研究背景：

骨关节炎（Osteoarthritis, OA）是一种最常见的慢性退行性关节疾病，可以导致老年人关节疼痛、活动能力丧失，甚至使老年人残疾，造成重大的社会经济负担。细胞外基质（ECM）稳态的破坏、软骨细胞代谢异常、衰老软骨细胞数量增加以及软骨细胞凋亡等是 OA 发生的关键步骤。Sirtuins（SIRTs）是一组烟酰胺腺嘌呤二核苷酸依赖性组蛋白脱乙酰酶，可以使某些蛋白质脱乙酰，在细胞生理和病理过程的调节中发挥关键作用。据报道，SIRTs 家族通过维持 ECM 稳态、调节软骨细胞代谢、防止软骨细胞衰老和减少软骨细胞凋亡来维持软骨细胞功能。SIRT6 是 SIRTs 家族的成员，有研究表明，人类软骨细胞中 SIRT6 的缺失会导致 DNA 损伤增加和端粒功能障碍，以及随后的过早衰老， SIRT6 的过度表达可以通过减少炎症反应和软骨细胞衰老来预防 OA 的发展。

OA 现有的治疗手段主要聚焦于缓解症状和改善关节功能，如非甾体抗炎药（Non steroidal anti-inflammatory drugs, NSAIDs）、物理治疗以及针对晚期 OA 的关节置换手术等，但这些方法均不能阻止疾病进展或修复已损伤的关节软骨，从根本上实现对 OA 的治愈。多项临床试验研究证实，间充质干细胞（Mesenchymal stem cells, MSCs）治疗 OA 疗效显著，甚至被认为是最具潜力的临床根治 OA 的疗法。然而临床上使用 MSCs 治疗 OA 仍存在诸多挑战，比如移植细胞的成瘤性与致瘤性、保存时间短等限制了临床使用。MSCs 主要是通过旁分泌作用发挥治疗作用，即通过 MSCs 分泌的细胞外囊泡（Extracellular vesicles, EVs）发挥作用。与 MSCs 相比，EVs 具有安全性更好、工业化制备更容易、可以长期储存等优点。因此，EVs 被认为可代替 MSCs 用于临床治疗。由于 EVs 产能及功能性与来源细胞相关，同一供体来源的 MSCs 数量有限从而导致分泌的 EVs 数量不能满足大规模临床试验研究，而不同供体 MSCs 分泌的 EVs 的质量存在差异，导致临床治疗效果不稳定。为了解决以上难题，本课题对人脐带间充质干细胞（human Umbilical Cord Mesenchymal Stem Cells，hUCMSCs）进行永生化处理，以大批量获取同一脐带来源的 hUCMSCs，从而大批量制备同一批次的 EVs，解决临床研究时需要质量稳定的大批量 EVs 的问题。为了进一步提高 EVs 治疗 OA 的功能性，希望通过工程化改造的可以获取更强治疗 OA 效果的 EVs。

研究目的：

本课题拟使用慢病毒感染基因修饰方法对 hUCMSCs 进行工程化改造，制备永生化过表达 SIRT6 的特异性 hUCMSCs（S6-hUCMSCs）及其 EVs（S6-EVs），以解决传统 EVs 由于同一供体来源种子细胞数量有限而导致获得量不足以及需要提高治疗 OA 疗效的问题，并通过 OA 小鼠体内实验与软骨细胞 OA 模型体外实验，对比 EVs/S6-EVs 对 OA 的治疗效果，初步探索其作用机制，希望为进一步探索 OA 的临床治疗及相关产品开发提供新思路。

研究方法：

1. 构建与检测 S6-hUCMSCs

从脐带组织中分离华通氏胶，进行组织块贴壁培养 9～13 天，分离获取原代 hUCMSCs，通过表达 TERT 基因的 puro 抗性慢病毒感染，药物筛选获取永生化 hUCMSCs（TERT-hUCMSCs）。然后通过表达 SIRT 基因的灭瘟素 S 抗性慢病毒再次感染筛选，获取过表达 SIRT6 永生化 hUCMSCs（S6-hUCMSCs）。并对 S6-hUCMSCs 进行鉴定，包括细胞标志物的、多向分化能力、TERT 和 SIRT6 mRNA 的表达水平、SIRT6 蛋白的表达水平、端粒酶长度、细胞累积增殖能力等检测。

2. 分离获取与检测 S6-EVs

通过差速超速离心法从 S6-hUCMSCs 培养上清中分离获取 S6-EVs，并采用粒径分析仪（NTA）、透射电镜（TEM）检测其形状、粒径大小及形态。采用 WB 鉴定其标志物及 SIRT6 蛋白的表达情况。

3. 荧光标记 S6-EVs 检测体内存留时间

使用 DIR 染料标记 S6-EVs，注射到小鼠膝关节腔内，通过活体成像仪每天观察荧光强度。

4. 对比研究 S6-EVs/EVs 对小鼠膝关节 OA 模型的治疗作用

通过前交叉韧带横断术（Anterior cruciate ligament transection, ACLT）构建小鼠膝关节 OA 模型，手术 1 周后，每周1次关节腔内注射 S6-EVs/EVs，共注射 6 次。取材小鼠膝关节进行 HE、番红-O 固绿染色，并进行免疫组织化学染色鉴定软骨细胞表达炎症因子、合成代谢相关因子、SIRT6 的表达情况，评价 EVs/S6-EVs 治疗小鼠 OA 的效果。

5. 对比研究 S6-EVs/EVs 对软骨细胞的生物学行为的影响

通过划痕实验检测 S6-EVs/EVs 对软骨细胞迁移的影响，CCK-8/Edu 检测 S6-EVs/EVs 对软骨细胞增殖的作用，使用 Annexin V PE/7-AAD 染色研究 S6-EVs/EVs 对软骨细胞凋亡的作用。

6. 对比研究 S6-EVs/EVs 对软骨细胞 OA 模型的作用

IL-1β 诱导体外软骨细胞 24h，使用 S6-EVs/EVs 干预 72 小时，通过  WB 、RT-qPCR检测软骨细胞表达ECM 合成代谢相关标志物、ECM 分解相关标志物及炎症因子的水平，检测活性氧（reactive oxygen species, ROS）、抗氧化物酶、ATP 水平。

7. 高通量测序结合生物信息学分析，探索作用机制

采用RNA-seq测序技术对 OA 模型的软骨细胞以及 S6-EVs/EVs 处理 OA 模型软骨细胞转录组进行测序。分析 S6-EVs 作用 OA 软骨细胞可能作用机制，并通过 WB 进行初步验证。

研究结果：

1. 成功分离获得 S6-hUCMSCs

流式检测显示 S6-hUCMSCs 高表达 MSCs 的标志物 CD73、CD90、CD105，不表达 CD34、CD45、CD19、CD14 和 HLA-DR，可定向诱导向软骨细胞、骨细胞和脂肪细胞方向分化。端粒酶长度显著高于原代 hUCMSCs，SIRT6 的 mRNA 以及 SIRT6 蛋白的表达水平显著高于原代 hUCMSCs，β-半乳糖苷酶染色显示比原代 hUCMSCs 衰老更慢。结果表明已成功构建永生化过表达 SIRT6 的 S6-hUCMSCs。

2. 成功分离获得 S6-EVs

结果显示 S6-EVs 呈球状，具有双层膜结构，直径约为 30～150nm，表达外囊泡标志物 CD63、CD9、CD81 和 TSG101。S6-EVs 装载 SIRT6 蛋白水平显著高于 EVs，上述结果表明已成功获取 S6-EVs。

3. S6-EVs 体内分布及存留时间

结果发现 S6-EVs 一直存留在小鼠关节腔内，随着时间延长荧光强度逐渐减弱，注射后第 7 天荧光信号几乎全部消失，猜测 S6-EVs 在关节腔的存留时间大概是 6 天，从而确定注射频率为每周注射一次。

4. 对比研究 S6-EVs/EVs 对 OA 小鼠膝关节软骨的保护作用。

ACLT 诱导小鼠 OA 模型，通过 H&E 染色、番红 O-固绿染色、甲苯胺蓝染色以及阿利新蓝染色，并利用 OARSI 分级标准对小鼠膝关节 OA 软骨磨损的程度进行评估，发现 S6-EVs 比 EVs 缓解 OA 小鼠膝关节软骨磨损效果更好。通过免疫组化染色发现 S6-EVs/EVs 均能促进 ECM 合成代谢指标 AGC/SOX9 水平，抑制 ECM 分解代谢相关指标 MMP-13、ADAMTS-5 水平，降低炎症因子 IL-6 和 TNF-α水平，S6-EVs 组软骨中 SIRT6 蛋白表达更高，证明 S6-EVs 相比 EVs 治疗 OA 效果更好。

5. 证明 S6-EVs 促进软骨细胞增殖、迁移，抑制软骨细胞凋亡

与 EVs 组相比，划痕实验结果表明，加入 S6-EVs 的软骨细胞迁移的数量明显更多。CCK-8/Edu 结果显示 S6-EVs 促进软骨细胞增殖能力更强。Annexin V PE/7-AAD 染色结果显示加入 S6-EVs 凋亡的软骨细胞数量明显减少。

6. 对比研究 S6-EVs/EVs 对 OA 软骨细胞模型的调控作用

OA 软骨细胞体外实验发现， S6-EVs/EVs 干预后，均能增强 OA 软骨细胞 ECM 合成代谢 COL2A1、ACAN 水平，降低 ECM 分解代谢 MMP-13、ADAMT-5 水平及降低炎症因子 IL-6 分泌水平，S6-EVs 比 EVs 作用效果更强。相比 EVs，S6-EVs 降低软骨细胞内 ROS 的生成、提高抗氧化物酶活性及促进 ATP 合成的作用更强。

7. S6-EVs 可能通过抑制 NF-κB 信号通路的激活保护 IL-1β 诱导的软骨细胞损伤

通过高通量测序结合生物信息学分析筛选出 S6-EVs/EVs 干预组的差异表达基因，发现 NF-κB 通路相关因子表达与模型组均存在显著性差异。WB 验证 NF-κB 通路相关因子发现，相比 OA 软骨细胞模型组，S6-EVs/EVs 干预组 P65 和 IKBα的磷酸化水平均明显降低，抑制了 NF-κB 信号通路的激活，且 S6-EVs 组抑制作用更为明显。

研究结论：

本课题制备的 S6-hUCMSCs 传代培养到 P60，未出现明显的衰老现象，解决了分泌 EVs 同一供体种子细胞不足的问题，同时 S6-hUCMSCs 表达 SIRT6 水平显著高于原代 hUCMSCs，制备获取的 S6-EVs 中 SIRT6 蛋白装载量显著高于原代 EVs，经过体内和体外验证，对 OA 的治疗作用优于 EVs，其治疗作用可能是通过抑制 ROS/NF-κB 信号通路的激活实现。本课题为开发治疗 OA 更好效果的产品提供了新的思路。

Background：

Osteoarthritis (OA) is the most common chronic degenerative joint disease, which results in joint pain, loss of mobility, and even disability in the elderly, causing a significant socio-economic burden. The breakdown of ECM homeostasis, abnormal chondrocyte metabolism, increase of senescent chondrocytes and apoptosis of chondrocytes are the key steps of OA. Sirtuins (SIRTs) are a group of nicotinamide adenine dinucleotide-dependent histone deacetylases that can deacetylate certain proteins and play a key role in the regulation of cellular physiological and pathological processes. The SIRTs family has been reported to maintain chondrocyte function by maintaining ECM homeostasis, regulating chondrocyte metabolism, preventing chondrocyte senescence, and reducing chondrocyte apoptosis. SIRT6 is a member of the SIRTs family, and studies have shown that the loss of SIRT6 in human chondrocytes can lead to increased DNA damage and telomere dysfunction, as well as subsequent premature aging, and that overexpression of SIRT6 can prevent the development of OA by reducing inflammatory response and chondrocyte aging.

Existing treatment methods for OA mainly focus on relieving symptoms and improving joint function, such as non steroidal anti-inflammatory drugs (NSAIDs), physiotherapy and joint replacement surgery for advanced OA, etc. However, none of these methods can prevent the progression of the disease or repair the damaged articular cartilage, and fundamentally cure OA. A number of clinical trials and studies have confirmed that Mesenchymal stem cells (MSCs) have significant therapeutic effects on OA, and are even considered as the most potential clinical therapy for the radical treatment of OA. However, there are still many challenges in the clinical use of MSCs in the treatment of OA, such as tumorigenicity of transplanted cells and short preservation time, which limit the clinical use. MSCs play a therapeutic role mainly through paracrine function, that is, through Extracellular vesicles (EVs) secreted by MSCs. Compared with MSCs, EVs has the advantages of better safety, easier industrial preparation and long-term storage. Therefore, EVs is considered to be a substitute for MSCs for clinical treatment. Because the capacity and function of EVs are related to the source cells, the number of MSCs from the same donor is limited, resulting in the number of EVs secreted by the same donor cannot meet the requirements of large-scale clinical trials, and the quality of EVs secreted by MSCs from different donors is different, resulting in unstable clinical therapeutic effects. In order to solve the above problems, this study immortalized human Umbilical Cord Mesenchymal Stem Cells (hUCMSCs) to obtain hUCMSCs from the same umbilical cord in large quantities, so as to prepare the same batch of EVs in large quantities to solve the problem of requiring large quantities of EVs with stable quality in clinical research. In order to further improve the function of EVs in OA treatment, it is hoped that EVs with stronger OA treatment effect can be obtained through engineering transformation.

Objects:

In this study, hUCMSCs were engineered by lentivirus infection gene modification to prepare specific hUCMSCs (S6-hUCMSCs) and its EVs (S6-EVs) that overexpressed SIRT6 in immortalization to solve the problem of insufficient amount of traditional EVs due to the limited number of seed cells from the same donor and the need to improve the efficacy of OA treatment. The therapeutic effects of EVs/S6-EVs on OA was compared in vivo experiment of OA mice and in vitro experiment of OA model of chondrocytes to explore its mechanism of action and provide new ideas for further exploration of clinical treatment of OA and related product development.

Methods:

Culture and identification of S6-hUCMSCs

Watton's gum of umbilical cord was isolated and tissue block culture was carried out for 8-11 days, primary hUCMSCs were isolated and obtained, and immortalized hUCMSCs (TERT-Hucmscs) were obtained through puro resistant lentivirus infection expressing TERT gene and drug screening. S6-hUCMSCs were identified, including cell markers, multidirectional differentiation ability, expression levels of TERT and SIRT6 mRNA, expression levels of SIRT6 protein, telomerase length, and cell accumulation and proliferation ability.

Extraction and identification of S6-EVs

S6-EVs were obtained from the culture supernatant of S6-hUCMSCs by differential ultra-centrifugal method. The shape and particle size of S6-EVS were determined by transmission electron microscopy (TEM) and particle size analyzer (NTA). The expression of SIRT6 protein and its markers were identified by WB.

Fluorescence labeled S6-EVs was used to detect the in vivo retention time

The EVs was labeled with DIR dye and injected into the knee cavity of mice. The fluorescence intensity was observed daily by a living imager.

To compare and study the therapeutic effects of S6-EVs/EVs on OA model of knee joint in mice

A mouse knee OA model was established by Anterior cruciate ligament transection (ACLT). After 1 week of modeling, S6-EVs/EVs was injected into the joint cavity once a week for a total of 6 weeks. HE and saffrane-O solid green staining were carried out in pathological sections of the knee joint of mice to observe the damage and repair of the articular surface cartilage, and immunohistochemical staining was carried out to identify the expression of chondrocyte markers, inflammatory factors and SIRT6, so as to evaluate the effects of EVs/S6-EVs in the treatment of OA in mice.

To compare and study the effects of S6-EVs/EVs on the biological behavior of chondrocytes

The effects of S6-EVs/EVs on chondrocyte proliferation was detected by CCK-8/Edu, the effects of S6-EVs/EVs on chondrocyte migration was detected by scratch assay, and the effects of S6-EVs/EVs on chondrocyte apoptosis was studied by Annexin V PE/7-AAD staining.

To compare and study the effects of S6-EVs/EVs on IL-1β-induced OA chondrocyte model

Chondrocytes were induced by IL-1β for 24h to construct an OA model of chondrocytes. After S6-EVs/EVs intervention for 72h, the expression levels of inflammatory cytokines secreted by chondrocytes, anabolic markers of ECM and markers of ECM decomposition, the levels of reactive oxygen species (ROS), antioxidant enzymes and ATP were detected by RT-qPCR and WB.

To explore the mechanism of action through High-throughput sequencing combined with bioinformatics analysis

The chondrocytes of OA model and chondrocytes transcriptome of OA model treated with S6-EVs/EVs were sequenced by high-throughput sequencing technology. The possible mechanism of chondrocyte action of S6-EVs on OA model was analyzed and preliminarily verified by WB.

Results：

S6-hUCMSCs were successfully isolated

Flow cytometry showed that S6-hUCMSCs highly expressed CD73, CD90 and CD105 markers of MSCs, but did not express CD34, CD45, CD19, CD14 and HLA-DR, which could induce differentiation toward chondrocytes, osteocytes and adipocytes. Telomerase length was significantly higher than that of primary hUCMSCs, mRNA and protein expression levels of SIRT6 were significantly higher than that of primary hUCMSCs, and β-galactosidase staining showed slower aging than that of primary hUCMSCs. The results showed that S6-hUCMSCs of immortalizedoverexpression SIRT6 were successfully constructed.

S6-EVs was successfully isolated

Ultrastructure showed that S6-EVs was spherical, with a typical bilayer membrane structure, a diameter of about 30 ~ 150nm, and expressed the external vesicle marker proteins CD9, CD63, CD81 and TSG101. The SIRT6 protein level of S6-EVs was significantly higher than that of EVs. The above results indicated that S6-EVs had been successfully obtained.

Distribution and duration of S6-EVs in vivo

The results showed that S6-EVs remained in the joint cavity of mice, the fluorescence intensity gradually weakened with the extension of time, and the fluorescence signal almost disappeared on the 7th day after injection. It was speculated that the retention time of S6-EVs in the joint cavity was about 6 days, so the injection frequency was determined to be once a week.

To compare and study the protective effects of S6-EVs/EVs on knee cartilage of OA mice

ACLT induced mouse OA model, H&E staining, saffranine O-solid green staining, toluidine blue staining and alcian blue staining were used to evaluate the degree of OA cartilage wear of the knee joint of mice by OARSI grading standard, and it was found that S6-EVs had a better effect on alleviating OA cartilage wear of the knee joint of mice than EVs. Immunohistochemical staining showed that S6-EVs/EVs could promote the level of ECM anabolic indicators AGC/SOX9, inhibit the levels of ECM catabolic indicators MMP-13 and ADAMTS-5, and reduce the levels of inflammatory factors IL-6 and TNF-α. The expression of SIRT6 protein in cartilage of S6-EVs group was higher. It is proved that S6-EVs is more effective than EVs in treating OA.

It was proved that S6-EVs promoted chondrocyte proliferation and migration, and inhibited chondrocyte apoptosis

Compared with EVs group, CCK-8/Edu results showed that S6-EVs had a stronger ability to promote chondrocyte proliferation. The results of the scratch asssay showed that the number of chondrocytes migrated significantly more when S6-EVs was added. Annexin V PE/7-AAD staining showed that the number of apoptotic chondrocytes added with S6-EVs significantly decreased compared to the EVs group.

To compare and study the regulation effects of S6-EVs/EVs on OA chondrocyte model

In vitro experiments of OA chondrocytes showed that after the intervention of S6-EVs/EVs, the levels of COL2A1 and ACAN in ECM anabolism in OA chondrocytes were enhanced, the levels of MMP-13 and ADAMT-5 in ECM catabolism and the secretion level of inflammatory factor IL-6 could be reduced, and the effect of S6-EVs was stronger than that of EVs. Compared with EVs, S6-EVs had a stronger effect on reducing ROS production, increasing antioxidant activity and promoting ATP synthesis in chondrocytes.

S6-EVs may protect IL-1β-induced chondrocyte injury by inhibiting the activation of NF-κB signaling pathway

The differentially expressed genes in the S6-EVs/EVs intervention group were screened by high-throughput sequencing combined with bioinformatics analysis, and significant differences in the expression of factors related to NF-κB pathway were found. WB verification of NF-κB pathway related factors showed that compared with OA chondrocyte model group, the phosphorylation levels of P65 and IKB-α were significantly reduced in S6-EVs/EVs intervention group, which inhibited the activation of NF-κB signaling pathway, and the inhibition effect was more obvious in S6-EVs group.

Conclusion:

The S6-hUCMSCs prepared in this study were cultured to P60 without obvious senescence phenomenon, which solved the problem of insufficient seed cells from the same donor that secreted EVs. Meanwhile, the SIRT6 expression level of S6-hUCMSCs was significantly higher than that of the primary hUCMSCs. The SIRT6 protein loading of prepared S6-EVs was significantly higher than that of primary EVs, and the therapeutic effect of prepared S6-EVs on OA was proved to be superior to EVs after in vivo and in vitro verification. The therapeutic effect may be realized by inhibiting the activation of ROS/NF-κB signaling pathway. This study provides a new idea for developing better products for OA treatment.