Effect of β-catenin gene silencing by shRNA on biologic characteristics of human esophageal carcinoma cells / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the effects of short hairpin RNA (shRNA) mediated gene silencing of β-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 β-catenin gene.</p><p><b>METHODS</b>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 β-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 β-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 β-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.</p><p><b>RESULTS</b>β-catenin expression levels were found markedly decreased in Eca-109 cells transfected with pGen-3-CTNNB1. In vivo, transfection with β-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 β-catenin in β-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).</p><p><b>CONCLUSIONS</b>There are abnormal expression of β-catenin and activation of Wnt signaling pathway in human esophageal carcinoma cell line Eca-109. RNA interference targeting β-catenin gene suppresses the growth of xenograft tumorigenesis in nude mouse and the invasiveness and metastatic capability of esophageal carcinoma cells.</p>

Comparison of mtDNA extraction from different parts of sarcosaphagous insects / 法医学杂志

OBJECTIVE@#To explore mitochondrial DNA (mtDNA) extraction effects of different parts from sarcosaphagous insects using improved cetyltriethylammnonium bromide (CTAB) method.@*METHODS@#Thirteen Lucilia sericata (Meigen) and 13 Nicrophorus fossor (Erichson) were collected from the corpses of rabbits placed on the outdoor lawn in Huhehot district. Four parts (head, chest muscle, legs and wings) of insect were collected, and the mtDNA of all samples were extracted using CTAB method. The purity and concentration were tested using protein and nucleic acid spectrophotometry. The integrity of the extracted mtDNA and PCR products were checked by agarose gel electrophoresis. The PCR products were sequenced and the obtained sequences were imputed into GenBank for comparison.@*RESULTS@#mtDNA were successfully extracted from 10 head samples, 6 legs samples, 4 wing samples and 13 chest muscle samples of the Lucilia sericata (Meigen). Also, mtDNA were successfully extracted from 5 head samples, 8 legs samples, 3 wing samples and 13 chest muscle samples of the Nicrophorus fossor (Erichson).@*CONCLUSION@#mtDNA can be obtained from chest muscle and other parts of sarcosaphagous insects using the improved CTAB method.

Forensic analysis of 74 tumor related medical malpractice cases / 法医学杂志

OBJECTIVE@#To analyze the causes of medical malpractice in patients with tumor, to determine the medical responsibility, and to recommend the related preventions.@*METHODS@#Seventy four medical malpractice cases, which were involved in tumor and collected from 2000 to 2009 in medicolegal expertise center of west China, were analyzed retrospectively.@*RESULTS@#The medical malpractice cases in the patients with tumor showed an increasing tendency in recent years. The main causes are missed diagnosis, misdiagnosis, improper chemotherapy and neglect of complications. The causes of medical malpractice were different in the different levels of medical services. The occurrence of medical malpractice in surgery and OB-GYN showed more frequent than the others.@*CONCLUSION@#Forensic pathology autopsy is important to resolve medical malpractice of tumor patients by finding out the cause of death and clarifying the medical responsibility. The occurrence of medical malpractice could be reduced by the clinical doctors through improving serve consciousness, obtaining the patients' trust, improving the medical treatment, following related laws and rules, fulfiling duty of medical careness.

Advances in the studies of postmortem interval estimation by the levels of chemical components in human vitreous humor after death / 法医学杂志

Estimation of postmortem interval (PMI) is one of the problems that need to be solved for forensic examination of the dead body. Accurate estimation of PMI has great values to criminal investigation and trial. The levels of chemical components in human vitreous humor are changed with time after death, which can help estimate the PMI. The levels of certain chemical components, such as potassium, magnesium, ammonia, urea, creatinine, uric acid, hypoxanthine, lactic acid and so on, in human vitreous humor will gradually increase with time after death, while others such as calcium, sodium, enzymes, glucose, vitamin C and so on will decrease. The updates and advances in those studies were reviewed in this article.

Role of activator protein-1 in pathogenesis of silicosis: an in-vitro study / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study the role of activator protein-1 (AP-1) in the up-regulation expression of tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta1(TGF-beta(1)) in silica-stimulated macrophage cells (RAW264.7).</p><p><b>METHODS</b>RAW264.7 cells were treated with AP-1 inhibitor Curcumin. The expression of c-jun and c-fos in nuclear protein was detected by western blotting. The level of TNF-alpha and TGF-beta(1) protein in the cell supernatant was measured using enzyme-linked immunoadsorbent assay (ELISA). Meanwhile the expression of TNF-alpha and TGF-beta(1) mRNA was also monitored by reverse transcriptase-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>The nucleoprotein expression of c-jun and c-fos in 10 and 20 micromol/L Curcumin prevention group (1.150 +/- 0.020, 1.010 +/- 0.108, 80.430 +/- 0.023, 0.256 +/- 0.015) were lower than those in silica-stimulated group (1.550 +/- 0.029, 0.860 +/- 0.036) (P < 0.01). In 20 micromol/L Curcumin prevention group and silica stimulated group, the expression of TNF-alpha protein were 23.58 +/- 45.78 and 32.12 +/- 5.34, and the expression of TGF-beta(1) protein were 1582.18 +/- 437.52 and 55.60 +/- 5.51 (P < 0.05 =; the expression of TNF-alpha, TGF-beta(1) mRNA were 0.74 +/- 0.01, 0.22 +/- 0.04 and 2.27 +/- 0.33, 2.96 +/- 0.15 (P < 0.05 =.</p><p><b>CONCLUSION</b>The expression of TNF-alpha, TGF-beta(1) mRNA and proteins is associated with activation of AP-1 in silica-stimulated macrophage cells.</p>

Effect of Twist gene on the migration and invasion of gastric carcinoma cells / 中南大学学报(医学版)

OBJECTIVE@#To explore the effect of Twist gene on the migration and invasion of human gastric carcinoma cells.@*METHODS@#MKN28 cells, a human gastric carcinoma cell line, were transfected with PcDNA3.1-Twist plasmid by lipofectamine transfecting technique. The transfected cells were selected with geneticin. Expressions of Twist,ecadherin and vimentin protein were detected by Western blot in cells transfected Twist gene. Matrigel invision chambers were performed to analyse the cell migration and invasion.@*RESULTS@#MKN28 cells transfected with PcDNA3.1-Twist plasmid showed stronger intracellular expression of Twist protein than MKN28 cells transfected with PcDNA3.1 and MKN28 cells without transfection. The expression of ecadherin protein in MKN28 cells transfected with PcDNA3.1-Twist plasmid was significantly decreased compared with that in MKN28 cells transfected with PcDNA3.1 and MKN28 cells without the transfection. However, The expression of vimentin protein in MKN28 cells transfected with PcDNA3.1-Twist plasmid was significantly increased compared with that in MKN28 cells transfected with PcDNA3.1 and MKN28 cells without transfection. The migration and invasion ability of Twist+ - MKN28 cells were stronger than that of MKN28 cells transfected with PcDNA3.1 and MKN28 cells without transfection.@*CONCLUSION@#Twist gene may promote the migration and invasion ability of gastric carcinoma cells through epithelial mesenchymal transition.

Effect of FGF-2 on survivin and subcellular location of Smac in human small cell lung cancer cell NCI-H446 / 中南大学学报(医学版)

OBJECTIVE@#To investigate the effect of basic fibroblast growth factor (FGF-2)on survivin and subcellular location of Smac in human small cell lung cancer (SCLC) cell NCI-H446.@*METHODS@#Western blot was used to detect the expression of survivin protein induced by FGF-2. The release of Smac from mitochondria to cytoplasm affected by FGF-2 was observed by Western blot and immunofluorescence. Apoptosis of NCI-H446 cells was detected with flow cytometry and Hoechst 33258 staining.@*RESULTS@#The expression of survivin could be up-regulated in response to FGF-2 treatment in NCI-H446 cells, and the level of survivin expression is related to the concentration and time of FGF-2 treatment. FGF-2 could inhibit the release of Smac from the mitochondria to cytoplasm induced by serum starving. FGF-2 could inhibit the apoptosis induced by serum starving.@*CONCLUSION@#FGF-2 up-regulates the expression of survivin protein in NCI-H446 cells, and blocks the release of Smac from mitochondria cytoplasm. Survivin and Smac might play important roles in the apoptosis inhibited by FGF-2.

Biological implication of PTEN gene expression in human gastric cancer and related molecular mechanisms / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the role of PTEN gene involved in the biological behavior of human gastric carcinoma cells and underlying molecular mechanisms.</p><p><b>METHODS</b>Gastric carcinoma cell line, SGC7901, was transfected with plasmid PBP-PTEN and stable high PTEN expression clones were selected by Western blot and cell immunohistochemistry screening. Cell proliferation rate and apoptosis index of transfected cells were investigated by growth curve analysis, colony-formation assay and flow cytometry (FCM). Expressions of vascular endothelial growth factor (VEGF), matrix metalloprotease-2 (MMP-2) and MMP-9 proteins in cell culture supernatant and cytoplasm were determined by ELISA, gelatin zymogram, Western blot and cell immunohistochemistry.</p><p><b>RESULTS</b>Stable clone with high level expression of PTEN was successfully established (PTEN-SGC7901). Cell doubling time of PTEN-SGC7901 was longer than that of the control cells (P < 0.05). The size and colony-forming efficiency of PTEN-SGC7901 cells deceased compared with those of the control. The relative colony-inhibition efficiency of PTEN-SGC7901 to SGC7901 (naïve untransfected) and PBP-SGC7901 (control vector transfected) cells were 69.8% and 64.8%, respectively. PTEN-SGC7901 clone had more cells at G1 phase (P < 0.05) compared with that of the control. However, the apoptosis index did not show significant differences among the three groups (P > 0.05). There were significantly less VEGF and MMP-9 protein expressions in the PTEN-SGC7901 culture supernatant and cytoplasm (P < 0.05). In contrast, the MMP-2 expression among three cell groups had no significant difference (P > 0.05).</p><p><b>CONCLUSIONS</b>PTEN expression suppresses the growth and proliferation of gastric carcinoma cell SGC7901, possibly through an inhibition of the expressions of VEGF and MMP-9.</p>

Expression and role of nuclear transcription factor Sp1 in macrophages stimulated by silicon dioxide / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study the expression and localization of nuclear transcription factor Sp1 in macrophages after stimulated by silicon dioxide in vivo and in vitro.</p><p><b>METHODS</b>Forty Sprague Dawley rats were randomly divided into the control group and the silica exposure group, 20 in each group. The rat silicosis models were established by direct tracheal instillation of silica into rat lung (0.2 g/kg) only once while the control group was instilled with equal amount of saline. Animals were killed at 1st, 7th, 14th, 21st and 28th day after instillation. Dynamic changes of Sp1 protein expression and its cellular localization were detected by immunohistochemistry in pulmonary macrophages. In vitro, Sp1 mRNA and protein expression and their dynamic changes were monitored by RT-PCR and western blotting after stimulated by silicon dioxide in cultured RAW264.7 macrophages respectively. Cellular localization of Sp1 protein was characterized by immunocytochemistry.</p><p><b>RESULTS</b>Compared to the control group, the Sp1 protein expression was increased in pulmonary macrophages and reached the peak at the 14th day in the silica exposure group. In vitro, the Sp1 mRNA level began to rise at 30 minutes after the administration of silicon dioxide and reached the peak at 240 minutes and then decreased to the minimal level at 960 minutes. The Sp1 total protein and nuclear protein also exhibited the similar trend. The former reached the peak at 240 minutes and the latter at 480 minutes. The significant nuclear translocation of Sp1 protein was observed at 120 minutes after the administration of silicon dioxide and became most significant at 480 minutes.</p><p><b>CONCLUSION</b>Silicon dioxide can activate nuclear transcription factor Sp1 in macrophages in vivo and in vitro. Sp1 might play an important pathogenic role in the development of silicosis.</p>

Effects of silicon dioxide on expression of alpha-smooth muscle actin in human lung fibroblasts / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the effects of SiO(2) on the expression of alpha-smooth muscle actin (alpha-SMA) in human lung fibroblasts in vitro and vivo.</p><p><b>METHODS</b>The experimental group comprised 32 rats while 32 rats were included in the control. In vivo, the expression of alpha-SMA in lung tissues of rats exposed to SiO(2), the supernate of RAW264.7 cells, SiO(2) and the growth factor beta(1) (TGF-beta(1)) were investigated, respectively.</p><p><b>RESULTS</b>(1) alpha-SMA positive myofibroblasts appeared in the lung tissues of the 28th day groups exposed to SiO(2). (2) The expression of alpha-SMA in HLF-02 cells was unregulated by TGF-beta(1) and supernate of RAW264.7 cells exposed to SiO(2). (3) The expression of alpha-SMA in HLF-02 cells was not induced by SiO(2).</p><p><b>CONCLUSION</b>Myofibroblasts related to silicosis, and the appearance of myofibroblasts (in vitro) are independent on direct stimulation by SiO(2), but related to the mediator (TGF-beta(1)) secreted by SiO(2) stimulated macrophages.</p>

Expressions of TGIF, MMP9 and VEGF proteins and their clinicopathological relationship in gastric cancer / 中南大学学报(医学版)

OBJECTIVE@#To detect the expressions of TG-interacting factor (TGIF), matrix metalloproteinase 9 (MMP9) and vascular endothelial growth factor (VEGF) proteins, and to analyze their clinicopathological relationship in gastric cancer.@*METHODS@#Vascular invasion and remote metastasis were examined in nude mice inoculated with SGC-7901 cells, vector control cells or TGIF transfected cells via serial sections, and sequentially the expressions of MMP2, MMP9 and VEGF proteins in tumor tissues from nude mice were detected by immunohistochemical staining. At the same time, TGIF, MMP9 and VEGF proteins were examined in 76 patients with gastric carcinoma, and the relationships between the expressions of the three proteins and clinicopathological features were analyzed.@*RESULTS@#Metastasis was not observed in nude mice inoculated with any cells. There were cancerous embolisms in nude mice tumor tissue inoculated with SGC-7901 cells and vector control cells, but not in TGIF transfected cells. The expressions of MMP9 and VEGF proteins were higher in the tumor tissues originated from SGC-7901 cells and vector control cells than that from TGIF transfected cells. The expression of MMP2 had no distinct difference among the tumor tissues originated from the three cells. The positive rate of TGIF protein in gastric carcinoma was 46.1% (35/76), obviously lower than that in surgical marginal mucosa (78.1%) (P < 0.05). Low expression of TGIF protein correlated significantly with the metastasis of lymph node. The positive rates of MMP9 and VEGF proteins in gastric carcinoma were 59.2% and 56.6%, respectively, obviously higher than that in surgical marginal mucosa (31.3%) (P < 0.05). High expressions of both MMP9 and VEGF also correlated significantly with the metastasis of lymph node. The expression level of VEGF protein was also associated with the invasion of gastric carcinoma (P < 0.05). The expression of TGIF protein reversely correlated with that of MMP9 and VEGF proteins (P < 0.05).@*CONCLUSION@#TGIF may inhibit the invasion and metastasis of gastric carcinoma via the downregulation of MMP9 and VEGF proteins.

Expression and significance of PTEN and nm23 in the metastasis of gallbladder cancer / 中南大学学报(医学版)

OBJECTIVE@#To explore the relationship between the expressions of PTEN and the metastasis of the gallbladder cancer.@*METHODS@#The expression of PTEN and nm23 were detected by immunohistochemical staining in 32 cases of gallbladder cancer with metastasis and the staining intensity was scored semi-quantitatively, compared with the cases without metastasis.@*RESULTS@#The intensity score of PTEN and nm23 in gallbladder cancer with metastasis was 8.9947+/-4.5590 and 10.2003+/-3.9031, respectively, which was lower than that in those without metastasis (12.9433+/-4.7618 and 15.8436+/-5.6917 respectively, P < 0.01 ). The expression of PTEN was correlative with that of nm23 ( Pearson = 0.370, P < 0.05).@*CONCLUSION@#The lower expressions of PTEN and nm23 are related to the metastasis of gallbladder cancer.

Growth suppression of colon cancer cells in vitro by DPC4 gene expression and its mechanism / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the effect of DPC4 gene expression on the growth of colon cancer cells and its mechanism.</p><p><b>METHODS</b>Expression plasmid pcDNA3.1-DPC4 was constructed and transfected into the colon cancer cell line SW620 by use of lipofectamine gene transfer technique. DPC4 protein expression was detected by Western blot and immunocytochemistry. The effect of DPC4 gene on the growth of SW620 cells was monitored by population doubling time (PDT) and cloning efficiency. The influence of DPC4 expression on p21WAF1 transcription was investigated by RT-PCR to detect p21WAF1 mRNA.</p><p><b>RESULTS</b>Successful expression of DPC4 protein was detected in the transfected SW620 cells. Compared with the control cells, PDT (74 h) of the DPC4 expressing cells was prolonged and the cloning efficiency (21%) decreased. In addition, the mRNA level of p21(WAF1) in DPC4 transfected cells was increased.</p><p><b>CONCLUSIONS</b>Overexpression of DPC4 gene inhibits the growth of colon cancer in vitro, and induction of p21(WAF1) expression may be an important functional aspect of DPC4.</p>