Chen Xiyan, Wang Qi, Gu Ke, Li Aonan, Fu Xucheng, Wang Ying, Gu Weiting, Wen Yong
Shandong Provincial Key Laboratory of Oral Tissue Regeneration, School of Stomatology, Shandong University, Jinan, Shandong Province, China.
School of Stomatology, Shandong University, Jinan, Shandong Province, China.
Stem Cells Int. 2019 Apr 1;2019:6804036. doi: 10.1155/2019/6804036. eCollection 2019.
To establish an immortalized human periodontal ligament stem cell line (hPDLSC) and investigate whether and how YAP mediates the establishment of the stem cell line.
Primary hPDLSCs were cultured and transfected with lentivirus containing the telomerase reverse transcriptase (TERT) gene. The expression of TERT was detected via the polymerase chain reaction (PCR) and real-time quantitative PCR (RT-PCR). Flow cytometry was employed to detect surface markers of hPDLSCs and TERT-hPDLSCs. The cell counting kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) methods were used to examine the proliferation ability of the cells. Flow cytometry and TUNEL staining were employed to examine the cell apoptosis rate. The -galactosidase staining assay was used to assess the rate of cell senescence. The osteogenic differentiation ability of the cells was detected via alkaline phosphatase (ALP) staining and Alizarin red staining assays. BALB/c mice were employed to determine the tumorigenicity of TERT-hPDLSCs. The expression levels of YAP and other proteins in the Hippo signaling pathway were detected by Western blotting. Verteporfin was used to inhibit the binding of YAP to the downstream target gene TEAD.
TERT-hPDLSCs showed stable high expression of TERT, even at the thirtieth passage after transfection with lentivirus containing the TERT gene. Compared with primary hPDLSCs, TERT-hPDLSCs exhibited a stronger proliferation ability and lower cell apoptosis and senescence rates while maintaining the same osteogenetic differentiation ability as primary hPDLSCs. The transfection of hPDLSCs with lentivirus containing the TERT gene did not lead to tumorigenesis in nude mice. The Hippo signaling pathway was inactivated in TERT-hPDLSCs compared to hPDLSCs. When treated with verteporfin, the proliferation of TERT-hPDLSCs decreased, while the apoptosis and senescence rates of these cells increased. However, TERT-hPDLSCs still showed a stronger proliferation ability and lower cell apoptosis and senescence rates than hPDLSCs treated with verteporfin at the same concentration.
Overexpression of TERT in hPDLSCs resulted in the successful establishment of an immortalized periodontal ligament stem cell line. TERT may regulate the biological characteristics of hPDLSCs through the Hippo/YAP signaling pathway. hPDLSCs could be a feasible resource for stem cell research and a promising resource for stem cell therapy.
建立永生化人牙周膜干细胞系（hPDLSC），并研究Yes相关蛋白（YAP）是否以及如何介导该干细胞系的建立。
培养原代hPDLSCs，并用含端粒酶逆转录酶（TERT）基因的慢病毒进行转染。通过聚合酶链反应（PCR）和实时定量PCR（RT-PCR）检测TERT的表达。采用流式细胞术检测hPDLSCs和TERT-hPDLSCs的表面标志物。使用细胞计数试剂盒-8（CCK-8）和5-乙炔基-2'-脱氧尿苷（EdU）方法检测细胞的增殖能力。采用流式细胞术和TUNEL染色检测细胞凋亡率。用β-半乳糖苷酶染色法评估细胞衰老率。通过碱性磷酸酶（ALP）染色和茜素红染色试验检测细胞的成骨分化能力。采用BALB/c小鼠确定TERT-hPDLSCs的致瘤性。通过蛋白质免疫印迹法检测Hippo信号通路中YAP和其他蛋白的表达水平。使用维替泊芬抑制YAP与下游靶基因TEAD的结合。
TERT-hPDLSCs即使在转染含TERT基因的慢病毒后第30代仍显示TERT稳定高表达。与原代hPDLSCs相比，TERT-hPDLSCs表现出更强的增殖能力、更低的细胞凋亡率和衰老率，同时保持与原代hPDLSCs相同的成骨分化能力。用含TERT基因的慢病毒转染hPDLSCs在裸鼠中未导致肿瘤发生。与hPDLSCs相比，TERT-hPDLSCs中的Hippo信号通路失活。用维替泊芬处理时，TERT-hPDLSCs的增殖减少，而这些细胞的凋亡率和衰老率增加。然而，TERT-hPDLSCs与相同浓度维替泊芬处理的hPDLSCs相比，仍表现出更强的增殖能力和更低的细胞凋亡率及衰老率。
hPDLSCs中TERT的过表达成功建立了永生化牙周膜干细胞系。TERT可能通过Hippo/YAP信号通路调节hPDLSCs的生物学特性。hPDLSCs可能是干细胞研究的可行资源和干细胞治疗的有前景资源。
