Aim To study the protective effects of sphingosine 1-phosphate (S1P) postconditioning on rat myocardial cells injured by hypoxia/reoxygenation in reperfusion injury salvage kinase ( RISK ) signal path-way. Methods The cultured rat H9c2 cells were ran-domly divided into seven groups: ( 1 ) control group;(2) hypoxia/reoxygenation (H/R) group; (3) S1P group;(4) S1P+LY294002 group(S1P+LY); (5) LY group; ( 6 ) S1 P +PD98059 group ( S1 P +PD );(7) PD group. The viability of H9c2 cells was detec-ted using MTT method. The content of MDA in the cultured medium and the activity of T-SOD and Mn-SOD were measured with colorimetry. The concentra-tion of intracellular free calcium ion was detected by confocal microscopy. The rate of cell apoptosis was de-termined by flow cytometric analysis. Western blot was used to assess phosphorylation of Akt and ERK1/2 in H9c2 cells. Results Compared with the H/R group, S1P significantly increased vaibility of cells, lowered the rate of apoptosis, decreased the content of MDA in the culture medium, increased the activity of T-SOD and Mn-SOD, reduced concentration of intracellular calcium and increased the phosphorylation of Akt and ERK1/2 . When added LY294002 or PD98059 , the effects of S1P above were inhibited. Conclusion S1P protects H9 c2 cells against hypoxia/reoxygenation inju-ry. The protection of S1P was inhibited by LY294002, the inhibitor of PI3 K/Akt and PD98059 , the inhibitor of ERK1/2 . S1 P protects H9 c2 cells against hypoxia/reoxygenation injury via RISK pathway.

The important progress of the relationship between genomics and cardiovascular diseases has elucidated several abnormalities of genes leading to familial cardiomyopathy and ion channel diseases.Investigations of genomics show that the difference including resistance and susceptivity to diseases among human is determined by over 3 million base pairs of all 3,000 million.Single nucleotide polymorphism plays an essential role in elucidating hereditary cardiovascular diseases.

Objective To investigate the effect of intrathecal(IT)mibefradil on the mechanical and thermal hyperalgesia following chronic compression of dorsal mot ganglion(CCD)in rats,trying to elucidate the role of T-type calcium channels in the nociceptive signal transmission at spinal level.Methods Forty-eight male SD rats weighing 230-250 g were randomly divided into 6 groups(n=8 each):group Ⅰ sham operation;group Ⅱ CCD;group Ⅲ CCD+normal saline(N.S.)and group Ⅳ,Ⅴ,Ⅵ CCD+mibefradil 50 ?g(Ⅳ),100 ?g (Ⅴ),200 ?g(Ⅵ).The animals were anesthetized with intraperitoneal(IP)1% pentobarbital 40 mg?kg~(-1). Intrathecal catheter was placed according to the technique described by Yaksh et al.Five days after IT catheter placement L_4 and I_5 dorsal root ganglion(DRG)compression(CCD)was performed in group Ⅱ-Ⅵ.In sham operation group(Ⅰ)only L_(4-5) transverse processes and intervertebral foramina were exposed but DRGs were not compressed.In group Ⅳ,Ⅴ,and Ⅵ 5 days after CCD model was established a bolus of mibefradil 50,100 and 200 ?g in 10 ?l NS was given IT.In group Ⅲ NS 10 ?l was given IT instead of mibefradil.Mechanical withdrawal threshold(MWT)using Von Frey filament and thermal withdrawal latency(TWL)using radiant heat applied to the plantar surface were measured before CCD(baseline)and 1,3,5,7,14 and 21 days after CCD in group Ⅰ and Ⅱ and before and 30,60,120,240 and 480 min after IT mibefradil in group Ⅲ-Ⅵ.Results The animals in group Ⅱ(CCD group)developed mechanical and thermal hyperalgesia from the 1st day after operation to the end of the experiment.IT mibefradil 50,100 and 200 ?g can all attenuate both mechanical and thermal hyperalgesia induced by CCD.Conclusion Spinal T-type calcium channel may play an important role in the pathogenesis of neuropathic pain.

OBJECTIVETo search and identify the non-steroid receptor binding cis-acting elements in the L-plastin promoter in prostate cancer, and the correlative regulation pathway and transcription factors.

METHODSOn the basis of construction of the L-plastin promoter luciferase vectors which were removed the steroid hormone receptor AR and ER binding elements, the promoter on the vector was nest-deleted by Exonuclease III and the relative luciferase plasmids were constructed. Transfected these twelve plasmids into prostate cancer cell line LNCaP under dihydrotestosterone-stimulated situation or not and test the intensity of luciferase, then we got the regulation message of every 200 bp part of the promoter in prostate cancer. After the analysis of relative programme, we got the possible regu- lation pathway of non-steroid hormone transcription factors. After removing the possible transcription factors binding site sequence by site-specific mutagenesis, the changes luciferase of activities proved our reasoning.

RESULTSWe succeed in segmental deletion of the L-plastin promoter, and constructing the relative plasmids containing part L-plastin promoter on luciferase vector pGL3-basic. After testing the luciferase activities of constructed plasmids, we found the sequence from 206 to 1 of L-plastin promoter had significant luciferase activity. The software TRANSFECT showed that there were binding elements for transcription factors AP-4 at seq-198 to 192 and SP-1 at seq-54 to 41 on the short part promoter (206 to 1). The recombinant plasmids deleted the AP-4 and SP-1 binding elements had lower luciferase activity than the wild-type.

CONCLUSIONThere are some other non-steroid hormone pathway to regulate the expression of L-plastin except the steroid hormone pathway in prostate cancer. The main binding sites of the non-steroid hormone regulator lies in the sequence from 206 to 1. Transcription factors AP4 and SP-1 may up-regulated the expression of L-plastin by binding these sites.

Animals ; DNA-Binding Proteins ; physiology ; Gene Expression Regulation, Neoplastic ; Luciferases ; metabolism ; Male ; Membrane Glycoproteins ; Mice ; Microfilament Proteins ; Phosphoproteins ; biosynthesis ; genetics ; Promoter Regions, Genetic ; genetics ; Prostatic Neoplasms ; metabolism ; Response Elements ; Transcription Factors ; physiology ; Transfection ; Tumor Cells, Cultured ; Up-Regulation

OBJECTIVETo study the radiobiological effects of fast neutron/photon mixed irradiation on human cancer cell in vitro and to discuss the mechanism in relation with cell cycle and apoptosis, thus to provide experimental support for the further application of fast neutron radiotherapy of cancer.

METHODSExponentially growing human nasopharyngeal cancer cell line CNE-1 was irradiated in vitro with 35 MeV p-->Be fast neutron and 6 MV-X ray in grading doses (0 cGy, 40 cGy, 80 cGy, 120 cGy, 160 cGy, 240 cGy, 320 cGy and 400 cGy for neutron, and 0 cGy, 100 cGy, 200 cGy, 300 cGy, 400 cGy, 600 cGy, 800 cGy and 1000 cGy for X ray). Clonogenic assay was performed, and relative biological effectiveness (RBE) of fast neutron was determined with D(10) by means of cell survival curves. Isoeffective doses of 35 MeV p-->Be fast neutron and 6 MV-X ray were obtained according to the RBE. The cells were assigned into two irradiation regimens, (1) the one-week-fractionation regimen, which adopted the radiation pattern of X x 5, N x 2 and X-N-X-X-N. After irradiation the clonogenic assay was performed to compare their survival fractions; (2) the two-dose regimen, with the radiation pattern of X + N, N + X and X + X. Flow cytometry was done at different time points after irradiation to analyze cell cycle distribution and apoptosis. Fast neutron dose was delivered on Tuesday and Friday, and all the other irradiation intervals were 24 h.

RESULTSThe RBE of fast neutron to X ray in CNE-1 cells according to the D(10) ratio was 2.40. The neutron isoeffective dose for a single dose of 200 cGy of 6 MV-X ray was approximately 80 cGy. In clonogenic assay, the cell survival fractions were significantly lower in X-N-X-X-N group (0.0079) than those in X x 5 (0.018) and N x 2 (0.017) groups. The flow cytometry suggested a higher percentage of apoptotic cells after mixed irradiation, and different sequence of X ray and neutron irradiations caused varying changes in cell cycle arrest.

CONCLUSIONMixed irradiation of fast neutron and X ray showed a synergic effect in vitro on CNE-1 cell killing. Cell cycle arrest and apoptosis may play some role in the radiation damage repair mechanisms of mixed beam irradiation.

Apoptosis ; radiation effects ; Carcinoma, Squamous Cell ; pathology ; Cell Cycle ; radiation effects ; Cell Line, Tumor ; Dose-Response Relationship, Radiation ; Fast Neutrons ; therapeutic use ; Humans ; Nasopharyngeal Neoplasms ; pathology ; Photons ; therapeutic use

In order to investigate the influence of cytokine combinations on proliferation and differentiation of human umbilical cord blood CD34(+) cells into megakaryocytes/platelets in vitro, the CD34(+) cells from human umbilical cord blood were amplified in serum-free medium StemSpan(SFEM) supplemented with several cytokine combinations by three-phase culture system. The effects of the cytokine combinations were compared. The results showed that at day 14 of the first culture phase, the CD34(+) cells cultured with cytokine combinations SCF + TPO + FL + IL-3 were amplified (11 000 ± 1 000) times, which were significantly higher than that of cells cultured with SCF + TPO + FL, but were not significantly different from that of cells cultured with SCF + TPO + IL-3 or SCF + TPO + FL + IL-3+ hydroxyl-corticosteroids. At day 7 of the second culture phase, the CD34(+) cells cultured with cytokine combination SCF + TPO + FL + IL-11 were amplified by (204666.7 ± 11718.9) times, which were significantly higher than that of cells cultured with SCF + TPO + FL + IL-3, but were not significantly different from that of cells cultured with SCF + TPO + FL + IL-11 + BMP4 + VEGF. At day 3 and day 6, the CD34(+) platelet-like cells accounted for about (39.8 ± 1.9)%, (39.7 ± 2.6)% and (25.5 ± 1.4)%, (23.1 ± 3.5)% cultured with SCF + TPO + FL + IL-11 and SCF + TPO + FL + IL-11 + BMP4 + VEGF, and significantly higher than that of the cells cultured with SCF + TPO + FL + IL-3. It is concluded that the cytokine combination of SCF + TPO + FL + IL-3 is most suitable cytokines combination for the amplification of CD34(+) hematopoietic progenitor cells. The cytokine combination of SCF + TPO + FL + IL-11 is preferred for the proliferation and differentiation of megakaryocytes, this study lays an experimental basis for investigating the proliferation and differentiation of CD34(+) into megakaryocytes/platelets in vitro.
Antigens, CD34 ; immunology ; Blood Platelets ; cytology ; Cell Differentiation ; Fetal Blood ; cytology ; immunology ; Humans ; Interleukin-11 ; pharmacology ; Interleukin-3 ; pharmacology ; Megakaryocytes ; cytology ; Stem Cell Factor ; pharmacology ; Thrombopoietin ; pharmacology

OBJECTIVETo explore the effects of Panax Quinquefolium Saponin (PQS) on phosphatidylinositol 3-kinase/serine threonine kinase (PI3K/Akt) pathway of neonatal rat myocardial cells subjected to hypoxia.

METHODSNeonatal rat myocardial cells were cultured in vitro. After the myocardial cell injury was induced by hypoxia, the cells were randomized into 5 groups: the normal group, the model group, the positive control group (Ciclosporin A, 2 µ mol/L), the low-dose PQS group (PQSL, 25mg/L), and the high-dose PQS group (PQSH, 50 mg/L). Morphology and behavior of myocardial cells were observed under an inverted microscope. Apoptosis rate and lactate dehydrogenase (LDH) leakage rate of myocardial cells were determined by colorimetry. Mitochondrial transmembrane potential was assessed using a fluorexon laser. Phospho-glycogen synthase kinase (GSK)-3β and phospho-Akt as well as cytochrome C were determined by Western blot

RESULTSLDH leakage in the Ciclosporin A group, PQSH group and PQSL group reduced progressively compared with the model group (P<0.05). Akt and GSK-3β was strongly phosphorylated after treatment with Ciclosporin A and PQS compared with the model group (P<0.05, P<0.01). Compared with the model group (16.41±1.74; 35.28±6.30), both the integrated optical density of mitochondrial permeability transition pore (MPTP) and the mitochondrial transmembrane potential significantly increased in the PQSH group (42.74±2.12; 71.36±6.54) and the PQSL group (39.58±1.49; 66.99±5.45; P<0.05, P<0.01). However, the protein of cytochrome C outside the mitochondrion decreased in the PQSH group (273.66±14.61) and the PQSL group (259.62±17.31) compared with the model group (502.41±17.76; P<0.05).

CONCLUSIONThrough activation of the PI3K/Akt pathway and inhibition of the MPTP, PQS might protect the heart against ischemia injury and apoptosis of myocardial cells.

Animals ; Animals, Newborn ; Cell Hypoxia ; drug effects ; Cell Shape ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; L-Lactate Dehydrogenase ; metabolism ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; drug effects ; metabolism ; Mitochondrial Membrane Transport Proteins ; metabolism ; Myocytes, Cardiac ; cytology ; drug effects ; enzymology ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; drug effects ; Protein-Serine-Threonine Kinases ; metabolism ; Rats, Sprague-Dawley ; Saponins ; pharmacology ; Signal Transduction ; drug effects

OBJECTIVETo get the cDNA clones which cover the whole genome of SARS-CoV PUMC2 strain.

METHODSUsing the SARS-CoV PUMC2 strain genomic RNA as the template, the cDNA fragments were amplified by RT-PCR, the PCR products were further purified and ligated into the pGEM-T vector, and all the clones obtained were sequenced.

RESULTSThe cDNA clones which cover the whole genome of SARS-CoV PUMC2 strain were obtained.

CONCLUSIONSThese cDNAs can be provided for the function study of SARS-CoV proteins and the construction of full-length infectious cDNA clone of SARS-CoV.

Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; genetics ; DNA, Viral ; genetics ; Genome, Viral ; Molecular Sequence Data ; Nucleic Acid Amplification Techniques ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; SARS Virus ; genetics ; isolation & purification ; Sequence Analysis, DNA ; Viral Proteins ; genetics

OBJECTIVETo investigate the effects of endothelia progenitor cells conditioned medium (EPC-CM) on the migration, adhesion and proliferation of vascular smooth muscle cells (VSMCs).

METHODSMononuclear cells were isolated from rat bone marrow by density gradient centrifugation,plated on dishes precoated with 5% fibronectin, and then cultured with complete M199 medium (including 15% fetal calf serum, 10 microg/L VEGF and 5 microg/L bFGF). EPC-CM was collected and used to incubate VSMCs isolated from rat arteriae aorta. After 24 h, VSMCs proliferation, adhesion and migration were assayed with CCK-8, adhesion test and modified Boyden chamber assay, respectively.

RESULTSThe proliferation, adhesion and migration of VSMCs were obviously decreased when the cells were cultured with EPC-CM.

CONCLUSIONEPC-CM could inhibit VSMC functions, which would be one of the mechanisms against atherosclerosis by EPCs.

Animals ; Cell Adhesion ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Culture Media, Conditioned ; chemistry ; Endothelial Cells ; cytology ; Male ; Muscle, Smooth, Vascular ; cytology ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; cytology

To develop a gene therapy strategy for treating bovine mastitis, a new mammary-specific vector containing human lysozyme (hLYZ) cDNA and kanamycin resistance gene was constructed for intramammary expression and clinical studies. After one time acupuncture or intracisternal infusion of healthy cows with 400 microg of the p215C3LYZ vector, over 2.0 microg/ml of rhLYZ could be detected by enzymatic assay for about 3 weeks in the milk samples. Western blotting showed that rhLYZ secreted into milk samples from the vector-injected cows had molecular weight similar to that of the natural hLYZ in human colostrums. Twenty days after the primary injection, the quarters were re-injected with the same vector by quarter acupuncture and even higher concentrations of rhLYZ could be detected. Indirect competitive ELISA of milk samples showed that the vector injection did not induce detectable humoral immune response against hLYZ. Clinical studies showed that twice acupuncture of quarters with the p215C3LYZ vector had overt therapeutic effect on clinical and subclinical mastitis previously treated with antibiotics, including disappearance of clinical symptoms and relatively high microbiological cure rates. These data provide a solid rationale for using the vector to develop gene therapy for treating bovine mastitis.
Acupuncture ; Animals ; Cattle ; Female ; Genetic Therapy ; methods ; veterinary ; Genetic Vectors ; genetics ; Mastitis, Bovine ; genetics ; therapy ; Milk ; chemistry ; Muramidase ; biosynthesis ; genetics ; metabolism