Effect Of Low-level Laser Irradiation On The Gene Expression Of Bone Marrow Stem Cells During The Osteogenic Process

Purpose:As has been reported, LLLR can show some positive photobio-stimulation effect when given to cells or tissues in vivo or in vitro. By means of reviewing papered related to this field, it has been seen that LLLR can stimulate cell growth, increase the ratio of cell proliferation, stimulate cell metabolism; also, it can regulate the regeneration of new vessels and bone turnover, control the process of inflammation. What is more, other data reported confirms LLLR can somehow accelerate the teeth movement during orthodontic treatment, improve the bone healing quality after fracture as well as reducing the pain accompanying. However, the mechanisms how LLLR works to get so many biologic effects are not clear and explanations from labs reported before they are not consistent. Meanwhile, human BMSCs when cultured with osteogenic medium can be induced to osteoblasts and all the process can be detected with lab techniques. The main purpose of this study, is to test whether by means of detecting changes of hBMSCs gene expression can help us to get more detailed information of how LLLR interacts with cells after LLLR treatment. Based on previous literature reviewed and reasonable factors involved in, we designed this project.This study aims to set up a scientific method of culturing hBMSCs by groping for a better way to isolate and subculture the cells; at the same time, store the seed cells for experiment of next steps.Low-level laser of635nm,808nm wavelengths has been used in this project, which were selected carefully due to the paper reviewing. Through changing the dosages of laser, we can get the reactions of cells by sieving genes whose expression show any difference compared with control groups using the technique of Microarray. What is more, pathway analysis can help to get more information using specific software. Afterwards, at both RNA and protein levels we can confirm the initial data from microarray and to further clarify the mechanism how LLLR stimulates the process of bone regeneration and help with explain questions that seem confusing to us derived from papers reported previously.Methods:1、Isolate hBMSCs from fresh human long bone marrow which was bought from US biology company and donated by healthy adult volunteer. The cells are cultured regularly following the protocol offered by Dr. Songtao Shi’s lab. Cells of P3-P5are used in the experiments following while some earlier passages are kept in nitrogen tank alive.2、After regular culturing, the cells are kept using osteogenic medium which refreshes every2or3days in6-well plates. Right after the first time of changing medium, irradiate the cells with lasers carefully designed in the protocol, totally9groups at this part of experiment with8test groups and1control group.2weeks after induction, all RNA are collected separately and sent out for microarray analysis to get the initial data of laser effect on hBMSCs gene expression and sieve the target genes involved in bone regeneration and remodeling.3、In view of the data from microarray, the last part of this project simplifies experiment conditions with only808nm wavelength and dosages of0J/cm2,0.5J/cm2,2.0J/cm2. After the same process of osteogenic induction, RNA and protein are harvested and get prepared for RT-PCR and western blot tests in order to confirm the data we get from microarray and further study the mechanism of LLLR interaction with cells.Results: 1. Isolate hBMSCs and set up the cell line successfully. Better the protocol of culturing hBMSCs that I got from Dr. Shi’s lab.2. By analyzing the RNA of the9groups’cells, data of microarray shows laser irradiation does serve some effects on gene expression of hBMSCs when induced with osteogenic medium. Any change of wavelengths and dosages can bring some change of gene expression, relatively. Obviously, we can see from the curve that the effect can be dose-dependent, especially at the808nm wavelength. Maps of signal pathway analysis also show there are some intersections among all the genes regulated by laser irradiation; when one gene can be affected by kinds of laser conditions, a specific laser irradiation also does effect to lots of genes.3. Based on results of microarray, the biostimulation of LLLR can be much stronger at the808nm wavelength, which was selected as the only wavelength used in the last part of experiment. Within the dosage of our project design, effect of LLLR on gene expression shows a relation linear to the laser dosages. To simplify our experiment, only two dosages of laser are used in later experiment,0.5J/cm2,2.0J/cm2, together with another control group, no laser irradiation. Results of RT-PCR show groups of cells they react differently when irradiated with lasers. Except no difference between the two test groups of OCN、RANKL, results of others do show some significantly Statistical difference. Meanwhile, it is easy to reach the conclusion that the up-regulation of gene expression depends on increasing the dosage of lasers as long as it is within the right dosage limit. Moreover, protein information from western blots also is consistent with what we have seen from microarray; IR irradiation up-regulates gene expression of hBMSCs and the more irradiation, the more change it will serve to the cells.Conclusions:1. Laser with specific wavelength, dosage, can stimulate specific gene expression of hBMSCs. And there are some genes that can show effects with similar tendencies even the irradiation conditions differ one from another. Even kinds of genes can show a similar trend toward each other, sometimes overlapping.2. At the wavelength of808nm, with a certain dosage of laser irradiation, gene expression related with tissue turnover manifests some characteristic dosage-dependent; ingenuity software analysis also shows regulations of laser irradiation can be overlapped among kinds of different genes.3. After808nm laser irradiation, during the process of bone regeneration, genes related both to osteogenic and osteoclastic, show some more transcription compared with control group that means no laser irradiation at all.However, due to the limit of time and other factors of this project design, there are still some uncertainties of how the laser affects gene expression during osteogenic process of hBMSCs. And as for the detailed mechanism, it still requires more effect from researchers internationally.