Biological effects of neuropeptide substance P on human dental pulp stem cells

doi:10.3877/cma.j.issn.1674-1366.2022.06.002

Abstract

Abstract:

Objective

To investigate the effects of neuropeptide substance P on the biological behavior of human dental pulp stem cells (hDPSCs) .

Methods

hDPSCs were isolated and cultured by tissue block method, and then identified by flow cytometry and alizarin red staining. hDPSCs were treated with different concentrations of neuropeptide substance P [0 (control group) , 0.1 nmol/L, 10 nmol/L, 1 μmol/L, 10 μmol/L]. Cell proliferation was detected by cell counting kit-8 (CCK-8) method at 24, 48, 72 and 96 h. At 48 h, while cell migration was detected by Transwell assay. After 14 days of intervention under angiogenic induction conditions, the tubule formation was detected by tubule formation assay. Vascular endothelial growth factor receptor 2 (VEGFR2) and vascular endothelial growth factor (VEGF) which are associated with tubule formation, were detected by quantitative real-time polymerase chain reaction (RT-PCR) . GraphPad Prism (v.8) was used to analyze the data.

Results

hDPSCs were successfully isolated and cultured, and the expression rates of cell surface markers CD29, CD90, CD34 and CD45 were 99.90%, 99.90%, 0.40% and 0.04%, respectively. Alizarin red staining results showed that the osteogenic induction group formed more mineralized nodules. The results of CCK-8 showed that the relative proliferation rates of the control group at 48, 72 and 96 h were (100.0 ± 0.4) %, (100.0 ± 0.7) % and (100.0 ± 1.9) %, respectively. The promotion of cell proliferation was observed at 48, 72 and 96 h for the experimental groups, which was statistically significant between groups (F_{48 h} = 50.1, P_{48 h}<0.001; F_{72 h} = 323.5, P_{72 h}<0.001; F_{96 h} = 253.6, P_{96 h}<0.001) . The optimal effect concentration of substance P was 10 μmol/L, and with its presence at this concentration, the cell proliferation rates at 48, 72 and 96 h were (119.0 ± 1.0) %, (148.4 ± 3.5) % and (140.8 ± 1.0) %. Transwell results showed that all the experimental groups had stronger promoting effect on the migration of hDPSCs than the control group (F = 310.3, P<0.001) , since the number of membrane penetration in the control group (5.0 ± 1.4) was less than that of the experimental group with 10 nmol/L substance P (87.6 ± 8.3) . After 14 days of angiogenesis induction, the results of tubule formation assay showed that, compared with the control group, each substance P group could promote the tubule formation of hDPSCs (F_{branch nodes} = 43.7, P_{branch nodes}<0.001, F_{tubule length} = 19.4, P_{tubule length} = 0.0001) . The results of RT-PCR showed that the expression of VEGFR2 in 0, 0.1 nmol/L, 10 nmol/L, 1 μmol/L, 10 μmol/L groups were (1.000 ± 0.023) , (1.129 ± 0.076) , (1.474 ± 0.101) , (1.285 ± 0.055) and (1.213 ± 0.160) , respectively. The expression levels of VEGF were (1.000 ± 0.026) , (1.211 ± 0.023) , (1.242 ± 0.070) , (1.679 ± 0.043) and (1.138 ± 0.156) , respectively. Compared with the control group, the difference was statistically significant (F_VEGFR2 = 10.43, P_VEGFR2 = 0.0014, F_VEGF = 29.87, P_VEGF<0.001) .

Conclusion

Substance P may have certain promoting effect on the proliferation, migration and angiogenesis of hDPSCs.

Key words: Neuropeptide, Substance P, Dental pulp stem cells, Biological behavior, Dental pulp regeneration

Mengzhu Chen, Xiaolang Wei, Wenxia Chen. Biological effects of neuropeptide substance P on human dental pulp stem cells[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2022, 16(06): 343-351.

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Figures/Tables 11

References 20

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基因名称	引物序列
VEGF	正向：5′-AGAAGGAGGAGGGCAGAATCATCAC-3′
	反向：5′-GGGCACACAGGATGGCTTGAAG-3′
VEGFR2	正向：5′-AGGGAGTCTGTGGCATCTGAAGG-3′
	反向：5′-GTGGTGTCTGTGTCATCGGAGT-3′
GAPDH	正向：5′-CAGCAAGGATACTGAGAGCAAGAG-3′
	反向：5′-GGATGGAATTGTGAGGGAGATG-3′

基因名称	引物序列
VEGF	正向：5′-AGAAGGAGGAGGGCAGAATCATCAC-3′
	反向：5′-GGGCACACAGGATGGCTTGAAG-3′
VEGFR2	正向：5′-AGGGAGTCTGTGGCATCTGAAGG-3′
	反向：5′-GTGGTGTCTGTGTCATCGGAGT-3′
GAPDH	正向：5′-CAGCAAGGATACTGAGAGCAAGAG-3′
	反向：5′-GGATGGAATTGTGAGGGAGATG-3′

组别	例数	24 h	48 h	72 h	96 h
对照组	3	100.0 ± 2.3	100.0 ± 0.4	100.0 ± 0.7	100.0 ± 1.9
0.1 nmol/L实验组	3	102.2 ± 3.7	105.9 ± 0.9^a	111.0 ± 1.0^a	111.9 ± 0.2^a
10 nmol/L实验组	3	94.5 ± 1.5^ab	99.3 ± 1.4^b	104.5 ± 0.7^ab	105.5 ± 2.5^ab
1 μmol/L实验组	3	104.2 ± 1.5^c	105.8 ± 3.8^ac	111.2 ± 1.7^ac	113.6 ± 2.0^ac
10 μmol/L实验组	3	99.9 ± 1.5^cd	119.0 ± 1.0^abcd	148.4 ± 3.5^abcd	140.8 ± 1.0^abcd
F值		7.6	50.1	323.5	253.6
P值		0.004 2	<0.001	<0.001	<0.001

组别	例数	24 h	48 h	72 h	96 h
对照组	3	100.0 ± 2.3	100.0 ± 0.4	100.0 ± 0.7	100.0 ± 1.9
0.1 nmol/L实验组	3	102.2 ± 3.7	105.9 ± 0.9^a	111.0 ± 1.0^a	111.9 ± 0.2^a
10 nmol/L实验组	3	94.5 ± 1.5^ab	99.3 ± 1.4^b	104.5 ± 0.7^ab	105.5 ± 2.5^ab
1 μmol/L实验组	3	104.2 ± 1.5^c	105.8 ± 3.8^ac	111.2 ± 1.7^ac	113.6 ± 2.0^ac
10 μmol/L实验组	3	99.9 ± 1.5^cd	119.0 ± 1.0^abcd	148.4 ± 3.5^abcd	140.8 ± 1.0^abcd
F值		7.6	50.1	323.5	253.6
P值		0.004 2	<0.001	<0.001	<0.001

组别	例数	小管分支点数（个）	相对小管长度（μm）
对照组	3	11 ± 3	1 751 ± 1 061
0.1 nmol/L实验组	3	32 ± 3^a	3 656 ± 193^a
10 nmol/L实验组	3	48 ± 3^ab	5 348 ± 471^ab
1 μmol/L实验组	3	40 ± 4^ab	5 061 ± 709^ab
10 μmol/L实验组	3	55 ± 7^abd	6 440 ± 794^ab
F值		43.7	19.4
P值		<0.001	0.0001

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