Low intensity He-Ne laser therapy (eg: intravascular laser irradiation on blood, ILIB) has been wildly used to treat with some illness in clinic but the mechanism remain obscure. Laser power, the irradiation time and the irradiation dose were changed. Laser scanning confocal microscope (LSCM) was used to real-time research the effort of low intensity irradiation on {Ca2+}I in mouse marcophage and single photon counter device (SPC) was used to detect the MCLA enhanced chemiluminescence of macrophage irradiation with low intensity He-Ne laser. Proper dose of low intensity He-Ne laser irradiation can increase the [Ca2+]I of the macrophage and immunocompetence of macrophage effectively. Laser power of 0.16mw is more effect to increase the [Ca2+]i than laser power of 0.40mw. The [Ca2+]i is markedly different even the irradiation dose or the irradiation time is same. Both the irradiation dose and laser power are key factor for the change of [Ca2+]i and immunocopetance of macrophage. Activating ammuno-system of body is one of the very important mechanisms of Low-intensity He-Ne laser therapy.
The green fluorescent protein (GFP), from the bioluminescent jellyfish Aequorea victoria, yields a bright green fluorescence when expressed in either eukaryotic or prokaryotic cells and illuminated by blue or UV light. The characteristic properties of GFP make this protein a good candidate for use as a molecular reporter to monitor patterns of protein localization, gene expression, and intracellular protein trafficking in living cells. In this study, the plasmid EGFP encoding GFP was used to transfect SWO cells (a cancer cell line of nerve gelatinous tissue) mediated by liposome: (1) The plasmid EGFP-C1, purchased from Clontech Co., propagated in suitable E. coli strain (JM 109), was extracted by Concert High Purity Plasmid Miniprep (Gibco). (2) SWO was cultured in RPMI 1640 (10% FCS and 25 mM HEPES), 37 degree(s)C, 5% CO2. Cancer cells were transfected in 6-cm tissue culture dishes by Lipofectin Reagent (Gibco) for 6-12 hr using 2 ug DNA. (3) Then, infected cells were collected in medium containing 800 ug/ml G418, and the resistant clones were harvested and subcloned in fresh culture medium maintaining 800 ug/ml G418. (4) The cells were examined by using Nikon fluorescent microscope (E600) and Bio-Rad confocal microscope (MRC 600). (5) Next step, the cancer cells, stably expressing GFP after in vivo transduction, were implanted by surgical orthotopic implantation (SOI) in nude mice. Tracking of these cancer cells will become more sensitive and rapid than the traditional procedure of histopathological examination or immunohistochemistry. This method demonstrates external, noninvasive, whole-body, real-time fluorescence optical imaging of internally growing tumors and metastases in transplanted animals.
Low level He-Ne laser irradiation (e.g., intravascular laser irradiation on blood, ILIB) has been widely used to treat some illness clinically. One of the action processing is by improving immunocompetence of the body, but the mechanism is still not clear. In this paper, we used mouse macrophages as study object for real-time observation of the concentration change of intracellular free calcium (abb. as [Ca2+]i) of macrophages and the distribution of the Ca2+ using LSCM (laser scanning confocal microscope) after irradiation with different dose low level He-Ne laser (the actual irradiation power (abb. as P) is 0.16 mw and the facular diameter (abb. as D) is 0.5 cm. We found interesting phenomena through the image of [Ca2+]i of macrophages. The distribution of Ca2+ in macrophages has no obvious change when the time of irradiation (abb. as Ti) is 10 min. The [Ca2+]i shows obvious increase and a concentrate phenomena in Ca2+ distribution appears when Ti reach 20 min. The change of the distribution of Ca2+ become more distinct: [Ca2+]i increase more visibly and the distribution of Ca2+ shows a spacial grads when Ti reach 30 min.
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