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Objective:To investigate the effect of pterostilbene on the growth, apoptosis and autophagy of a human papillomavirus type 16 (HPV-16) -immortalized cervical epithelial cell line H8.Methods:H8 cells were treated with pterostilbene at different concentrations of 0 (control group) , 25, 50, 75, 100 μmol/L for 24 and 48 hours. Cell counting kit-8 (CCK8) assay was performed to evaluate the cellular proliferative activity, flow cytometry was conducted to detect apoptosis and cell cycle, monodansylcadaverine (MDC) staining and fluorescence microscopy were performed to detect autophagy, and Western blot analysis was conducted to determine the expression of the cell cycle-related protein cyclinD1, apoptosis-related proteins caspase-3 and caspase-9, autophagy-related proteins Beclin1, microtubule-associated protein 1 light chain 3 (LC3) -Ⅱ/Ⅰ, ATG5 and P62, as well as HPV oncoproteins E6 and E7. Statistical analysis was carried out by using one-way analysis of variance, repeated measures analysis of variance and least significant difference- t test. Results:After 48-hour treatment with pterostilbene at different concentrations of 0, 25, 50, 75, 100 μmol/L, the relative cellular proliferation rate significantly differed among the groups (100.00% ± 1.56%, 99.02% ± 4.97%, 93.59% ± 2.01%, 81.28% ± 4.90%, 69.17% ± 7.56%, respectively; F = 77.22, P < 0.05) , and gradually decreased along with the increase in the concentration of pterostilbene; compared with the control group, the pterostilbene groups all showed significantly decreased cellular proliferation rate (all P < 0.05) . After 24-hour treatment with pterostilbene, the proportions of H8 cells at G1, G2 and S phases significantly differed among the above groups ( F = 7 845.00, 51.14, 266.50, respectively, all P < 0.05) ; compared with the control group, the pterostilbene groups showed significantly increased proportions of H8 cells at G1 and G2 phases (all P < 0.05) , but significantly decreased proportions of H8 cells at S phase ( P < 0.05) . After 48-hour treatment with pterostilbene, the apoptosis rate was significantly higher in the 25-, 50-, 75- and 100-μmol/L pterostilbene groups (14.66% ± 0.22%, 13.50% ± 0.49%, 14.56% ± 0.19%, 15.30% ± 0.76%, respectively) than in the control group (11.58% ± 0.50%, all P < 0.05) . After 24-hour treatment with pterostilbene, MDC staining showed only a small number of H8 cells with bright dot-like fluorescence in the control group, but increased number of autophagosome-positive H8 cells with bright dot-like fluorescence in the pterostilbene groups. Western blot analysis revealed that there were significant differences in the protein expression of cyclin D1, caspase-3, caspase-9, Beclin1, LC3-Ⅱ/Ⅰ, ATG5, P62, E6 and E7 among the control and pterostilbene groups after 24- and 48-hour treatment with pterostilbene (all P < 0.05) . The treatment with pterostilbene could down-regulate the expression of cyclin D1, E6 and E7, and up-regulate the expression of caspase-3, caspase-9, Beclin1, LC3-Ⅱ/Ⅰ, ATG5 and P62, with significant differences between the control group and most pterostilbene groups in expression of the above proteins (all P < 0.05) . Conclusion:Pterostilbene can inhibit the proliferation of H8 cells, promote their apoptosis and autophagy, and down-regulate the expression of oncogenes E6 and E7.
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Objective: To compare the mutation status of epidermal growth factor receptor (EGFR) between different lesions and clini-cal characteristics of synchronous multiple ground-glass nodules (SMGGNs). Methods: A retrospective analysis was conducted using clinical data from 35 patients with SMGGNs who were admitted to and received surgery at The Fourth Hospital of Hebei Medical Uni-versity Hospital from January 2017 to December 2018. Next generation sequencing (NGS) was performed for all surgical specimens to detect the mutation status of exons 18, 19, 20, and 21 of the EGFR gene to analyze the relationship between the EGFR mutation sta-tus of the lesions and patient gender, age, lesion location, imaging manifestation of nodules, and adenocarcinoma pathological type . Results: The EGFR mutation rate was 65.7% (23/35 patients). Non-smoking patients and females had higher EGFR mutation rates (P=0.015, P<0.001). The EGFR mutation rate of invasive adenocarcinoma nodules was higher than those of atypical adenomatous hyper-plasia, adenocarcinoma in situ, and minimally invasive adenocarcinoma ( P<0.001). Exon 19 deletion and L858R mutation were the most common mutations of the EGFR gene. There was no significant difference between the pathological subtypes of adenocarcino-ma and the EGFR mutant subtype (P=0.707). Among the 27 patients with multiple nodules with detectable EGFR mutations, the EGFR mutation rate was 85.2% (23/27 patients). Conclusions: The EGFR gene mutation status is different in patients with multiple pulmo-nary ground-glass nodules, suggesting that the occurrence and development of each nodule are independent events. EGFR gene muta-tion is closely related to the development of ground-glass nodules, especially in the invasion of tumors.
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Objective@#To investigate the effect of extensive retraction clefts (RC, >20% of tumor volume) on prognosis in invasive breast carcinoma of no specific type (IBC-NST).@*Methods@#A total of 2 184 cases of IBC-NST diagnosed at the Fourth Hospital of Hebei Medical University from January 2006 to December 2008 were collected. All the cases were diagnosed according to the latest guideline and standard. After excluding cases of shrinkage due to tissue fixation, 483 cases with RC were identified, and the clinical and pathological features were retrospectively analyzed.@*Results@#Among the 483 cases, the mean tumor size was 2.0 cm (range 0.8 to 4.8 cm). Two hundred and thirty-two cases were moderately differentiated (48.0%), 97 were well differentiated (20.1%), 154 were poorly differentiated (31.9%); 382 (79.1%) cases were of stages Ⅰ and Ⅱ. A total of 177 cases (36.7%) had lymphatic invasion; nodal metastasis were found in 202 cases (41.8%). Extensive RC was found in 237 of 483 cases (49.1%). Follow-up information was available in 407 patients, and 46 died of breast cancer with survival time from 37 to 103 months. Multivariate analysis of extensive RC showed that tumor size, histological grade and nodal metastasis were risk factors of patients with IBC-NST (P<0.05). Lymphatic invasion and nodal metastasis were risk factors for extensive RCs in patients with IBC-NST (P<0.05). There was a high probability of lymph node metastasis in patients of extensive RC without lymphatic invasion, and the difference was statistically significant (P<0.05).@*Conclusions@#Both lymphatic invasion and nodal metastasis are risk factors of extensive RC. The presence of extensive RC in IBC-NST patients is correlated with poor outcome. Tumors with lymphatic invasion are more likely to show extensive RC.
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Objective To evaluate the protective effect of pterostilbene against ultraviolet B (UVB)-induced acute damage in HaCaT cells,and to explore related mechanisms.Methods The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazo1ium (MTS) assay and flow cytometry were performed to estimate the proliferative activity and the apoptosis and necrosis rate of HaCaT cells treated with different concentrations of pterostilbene respectively,so as to screen the non-toxic concentration of pterostilbene.HaCaT cells were randomly divided into several groups:normal control group receiving no treatment,UVB group irradiated with 57 mJ/cm2 UVB,3 pterostilbene groups treated with 2.44,4.88 and 9.75 μmol/L pterostilbene respectively for 24 hours,3 pterostilbene + UVB groups treated with 2.44,4.88 and 9.75 μmol/L pterostilbene respectively for 24 hours followed by UVB radiation.Western blot analysis was conducted to detect changes of the transcription factor NF-E2-related factor 2 (Nrf2) expression in cell nuclei and cytoplasm before and after the treatment with pterostilbene and UVB,quantitative PCR to determine the mRNA expression of catalase and superoxide dismutase in the HaCaT cells,and enzyme-linked immunosorbent assay (ELISA) to evaluate the activity of catalase and superoxide dismutase.Results MTS assay and flow cytometry showed that 2.44,4.88 and 9.75 μmol/L pterostilbene had non-toxic effect on HaCaT cells.The protein expression of Nrf2 in the nuclei and cytoplasm in the normal control group was 1.03 ± 0.08 and 1.04 ± 0.11 respectively.Compared with the normal control group,the protein expression of Nrf2 in the nuclei and cytoplasm experienced no significant changes in the 2.44-,4.88-and 9.75-μmol/L pterostilbene groups,and the UVB group showed similar protein expression of Nrf2 in the cytoplasm,but significantly increased protein expression of Nrf2 in the nuclei (1.77 ± 0.08,q =17.24,P ＜ 0.01).Compared with the normal control group and UVB group,the 2.44-,4.88-and 9.75-μmol/L pterostilbene + UVB groups all showed significantly lower protein expression of Nrf2 in the cytoplasm (0.86 ± 0.10,0.87 ± 0.11 and 0.46 ± 0.11 respectively,all P ＜ 0.05),but significantly higher protein expression of Nrf2 in the nuclei (2.38 ± 0.11,2.57 ± 0.11 and 2.07 ± 0.13,all P ＜ 0.01).As qPCR showed,UVB radiation could significantly inhibit the mRNA expression of CAT (P ＜ 0.05),but had no obvious effect on the mRNA expression of SOD (P ＞ 0.05).The mRNA expression of CAT and SOD experienced no significant changes in the 2.44-,4.88-and 9.75-μmol/L pterostilbene groups compared with the normal control group (P ＞ 0.05).However,2.44,4.88 and 9.75 μmol/L pterostilbene could significantly reduce the inhibitory effect of UVB radiation on the mRNA expression of CAT (P ＜ 0.05) and up-regulate the mRNA expression of SOD in the pterostilbene + UVB groups (P ＜ 0.05).ELISA revealed that UVB radiation could inhibit the activity of CAT and SOD in the HaCaT cells (both P ＜ 0.001),while 2.44,4.88 and 9.75 μmol/L pterostilbene could reduce the inhibitory effect of UVB radiation on the activity of CAT and SOD (all P ＜ 0.05).However,the activity of CAT and SOD were still lower in the 2.44-,4.88-and 9.75-μmol/L pterostilbene + UVB groups than in the normal control group (P ＜ 0.05).Conclusion Pterostilbene can prevent UVB-induced acute damage in HaCaT cells by activating the Nrf2 pathway and up-regulating the expression of the downstream antioxidant enzymes CAT and SOD.
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Objective@#To investigate the grading system for lymph vessel tumor emboli and its prognostic value in patients with invasive carcinomas of no special type (ICNST) of the breast.@*Methods@#Clinical data of 466patients with ICNST were collected from January 2006 to December 2008 in the Fourth Hospital of Hebei Medical University. The expression levels of D2-40, estrogen receptor(ER), progesterone receptor(PR) and human epidermal growth factor receptor 2 (HER-2) were analyzed using immunohistochemical staining. Grades for lymph vessel tumoremboli were classified based on the number of mitotic and apoptotic figures in tumor cells under a high-power field. Correlation analysis was performed using Spearman rank correlation test. Kaplan-meier curves and Log-rank tests were used to analyze the survival rate. Multivariate Cox proportional hazard model was used to analyze the prognostic factors.@*Results@#Among the 466 patients, grades for lymph vessel tumor emboli were categorized as follows: 280 cases were grade 0 (60.1%); 112 cases were grade 1 (24.0%); 58 cases were grade 2 (12.5%); 16 cases were grade 3 (3.4%). Correlation analyses showed that lymph vessel tumor emboli grading system was positively correlated with lymph node metastasis (r=0.365, P<0.001). Kaplan-Meier univariant analysis showed that histological grading, lymph vessel tumor emboli grading system, lymph node metastasis, the expression levels of ER, PR and HER-2 and molecular typing were associated with prognosis of patients (P<0.05 for all). Multivariate analysis of Cox proportional hazard model showed that lymph vessel tumor emboli grading system and lymph node metastasis were independent prognostic factors in patients with ICNST(P<0.05 for all).@*Conclusion@#Grading system for lymph vessel tumor emboli canpredict the clinical outcome of patients with ICNST.
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Objective To evaluate effects of tea polyphenols on the mRNA and nucleoprotein expression of Nrf2/Bach1 in human skin fibroblasts (HSFs).Methods Some HSFs were incubated with tea polyphenols at different concentrations of 0,2.5,5,10,20 and 40 mg/L for 24 hours.Methyl thiazolyl tetrazolium (MTT) assay was conducted to evaluate the proliferative activity of HSFs to screen the optimal concentration of tea polyphenols.Then,some other HSFs were treated with tea polyphenols at this optimal concentration for 24 hours.Real-time quantitative PCR (RT-qPCR) was performed to determine mRNA expression of Nrf2 and Bach1,Western blot analysis to measure nuclear expression of Nrf2 and Bach1 proteins,and immunofluorescence assay to determine the distribution of Nrf2 and Bach1 protein in the cell nucleus.Results MTT assay showed that 5 mg/L tea polyphenols had no obvious effects on the proliferation of HSFs,so 5 mg/L was chosen as the optimal concentration of tea polyphenols for subsequent experiments.HSFs cultured without tea polyphenols served as control group.After the treatment,the 5-mg/L tea polyphenol group showed significantly decreased mRNA and nuclear protein expression of Bach 1 (mRNA:0.629 ± 0.077 vs.0.940 ± 0.033,t =6.397,P ＜ 0.05;protein:1.424 ± 0.171 vs.16.966 ± 1.702,t =15.730,P ＜ 0.05),but significantly increased mRNA and nuclear protein expression of Nrf2 (mRNA:1.467 ± 0.076 vs.0.977 ± 0.091,t =7.133,P ＜ 0.05;protein:6.929 ± 0.121 vs.3.537 ± 0.126,t =33.636,P ＜ 0.05) compared with the control group.Immunofluorescence assay showed increased accumulation of Nrf2 protein,but decreased accumulation of Bach1 protein in the nucleus.Conclusion Tea polyphenols can promote the mRNA and nuclear protein expression as well as nuclear distribution of Nrf2,but suppress the mRNA and nuclear protein expression as well as nuclear distribution of Bach 1,finally exerting antioxidative effects.
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Objective To investigate the clinicopathological features and risk factors of lymph node metastasis of gastrointestinal neuroendocrine neoplasms (GI-NENs).Methods The retrospective case-control study was conducted.The clinicopathological data of 467 patients with GI-NENs who were admitted to the Fourth Hospital of Hebei Medical University from January 2006 to December 2015 were collected.Observation indicators:(1) occurrence sites and pathological classification of GI-NENs;(2) pathological characteristics of surgical specimens of GI-NENs;(3) univariate analysis and multivariate analysis affecting lymph node metastasis of GI-NENs:sex,age,tumor location,tumor diameter,pathological classification,pathological stage and tumor invasive depth.The univariate analysis and multivariate analysis were respectively done using the chi-square test and Logistic regression model.Results (1) Occurrence sites and pathological classification of GI-NENs:of 467 patients with GI-NENs,tumors of 304,15,7,14 and 127 patients were located at stomach,duodenum,small intestine,colon and rectum,respectively.Tumor diameter was 0.3-12.0 cm,with an average diameter of 2.2 cm.Of 467 patients with GI-NENs,G1 and G2 of neuroendocrine tumors (NETs),G3 of neumendocfine carcinomas (NECs) and mixed adenoneuroendocfine carcinomas (MANECs) were respectively detected in 209,64,146 and 48 patients.Lymph node metastasis rate of GI-NENs was 31.48％ (147/467).(2) Pathological characteristics of surgical specimens of GI-NENs:NETs were high-differentiated NENs.Ceils of NETs were solid and nest-,trabeculum-and tubular-shaped,and consisted of small or medium cells,with moderate amount or massive cytoplasms,round or oval nucleus,particle-shaped chromatin,unobvious nucleolus and positive endocrine markers.There were abundant of small blood vessels and surrounding fibrous stroma in peripheral tumor cell nests.NECs were low-differentiated NENs and included small cell carcinoma and large cell NEC.Cells of small cell carcinoma were small round or oval and looked similar to lymphocytes,with few amount cytoplasms,fine granularshaped or hyperchromatic nucleus and common mitosis figures.Cells of large cell NEC were large and greater than 3 lymphocytes,arrayed in organoid-or chrysanthemum-shape,with massive cytoplasms,coarse particle-shaped chromatin,obvious nucleus,clear mitosis figures and large laminar-shaped necrosis.There were different positive expressions of endocrine markers between small cell carcinoma and large cell NEC.MANECs had the characteristics of glandular cavity formation of traditional adenocarcinoma and NENs.Results of immunohistochemical staining in 467 patients showed that Ki-67 of 467 patients was positive;CD56 in 379 of 428 with CD56 test was positive;synaptophysin (Syn) in 416 of 422 with Syn test was positive;cytokeratin (CK) in 354 of 396 with CK test was positive;chromogranin (CgA)in 264 of 388 with CgA test was positive;neuron specific enolase (NSE) in 287 of 346 with NSE test was positive.(3) Univariate analysis and multivariate analysis affecting lymph node metastasis of GI-NENs:results of univariate analysis showed that sex,tumor location,tumor diameter,pathological classification,pathological satge and tumor invasive depth were related factors affecting lymph node metastasis of patients with GI-NENs (X2 =20.654,18.182,26.788,184.709,163.738,195.391,P＜ 0.05).Results of multivariate analysis showed that pathological classification and pathological stage were independent influenced factors affecting lymph node metastasis of patients with GI-NENs (HR =2.129,7.171,95％ confidence interval:1.273-3.561,-2.327-22.098,P＜0.05).Conclusions GI-NENs are mostly located on the stomach and rectum.Results of immunohistochenical staining could help diagnosis of GI-NENs.Pathological classification and pathological stage are independent influenced factors affecting lymph node metastasis of patients with GI-NENs.
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<p><b>OBJECTIVE</b>To detect the mutation of PORCN gene in a patient with focal dermal hypoplasia and study the genotype-phenotype correlation.</p><p><b>METHODS</b>Peripheral blood samples were obtained from the family members and control subjects. PCR was carried out to amplify all the exons and adjacent splice sites of PORCN gene and mutation was detected by bidirectional sequencing.</p><p><b>RESULTS</b>A G149C mutation was found at exon 2 of the PORCN gene in the patient, which caused a change from Alanine to Proline at codon 38 (A38P). The patient presented mild clinical manifestations.</p><p><b>CONCLUSION</b>A new missense mutation (A38P) in the PORCN was detected in the patient, which maybe one of the molecular mechanisms in the pathogenesis of the disease. The relationship between G149C genotype and moderate phenotype might be attributed to the influence of A38P missense mutation towards the corresponding protein, which is different from previous results.</p>
Subject(s)
Child , Female , Humans , Acyltransferases , Base Sequence , DNA Mutational Analysis , Focal Dermal Hypoplasia , Genetics , Pathology , Membrane Proteins , Genetics , Mutation , GeneticsABSTRACT
Objective:To construct GJB2 gene mutaitons common in Chinese EGFP fusion protein vectors, and to search for better way to study the mechanism of deletion mutaitons in GJB2 gene. Method: Non-fusion protein vectors of 235delC, 299-300 del AT and 176 del 16 bp were first made by point mutaiton methods in vitro. Then expression part of the upper 3 mutations were amplified by PCR and the PCR products were cloned into TA cloning vector. After cutting by restriction enzymes EcoRI/BamHI, three deletion mutaions were inserted into pEG-FP-N1 vector. Sequencing was used to verify the validity of the fusion protein vectors. HEK293 cells were trans-fected with the recombinant DNA samples by the liposome complex method. Results The recombined plasmids were highly expressed in HEK293 cells. Green fluorescence singals were distributed uniformly in cytoplasm. Conclusion; GJB2 mutations common in Chinese EGFP fusion protein vectors were constructed successfully. It may provide a better way to explore the reasons of nonsyndromic hearing loss common in Chinese.
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OBJECTIVE@#To construct GJB2 gene mutations common in Chinese EGFP fusion protein vectors, and to search for better way to study the mechanism of deletion mutations in GJB2 gene.@*METHOD@#Non-fusion protein vectors of 235delC, 299-300 del AT and 176 del 16 bp were first made by point mutation methods in vitro. Then expression part of the upper 3 mutations were amplified by PCR and the PCR products were cloned into TA cloning vector. After cutting by restriction enzymes EcoRI/BamHI, three deletion mutations were inserted into pEGFP-N1 vector. Sequencing was used to verify the validity of the fusion protein vectors. HEK293 cells were transfected with the recombinant DNA samples by the liposome complex method.@*RESULT@#The recombined plasmids were highly expressed in HEK293 cells. Green fluorescence signals were distributed uniformly in cytoplasm.@*CONCLUSION@#GJB2 mutations common in Chinese EGFP fusion protein vectors were constructed successfully. It may provide a better way to explore the reasons of nonsyndromic hearing loss common in Chinese.