistochemistry among the four groups.(2)The expression of Dyrk1b in HeLa and SiHa cells were detected by western blot method and the expression of Dyrk1b protein were also detected after treatment of AZ191 (5, 10 μmol/L) for 48 hours in HeLa and SiHa cells.(3)The cellular survival and proliferation of HeLa and SiHa cells treated by different concentrations of AZ191(2.5, 5, 10, 25, 50, 100 μmol/L)for 48 hours were detected by methyl thiazolyl tetrazolium (MTT) assay.(4)The rate of apoptosis of HeLa and SiHa cells was detected by flowcytometry after treatment of AZ191 (5, 10μmol/L) for 48 hours. Results (1)The positive rates of Dyrk1b protein in chronic cervicitis, LSIL, HSIL and cervical squamous cancer by immunohistochemistry were 11%(3/28), 1/12, 42%(17/40)and 71%(53/75), respectively. The expression of Dyrk1b in cervical squamous cancer and HISL were higher than those in LSIL and chronic cervicitis (P0.05). Expression of Dyrk1b was correlated with stromal invasion depth of cervical cancer (P0.05). (2) Dyrk1b protein was expressed in different levels in HeLa and SiHa cells, and the expression of Dyrk1b was decreased gradually as the increased of the concentration of AZ191 in both HeLa and SiHa cells by treatment of AZ191 for 48 hours. (3) Different concentration of AZ191 treated on cervical cancer cells could inhibit the cellular proliferation and induce cell apoptosis in a concentration-dependent manner(P<0.01), concomitant to the decreased cell survival rate. The apoptosis rate of HeLa and SiHa were increased significantly after 10μmol/L AZ191-treatment for 48 hours, but no any difference induced by 5 μmol/L AZ191-treatment compared to control group. Also,there was no any difference between Hela and SiHa cells in either inhibitory effect or apoptosis rate induced by AZ191. Conclusions Dyrk1b is over-expressed in either specimens or cells of cervical cancer. The expression of Dyrk1b protein in cervical lesions is increased as the progression of disease. Dyrk1b inhibitor AZ191 could inhibit cellular proliferation and induce apoptosis in a concentration-dependent manner in cervical cancer cells.
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Objective To explore the clinical effects of arginine-rich enteral nutritional support therapy in patients undergoing gastrectomy.Methods The clinical data of 84 patients with gastric carcinoma who were admitted to the Wuhan Central Hospital from January 2013 to December 2014 were retrospectively analyzed.Mter gastrectomy,42 patients undergoing arginine-rich enteral nutritional support therapy were allocated to the argininerich enteral nutrition (AEN) group and 42 patients undergoing standardized enteral nutritional support therapy were allocated to the enteral nutrition (EN) group.The indexes of nutrition [BMI of patients were calculated and serum total protein (TP),albumin (Alb) and prealbumin (PAB) were detected] and immunology [T-lymphocyte subsets,total number of lymphocyte,white blood cell (WBC) and CD4 +/CD8 + were detected by flow cytometry],postoperative complications and indexes of prognosis were analyzed.The measurment data with normal distributionwere presented as (x) ± s.The comparision between groups were evaluated with the t test and repeated measures ANOVA.The count data were analyzed using the chi-square test.Results The value of TP from preoperation to postoperative day 7 was from (64 ± 16)g/L to (55 ± 13)g/L in the AEN group and from (65 ± 21)g/L to (52 ±11) g/L in the EN group,with no significant difference between the 2 groups (F =29.653,P ＞ 0.05).The values of Alb and PAB from pre-operation to postoperative day 7 were from (33 ± 17) g/L to (32 ± 3) g/L and from (0.20 ± 0.01) g/L to (0.26 ± 0.06) g/L in the AEN group and from (32 ± 19) g/L to (27 ± 5) g/L and from (0.20 ±0.03)g/L to (0.22 ±0.03) g/L in the EN group,respectively,with significant differences in the Alb and PAB between the 2 groups (F =21.784,10.653,P ＜ 0.05).The number of WBC,number of lymphocyte and percentage of CD8 + T-cell in the AEN group were (5.3 ± 0.7) × 109/L,(1.39 ± 0.06) × 109/L and 17.3 ％ ±3.5％ before operation and (5.9 ±0.7) × 109/L,(1.33 ± 0.03) × 109/L and 18.4％ ± 3.8％ after operation.The number of WBC,number of lymphocyte and percentage of CD8 + T-cell in the EN group (5.1 ± 0.5) × 109/L,(1.40±0.04) × 109/L and 16.4％±2.8％ before operation and (5.4±0.5) ×109/L,(1.23±0.04) ×109/L and 17.3％ ± 3.1％ after operation.There was no significant difference in the above indexes between the 2 groups (F =17.429,20.461,38.820,P ＞ 0.05).The percentages of CD4 + T-cell and CD4 +/CD8 + before operation and at postoperative day 10 were 34.7％ ± 5.4％,39.5％ ± 3.9％ and 1.80 ± 0.29,2.23 ± 0.32 in the AEN group,and 33.2％±4.6％,34.6％±2.4％ and 1.73 ±0.26,1.82 ±0.42 in the EN group,respectively,with no significant difference in the above indexes between the 2 groups (F =14.398,7.473,P ＜ 0.05).The incidences of postoperative complication in the AEN group and in the EN group were 9.5％ (4/42) and 31.0％ (13/42),showing a significant difference between the 2 groups (x2=4.459,P ＜ 0.05).The duration of postoperative systemic inflammatory response syndrome (SIRS) were (1.3 ± 0.6)days in the AEN group and (2.4 ± 1.0) days in the EN group,with a significant difference between the 2 groups (t =6.360,P ＜ 0.05).The time to anal exsufflation and time of bowel movements were (2.6 ± 0.3) days and (3.6 ± 0.5) days in the AEN group and (2.5 ± 0.3) days and (3.5 ± 0.5) days in the EN group,respectively,with no significant difference between the 2 groups (t =1.570,0.897,P ＞ 0.05) . There was no perioperative death.The hospital expenses were (17 000 ±4 000) yuan in the AEN group and (22 000 ±5 000) yuan in the EN group,with a significant difference between the 2 groups (t =3.860,P ＜ 0.05).Conclusion Arginine-rich enteral nutritional support therapy is superior to standardized enteral nutrition in effectively improving postoperative nutrition status,immune function and prognosis of patients undergoing gastrectomy.