【摘要】：目的： 近些年,低能量激光照射(low level laser irradiation, LLLR)陸續(xù)被報道具有良好的生物學(xué)作用,包括光生物學(xué)促進(jìn)作用、光生物學(xué)調(diào)節(jié)功能等。通過文獻(xiàn)回顧可知,LLLR不僅能夠促進(jìn)細(xì)胞生長,增強細(xì)胞代謝和細(xì)胞增殖,而且可以調(diào)節(jié)新生血管的生成、改善炎癥狀態(tài)。另外,實驗室研究結(jié)果顯示,通過照射LLLR成骨細(xì)胞的增殖以及成骨分化增加；動物實驗證實,照射低能量激光可以提高牙齒移動速度,促進(jìn)骨折區(qū)新生骨生成,加速骨愈合,另外可以降低疼痛。低能量激光所指指的可見紅光和紅外光,穿透能力較強,產(chǎn)熱較少,照射過程中熱損傷效應(yīng)比較低。低能量激光廣泛的生物學(xué)作用以及創(chuàng)傷、疼痛等副作用少,可以對治療某些疾病產(chǎn)生優(yōu)越的作用。但是不同的文獻(xiàn)對于低能量激光對成骨過程的具體影響闡述并不完全一致,甚至呈現(xiàn)相反的效應(yīng)模式；對于成骨分化過程中的人骨髓來源間充質(zhì)干細(xì)胞(human bone marrow mesenchymal stem cells, hBMSCs)受到LLLR照射后其具體的生物學(xué)效用如何,LLLR通過何種生物學(xué)機制發(fā)揮作用,國內(nèi)外文獻(xiàn)報道不一�；谇捌诘膶嶒瀳蟮阑仡櫼约昂侠淼膶嶒炓蛩乜刂�,設(shè)計了本實驗。本研究通過分離人骨髓來源間充質(zhì)干細(xì)胞,探究分離以及培養(yǎng)該細(xì)胞的正確方法,摸索科學(xué)的實驗?zāi)Ｊ?建立(?)BMSCs細(xì)胞系,為將來與hBMSC相關(guān)的實驗項目做好細(xì)胞儲備。 利用635nm可見紅光(visible red, VR)和808nm紅外光(invisible red, IR)兩種不同波長的低能量激光,利用而激光管將低能量激光作為實驗因素導(dǎo)入,通過改變光照時間調(diào)節(jié)光照計量,探究人骨髓來源的干細(xì)胞在不同劑量不同波長的低能量激光照射下的反應(yīng),使用基因芯片技術(shù)(Microarray)篩選LLLR可以調(diào)節(jié)的成骨相關(guān)基因以及細(xì)胞信號通路,之后運用RT-PCR技術(shù)、western技術(shù)等在RNA水平、蛋白水平分析驗證成骨性基因的表達(dá)與激光照射之間是否存在如Microarray所示的關(guān)系,探究LLLR調(diào)節(jié)hBMSC成骨分化過程中基因表達(dá)的具體機制,為解釋LLLR可以促進(jìn)正畸治療過程中的牙齒移動以及成骨愈合提供新的證據(jù)和更好的解釋。 方法： 1、利用密度梯度離心法分離人骨髓來源的間充質(zhì)干細(xì)胞(新鮮人長骨骨髓購買于生物公司),并進(jìn)行常規(guī)貼壁培養(yǎng),P3～P5細(xì)胞用于實驗中。部分早期細(xì)胞(P1～P3)進(jìn)行低溫冷凍儲存于液氮罐中以備后期實驗使用。 2、第二實驗階段,復(fù)蘇凍存BMSCs細(xì)胞,待細(xì)胞貼壁后將培養(yǎng)細(xì)胞所使用的常規(guī)培養(yǎng)液更換為成骨誘導(dǎo)液連續(xù)培養(yǎng)2week,每隔1至2天換液一次。初次換液后即刻對細(xì)胞進(jìn)行LLLR照射處理。低能量激光波長分別為635nm,808nm,照射劑量分別為0.5J/cm2,1.0J/cm2,1.5J/cm2,2.0J/cm2,計8個實驗組；另夕BMSCs無光照處理組為對照組,共9組。加入誘導(dǎo)液2周后,收集被誘導(dǎo)分化的成骨細(xì)胞,應(yīng)用基因芯片技術(shù)(Microarray技術(shù))檢測外顯子水平上不同條件下各種基因的表達(dá)差異,篩選受LLLR影響其表達(dá)與對照相比呈現(xiàn)統(tǒng)計性差異的基因,探究骨相關(guān)基因在激光照射下的表達(dá)差異。 3、基于Microarray的實驗結(jié)果,簡化實驗條件,采用808nm激光以及0,0.5J/cm2,2.0J/cm2三種激光劑量,利用RT-PCR和western技術(shù)驗證其成骨過程相關(guān)基因表達(dá)和蛋白合成的差異,以驗證低能量激光對干細(xì)胞成骨基因分化表達(dá)的影響作用。 結(jié)果： 1、從新鮮長骨骨髓中成功分離出人骨髓間充質(zhì)干細(xì)胞并子啊實驗室建立該細(xì)胞系,部分早期hBMSCs細(xì)胞凍存?zhèn)溆谩?2、通過收集9種條件下各個實驗組以及對照組的全細(xì)胞RNA,利用基因芯片技術(shù)分析外顯子水平的基因表達(dá)情況。可知,不同波長、不同照射計量的激光對骨髓來源間充質(zhì)干細(xì)胞成骨分化過程的基因表達(dá)影響不同,隨著改變激光照射條件,基因表達(dá)呈現(xiàn)明顯劑量依賴性,其中RANKL、IL-6、OPG、CTSK等基因的差異最為明顯,且在紅外波段表現(xiàn)出線性特點。同時,由基因芯片專業(yè)軟件繪制信號通路圖譜分析,不論635nm波段還是808nm波段的激光處理,均可以對不同的信號通路產(chǎn)生生物學(xué)效應(yīng),有的呈現(xiàn)因激光照射表達(dá)增加的上調(diào)效應(yīng),有的則呈現(xiàn)下調(diào)效應(yīng)表達(dá)相應(yīng)的減少；尤其以紅外波段808nm激光照射條件下,TGF-β1信號通路的上調(diào)效應(yīng)最為顯著, 3、基于基因芯片的實驗結(jié)果,紅外波段激光照射生物學(xué)效應(yīng)較可見光波段更為明顯。在實驗設(shè)計范圍內(nèi),LLLR對不同基因的表達(dá)調(diào)節(jié)作用呈現(xiàn)線性調(diào)節(jié)作用,故而選用0.5J/cm2,2.0J/cm2兩個實驗劑量以及0J/cm2作為空白對照設(shè)計實驗,處理成骨誘導(dǎo)過程中的hBMSCs, RT-PCR實驗結(jié)果顯示,對于不同的激光照射劑量,除了OCN、RANKL兩種基因的表達(dá)在實驗組內(nèi)部不存在統(tǒng)計性差異以外,Runx2、ALP、OCN以及RANKL的表達(dá)任兩組間相比較即存在統(tǒng)計學(xué)差異,照射時間增加激光的正向調(diào)節(jié)更為明顯。蛋白水平上,Western實驗進(jìn)一步驗證808nm的低能量激光對骨相關(guān)基因具有明顯的上調(diào)作用,與RT-PCR的驗證結(jié)果相一致,共同驗證了808nm特定波長的激光能夠促進(jìn)成骨相關(guān)基因表達(dá)。 結(jié)論： 1、特定波長特定能量的低能量激光照射,都可以激發(fā)一系列不同的基因差異性表達(dá)。在眾多的基因中,部分基因可以對各種不同條件激光的照射均產(chǎn)生生物學(xué)效應(yīng)；部分則只能對某種特定激光照射條件有反應(yīng)。類似的是,另有部分基因在不同激光劑量的條件下,也存在互相重疊的現(xiàn)象。 2、808nm的紅外波長條件下,在一定的激光劑量內(nèi),組織再生相關(guān)基因呈現(xiàn)出照射劑量依賴性特征,而這一特點在具有反向調(diào)節(jié)作用的基因表達(dá)上特征不明顯。Ingenuity軟件分析,基因之間相互調(diào)節(jié)各種作用互相交叉,激光照射可以起到各個通路的調(diào)控作用,包括上調(diào)和下調(diào)。 3、808nm紅外光譜照射后,BMSCs細(xì)胞誘導(dǎo)成骨過程中,其成骨相關(guān)基因以及破骨相關(guān)基因表達(dá)都有增加,進(jìn)一步證明激光對于成骨、破骨過程的生物學(xué)調(diào)控作用。但是鑒于實驗的設(shè)計以及時間的限制,尚且無法得出確定性結(jié)論激光如何準(zhǔn)確的調(diào)控成骨相關(guān)通路,這一機制值得繼續(xù)研究。
[Abstract]:Objective:
In recent years, low-level laser irradiation (LLLR) has been reported to have good biological effects, including photobiological promotion, photobiological regulation and so on. In addition, laboratory studies have shown that irradiation of LLLR osteoblasts increases proliferation and osteogenic differentiation; animal experiments have shown that irradiation of low-energy laser can improve tooth movement, promote bone regeneration in fracture areas, accelerate bone healing, and reduce pain. Low-energy laser has a wide range of biological effects, as well as less side effects such as trauma and pain. It can have a superior effect on the treatment of some diseases. However, the specific effects of low-energy laser on the osteogenesis process are described and discussed in different literatures. The biological effects of human bone marrow mesenchymal stem cells (hBMSCs) irradiated by LLLR are inconsistent or even reversed. There are different reports on the biological mechanism of LLLR. In this study, we isolated human bone marrow-derived mesenchymal stem cells, explored the correct methods of isolation and culture of the cells, explored the scientific experimental model, established (?) BMSCs cell lines, and made a good cell reserve for future experimental projects related to hBMSC.
Using 635 nm visible red light (VR) and 808 nm infrared light (IR) as two different wavelengths of low-energy laser, and using laser tube as experimental factors, the low-energy laser was introduced, through changing the light time to adjust the light metering, to explore the human bone marrow stem cells at different doses and wavelengths of low-energy laser irradiation. In response to irradiation, LLLR-regulated osteogenesis-related genes and cell signaling pathways were screened by microarray. Then, RT-PCR and Western techniques were used to detect the relationship between the expression of osteogenic genes and laser irradiation at RNA level. Protein level analysis was used to verify the relationship between the expression of osteogenic genes and laser irradiation as shown in Microarray. The specific mechanism of gene expression during osteogenic differentiation of ganglion hBMSC provides new evidence and better explanation for LLLR promoting tooth movement and bone healing during orthodontic treatment.
Method:
1. Human bone marrow-derived mesenchymal stem cells (fresh human long bone marrow was purchased from a biological company) were isolated by density gradient centrifugation and cultured in adherent culture. P3-P5 cells were used in the experiment. Some of the early cells (P1-P3) were cryopreserved in liquid nitrogen tanks for later experiments.
2. In the second experimental stage, BMSCs cells were cryopreserved after resuscitation. After adherence, the conventional culture medium was replaced by osteogenic induction medium for 2 weeks, and the culture medium was changed every 1 to 2 days. The cells were irradiated by LLLR immediately after the first liquid exchange. The low energy laser wavelengths were 635 nm, 808 nm, and the irradiation doses were 0.5 J/cm 2, respectively. 1.0J/cm2,1.5J/cm2,2.0J/cm2,8 experimental groups and 9 control groups were treated with BMSCs without illumination on the other day.After adding induction solution for 2 weeks, the differentiated osteoblasts were collected and the expression of various genes at different exon levels was detected by microarray technique. Compared with the genes showing statistical differences, the difference in expression of bone related genes under laser irradiation is explored.
3. Based on the experimental results of Microarray, the experimental conditions were simplified. Using 808 nm laser and 0,0.5J/cm2,2.0J/cm2 laser doses, the differences of gene expression and protein synthesis related to osteogenesis were verified by RT-PCR and Western technique, in order to verify the effect of low-energy laser on the expression of osteogenic genes.
Result:
1. Human bone marrow mesenchymal stem cells were successfully isolated from fresh long bone marrow and established in laboratory. Some early hBMSCs cells were cryopreserved.
2. By collecting the whole cell RNA of each experimental group and control group under nine conditions, the gene expression at the exon level was analyzed by gene chip technology. Gene expression was dose-dependent, in which RANKL, IL-6, OPG, CTSK and other genes showed the most obvious differences, and showed linear characteristics in the infrared band. At the same time, the signal pathway map was drawn by the professional software of gene chip, which could produce biology for different signal pathways, regardless of 635 nm or 808 nm band laser treatment. Some of them showed up-regulation effect due to the increase of laser irradiation expression, while others showed down-regulation effect, especially under the condition of 808 nm infrared laser irradiation, the up-regulation effect of TGF-beta 1 signaling pathway was the most significant.
3. Based on the experimental results of gene chip, the biological effect of laser irradiation in infrared band is more obvious than that in visible band. In the experimental design range, LLLR regulates the expression of different genes linearly. Therefore, two experimental doses of 0.5J/cm2, 2.0J/cm2 and 0J/cm2 were selected as the blank control design experiment to treat osteogenesis. The results of hBMSCs and RT-PCR showed that the expression of Runx2, ALP, OCN and RANKL were significantly different between the two groups except the expression of OCN and RANKL genes in the experimental group at different laser irradiation doses. At the protein level, Western assay further confirmed that 808 nm low-energy laser had an obvious up-regulation effect on bone-related genes, which was consistent with the results of RT-PCR. It was also verified that 808 nm specific wavelength laser could promote the expression of bone-related genes.
Conclusion:
1. Low-energy laser irradiation at specific wavelengths and specific energies can stimulate a series of different gene expression. Among many genes, some genes can produce biological effects under different laser irradiation conditions; others can only respond to certain laser irradiation conditions. There are overlapping phenomena under different laser doses.
Under the condition of infrared wavelength of 2,808 nm, the gene related to tissue regeneration showed dose-dependent characteristics in a certain laser dose, but this characteristic was not obvious in the gene expression with reverse regulation. The regulatory roles of pathways include up regulation and downregulation.
The expression of osteogenesis-related genes and osteoclast-related genes in BMSCs cells during osteogenesis induced by 3,808 nm infrared spectroscopy has been increased, which further proves the biological regulation of laser on osteogenesis and osteoclast. However, it is not possible to draw a definite conclusion on how accurate laser is in view of the design of the experiment and the limitation of time. The mechanism of regulating osteogenesis related pathways is worthy of further study.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R329

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本文編號：2225582