[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].
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.
Qiao
Li-Fen
LF
Department of Respiratory Medicine, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Xu
Yong-Jian
YJ
Liu
Xian-Sheng
XS
Xie
Jun-Gang
JG
Du
Chun-Ling
CL
Zhang
Jian
J
Ni
Wang
W
Chen
Shi-Xin
SX
D000818:Animals; D001249:Asthma / Q000473:pathology*; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects*; D003062:Codon / Q000235:genetics / Q000494:pharmacology; D019938:Cyclin D1 / Q000819:agonists / Q000037:antagonists & inhibitors* / Q000235:genetics; D016373:DNA, Antisense / Q000235:genetics / Q000494:pharmacology*; D004195:Disease Models, Animal; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D008297:Male; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000473:pathology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011995:Recombination, Genetic / Q000235:genetics; D012137:Respiratory System; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014161:Transduction, Genetic; D014162:Transfection
D003062:Codon; D016373:DNA, Antisense; D019938:Cyclin D1