Screening Of The Active Ingredients Of Traumatic Healing And Its Mechanism In The Preserving Stage Of The Body

Objective:On the basis of pre-experiment research and the hypothesis of "Amputated lumbricus", to screen the optimum broken time point of earthworm, and study on the influenceing factors of the stability of the earthworm extract.Research the detach earthworm active ingredient, to explore the active components of earthworm and promote the mechanism of wound healing.Method:1.Screening and validate the optimum broken time points (0-7d) on HaCaT cells using the CCK-8 method.Using the HPCE and SDS-PAGE method to compare the different between earthworm samples broken in different time.2. Establish tissue repair cells (fibroblasts, epidermal cells, vascular endothelial cells) culture method and research the influence of the sample on the growth, examined the cells under the microscopic and use of the CCK-8 assay and measurement the Cell viability, the calculation of the rate of each sample to repair cells proliferation, and draw the proliferation rate curve, analysis of samples of cells to repair the expression.3. Compared with the activity of samples treated by different pH on NIH3T3 cells by CCK-8 method, draw the pH and proliferation rate curve; Compared with the activity of samples treated by different temperature on NIH3T3 cells by CCK-8 method, drawing temperature and proliferation rate curve observed range of temperature tolerance of protein activity.4.The earthworm samples has been salting out into 4 parts, through the comparison of the rate of the proliferation of HaCaT cells and NIH3T3 cells, choose the effective parts, using mouse skin linear trauma in vivo screening, to observe the wound healing process between the treatment group and control group, and observed by HE staining and immuno histochemistry to observe the wound tissues of healing,through and analysis the expression of FGF and VEGF, explore the earthworm and promote the mechanism of wound healing.Result:1.Broken earthworm extraction liquid played a role in the repair material of HaCaT cells, and in the 0-5 days, the proliferation effection was better, after 6 days, the proliferation effection turned weakened, the repair material produced in different time was different, the effect of DL3 was the best. This part of the study of brokon body on different days of the earthworm extract differences in composition, through the HPCE and SDS-PAGE results, the earthworm extract liquid on different days are some different in composition and content, however, the specific components of any likelihood to be further studied.2. Broken body of Earthworm have certain proliferation on NIH3T3 cells and HaCaT cells, may be related to the promote wound healing in a certain relationship. While the earthworm extract had no obvious effect on the proliferation of HUVEC cells.3. In order to maintain the stability of the earthworm extract, the earthworm extract at low temperature, neutral pH environment preservation. The earthworm future into processing and storage of resistance to do a preliminary research,it is useful for the large number of production and storage laid the foundation.4.Through the result of HE and immuno histochemistry, the two parts which were DL3-1 and DL3-4 made the mice recovered well and expression of FGF and VEGF were significantly different compared with the control group, then use SDS-PAGE to analysis the protein molecular weight range of DL3-1 and DL3-4.