The main factors which affected the isolation, purification and cultivation of Pinellia cordata protoplasts from leaves were studied. The results indicated that the optimum enzyme solution for P. cordata leaves was 13% CPW + 1.0% Cellulose +0.1% Pectolase, at pH 6.0, temperature (25-28 degrees C ) for 4 h. The sucrose density gradient centrifugation was adopted to purificate the protoplasts collected, when 25% sucrose was used as mediator, centrifugating at 500 rpm for 10 min. When the protoplasts were shallow liquid and liquid-solid double layer cultured on the medium of MS + 0.5 mg x L(-1) 6-BA + 0.25 mg x L(-1) NAA + 13% mannitol at the density of 2.5 x 104 protoplasts/mL, or fed and nursed cultured at the density of 100-500 protoplasts/mL, cell division could be observed for 3 days; granular calli appeared for 30 days. Calli was proliferated on the medium of MS + 0.5 mg x L(-1) 6-BA + 0.25 mg x L(-1) NAA solidified by 0.55% agar, and differentiated and regenerated after 5-6 months. Plant generation of P. cordata is successfully established.
Cell Separation ; methods ; Culture Media ; Pinellia ; physiology ; Protoplasts ; physiology ; Regeneration

AIMTo investigate the effects of blockers of the three kinds of potassium channels: voltage-dependent K+ channel(KV), calcium-activated K+ channel(KCa) and ATP-sensitive K+ channel(KATP), on the proliferation of rat bronchial smooth muscle cells (BSMCs).

METHODSThe effects of three kinds of potassium channel blockers on the proliferation of BSMCs were detected by MTT method, immunocytochemistry staining and flow-cytometry. Their effects on the dynamic changes of Ca2+ concentration in BSMCs were investigated by the fluorophotometry of Fura-2/AM.

RESULTSThe KV blocker 4-aminopyridine (4-AP) was shown to significantly increase the expression of proliferating cell nucleus antigen in cultured rat BSMCs (P < 0.01), but the KCa blocker tetraethylammonium (TEA) and KATP blocker glibenclamide(Glib) did not show such effect (P > 0.05). 4-AP was found to significantly increase the optical density value of the cultured BSMCs (P < 0.01) by MTT method and the numbers of S + G2M BSMCs(P < 0.05) by flow-cytometry. TEA and Glib did not show such effects. 4-AP significantly increased the Ca2+ concentration in cultured BSMCs(P < 0.01). TEA and Glib did not show such effects.

CONCLUSIONThis result suggests that inhibition of KV activity can increase intracellular Ca2+ and proliferation of rat BSMCs, but inhibition of KCa and KATP showed no effect.

4-Aminopyridine ; pharmacology ; Animals ; Bronchi ; cytology ; Calcium ; metabolism ; Cell Division ; drug effects ; Cells, Cultured ; Glyburide ; pharmacology ; Muscle, Smooth ; drug effects ; metabolism ; Potassium Channel Blockers ; pharmacology ; Potassium Channels, Calcium-Activated ; antagonists & inhibitors ; Potassium Channels, Voltage-Gated ; antagonists & inhibitors ; Proliferating Cell Nuclear Antigen ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tetraethylammonium ; pharmacology

Amino Acid Sequence ; Base Sequence ; Genes, Bacterial ; Humans ; Molecular Sequence Data ; Polymerase Chain Reaction ; Staphylococcus epidermidis ; enzymology ; genetics ; beta-Lactamases ; chemistry ; genetics

Adult ; Aged ; Coronary Disease ; therapy ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Sirolimus ; administration & dosage ; Stents ; adverse effects ; Thrombosis ; etiology

This study is to investigate the expression of CyclinD1 in asthmatic rats and construct expression plasmids of sense and antisense CyclinD1 gene and transfect them to asthmatic airway smooth muscle cell to study the effects of CyclinD1 on the proliferation of airway smooth muscle cells in asthmatic rats. CyclinD1 cDNA was obtained by RT-PCR of total RNA extracted from the airway smooth muscle in asthmatic rats. The sequence was inserted into eukaryotic expression vector pcDNA3.1 (+) to recombinate the sense and antisense pcDNA3.1-CyclinD1 eukaryotic expression vector. The two recombinations and vector were then separately transfected into airway smooth muscle cell in asthmatic rats by using liposome. The expression level of CyclinD1 was certificated by Western blotting analysis. The proliferations of ASMCs isolated from asthmatic rats were examined with cell cycle analysis, MTT colorimetric assay and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. Results showed (1) Compared with control group, the content of CyclinD1 was significantly increased; (2) It was comformed by restriction endonucleasa digestion and DNA sequence analysis that the expression plasmid of sense and antisense CyclinD1 were successfully recombinated. There was significant change of CyclinD1 expression between vector and sense CyclinD1 transfected cells, and the expression level of CyclinD1 in ASMC transfected with antisense CyclinD1 was lower than that in vector transfected cells (P <0.01); (3) In the asthmatic groups, compared with the vecter group, the percentage of S + G2M phase, absorbance A value of MTT and the expression rate of PCNA protein in ASMC transfected with pcDNA3. 1-CyclinD1 vector significantly increased. The values decreased remarkably in the pcDNA3,1-as CyclinD1 group. Statistical analysis revealed that there were significant differences in these indicators of cell proliferation in three groups (P <0.01). In the normal groups, statistical analysis revealed that there were significant differences in the percentage of S + G2M phase, a value of MTT and the expression rate of PCNA protein in three groups (P <0.01). Sense CyclinD1 eukaryotic expression vectors could have a positive effect on the proliferation of ASMC, however the antisence one have a negative effect, which implicated that CyclinD1 might contribute to the process of airway smooth muscle cell proliferation.
Animals ; Asthma ; pathology ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Codon ; genetics ; pharmacology ; Cyclin D1 ; agonists ; antagonists & inhibitors ; genetics ; DNA, Antisense ; genetics ; pharmacology ; Disease Models, Animal ; Gene Expression ; Genetic Vectors ; genetics ; Male ; Myocytes, Smooth Muscle ; drug effects ; pathology ; Rats ; Rats, Sprague-Dawley ; Recombination, Genetic ; genetics ; Respiratory System ; Reverse Transcriptase Polymerase Chain Reaction ; Transduction, Genetic ; Transfection

BACKGROUNDAirway smooth muscle (ASM) is suspected to be a determining factor in the structural change of asthma. However, the role of protein kinase C alpha (PKCalpha) and cyclin D1 involved in the dysfunction of ASM leading to asthmatic symptoms is not clear. In this study, the central role of PKCalpha and cyclin D1 in ASM proliferation in asthmatic rats was explored.

METHODSThirty-six pathogen-free male Brown Norway (BN) rats were randomly divided into 2 groups: control groups (group N1, N2 and N3) and asthmatic groups (group A1, A2, and A3). Groups A1, A2 and A3 were challenged with ovalbumin (OA) for 2 weeks, 4 weeks and 8 weeks respectively. Control animals were exposed to an aerosolized sterile phosphate buffered saline (PBS). The ASM mass and nucleus numbers were studied to estimate the degree of airway remodeling by the hematoxylin-eosin staining method. PKCalpha and cyclin D1 expression in the ASM cells was detected by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The relation between PKCalpha and cyclin D1 was assessed by linear regression analysis. PKC agonist phorbol 12-myristate 13-acetate (PMA), PKC inhibitor Ro31-8220 and an antisense oligonucleotide against cyclin D1 (ASOND) were used to treat ASM cells (ASMCs) obtained from the 2 weeks asthmatic rats. The cyclin D1 protein expression level was detected by Western blotting.

RESULTSCompared with the control group, the PKCalpha and cyclin D1 mRNA levels were increased in the asthmatic group. Similar to RT-PCR results, immunohistochemistry analysis for PKCalpha and cyclin D1 expression revealed an increased production in ASMCs after allergen treatment for 2, 4 and 8 weeks compared with the respective control groups. No difference in expression of PKCalpha and cyclin D1 in ASM were found in the 2, 4 or 8 weeks asthmatic rats. There were significant positive correlations between PKCalpha and cyclin D1 expression, both transcriptionally (r = 0.944, P < 0.01) and translationally (r = 0.826, P < 0.01), in ASM. The content of cyclin D1 in asthmatic ASMCs increased after being stimulated by PMA, and decreased when induced by Ro31-8220. ASOND targeting for cyclin D1 lowered the expression of cyclin D1 induced by PMA.

CONCLUSIONSIncreased expression of PKCalpha and cyclin D1 in ASM along with smooth muscle structure changes might implicate PKCalpha and cyclin D1 participation in the proliferation of ASM and contribute to the pathogenesis of asthma after repeated allergen exposure in rats. The results suggested that cyclin D1 might be downstream of PKC signal transduction pathway.

Animals ; Asthma ; pathology ; Cell Proliferation ; Cyclin D1 ; genetics ; physiology ; Lung ; pathology ; Male ; Myocytes, Smooth Muscle ; pathology ; Protein Kinase C-alpha ; genetics ; physiology ; RNA, Messenger ; analysis ; Rats ; Rats, Inbred BN

BACKGROUNDIt has been demonstrated that only 10%-20% cigarette smokers finally suffer chronic obstructive pulmonary disease (COPD). The underlying mechanism of development remains uncertain so far. Nitric oxide (NO) has been found to be closely associated with the pathogenesis of COPD, the alteration of NO synthase (NOS) expression need to be revealed. The study aimed to investigate the alterations of NOS isoforms expressions between smokers with and without COPD, which might be helpful for identifying the susceptibility of smokers developing into COPD.

METHODSPeripheral lung tissues were obtained from 10 nonsmoker control subjects, 15 non-COPD smokers, and 15 smokers with COPD. Neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS) mRNA and protein levels were measured in each sample by using real-time polymerase chain reaction and Western blotting.

RESULTSINOS mRNA was significantly increased in patients with COPD compared with nonsmokers and smokers with normal lung function (P < 0.001, P = 0.001, respectively). iNOS protein was also higher in COPD patients than nonsmokers and smokers with normal lung function (P < 0.01 and P = 0.01, respectively). However, expressions of nNOS and eNOS did not differ among nonsmokers, smokers with and without COPD. Furthermore, there was a negative correlation between iNOS protein level and lung function parameters forced expiratory volume in 1 s (FEV₁) (% predicted) (r = -0.549, P = 0.001) and FEV₁/forced vital capacity (%, r = -0.535, P = 0.001).

CONCLUSIONSThe expression of iNOS significantly increased in smokers with COPD compared with that in nonsmokers or smokers without COPD. The results suggest that iNOS might be involved in the pathogenesis of COPD, and may be a potential marker to identify the smokers who have more liability to suffer COPD.

Adult ; Aged ; Blotting, Western ; Female ; Humans ; Isoenzymes ; genetics ; metabolism ; Lung ; enzymology ; pathology ; Male ; Middle Aged ; Nitric Oxide Synthase ; genetics ; metabolism ; Pulmonary Disease, Chronic Obstructive ; enzymology ; pathology ; Real-Time Polymerase Chain Reaction

To investigate the regulatory effect of extracellular signal-regulated kinase (ERK) signaling pathway on airway smooth muscle cell (ASMC) proliferation in chronic asthmatic rats, the rat model of chronic asthma was established, and ERK agonist epidermal growth factor (EGF) and inhibitor PD98059 were used in the cell culture. ASMC proliferation was examined by flow cytometry analysis, methyl thiazolyl tetrazolium (MTT) colorimetric assay, [(3)H]-thymidine (TdR) incorporation and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. The expressions of ERK mRNA, ERK protein, phosphorylated ERK1/2 (p-ERK1/2) protein were observed by RT-PCR and Western blot. The results showed that in chronic asthmatic group, compared with that in the control group, the percentage of cells at G(0)/G(1) phase was significantly decreased and the percentage of cells at S+G(2)/M phase was significantly increased. Absorbance (A(490)), DNA synthesis and the expression of PCNA protein in ASMCs in chronic asthmatic group were significantly increased. The expressions of ERK mRNA, ERK1/2 protein, p-ERK1/2 protein and the activation ratio of ERK in ASMCs in chronic asthmatic group were significantly increased compared with those in the control group. After treatment with PD98059, the percentage of cells at S+G(2)/M phase, A(490), DNA synthesis and the expression of PCNA protein in ASMCs in chronic asthmatic group were significantly decreased; the expressions of ERK mRNA, ERK1/2 protein, p-ERK1/2 protein and the activation ratio of ERK in ASMCs in chronic asthmatic group were significantly decreased compared with those in the control group. After treatment with EGF, the percentage of cells at S+G(2)/M phase, A(490), DNA synthesis and the expression of PCNA protein in ASMCs in chronic asthmatic group were significantly increased compared with those before treatment; and PD98059 markedly inhibited the effect of EGF. These results suggest that the endogenous proliferation activity of ASMCs in chronic asthmatic rats significantly increases compared with that in the control rats, and ERK1/2 participates in this process. The ERK signaling pathway might play an important role in regulating ASMC proliferation, leading to asthmatic airway remodeling.
Animals ; Asthma ; enzymology ; pathology ; Bronchi ; pathology ; Cell Proliferation ; Cells, Cultured ; Chronic Disease ; Colorimetry ; Enzyme Activation ; Extracellular Signal-Regulated MAP Kinases ; analysis ; genetics ; physiology ; Flow Cytometry ; Myocytes, Smooth Muscle ; pathology ; Rats

This work was designed to explore the role of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway in migration of bronchial smooth muscle cells (BSMCs) of chronic asthmatic rats. To make chronic asthma model, Wistar rats underwent ovabumin (OVA) injection and eight-week inhalation. BSMCs were cultured in vitro. The expression of ERK1/2 in BSMCs was analyzed by immunocytochemistry, Western blot and RT-PCR. Migration of BSMCs was detected by both plate test and Boyden cell test. Results showed: (1) With Western blot technique, the ratio of p-ERK1/2 to total ERK1/2 in chronic asthmatic group was obviously higher than that in the control group (0.55 +/- 0.05 vs 0.48 +/- 0.04, n=10, P<0.01). (2) With RT-PCR, the relative A values of ERK1 and ERK2 mRNA in airways of chronic asthmatic rats were 1.83 +/- 0.24 and 1.07 +/- 0.11, respectively, which were significantly increased compared with that in the control group (0.58 +/- 0.14 and 0.51 +/- 0.12, n=10, P<0.01). (3) In plate test, the migration of BSMCs of chronic asthmatic rats was 2.9 times of that in the control group and reached 5.0 times by epidermal growth factor (EGF) stimulation, but decreased to 1.7 times by 30 mumol/L PD98059. (4) In Boyden cell test, the migration of BSMCs of chronic asthmatic rats was 1.9 times of that in the control group, and reached 3.1 times by EGF stimulation, but decreased to 1.45 times by 30 mumol/L PD98059. Our results indicate that the migration ability of BSMCs of chronic asthmatic rats increases, and ERK1/2 signaling pathway may play an important role in this process.
Animals ; Asthma ; chemically induced ; physiopathology ; Bronchi ; pathology ; Cell Movement ; physiology ; Cells, Cultured ; Male ; Mitogen-Activated Protein Kinase 1 ; metabolism ; physiology ; Mitogen-Activated Protein Kinase 3 ; metabolism ; physiology ; Myocytes, Smooth Muscle ; pathology ; Ovalbumin ; Rats ; Rats, Wistar ; Signal Transduction ; physiology