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Mucociliary clearance plays an important role in prlmonary defense
mechanism. Abnormalities in mucociliary clearance are directly or
indirectly related to various pulmonary desorders such se chromic
bromchitis, bronchiectasis, cystic fibrosis, boinchial asthma, pneumonia
and even lung cancer. There are two major functions of human respiratory
epithelium: sceretion of viscoelastic mucus which can trap the inhaled
potentially harmful particles and ciliary escalation which removes these
foreign particles.
At least eight different types of cells have been identified in human
airway epithelium. Among them. mucous cell, ciliated cells and basal
cells are the predominant three cell types. The mucous cells and ciliated
cells are differentiated cells which contribute to the mucus secretion and
ciliary transport. While the basal cells are the stem cells of the
respiratory epithelium. When the respiratory mucous membranes are
injured,the basal cells start to proliferate and differentiate into mucous
cells and ciliated cells to replace the damaged epithelium. Althouge
extensive works have been done in animal airway epithelium, the comtrol
mechanism of mucus secretion and ciliary movement, as well as the
regulation of basal cell proliferation and differentiation are still
poorly understood.
Wu and Lee first reported a serum-free, hormone-supplemented culture
system for hamster and rabbit respiratory epithelium in 1982. In this
culture system, the cultured respiratory epithelial cells are able to
maintain mucus secretion and ciliary differentiation. We adapted this
culture system and apply to the culture of human bromchial epithelial
cells. We used this system to study the regulatory mechanisms of ciliary
activity and mucus secretion.
A total of 40 surgical specimens of normal human bromchial tissues were
used in this study. The bronchial epithelial cells were dissociated by
protease and cultured with F12 medium supplemented with insulin,
transferren, epidermal growth factor, bovine hypothalamic extract, cholera
toxin and hydrocortisone. The cultured bronchial epithelial cells were
able to maintain mucous and ciliary differentiation when they are grown on
the collagen substrata in the presence of vitamin A. The transmission
electron microscopic and immunocytochemical studies with mucin monoclonal
antibody confirmed the differentiated phenotypes in this culture model.
* Regulation of Ciliary Activity in Cultured Human Bronchial Epithelial
Cells
Human bronchial epithelial cells cultured on collagen gel substrata in a
serum-free F12 medium maintained differentiated phenotype with beating
cilia. The ciliary beating frequency(CBF) could be measured with a
computer assisted image analyzing system. We studied the ciliary activity
of cultured bronchial epithelial cells from 21 adults and one 3 month-old
fetus. The CBFs varied fron 8.1±1.0 Hz to 13.1±0.6 Hz and could last
2-3 weeks in culture. The CBF increased with the increase of temperature
at a rate of 15 ±3 beats /℃ and reached a plateau at 40-45℃. The
environment pH and presence of local anesthetic agents would affect on the
ciliary activity. Isoproterenol (10-7M), terbutalina (10-6M), forskolin
(10-5M), cAMP(10-6M), and isobutylmethylxanthine (10-4M), could stimulate
ciliary activity and the increase of CBF was associated with the increase
in intracellular cAMP level. Propranlol (10-6M) blocked the stinulatory
effect caused by isoproternol. Renoual of calcium ion decreased 40% of
CBF. These results suggest that calcium and cAMP are two important
regulators for control of ciliary activity in cultured human bronchial
epithelial cells. The single cilia beating pattern analysis by asymmetric
illumination technique showed that the ciliary movement in culture
composed of a recovery stroke and effective stroke with the duration ratio
of 2:1. The cilia swipt an angle of 110。 (at 25℃, CBF 5Hz)which
increased to 150。 after the stimulation of isoproterenol 10-6M (CBF
7.5Hz). This in vitro model provides an ideal system in the study of the
regulation of human cilia kinetics.
* Regulation of Mucus Secretion in Cultured Human
Bronchial Epithelial Cells
We established an ELISA system for mucin quantitation by monoclonal
antibody against mucin (17Q2). This ELISA system was sensitive to detect
0.2 ng of mucin antigen. Using this ELISA technique, we are able to study
the regulatory mechanisms of mucin production in cultured bronchial cells.
We found that beta adrenergic agents could stimulate mucin production
while propranolol inhibit mucin secretion. Atropine and acetylcholine had
no effect on mucus secretion in cultured mucous cells. Pseudomonas
ndotoxin, histamine, prostaglandin E2. prostaglandin F2 α, leukotriene C4
and D4 could also stimulate mucin production at high pharmacologic
concentration. This in vitro moedl in conjunction with the mucin ELISA
technique was a powerful tool to study the control of mucous cell
secretion in human respiratory epithelium.
* Growth and Differentiation of Lung Adenocarcinoma Cell Lines
We nodified the culture methods for normal bronchial epithelial cells and
established a culture system for human lung adenocarcinoma. The basal F12
medium was supplomented with selenium, insulin, transferrin, bovine
hypothalamic extract, cholera toxin, epidernal growth factor and
hydrocortisone. We have succeeded in the establishment of four
adenocarcinoma cell lines from 12 clinical specimen. Three of the cell
lines maintianed differentiated phenotype with glandular structure and
mucin production.
Using 3H-glucosamine as a mucin precursor to label the biosynthesis of
mucin molecule, we had confirmed that CL2 lung adenocarcinoma cell lines
could synthesize mucin. The intact mucin molecules were identified by
chromatographic characteristics, enzyme digestion and immunoprecipitation.
Although the CL2 cells could synthesize mucin, the mucin was
cell-associated and did not release into the culture medium. The mucin
production by CL2 cells be stimulated by 10-6M vitamin A (retinoic acid).
Meanwhile, the growth of CL2 cells were inhibited in this concentration of
vitamin A. The DNA synthesis by 3H-thymidine incoporation showed that
vitamin A inhibited DNA synthesis by CL2 cells and there was a 48 hour
latency period. The DNA flow cytometry study showed that the cell cycle
distribution shifted in vitamin A treated CL2 cells. The S phase and G2-M
phase cells were shifted to G0-G1 phase after treatment with vitamin A.
These results indicate that vitamin Ainhibit CL2 cell growth by induction
of mucin differentiation.
* Basal cell Markers Studied by Monoclonal Antibodies Approach
Basal cells of epithelium possess some biologic characteristics which are
similar to transformed cancer cells, such as undifferentiation, ability to
proliferate and unlimited life span. To fascilitate the study of
proliferation and differentiation of basal cells, wedeveloped two
monoclonal antibodies as basal cell marker with hybridoma technique. The
immunogens were living CL2 cells. Immunofluorescense study showed that
these two antibodies, S4 and M2, were specific for basal cells of skin,
trachea and esophageal epithelium. These two antibodies could also be
stained on the cancer cell lines and frozen tumor tissue sections of the
lung and esophagus. Western blot analysis showed S4 antigen was a 160-180
kD protein and M2 antigen, 50 kD. Both M2 and S4 antigens were localized
on the cell surface as confirmed by membrane immunofluorescence staining
and surface protein labeling by 125I-lactoperoxidase system. Functional
study of S4 antibody revealed that S4 antibody inhibited cell attachment
and colony formation; while M2 antibody had no effect on cell attachment.
The M2 antigens were preferentially expressed on lung cancer cells and
viral transformed cells. These two monoclonal antibodies are useful
markers to study the interaction between cell and cell, cell and matrix,
as well as the growth and differentiation of basal cells.
* Future Works
The preferential expression of S4 and M2 antigens in undifferentiated
basal cells and cancer cells suggests that these antigens are related to
undifferentiation and cell proliferation. We are now interested in the
gene expression of these antigens. Recently, we have identified a 1.5 kb
cDNA clone by immunoscreening with M2 antibodies. Preliminary data of in
situ hybridization using anti-sense RNA probe confirmed the basal
localization of the complimentary mRNA. The Northern hybridization
revraled a 2 to 4-fold increase of 2.0 kb mRNA expression in lung cancer
cell lines as compared with the normal bronchial epithelial cells.
Further studies are needed to confirm this cDNA clone.
In conclusion, we have established an in vitro model to study the growth
and differentiation of human bronchial epithelium. The ciliary kinetics
can be studied by this clture system with a computed asisted image
processing. The regulation of mucin production by mucous cells can be
studied by this culture techmique with mucin ELISA assay. A systen for
culturing lung abenocarcinoma cell line is also developed with a suscess
rate of 30%. We also demonstrated that vitamin A retinoic acid can
modulated cell growlth and mucin differentiation in a well differentiated
lung adenocarcinoma cell line. This cell line can be used as a model to
study the anti-tumor effect of vitamin A. Finally, we developed two
monoclonal antibodies specific for undifferentiated basal cells. These
two surface markers basal cell of may be potentially useful in the study
of the control of differentiation and proliferation of basal stem cells.
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