构建高效载体OPEI沉默TRAF6促进骨关节炎软骨再生的研究

doi:10.3969/j.issn.1674-8115.2022.07.002

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (7): 846-857.doi: 10.3969/j.issn.1674-8115.2022.07.002

• 论著 · 基础研究 • 上一篇

构建高效载体OPEI沉默TRAF6促进骨关节炎软骨再生的研究

刘宏强¹(), 陆艳青², 高宇轩³, 王一云², 王传东², 张晓玲²()

^1.山西大学体育学院，太原 030006
^2.上海交通大学医学院附属新华医院骨科，上海 200092
^3.山西医科大学第二医院骨科，太原 030001

收稿日期:2022-01-06 接受日期:2022-06-18 出版日期:2022-07-28 发布日期:2022-09-04
通讯作者: 张晓玲 E-mail:liuhq1969@sxu.edu.cn;xlzhang@shsmu.edu.cn
作者简介:刘宏强(1969—)，男，副教授，硕士；电子信箱：liuhq1969@sxu.edu.cn。
基金资助:
国家自然科学基金(81772432);山西省重点研发计划项目(201903D321097)

Construction of OPEI vector for silencing TRAF6 to promote cartilage regeneration in inflammatory environment

LIU Hongqiang¹(), LU Yanqing², GAO Yuxuan³, WANG Yiyun², WANG Chuandong², ZHANG Xiaoling²()

^1.School of Physical Education, Shanxi University, Taiyuan 030006, China
^2.Department of Orthopedic Surgery, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China
^3.Second Hospital of Shanxi Medical University, Taiyuan 030001, China

Received:2022-01-06 Accepted:2022-06-18 Online:2022-07-28 Published:2022-09-04
Contact: ZHANG Xiaoling E-mail:liuhq1969@sxu.edu.cn;xlzhang@shsmu.edu.cn
Supported by:
National Natural Science Foundation of China(81772432);Key Research and Development Program of Shanxi Province(201903D321097)

摘要/Abstract

摘要：

目的·构建低毒性的关节滑膜高效率siRNA转染载体OPEI，抑制肿瘤坏死因子受体相关因子6（tumor necrosis factor receptor-associated factor 6，TRAF6），观察其对骨关节炎（osteoarthritis，OA）模型骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）成软骨能力的影响。方法·通过内侧半月板切除术建立SD大鼠OA模型（OA组，n=20）；另设假手术组（n=10），半月板保持完好。造模后3个月后采集手术部位软骨和滑膜，免疫组织化学法检测TRAF6的表达，Western blotting方法检测白介素-1β（interleukin-1β，IL-1β）诱导的大鼠原代滑膜细胞中MMP13、TRAF6、p-p65的表达。在无水厌氧环境中合成小分子聚乙烯亚胺（polyethylenimine，PEI）衍生物OPEI，琼脂糖凝胶电泳检测OPEI包裹siRNA的能力，动态光散射测量形成的OPEI/siRNA复合物的粒径和Zeta电位，MTT法检测形成的复合物对大鼠原代滑膜细胞的细胞毒性，流式细胞术分析复合物对滑膜细胞凋亡的影响，并利用激光共聚焦技术及荧光显微镜观测体内外OPEI在滑膜细胞中转染siRNA的效率，阿尔新蓝染色检测软骨细胞基质蛋白聚糖含量。结果·与假手术组相比，TRAF6在大鼠OA模型的滑膜及软骨中的表达显著升高；抑制TRAF6表达可显著降低IL-1β刺激的原代滑膜细胞中MMP13和p-p65的表达。构建的OPEI载体在大鼠原代滑膜细胞中的siRNA转染效率高达99.33%，在大鼠膝关节腔内注射OPEI/Cy3-siRNA后第3日、第7日均显示大量滑膜细胞摄取siRNA。OPEI/siTRAF6复合物转染大鼠原代滑膜细胞2 d，Western blotting结果显示OPEI/siTRAF6组在质量比值为3∶1、4∶1和5∶1时，TRAF6基因敲除效率分别为49.05%、74.61%和83.18%。OPEI/siTRAF6沉默TRAF6基因后，与对照组（OPEI/siNC）相比，IL-1β刺激条件下软骨细胞阿尔新蓝染色显著增强。结论·OPEI是低毒性高效率siRNA转染载体，在滑膜细胞中沉默TRAF6可促进骨关节炎软骨再生。

关键词: OPEI, siRNA, 滑膜细胞, 肿瘤坏死因子受体相关因子6, 骨髓间充质干细胞, 软骨再生, 骨关节炎

Abstract:

Objective·To construct a low toxicity and high-efficiency joint synovial siRNA transfection vector OPEI, and inhibit tumor necrosis factor receptor-associated factor 6 (TRAF6) to rescue the chondrogenic ability of bone marrow mesenchymal stem cells (BMSCs) under inflammatory conditions.

Methods·The osteoarthritis (OA) models of SD rats (n=20) were established by medial meniscectomy of knee. Another sham operation group (n=10) was established, and the meniscus remained intact. The cartilage and synovium were collected 3 months after surgery. The expression of TRAF6 was detected by immunohistochemistry, and Western blotting was used to detect the expression of MMP13, TRAF6 and p-p65 in primary rat synovial cells induced by interleukin-1β (IL-1β). Further, the small molecule polyethylenimine (PEI) derivative OPEI was synthesized in anhydrous anaerobic environment, and the ability of OPEI to encapsulate siRNA was detected by agarose gel electrophoresis. The particle size and Zeta potential of OPEI/siRNA complex were measured by dynamic light scattering. The cytotoxicity of the formed complex to rat primary synovial cells was detected by MTT method. The effect of the complex on synovial cell apoptosis was analyzed by flow cytometry. The transfection efficiency of siRNA by OPEI in synovial cells in vivo and in vitro was detected by laser confocal technique and fluorescence microscopy. The proteoglycan content of chondrocyte matrix was detected by alcian blue staining.

Results·Compared with the sham operation group, TRAF6 was highly expressed in synovium and cartilage of the rat OA models, and inhibition of TRAF6 could significantly reduce the expression of MMP13 and p-p65 in IL-1β-stimulated primary synovial cells. The siRNA transfection efficiency of OPEI in the rat primary synovial cells was as high as 99.33%. A large number of synovial cells ingested siRNA on the 3rd and the 7th day after injection of OPEI / Cy3-siRNA into rat knee joints. Two days after OPEI / siTRAF6 complex with different w/w ratios was transfected into rat primary synovial cells, the results of TRAF6 protein expression showed that the knockout efficiency of TRAF6 gene in the OPEI / siTRAF6 group was 49.05%, 74.61% and 83.18% respectively when the mass ratio was 3∶1, 4∶1 and 5∶1. After TRAF6 gene was silenced by OPEI/siTRAF6, compared with the control group (OPEI/siNC), alcian blue staining of chondrocytes was significantly enhanced under IL-1β stimulation.

Conclusion·OPEI is a low toxicity and high efficiency siRNA transfection vector, silencing TRAF6 in synovial cells could promote OA cartilage regeneration.

Key words: OPEI, siRNA, synoviocytes, tumor necrosis factor receptor-associated factor 6 (TRAF6), bone marrow mesenchymal stem cells (BMSCs), cartilage regeneration, osteoarthritis (OA)

中图分类号:

刘宏强, 陆艳青, 高宇轩, 王一云, 王传东, 张晓玲. 构建高效载体OPEI沉默TRAF6促进骨关节炎软骨再生的研究[J]. 上海交通大学学报（医学版）, 2022, 42(7): 846-857.

LIU Hongqiang, LU Yanqing, GAO Yuxuan, WANG Yiyun, WANG Chuandong, ZHANG Xiaoling. Construction of OPEI vector for silencing TRAF6 to promote cartilage regeneration in inflammatory environment[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(7): 846-857.

图/表 7

图1 大鼠OA模型及IL-1β刺激下原代滑膜细胞中TRAF6的表达Note： A. Immunohistochemical method. B—F. Western blotting. ①P=0.000, compared with Lipo2000/siNC.

Fig 1 Expression of TRAF6 in OA models and IL-1β-stimulated primary synovial cells

图2 OPEI/siRNA复合物的表征Note： A. Agarose gel electrophoresis. B/C. Dynamic light scattering. D/E. Transmission electron microscope.

Fig 2 Characterization of OPEI/siRNA polyplex

图3 OPEI和OPEI/siRNA复合物对大鼠原代滑膜细胞的生物相容性和细胞毒性Note： A/B. Cell viability measured by MTT assay. C. Detection of cell proliferation by PrestoBlue. D?H. Detection of apoptosis by flow cytometry. ①P=0.020, ②P=0.006, ③P=0.010, ④P=0.000, ⑤P=0.010, ⑥P=0.012, ⑦P=0.008, ⑧P=0.012, ⑨P=0.009, compared with PEI25kDa.

Fig 3 Biocompatibility and cytotoxicity of OPEI and OPEI/siRNA polyplexes on rat primary synoviocytes

图4 OPEI/siRNA复合物在体外和体内的细胞转染效率Note： A. Flow cytometry. B. Laser confocal technology. C. Fluorescence microscopy.

Fig 4 Cell transfection efficiency of OPEI/siRNA polyplexs in vitro and in vivo

图5 OPEI/siTRAF6复合物转染大鼠原代滑膜细胞的 TRAF6 基因敲除效率Note： A/B. Real-time PCR. C/D. Western blotting. ①P=0.001, ②P=0.016, ③P=0.000,④P=0.013, ⑤P=0.001, ⑥P=0.002, ⑦P=0.004, ⑧P=0.002, compared with NC.

Fig 5 TRAF6 knockdown efficiency of OPEI/siTRAF6 polyplexes in rat primary synoviocytes

图6 TRAF6 基因敲除对IL-1β诱导的大鼠滑膜细胞炎症的影响Note： ①P=0.001, ②P=0.003, ③P=0.000, compared with naked siTRAF6.

Fig 6 Effect of TRAF6 knockdown on IL-1β-induced inflammation in rat primary synoviocytes

图7 TRAF6 基因敲除增强BMSCs在IL-1β诱导的炎症条件下的软骨形成能力Note： A. Alcian blue staining. B—D.Western blotting. BM—basic medium;CM—cartilage differentiation medium. ①P=0.005, compared with OPEI/siNC+IL-1β.

Fig 7 TRAF6 knockdown restores the chondrogenic ability of rBMSCs under IL-1β-induced inflammation.

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构建高效载体OPEI沉默TRAF6促进骨关节炎软骨再生的研究

Construction of OPEI vector for silencing TRAF6 to promote cartilage regeneration in inflammatory environment

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