J Korean Neurosurg Soc.  2019 Nov;62(6):626-634. 10.3340/jkns.2019.0081.

Mouse Nerve Growth Factor Facilitates the Growth of Interspinal Schwannoma Cells by Activating NGF Receptors

• Liu SY
• Liu SZ
• Li Y
• Chen S

• KMID: 2463654
• DOI: http://doi.org/10.3340/jkns.2019.0081

Abstract

OBJECTIVE
Nerve growth factor (NGF) is a member of the neurotrophic factor family and plays a vital role in the physiological processes of organisms, especially in the nervous system. Many recent studies have reported that NGF is also involved in the regulation of tumourigenesis by either promoting or suppressing tumor growth, which depends on the location and type of tumor. However, little is known regarding the effect of NGF on interspinal schwannoma (IS). In the present study, we aimed to explored whether mouse nerve growth factor (mNGF), which is widely used in the clinic, can influence the growth of interspinal schwannoma cells (ISCs) isolated from IS in vitro.
METHODS
ISCs were isolated, cultured and identified by S-100 with immunofluorescence analysis. S-100-positive cells were divided into five groups, and separately cultured with various concentrations of mNGF (0 [phosphate buffered saline, PBS], 40, 80, 160, and 320 ng/mL) for 24 hours. Western blot and quantantive real time polymerase chain reaction (PCR) were applied to detect tyrosine kinase A (TrkA) receptor and p75 neurotrophin receptor (p75(NTR)) in each group. Crystal violet staining was selected to assess the effect of mNGF (160 ng/mL) on ISCs growth.
RESULTS
ISCs growth was enhanced by mNGF in a dose-dependent manner. The result of crystal violet staining revealed that it was significantly strengthened the cells growth kinetics when cultured with 160 ng/mL mNGF compared to PBS group. Western blot and quantantive real time PCR discovered that TrkA receptor and mRNA expression were both up-regualated under the condition of mNGF, expecially in 160 ng/mL, while the exoression of p75(NTR) demonstrated no difference among groups.
CONCLUSION
From these data, we conclude that exogenous mNGF can facilitate ISC growth by activating both TrkA receptor and p75(NTR). In addition, patients who are suffering from IS should not be administered mNGF in the clinic.

Keyword

Schwannoma; Mouse nerve growth factor; TrkA; p75(NTR)

MeSH Terms

Animals
Blotting, Western
Fluorescent Antibody Technique
Gentian Violet
Humans
In Vitro Techniques
Kinetics
Mice*
Nerve Growth Factor*
Nervous System
Neurilemmoma*
Physiological Processes
Protein-Tyrosine Kinases
Real-Time Polymerase Chain Reaction
Receptor, Nerve Growth Factor*
Receptor, trkA
Receptors, Nerve Growth Factor*
RNA, Messenger
Gentian Violet
Nerve Growth Factor
Protein-Tyrosine Kinases
RNA, Messenger
Receptor, Nerve Growth Factor
Receptor, trkA
Receptors, Nerve Growth Factor

Figure

• 

  Fig. 1. MRI and pathological characteristics of IS. A : Contast enhanced sagittal T1A weighted MRI showing the tumor. B : Tumor tissue was stained by HE staining (×100). C : Tumor tissue was stained for S-100 (brown; ×100). D : Tumor tissue was stained for GFAP (brown; ×100). MRI : magnetic resonance imaging, IS : interspinal schwannoma, HE : hematoxylin-eosin, GFAP : glial fbrillary acidic protein.

• 

  Fig. 2. Morphological characteristics of the primary cultured ISCs. A : ISCs were isolated from tumor tissue blocks after 48 hours of primary culture and demonstrated typical bipolar or tripolar morphologies (×100). B : ISCs were brightly stained for S-100 (green; ×100). C : ISCs and fibroblasts (white arrow) nuclei counterstained by DAPI (blue; ×100). D : Merged image and fibroblasts were not stained for S-100 (white arrow). Scale bars, 100 μm. ISCs : interspinal schwannoma cells, DAPI : 4',6-diamidino-2-phenylindole, dihydrochloride.

• 

  Fig. 3. mNGF can accelerate the growth of ISCs. A : The effect of different mNGF concentrations on ISCs was dose-dependent, and the peak effect was observed for the concentration of 160 ng/mL. B : ISC growth curve studies using crystal violet with and without daily 160 ng/mL mNGF stimulation for 12, 24, 36, and 48 hours. The data represent the mean±standard error. *p<0.001. †p<0.05. ‡p<0.01. mNGF : mouse nerve growth factor, ISCs : interspinal schwannoma cells.

• 

  Fig. 4. mNGF can promote the expression of TrkA. A : Western blot for evaluating TrkA and p75NTR expression in ISCs at 24 hours. TrkA gradually increased with increasing concentrations of mNGF, especially for the 160 ng/mL condition, compared with the control group (PBS), whereas the p75NTR did not significantly change between the treated group and control group. B : The statistical analysis of the TrkA and p75NTR level. The data represent the mean±standard error. *p<0.01. mNGF : mouse nerve growth factor, TrkA : tyrosine kinase A, GAPDH : glyceraldehyde-3-phosphate dehydrogenase, p75NTR : p75 neurotrophin receptor, ns : nonsignificant, PBS : phosphate buffered saline.

• 

  Fig. 5. Real-time TaqMan qPCR for the mRNA expression levels in the ISCs at 24 hours. Each group normalized to GAPDH and calibrated to the sham for increasing mNGF concentrations. The mRNA levels of TrkA were significantly increased for the 160 ng/mL condition compared to the control group (PBS), but the mRNA levels of p75NTR did not significantly differ among groups, which were consistent with the results of the western blot. The data represent the mean±standard error. *p<0.05. †p< 0.01. TrkA : tyrosine kinase A, ISC : interspinal schwannoma cell, p75NTR : p75 neurotrophin receptor, ns : nonsignificant, qPCR : quantitative polymerase chain reaction, mRNA : messenger-ribonucleic acid, GAPDH : glyceraldehyde-3-phosphate dehydrogenase, mNGF : mouse nerve growth factor, PBS : phosphate buffered saline.

Reference

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