Abstract
目的
比较强直性脊柱炎患者间充质干细胞（ASMSCs）与健康人间充质干细胞（HDMSCs）自噬水平的差异及其机制。
方法
将ASMSCs和HDMSCs种于6孔板，细胞贴壁后，实验组加入25 ng/mL α-肿瘤坏死因子（TNF-α）及培养液，对照组单纯加入培养液，诱导6 h后进行后续实验。用GFP-LC3B免疫荧光染色检测自噬情况，并通过检测LC3 II/LC3 I及P62的蛋白表达进一步确认。通过qRT-PCR检测基因表达水平，通过Western blot检测蛋白表达水平。使用TG100713特异性阻断PI3K的磷酸化以明确PI3K/AKT/mTOR通路与ASMSCs自噬减弱的关系。
结果
ASMSCs的LC3 II/LC3 I表达水平和GFP-LC3B免疫荧光斑点明显弱于HDMSCs（P < 0.05），而P62表达水平明显高于HDMSCs（P < 0.05）。在自噬过程中，ASMSCs中p-PI3K/PI3K、p-AKT/AKT和p-mTOR/mTOR的表达明显高于HDMSCs（P < 0.05）。阻断PI3K磷酸化后，p-AKT/AKT和p-mTOR/mTOR的表达及ASMSCs的自噬可恢复至HDMSCs的水平。
结论
TNF-α诱导下，PI3K/AKT/mTOR信号通路异常是导致ASMSCs自噬减弱的关键，可能参与AS慢性炎症的发生。
Keywords: 强直性脊柱炎, 间充质干细胞, 肿瘤坏死因子-α, 自噬
Abstract
Objective
To investigate the changes in autophagy of mesenchymal stem cells (MSCs) from patients with ankylosing spondylitis and explore the mechanism for decreased autophagy in ASMSCs.
Methods
MSCs collected from 14 patients with AS (ASMSCs) and from 15 healthy donors (HDMSCs) were cultured in the absence or presence of 25 ng/mL TNF-α for 6 h. Autophagy of the cells was determined by immunofluorescence staining of GFP-LC3B, and the results were confirmed by detecting the protein expressions of autophagy markers LC3 II/LC3 I and P62. The mRNA expressions of the related genes were detected using qRT-PCR, and the protein expressions of the autophagy markers and signaling pathway-related molecules were determined with Western blotting. TG100713 was used to block the PI3K/AKT/mTOR signal pathway, and its effect on autophagy of ASMSCs was evaluated.
Results
ASMSCs showed significantly weaker GFP-LC3B puncta staining and lower protein expression levels of LC3 II/LC3 I but higher levels of P62 protein (P < 0.05), indicating a decreased autophagy capacity as compared with HDMSCs. TNF-α-induced ASMSCs showed significantly higher protein expressions of p-PI3K/ PI3K, p-AKT/AKT and p-mTOR/mTOR than HDMSCs (P < 0.05), suggesting hyperactivation of the PI3K/AKT/mTOR signaling pathway in ASMSCs. Blocking PI3K/AKT/mTOR signaling with TG100713 eliminated the difference in TNF-α-induced autophagy between HDMSCs and ASMSCs.
Conclusion
In patients with AS, hyperactivation of the PI3K/AKT/mTOR signaling pathway results in decreased autophagy of the MSCs and potentially contributes to chronic inflammation.
Keywords: ankylosing spondylitis, mesenchymal stem cells, tumor necrosis factor-α, autophagy
强直性脊柱炎（AS）是一种常见的自身免疫性疾病，我国AS的患病率高达0.2%~0.54%[1, 2]。AS以炎性腰背痛及脊柱关节僵硬为主要临床表现，后期可导致患者丧失劳动能力及生活能力，严重影响患者生活质量[3]。但是，AS的发病机制尚不明确，给临床诊疗带来了极大的困难。
间充质干细胞（MSCs）是一种具有强大免疫调节能力和多向分化潜能的多能干细胞[4]。MSCs既能通过影响体内的免疫调节能力参与炎症反应，又能通过自身的成骨分化能力参与成骨的发生，与AS的发病密切相关[5]。AS患者MSCs成骨分化功能增强而抑制破骨分化的能力减弱，是导致AS异常成骨的关键因素之一[6, 7]。因此，深入研究ASMSCs的功能有助于明确AS的发病机制。肿瘤坏死因子-α（TNF-α）是AS患者体内最重的炎症因子之一，不但参与炎症反应，还可诱导细胞凋亡、自噬，在AS的发病中扮演着重要的角色[8]。有研究进一步指出，TNF-α可通过调控MSCs的功能参与AS的发病[5]，但具体的调控机制不明。
自噬是细胞内的一种自食现象，具有调节细胞内环境稳态及免疫平衡的作用[9]。研究表明，细胞自噬水平与体内炎症反应及骨代谢密切相关，而MSCs自噬失调与多种自身免疫性疾病的发病密切相关[10, 11]。那么，AS患者MSCs（ASMSCs）是否存在自噬失调并参与AS的发病呢？目前尚不明确。
本研究拟探讨TNF-α刺激下，ASMSCs与正常人MSCs（HDMSCs）的自噬水平是否存在差异并探究其内在机制，为AS的发病机制及治疗提供新的思路和方法。
1. 资料和方法
1.1. 病例纳入
选取2017年7月~2020年7月在我院诊断为AS的患者共14例（参照1984年纽约修订版AS诊断标准进行诊断[12]），健康对照组共15例。所有患者均同意参加实验，并签署知情同意书，伦理证明号（LAEC- 2021-174）。两组入选病例一般情况见表 1。
表 1.
两组一般情况比较
Baseline characteristics of the study subjects[Mean±SD, n(%)]
| Items | Healthy donors | AS patients |
| AS: Ankylosing spondylitis; HLA-B27: Human leukocyte antigen B27; CRP: C-reactive protein; ESR: Erythrocyte sedimentation rate; BASDAI: The bath ankylosing spondylitis disease activity index. *P < 0.05 compared to healthy donors. | ||
| Number | 15 | 14 |
| Age (year) | 28.3±9.4 | 29.7±10.5 |
| Male no. (%) | 11 (73.3%) | 11 (78.6%) |
| HLA-B27 positive no. (%) | 0 | 12 (85.7%)* |
| Disease duration (year) | - | 4.36±3.5 |
| CRP (mg/L) | 4.2±1.8 | 24.1±12.7* |
| ESR (mm/h) | 8.9±4.4 | 31.5±16.8* |
| BASDAI | 0.98±0.81 | 5.13±1.52* |
1.2. MSCs的分离、培养和鉴定
按既往报道的方法，经髂后上棘穿刺抽取患者及健康志愿者骨髓，用密度梯度离心法分离MSCs[13]。用含10%胎牛血清（四季青）的DMEM培养液（Gibco）于25 cm2的培养瓶中培养，贴壁后定期换液、传代，将第4代细胞用于实验。用流式细胞学技术检测细胞的表面抗体[CD14-APC、CD29-PE、CD44-FITC、CD45-APC、CD105-FITC和HLA DR（BD）]。
1.3. 细胞增殖能力检测
将健康志愿者和患者的第4代MSCs种于96孔板上（5×104/孔），用含10%胎牛血清的DMEM培养，将无细胞孔作为阴性对照。采用CCK-8试剂盒（Dojindo）检测不同时间点HDMSCs和ASMSCs的增殖情况。
1.4. TNF-α诱导自噬
将HDMSCs及ASMSCs分别种于6孔板（1×106/孔），贴壁培养24 h后，实验组加入TNF- α（Sigma Aldrich，25 ng/mL）及培养液，对照组单纯加入培养液，诱导6 h后进行后续实验。
1.5. 定量PCR（qRT-PCR）
先用TRIzol（Invitrogen）按步骤提取RNA，接着用PrimeScriptTM RT Master Mix(Takara)试剂盒将RNA转录为cDNA。按步骤设计并合成引物（Invitrogen）（引物序列详见表 2），在LightCycler®480 PCR System (Roche)上进行qRT-PCR实验。选取GAPDH作为内参，目标基因Ct值以GAPDH的Ct值标准化，用2–ΔCt的方法计算基因的相对表达量。qRT-PCR所用引物见表 2。
表 2.
qRT-PCR引物序列
Primers used for qRT-PCR
| Gene | Forward primer (5'-3') | Reverse primer (5'-3') | |
| GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; PI3K: Phosphatidylinositol 3-kinase; AKT: Serine/threonine kinase; mTOR: Mammalian target of rapamycin kinase. | |||
| GAPDH | GGAGCGAGATCCCTCCAAAAT | GGCTGTTGTCATACTTCTCATGG | |
| PI3K | TCTGATGGATATTCCCGAAAGCC | CTCACCCACTGGAAGTTTTTGAT | |
| AKT | GGACAACCGCCATCCAGACT | GCCAGGGACACCTCCATCTC | |
| mTOR | CCTCCATCCACCTCATCA | GACGCCAAGACACAGTAG | |
1.6. Western blot
按步骤提取蛋白并依次定量、变性，将变性后的蛋白与SDS混合物进行电泳并转至PVDF膜（Millipore）上。将PVDF膜洗涤后封闭1 h，4 ℃下孵育一抗过夜（GAPDH、LC3B、P62、p-PI3K、PI3K、p-AKT、AKT、pmTOR、mTOR，CST），洗膜后室温孵育二抗（Santa Cruz）1.5 h。再次洗膜后加入显影液（Millipore），放入Chem Studio PLUS Motorized成像系统曝光。Image J软件测量目标蛋白灰度值，将GAPDH设为内参，目标蛋白的相对表达量=目标蛋白灰度值/GAPDH灰度值。
1.7. 绿色荧光蛋白和轻链3B融合蛋白（GFP-LC3B）分析
将MSCs种于6孔板，贴壁培养24 h后，将含有绿色荧光蛋白轻链3B的慢病毒载体（GFP LC3B，吉玛基因）加入MSCs中。再次培养24 h后，更换培养液，加入TNF-α诱导，以不加入TNF-α诱导孔为对照。转染72 h后，通过荧光显微镜观察诱导前后MSCs中GFP-LC3B的表达。自噬越强，细胞中GFP-LC3B荧光点更多更亮。用Image Pro软件计算GFP-LC3B荧光点的数量。
1.8. 统计学分析
采用SPSS 22.0统计软件进行统计学分析。两组之间比较采用独立样本t检验，多组之间比较用方差分析。数据以均数±标准差来表示，P < 0.05认为差异有统计学意义。
2. 结果
2.1. ASMSCs与HDMSCs具有相同的形态、表型和增殖能力
在显微镜下，HDMSCs与ASMSCs均为长梭形贴壁生长的细胞。HDMSCs与ASMSCs均持续表达CD29、CD44和CD105，但不表达CD14、CD45和HLA-DR。在1~7 d的培养过程中，HDMSCs与ASMSCs增殖能力无明显差异（图 1）。
图 1.
ASMSCs与HDMSCs具有相同的形态、表型和增殖能力
ASMSCs and HDMSCs have identical morphology, phenotype and proliferation rate. A: Both HDMSCs and ASMSCs are plastic-adherent, spindle-shaped cells. B: The cells express CD29, CD44, and CD105 but not CD14, CD45, or HLA-DR. C: HDMSCs and ASMSCs have similar proliferation rate when cultured in DMEM with 10% FBS for 1-7 days.
2.2. TNF-α诱导下，ASMSCs自噬水平低于HDMSCs TNF-α诱导6 h后，ASMSCs中LC3 II/LC3 I的蛋
白表达水平明显低于HDMSCs，而P62的表达水平明显高于HDMSCs（图 2）。进一步通过荧光显微镜观察，我们发现自噬诱导后ASMSCs的GFP-LC3B荧光斑点明显弱于HDMSCs（图 2）。
图 2.
TNF-α诱导下，ASMSCs自噬水平低于HDMSCs
ASMSCs show decreased autophagy after treatment with TNF-α. ASMSCs have a lower LC3 II/LC3 I protein expression ratio but a higher P62 protein expression than HDMSCs. Fluorescence microscopy further reveals a significantly weaker and more diffuse GFP-LC3 puncta staining in ASMSCs than in HDMSCs. *P < 0.05.
2.3. ASMSCs自噬过程中PI3K/AKT/mTOR信号通道异常激活
尽管自噬过程中ASMSCs与HDMSCs的PI3K、AKT及mTOR基因表达水平无明显差异（图 3A），但ASMSCs的p-PI3K/PI3K、p-AKT/AKT及p-mTOR/ mTOR蛋白表达水平明显高于HDMSCs（图 3B）。
图 3.
ASMSCs自噬过程中PI3K/AKT/mTOR信号通道异常激活
The PI3K/Akt-mTOR signaling pathway is hyperactivated in ASMSCs during TNF-α-induced autophagy. A: Expressions of PI3K, AKT and mTOR mRNA are comparable between HDMSCs and ASMSCs. B: Protein expressions of p-PI3K/PI3K, p-AKT/ AKT and p-mTOR/mTOR are significantly higher in ASMSCs than in HDMSCs. *P < 0.05.
2.4. 阻断PI3K/AKT/mTOR信号通路可恢复ASMSCs自噬水平
400 nmol/L的TG100713可有效阻断PI3K的磷酸化（图 4A）。阻断PI3K磷酸化后，ASMSCs中p-AKT/ AKT、p-mTOR/mTOR、LC3 II/LC3 I及P62的表达恢复至HDMSCs水平，而GFP- LC3B荧光斑点强度也与HDMSCs相当（图 4B）。
图 4.
阻断PI3K/AKT/mTOR信号通路可将ASMSCs自噬水平恢复至HDMSCs的水平
Blocking PI3K/AKT/mTOR signaling pathway eliminates the difference in autophagy between ASMSCs and HDMSCs. A: TG100713 (400 nmol/L) effectively eliminates the difference in p-PI3K/PI3K expression. B: TG100713 eliminates the differences in the puncta staining (GFP-LC3B) and protein expressions (p-AKT/AKT, p-mTOR/mTOR, LC3 II/LC3 I and P62) between HDMSCs and ASMSCs. *P < 0.05 vs healthy donors.
3. 讨论
TNF-α是一种多功能细胞因子，广泛参与体内的病理生理过程，如：增殖、分化、凋亡、自噬等[14]。研究表明，TNF-α与AS的发病密切相关。首先，TNF-α在-238，-308，-850，-1031和rs769178位点上的基因多态性与AS的易感性密切相关[15]；其次，AS患者血清及骶髂关节中TNF-α水平明显升高[16, 17]，且血清中TNF-α水平与AS疾病活动度成正相关[18]；此外，TNF-α抑制剂可有效治疗AS[19]。但是，TNF-α参与AS发病的具体机制尚未完全明确。最近多项研究进一步指出，TNF-α可通过调控MSC的功能参与AS的发生发展。TNF-α介导ELMO1的m6A甲基化修饰可诱导AS患者MSC定向迁移[20]；TNF-α可通过自噬调控MSC凋亡，影响MSC的免疫调节能力[21]。我们前期研究表明，激活自噬可提高MSC的抑炎能力，反之亦然[22]。因此，我们推测TNF-α可能通过调控MSC自噬参与AS的发病。
细胞自噬与内环境稳态密切相关，自噬水平异常可诱发炎症反应并参与自身免疫性疾病的发病[10]。B细胞自噬增多而T细胞自噬减少与系统性红斑狼疮体内慢性炎症密切相关[23, 24]；滑膜成纤维细胞自噬增多及成骨细胞/破骨细胞自噬失调参与了类风湿性关节炎的发生发展[25]；肌成纤维细胞自噬水平下降是系统性硬化症的重要发病机制[26]。在本研究中，我们发现TNF-α诱导下ASMSCs的自噬水平明显低于HDMSCs。ASMSCs自噬水平下降可导致其抑炎能力降低、体内炎症水平升高，这可能是AS慢性炎症的发病机制之一。
细胞自噬是一个复杂的病理生理过程，多种细胞因子及信号通路参与其中，PI3K/AKT/mTOR信号通路是其中最重要的调控通路之一[27, 28]。PI3K/AKT/mTOR信号通路激活后，既可直接抑制自噬，又可通过调控P70S6K、AMBRA1、ULK1和BECLIN1等因子的表达抑制自噬小体的形成，负向调控细胞自噬[29, 30]。本研究结果显示，在ASMSCs自噬过程中，PI3K/AKT/mTOR表达明显升高；抑制PI3K磷酸化后，AKT及mTOR的表达及ASMSCs自噬均恢复至HDMSCs水平。说明PI3K/AKT/mTOR信号通路异常激活是导致ASMSCs自噬减弱的关键机制。
本研究中，我们发现TNF-α诱导下，PI3K/AKT/ mTOR信号通路异常激活是导致ASMSCs自噬减弱的关键。ASMSCs自噬减弱可导致其抑炎能力下降并诱发或加重AS患者体内慢性炎症。但是，PI3K/AKT/ mTOR信号通路异常激活的关键分子究竟是什么？在AS患者体内是否同样存在这种自噬减弱的情况？上述问题仍有待进一步研究。
Biography
刘振华，博士，主治医师，E-mail: 179474969@qq.com
Funding Statement
广东省自然科学基金（2018A0303130258）
Contributor Information
刘 振华 (Zhenhua LIU), Email: 179474969@qq.com.
邱 素均 (Sujun QIU), Email: 124131852@qq.com.
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