本文選題：低能量激光 + 人臍帶間充質(zhì)干細(xì)胞��； 參考：《北京協(xié)和醫(yī)學(xué)院》2016年博士論文

【摘要】：目的：低能量激光療法(LLLT)具有促進(jìn)干細(xì)胞增殖分化和組織修復(fù)的生物學(xué)效應(yīng),可用于再生醫(yī)學(xué)方面的治療。然而,LLLT的治療機(jī)制尚無(wú)定論,阻礙了其在臨床的應(yīng)用和發(fā)展。因此,本研究將探索雙波長(zhǎng)LLLT增強(qiáng)干細(xì)胞活力的機(jī)制及其在脊髓損傷和抗輻射方面的應(yīng)用,同時(shí),從細(xì)胞和分子水平探討LLLT改善高糖環(huán)境下成纖維細(xì)胞功能損傷的作用機(jī)制,探索LLLT在組織修復(fù)中的應(yīng)用研究,為雙波長(zhǎng)激光的臨床治療和推廣提供理論依據(jù)。方法：體外培養(yǎng)人臍帶間充質(zhì)干細(xì)胞和人成纖維細(xì)胞,采用635/808 nm雙波長(zhǎng)半導(dǎo)體激光器,照光劑量為20mW/cm2、0-24 J/cm2,分別檢測(cè)細(xì)胞增殖活力、活性氧產(chǎn)量、抗氧化酶活性(CAT、SOD和tGPx)、脂質(zhì)氧化能力(MDA)以及細(xì)胞生長(zhǎng)因子和相關(guān)蛋白表達(dá)量。建立大鼠脊髓損傷模型,LLLT聯(lián)合干細(xì)胞移植對(duì)其進(jìn)行治療,對(duì)模型鼠進(jìn)行行為學(xué)評(píng)價(jià),取材脊髓組織進(jìn)行組織病理學(xué)評(píng)價(jià)以及采用DTI技術(shù)追蹤脊髓神經(jīng)纖維。結(jié)果：1、635 nm和雙波長(zhǎng)LLLT作用后hUCMSCs增殖速度明顯大于對(duì)照組；LLLT促進(jìn)ROS低水平表達(dá)；CAT、SOD和tGPx酶活力提高；808 nm低能量激光促進(jìn)MDA增加；LLLT使IL-1、IL6和NFκB分泌量顯著增多,其中雙波長(zhǎng)效果最好。2、LLLT聯(lián)合hUCMSCs促進(jìn)大鼠脊髓損傷部位的神經(jīng)恢復(fù),行為學(xué)評(píng)分高于對(duì)照組；NF-200和GFAP的表達(dá)量增加；尼氏小體染色變深、數(shù)量變多；DTI結(jié)果顯示對(duì)照組神經(jīng)纖維斷裂,治療組損傷處纖維束相互交錯(cuò)。單純的LLLT組是最晚出現(xiàn)大鼠死亡和死亡率最低的一組；3、γ射線照射處理對(duì)DNA有明顯的損傷,激光照射聯(lián)合Y射線處理組比對(duì)照組彗星尾部DNA量增多,但相比γ射線組有所降低；LLLI預(yù)處理降低了電離輻射引起的氧化應(yīng)激水平,提高了抗氧化酶活力。4、LLLT提高成纖維細(xì)胞的增殖能力,改善了高糖環(huán)境對(duì)細(xì)胞增殖的抑制作用；促進(jìn)了ROS的表達(dá),提升了抗氧化酶活力,刺激細(xì)胞分泌多種細(xì)胞因子(FGF、NGF、 IGF、TGF、IL-1、IL-6、NFκB和VEGF等)；下調(diào)MMPs的表達(dá),上調(diào)TIMPs的表達(dá),扭轉(zhuǎn)高糖環(huán)境引起的MMPs/TIMPs的高表達(dá),提高膠原蛋白的表達(dá)量。結(jié)論：LLLT誘導(dǎo)產(chǎn)生的低水平氧化應(yīng)激反應(yīng)可促進(jìn)細(xì)胞的增殖,提升細(xì)胞抗氧化酶活力,增強(qiáng)細(xì)胞對(duì)外界環(huán)境的抵抗能力,降低輻射敏感性,改善高糖環(huán)境對(duì)細(xì)胞增殖的抑制作用。LLLT刺激細(xì)胞分泌多種細(xì)胞因子,調(diào)控創(chuàng)傷愈合的進(jìn)程。LLLT聯(lián)合干細(xì)胞促進(jìn)受損神經(jīng)纖維的再生與功能修復(fù)。紅光或近紅外光對(duì)細(xì)胞的刺激效應(yīng)不同,雙波長(zhǎng)激光能聯(lián)合兩種波長(zhǎng)激光的生物效應(yīng),對(duì)干細(xì)胞移植、脊髓損傷和創(chuàng)傷修復(fù)有更好的療效。
[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. 緯 -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 緯 -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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R454.2

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