Response Of Mast Cell To The Physical Stimuli And The Effective Mass Density Of Liquid Composite

As a safe, convenient and cheap method to cure many kinds of diseases, acupunture has been accepted by more and more west countries. With the development of modern science and technology, acupuncture expands to a new area such as laser acupuncture. However, neither the needle acupuncture nor the laser one experiences a hard way to the West Country. One of the main reasons is that the mechanism how acupuncture works is still unknown. Among the researches relative to the mechanism, the mast cell gets much attention. The past researches, e.g., mast cell and calcium accumulating in acupoints, and degranulation of mast cell increasing with the acupuncture needle stimulated in the acupoint, and the analgesic effect of acupuncture decreasing when the degranulation of mast cell inhibited by sodium cromoglycate, showed that mast cell and calcium were related to the effect of acupuncture. But by far, it is lack of the direct evidence to prove the degranulation of mast cell induced by laser irradiation and shear stress.To achieve the "needle grasp (deqi)", the needle manipulation is necessary, which is composed of pulling, inserting and rotating the needle. Essentially, the manipulation is the shear stress exerted on the tissue and cells in the acupoint. So, in this paper, with the scanning Confocal microscopy and fluorescent spectra, the effects and mechanism of laser irradiation and shear stress on the intracellular calcium ([Ca²⁺]_i) and histamine release in RBL-2H3 mast cell were studied. On the other hand, due to the vessel relaxation of histamine released by mast cell degranulation, the blood plasma and cytoplasma of mast cell are seeped and mixed with the interstitial fluid, and then the directional flow of the interstitial fluid will be influenced. So the effective mass density of the liquid composite was studied. The details are as follows:Firstly, RBL-2H3 cells were co-incubated with the calcium fluorescent probe, Fluo-3/AM, and a laser with wavelength of 405 nm was irradiated on the single cell. One minute later, the [Ca²⁺]_i, was observed. The result shows that [Ca²⁺]_i concentration increases significantly, with a change ratio of 56.6% compared with the baseline. To assay the histamine release, the RBL-2H3 cell in 96-well plate was irradiated with different time and different power, and the supernatant was mixed with o-phthalaldehyde (OPA) and the fluorescent spectra of the mixture were measured. The spectrum results show that the histamine in the supernatant enhances markedly in a dose-effect manner. The laser, with the irradiation time and power used in the present experiment, has no effect on the cell viability, which shows that the change of histamine in the extracellular solution does not come from the leakage of the cell membrane due to the damaged cells. So, the laser irradiation can induce the increase of [Ca²⁺]_i and degranulation to release histamine in the RBL-2H3 mast cell.Secondly, to study the response of RBL-2H3 to the shear stress, a plate-plate chamber was used. The shear stress was produced by the rotor, which was driven by the stepping motor. The experiment result shows that, under the shear stress, the fluorescence intensity of the [Ca²⁺]_i increases gradually and goes to the stabilization. The increase of the [Ca²⁺]_i is dependent on the extracellular calcium. When the extracellular solution is Ca-free, the [Ca²⁺]_i, no more increases, although the same shear stress exerted. This shows that the increase of [Ca²⁺]_i results from the influx of the extracellular calcium. The histamine spectra was conducted with the same method as that of laser irradiation, and the result shows that shear stress can induce the RBL-2H3 cell degranulate to release histamine in a shear rate-effect manner too.Thirdly, the mechanism of the cell effects (intracellular calcium and degranulation to release histamine) induced by laser irradiation and shear stress was compared. Theoretical calculation result shows that the optical stress of the laser irradiation on the single cell surface is 5 orders smaller than that of the shear stress, so, the cell effects of laser irradiation should not come from the optical stress directly. To further study the mechanism of laser irradiation, the transient receptor potential vanilloid 4(TRPV4) channel in the RBL-2H3 cell was verified by immunocytochemistry. The result indicates abundant of TRPV4 expresses on the plasma membrane (nuclear membrane included). The TRPV4 is a non-selective cation, especially for calcium, channel activated by many physical and chemical stimuli. So, it is very possible that the TRPV4 channel is one of the targets responses to the laser and shear stress.Then, the research whether the TRPV4 involves in the response of RBL-2H3 to the laser irradiation and shear stress was conducted. When ruthenium red (RR), the blocker of TRPV4, was added into the extracellular solution, and then the laser irradiation and shear stress were exerted on the RBL-2H3 cells, it was found that the histamine release was inhibited by RR in a dose-effect manner. So, it is concluded that TRPV4 channel plays an important role in the response of RBL-2H3 to the laser irradiation and shear stress and the pathway is: by the laser irradiation and shear stress, the TRPV4 is open and the extracellular calcium influx to increase the [Ca²⁺]_i concentration, then the increase of [Ca²⁺]_i induces the RBL-2H3 cell degranulation to release histamine.Finally, started from the effective medium concept, new methods to predict the effective mass density of liquid composites were presented too. At first, three solutions (deioned water, ethanol and dimethyl sulphoxide (DMSO) were used to study the effective mass density of every two different liquids with different mass ratio, or, the mass fraction of one component being fixed, and the other two adjusted accordingly with various mass fraction. Secondly, started from the effective medium concept, the Maxwell-Garnett and Bruggeman formula were transformed and by mathematical analog, the new formulas of liquid composites were presented. In the new formula, an effective medium parameter d was introduced. The effective medium parameter was a phenomenogical parameter and depended on the chemical nature of components and the operating temperature as well as the pressure of air. Then, the experiment results were fitted by the new formula and the traditional VAMD. When fitted by the new formula, one experimental data was chosen to extract an effective medium parameter d. Then, by using the same d, all the other data were calculated and compared with the results of VAMD. The results show that, for liquid composites, the new method can predict the effective mass density very well.In summary, through the TRP channel, the physical stiluli, such as laser irradiation and shear stress, exert on the mast cell and induce the intracellular calcium concentration increasing, and then the degranulation and histamine released are followed.