Objoctive:To investigate the expression of transforming growth factor-β(TGF-β)isoforms in colonic mucosa of ulcerative colitis(UC),and its role in the incidence of UC thereof.Methods:The expression of TGF-β isoforms was detected in colonic mucosa by immunohistochemical Envision method in UC patients,and the patients with irritable bowel syndrome (IBS)were used as controls.The relationship was analyzed between the expression of TGF-β isoforms and the degree of severity and extent of the disease.Results:There were statistical differences in the expression of TGF-β1 and TGF-β2 between the active stage UC,remission stage UC and IBS groups(P ＜ 0.01).The expression was significantly higher in active stage UC group than that in remission stage UC and 1BS groups(P＜ 0.01).The expression was significantly higher in remission stage UC group than that in IBS group(P ＜ 0.01).However,there was no statistical difference in the expression of TGF-β3 between active stage UC,remission stage UC and IBS groups(P＞ 0.05).The expression of TGF-β1 and TGF-β2 had positive correlation with the degree of sevefity(P ＜ 0.05).However,the expression of TGF-β3 had no linear correlation with the degree of severity(P ＞ 0.05).There was no linear correlation on the expressions of TGF-β1,TGF-β2 and TGF-β3 with extent of the disease(P ＞ 0.05).The degree of severity had positive correlation with extent of the disease(P ＜ 0.05).Conclusion:The expressions of TGF-β1 and TGF-β2 were enhanced in colonic mucosa of UC,and were correlated with the pathogenesis of UC.The expressions of TGFβ1 and TGF-β2 may serve as markers for assessing disease activity of UC.

Aged ; Giant Cells ; pathology ; Humans ; Male ; Stomach Neoplasms ; pathology

Objective To prepare hepatocyte growth factor(HGF) recombinant protein and confirm its activity preliminarily according to building HGF gene prokaryotic expression vector and transforming into E.coli.Methods Clone HGF inserted into the vector pET-26b(+) to construct prokaryotic expression vector pET-26b(+)-HGF and transform into E.coli Rosseta(DE3).The transformed bacteria induced by IPTG was purified through Ni-NTA resin affinity chromatography frozen-drying after renaturation.Results HGF gene recombinant prokaryotic expression vector pET-26b(+)-HGF was constructed successfully.E.coli Rosseta(DE3) which was transformed into pET-26b(+)-HGF expresses the target protein as the form of inclusion bodies,accounting for 38％ of the total bacterial proteins,and confirmed by Western blot.HGF protein which was purified by Ni-NTA resin affinity chromatography,has a purity of about 95％,and can promote proliferation,migration,and inhibition of apoptosis for human non-small cell lung cancer cell line A549 cells after interaction.Conclusion HGF gene recombinant prokaryotic expression vector pET-26b (+)-HGF was constructed and expressed in transformed E.coli Rosseta(DE3) successfully.They resumed their recombinant HGF protein structure after purification and renaturation,and had biological activity confirmed by in vitro studies.

AIMTo study the antitumor effect of baicalein on human leukemia K562 cell and its mechanism.

METHODSThe IC50 value and cytotoxity of K562 cell were detected by MTT method. The apoptotic cell was analyzed by FCM using Annexin V FITC--PI staining method. Sub-G1 peak was also measured by FCM. Protein expressions of Bcl-2, Fas, Caspase 3 were evaluated with FCM.

RESULTSBaicalein was shown to significantly inhibit the proliferation of K562 cell in a dose-dependent manner and selectively induce apoptosis of human leukemia K562 cells. Flow cytometric analysis showed that baicalein arrested K562 cells in the S phase. In addition, protein expression of Fas, Caspase 3 of K562 cells increased after exposure to baicalein, but Bcl-2 was unchanged.

CONCLUSIONBaicalein can selectively induce apoptosis of human leukemia K562 cell dose and time dependently through up-regulation of caspase-3 and fas gene expression level.

Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Caspase 3 ; Caspases ; metabolism ; Cell Cycle ; drug effects ; Flavanones ; Flavonoids ; pharmacology ; Humans ; K562 Cells ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Up-Regulation

This study is to investigate the influence and mechanism of action of asymmetrical dimethylarginine (ADMA) and the induced oxidative stress level on Alzheimer's disease (AD) incidence. ADMA concentration, nitric oxide, Abeta(40)/Abeta(42) ratio, inducible NO synthase (iNOS) activity and the concentrations of the induced free radicals including malondialdehyde (MDA), 3-nitrotyrosine (3-NT) and peroxynitrite (ONOO-) in the cerebrospinal fluid (CSF) from 34 neurologically normal controls and 37 AD patients were quantitatively determined and statistically compared. The results showed that the ADMA concentration significantly decreased in AD patients, and it showed negative correlation with the NO, iNOS activity, and showed positive correlation with MMSE score. ADMA concentration was negatively correlated with Abeta(40)/Abeta(42) ratio (P<0.01) with the observation that Abeta(40)/Abeta(42) ratio increased while ADMA level decreased in CSF in AD patients. The concentration levels of MDA, 3-NT and ROS significantly increased compared with the control with all the P values less than 0.05. These findings suggested that the ADMA disorder and the oxidative damage effect of the induced free radicals in CSF of AD patients are an important mechanism of AD incidence, and their joint regulation may provide new idea for the prevention and clinical treatment of AD.
Aged ; Alzheimer Disease ; cerebrospinal fluid ; Amyloid beta-Peptides ; cerebrospinal fluid ; Arginine ; analogs & derivatives ; cerebrospinal fluid ; Female ; Humans ; Male ; Malondialdehyde ; cerebrospinal fluid ; Middle Aged ; Nitric Oxide ; cerebrospinal fluid ; Nitric Oxide Synthase Type II ; cerebrospinal fluid ; Oxidative Stress ; Peptide Fragments ; cerebrospinal fluid ; Peroxynitrous Acid ; cerebrospinal fluid ; Reactive Oxygen Species ; cerebrospinal fluid ; Tyrosine ; analogs & derivatives ; cerebrospinal fluid

OBJECTIVETo investigate the role of plasma tissue factor (TF) and tissue factor pathway inhibitor-1 (TFPI-1) level and to observe the effect of extrinsic TFPI-1 on no-reflow (NR) in a rabbit model of ischemia/reperfusion.

METHODSRabbits were randomized into four groups (n = 10 each): ischemic- reperfusion group (IR, subjected to 120 minutes of coronary artery occlusion and followed by 60 minutes of reperfusion); ischemic- reperfusion TFPI-1 group (100 ng/kg bolus and 1 ng x kg(-1) x min(-1) infusion during reperfusion); ischemic group (subjected to 180 minutes of coronary artery occlusion) and sham group. The NR area and ischemic area were determined by thioflavin S and Evan's blue staining in vivo. Plasma TF and TFPI-1 levels were measured before operation, before and at 120 minutes post coronary artery ligation, 10 and 60 minutes after reperfusion by ELISA.

RESULTSPlasma TF and TFPI-1 levels before and at 120 minutes post coronary artery ligation were similar among the four groups (all P > 0.05). At 10 and 60 minutes after reperfusion, the plasma TF levels in the IR group was significantly higher than those in ischemic group and sham group [10 minutes: (20.7 + or - 4.1) pg/ml vs. (13.9 + or - 2.2) pg/ml (P < 0.001), (20.7 + or - 4.1) pg/ml vs. (13.2 + or - 2.6) pg/ml (P < 0.001); 60 minutes: (15.8 + or - 2.6) pg/ml vs. (13.5 + or - 1.6) pg/ml (P < 0.05), (15.8 + or - 2.6) pg/ml vs. (12.1 + or - 0.7) pg/ml (P < 0.001)] while the plasma TFPI-1 levels were similar among IR, ischemic and sham groups at 10 minutes after reperfusion and at 60 minutes after reperfusion (all P > 0.05). TFPI-1 level [(9.7 + or - 1.6) ng/ml] was significantly lower in the IR group than in the ischemic group [(11.6 + or - 1.6) ng/ml, P < 0.05] and sham group [(10.1 + or - 1.3) ng/ml, P < 0.01]. TF mRNA expression in the NR area in IR group was significantly up-regulated compared to the ischemic group (P < 0.05) and sham group (P < 0.001) while TFPI-1 mRNA expression was similar between IR group and ischemic group (P > 0.05). NR severity in the ischemic-reperfusion TFPI-1 group was significantly attenuated compared to IR group (0.39 + or - 0.11 vs. 0.54 + or - 0.06, P < 0.01).

CONCLUSIONUpregulated TF mRNA expression in the NR area and increased plasma TF level during reperfusion period, reduced plasma TFPI-1 level during reperfusion period as well as attenuated NR severity by extrinsic application of human rTFPI-1 in this model suggested an important role in the pathogenesis of the NR phenomenon.

Animals ; Blood Proteins ; metabolism ; Lipoproteins ; blood ; Myocardial Reperfusion Injury ; blood ; Rabbits ; Thromboplastin ; metabolism

OBJECTIVETo modify the current PCR-based method for rapid and efficient preparation of small interfering RNA (siRNA) expression cassette to improve the efficiency of RNA interference.

METHODSThe U6 promoter sequence was amplified by PCR using the genomic DNA of K562 cells as the template, and cloned into pMD18-T vector which served as the template for further PCR amplification with the primers on the plasmid. The amplified product was directly used as the template for preparing siRNA expression cassette. The siRNA expression cassette targeting p53 gene was amplified, verified by sequencing, and transfected into SH-SY5Y cells. After a 48-hour transfection, the cells were harvested and the total RNA was for RT-PCR for evaluating the effect of RNA interference.

RESULTSThe sequencing result confirmed the correct U6 promoter sequence cloned from K562 cells. After transfection of SH-SY5Y cells for 48 h with siRNA expression cassette, the p53 gene expression was inhibited at the mRNA level in comparison with the control cells as demonstrated by RT-PCR detection.

CONCLUSIONThe siRNA expression cassette prepared using the established method described hereby can be well applicable in RNA interference research.

Gene Silencing ; Gene Targeting ; methods ; Genetic Vectors ; genetics ; Humans ; K562 Cells ; Polymerase Chain Reaction ; RNA, Small Interfering ; biosynthesis ; genetics ; RNA, Small Nuclear ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics

OBJECTIVETo study the occurrence, management and prognosis of fatal pulmonary embolism in patients underwent coronary intervention in our department.

METHODSeven patients had fatal pulmonary embolism after coronary intervention in six years, we analysis each patient by the occurrence, prognosis, management of the disease.

RESULTSDuring the last 6 years, 7 [five males, mean age (55.9 +/- 11.7) years, 5 after coronary angiography and 2 after percutaneous coronary intervention] patients developed fatal pulmonary embolism after PCI. All 7 patients presented respiratory and cardiac arrest within 24 hours post coronary intervention. Three patients died, one patient experienced brain death and another three patients survived and are alive without complication till now.

CONCLUSIONThe fatal pulmonary embolism is a scarce complication after coronary intervention with high acute mortality and satisfactory outcome for survivors.

Adult ; Aged ; Angioplasty, Balloon, Coronary ; adverse effects ; Humans ; Male ; Middle Aged ; Prognosis ; Pulmonary Embolism ; diagnosis ; etiology ; Treatment Outcome

OBJECTIVETo investigate the impact of the stents coated with sirolimus and anti-CD34 antibody on the short-term re-endothelialization and the long-term restenosis in Chinese Minipigs.

METHODSThree different types of stents [bare-metal stent (BMS), sirolimus-eluting stent (SES) and anti-CD34 antibody and sirolimus-coated stent (ASES)] were randomly implanted in the coronary arteries of 22 Chinese Minipigs. At two weeks after stenting, coronary angiography and optical coherence tomography (OCT) were performed in 10 experimental animals. At three months after stenting, coronary angiography and OCT were performed in the remaining 12 experimental animals. Histopathologic examination was performed on the coronary artery segments containing stent after the animals were executed.

RESULTS(1) No in-stent thrombosis and parietal thrombus were found by coronary angiography, OCT and histopathologic examination at two weeks post stenting. OCT analysis showed that the covered ratio of stent struts by neointima in ASES group was higher than in SES group [(55.56 ± 35.27)% vs. (41.82 ± 23.28)%, P < 0.05]. The mean thickness of neointima in ASES group was significantly higher than in SES group [(89.0 ± 5.0) µm vs. (32.0 ± 4.9) µm, P < 0.01] and BMS group [(89.0 ± 5.0) µm vs. (44.0 ± 7.2) µm, P < 0.01]. Histopathologic and scanning electron microscopy examinations demonstrated that the covering level and quality of stent struts by neointima in BMS and ASES group were both better than in SES group. (2) At three months follow-up, quantitative coronary angiography analysis found that late in-stent lumen loss in ASES group was significantly lower than in BMS group [(0.18 ± 0.06) mm vs.(0.35 ± 0.06) mm, P < 0.05]. OCT analysis showed that the percent neointimal hyperplasia in ASES and SES group was significantly lower than in BMS group [(34.75 ± 2.64)% and (35.63 ± 2.07)% vs. (48.28 ± 3.25)%, both P < 0.01]. Histopathologic analysis demonstrated that the percent areal restenosis of ASES and SES group were both significantly lower than that of BMS group [(28.65 ± 5.64)% and (29.33 ± 6.07)% vs. (46.18 ± 8.25)%, both P < 0.05].

CONCLUSIONThe stents coated with anti-CD34 antibody and sirolimus can attenuate the inhibitory effect of sirolimus on the re-endothelialization at two weeks after stenting and the anti-hyperplasia effect of sirolimus at three months after stenting.

Animals ; Antibodies ; administration & dosage ; therapeutic use ; Antigens, CD34 ; immunology ; Drug-Eluting Stents ; Male ; Sirolimus ; administration & dosage ; therapeutic use ; Swine ; Swine, Miniature ; Treatment Outcome

OBJECTIVETo study gene expression of collagen types IX and X in human lumbar intervertebral discs during aging and degeneration and to explore the role of collagen types IX and X in disc degeneration.

METHODSFetal, adult and pathologic specimens were subjected to in situ hybridization with cDNA probes to investigate mRNA-expressions of types IX and X collagen gene.

RESULTSIn fetal intervertebral discs, positive mRNA hybridization signals of type IX collagen were concentrated in the nucleus pulposus and the inner layer of anulus fibrosus. Interstitial matrix of the nucleus pulposus also showed positive type X collagen staining. Positive mRNA hybridization signals of types IX and X were not detected in the middle and outer layers of anulus fibrosus. In adult specimens, expression of type IX collagen mRNA was markedly decreased. No hybridization signals of type X collagen was observed. As for pathological specimens, there was no gene expression of type IX collagen. In severe degenerated discs from adults, there were focal positive expressions of type X collagen.

CONCLUSIONSObvious changes of collagen gene expression occur with aging. Expression of type IX collagen decreases in adult and pathological discs. Results of type X collagen expression suggest that type X collagen is expressed only in older adult and senile discs (i.e., when disc degeneration has already reached a terminal stage), indicating the terminal stage of degeneration.

Adolescent ; Adult ; Collagen Type IX ; metabolism ; Collagen Type X ; metabolism ; Female ; Gene Expression ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Intervertebral Disc ; embryology ; metabolism ; Lumbar Vertebrae ; Male