低能量激光对骨髓来源干细胞成骨分化过程基因表达的影响
发布时间：2018-09-06 06:51
【摘要】：目的： 近些年,低能量激光照射(low level laser irradiation, LLLR)陆续被报道具有良好的生物学作用,包括光生物学促进作用、光生物学调节功能等。通过文献回顾可知,LLLR不仅能够促进细胞生长,增强细胞代谢和细胞增殖,而且可以调节新生血管的生成、改善炎症状态。另外,实验室研究结果显示,通过照射LLLR成骨细胞的增殖以及成骨分化增加；动物实验证实,照射低能量激光可以提高牙齿移动速度,促进骨折区新生骨生成,加速骨愈合,另外可以降低疼痛。低能量激光所指指的可见红光和红外光,穿透能力较强,产热较少,照射过程中热损伤效应比较低。低能量激光广泛的生物学作用以及创伤、疼痛等副作用少,可以对治疗某些疾病产生优越的作用。但是不同的文献对于低能量激光对成骨过程的具体影响阐述并不完全一致,甚至呈现相反的效应模式；对于成骨分化过程中的人骨髓来源间充质干细胞(human bone marrow mesenchymal stem cells, hBMSCs)受到LLLR照射后其具体的生物学效用如何,LLLR通过何种生物学机制发挥作用,国内外文献报道不一。基于前期的实验报道回顾以及合理的实验因素控制,设计了本实验。本研究通过分离人骨髓来源间充质干细胞,探究分离以及培养该细胞的正确方法,摸索科学的实验模式,建立(?)BMSCs细胞系,为将来与hBMSC相关的实验项目做好细胞储备。 利用635nm可见红光(visible red, VR)和808nm红外光(invisible red, IR)两种不同波长的低能量激光,利用而激光管将低能量激光作为实验因素导入,通过改变光照时间调节光照计量,探究人骨髓来源的干细胞在不同剂量不同波长的低能量激光照射下的反应,使用基因芯片技术(Microarray)筛选LLLR可以调节的成骨相关基因以及细胞信号通路,之后运用RT-PCR技术、western技术等在RNA水平、蛋白水平分析验证成骨性基因的表达与激光照射之间是否存在如Microarray所示的关系,探究LLLR调节hBMSC成骨分化过程中基因表达的具体机制,为解释LLLR可以促进正畸治疗过程中的牙齿移动以及成骨愈合提供新的证据和更好的解释。 方法： 1、利用密度梯度离心法分离人骨髓来源的间充质干细胞(新鲜人长骨骨髓购买于生物公司),并进行常规贴壁培养,P3～P5细胞用于实验中。部分早期细胞(P1～P3)进行低温冷冻储存于液氮罐中以备后期实验使用。 2、第二实验阶段,复苏冻存BMSCs细胞,待细胞贴壁后将培养细胞所使用的常规培养液更换为成骨诱导液连续培养2week,每隔1至2天换液一次。初次换液后即刻对细胞进行LLLR照射处理。低能量激光波长分别为635nm,808nm,照射剂量分别为0.5J/cm2,1.0J/cm2,1.5J/cm2,2.0J/cm2,计8个实验组；另夕BMSCs无光照处理组为对照组,共9组。加入诱导液2周后,收集被诱导分化的成骨细胞,应用基因芯片技术(Microarray技术)检测外显子水平上不同条件下各种基因的表达差异,筛选受LLLR影响其表达与对照相比呈现统计性差异的基因,探究骨相关基因在激光照射下的表达差异。 3、基于Microarray的实验结果,简化实验条件,采用808nm激光以及0,0.5J/cm2,2.0J/cm2三种激光剂量,利用RT-PCR和western技术验证其成骨过程相关基因表达和蛋白合成的差异,以验证低能量激光对干细胞成骨基因分化表达的影响作用。 结果： 1、从新鲜长骨骨髓中成功分离出人骨髓间充质干细胞并子啊实验室建立该细胞系,部分早期hBMSCs细胞冻存备用。 2、通过收集9种条件下各个实验组以及对照组的全细胞RNA,利用基因芯片技术分析外显子水平的基因表达情况。可知,不同波长、不同照射计量的激光对骨髓来源间充质干细胞成骨分化过程的基因表达影响不同,随着改变激光照射条件,基因表达呈现明显剂量依赖性,其中RANKL、IL-6、OPG、CTSK等基因的差异最为明显,且在红外波段表现出线性特点。同时,由基因芯片专业软件绘制信号通路图谱分析,不论635nm波段还是808nm波段的激光处理,均可以对不同的信号通路产生生物学效应,有的呈现因激光照射表达增加的上调效应,有的则呈现下调效应表达相应的减少；尤其以红外波段808nm激光照射条件下,TGF-β1信号通路的上调效应最为显著, 3、基于基因芯片的实验结果,红外波段激光照射生物学效应较可见光波段更为明显。在实验设计范围内,LLLR对不同基因的表达调节作用呈现线性调节作用,故而选用0.5J/cm2,2.0J/cm2两个实验剂量以及0J/cm2作为空白对照设计实验,处理成骨诱导过程中的hBMSCs, RT-PCR实验结果显示,对于不同的激光照射剂量,除了OCN、RANKL两种基因的表达在实验组内部不存在统计性差异以外,Runx2、ALP、OCN以及RANKL的表达任两组间相比较即存在统计学差异,照射时间增加激光的正向调节更为明显。蛋白水平上,Western实验进一步验证808nm的低能量激光对骨相关基因具有明显的上调作用,与RT-PCR的验证结果相一致,共同验证了808nm特定波长的激光能够促进成骨相关基因表达。 结论： 1、特定波长特定能量的低能量激光照射,都可以激发一系列不同的基因差异性表达。在众多的基因中,部分基因可以对各种不同条件激光的照射均产生生物学效应；部分则只能对某种特定激光照射条件有反应。类似的是,另有部分基因在不同激光剂量的条件下,也存在互相重叠的现象。 2、808nm的红外波长条件下,在一定的激光剂量内,组织再生相关基因呈现出照射剂量依赖性特征,而这一特点在具有反向调节作用的基因表达上特征不明显。Ingenuity软件分析,基因之间相互调节各种作用互相交叉,激光照射可以起到各个通路的调控作用,包括上调和下调。 3、808nm红外光谱照射后,BMSCs细胞诱导成骨过程中,其成骨相关基因以及破骨相关基因表达都有增加,进一步证明激光对于成骨、破骨过程的生物学调控作用。但是鉴于实验的设计以及时间的限制,尚且无法得出确定性结论激光如何准确的调控成骨相关通路,这一机制值得继续研究。
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R329
本文编号：2225582
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R329
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