神经肽P物质对人牙髓干细胞生物学行为的影响

目的探讨神经肽P物质对人牙髓干细胞（hDPSC）生物学行为的影响。 方法组织块法分离、培养hDPSC，通过流式细胞术和茜素红染色鉴定hDPSC。用不同浓度的神经肽P物质0（对照组）、0.1 nmol/L、10 nmol/L、1 μmol/L、10 μmol/L]干预hDPSC，在第24、48、72和96 h通过细胞计数试剂盒8（CCK-8）法检测细胞增殖能力；在48 h时通过Transwell实验检测细胞迁移能力。成血管诱导条件下干预14 d后通过小管形成实验检测细胞成管情况；通过实时荧光定量反转录聚合酶链反应（RT-PCR）检测成管相关基因血管内皮生长因子受体2（VEGFR2）和血管内皮生长因子（VEGF）的表达情况。采用GraphPad Prism 8软件对数据进行统计学分析。 结果成功分离、培养hDPSC。流式细胞术结果显示，细胞表面标记物CD29、CD90、CD34和CD45表达率分别为99.90%、99.90%、0.40%和0.04%；茜素红结果显示，成骨诱导组形成较明显的矿化结节。CCK-8结果显示，对照组在48、72、96 h的相对增值率分别为（100.0 ± 0.4）%、（100.0 ± 0.7）%和（100.0 ± 1.9）%，实验组在48 h时开始促进细胞增殖，72、96 h各浓度实验组均显著促进hDPSC的增殖，差异具有统计学意义（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），最佳浓度10 μmol/L时，48、72和96 h的细胞增殖率分别为（119.0 ± 1.0）%、（148.4 ± 3.5）%和（140.8 ± 1.0）%；Transwell结果显示，与对照组穿膜数量（5.0 ± 1.4）相比，实验组均对hDPSC的迁移具有促进作用（F = 310.3，P<0.001），最佳效应浓度为10 nmol/L，穿膜数量为（87.6 ± 8.3）；成血管诱导14 d后，小管形成实验结果显示，各浓度的P物质实验组与对照组相比均可促进hDPSC小管形成情况（F_{小管分支点数} = 43.7，P_{小管分支点数}<0.001；F_{相对小管长度} = 19.4，P_{相对小管长度} = 0.0001）；RT-PCR结果显示，0、0.1 nmol/L、10 nmol/L、1 μmol/L、10 μmol/L实验组VEGFR2的相对表达量分别为（1.000 ± 0.023）、（1.129 ± 0.076）、（1.474 ± 0.101）、（1.285 ± 0.055）和（1.213 ± 0.160）；VEGF的相对表达量分别为（1.000 ± 0.026）、（1.211 ± 0.023）、（1.242 ± 0.070）、（1.679 ± 0.043）和（1.138 ± 0.156），P物质组与对照组相比，差异均有统计学意义（F_VEGFR2 = 10.43，P_VEGFR2 = 0.001 4；F_VEGF = 29.87，P_VEGF<0.001）。 结论P物质对hDPSC的增殖、迁移和成血管能力具有一定促进作用。

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

ObjectiveTo investigate the effects of neuropeptide substance P on the biological behavior of human dental pulp stem cells (hDPSCs) . MethodshDPSCs 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. ResultshDPSCs 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) . ConclusionSubstance P may have certain promoting effect on the proliferation, migration and angiogenesis of hDPSCs.