中国组织工程研究

中国组织工程研究 ›› 2018, Vol. 22 ›› Issue (1): 113-118.doi: 10.3969/j.issn.2095-4344.0420

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

低氧诱导因子1α联合Scleraxis修饰人羊膜间充质干细胞促进腱骨愈合的体外实验

朱喜忠1，刘子铭2，刘 毅1，熊华章1，杨继滨1，李豫皖1，金 瑛1，吴术红1   

  1. 1遵义医学院附属医院骨科，贵州省遵义市 563000；2重庆医科大学第一附属医院关节外科，重庆市 400000
  • 修回日期:2017-11-20 出版日期:2018-01-08 发布日期:2018-01-08
  • 通讯作者: 吴术红，硕士，主任医师，遵义医学院附属医院骨科，贵州省遵义市 563000
  • 作者简介:朱喜忠，男，1993年生，河南省开封市人，汉族，遵义医学院在读硕士，主要从事干细胞与组织工程，运动医学的研究。
  • 基金资助:

    贵州省科技厅联合基金(黔省专合字(2012)172号；黔科合LH字[2016]7477号；黔科合支撑[2017]2882号)

An in vitro study of enhancing tendon-bone healing by human amniotic mesenchymal stem cells co-modified with hypoxia-inducible factor 1 alpha and Scleraxis gene

Zhu Xi-zhong1, Liu Zi-ming2, Liu Yi1, Xiong Hua-zhang1, Yang Ji-bin1, Li Yu-wan1, Jin Ying1, Wu Shu-hong1   

  1. 1Department of Orthopedics, the Affiliated Hospital of Zunyi Medical University, Zunyi 563000, Guizhou Province, China; 2Department of Orthopaedics, First Affiliated Hospital of Chongqing Medical University, Chongqing 400000, China
  • Revised:2017-11-20 Online:2018-01-08 Published:2018-01-08
  • Contact: Wu Shu-hong, Master, Chief physician, Department of Orthopedics, the Affiliated Hospital of Zunyi Medical University, Zunyi 563000, Guizhou Province, China
  • About author:Zhu Xi-zhong, Studying for master’s degree, Department of Orthopedics, the Affiliated Hospital of Zunyi Medical University, Zunyi 563000, Guizhou Province, China
  • Supported by:

    the Joint Foundation of Guizhou Provincial Science and Technology Department, No. (2012)172, [2016]7477; [2017]2882

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摘要：

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文题释义：
低氧诱导因子1：由Semenza和Wang于1992年发现，普遍存在于人的细胞内并稳定表达于缺氧条件下。其能够促进多种多样靶基因的表达，如血管内皮生长因子、血小板源性生长因子及乳酸脱氢酶A，因此对于细胞、组织在缺氧条件下内环境的稳定具有重要意义。腱骨连接部位常因血供差等因素不能够有效修复损伤，研究低氧诱导因子的作用和机制可为促进腱骨愈合提供新的思路。
腺病毒载体：其通过受体介导的内吞作用进入细胞内使目的基因转移至细胞核且维持于染色体外，实现目的基因在宿主细胞中高表达的需求。具有操作简便、转染效率高及靶细胞范围广等优点。实验使用腺病毒载体的方法使两种不同的目的基因同时或单一表达于同一靶细胞中，观察并检测相关指标，为将来临床损伤的应用和治疗提供理论依据。

 

摘要
背景：由于肌腱与骨的愈合缓慢而复杂，往往影响临床治疗效果，提高腱骨接触面细胞活性是损伤治疗的新策略。Scleraxis 是肌腱细胞的特异性标志分子，低氧诱导因子1α(hypoxia inducible factor 1α，HIF-1α)可通过多种途径诱导血管和骨组织的形成。但HIF-1α能否增强Scleraxis诱导人羊膜间充质干细胞分化并用于腱骨损伤的修复还尚未研究。
目的：探讨HIF-1α联合Scleraxis修饰人羊膜间充质干细胞在体外促进腱骨愈合的能力并研究其分子机制。
方法：取第3代人羊膜间充质干细胞，分别经AdHIF-1α、AdScx、AdGFP和AdHIF-1α+AdScx腺病毒载体感染第3天和第7天后检测肌腱、软骨和骨组织相关基因的表达。
结果与结论：①倒置相差显微镜观察显示，第3代人羊膜间充质干细胞呈长梭形、漩涡状贴壁生长；②透射电镜观察显示，人羊膜间充质干细胞呈椭圆形，结构清晰，胞浆含丰富的内质网及线粒体；③荧光显微镜观察显示，腺病毒感染24 h后，AdHIF-1α组表达红色荧光，荧光表达量约为50%，AdScx组和AdGFP组均表达绿色荧光，荧光表达量约为70%；④实时荧光定量PCR显示，感染第3天和第7天时，AdHIF-1α+AdScx组、AdHIF-1α组和AdScx组Ⅰ型胶原、Fibronectin、RUNX2、VEGF和ALP的mRNA表达量均高于AdGFP组(P < 0.05)，AdHIF-1α+AdScx组Ⅰ型胶原、Fibronectin、RUNX2、VEGF和ALP的mRNA表达量均显著性高于AdScx组(P < 0.05)；⑤荧光免疫组化分析显示，AdHIF-1α+AdScx组感染第7天时Ⅰ型胶原表达量高于感染第3天；⑥结果表明，体外HIF-1α和Scleraxis基因联合修饰后可促进人羊膜间充质干细胞表达肌腱细胞、软骨细胞和骨细胞的标志分子，具有促进腱骨愈合的能力。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
ORCID: 0000-0003-2780-209X(朱喜忠)

关键词: 低氧诱导因子1α, 人羊膜间充质干细胞, Scleraxis, 腱骨愈合, 韧带组织工程, 干细胞

Abstract:

BACKGROUND: Tendon-to-bone healing is a complex and slow process, which often hinders the therapeutic outcomes. To enhance the tendon-to-bone healing, increasing cell bioactivity on the contact surface is a new strategy. Hypoxia-inducible factor 1α (HIF-1α) can induce the formation of blood vessel and bone tissue via many ways. However, it is unclear whether HIF-1α can strengthen differentiation of human amniotic mesenchymal stem cells (hAMSCs) induced by Scleraxis, a specific marker of tendon, and can be used in tendon and bone repair.
OBJECTIVE: To investigate the ability of HIF-1α in enhancing Scleraxis to modify hAMSCs aiming to promote tendon-to-bone healing and its molecular mechanism.
METHODS: Passage 3 hAMSCs were infected with AdHIF-1α, AdScx, AdGFP and AdHIF-1α+AdScx. The expressions of related genes of tendon, cartilage and bone tissue were detected at 3 and 7 days after infection.
RESULTS AND CONCLUSION: Under the inverted phase contrast microscopy, the passage 3 hAMSCs were in spindle shape and presented with vortex-like adherent growth. Under the transmission electron microscopy, hAMSCs were in oval shape and had clear structure, with abundant endoplasmic reticulum and mitochondria. Fluorescence photographs were taken at 24 hours after adenovirus infection, showing about 50% red fluorescence expression in the AdHIF-1α group, while about 70% green fluorescence expression in the AdScx and AdGFP groups. Real time-PCR results showed that at 3 and 7 days after infection, the mRNA expressions of collagen type I, Fibronectin, RUNX2, VEGF and ALP in the AdHIF-1α+AdScx group, AdHIF-1α group and AdScx group were higher than those in the AdGFP group (P < 0.05); the mRNA expressions of collagen type I, Fibronectin, RUNX2, VEGF and ALP in the AdHIF-1α+AdScx group were significantly higher than those in the AdScx group (P < 0.05). Fluorescence immunohistochemistry results showed that the expression of collagen type I in the AdHIF-1α+AdScx group at 7 days after infection was higher than that at 3 days after infection. To conclude, HIF-1α can enhance the ability of Scleraxis to modify hAMSCs to promote tendon-to-bone healing by upregulating the expressions of molecular markers of tenocytes, chondrocytes and osteocytes.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Amnion, Mesenchymal Stem Cells, Hypoxia-Inducible Factor 1, alpha Subunit, Tissue Engineering

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