[Isolation and characterization of human alveolar type II cells and phenotypes maintaining study].

Objective: To establish a method of isolate, purify, primary culture and identify human alveolar type II cells (AT II ) in vitro, as well as its possible maintaining phenotype characteristics.

Methods: The marginal lung tissue was collected. AT II cells were isolated with trypsin and elastase, purified by a series of steps, such as, cell sieve filtration, differential adhesion, gradient separation and anti-CD14 beads separation. AT II cells were identified with immunofluorescence of human pro-surfactant-associated protein C (pro-SP-C), Green DND-26 probe and electron microscope. The purity of AT II cells was measured by immunofluorescence of human pro-SP-C and Green DND-26 probe. The viability of AT II cells was measured by trypan blue staining. The phenotypes (SP-A, SP-B,SP-C, SP-D) were monitored with reverse transcription-polymerase chain reaction (RT-PCR) at different time points.

Results: The output of AT II cells from lung tissue was (5-10) x 105/g, and the cell viability was (93 ± 2)% with trypan blue staining, the cell purity was about 98% with pro-SP-C immunofluorescence and Green DND-26 fluorescent probe, the lamellar bodies were clearly observed with transmission electron microscope. In the aspect of phenotypes maintaining, the time of surfactant expression was about 24 days [SP-A: 0.52 + 0.03 (day 16), 0.35 + 0.02 (day 20),0.26 ± 0.01 (day 24), 0.10 + 0.08 (day 28); SP-C: 0.68 0.16 (day l6), 0.31 + 0.04 (day 20), 0.18 + 0.06 (day 24), 0.14 + 0.09 (day 28)], and the longest one was more than 28 days [SP-B: 1.05 + 0.17 (day 16), 0.76 + 0.35(day 20), 0.55 0.15 (day 24), 0.36 0.19 (day 28); SP-D: 0.52 0.19 (day 16), 0.33 + 0.12 (day 20), 0.31 +0.04 (day 24), 0.23 ± 0.02 (day 28)).

Conclusion: We successfully established a procedure to separate, purify,identify of AT II cells, which retain primary phenotypic characteristics over long period.