ABSTRACT
In order to confirm the alteration and significance of cigarette smoke exposure on SP-A in rats, 20 Wistar rats were assigned randomly to two groups: an N group (n=10), and an S group (n=10). The ultra-structural change was observed by electron microscopy. The number of cells positive for SPA was by immunohistochemically measured. The mRNA expression in the lung tissues was determined by reverse transcription polymerase chain reaction (RT-PCR). The number of cells positive for SPA of the S group (0.52 +/- 0.05) was lower than that of the N group (0.72+/-0.06) (P<0.05). The levels of mRNA of SPA in the lung tissues of the S group (0.3522+/-0.0512) was significantly lower than that of the N group (0.4432+/-0.05628) (P<0.05). It is concluded that cigarette smoke alone decreased the level of SP-A and that might have an important effect on surfactant metabolism and the host defense functions of surfactant in the peripheral airways, which might play a crucial role in the development of chronic obstructive lung disease.
Subject(s)
Gene Expression Regulation , Immunohistochemistry/methods , Lung/metabolism , Microscopy, Electron , Pulmonary Surfactant-Associated Protein A/biosynthesis , RNA, Messenger/metabolism , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Smoking/adverse effects , Tobacco Smoke Pollution/adverse effectsABSTRACT
Objective To construct the eukaryotic expression vector harboring the fragment of Alia gene, and to investigate the effects of it on the signal of quorum sensing and virulence factors producted by Pseudomonas aeruginosa(Pa). Methods The plasmid pET-AiiA was cutted by Nhe Ⅰ and Xho Ⅰ , then the AiiA fragment was cloned into eukaryotic expression vector pEGFP-N2. After the plasmid was transfected into A549 cells, the protein was extracted and AiiA protein was found in it by Western blot. After the extrac- tion was admixed into the LB broth, from culture supernatant extracts of Pa, the N-acylhomoserine lactone (AHL) was detected by bioassay, and the expression of pyocyanin and elastase were assayed by RT-PCR and optical density. Results The fragment of AiiA gene was cutted and then cloned into pEGFP-N2. AiiA protein was found in the transfected cells. After admixed with the extract harboring AiiA protein, in Pa medium, the AHL was hydrolyzed, and the expression of pyocyanin and elastase were reduced. Conclusion The virulence factors synthesized by Pa were reduced by the AiiA protein expressed in eukaryotic cell.
ABSTRACT
To establish a better method of primary culture for alveolar epithelial type Ⅱ cells (AEC Ⅱ) and to study its bionomics, alveolar epithelial type Ⅱ cells were isolated by digestion with tryp- sin and collagenase, which were then purified by plated into culture flask coated with rat immu- noglobulin G. The purified AEC Ⅱ were identified by alkaline phosphatase staining, electron mi-croscopy, immunocytochemical staining of pulmonary surfactant protein A (SPA). The SPA expres-sion and transfection characteristics were compared with those of A549 cell line. The results showed that AEC Ⅱ could be isolated by digestion with trysin and collagenase and purified by adhesive pu- rification by using IgG, with a yield of about 2-3×107, and a purity of about 75%-84 %. Cells could be quickly identified with AKP staining. AEC Ⅱ were different from A549 cell line in terms of SPA expression and transfection characteristics. It is concluded that adhesive purification with IgG can improve the purity of AEC Ⅱ, and AKP staining is simple in cell identification. AEC Ⅱ can not be completely replaced by A549 cells in some studies because the differences between them, such as SPA expression.
ABSTRACT
To establish a better method of primary culture for alveolar epithelial type II cells (AEC II) and to study its bionomics, alveolar epithelial type II cells were isolated by digestion with trypsin and collagenase, which were then purified by plated into culture flask coated with rat immunoglobulin G. The purified AEC II were identified by alkaline phosphatase staining, electron microscopy, immunocytochemical staining of pulmonary surfactant protein A (SPA). The SPA expression and transfection characteristics were compared with those of A549 cell line. The results showed that AEC II could be isolated by digestion with trysin and collagenase and purified by adhesive purification by using IgG, with a yield of about 2-3 x 10(7), and a purity of about 75%-84%. Cells could be quickly identified with AKP staining. AEC II were different from A549 cell line in terms of SPA expression and transfection characteristics. It is concluded that adhesive purification with IgG can improve the purity of AEC II, and AKP staining is simple in cell identification. AEC II can not be completely replaced by A549 cells in some studies because the differences between them, such as SPA expression.
Subject(s)
Cell Culture Techniques/methods , Cell Separation/methods , Cells, Cultured , Ecology , Epithelial Cells/cytology , Immunoglobulin G/pharmacology , Pulmonary Alveoli/cytology , Pulmonary Surfactant-Associated Protein A/biosynthesis , Rats, WistarABSTRACT
AIM:To clone the SPA gene promoter and construct its luciferase report vector of SPA gene and to study its transcriptional targeting activity.METHODS:① The SPA gene sequence was acquired from GenBank,of which the upstream was analyzed according to bioinformatics.The results showed that the upstream region of SPA gene sequence about 163bp has the function of promoter.② The SPA gene promoter fragment was generated by polymerase chain reaction and then subcloned into the multiple clone site(MCS)of luciferase report gene vector pGL3-basic to generate the recombined plasmid pGL3-SPA.This fragment was also subcloned into pGL3-control to generate recombined plasmid pGL3-SPA-enhancer by replacing its primary SV40 promoter.③ pGL3-SPA,pGL3-SPA-enhancer,pGL3-control,pGL3-basic were cotransfected with pRL-TK into A549 cells and H441 cells.The luciferase activities were measured by dual luciferase reportor(DLR)system.RESULTS:Sequencing and restricted digestive results showed that SPA gene promoter was successfully cloned and identified,and also correctly subcloned into plasmid pGL3-basic and pGL3-control to construct its luciferase report plasmid pGL3-SPA and pGL3-SPA-enhancer,respectively.The transcriptional activity was high in H441 cells.CONCLUSION:The luciferase report system of SPA gene promoter is successfully constructed with high transcriptional activity.