Molecular epidemiological characteristics of carbapenem-resistant Pseudomonas aeruginosa clinical isolates in southeast Shanxi, China.

OBJECTIVES: Infection by carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a serious clinical problem worldwide. However, the molecular epidemiology of the clinical isolates varies depending on the region. This study was conducted to analyse the resistance phenotype and clarify the genetic and epidemiological properties of CRPA clinical isolates from southeast Shanxi, China. METHODS: Fifty-seven isolates of CRPA were collected from a hospital in this region. These isolates were reidentified by MALDI-TOF and subjected to whole-genome sequencing by next-generation sequencing. Phylogenetic trees were constructed based on single nucleotide polymorphisms (SNPs), after which multilocus sequence typing (MLST) was performed and antimicrobial resistance genes were identified. RESULTS: All the 57 CRPA isolates carried at least one kind of gene encoding carbapenemase, such as blaIMP-1, blaIMP-10, blaOXA-10, blaOXA-395, blaOXA-396, blaOXA-485, blaOXA-486, blaOXA-488, blaOXA-494, and blaOXA-50. The isolates harboured AIM-1, CMY-51, mecD, and NmcR genes and carried one kind of Pseudomonas-derived cephalosporinase (PDC) ß-lactamase-encoding gene, such as blaPCD-1 to blaPCD-3, blaPCD-5, or blaPCD-7 to blaPCD-10. Two isolates were found to harbour the aminoglycoside-modifying enzyme genes aadA1 and aadA7; however, no isolates were found to harbour genes encoding 16S rRNA methylase or quinolone resistance-related genes. These CRPA isolates belonged to various sequence types (STs), two of which, namely, ST235 and ST277, were high-risk types. CONCLUSIONS: Our findings indicate that CRPA isolates carrying resistance genes with unique regional characteristics are spreading in this region, with a high diversity of STs, especially in high-risk clones. These findings highlight the necessity for further measures to prevent CRPA spread in Shanxi.

Vitamin D receptor induces oxidative stress to promote esophageal squamous cell carcinoma proliferation via the p53 signaling pathway.

Background: Esophageal squamous cell carcinoma (ESCC) is a common pathological esophageal cancer with poor prognosis. Vitamin D deficiency reportedly occurs in ESCC patients, and this is related to single nucleotide polymorphism of vitamin D receptor (VDR). Objective: We investigated the effect of VDR on ESCC proliferation, invasion, and metastasis and its potential mechanism. Methods: ESCC and normal tissues were collected from 20 ESCC patients. The ESCC tissue microarray contained 116 pairs of ESCC and normal tissues and 73 single ESCC tissues. VDR expression and its clinicopathological role were determined by real-time quantitative polymerase chain reaction, Western blot, and immunohistochemistry staining. sh-VDR and VDR overexpression were used to validate the effect of VDR on ESCC cell phenotype, and tandem mass tag-based quantitative proteomics and bioinformatics methods identified differential VDR-related proteins. The downstream pathway and regulatory effect were analyzed using ingenuity pathway analysis (IPA). Differentially expressed proteins were verified through parallel reaction monitoring and Western blot. In vivo imaging visualized subcutaneous tumor growth following tail vein injection of VDR-deficient ESCC cells. Results: High VDR expression was observed in ESCC tissues and cells. Gender, T stage, and TNM stage were related to VDR expression, which was the independent prognostic factor related to ESCC. VDR downregulation repressed ESCC cell proliferation, invasion, and migration in vitro and subcutaneous tumor growth and lung metastases in vivo. The cell phenotype changes were reversed upon VDR upregulation, and differential proteins were mainly enriched in the p53 signaling pathway. TP53 cooperated with ABCG2, APOE, FTH1, GCLM, GPX1, HMOX1, JUN, PRDX5, and SOD2 and may activate apoptosis and inhibit oxidative stress, cell metastasis, and proliferation. TP53 was upregulated after VDR knockdown, and TP53 downregulation reversed VDR knockdown-induced cell phenotype changes. Conclusions: VDR may inhibit p53 signaling pathway activation and induce ESCC proliferation, invasion, and metastasis by activating oxidative stress.

[The relationship between C20orf54 gene rs3746804 position single nucleotide polymorphism and susceptibility to esophageal squamous cell carcinoma].

OBJECTIVE: To explore the association of C20orf54 gene rs3746804 position single nucleotide polymorphism and susceptibility to esophageal squamous cell carcinoma (ESCC). METHODS: Purification of genomic DNA from whole blood was used the Maxwell(16) System. rs3746804 in C20orf54 was detected by direct sequencing in 434 ESCC patients from Changzhi (Shanxi province) and Linzhou (Henan province) and 554 healthy controls from Changzhi, Linzhou and including immigrators from Linzhou to Changzhi. RESULTS: For rs3746804, the genotypic frequencies of CT (37.5% vs 51.0%, 37.5% vs 52.0%), CC (44.2% vs 34.8%, 44.2% vs 33.0%) in Changzhi ESCC patients showed significant differences with healthy Changzhi controls and the healthy immigrator controls (all P < 0.05), and the frequencies of TT (18.3% vs 4.1%) and CC (44.2% vs 54.6%) in Changzhi ESCC patients showed significant differences with Linzhou ESCC patients (all P < 0.05). The genotypic frequencies of TT (4.1% vs 15.0%), CT (41.2% vs 52.0%) and CC(54.6% vs 33.0%) showed significant differences between Linzhou ESCC patients and the healthy immigrator controls (all P < 0.05), and the frequencies of TT (4.1% vs 14.1%) and CC(54.6% vs 34.8%) showed significant differences between Linzhou ESCC patients and Changzhi healthy controls (all P < 0.01). Meanwhile, there were significant differences between ESCC patients (including Changzhi and Linzhou ESCC patients) and healthy controls (including the healthy Changzhi, Linzhou and immigrator controls) in genotypic frequencies of CT (39.2% vs 48.7%) and CC (48.8% vs 38.2%) (all P < 0.01). CT and CT + TT genotype could decrease the risk of ESCC compared with the CC genotype (OR = 0.630, 95%CI 0.481 - 0.826; OR = 0.654, 95%CI 0.507 - 0.844). CONCLUSION: There is a closed relationship between SNP rs3746804 in C20orf54 and susceptibility to ESCC.

[A comparison and significance of plasma riboflavin levels in patients with esophageal squamous cell carcinoma versus Linzhou healthy migrants in Changzhi of Shanxi].

OBJECTIVE: To study the relationship between plasma riboflavin levels and esophageal squamous cell carcinoma. METHODS: We detected and compared plasma concentrations of riboflavin in patients with esophageal squamous cell carcinoma (ESCC) and immigrants of Linzhou living in Changzhi. Plasma riboflavin levels were quantified in 445 ESCC patients, 689 healthy control subjects and 347 immigrants of Linzhou living in Changzhi by using enzyme-linked immunosorbent assay. RESULTS: The plasma riboflavin levels in patients with ESCC were significantly lower than those in the healthy controls and immigrants of Linzhou living in Changzhi [(731.69 ± 330.67) µg/L vs (1090.43 ± 445.08) µg/L, (731.69 ± 330.67) µg/L vs (897.58 ± 177.78) µg/L, respectively, all P < 0.05], and the plasma riboflavin levels of the healthy controls were higher than those in the immigrants of Linzhou living in Changzhi (P < 0.05). CONCLUSION: Patients with ESCC have decreased plasma riboflavin levels as compared with the healthy controls and immigrants of Linzhou living in Changzhi, there exists a lack of riboflavin in ESCC patients, but the specific mechanism needs further study.

[Effect of ß-catenin gene silencing by shRNA on biologic characteristics of human esophageal carcinoma cells].

OBJECTIVE: To study the effects of short hairpin RNA (shRNA) mediated gene silencing of ß-catenin on the biological characteristics of esophageal carcinoma cells, and to provide theoretical and experimental evidence for the gene therapy of esophageal carcinoma through target inhibition of ß-catenin gene. METHODS: Single strand DNA was synthesized according to the hairpin RNA sequence, and then subcloned into eukaryotic expression vector pGenesil-3 to construct a shRNA-expression pDNAs driven by human U6 promoter of ß-catenin (pGen-3-CTNNB1). One additional construct of random siRNA (pGen-3-con) without homologous to any human genes was constructed in a similar fashion as control.Positive clones were identified and verified by restriction cleavage and DNA sequencing analyses. pGen-3-CTNNB1 and pGen-3-con were then transfected into esophageal carcinoma cell line Eca-109 with liposome, respectively. Positive colonies were selected with G418. Expression of ß-catenin protein and mRNA in the transfected and nontransfected Eca-109 cells were examined by Western blotting, immunofluorescence and RT-PCR, respectively. Xenograft tumor model was used to compare the tumorigenesis of three different cells.Expressions of ß-catenin in all tumor tissues were examined by immunohistochemistry staining. The invasive abilities of three different cells were examined with transwell invasion filter and Matrigel. RESULTS: ß-catenin expression levels were found markedly decreased in Eca-109 cells transfected with pGen-3-CTNNB1. In vivo, transfection with ß-catenin shRNA greatly impeded the tumor growth, pGen-3-con (1.18 ± 0.13) g, Eca-109 (1.38 ± 0.21) g, pGen-3-CTNNB1 (0.42 ± 0.09) g, P < 0.05. Immunohistochemistry staining showed a significantly decreased expression of ß-catenin in ß-catenin shRNA transfected cells than in random shRNA transfected and nontransfected cells (P < 0.05). The infiltration abilities of esophageal carcinoma cells were significantly suppressed, pGen-3-con (81 ± 5)/HPF, Eca-109 (77 ± 6)/HPF, pGen-3-CTNNB1 (41 ± 4)/HPF, P < 0.01; along with significantly decreased migration abilities, pGen-3-con (73 ± 5)/HPF, Eca-109 (69 ± 5)/HPF, pGen-3-CTNNB1 (38 ± 4)/HPF (P < 0.05). CONCLUSIONS: There are abnormal expression of ß-catenin and activation of Wnt signaling pathway in human esophageal carcinoma cell line Eca-109. RNA interference targeting ß-catenin gene suppresses the growth of xenograft tumorigenesis in nude mouse and the invasiveness and metastatic capability of esophageal carcinoma cells.

Clinical and prognostic effects of adjuvant therapy on less advanced esophageal squamous cell carcinoma patients.

BACKGROUND: This study aimed to clarify whether adjuvant therapy is suitable for less advanced esophageal squamous cell carcinoma (ESCC) patients postoperatively. METHODS: Data from 973 patients were collected. The prognosis and clinicopathological traits of these patients were calculated in both the TNM I and IIA stages. Meanwhile, 2 separate nomograms were applied in terms of the variables with a multivariate P value <0.05 in the Cox proportional hazard regression model. RESULTS: There were 471 and 502 patients in the I and IIA stage respectively; among all enrolled patients, 641, 130, 73, and 129 patients were in the no-treatment, chemotherapy, radiotherapy, and chemoradiotherapy groups, respectively. Adjuvant therapy was drawn as the independent prognostic factors for stage I (P=0.026; HR =1.081, 95% CI: 1.093, 1.308) and IIA patients (P<0.001; HR =0.788, 95% CI: 0.693, 0.896). Radiotherapy consistently obtained the best prognosis for patients when compared with the other 3 groups in the I and IIA stage. Patients in I stage had the worst prognosis after receiving chemoradiotherapy, and still, the small survival benefits of chemoradiotherapy were seen in patients of IIA stage. Two separate nomograms for the I and IIA stage were constructed. The C-index was 0.665 (95% CI: 0.569, 0.761) in the I stage and 0.645 (95% CI: 0.567, 0.723) in the IIA stage. Meanwhile, the calibration curves for predicting 3-year and 5-year survival for I and IIA patients agreed well with the actual observations. CONCLUSIONS: Among less advanced ESCC patients, adjuvant therapy was not only found to be an independent factor but also proved to be of importance in patient prognosis, and radiotherapy is recommended.

Clinical and prognostic significance of preoperative lymphocyte-monocyte ratio, neutrophil-lymphocyte ratio and neutrophil-monocyte ratio on esophageal squamous cell carcinoma patients.

BACKGROUND: The interaction between tumor cells and inflammatory cells has not been systematically investigated in esophageal squamous cell carcinoma (ESCC). The aim of the present study was to evaluate whether preoperative lymphocyte-monocyte ratio (LMR), neutrophil-lymphocyte ratio (NLR), and neutrophil-monocyte ratio (NMR) could predict the prognosis of ESCC patients undergoing esophagectomy. METHODS: A total of 1,883 patients with histologically diagnosed ESCC who underwent radical esophagectomy from May 2005 to May 2015 were retrospectively reviewed. Besides clinicopathological factors, "Survminer" package in R® was applied to determine the optimal cut-off point for LMR, NLR and NMR. Meanwhile, we evaluated the prognostic value of LMR, NLR, and PLR using Kaplan-Meier curves and Cox regression models. RESULTS: The median follow-up was 28.77 months (range, 1.60-247.90 months). The optimal cut-off point of LMR, NLR and NMR is 3.83, 2.06 and 7.21, respectively. Kaplan-Meier survival analysis of patients with low preoperative LMR demonstrated a significant worse prognosis for 5-year OS (P<0.001) than those with high preoperative LMR. The high NLR cohort had lower 5-year OS (P<0.001). No significant difference with 5-year OS was found in NMR (P=0.405). On multivariate analysis, preoperative LMR (P=0.018; HR =0.786, 95% CI: 0.645, 0.959) and NLR (P=0.028; HR =1.247, 95% CI: 1.024, 1.519) were the independent prognostic factors in ESCC patients. Integrating LMR and NLR, we divided the ESCC patients in four groups according to their cut-off points and we found the patients in LMR ≥3.83 and NLR <2.06 group received the best prognosis while the prognosis of patients in LMR<3.83 and NLR ≥2.06 group was the worst. The difference was statistically significant. CONCLUSIONS: Preoperative LMR and NLR better predicts cancer survival in patients with ESCC undergoing esophagectomy, especially under the circumstances of LMR ≥3.83 and NLR <2.06.

Prognostic significance and role of thoracic lymph node metastasis based on Chinese expert consensus in esophageal cancer.

BACKGROUND: The Chinese expert consensus on thoracic lymph node (LN) dissection in radical esophagectomy (Chinese Criteria, 2017 edition) was newly promoted. This study examined the prognostic significance and role of thoracic LN metastasis based on the Chinese Criteria for esophageal cancer. METHODS: Data of patients with thoracic esophageal squamous cell carcinoma (ESCC) who underwent curative esophagectomy in the West China Hospital from May 2005 to May 2015 were retrospectively analyzed. Patients' prognosis and clinicopathological features were compared to determine the role of Chinese Criteria and their relationship with Union for International Cancer Control (UICC)/American Joint Committee on Cancer (AJCC) 8th TNM staging. RESULTS: Overall, 2,285 qualified patients were divided into the no (n=1,148), skip (n=156), local (n=665), and mediastinal (n=316) metastasis groups according to the Chinese Criteria. Significant prognostic differences occurred among the four groups in all the thoracic and lower mediastinal ESCC patients (both P<0.001). The Chinese Criteria grouping was an independent prognostic factor for all thoracic [P<0.001; hazard ratio (HR) =1.261, 95% confidence interval (CI): 1.103-1.441], upper (P<0.001; HR =1.391, 95% CI: 1.264-1.530), lower mediastinal thoracic ESCC patients (P<0.001; HR =1.312, 95% CI: 1.257-1.370) and all thoracic ESCC after adjuvant therapy (P<0.001; HR =1.303, 95% CI: 1.221-1.390). Significant prognostic differences among Chinese Criteria groups occurred with N1 (P=0.014) and N2 (P=0.018) stages only. Significant differences in survival among N stages were found in local (P<0.001) and mediastinal (P=0.009) metastasis groups. CONCLUSIONS: Our study was the first to report the Chinese Criteria in measuring the degree of thoracic LN metastasis. Similar to N-stage, the Chinese Criteria were confirmed as an independent prognostic factor for thoracic ESCC. Further confirmation of our findings is warranted.

Effects of bone marrow-derived mesenchymal stem cells transplanted via the portal vein or tail vein on liver injury in rats with liver cirrhosis.

The aim of the present study was to compare the effects of bone marrow-derived mesenchymal stem cells (BMSCs) transplanted via the portal vein or tail vein on liver injury in rats with liver cirrhosis. BMSCs were isolated from rat bone marrow and labeled with green fluorescent protein (GFP). Then, the labeled BMSCs were injected into rats with liver injury via the portal vein or tail vein. Two weeks after transplantation, three rats in each group were sacrificed to test the distribution of GFP in the liver and the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and albumin. Six weeks later, the remaining rats were sacrificed, and serum ALT, AST, albumin, hyaluronic acid (HA), laminin (LN) and procollagen type III (PC-III) levels were measured. The expression of albumin in the liver was analyzed by immunohistochemistry. Two weeks after BMSC transplantation, GFP-positive cells were detected in the livers of rats with BMSCs transplanted via the portal vein and tail vein. Compared with pre-transplantation levels, the ALT levels of the groups with BMSC transplantation via the portal vein and tail vein were significantly decreased after two and six weeks of BMSC transplantation (P<0.05), whereas the AST and albumin levels were not significantly different at two weeks after BMSC transplantation in the two groups (all P>0.05). However, the AST and albumin levels were significantly reduced at six weeks after BMSC transplantation (all P<0.05). At six weeks after BMSC transplantation, the serum HA, LN and PC-III levels in rats transplanted with BMSCs via the portal vein or tail vein had decreased significantly (all P<0.05), as compared with the levels prior to BMSC transplantation. BMSCs transplanted via the portal vein and tail vein achieved similar improvements in liver function in rats with liver cirrhosis, which suggests that peripheral venous administration is a convenient and effective route for BMSC transplantation.

Association between Ras association domain family 1A promoter methylation and esophageal squamous cell carcinoma: a meta-analysis.

RASSF1A has been reported to be a candidate tumor suppressor in esophageal squamous cell carcinoma (ESCC). However, the association between RASSF1A promoter methylation and ESCC remains unclear. Eligible studies were identified through searching PubMed, Medline, Web of Science, and the China National Knowledge Infrastucture database. Studies were pooled and odds ratios (ORs) with corresponding confidence intervals (CIs) were calculated. Funnel plots were also performed to evaluate publication bias. Twelve studies involving 859 cases and 675 controls were included in this meta-analysis. A significant association was observed between RASSF1A methylation and ESCC overall (OR = 11.7, 95% CI: 6.59-20.9, z=8.36, P<0.00001). Subgroup analysis showed that the OR for heterogeneous tissues was 5.35 (95% CI = 2.95-9.71) while for autologous tissues it was 16.0 (8.31-30.96). For patient sample size, the OR for the <50 subgroup was 9.92 (95% CI = 2.88-34.2) and for the 50 case group was 13.1 (95% CI = 6.59-25.91). The OR for a relationship between RASSF1A methylation and TNM stages was 0.27 (95% CI=0.10-0.77), whereas there were no significant differences in RASSF1A methylation in relation to gender and differentiation among ESCC cases. This meta-analysis suggests a significant association between RASSF1A methylation and ESCC.

Effects of PLCE1 gene silencing by RNA interference on cell cycling and apoptosis in esophageal carcinoma cells.

Esophageal squamous cell carcinoma (ESCC) is one of the most malignancies with a poor prognosis. The phospholipase C? gene (PLCE1) encodes a novel ras-related protein effector mediating the effects of R-Ras on the actin cytoskeleton and membrane protrusion. However, molecular mechanisms pertinent to ESCC are unclear. We therefore designed PLCE1-special small interfering RNA and transfected to esophageal squamous cell (EC) 9706 cells to investigate the effects of PLCE1 gene silencing on the cell cycle and apoptosis of ESCC and indicate its important role in the development of ESCC. Esophageal cancer tissue specimens and normal esophageal mucosa were obtained and assayed by immunohistochemical staining to confirm overexpression of PLCE1 in neoplasias. Fluorescence microscopy was used to examine transfection efficiency, while the result of PLCE1 silencing was examined by reverse transcription (RT-PCR). Flow cytometry and annexin V apoptosis assays were used to assess the cell cycle and apoptosis, respectively. Expression of cyclin D1 and caspase-3 was detected by Western-blotting. The level of PLCE1 protein in esophageal cancer tissue was significantly higher than that in normal tissue. After transfection, the expression of PLCE1 mRNA in EC 9706 was significantly reduced, compared with the control group. Furthermore, flow cytometry results suggested that the PLCE1 gene silencing arrested the cell cycle in the G0/G1 phase; apoptosis was significantly higher than in the negative control group and mock group. PLCE1 gene silencing by RNAi resulted in decreased expression of cyclin D1 and increased expression of caspase-3. Our study suggests that PLCE1 may be an oncogene and play an important role in esophageal carcinogenesis through regulating proteins which control cell cycling and apoptosis.

[Inducing-apoptosis effect of brucine on human monocytic leukemia cell line THP-1 and its mechanism].

This study was aimed to investigate the inducing-apoptosis effect of brucine on human monocytic leukemia cell line THP-1 cells and its possible mechanism. The inhibition effect of brucine on growth of THP-1 cells was measured by CCK-8 method. Morphological changes of THP-1 cells treated with brucine was detected by acridine orange/ethidium bromide (AO/EB)double staining. Annexin-V/PI double labeling method was used to assay the apoptosis rate of THP-1 cells. The effect of brucine on THP-1 cell cycle distribution was detected by PI single staining. RT-PCR was used to detect the expression of BCL-2 and BAX. The results showed that the brucine could inhibit the THP-1 cell growth in concentration and time-dependent manners at the range of 50 to 400 µg/ml. The cells stained with AO/EB revealed that the brucine induced the nuclear chromatin condensation. After the THP-1 cells were treated with brucine of 400µg/ml for 48 hours, most nucleic were stained as orange-red, and condensed, displaying the late apoptotic cell morphology. Annexin-V/PI detection showed that brucine could induce apoptosis of THP-1 cells in a concentration-dependent manner. Compared with the control group, more cells in brucine-treated group were arrested at G0/G1 phase in a concentration-dependent manner. RT-PCR detection revealed that the expression of BCL-2 was down-regulated strikingly and BAX was up-regulated. It is concluded that brucine can efficiently inhibit cell growth and block THP-1 cells in G0/G1 phase. The mechanism of THP-1 cell apoptosis induced by brucine may be related to the inhibition of BCL-2 and activation of BAX.

[A new photodynamic therapy drug toward gastric cancer MGC803 cell].

OBJECTIVE: To investigate the treatment efficiency of a new photodynamic therapeutic(PDT) drug synthesized by our laboratory toward MGC803 cells and related mechanisms. METHODS: Bleaching method was used to evaluate the photostability of drug upon repetitive illumination. MTT assay was used to determine the ability of new drug killing MGC803 cells after PDT. Laser scanning confocal microscopy (LSCM) was applied to investigate the subcellular localization of drug in MGC803 cells (mitochondria and/or lysosomes). Hoechst staining and flow cytometry(Annexin V/PI double-staining) were performed to detect the death mode of MGC803 cells after PDT. RESULTS: This new PDT drug had good stability to light irradiation after repetitive illumination. MTT assay showed no cytotoxicity towards MGC803 cells only by drug or only by irradiation(P>0.05), but intense lethal effect was observed with drug and light combination(P<0.05). The phototoxicity of medicine increased with the elevation of concentration, the LD50 was 1.74 µmol/L, and reaching plateau at the concentration of 3.12 µmol/L, even increasing the concentration. LSCM found that drug localized in lysosomes of MGC803 cells. Hoechst staining showed that the death mode of cells was mainly necrosis and Annexin V/PI double-staining proved this result further. CONCLUSION: This new PDT drug is an effective PDT sensitizer for MGC803 cells and the death mode of cells is mainly necrosis.

Gene silencing of ß-catenin by RNAi inhibits proliferation of human esophageal cancer cells by inducing G0/G1 cell cycle arrest.

OBJECTIVES: The aim of the present study was to explore mechanisms underlying the effects of down-regulating ß-catenin expression on esophageal carcinoma (EC) cells. METHODS: Cell cycle distribution and apoptosis were determined using flow cytometry and annexin V apoptosis assay, respectively. Transmission electron microscopy (TEM) was used to examine changes in ultrastructure, while expression of cyclin D1 protein and mRNA was detected by western blot and real-time PCR. Proliferating cell nuclear antigen (PCNA) and extracellular signal-regulated kinase (ERK) 1/2 were evaluated by Western blot analysis. PCNA labeling index (LI) was determined by immunocytochemistry. RESULTS: Compared with pGen-3-con transfected and Eca-109 cells, the percentage of G0/G1-phase pGen-3-CTNNB1 transfected cells was obviously increased (P<0.05), with no significant difference among the three groups with regard to apoptosis (P>0.05). pGen-3-CTNNB1 transfected cells exhibited obvious decrease in cyclin D1 mRNA and protein expression (P<0.05) and the ultrastructure of Eca-109 cells underwent a significant change after being transfected with pGen-3-CTNNB1, suggesting that down-regulating ß-catenin expression can promote the differentiation and maturation. The expression of PCNA and the ERKI/2 phosphorylation state were also down-regulated in pGen-3-CTNNB1 transfected cells (P<0.05). At the same time, the PCNA labeling index was decreased accordingly (P<0.05). CONCLUSION: Inhibition of EC Eca-109 cellproliferation by down-regulating ß-catenin expression could improve cell ultrastructure by mediating blockade in G0/G1 through inhibiting cyclin D1, PCNA and the MAPK pathway (p-ERK1/2).

[Effect of brucine on secretion function and proliferation capability of T lymphocytes in patients with aplastic anemia].

The aim of this study was to investigate the effect of brucine on secretion of TNF-α, IFN-γ, IL-4 and proliferation of T lymphocytes in patients with aplastic anemia (AA), and to explore its mechanism. Peripheral blood T lymphocytes from 10 patients with AA and 10 healthy volunteers were isolated, purified and cultured. T lymphocytes from the patients were divided into 0, 100, 200 and 400 µg/ml brucine-treated groups. T lymphocytes from healthy volunteers were used as control group. After being cultured for 72 hours, the levels of TNF-α, IFN-γ, IL-4 in the supernatant of cultured T lymphocytes from AA patients were detected by ELISA, and the proliferation of T lymphocytes from AA patients was detected by MTT. The results showed that compared with the normal control group, the levels of TNF-α and IFN-γ in the culture supernatant significantly increased, and IL-4 was significantly decreased. The levels of TNF-α and IFN-γ in the culture supernatant of brucine treated groups were lower, and were dependent on the concentration of brucine. However, the levels of IL-4 were found to be not obviously changed. The inhibition rate of T lymphocytes in 100, 200 and 400µg/ml brucine-treated groups were (13.61 ± 4.31)%, (14.28 ± 4.31)% and (15.12 ± 4.56)% respectively, among which the differences were not statistically significant. It is concluded that the brucine can reduce the levels of TNF-α and IFN-γ through inhibiting the proliferation of T lymphocytes in AA patients, which provides experimental basis for therapy of AA patients.