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Simultaneous
visualization for coexpression of multiple neurotrophic
factors in living Schwann cells
Jing Chen*,
Yanfei Chu, Jianmin Wang and Zaiyun Long
State Key Laboratory of Trauma, Burns and Combined Injury,
Institute of Surgery Research, Daping Hospital, Third
Military Medical University, Chongqing 400042, China.
*Corresponding author. E-mail:
yi_yi0809@126.com.
Abbreviations: PNS,
Peripheral nervous system;
NTFs,
neurotrophic factors; DRG,
dorsal root ganglion; NGF, nerve growth factor;
CNTF, ciliary neurotrophic factor; GDNF, glial
cell line-derived neurotrophic factor; CLS, confocal
laser scanning; RT-PCR, reverse transcriptase-
polymerase chain reaction; DMEM, dulbecco’s modified
eagle’s medium; FBS, fetal bovine serum; GGF,
glial growth factor; FISH, fluorescence in situ
hybridization; PBS, phosphate-buffered saline; BSA,
bovine serum albumin.
Accepted 22 December, 2009 |
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Schwann cells, as specialized glial cells found in the
peripheral nervous system (PNS), produce a variety of
neurotrophic factors (NTFs) and play a vital role in
maintaining PNS functions. The combined biological effects
of multiple NTFs are strongly associated with their
coexpression characteristics in the physiological
environment of living cells. In this study, the method for
visualizing coexpression of multiple NTFs in living Schwann
cells was investigated. We isolated Schwann cells from rat
sciatic nerve and co-cultured them with dorsal root ganglion
(DRG) neurons. The DRG neurons were removed, following which
the Schwann cells were hybridized with fluorescence-labeled
oligonucleotide probes for nerve growth factor (NGF),
ciliary neurotrophic factor (CNTF) and glial cell
line-derived neurotrophic factor (GDNF) introduced by
electroporation and observed under confocal laser scanning (CLS)
microscope. Our experimental results revealed that the
following factors were crucial for visualizing the
coexpression of multiple NTFs in living cells: (1) probe
design and labeling, (2) probe specificity, (3)
electroporation parameters for introducing probes into
living cells and (4) hybridization signal detection. Our
study may provide further insights into the synergistic
effects of these factors on neurons.
Key words:
Coexpression, neurotrophic factor, Schwann cell,
visualization. |
