脐静脉内皮细胞对沉默整合素α6的人牙髓干细胞增殖和干性的影响

doi:10.13591/j.cnki.kqyx.2025.04.002

摘要/Abstract

摘要：

目的探讨人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)对沉默整合素α6(integrin α6,ITGA6)的人牙髓干细胞(human dental pulp stem cells,hDPSCs)增殖和干性的影响。方法慢病毒干扰沉默hDPSCs的ITGA6表达,并验证其沉默效率。实验分为sh-NC、sh-ITGA6、sh-NC+HUVECs、sh-ITGA6+HUVECs四组,sh-NC组、sh-ITGA6组hDPSCs分别转染sh-NC、sh-ITGA6,sh-NC+HUVECs组、sh-ITGA6+HUVECs组分别将转染sh-NC、sh-ITGA6的hDPSCs与HUVECs共培养。采用CCK-8及EdU法检测各组hDPSCs增殖能力,免疫荧光法检测Stro-1表达,Real-time PCR检测各组Oct4与Nanog表达。结果 ①荧光显微镜下可见慢病毒转染效率约为80%;Real-time PCR及Western blot结果显示慢病毒有效干扰hDPSCs的ITGA6表达。②CCK-8结果显示,共培养第5天时,sh-ITGA6+HUVECs组的增殖能力优于sh-ITGA6组(P<0.05);第7天时,共培养组的增殖能力优于单一培养组(P<0.05);EdU结果显示共培养组的hDPSCs DNA合成能力显著优于单一培养组(P<0.05)。③免疫荧光染色显示共培养组的Stro-1表达强于单一培养组(P<0.05)。④Real-time PCR结果显示,共培养组的Oct4表达量高于单一培养组(P<0.05);相比sh-ITGA6组,sh-ITGA6+HUVECs组的Nanog表达上调(P<0.05)。结论 HUVECs可以显著增强沉默整合素α6的hDPSCs的增殖和干性。

关键词: 整合素α6, 人牙髓干细胞, 脐静脉内皮细胞, 增殖, 干性

Abstract:

Objective To investigate the effect of human umbilical vein endothelial cells (HUVECs) on the proliferation and stemness of human dental pulp stem cells (hDPSCs) silencing with integrin α6 (ITGA6). Methods ITGA6 silencing lentivirus was used to interfere the ITGA6 expression of hDPSCs, and its silencing efficiency was detected. Then HUVECs were added to the chambers to co-culture, and the experiments were divided into four groups (sh-NC, sh-ITGA6, sh-NC+HUVECs and sh-ITGA6+HUVECs). hDPSCs in the sh-NC and sh-ITGA6 groups were transfected with sh-NC and sh-ITGA6 respectively. hDPSCs transfected with sh-NC and sh-ITGA6 were co-cultured with HUVECs in the sh-NC+HUVECs group and sh-ITGA6+HUVECs group respectively. The proliferation capacity of hDPSCs of each group was examined by CCK-8 and EdU on day 7. Immunofluorescence detected the expression of Stro-1, and Real-time PCR was used to detect the expression of Oct4 and Nanog. Results ①Fluorescence microscopy showed that the transfection efficiency was about 80%. Real-time PCR and Western blot results showed that sh-ITGA6 lentivirus effectively interfered with ITGA6 expression in hDPSCs. ②CCK-8 results showed that on day 5 of co-culture, the proliferation ability of the sh-ITGA6+HUVECs group was superior to that of the sh-ITGA6 group (P<0.05); on day 7, the proliferation ability of the sh-NC+HUVECs and sh-ITGA6+HUVECs group was superior to that of the sh-NC and sh-ITGA6 group (P<0.05). EdU results showed that the DNA synthesis ability of hDPSCs in the co-culture group was significantly stronger than that in the single-culture group (P<0.05). ③Immunofluorescence staining revealed that the expression of Stro-1 in the co-culture group was significantly stronger than that in the single-culture group. ④Real-time PCR results showed that the expression of Oct4 in the co-culture group was higher than that in the single-culture group (P<0.05); the expression of Nanog in hDPSCs with sh-ITGA6 was elevated by the addition of HUVECs co-culture (P<0.05). Conclusion HUVECs significantly enhance the proliferation and stemness of hDPSCs silencing integrin α6.

Key words: integrin α6, human dental pulp stem cells, umbilical vein endothelial cells, proliferation, stemness

中图分类号:

R783.1

安琪, 张巍巍, 何丽娜, 李艳萍, 潘爽, 牛玉梅. 脐静脉内皮细胞对沉默整合素α6的人牙髓干细胞增殖和干性的影响[J]. 口腔医学, 2025, 45(4): 248-253.

AN Qi, ZHANG Weiwei, HE Lina, LI Yanping, PAN Shuang, NIU Yumei. Effect of HUVECs on proliferation and stemness of hDPSCs silencing integrin α6[J]. Stomatology, 2025, 45(4): 248-253.

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基因	引物序列(5'→3')
Nanog	F:CCAACATCCTGAACCTCAGCTAC R:GCCTTCTGCGTCACACCATT
Oct4	F:GGTGGAGGAAGCTGACAACAATG R:TTGCCTCTCACTCGGTTCTCG
ITGA6	F:GAGCATGATGAAAGTCTCGTTC R:ACTTCATGTCTCTCTTCAGCAA
GAPDH	F:ACACCCACTCCTCCACCTTTG R:TCCACCACCCTGTTGCTGTAG