低能量激光在组织修复中的应用研究

发布时间：2018-06-28 12:18

本文选题：低能量激光 + 人脐带间充质干细胞　；参考：《北京协和医学院》2016年博士论文

【摘要】：目的：低能量激光疗法(LLLT)具有促进干细胞增殖分化和组织修复的生物学效应,可用于再生医学方面的治疗。然而,LLLT的治疗机制尚无定论,阻碍了其在临床的应用和发展。因此,本研究将探索双波长LLLT增强干细胞活力的机制及其在脊髓损伤和抗辐射方面的应用,同时,从细胞和分子水平探讨LLLT改善高糖环境下成纤维细胞功能损伤的作用机制,探索LLLT在组织修复中的应用研究,为双波长激光的临床治疗和推广提供理论依据。方法：体外培养人脐带间充质干细胞和人成纤维细胞,采用635/808 nm双波长半导体激光器,照光剂量为20mW/cm2、0-24 J/cm2,分别检测细胞增殖活力、活性氧产量、抗氧化酶活性(CAT、SOD和tGPx)、脂质氧化能力(MDA)以及细胞生长因子和相关蛋白表达量。建立大鼠脊髓损伤模型,LLLT联合干细胞移植对其进行治疗,对模型鼠进行行为学评价,取材脊髓组织进行组织病理学评价以及采用DTI技术追踪脊髓神经纤维。结果：1、635 nm和双波长LLLT作用后hUCMSCs增殖速度明显大于对照组；LLLT促进ROS低水平表达；CAT、SOD和tGPx酶活力提高；808 nm低能量激光促进MDA增加；LLLT使IL-1、IL6和NFκB分泌量显著增多,其中双波长效果最好。2、LLLT联合hUCMSCs促进大鼠脊髓损伤部位的神经恢复,行为学评分高于对照组；NF-200和GFAP的表达量增加；尼氏小体染色变深、数量变多；DTI结果显示对照组神经纤维断裂,治疗组损伤处纤维束相互交错。单纯的LLLT组是最晚出现大鼠死亡和死亡率最低的一组；3、γ射线照射处理对DNA有明显的损伤,激光照射联合Y射线处理组比对照组彗星尾部DNA量增多,但相比γ射线组有所降低；LLLI预处理降低了电离辐射引起的氧化应激水平,提高了抗氧化酶活力。4、LLLT提高成纤维细胞的增殖能力,改善了高糖环境对细胞增殖的抑制作用；促进了ROS的表达,提升了抗氧化酶活力,刺激细胞分泌多种细胞因子(FGF、NGF、 IGF、TGF、IL-1、IL-6、NFκB和VEGF等)；下调MMPs的表达,上调TIMPs的表达,扭转高糖环境引起的MMPs/TIMPs的高表达,提高胶原蛋白的表达量。结论：LLLT诱导产生的低水平氧化应激反应可促进细胞的增殖,提升细胞抗氧化酶活力,增强细胞对外界环境的抵抗能力,降低辐射敏感性,改善高糖环境对细胞增殖的抑制作用。LLLT刺激细胞分泌多种细胞因子,调控创伤愈合的进程。LLLT联合干细胞促进受损神经纤维的再生与功能修复。红光或近红外光对细胞的刺激效应不同,双波长激光能联合两种波长激光的生物效应,对干细胞移植、脊髓损伤和创伤修复有更好的疗效。
[Abstract]:Objective: low energy laser therapy (LLLT) has the biological effect of promoting stem cell proliferation, differentiation and tissue repair, and can be used in regenerative medicine. However, the mechanism of LLLT has not been determined, which hinders its clinical application and development. Therefore, this study will explore the mechanism of double wavelength LLLT in enhancing stem cell viability and its application in spinal cord injury and radiation resistance. At the same time, we will explore the mechanism of LLLT in improving fibroblast function injury in high glucose environment at the cellular and molecular level. To explore the application of LLLT in tissue repair and provide theoretical basis for clinical treatment and popularization of double wavelength laser. Methods: human umbilical cord mesenchymal stem cells and human fibroblasts were cultured in vitro. 635 / 808 nm dual-wavelength semiconductor laser was used at a dose of 20 MW / cm ~ (2) and 0-24 J / cm ~ (2) to detect the proliferation activity and the production of reactive oxygen species. The activity of antioxidant enzymes (SOD and tGPx), lipid oxidation ability (MDA), and the expression of cell growth factor and related protein. Results the proliferation rate of hUCMSCs treated with 1: 1635 nm and double wavelength LLLT was significantly higher than that of control group. The activity of CATX SOD and tGPx enzyme in low Ros expression level was increased by low energy laser irradiation at 808 nm, and the secretion of IL 1, IL 6 and NF 魏 B was significantly increased by low energy laser irradiation at 80 8 nm, and the secretion of IL 1, IL 6 and NF 魏 B was significantly increased after treatment with 1: 1635 nm and double wavelength LLLT. Two-wavelength LLLT combined with hUCMSCs promoted the recovery of spinal cord nerve in rats. The behavioral score was higher than that of the control group, the expression of NF-200 and GFAP was increased, the staining of Nissl corpuscles became deeper and more numerous, and the expression of NF-200 and GFAP in the control group was higher than that in the control group. DTI results showed that the nerve fibers in the control group were broken, and the nerve bundles in the treatment group were interlaced. Single LLLT group was the group with the lowest mortality and death rate at the latest. 纬 -ray irradiation had obvious damage to DNA. The amount of DNA in the tail of comet in the laser irradiation combined with Y-ray treatment group was higher than that in the control group, but it was lower than that in the 纬 -ray group. LLLI pretreatment decreased the level of oxidative stress induced by ionizing radiation, increased the activity of antioxidant enzymes. LLLT increased the proliferation of fibroblasts, improved the inhibitory effect of high glucose environment on cell proliferation, and promoted the expression of Ros. It enhanced the activity of antioxidant enzymes, stimulated the secretion of various cytokines (FGFNGF, IGFFTGF-1, IL-6, NF- 魏 B and VEGF), down-regulated the expression of MMPs, upregulated the expression of TIMPs, reversed the high expression of MMPs / TIMPs induced by high glucose environment, and increased the expression of collagen. Conclusion the low level oxidative stress induced by WL LLLT can promote cell proliferation, enhance the activity of antioxidant enzymes, enhance the resistance of cells to the external environment, and reduce the radiosensitivity. To improve the inhibitory effect of high glucose environment on cell proliferation. LLLT stimulates the secretion of many cytokines and regulates the process of wound healing. LLLT combined with stem cells promotes the regeneration and functional repair of damaged nerve fibers. The stimulative effect of red light or near infrared light on cells is different. Dual wavelength laser can combine the biological effect of two kinds of wavelength laser, which has better effect on stem cell transplantation, spinal cord injury and wound repair.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R454.2

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