Apoptosis of Tca8113 cell induced by antitumor component-I from Agkistrodon acutus venom through CHOP/Caspase-12 pathway / 中国临床药理学与治疗学

AIM: To investigate the effect of endoplasmic reticulum stress pathway on the apoptosis of tongue squamous cancer Tca8113 cells induced by antitumor component-I from Agkistrodon acutus venom (AAVC-I). METHODS: The in vitro experiments were performed on subculture tongue squamous cancer Tca8113 cells in their growth period. A normal control group, a DL-dithiothreitol (DTT) positive control group and different AAVC-I concentrations were set according to the experiment objective. MTT assay was used to detect the proliferation inhibition of Tca8113 cells after been treated with different concentrations of DTT and AAVC-I for 24 h. The results were used to choose appropriate concentrations of DTT and AAVC-I in DTT positive control group and AAVC-I treated group, respectively. HE staining and Annexin V-FITC/PI double fluorescence staining were used to monitor the apoptosis of Tca8113 cells. Western blot was used to identify the expression levels of apoptosis-related proteins including endoplasmic reticulum stress glucose-regulatory protein 78 (GRP78), enhance-binding protein-homologousprotein (CHOP), cysteine-containing aspartate specific protease-12 (Caspase-12), cysteine-containing aspartate specific protease-9 (Caspase-9) and cysteine-containing aspartate specific protease-3 (Caspase-3).RESULTS:The proliferation inhibition of Tca8113 cells increased with an increased concentration of AAVC-I concentration (P<0.05), causing cell shrinkage, increased cell gaps, cytonuclear condensation, cell fragmentation, the appearance of apoptotic bodies, and increased rate of apoptosis (P<0.05). In addition, the expression level of GRP78 protein, CHOP protein, proteins of Caspase-12, Caspase-9 and Caspase-3 were increased (P<0.05).CONCLUSION: Endoplasmic reticulum stress CHOP/Caspase-12 pathway plays an important role in AAVC-I induced Tca8113 cells apoptosis.

Tumoral calcinosis misdiagnosed

Tumoral calcinosis is an uncommon condition which has been described to exist in primary and secondary forms. A lack of awareness of this entity can lead to unnecessary procedures and incorrect management. We report a case of a patient on peritoneal dialysis who presented with multiple painful joint swellings to the orthopaedic department. An initial diagnosis of septic arthritis was made, then revised to chronic tophaceous gout and referred to the rheumatology unit.

Effects of Target-controlled Infusion of Etomidate Combined with Remifentanil on Immune and Stress Re-sponse Indexes in Elderly Surgery Patients / 中国药房

OBJECTIVE:To investigate the effects of target-controlled infusion of etomidate combined with remifentanil on in-dexes of immune and stress response in elderly surgery patients. METHODS:Totally 60 patients undergoing elective surgery were randomly divided into control group(n=30)and observation group(n=30). Control group was given Propofol injection 1.5-2 mg/kg intravenously. Observation group was given Remifentanil hydrochloride for injection with pump volume of 0.5 μg/(kg·min), and then target controlled infusion of Etomidate injection 0.1-0.3 mg/kg;the dose of etomidate increased by 0.05-0.1 mg/kg accord-ing to physical activity during surgery. Postoperative eye opening time,recovery time of orientation and extubation time were com-pared between 2 groups as well as the levels of immune indexes(CD4+,CD8+,CD4+/CD8+),stress response indexes [serum norepi-nephrine(NE),adrenaline(E)and cortisol(Cor)]. The occurrence of ADR was recorded during surgery. RESULTS:Postopera-tive eye opening time,recovery time of orientation and extubation time in observation group were significantly shorter than control group,with statistical significance(P0.05). After surgery,CD4+ and CD8+ of 2 groups were significantly higher than before surgery,and the ob-servation group was significantly higher than the control group,CD4+/CD8+ of 2 groups was significantly lower than before,and the observation group was significantly lower than control group,with statistical significance(P0.05);after surgery,the levels of NE,E and Cor in 2 groups were significantly higher than before surgery,but the observation group was significantly lower than the control group, with statistical significance(P<0.05). There were no obvious adverse reaction occurred in 2 groups during the surgery. CONCLU-SIONS:The target-controlled infusion of etomidate combined with remifentanil is ideal for perioperative anaesthesia in the elderly patients and effectively improves related immune indexes and stress response indexes with good safety.

Three-Dimensional Cell Culture At The Frontiers Of In Vitro Cancer Research: Present Perspectives

In recent years, three-dimensional (3D) in vitro cell culture models have earned great attention, especiallyin the field of human cancer disease modelling research as they provide a promising alternative towardsthe conventional two-dimensional (2D) monolayer culture of cells with improved tissue organization. In2D cell culture systems, the complexity of cells on a planar surface does not accurately reflects the invivo cellular microenvironment. Cells propagated in 3D cell culture model, on the other hand, exhibitphysiologically relevant cell-to-cell interactions and cell-to-extracellular matrix (ECM) interactions,important in maintaining a normal homeostasis and specificity of tissues. This review gives an overviewon 2D models and their limitations, followed by 3D cell culture models, their advantages, drawbacks andchallenges in present perspectives. The review also highlights the dissimilarities of 2D and 3D modelsand the applicability of 3D models in current cancer research.

Plasma α1-antitrypsin: a neglected predictor of angiographic severity in patients with stable angina pectoris / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>As an acute phase protein, α1-antitrypsin (AAT) has been extensively studied in acute coronary syndrome, but it is unclear whether a relationship exists between AAT and stable angina pectoris (SAP). The purpose of the present study was to investigate the association between AAT plasma levels and SAP.</p><p><b>METHODS</b>Overall, 103 SAP patients diagnosed by coronary angiography and clinical manifestations and 118 control subjects matched for age and gender were enrolled in this case-control study. Plasma levels of AAT, high-sensitivity C-reactive protein (hsCRP), lipid profiles and other clinical parameters were assayed for all participants. The severity of coronary lesions was evaluated based on the Gensini score (GS) assessed by coronary angiography.</p><p><b>RESULTS</b>Positively correlated with the GS (r = 0.564, P < 0.001), the plasma AAT level in the SAP group was significantly higher than that in the control group (142.08 ± 19.61 mg/dl vs. 125.50 ± 19.67 mg/dl, P < 0.001). The plasma AAT level was an independent predictor for both SAP (odds ratio [OR] = 1.037, 95% confidence interval [CI]: 1.020-1.054, P < 0.001) and a high GS (OR = 1.087, 95% CI: 1.051-1.124, P < 0.001) in a multivariate logistic regression model. In the receiver operating characteristic curve analysis, plasma AAT level was found to have a larger area under the curve (AUC) for predicting a high GS (AUC = 0.858, 95% CI: 0.788-0.929, P < 0.001) than that of hsCRP (AUC = 0.665, 95% CI: 0.557-0.773, P = 0.006; Z = 2.9363, P < 0.001), with an optimal cut-off value of 137.85 mg/dl (sensitivity: 94.3%, specificity: 68.2%).</p><p><b>CONCLUSIONS</b>Plasma AAT levels correlate with both the presence and severity of coronary stenosis in patients with SAP, suggesting that it could be a potential predictive marker of severe stenosis in SAP patients.</p>

Mammalian target of rapamycin inhibitor abrogates abnormal osteoclastogenesis in neurofibromatosis type 1 / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Neurofibromatosis type 1 (NF1) is the most common genetic syndrome predisposing patients to various tumors due to dysregulation of the Ras signaling pathway. Recent research has shown NF1 patients also suffer a spectrum of bone pathologies. The pathogenesis of NF1 bone diseases is largely unknown. There is no current treatment. By Nf1 heterozygote (Nf1+/-) mice and Nf1 conditional knockout mice, we and other groups demonstrated abnormal osteoblast and osteoclast function due to dysregulation of Ras signaling. However, the specific downstream effector pathways linked to NF1 abnormal osteoblastogenesis and osteoclastogenesis have not been defined. In this study, we investigated the Ras downstream effector related with NF1 bone disease.</p><p><b>METHODS</b>We used Nf1+/+ and Nf1+/- mice as normal and NF1 models. Bone stromal cells extracted from Nf1+/+ and Nf1+/- mice were induced osteoclasts. The osteoclast cell was stained by tartrate resistant acid phosphatase staining. The osteoclast cell number was counted and the surface area of osteoclast cells was calculated under the microscope. The mRNA of mammalian target of rapamycin (mTOR) was determined by quantitative reverse-transcription-polymerase chain reaction. The presence of ribosomal protein S6 kinase was determined by Western blotting.</p><p><b>RESULTS</b>Compared with Nf1+/+ mice, Nf1+/- mice had about 20% more of osteoclast cells. These osteoclast cells were larger in size with more nuclei. Hyperactive mTOR was detected in Nf1+/- osteoclast cells. Inhibition of mTOR signaling by rapamycin in Nf1+/- osteoclasts abrogated abnormalities in cellular size and number.</p><p><b>CONCLUSION</b>mTOR pathway inhibition may represent a viable therapy for NF1 bone diseases.</p>

Correlation between polymorphism of tumor necrosis factor-related apoptosis-inducing ligand gene 1525 locus polymorphism and nodular thyroid disease / 中国地方病学杂志

ObjectiveTo study tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) 1525 locus polymorphism in patients with nodular thyroid disease and investigate the relation between individual gene polymorphism and thyroid diseases.Methods A total of 125 patients were diagnosed with nodular thyroid disease at the Department of Endocrinology,the First Affiliated Hospital of Inner Mongolia Medical College.Among these patients,67 cases were nodular thyroid goiter and 58 cases were nodular thyroid adenoma,54 males,71 females,and average age was 41.05 ± 14.42. Patients with nodular thyroid goiter were grouped into toxic and non-toxic and thyroid adenoma were grouped into high-functioning or non-high-functioning.A total of 100 healthy subjects.47 males,53 females,average age 42.35 ± 16.52 were as control group.According to the principle of informed consent,venous blood was collected,polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for TRAIL gene 1525 locus polymorphism determination was performed to calculate genotype of the TRAIL gene (homozygous GG,heterozygous GA,mutationhomozygous AA) and the gene frequency (G,A),and relative degree of risk(odd ratio,OR) was compared.ResultsNodular goiter group TRAIL gene 1525 locus genotype frequencies(GG:40.3％,AG:44.8％,AA:14.9％),allele frequencies(G:62.7％,A:37.3％) were compared with that of the control group(GG:17.0％,AG:65.0％,AA:18.0％; G:49.5％,A:50.5％),the differences were statistically significant(x2 =11.376,5.633,P ＜ 0.01 or ＜ 0.05 ).Adenomas group 1525 locus genotype frequencies of the TRAIL gene(GG:44.8％,AG:38.0％,AA:17.2％),allele frequencies(G:63.8％,A:36.2％) were compared with that of the control group,the differences were statistically significant(x2 =15.342,6.054,P ＜ 0.01 or ＜ 0.05).Allele frequencies of thyroid goiter group and denoma group were compared with that of the control group,OR values were 1.714 and 1.797(all P ＜ 0.05) and 95％ confidence intervals were 1.097 - 2.679 and 1.124 - 2.874.The difference of 1525 locus genotype or allele frequency distribution in toxic and non-toxic nodular group,high functioning and non-high-functioning adenomas group were not statistically significant (x2 =3.714,2.792; 1.103,2.020; all P ＞ 0.05).ConclusionTRAIL gene 1525 locus polymorphism is significantly associated with nodular thyroid disease.

Histone deacetylase inhibitors inducing human cervical cancer cell apoptosis by decreasing DNA-methyltransferase 3B / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Histone deacetylase (HDAC) inhibitors are a group of small chemical molecules that inhibit histone deacetylase. At cell level, HDAC inhibitors have multiple biological effects such as cell cycle arrest, apoptosis, cell differentiation and auotophagy. At molecular level, HDAC inhibitors cause histone and nonhistone acetylation and induce gene expression. HDAC inhibitors are widely used in cancer therapy because of its function of inducing apoptosis. However, the mechanisms of apoptosis effect are not fully understood. TSA is a classical HDAC inhibitor and widely used in epigenetic and anti-cancer research. In this study, we selected Trichostatin A (TSA) to investigate the mechanisms of HDAC inhibitors apoptotic effect on cancer cells.</p><p><b>METHODS</b>Cervical cancer cell lines such as Hela, Caski and normal human keratinocyte line HaCaT were treated with various concentrations of TSA. Crystal violent assay and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were performed to determine cell number. PARP cleavage and FITC-AnexinV were performed to determine apoptosis. DNA-methyltransferase (DNMT)1, DNMT3A and DNMT3B were determined by regular PCR, qPCR and Western Blotting. Small interfering RNA (SiRNAi) was used to knock down DNMT3B.</p><p><b>RESULTS</b>HDAC inhibitors only induce cervical cancer cell apoptosis. At 1 µmol/L of TSA, 86% of Hela cell and 76% of Caski went apoptosis. For normal cells, HDAC inhibitors have no cytotoxic effect at therapeutic dosage, (90.0 ± 8.4)% of normal cell survive after treated with 1 µmol/L of TSA. We compared 1 µmol/L group with untreated control with t-test. There was no significance between 1 µmol/L group and untreated control for normal cell (P > 0.05). HDAC inhibitors decreased DNMT3B in cancer cell but not in normal cell. Manually knock-down of DNMT3B induced Hela and Caski cell apoptosis. More than 99% of Hela and Caski cell went apoptosis after deprived of DNMT3B.</p><p><b>CONCLUSIONS</b>DNMT3B was essential to cervical cancer cell survival. Down-regulated DNMT3B by HDAC inhibitors may play an important role in the toxicity of HDAC inhibitors on cervical cancer cells.</p>

Modulatory effect of Rac1 protein on epidermal stem cells migration during wound healing / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate modulatory role of Rac1 protein in epidermal stem cell (ESC) migration during wound healing, in order to provide a reference for enriching basic theory of wound healing and guiding clinical application.</p><p><b>METHODS</b>Constitutively active mutant of Rac1 protein (Rac1Q61L) or dominant negative isoform of Rac1 protein (Rac1T17N) was transfected into ESC using a retroviral vector FUGW, and retroviral vector FUGW transfected into ESC in singles was used as blank control. The cells were divided into 3 parts according to the random number table and treated as follows. First, equal numbers of cells were inoculated into 24-well plates coated with collagen I (20 µg/mL), collagen IV (20 µg/mL) or fibronectin (10 µg/mL). Cells adhered to above matrices were quantitated using CytoTox 96 colorimetric kit. Second, 1000 cells adhered to collagen IV, after being stained with tetramethyl rhodamine isothiocyanate-phalloidin, were collected for observation of cell morphology and comparison of spreading area under confocal laser scanning microscope. Third, ESC with density of 2 × 10(5) cells per well were placed in upper compartment of Transwell chamber, DK-SFM culture medium alone or that containing stromal cell derived factor 1 (SDF-1) was added into lower compartment of Transwell chamber. Migration of ESC was observed using inverted phase contrast microscope, and the result was denoted as migration rate. Lastly, ESC with density of 7.5 × 10(5) cells per well was inoculated into 6-well plates for 12 hours, and treated with 4 µg/mL mitomycin C for 2 hours. The remaining scratch width of monolayer was respectively measured 6 hours or 12 hours after scratching to calculate the percentage of remaining scratch width. Data were processed with t test.</p><p><b>RESULTS</b>Compared with that of blank control, the number of Rac1Q61L-transfected cells adhered to collagen I was significantly increased (t = 5.302,P < 0.05), while the number of Rac1T17N-transfected cells adhered to collagen I, IV, and fibronectin were all obviously decreased (with t value respectively 13.741, 15.676, 8.256, P values all below 0.05). Confocal laser scanning microscope showed that spreading area of Rac1Q61L-transfected ESC (with laminate pseudopodia on edge) and Rac1T17N-transfected ESC was respectively larger and smaller as compared with that of blank control. With SDF-1 effect, the migration rate of Rac1T17N-transfected ESC was decreased by 78.0% and Rac1Q61L-transfected ESC was increased by 43.4% as compared with that of blank control. Without SDF-1 effect, the migration rate of Rac1T17N-transfected ESC was decreased by 55.2%, while the migration rate of Rac1Q61L-transfected ESC was close to that of blank control. Six or 12 hours after scratching, the percentage of remaining scratch width in Rac1Q61L-transfected ESC was lower as compared with that in blank control [(39 ± 9)% vs. (43 ± 5)%, (6 ± 5)% vs. (18 ± 7)%, with t value respectively 1.027, 4.389, with P value respectively above and below 0.05], while that in Rac1T17N-transfected ESC [(81 ± 9)%, (71 ± 11)%, respectively] was obviously higher as compared with that in blank control (with t value respectively 11.386, 11.726, P values all below 0.05).</p><p><b>CONCLUSIONS</b>Rac1 protein may control the migration of ESC by regulating its adhesion, spreading, and chemotaxis, and it plays an active role in wound healing accelerated by ESC.</p>

Promotion effect of stromal cell-derived factor 1 on the migration of epidermal stem cells in the healing process of frostbite-wound model ex vivo / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study the promotion effect of stromal cell-derived factor 1 (SDF-1) on the migration of epidermal stem cells (ESC) in the healing process of frostbite-wound model ex vivo.</p><p><b>METHODS</b>A three-dimensional model of full-thickness frostbite of skin was constructed (with slot-like wound) out of skin equivalent. The expression of SDF-1 in wound stroma was observed with immunohistochemistry staining on post injury days (PID) 3 and 7. The model frostbite wounds were divided into control group (treated with PBS 50 microL per wound), SDF-1 group (treated with 100 ng/mL SDF-1, 50 microL per wound), and AMD3100 group [treated with 100 ng/mL AMD3100 (50 microL per wound) for 30 minutes, and then SDF-1 50 microL was added per wound]. The redistribution of ESC around wound was observed.</p><p><b>RESULTS</b>The expression of SDF-1 in wound stroma increased gradually on PID 3 and 7. Compared with those in control and AMD3100 groups, there were more ESC and epithelial cell layers, and more integrin beta(1)-positive cells appeared at the basal layer of wound in SDF-1 group, and some of the positive cells migrated upward to epidermis.</p><p><b>CONCLUSIONS</b>SDF-1 contributes to wound repair through promoting ESC to migrate toward and gather around wound edge. This may be one of the mechanisms of ESC participating in wound repair.</p>