Syndecan-1 expression in the lung during Pneumocystis infection.
Syndecan-1 expression in the lung during Pneumocystis infection.
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肺孢子虫感染期间，Syndecan-1 在肺部表达。
DOI：
10.1111/j.1550-7408.2006.00198.x
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Lee,Chao-Hung
中科院分区：
文献类型：
--
作者：
Wang,Shao-Hung;Zhang,Chen;Liao,Chung-Ping;Lasbury,MarkE;Durant,PamelaJ;Tschang,Dennis;Lee,Chao-Hung
MATERIALS AND METHODSEight-wk-old Rag-1À/À mice were infected with Pneumocystis murina by transtracheal inoculation as previously described (Zhang et al. 2006). Sprague–Dawley rats (Harlan, Indianapolis, IN) were immunosuppressed with 1.8 mg/L dexamethasone in drinking water and then transtracheally inoculated with P. carinii as previously described (Bartlett et al. 1988). To quantify the Sdc-1 protein in bronchoalveolar lavage (BAL) fluids, the first 1ml of BAL fluid was used. The cell-free BAL fluid was acidified using NaOAc (pH 4.5) and then NaCl and Tween 20 were added (final concentrations were 50, 150 mM, and 0.1%(v/v), respectively). Samples were spotted onto a Nytran SPC membrane and then reacted with an anti-Sdc-1 antibody (281-2)(BD Pharmingen, San Diego, CA). The results were analyzed by the ImageJ software. For Western blotting, the cell-free BAL fluids were concentrated, and then subjected to electrophoresis on a 10% SDS-polyacrylamide gel. The blotted membrane of the gel was probed with anti-Sdc-1 antibody (281-2). To detect Sdc-1 in situ, lung sections were reacted with anti-Sdc-1 antibody (281-2), and the reaction signals were developed with the Vectastain ABC-AP KIT (Vector Lab, Burlingame, CA). Total RNA was purified from lung tissue and then reverse transcribed to cDNA. The cDNA was used as templates for PCR to detect the mRNAs of MMP2, MMP12, MMP14, and the ribosomal protein subunit 8 (Rps8) genes using the following primers: MMP2 (forward: 50-TCCGGAGATCTGCAAACAGGACAT-30; reverse: 50-ATTCCAGGAGTCTGCGATGAGCTT-30), MMP12 (forward: 50-TTGGGCTAGAAGCAAATGGGCAAC-30; reverse: 50-AGCATGGGCTAGTGTACCACCTTT-30), MMP14 (forward: 50-AGATCAAGGCCAATGTTCGGAGGA-30; reverse: 50-TGGCGGAGGGATCGTTAGAATGTT-30); Rps8 (forward: 50-AGTCCGAGTTCGAGGAGGCAATAA-30; reverse: 50-TTGCCTTTCCGGGCTTTGATCTTC-30). The RPS8 gene was used as the internal control because its expression was found not affected by immunosuppression or Pneumocystis infection (Zhang et al. 2006). For quantification of Sdc-1 mRNA, real-time RT-PCR was performed using the TaqMans Gene Expression Assay kit (Applied Biosystems, Foster City, CA).
