Objective To study the efficacy and safety of intravenous and intra-arterial thrombolytic therapy in acute cerebral infarction(ACI).Methods 65 patients with ACI were randomly divided in two groups:intra-arterial thrombolytic group (n=21) were intra-arterially administed with 250 000-500 000 units urokinase through femoral artery and intravenous thrombolytic group (n=44) were intravenously administed with 1.5 million units urokinase.Before and after treating, their neurological deficit scales were evaluated. The thrombin time (TT), prothrombin time (PT) and fibrinogen were detected before and after thrombolysis 6 h,3 d and 7 d.The CT scan was rechecked after thrombolysis 24 h.Results (1)ESS scales were higher in the intra-arterial infusion group than those in the intravenous infusion group at 2h, 6h, 24h, and 3d (all P

Accurate knowledge,diagnosis and treatment of breast ductal carcinoma in situ(DCIS),are crucial in controlling the development of breast cancer.In the diagnosis phase,breast ultrasound is commonly used as a screening tool,and a clear diagnosis can be made by mammography.Meanwhile,serological tests contribute to the detection of DCIS in early stages.In the treatment,the optimal surgical operation method remains debatable.It is widely acknowledged that the radiotherapy of postoperative patients should become more individualized.In addition,corresponding endocrine therapy helps those ER positive patients to reduce the recurrence.In the development of DCIS to invasive cancer,there are changes in gene and protein expressions,which may be a potential direction for further research.

BACKGROUND:Human mesenchymal stem cells (hMSCs) become aging and even die after several passages. Some investigations have shown that telomere has a close correlation with life span of the cells. Whether the ectopic expression of human telomerase reverse transcriptase (hTERT) could induce the activity of the telomerase, maintain the length of telomere, and finally prolong the life cycle of MSCs without losing their multipotent differentiation capacity is still uncertain.OBJECTIVE: To observe the influence of the ectopic expression of hTERT on the telomerase activity and cell life cycles of hMSCs.DESIGN: Repetitive measurement trails.SETTING: Research Center of Stem Cell, Zhengzhou University Medical College.MATERIALS: The experiment was conducted in the Research Center of Stem Cell, Zhengzhou University Medical College from October 2003 to December 2005. hMSCs were obtained from 20 healthy donators from the Department of Pediatric Surgery and Outpatient, the Third and First Affiliated Hospitals of Zhengzhou University. Enhanced green fluorescent protein plasmid (pEGFP-C1) and pEGFP-hTERT were provided by Dr. Chantal Autexier of Canada. DH5α strain provided by Dr. Hou Wei-hong, the Key Molecular Medical Laboratory of Zhengzhou University Medical College.METHODS.: Under sterile condition, 2 mL bone marrow of sternum of healthy donors were harvested, and prepared after centrifugalization,dilution and passage.① Transfection of pEGFP-hTERT into hMSCs and the screening and amplification of resistance cloning:The 5th passage cells were seeded in a 24-well plate,and transfected by pEGFP-hTERT with lipofectamine method.The cells were divided into four groups including untransfected group,lipofectamine group,pEGFP-C1 group and pEGFP-hTERT group. Resistance cloning screen and amplification was performed by G418. ②hTERT mRNA expression and detection of telomerase activity:RT-PCR and PCR-ELISA were used to detect the hTERT mRNA expressions of the fifth passage hMSCs transfected with pEGFP-hTERT, and pEGFP-C1, the untransfected tenth passage hMSCs and K562 cells (positive control), and the telomerase activity of the fifth and thirtieth passage hMSCs transfected with pEGFP-hTERT,the fifth pEGFP-C1-transfected cells and the tenth passage untransfected cells. ③Karyotype analysis of hTERT-transfected MSCs: Chromosome analysis was performed by conventional Giemsa staining.④Inducement of differentiation from telomerase-positive MSCs into neuron-like cells and RE-PCR identification:The transfected MSCs were cultured in a medium containing epidermal growth factor and basic fibroblast growth factor, which could induce the cells differentiate into neuron-like cells. The culture solution was changed every 3 days, and the changes in cell growth condition and morphologic characteristics were observed under an inverted microscope. The microtubule associate protein (MAP2) and neurofliament subunit M (NF-M) were identified by RT-PCR.MAIN OUTCOME MEASURES:①hMSCs transfection with different kathion liposomes and the screening and amplification of resistance cloning; ②hTERT mRNA expressions of each group and detection of telomerase activity; ③Karyotype analysis of pEGFP-hTERT-transfected MSCs; ④ Induction of differentiation from telomerase-positive MSCs into neuron-like cells and RE-PCR identification.RESULTS: ①With the decrease of G418 concentration, the cells in the untransfected and lipofectamine groups died, and stably EGFP expressed MSCs were obtained; after G418 screening, there was a cell clone undergone 35 passages and continued to proliferate, whose appearance and growth characteristics were similar to the untransfected MSCs observed under inverted microscope. ②The fifth passage pEGFP-C1-transfected hMSCs and tenth passage untransfected hMSCs remained telomerase-negative, but the K562 and fifth passage hTERT-transfected cells showed positive telomerase activity. ③The telomerase activity of the fifth and thirtieth passage hTERT-transfected cells was positive. ④The hTERT-MSCs at passage 10, 20 and 30 had 23 pairs of chromosomes, and two X chromosomes. So they were still normal diploid with normal chromosome appearance and number. ⑤Many hTERT-transfected MSCs had the typical appearance of neuron-like cells. RT-PCR analysis showed that th expressions of MAP2 and NF-M were increased.CONCLUSION:Ectopic expression of the hTERT gene is found in hMSCs,and can induce the telomerase activity of hMSCs.The ectopic expression of the hTERT gene in hMSCs could extend the life spans of cells and maintain their multipotent differentiation capacity.

Objective:To determine the correlation of BCG concentration in Freund complete adjuvant ( FCA) and severity of arthritis during arthritis establishment in DBA 1/j male mice induced by bovine type Ⅱcollagen.Methods:CIA was induced by the im-munization of DBA1/j mice with bovine type Ⅱcollagen and BCG of various concentrations dissolved in FCA.To ascertain the effects administering the collagen booster,CIA-related features(including body weight,clinical scoring of arthritis),TNF-αin serum and the histopathological changes in the spleen and the joints regions were measured.Results:4 mg/ml BCG induced more serious arthritis than 1 mg/ml in DBA1/j mice after collagen exposure.Conclusion:There is a positive correlation of arthritis severity with BCG.Which indicates that selection of adjuvant with suitable BCG concentration could determine pathological outcome in CIA mouse model .

Objective To investigate the level of MEF2C phosphorylation (activation) and protein expression, and to further clarify the possible mechanism following ischemia-reperfusion in hippocampal CA1 region of rat. MethodsBrain ischemia was induced by four-vessel occlusion in SD rats. Protein level was determined by Western blotting. Results MEF2C was significantly activated with a peak at 6 h of reperfusion, but its protein expression decreased in late phase of reperfusion (3～5 d). The elevation of activated (17 ku) and the inactivated forms (32 ku) of caspase-3 proteases were remarkable during 1～5 d of reperfusion. In addition, Ac-DEVD-CHO, a specific inhibitor of caspase-3, up-regulated MEF2C protein level of 3 d reperfusion. SB202190 (an inhibitor of P38), but not ERK5-antisense oligonucleotides, not only inhibited MEF2C activation of 6 h reperfusion but also apparently prevented the increase of caspase-3 activation caused by 3 d reperfusion. Conclusion P38/caspase-3 mediated MEF2C pathway may function in the injuries of hippocampal CA1 region of rats following ischemia/reperfusion.

Objective To identify differentially expressed N-linked glycoproteins between hepatocellular carcinoma ( HCC) and adjacent non-tumorous liver tissues .Methods N-linked glycoproteome was extracted by multi-lectin affinity chromatography comprising concanavalin A (ConA), lentil lectin (LCH), and snowdrop lectin (GNA) and subsequently subjected to two-dimensional electrophoresis ( 2DE ) and mass spectrometry ( MS ) for identification of differential glycoproteins between 10 pairs of HCC and adjacent non-cancer tissue .Western blotting was used to verify different expression of human liver carboxylesterase 1 (hCE1), haptoglobin (HP)and cathepsin D (CD).Invasion potential in vitro was examined after si-RNA mediated CD gene scilencing .Results LC-ESI-MS/MS identified a total of 28 differentially expressed glycoproteins (14 up-regulation and 14 down-regulated).Western blotting detected consistent down-regulation of hCE1 and HP, and up-regulation of pro-cathepsin D (pCD) in HCC.Up-regulation of ConA-binding CD (ConA-CD), however , was verified in HCC only after ConA-CD enrichment by ConA chromatography .Down-regulation of CD expression mediated by CD-siRNA markedly inhibited the in vitro invasive potential of SNU449 and SNU473.Conclusion Dysregulation of HP , hCE1 expression and alteration of glycans linked to CD may play crucial roles in pathogenesis of HCC.

Objective To investigate the expression of TIPE2 in splenic lymphocytes derived from the mouse model of cattle collagen Ⅱ-induced rheumatoid arthritis (CIA) and from peripheral blood of patients with rheumatoid arthritis (RA) for its role in the pathogenesis of RA.Methods The CIA mouse model was established by immunization of DBA-1/J mice with cattle collagen Ⅱ.Peripheral blood lymphocytes were collected from RA patients and healthy donors.The TIPE2 mRNA and protein expression in splenocytes of CIA and control mice were measured by semi-quantitative RT-PCR and Western blot,respectively.In addition,fluorescence quantitative RT-PCR and Western blot were used to determine the TIPE2 mRNA and protein expression in peripheral blood lymphocytes derived from patients with RA and healthy donors.Results Both mRNA and protein expression of TIPE2 were higher in splenocytes of CIA mice and lymphocytes of peripheral blood from RA patients compared with corresponding controls.Furthermore,the mRNA and protein expression of TIPE2 increased significantly in patients with active RA.TIPE2 expression were positively correlated with DAS28 scores (P＜0.001).Conclusion Our results suggest that TIPE2 plays a role in the RA pathogenesis.The level of TIPE2 may also indicate the severity of rheumatoid arthritis.

Objective To discuss the diagnostic value of serum sialic acid for glioma.Methods A retrospective study was conducted.The levels of sialic acid in serum samples of 95 glioma patients, 175 patients with brain benign tumor and 400 normal persons from October 2014 to March 2015 were detected by automatic biochemistry analyzer using enzymic method.The SNK-q test and analysis of variance were used to compare the difference of the groups.By making receiver operating characteristic ( ROC) curve, the cut-off value, sensitivity, specificity, positive predictive value and negative predictive value were calculated to assess the diagnostic value of serum salivary acid.Then the cut-off value was validated by using the serums of 30 glioma patients and 30 normal persons who were out-patients and healthy controls.Results The levels of serum sialic acid in patients with gliomas, patients with brain benign tumor and healthy individuals were (0.66 ±0.14 ) g/L, (0.61 ±0.09 )g/L, (0.54 ±0.07 )g/L.The serum salivary acid of glioma patients were higher than brain benign tumor patients (q=6.74,P<0.05) and normal persons (q=16.42,P<0.05), and there was no significant difference (F=1.67,P>0.05) among the glioma patients of different grades (8 of gradeⅠ,32 of gradeⅡ,24 of grade Ⅲ,31 of grade Ⅳ).There was no significant difference between the low grade patients (gradeⅠandⅡ) and the high grade patients (gradeⅢandⅣ) (t=0.55, P>0.05), but the level of serum sialic acid of high grade group had an increasing trend than the low grade group.The area under the ROC curves was 0.79.The cut-off value of serum salivary acid for diagnosing glioma was 0.61 g/L.The sensitivity was 67.74%, and the specificity was 80.60%.The positive predictive value was 44.68%, and the negative predictive value was 90.76%.Then the cut-off value was used as a diagnostic criteria, and the detected results of 30 glioma patients and 30 normal persons showed that the sensitivity was 63.30% and the specificity was 83.30%.Conclusions The serum sialic acid has good specificity and negative predictive value for diagnosing glioma.It may be a valuable diagnostic marker.

Objective:To determine the effects of enforced expression of Foxp3 in lung cancer cell with regards to proliferation and tumorgeneity. Methods: A stable subline NCIH-hFoxp3 was established by liopfectamin-mediated pcDNA plasmid transfection carrying exogenous hFoxp3. The growth curve and secrection of IL-8 and IL-10 of NCIH-hFoxp3 were evaluated using MTT and ELISA, respectively. The in vivo tumorigeneity was assessed as well by inoculation of NCIH-hFoxp3 subcutaneously in nude mice. Results:Lung cancer cell NCIH-hFoxp3 with enforced expression of Foxp3 proliferated slowly but exihited increased in vivo tumorgeneity compared with corresponding control subline. In addition,increased expression of hFoxp3 in NCIH-hFoxp3 augmented secretion and at-tenuated secretion of IL-8 and IL-10,respectively. Conclusion:Increased expression of Foxp3 may promote progression of lung cancer cell by change of cellular microenvironment and evasion of immune surveillance.

Objective To explore the neuroprotective role of Genistein (GEN) on hippocampal CA1 neurons and the possible mechanism following global cerebral ischemia ( GCI) in rats.Methods Seventy five rats were subjected to global cerebral ischemia ( GCI ) by four-vessel occlusion and randomly divided into five groups , sham, ischemia/reperfusion (I/R), GEN, ICI 182,780 and vehicle groups.Fluoro-Jade B and neuron-specific nuclear-binding protein ( NeuN) staining was used to observe CA 1 neuronal survival .TUNEL was used to detect apoptotic neurons .Spatial learning and memory function of the rats were evaluated by Morris water maze .Results The best dose of neuroprotective role of GEN was 1.0mg/kg body weight.Compared with sham, TUNEL-positive neurons in the hippocampal CA1 region increased significantly in I/R and vehicle groups (P<0.01), while post-treatment with GEN (1.0mg/kg) at 5min after ischemia by tail vein injection decreased markedly (P<0.01).Treatment of 1.0mg/kg GEN markedly attenuated spatial learning and memory deficits of the rats after ischemic insult compared to I /R group.Furthermore, ICI 182,780 significantly abolished the neuroprotective role of GEN (P <0.01).Conclusion The low-dose (1.0mg/kg) GEN significantly attenuates neuronal damage and cognitive deficits following GCI in rats , and the mechanism may be involved in estrogen receptor activity.