Role of protein kinase C alpha and cyclin D1 in the proliferation of airway smooth muscle in asthmatic rats / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Airway 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.</p><p><b>METHODS</b>Thirty-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.</p><p><b>RESULTS</b>Compared 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.</p><p><b>CONCLUSIONS</b>Increased 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.</p>

Recombination and identification of sense and antisence CyclinD1 eukaryotic expression vectors and the effects of the vectors on the proliferation of airway smooth muscle cell in asthmatic rats / 药学学报

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.

Structure-based design and biosynthesis of collagen proteins / 生物工程学报

Collagen is the most abundant protein in human body and a periodic helix, i. e. , triple helix, fibrous protein, which provides the scaffold structures for the cell adhesion and macromolecule aggregation, etc. With the development of gene engineering and biomaterial technologies, and the incessant studies on the technique to obtain the proteins with special functions, the collagen protein has been one of the third generation biomaterials that attract more attention than others. In this paper, we reviewed the recent structure-based design and biosynthesis of collagen.