OBJECTIVETo investigate the apoptosis effect of diffuse large B-cell lymphoma cell line (DLBCL) SUDHL-4 induced by IL-21 and its related mechanism.

METHODSSUDHL-4 cells were treated with IL-21 at different concentration (1000 ng/ml, 100 ng/ml, 10 ng/ml, 1 ng/ml) for 24 h, 48 h, 72 h, respectively. The inhibitory rate of cell proliferation was detected by CCK-8 assay. The cell growth curves were drawn and half inhibitory concentration (IC(50)) values were calculated. The cell apoptosis were detected by flow cytometry (FCM), the expression of the caspase-9, caspase-3, cleaved caspase-3, Bcl-2, Bcl-XL, Bid, Bax and c-myc protein in SUDHL-4 cells treated with IL-21 by western blot, the mRNA expression of Bcl-2, Bcl-XL, Bid, Bax, c-myc by Survivin gene with RT-PCR.

RESULTSIL-21 markedly inhibited SUDHL-4 cell growth in a time- and dose-dependent manner. The 48 hIC(50) was 140.9ng/ml; The FCM showed that the apoptosis proportion of SUDHL-4 cells treated with 100 ng/ml of IL-21 apoptosis (AnnexinV-FITC(+) positive cells) gradually increased (48 h: 19.7 ± 2.3%). The protein expression of caspase-9, caspase-3, Bcl-2 and Bcl-XL decreased in a time-dependent manner. The Bax and c-myc protein markedly increased, but the Bid protein level did not change. IL-21 up regulated c-myc and Bax gene expression, however down regulated Bcl-2 and BCL-XL gene expression, but the gene expression of Bid and Survivin hadn't been changed significantly.

CONCLUSIONSIL-21 can inhibit proliferation and induce apoptosis of SUDHL-4 cell. The mechanism may involve in endogenous mitochondrial pathway mediated by the c-myc and the Bcl-2 genes.

Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Line, Tumor ; Humans ; Interleukins ; administration & dosage ; pharmacology ; Lymphoma, Large B-Cell, Diffuse ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-X Protein ; metabolism

This study was aimed to investigate the impact of specific siRNA on survivin gene in transfected lymphoma cell line and provide experimental evidences for future treatment of mantle cell lymphoma. The small interfering RNA (siRNA) targeted survivin mRNA was synthesized in vitro and was transfected into Jeko-1 that showed high survivin expression in mRNA level. The levels of survivin mRNA and protein expression were detected by quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot respectively. The apoptosis effect was examined by calculating the ratio of Annexin V-FITC/PI positive cells using flow cytometry. The inhibition of cell proliferation was assayed with CCK-8 reagent after transfection. The results showed that expression of survivin mRNA was markedly suppressed by the siRNA. The relative expression levels were 0.49 ± 0.03, 0.38 ± 0.02 and 0.17 ± 0.02 at time points of 24, 48 and 72 h respectively, compared with the control group; the inhibitive rates of cell proliferation were (31.2 ± 2.1)%, (43.3 ± 3.4)% and (52.6 ± 2.5)%; the apoptotic rates of cells were (6.3 ± 0.5)%, (13.5 ± 1.1)% and (23.6 ± 1.6)% respectively; survivin protein expression levels were gradually reduced. It is concluded that the siRNA targeting survivin down-regulates the expressions of survivin mRNA and protein evidently. The siRNA of survivin displays the potent ability to inhibit the proliferation of lymphoma cell line Jeko-1; survivin may become a potential molecular target for the therapy of lymphoma in the future.
Cell Line, Tumor ; Cell Proliferation ; Gene Silencing ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo investigate the effects of lentivirus-mediated RNA interference targeting vascular endothelial growth factor receptor 1 (VEGFR1) gene on the proliferation, migration and apoptosis of leukemic cell line U937.

METHODSShort hairpin RNAs (shRNA) targeting VEGFR-1 was synthesized and cloned into pRNAT-U6.2 lentiviral vector. The expression vectors were transfected into 293T cell line to produce packaged lentivirus. After infected with the packaged lentivirus, the expression of VEGFR-1 gene of U937 cells at mRNA and protein level was detected by real-time PCR and Western blot. VEGF production by the cells was determined by ELISA. Cell proliferation and survival under regular culture and in the presence of cytarabine (Ara-C) was determined by CCK-8 assay. Migration assays were performed by 5 µm pore transwell inserts.

RESULTSThe lentiviral shRNA vector targeting VEGFR-1 was successfully constructed and transfected into U937 cells. The shRNA vector effectively inhibited the expression of VEGFR-1 gene in U937 cell line at mRNA and protein levels. As compared to that of the control, the proliferation rate of U937-shVEGFR-1 cells reduced; The VEGF production and migrated cell number of U937-shVEGFR-1 cells decreased dramatically. After treated with Ara-C, the inhibition rate and apoptotic rate of U937-shVEGFR-1 cells increased significantly. The number of migrated cells in the KD group under regular culture and in the presence of VEGF was markedly lower than that in the NC group and CON group. Bevacizumab could decrease the number of migrated cells in the NC group and CON group, but could not in the KD group.

CONCLUSIONSLentivirus-mediated RNA interference targeting VEGFR1 gene reduces the proliferation, migration of U937 cell line and enhances its sensitivity to Ara-C.

Apoptosis ; genetics ; Cell Line, Tumor ; Cell Proliferation ; Gene Silencing ; Humans ; RNA Interference ; RNA, Small Interfering ; genetics ; U937 Cells ; Vascular Endothelial Growth Factor A ; metabolism

Objective To observe the differentiation of bone mesenehymal stem cells(BMSCs)cocultured with purified sinoatrial node cells(SNC)of neonate rata.Methods SNC from neonatal SD rat were cuhured and Durifted with differential attachment method and labded with BrdU.Rat BMSCs Were isolated by a Percoll's gradient solution and cultared in DMEM.After 2 passages,these BMSCs were transfected with pEGFP-N1 by Lipofectamin and labeled with GFP.EGFP-BMSC were co-cultured with SNC in a rate of 1:5 for 1 week.EGFP-BMSC cultured in SNC culture medium served as controls.SNC marker hyperpolarization activated cyclic nueleotide gated cation channel 4(HCN4) and connexin 45 (Cx45) expressions were determined by inunanofluorescence staining.Result Positive immunonuorescence staining against HCN4 and Cx45 were detected in EGFP-BMSC co-cultured with SNC but not in EGFP-BMSC cultured in SNC cuhure medium.Conclusion Direct cell-to-cell contact between BMSCs and SNC ceils may induce BMSCs differentiation into sinus node-like cells.

A sensitive high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) method was established for the determination of eplerenone (EP) in human plasma. The plasma samples of EP were extracted with ethyl acetate and separated by HPLC on a reversed phase C18 column with a mobile phase of 10 mmol x L(-1) ammonium acetate water solution-methanol (30 : 70, v/v). EP was determined with electrospray ionization-mass spectrometry (ESI-MS) in the selected ion monitoring (SIM) mode. The calibration curves were linear over the range of 2-4 000 ng x mL(-1) for EP. The lower limit of quantification was 2 ng x mL(-1). The method has been successfully applied in the pharmacokinetic study of the EP tablets. The main pharmacokinetic parameters of EP after oral administration of 25 mg, 50 mg, 100 mg were as follows, t1/2: (4.9 +/- 2.1), (4.7 +/- 1.5), (5.9 +/- 1.2) h; AUC(0-infinity): (4 402 +/- 1 735), (8 150 +/- 2 509), (13 783 +/- 4 102) microg x h x L(-1); and MRT: (6.2 +/- 2.1), (6.6 +/- 1.3), and (7.2 +/- 1.6) h. Parameters of EP after oral administration of multiple doses of 50 mg were as follows, t1/2: (6.1 +/- 1.7) h; AUC(ss): (10 071 +/- 4220) microg x h x L(-1); MRT: (8.1 +/- 2.3) h; and DF: (3.2 +/- 1.0).
Chromatography, High Pressure Liquid ; methods ; Humans ; Spectrometry, Mass, Electrospray Ionization ; methods ; Spironolactone ; analogs & derivatives ; blood ; pharmacokinetics

OBJECTIVETo investigate the clinical relevant factors causing laryngeal stenosis after partial laryngectomy.

METHODSA retrospective study was carried out to review the history clinical data from 138 patients of partial laryngectomy in the First Affiliated Hospital of Sun Yat-Sen University between January 1994 to October 2004. The clinical relevant factors causing laryngeal stenosis were included as follows: age, sex, TNM stage, tumor site, extension of thyroid cartilage defect, extension of larynx parenchyma defect, reconstruction method, laryngeal dilator, duration of using antibiotics, postoperative radiotherapy, lung infection, gastroesophageal reflux, diabetes. Multivariate stepwise logistic regression model was used for the analysis.

RESULTSOf 138 cases after partial laryngectomy, stenosis developed in 25 cases. The occurrence rate was 18.1%. In multivariate analysis, it was confirmed that the following factors correlated to laryngeal stenosis, i. e, extension of thyroid cartilage defect (chi2 = 4.323, P = 0.038), postoperative radiotherapy (chi2 = 6.002, P = 0.014), lung infection (chi2 = 4.220, P = 0.040), and gastroesophageal reflux (chi2 = 5.614, P = 0.018).

CONCLUSIONSThe clinical relevant factors causing laryngeal stenosis after partial laryngectomy were multiple. Statistical analysis showed that extension of thyroid cartilage defect, postoperative radiotherapy, lung infection and gastroesophageal reflux were the risk factors which may cause laryngeal stenosis.

Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Laryngeal Neoplasms ; pathology ; surgery ; Laryngectomy ; adverse effects ; Laryngostenosis ; etiology ; pathology ; Logistic Models ; Male ; Middle Aged ; Neoplasm Staging ; Postoperative Complications ; Retrospective Studies ; Risk Factors

OBJECTIVETo investigate the risk factors causing tracheal stenosis after tracheotomy for mechanical ventilation.

METHODSA retrospective study was carried out to review the clinical data of 560 patients who had been tracheotomy for mechanical ventilation in the First Affiliated Hospital of Sun Yat-sen University from 1990 to 2006. The clinical relevant factors causing tracheal stenosis included age, sex, preoperative intubation, preoperative intubation time, postoperative mechanical ventilation duration, airway infection, multiple changes of intubation tube, cricothyroidotomy, previous tracheotomy, gastroesophageal reflux, diabetes, etc. Multivariate stepwise logistic regression model was used for the analysis.

RESULTSFifty-four cases (9.6%) presented tracheal stenosis in 560 patients after tracheotomy. With multivariate analysis, it was confirmed that the following variable correlated to tracheal stenosis. i.e, preoperative intubation time (chi2 = 4.323, P = 0.038), postoperative mechanical ventilation duration (chi2 = 14.062, P = 0.000), airway infection (chi2 = 8.604, P = 0.004), diabetes (chi2 = 5.237, P = 0.014). The effect degree of these risk factors was as below, postoperative mechanical ventilation duration (OR = 10.818), airway infection (OR = 6.349), diabetes (OR = 3.019), intubation time preoperative (OR = 2.156).

CONCLUSIONSAmong patients who received tracheotomy for mechanical ventilation, the clinical relevant factors causing tracheal stenosis were various. Statistical analysis showed that preoperative intubation time, postoperative mechanical ventilation duration, diabetes, airway infection were main risky factors which may cause tracheal stenosis.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Logistic Models ; Male ; Middle Aged ; Multivariate Analysis ; Respiration, Artificial ; adverse effects ; Retrospective Studies ; Risk Factors ; Tracheal Stenosis ; etiology ; Tracheotomy ; adverse effects ; Young Adult

AIMTo explore the role of femoral nerves section (FNS) on the protection of limb ischemic preconditioning (LIP) against cerebral ischemia/reperfusion injuries.

METHODSModel of brain ischemia induced by Four-vessel occlusion was used. LIP was performed by clamping the bilateral femoral arteries for 10 min 3 times in a interval of 10 min. Rats with vertebral arteries permanently occluded were divided into sham group, cerebral ischemic group, FNS + cerebral ischemic group, LIP + cerebral ischemic group, FNS + LIP + cerebral ischemic group. The changes of neural density (ND) in the CA1 hippocampus were observed 7d after the sham operation or brain ischemia under thionin staining. The expression of c-Fos in the CA1 hippocampus was measured 6 h after the sham operation or brain ischemia under immunohistochemistry method.

RESULTSThionin staining revealed that serious neuronal damage was visualized in the CA1 hippocampus in both cerebral ischemic group and FNS + cerebral ischemic group as compared with sham group. LIP attenuated the neuronal damage of the CA1 subfield induced normally by cerebral ischemia/reperfusion, and ND in LIP + cerebral ischemic group was significantly higher than that in cerebral ischemic group (P < 0.01). But obvious neuronal damage of the CA1 subfield was found in FNS+ LIP + cerebral ischemic group, and ND was significantly decreased as compared with LIP + cerebral ischemic group (P < 0.01). These results suggested that the protection of LIP against cerebral ischemia/reperfusion injuries might be cancelled by preceding section of femoral nerve. It was found that there was almost no c-Fos expression in the CA1 hippocampus in sham group. Changes of c-Fos expression in the CA1 subfield in cerebral ischemic group were similar to that in sham group. But in LIP + cerebral ischemic group, c-Fos expression in the CA1 subfield was markedly increased and the number of positive cells and optical density of c-Fos expression were significantly higher than those in sham and cerebral ischemic group. c-Fos expression in the CA1 subfield was again decreased in FNS + LIP + cerebral ischemic group, and the number of positive cells and optical density of c-Fos expression were significantly lower than those in LIP + cerebral ischemic group.

CONCLUSIONNeural pathway participated in the protective effect of LIP on brain, and increased c-Fos expression in the CA1 hippocampus by LIP after cerebral ischemia/reperfusion, might be a part of neural pathway by which LIP induced brain ischemic tolerance.

Animals ; Brain Ischemia ; physiopathology ; Extremities ; blood supply ; Ischemic Preconditioning ; methods ; Male ; Neural Pathways ; physiopathology ; Rats ; Rats, Wistar ; Reperfusion Injury ; physiopathology

In the spinal cord, nitric oxide (NO) pathway is involved in pain and hyperalgesia, and nitric oxide synthase (NOS) expression and NO production are upregulated following several noxious and lesion stimuli. However, the mechanism of the increases is yet not well understood. The present study was designed to address the question of whether substance P (SP) released in the spinal cord enhances NOS expression and NO production of the spinal cord in rats. [Sar(9), Met(O2)(11)]-substance P (Sar-SP), a neurokinin-1 (NK-1) receptor agonist, was administered by intrathecal injection via L(5)-L(6) intervertebral space to induce nociception. The pain threshold was determined by hot water induced tail flick test. NOS expression of the L(5) segment of the spinal cord was determined using NADPH-d histochemical staining. NO production of the lumbar enlargement of the spinal cord was determined by assaying NO3(-) and NO2(-), the end product of NO metabolism, using the method of aqua fortis reduction. We found that (1) intrathecal injection of Sar-SP (6.5 nmol) elicited a characteristic, caudally directed, nociceptive behavioural response consisting of intense biting, licking and scratching episodes. Tail flick test showed decrease in pain threshold. (2) following the behavioural responses, the NOS expression level, including the number and the staining density of the NADPH-d reactive cells, increased in the superficial portion of the dorsal horn (Laminae I-II) and the grey matter surrounding the central canal (LaminaX) of the L(5) segment of the spinal cord after the Sar-SP intrathecal injection. At the same time, NO production in the enlargement of the spinal cord increased. (3) The decreased pain threshold and the increases in NOS expression and NO production could be substantially inhibited by intrathecal injection of [[D-Arg(1), D-Trp(7,9), Leu(11)]-substance P] (spantide) (5 microg), a non-selective antagonist of NK-1 receptor, 5 min prior to the Sar-SP injection. It might be concluded that the release of SP resulted from nociceptive afferents increased NOS expression and NO production of the rat spinal cord.
Animals ; Female ; Hyperalgesia ; Injections, Spinal ; Male ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase ; biosynthesis ; Nociceptors ; drug effects ; Pain Threshold ; drug effects ; Peptide Fragments ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Neurokinin-1 ; agonists ; Spinal Cord ; metabolism ; Substance P ; analogs & derivatives ; pharmacology

OBJECTIVETo evaluate the expression of vascular endothelial growth factor C (VEGF-C) and peroxisome proliferators-activated receptors (PPARgamma) in extrahepatic cholangioadenocarcinoma (EHCAC) and to elucidate its correlation with clinicopathological factors and their significance in prognosis.

METHODSThe expressions of PPARgamma and VEGF-C were detected by immunohistochemistry in 69 cases of EHCAC, 12 cases of non-tumor bile duct epithelium, and their relationship to clinicopathological parameters and follow-up were analyzed.

RESULTSThe positive rate of PPARgamma expression in 69 cases of EHCAC was 59.4%, significantly higher than that in 12 cases of non-tumor bile duct epithelium (0%), (P < 0.01). The positive rate of VEGF-C in 69 cases of EHCAC was 84.1%, also significantly higher than 16.7% in 12 cases of benign bile duct epithelium (P < 0.05). PPARgamma expression was associated with clinical TNM stage and lymph node metastasis. VEGF-C expression was associated with lymph node metastasis. Cox analysis results showed that portal vein and/or hepatic artery invasion, lymph node metastasis and VEGF-C expression were independent prognostic factors of EHCAC (P < 0.05).

CONCLUSIONPPARgamma expression may play an important role during tumorigenesis of extrahepatic cholangioadenocarcinoma. The expressions of PPARgamma and VEGF-C are significantly correlated with the clinicopathological characteristics and biological behavior of EHCAC. Expression of VEGF-C is an independent prognosis factors in EHCAC. The detection of PPARgamma and VEGF-C is valuable for evaluation of prognosis of EHCAC.

Adult ; Aged ; Aged, 80 and over ; Bile Duct Neoplasms ; metabolism ; pathology ; Bile Ducts, Intrahepatic ; Cholangiocarcinoma ; metabolism ; pathology ; Female ; Follow-Up Studies ; Humans ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Staging ; PPAR gamma ; metabolism ; Proportional Hazards Models ; Survival Rate ; Vascular Endothelial Growth Factor C ; metabolism