Effects of different concentrations of γ-secretase inhibitor on osteogenic differentiation of human periodontal ligament stem cells

doi:10.3877/cma.j.issn.1674-1366.2018.06.002

Abstract

Abstract:

Objective

To evaluate the effect of gamma secretase inhibitor (DAPT) on osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in different concentrations.

Methods

hPDLSCs cultured in vitro were randomly divided into, (1) two control groups: hPDLSCs group (without DAPT) and 0.173 25 μL/L dimethyl sulfoxide (DMSO) group, and (2) three experimental groups: 25, 50 and 75 μL/L DAPT groups. The effects of different concentrations of DAPT on the proliferation of hPDLSCs were evaluated by cell counting kit (CCK-8) in control group and three experimental groups (25, 50 and 75 μmol/L DAPT) . Notch letters of different concentrations of DAPT on hPDLSCs were detected by alizarin red staining, real-time fluorescence quantitative polymerase chain reaction (PCR) and western blot. SPSS 21.0 was used for statistical description and analysis, when α= 0.05 was set as the statistical significance level.

Results

CCK-8 results showed that DMSO concentration of 0.17325 ul/L had no significant effect on the proliferation of periodontal ligament stem cells, and DAPT at different concentrations inhibited the proliferation of hPDLSCs, and DAPT concentration of 50 μmol/L had the strongest inhibitory effect on cells (P<0.001) ; Alizarin red staining results showed that the mineralized nodules in SC and DMSO groups were large and dark. There was no significant difference between the two groups. In contrast, the mineralized nodules formed in the three experimental groups were less. Specifically, the mineralized nodules formed in the group of 50 μmol/L DAPT were less and smaller than that in the groups of 25 and 75 μmol/L. The results of real-time PCR showed that the expressions of CAP, OCN and Runt-related transcription factor 2 (Runx2) were 0.574 ± 0.182, 0.269 ± 0.100 and 0.470 ± 0.080, respectively, in the group of 50 μmol/L DAPT, less than that in other groups (P<0.05) . The expression levels of Hes-1 and Notch signaling pathway related molecules Notch1 and Hes-1 were 0.467 ± 0.118 (P<0.001) and 0.318 ± 0.015 (P<0.024) , respectively, the lowest among the groups. The expression of Notch protein, Hes-1 and Runx2 in the control group was higher than that of the three experimental groups, and the expression of Runx2 protein in the DAPT group at 50 μmol/L was 0.116 ± 0.006, which was significantly lower than that in the other groups (P<0.001) .

Conclusions

DMSO concentration of 0.173 25 μL/L had no significant effect on the proliferation and bone differentiation of periodontal ligament stem cells. DAPT may inhibit the proliferation and bone differentiation of hPDLSCs by blocking Notch signaling pathway. Such an inhibitory effect was found to be strongest in the group of 50 μmol/L DAPT.

Key words: Periodontal ligament, Stem cells, Osteoblasts, Notch signal pathway

Shencai Qiu, Xiaowei Dai, Yan Long, Xiaoyan Chen, Peiling Wu. Effects of different concentrations of γ-secretase inhibitor on osteogenic differentiation of human periodontal ligament stem cells[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2018, 12(06): 335-341.

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URL: https://zhkqyxyjzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.1674-1366.2018.06.002

https://zhkqyxyjzz.cma-cmc.com.cn/EN/Y2018/V12/I06/335

Figures/Tables 12

References 18

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基因名称	引物序列
CAP	F：5′-CTGGCTCACCTTCTACGACA-3′
?	R：5′-TACCTCAAGCAAGGCAAATG-3′
OCN	F：5′-CTCACACTCCTCGCCCTATT-3′
?	R：5′-GGCTCCCAGCCATTGATACA-3′
Runx2	F：5′-CGCCTCACAAACAACCACAG-3′
?	R：5′-TCACTGTGCTGAAGAGGCTG-3′
Notch1	F：5′-TGCGAGACCAACATCAACGAGTG-3′
?	R：5′-TCAGGCAGAAGCAGAGGTAGGC-3′
Hes-1	F：5′-ATGACAGTGAAGCACCTCCG-3′
?	R：5′-CGTTCATGCACTCGCTGAAG-3′
GADPH	F：5′-TCAAGGCTGAGAACGGGAAG-3′
?	R：5′-TGGACTCCACGACGTACTCA-3′

基因名称	引物序列
CAP	F：5′-CTGGCTCACCTTCTACGACA-3′
?	R：5′-TACCTCAAGCAAGGCAAATG-3′
OCN	F：5′-CTCACACTCCTCGCCCTATT-3′
?	R：5′-GGCTCCCAGCCATTGATACA-3′
Runx2	F：5′-CGCCTCACAAACAACCACAG-3′
?	R：5′-TCACTGTGCTGAAGAGGCTG-3′
Notch1	F：5′-TGCGAGACCAACATCAACGAGTG-3′
?	R：5′-TCAGGCAGAAGCAGAGGTAGGC-3′
Hes-1	F：5′-ATGACAGTGAAGCACCTCCG-3′
?	R：5′-CGTTCATGCACTCGCTGAAG-3′
GADPH	F：5′-TCAAGGCTGAGAACGGGAAG-3′
?	R：5′-TGGACTCCACGACGTACTCA-3′

组别	第1天	第2天	第3天	第4天	第5天	第6天	第7天
hPDLSC组	0.143 ± 0.041	0.191 ± 0.019	0.236 ± 0.022	0.430 ± 0.028	0.583 ± 0.022	0.749 ± 0.011	0.770 ± 0.019
DMSO组	0.132 ± 0.017	0.183 ± 0.023	0.223 ± 0.021	0.449 ± 0.017	0.600 ± 0.018	0.743 ± 0.021	0.773 ± 0.035
F值	1.183	0.431	0.051	0.208	0.308	1.979	0.001
P值	0.338	0.548	0.832	0.672	0.608	0.232	0.972

组别	第1天	第2天	第3天	第4天	第5天	第6天	第7天
hPDLSC组	0.143 ± 0.041	0.191 ± 0.019	0.236 ± 0.022	0.430 ± 0.028	0.583 ± 0.022	0.749 ± 0.011	0.770 ± 0.019
DMSO组	0.132 ± 0.017	0.183 ± 0.023	0.223 ± 0.021	0.449 ± 0.017	0.600 ± 0.018	0.743 ± 0.021	0.773 ± 0.035
F值	1.183	0.431	0.051	0.208	0.308	1.979	0.001
P值	0.338	0.548	0.832	0.672	0.608	0.232	0.972

组别	第1天	第2天	第3天	第4天	第5天	第6天	第7天
hPDLSC组	0.143 ± 0.041	0.191 ± 0.019	0.236 ± 0.022	0.430 ± 0.028^bcd	0.583 ± 0.022^bcd	0.749 ± 0.011^bcd	0.770 ± 0.019^bcd
DAPT25组	0.145 ± 0.013	0.182 ± 0.014	0.217 ± 0.020	0.378 ± 0.008^acd	0.525 ± 0.020^acd	0.701 ± 0.020^acd	0.711 ± 0.034^acd
DAPT50组	0.145 ± 0.006	0.179 ± 0.015	0.202 ± 0.015	0.279 ± 0.001^abd	0.412 ± 0.026^abd	0.514 ± 0.027^abd	0.534 ± 0.031^abd
DAPT75组	0.143 ± 0.015	0.176 ± 0.020	0.206 ± 0.018	0.331 ± 0.020^abc	0.468 ± 0.009^abc	0.604 ± 0.016^abc	0.633 ± 0.026^abc
F值	0.009	0.382	1.966	40.370	39.665	85.858	39.789
P值	0.999	0.769	0.198	<0.001	<0.001	<0.001	<0.001

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