过表达甲基转移酶样3修复炎症来源牙周膜干细胞的成骨能力

目的探讨N⁶-甲基腺嘌呤（m⁶A）修饰与甲基转移酶样3（METTL3）在牙周膜干细胞（PDLSC）成骨分化和成脂分化中的作用。 方法使用流式细胞术、细胞集落形成实验分别鉴定PDLSC的表面标志物和增殖能力。通过茜素红染色和油红O染色分别检测PDLSC的成骨和成脂分化潜能。在人牙周膜干细胞（hPDLSC）和炎症来源牙周膜干细胞（pPDLSC）中分别构建METTL3过表达和敲低模型，成骨诱导一定时间，通过实时定量反转录聚合酶链反应（RT-PCR）、蛋白免疫印迹（Western blot）、茜素红染色和油红O染色分别在mRNA水平、蛋白水平和宏观水平检测成骨和成脂的变化。两样本比较使用独立样本t检验，多组样本比较使用单因素方差分析。 结果（1）流式细胞术结果显示，hPDLSC和pPDLSC均阳性表达CD29（100.0%，98.0%）、CD105（100.0%，99.5%）和CD146（31.5%，17.8%），阴性表达CD34（1.3%，0.4%）和CD45（1.4%，0.4%）。（2）细胞集落形成实验结果显示，hPDLSC和pPDLSC的集落形成数量分别为55±5和72±8，hPDLSC的细胞增殖能力较pPDLSC低（t = 3.16，P = 0.034）。（3）茜素红染色和油红O染色说明，两种细胞均具有成骨和成脂分化能力，hPDLSC具有更强的成骨分化能力（t = 27.77，P<0.001），而pPDLSC具有更强的成脂分化能力（t = 5.02，P = 0.007）。（4）成骨诱导培养7 d后的慢病毒转染模型中，METTL3过表达组比过表达对照组的成骨关键基因Runx2的mRNA表达水平高，在hPDLSC和pPDLSC中的METTL3过表达组的表达量是3.63 ± 1.15和1.61 ± 0.38，分别是其过表达对照组的3.39倍（t = 3.777，P = 0.020）和1.71倍（t = 2.948，P = 0.042）；而在METTL3敲低组较敲低对照组低，在hPDLSC和pPDLSC中的METTL3敲低组的表达量是0.16 ± 0.03和0.26 ± 0.07，分别是其敲低对照组的0.15倍（t = 9.669，P<0.001）和0.26倍（t = 8.767，P<0.001）。同样地，Runx2的蛋白表达水平也发生相同改变。将转染后的细胞进行21 d的成骨诱导培养后进行茜素红染色，结果显示METTL3过表达组较过表达对照组染色深且钙化结节较大，定量分析结果显示在hPDLSC和pPDLSC中的METTL3过表达组是28.47% ± 3.82%和8.55% ± 0.43%，分别是其过表达对照组的1.78倍（t = 5.012，P = 0.007）和1.76倍（t = 7.293，P = 0.002），而在METTL3敲低组较敲低对照组染色浅且钙化结节较小，定量分析结果显示在hPDLSC和pPDLSC中的METTL3敲低组是6.36% ± 2.00%和3.78% ± 0.56%，分别是其敲低对照组的0.35倍（t = 4.444，P = 0.011）和0.43倍（t = 5.337，P = 0.006）；将转染后的细胞进行21 d的成脂诱导培养，再进行油红O染色，结果显示脂滴的大小及数量在METTL3过表达组较过表达对照组少，定量分析结果显示在hPDLSC和pPDLSC中的METTL3过表达组是0.89% ± 0.11%和1.10% ± 1.15%，分别是其过表达对照组的0.24倍（t = 5.454，P = 0.006）和0.49倍（t = 2.935，P = 0.043），而在METTL3敲低组则比敲低对照组的脂滴更大且多，定量分析结果显示在hPDLSC和pPDLSC中的METTL3敲低组是3.60% ± 1.08%和5.34% ± 1.31%，分别是其敲低对照组的1.94倍（t = 2.794，P = 0.049）和2.93倍（t = 4.131，P = 0.015）。 结论pPDLSC的METTL3表达水平低于hPDLSC；METTL3的过表达可促进hPDLSC和pPDLSC的成骨分化，并抑制两者的成脂分化。

Overexpression of methyltransferase-like 3 repairs the osteogenic ability of periodontal mesenchymal stem cells in patients with periodontitis

ObjectiveTo investigate the role of methyltransferase-like 3 (METTL3) in osteogenic and adipogenic differentiation of periodontal mesenchymal stem cells (PDLSCs) . MethodsFlow cytometry and colony formation assay were used to identify the surface markers and proliferation ability of PDLSCs. The osteogenic and adipogenic differentiation potential of PDLSCs was detected by alizarin red staining and oil red O staining, respectively. Human periodontal mesenchymal stem cells (hPDLSCs) and periodontal mesenchymal stem cells in patients with periodontitis (pPDLSCs) were used to construct METTL3 overexpression and knockdown models, respectively. The changes of osteogenesis and adipogenesis were detected by RT-PCR, Western blot, alizarin red staining and oil red O staining at mRNA level, protein level and macroscopic level, respectively. Two samples were compared using independent sample t test, and multiple groups were compared using One-Way ANOVA. ResultsFlow cytometry showed that hPDLSCs and pPDLSCs were positive for CD29 (100.0%, 98.0%), CD105 (100.0%, 99.5%) and CD146 (31.5%, 17.8%), and negative for CD34 (1.3%, 0.4%) and CD45 (1.4%, 0.4%). The results of colony formation experiment showed that the number of colonies formed of hPDLSCs and pPDLSCs was 55 ± 5 and 72 ± 8, respectively, and the cell proliferation ability of hPDLSCs was lower than that of pPDLSCs (t = 3.16, P = 0.034). Alizarin red staining and oil red O staining showed that both cells had osteogenic and adipogenic differentiation ability. hPDLSCs had stronger osteogenic differentiation ability (t = 27.77, P<0.001), while pPDLSCs had stronger adipogenic differentiation ability (t = 5.02, P = 0.007). In the lentivirus transfection model, after 7 days of osteogenic induction culture, the mRNA expression level of Runx2 in the METTL3 overexpression group was higher than that in the overexpression control group. The expression level of Runx2 in the METTL3 overexpression group was 3.63 ± 1.15 and 1.61 ± 0.38 for hPDLSCs and pPDLSCs, respectively, which was 3.39 times (t = 3.777, P = 0.020) and 1.71 times (t = 2.948, P = 0.042) of the control group. The expression level of Runx2 in the METTL3 knockdown group was 0.16 ± 0.03 and 0.26 ± 0.07 for hPDLSCs and pPDLSCs, respectively, which was 0.15 times (t = 9.669, P<0.001) and 0.26 times (t = 8.767, P<0.001) of the control group. Runx2 protein expression level changed in the same way. After 21 days of osteogenic induction culture, the transfected cells were stained with alizarin red. The results showed that the METTL3 overexpression group had deeper staining and larger calcified nodules than the overexpression control group. The quantitative analysis results showed that the values of METTL3 overexpression group in hPDLSCs and pPDLSCs were 28.47% ± 3.82% and 8.55% ± 0.43%, which were 1.78 times (t = 5.012, P = 0.007) and 1.76 times (t = 7.293, P = 0.002) of the METTL3 overexpression control group. The staining was lighter and the calcified nodules were smaller in the knockdown group. The results of quantitative analysis showed that Runx2 protein expression level in the METTL3 knockdown group was 6.36% ± 2.00% and 3.78% ± 0.56% for hPDLSCs and pPDLSCs, respectively, which was 0.35 times (t = 4.444, P = 0.011) and 0.43 times (t = 5.337, P = 0.006) of the knockdown control group. The transfected cells were cultured for lipid induction for 21 days, and then stained with oil red O. The results showed that the size and number of lipid droplets in the METTL3 overexpression group were less than that in the overexpression control group. The quantitative analysis results showed that the values of lipid droplets in the METTL3 overexpression group in hPDLSCs and pPDLSCs were 0.89% ± 0.11% and 1.10% ± 1.15%, which were 0.24 times (t = 5.454, P = 0.006) and 0.49 times (t = 2.935, P = 0.043) of the control. The lipid droplets in the METTL3 knockdown group were larger and more than those in the knockdown control group. The quantitative analysis results showed that the values of lipid droplets in the METTL3 knockdown group in hPDLSCs and pPDLSCs were 3.60% ± 1.08% and 5.34% ± 1.31%, which were 1.94 times (t = 2.794, P = 0.049) and 2.93 times (t = 4.131, P = 0.015) of the control group, respectively. ConclusionsThe METTL3 expression level in pPDLSCs was lower than that in hPDLSCs. Overexpression of METTL3 can promote the osteogenic differentiation of hPDLSCs and pPDLSCs, and inhibit the adipogenic differentiation.