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Bone marrow multipotent stem cells facilitate fluid resolution via activating epithelial sodium channels in injured lungs
China (IFMSA-China) - China Medical University, Shenyang
Department of Stem Cells and Regenerative Medicine
Type of Research Project
- Basic science
What is the background of the project?
Acute lung injury is characterized by severe lung edema at the early exudative phase, resulting from the imbalance of alveolar fluid turnover and clearance. Mesenchymal stem cells (MSCs) are multipotent stem cells, which have shown potential therapeutic effects on acute lung injury. MSCs culture medium (MSCs-CM) improved alveolar fluid clearance in vivo. However, the mechanisms for MSC-CM to benefit edema fluid resolution are incompletely clear. We thus aim to explore the roles of a major pathway for transalveolar ion transport, epithelial sodium channels in MSCs-mediated recovery of impaired alveolar fluid clearance.
What is the aim of the project?
The aim of the project is to explore a “cell-free therapy” method for alleviating the severity of pulmonary edema during acute lung injury by administrating with MSCs-CM, by analyzing the protective immunomodulatory paracrine factors of MSCs.
What techniques and methods are used?
The effects of MSCs-CM on in vivo alveolar fluid clearance were analyzed through bovine serum albumin protein assays by xylene brilliant cyanin G method. The activity of epithelial sodium channels were detected by transepithelial short-circuit currents in H441 monolayers with an Ussing chamber setup. Epithelial sodium channel expression was analyzed with real-time polymerase chain reaction and immunoblotting assay. • In vivo alveolar fluid clearance 8-10 weeks old C57 male mice were anaesthetized and ventilated with 100% O2 via an animal ventilator (Chengdu Taimeng Co. LTD). 200 μl 5% bovine serum album (BSA) solution with or without amiloride (1 mM) was instilled intratracheally. To mice treated with LPS (5 mg/kg, 24 h, intraperitoneally), 200 μl FBS-deprived DMEM/F12 medium or MSCs-CM containing 5% BSA was instilled as described above. After instillation, mice were ventilated for 30 min, and the alveolar fluid was aspirated. AFC was calculated as follows: AFC = (Vi-Vf)/Vi × 100, where Vi and Vf are the volume of the instilled and aspirated alveolar fluid, respectively. • Ussing chamber assay H441 monolayers were mounted in Ussing chambers (Physiologic Instrument) and then bathed on both sides with a solution containing (in mM): 120 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.83 K2HPO4, 1.2 CaCl2, 1.2 MgCl2, 10 HEPES sodium salt and either 10 mannitol for apical compartment or 10 D-glucose for basolateral compartment. The pH of each solution was adjusted to 7.4 and the osmolality was 290-300 mOsm/kg. The solutions of both sides were bubbled with mixed gas containing 95% O2 and 5% CO2 at 37 ºC. Short-circuit currents was measured with Ag-AgCl electrodes filled with 4% agar in 3 M KCl. The monolayers were short-circuited to 0 mV, and a 10 mV pulse of 1s was applied to every 10 s to monitor the resistance of transepithelium. Acquire and Analyze 2.3 program was used to collect data. Data were expressed as the mean ± SE. One-way ANOVA computations were used to analyze the difference of the means for normally distributed data. Mann-Whitney test was applied for nonparametric data.
What is the role of the student?
- The student will mainly observe
- The student will observe the practical experiments but will be highly involved in the analysis of the results
- The tasks of the student will be performed on his/her own
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
Isolation and culture of bone marrow multipotent stem cells. Analysis of in vivo alveolar fluid clearance by xylene brilliant cyanin G method. Detecting the transepithelial short-circuit currents in H441 monolayers with an Ussing chamber setup. mRNA and protein expression analysis of epithelial sodium channels with real-time polymerase chain reaction and immunoblotting assay, respectively. Data analysis and quantification: data will be analyzed with SPSS (statistical software system).
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student’s name will be mentioned in a future publication - The student will have the opportunity to present the results together with the supervisor at a conference
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Interests in the basic research activities.
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Pre-Medical students from the American-British system - Students in biomedical fields - Dental medicine students (IADS members)
- Ding Y; Zhao R; Zhao X; Matthay MA; Nie HG; Ji HL. ENaCs as both effectors and regulators of miRNAs in lung epithelial development and regeneration. Cell Physiol Biochem. 2017; 44(3): 1120-32
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