Background and purpose:It was reported that nuclear factor of activated T cells (NFAT) is closely related with carcinomas. Esophageal squamous cell carcinoma (ESCC) is one of the most common carcinomas in China. The present study investigated the expression and clinical significance of NFAT isoforms in ESCC. Methods:The expression of NFAT isoforms and the differences in different pathological levels of ESCC were detected in 104 specimens of human ESCC tissues and normal esophageal tissues by immunohistochemistry. Results:This study found that the positive rates of NFAT1 (53.8%), NFAT2 (10.6%), NFAT3 (26.9%), NFAT4 (45.2%) expression were significantly higher in tumor tissues than in adjacent normal esophageal tissues (P<0.001), respectively. The positive rate of NFAT1 expression was significant-ly higher in drinkers (62.3%) than nondrinkers (37.1%, P=0.01), and also higher in patients with lymph node metastasis (68.4% vs 5.5%, P=0.002) and with late stage (58.7% vs 36.2%, P=0.02). Multivariate analysis showed that NFAT1 expression was correlated with lymph node metastasis. The positive rate of NFAT3 was significantly higher in patients with lymph node metastasis (39.4%) than in those without lymph node metastasis (19.7%, P=0.03). Conclusion:These results suggest that the overexpression of NFAT1 and NFAT3 is associated with lymph node metastasis in ESCC.

Objective To discuss some expectation on reform of classroom teaching method for eight-year program medical education, in order to provide a basis for the evaluation and reform of classroom teaching method. Methods Investigation that mainly included expectation on reform of classroom teaching method, classroom teaching method of most expected application, and ex-pected value of classroom teaching method was investigated and analyzed with self-made question-naire, to total 78 medical students (22 of Grade 2008, 20 of Grade 2009, 20 of Grade 2010, 16 of Grade 2011) of eight-year program who had completed the stage before medical education in the uni-versity, and related 32 teachers. Software of SPSS 20.0 was used to make statistical analysis. Results In the survey, 55.8% students and 78.1% teachers hoped to reform the current classroom teaching methods (χ2=4.782,P=0.029). 61%students and 90.6% teachers expected comprehensive application of diverse method(χ2=15.464,P=0.017), including instruction combined with writing on blackboard, teaching by multimedia, PBL, inversion-type class, independent learning, a lot of questions for exami-nation, and so on. The value expectation (>60%) of classroom teaching method was multivariate, in-cluding to stimulate students' interest in learning, to cultivate ability of finding and solving problems, conducive to understanding of knowledge and memory, conducive to comprehension and memory of knowledge, conducive to cultivation of learning method, able to improve ability of creativity and criti-cal thinking, conducive to strengthening interaction and communication of teachers and students, to improve the subject consciousness of student in classroom, helpful to cultivate the spirit of teamwork, to improve the efficiency of teaching, and able to make full use of information technology. Conclusions For the diversified value demand and the highly expectation on reform of current classroom teaching method, more attention should be paid to evaluation and guidance on reform of classroom teaching method for eight-year program medical education.

Objective To investigate the expression of Hippo pathway component in hepatic cancer tissues and investigate its effects on the tumor recurrence after Iiver transplantation.Methods The clinical data of 105 patients with liver cancer who were admitted to the Third Affiliated Hospital of Sun Yat-Sen University from July 2004 to September 2009 were retrospectively analyzed.The samples of liver cancer tissues were collected.The maximum diameter,number of foci,blood vessel involvement,preoperative level of alpha-fetoprotein (AFP),results of postoperative pathological examination were analyzed.All the patients were followed up via out-patient examination,mail and phone call.Patients were followed up once a week within the first month after operation,and once a month within the 6 months after operation,and then once every 3 months at 1 year later.The follow-up ended in December 2012 or tumor recurrence.The disease-free survival time began at the date of operation and ended at the time of tumor recurrence.The expressions of Yes-associated protein (YAP),phosphorylated YAP,Hippo pathway component (Lats1/2,pLats1/2,Mst1,pMst1/2) were detected by immunohistochemical staining.All data were analyzed using the chi-square test or Student t test.Factors might influence the postoperative tumorfree survival time after liver transplantation were analyzed using the Cox regression model.The survival curve was drawn by Kaplan-Meier method,and the disease-free survival was analyzed using Log-rank test.Results Positive expressions of YAP and phosphorylated YAP were detected in the nucleus and cytoplasm,and the positive expressions of Lats1/2,pLats1/2,Mst1 and pMst1/2 were detected in the cytoplasm.The positive expressions of YAP,phosphorylated YAP,Latsl/2,pLats1/2,Mst1 and pMst1/2 protein were 51.43％ (54/105),55.24％ (58/105),45.71％ (48/105),9.52％ (10/105),64.76％ (68/105) and 20.00％ (21/105),respectively.The positive expression of YAP was correlated with the tumor diameter,venous infiltration and AJCC stage (x2=4.173,9.611,7.233,P ＜ 0.05).The positive expression of Lats1/2 protein was correlated with tumor diameter and AJCC stages (x2=14.413,7.969,P ＜ 0.05).The positive expression of Mst1 protein was correlated with the tumor diameter (x2=4,129,P ＜0.05).The results of univariate analysis showed that the protein expressions of YAP,Lats1/2,pMst1/2,age,tumor diameter,tissue differentiation,preoperative level of AFP,venous infiltration and AJCC stages were risk factors influencing tumor recurrence after liver transplantation (HR =2.603,0.502,1.802,0.955,3.559,2.395,2.414,2.915,2.086,95％ CI:1.452-4.666,0.287-0.880,1.040-3.123,0.931-0.981,1.921-6.595,1.475-3.889,1.313-4.337,1.604-5.229,1.370-3.176,P ＜ 0.05).The results of multivariate analysis showed that the positive expression of YAP,tumor diameter ＞ 5 cm,low differentiation of tissue and AJCC stages Ⅲ were independent risk factors influencing tumor recurrence after liver transplantation (HR=2.011,2.176,2.390,1.574,95％CI:1.115-3.628,1.125-4.206,1.448-3.945,1.041-2.381,P ＜ 0.05).The median time of follow-up was 13.0 months (range,1.0-96.0 months).Eight patients missed follow-up.Fifty-four patients had tumor recurrence,and the mean time of tumor recurrence was 6.7 months (range,1.0-41.0 months).The disease-free survival time of patients with positive expression of YAP were significantly shorter than those with negative expression of YAP (Log-rank value =12.890,P ＜ 0.05).Conclusions Positive expressions of YAP and phosphorylated YAP were detected in the nucleus and cytoplasm,and the positive expressions of Lats1/2,pLats1/2,Mst1 and pMst1/2 were detected in the cytoplasm.The positive expression of YAP is the independent risk factor for tumor recurrence after liver transplantation.

Objective To assess roles of vascular endothelial growth factor(VEGF)and plateletderived growth factor(PDGF)in the mechanisms of lymphangiogenesis in epithelial ovarian carcinoma.Methods (1)Expression of Proxl,a newly described lymphatic endothelial cell nucleus marker,VEGF-A,VEGF-C,VEGF-D and PDGF-A,PDGF-B,PDGF-C,PDGF-D were detected bv RT-PCR in SKOV3 cell line and in 90 ovarian tissue samples,included 15 benigh tumors 10 borderline tumors, 45 malignant tumors and 20 normal ovarian samples.(2)Expression levels of Proxl,VEGF-A,-C,-D and PDGF-A,-B,-C,-D were detected in 90 ovarian tissue sample mentioned above by real-time quantitative PCR(RTQPCR).Resuls (1)Proxl was expressed in ovarian samples mentioned above,while not detected in SKOV3 cell. VEGF-A,-C,-D and PDGF-A,-B,-C,-D were found in SKOV3 cell and various ovarian tissues.(2)Expression levels of Proxl(2.2±1.3,P＜0.01),VEGF-A(3.5±1.5,P＜0.01),VEGF-C(19±14.P＜0.01),VEGF-D(3.0±1.8,P＜0.01)and PDGF-A(3.3±3.3,P＜0.05),PDGF-C(6.9±4.6,P＜0.01)in malignant group were found to be significantly higher than those in borderline group and benign group.(3)The expression levels of Proxl,VEGF-A and PDGF-A were significantly greater in samples from the patients with lymph node metastasis(Proxl:3.0±1.4,VEGF-A:4.1± 1.7,PDGF-A:4.9±4.1),peritoneum metastasis(Proxl:2.8±0.9,VEGF-A:4.0±1. 8,PDGF-A:4.5±4.0)and in stage Ⅲ-Ⅳ(Proxl:2.6±1.3,VEGF-A:4.0±1.4,PDGF-A:4.1±3.7)than those without lymph node metastasis,without peritoneum metastasis and in stage Ⅰ-Ⅱ.There was a significant increased in the degree of VEGF-C and VEGF-D expression in positive lymph node metastasis group(VEGF-C:24±13,VEGF-D:3.9±2.0)compared with negative group(P＜0.05).(4)There were significant positive correlations between the expression levels of Proxl and VEGF-D(r=0.62,P＜0.01),PDGF-C(r=0.91,P＜0.01)or PDGF-D(r=0.61.P＜0.01).Conclusions VEGF-A,VEGF-C and PDGF-A may promote lymphatic metastasis in epithelial ovarian carcinoma though else mechanisms other than lymphangiogenesis.VEGF-D may facilitate lymphangiogenesis and lymph node metastasis in epithelial ovarian cancer.There is no significant correlation between the expression of PDGF-B and lymphangiogenesis and lymph node metastasis.PCGF-C and PDGF-D may motivate lymphangiogenesis,but could not participate in lymph node metastasis in ovarian carcinoma.

Objective To investigate the correlation between the Yes-associated protein (YAP) expression and epithelial-mesenchymal transition (EMT) by transiently transfecting with YAP small-interfering RNA (siRNA) in MHCC97H and MHCC97L cells.Methods MHCC97H and MHCC97L cells were transiently transfected by YAP siRNA.Furthermore,protein expressions and mRNA levels of characteristic markers of EMT (E-cadherin,N-cadherin) were examined by Western blotting and real-time polymerase chain reaction.Transwell invasion assay was used to detect changes of invasiveness of MHCC97H and MHCC97L cells.Results The YAP siRNA transfected group in MHCC97H was examined after 72 hours by Western blotting.The result showed obviously higher expression of E-cadherin in transfected group compared with the control group (P ＜ 0.05),and lower expression of N-cadherin (P ＜ 0.05).In MHCC97L cells,the expression of E-cadherin was also significantly increased (P ＜ 0.05),however,N-cadherin expression did not significantly change (P ＞ 0.05).Moreover,compared with the control group,transwell invasion assay showed that the number of the transfected group cells significantly decreased in MHCC97H(66 ± 6.89 vs 117 ± 7.23,P ＜ 0.05),and compared with the control group,the number of the transfected group also significantly decreased in MHCC97L (40 ±2.65 vs 77 ±4.33,P ＜0.05).The result of real-time polymerase chain reaction indicated that mRNA levels of E-cadherin increased (P ＜ 0.05),but mRNA levels of N-cadherin did not significantly change (P ＞ 0.05).This is considered as post-transcriptional regulation after silencing YAP in MHCC97H and MHCC97L.Conclusions YAP silencing is able to inhibit EMT in MHCC97H and MHCC97L cells by modulating the characteristic markers of EMT.The inhibition of YAP expression can reduce the invasion ability of hepatocellular carcinoma cells.

Objective To find out related proteins that could possibly predict curative effects of neoadjuvant chemotherapy(NACT) by applying surface-enhanced laser desorption/ionization time-of-flight mass spectrometry(SELDI-TOF MS)to detect the difference in protein spectrum of breast cancer tissues.Methods Cancer tissue samples from 30 cases of breast cancer patients were detected by SELDI-TOF MS before NACT to get protein spectrum of breast cancer tissues.After NACT,the curative effects were evaluated according to RECIST standard and patients were divided into effective group(19 cases with complete response or partial response)and invalid group (11 cases with stable disease or progressive disease).Results 11 different protein peaks (P ＜ 0.05) were screened out:3491 Da(t =3.189 P =0.004),5158 Da(t =3.897 P =0.001),5360 Da(t =2.157 P =0.04),7549 Da(t =2.173 P=0.038),8451 Da(t =2.258 P =0.032),8694 Da(t =3.234 P =0.003),9089 Da (t =2.653 P =0.013),10528 Da(t =3.127 P =0.004),13445 Da(t =2.231 P =0.034),15118 Da(t =3.255 P =0.003),and 44065 Da(t =2.554 P =0.017).They were all expressed higher in effective group than in invalid group.The difference had statistical significance(P ＜ 0.05).Conclusion SELDI-TOF MS technology can screen out protein spectrum sensitive to NACT,providing evidences for breast cancer patients whether or not taking NACT.

Objective To explore ideal surgical axillary management of early breast cancer,and to determine the feasibility of reducing false negative rate of sentinel lymph node biopsy(SLNB)by combination of axillary suspicious lymph node biopsy and SLNB.Methods From Jan.2008 to Oct.2009,42 consecutive cases with early breast cancer were enrolled.All patients underwent suspicious node hook-wire location by doppler ultrasonography before operation.SLNB and suspicious lymph node biopsy were performed during operation.Complete axillary nodes dissection(ALND)or level II dissection would be conducted according to the biopsy result.The difference of node status prediction between SLNB and SLNB with axillary suspicious lymph node biopsy and was compared.Results All the 42 cases successfully underwent SLNB (100%).There were 2 false negatives occurred in SLNB,resulting in false negative rate of 11%,sensitivity of 88.9%and accuracy of 95.2%in predicting axillary nodes status.By contrast,SLNB with axillary suspicious node biopsy showed a false-negative rate of 0%,sensitivity of 100%,and accuracy of 100%.Conclusions Compared to SLNB in early breast cancer,combination of suspicious node biopsy and SLAB has a tendency of reducing false negative rate.However,Because of the limited samples,the difference has no statistical significance(P=0.2500).

Objective:To observe the relationship between the occurrence of symptomatic hypocalcemia (SH) and various potential influencing factors in patients after thyroidectomy, stratify according to the scope of thyroidectomy, and explore the predictive value of intact parathyroid hormone (iPTH) for postoperative SH.Methods:Among 3 379 patients with thyroidectomy who admitted into the First Affiliated Hospital of Zhengzhou University from January 2020 to February 2021, 122 patients with SH after thyroidectomy were collected retrospectively and set as SH group. 100 patients of the remaining 3 200 patients who did not suffer from SH in the same year were selected by systematic sampling method and set as control group. Pearson correlation analysis was used to analyze the potential influencing factors such as age, preoperative calcium, postoperative calcium, preoperative iPTH, postoperative iPTH, central lymph node number, blood loss, operation duration, gender, lymph node dissection method, thyroidectomy range, postoperative pathological type and other. Among them, the measurement data of normal distribution were expressed by mean±standard deviation( Mean± SD), t-test was used for the comparison between the two groups, and Chi-square test was used for count data. By drawing the receiver operating characteristic curve (ROC), the iPTH levels in patients with and without SH before/after operation (different surgical methods) were studied, and the diagnostic threshold, sensitivity and specificity of iPTH were predicted. Results:Among 3 379 patients, 122 patients suffered from SH after thyroidectomy, with the incidence rate of 3.6%. There were significant differences in gender (8 males and 114 females in SH group; 27 males and 73 females in control group), whether lateral area dissection was performed (58 cases with dissection and 64 cases without dissection in SH group; 7 cases with dissection and 93 cases without dissection in control group), thyroidectomy range (14 cases with one side and 108 cases with both sides in SH group; 73 cases with one side and 27 cases with both sides in control group), age (40.1 years old vs 43.2 years old), dissection number of central lymph nodes (8.6 vs 4.6), dissection number of cervical lymph nodes (12.3 vs 0.7), blood loss (22.8 mL vs 11.0 mL), operation duration (1.7 h vs 0.8 h), postoperative iPTH (16.4 pg/mL vs 41.9 pg/mL), preoperative iPTH (39.4 pg/mL vs 47.8 pg/mL) in SH group; and postoperative calcium level (1.9 mmol/L vs 2.2 mmol/L). There was significant differences between the two groups ( P<0.05). However, there was no significant differences between them with postoperative pathological type (4 cases with toxic goiter, 3 cases with medullary thyroid carcinoma, 1 case with thyroid follicular carcinoma, 114 cases with papillary thyroid carcinoma in SH group; 1 case with medullary thyroid carcinoma, 1 case of thyroid follicular carcinoma, 98 cases with papillary thyroid carcinoma in control group, P=0.25) and preoperative calcium (2.3 mmol/L vs 2.3 mmol/L, P=0.10). For patients with bilateral thyroidectomy, SH was easy to occur when postoperative iPTH < 20.08 pg/mL, and its sensitivity and specificity were 74.07% and 96.30%; however, for patients with unilateral thyroidectomy, SH was easy to occur when iPTH < 24.00 pg/mL after operation. Conclusions:Gender, age, postoperative calcium, preoperative iPTH, postoperative iPTH, central lymph node number, blood loss, operation duration, lymph node dissection method and thyroidectomy range are important factors affecting the occurrence of SH after thyroidectomy. With the expansion of surgical range, the postoperative iPTH level gradually decreases, which predicts the occurrence of symptomatic hypocalcemia. In order to avoid the occurrence of symptomatic hypocalcemia after operation, it is necessary to supplement calcium in time according to the range of operation and postoperative iPTH level.

Objective To evaluate the effect of lipopolysaccharide (LPS) on the expression of ATP-binding cassette transporter A1 (ABCA1) in alveolar macrophage cells of rats.Methods The alveolar macrophage cells of rats NR8383 were seeded in 6-well plates at a density of 1 × 106 cells/ml (2 ml/well) and randomly divided into 6 groups:control group (group C,n =24),0.2 μg/L LPS group (group L0.2,n =12),2.0 μg/L LPS group (group L2.0,n =12),20.0 μg/L LPS group (group L20.0,n =60),200.0 pg/L LPS group (group L200.0,n =12),and 20.0 μg/L LPS + ABCA1 siRNA group (group L20.0 + siRNA,n =12).The cells were routinely cultured in group C.LPS with the final concentrations of 0.2,2.0,20.0 and 200.0μg/L was added to the culture medium in L0.2,L2.0,L20.0 and I200.0 groups,respectively.In group L20.0 + siRNA,siRNA 50 nmol/L was added to the culture medium and 12 h later LPS 20.0 μg/L was added.In group C,6 wells were chosen for determination of ABCA1 mRNA and protein expression.At 24 h of incubation with LPS 0.2,2.0 and 200.0 μg/L,or at 2,6,12 and 24 h of incubation with LPS 20.0 μg/L,6 wells were chosen and the cell suspension was obtained for measurement of ABCA1 mRNA expression (by real-time fluorescent quantitative PCR),and ABCA1 expression (by flow cytometry).At 12 h of incubation with 20.0 μg/L LPS or with 20.0 μg/L LPS + 50 nmol/L siRNA,6 wells were chosen and the cell suspension was obtained for measurement of TLR4 mRNA expression (by real-time fluorescent quantitative PCR) and TLR4 expression (by flow cytometry).Results Compared with group C,the expression of ABCA1 mRNA and protein was significantly down-regulated in L0.2,L2.0,L20.0 and L200.0 groups,and the expression of ABCA1 mRNA and protein was up-regulated in L20.0 and L20.0 + siRNA groups.In L20.0 group,the expression of ABCA1 mRNA and protein was gradually down-regulated with the prolonging time of incubation with LPS.Compared with group L20.0,the expression of TLR4 mRNA and protein was significantly up-regulated in group L20.0 + siRNA.Conclusion LPS can down-regulate the expression of ABCA1 in alveolar macrophage cells of rats,however,ABCA1 can inhibit the synthesis of TLR4.

Objective To investigate the relationship between human leukocyte antigen-G( HLA-G) gene Exon 8 14 bp deletion polymorphism and the pathogenesis of severe pre-eclampsia.Methods Forty-two pregnant women with severe pre-eclampsia,who admitted to the Third Affiliated Hospital of Zhengzhou University from October 2008 to February 2009,and their newborns were chosen as the severe pre-eclampsia group.Another 45 healthy gravidas at the third trimester and their newborns were chosen as the control.All gravidas in both groups were Han Nationality.HLA-G Exon 8 genotyping was detected by PCR in both groups and the allele frequencies and genotype frequencies were compared between the two groups.The genotype frequencies of maternal-neonatal pairs were also analyzed.Results ( 1 ) In the severe pre-eclampsia group,14% of the maternal-neonatal pairs were homozygote of 14 bp deletion,and significantly higher frequency 33% (15/45) was found in the control group (P =0.038).(2) No significant difference was found in the allele frequencies and genotype frequencies of HLA-G 14 bp deletion polymorphism among all the mothers between the two groups (P >0.05).(3) The + 14 bp and-14 bp allele frequencies of HLA-G 14 bp deletion polymorphism in newborns in the severe pre-eclampsia group were 44% (37/84) and56% (47/84),respectively,and 30% (27/90) and 70% (63/90) in the control group.Although there was no significant difference between the two groups,but differences in trends was identified (χ2= 3.678 P = 0.055) ; The genotype (-14 bp/-14 bp) frequency of neonates in the severe pre-eclampsia group showed no difference compared with that in the control group[29% (12/42) vs 49% (22/45)],but differences in trends was also found (P =0.052).Conclusions HLA-G 14 bp deletion polymorphism is associated with the susceptibility of severe pre-eclampsia in Chinese Han nationality.Maternal-fetal genotype pairs of-14 bp/-14 bp may have reduced risk of severe pre-eclampsia.