The Composite Skin Wound Healing And Fibroblasts. The Main Ingredients Of Astragalus On Immune Suppression

Astragali is " panacea of the sore family " ,which promote vital energy and elimination the poison to promote tissue regeneration, the favourable water subsides swelling, benefit defends efficiency such as the firm form. Treat qi and blood on clinic insufficiently, suppuration falling into ulcer of sore becomes the routed or routed chronic wound not held back for a long time. Cell proliferation-promoting and collagenic supersession play an essential role not normally in the course of healing in wound. The wound surface formates ability to reduce collagenically and decompose collagenically excessively inside, become the difficult important reason to heal of wound surface. Clinic and animal's trial prove traditional Chinese medicine treated the curative effect of ulcer of chronic skin prominently. In order to explore the mechanism Astragali promote wound healing ,this lecture use oral astragali and APS external application,and base on NIH3T3cell,explore the effect on proliferation promoting and synthesis of cllogan.In order to established the principle and scientific evidence of Chinese Medicine of cure chronic wound.The thesis is studied and divided into two following parts to go on:1 Experiment in vivo1.1 The wound with immunosuppressive mice induced by systemic hydrocortisone Characteristic with Yang and Cold Deficiency in TCMThis thesis prepares immunity and ISW animal model of the skin wound at first. Cause the immunity to inhibit the state with the Balb/c mouse of cortisone treatment of hydrogenation. Adopt thick cover excise skill on the back. And the general situation before and after observing Balb/c 's wound determines the healing rate of wound area dynamically.Fetch the wound tissue and observed the histologic change scarletly by HE and bitter acid-staining. The result demonstrate:(1)After injecting cortisone of hydrogenation intramuscularly for 7 days, group's Balb/c mouse's general situation of the model is relatively bad, appear weight decline, maos of color matt dispirited hunchbacked to move, be slow in reaction. The Balb/c mouse of normal group is generally in sound condition, do not have the above-mentioned symptoms. Compound behind the wound, model group little mouse above symptom aggravate , each other hoarse to grip, even a few dies. (2)When 3 days after we practice the surgery,control group and healing rate of group's wound area of model do not have obvious difference; wound area of the model is obviously greater than the control group, the healing rate of wound area has obvious difference (P<0.05).The healing rate of wound of control group is obviously higher than the model group.(3) Observe under the microscope: When 3 days after we practice the surgery, HE and observe control group wound have a large number of inflammation nature cell soak, model group wound surface inflammatory cell relatively sparse in Balb/c mouse;The control group can seeⅠtype collagenic and green type collagenic day, model group type be obviously less than the control group; When 7 days after we practice the surgery,HE and bitter acid-staining observe control group peripheral inflammation cell soak, have appeared granulation organize. Control group and model group type and increase collagenically, group'sⅢtype of the model is less than the control group collagenically. The result suggest the mice is in a immunosuppressive status after skin wound ,the mice is still in a status of suppressive, characteristic with Yang and cold deficiency in TCM.1.2 The effects of APS on wounding healing of mice and the mechanism studiesThe experiment chooses 40 Bal/c mouse, is divided into control group, model group, high dosage at randomly ,Measure the wounding area interval two days until the wound healing. The result demonstrate: When 3 days after we practice the surgery,there is no obvious different between conrol grouP and model group on the area of wound. After 6 days after we practice the surgery, it has obvious different between conrol group and model group(P<0.05).The rate of wounding rate control group is higer than the model group. After 6 days after we practice the surgery, it has obvious different between conrol group and model group(P<0.05).The rate of wounding rate control group is higer than the model group.The result of observation under microscope: When the wound surface of control group heals, wound surface of the model has inflammation nature cells to soak,Astragali water decoction high dosage group, hit dosage group, low dosage group's wound surface can see the new born capillary and become fibrous cells, take shape with the new born epidermis, particularly obvious with the high dosage.1.3 The effect on wound healing with APS external applicationThis experiment chooses 40 Bal/c little mouse, is divided into control group, model group, high dosage group (0.8g/L) at random ,moderate dosage group (0.4g/L) , low dosage group(0.2g/L) .The result demonstrate: The day we practice the surgery , it has no obvious different between control group and the model group in wound area. When 3 days after we practice the surgery,there is no obvious different between conrol group and model group on the area of wound. After 6 days after we practice the surgery, it has obvious different between conrol group and model group(P<0.05).The rate of wounding rate control group is higer than the model group. After 12 days after we practice the surgery, it has obvious different between conrol group and model group(P<0.001).The rate of wounding rate control group is higer than the model group. The result of observation under microscope: Model group has a little inflammation nature cells to soak; Astragali many candy high dosage have inflammation nature cell soak, can see angle take layer; When China's dosage heals, the epidermis structure has already basically taken shape, has had a large number of inflammation nature cells; It is collagenic that there are new students in the low dosage group.2 Experiment in vitro2.1 The effects of APS on fibroblast proliferation-promoting and immigrationThe experiment is base on NIH3T3 cell model ,use the MTT method test proliferation-promoting.After cultured with the tested drugs for 48 hours, the concentration of AstragalosideⅣis higer than 156μg/ml,it has an distinctly different effect of inhibition(P<0.001);When the concentration APS is between 9.75μg/ml and 19.5μg/ml,it has an distinctly different effect of promoting (P<0.01) When the concentration APS is 19.5μg/ml the proliferation-promoting rate is 23.06%; After cultured with the tested drugs for 24 hours, When the concentration APS is between 19.5μg/ml and 78μg/ml,it has an distinctly different effect of promoting(P<0.01)When the concentration APS is 39μg/ml the proliferation-promoting rate is 30.51%,it is the peak of proliferation promoting rate;The group of bFGF has an distinctly different effect of promoting. After cultured with the tested drugs for 72 hours, when the concentration APS is 312μg/ml,it has an distinctly different compare with the control group(P<0.001),it has an effect of inhibition predominantly.In the range of concentration ,it has no distinctly different. The above-mentioned result demonstrate: it has dose-effect relationship between the concentration APS and fibroblast proliferation promoting.The result hint we shoud paid attention to time and concentration when we use Astragali in clinical.2.2 The effects AstragalosideⅣon fibroblast proliferation- -promotingWe use MTT method to measure the effection AstragalosideⅣon fibroblast proliferation-promoting. Add with MTT law medicine at the 48h,the concentration of AstragalosideⅣis 10μg/ml and 5μg/ml, the OD value of AstragalosideⅣgroup is higer than the contol group.But there no distinctly different between the contol group and AstragalosideⅣgroup. After 72h we add AstragalosideⅣ, the concentration of AstragalosideⅣis 20μg/ml , the OD value of AstragalosideⅣgroup is distinctly higer than the contol DMSO group(P<0.05), the concentration of AstragalosideⅣis 10μg/ml and 5μg/ml, the OD value of AstragalosideⅣgroup is higer than the contol group. Combination the result of 48h and 72h,The power of AstragalosideⅣon fibroblast proliferation promoting is increasing accompany time is going.When the concentration of AstragalosideⅣis increasing, the power of AstragalosideⅣon fibroblast proliferation-promoting is decreasing.2.3 The effects of APS and AstragalosideⅣon synthesis of collagenMeasure the Radix Astragali impact on NIH3T3 cell's collagenic synthesis of many candies with the hydroxyproline reagent box. After add AstragalosideⅣ48h, synthesis of cllogan-promoting concentrations on 2.44μg/mL～9.75μg/mL. When the concentration of AstragalosideⅣis 20μg/ml,the OD value of AstragalosideⅣgroup is higer than the contol group(P<0.05). When the concentration of AstragalosideⅣis 10μg/ml and 5μg/ml, the OD value of AstragalosideⅣgroup is higer than the contol group.But there no distinct different between the contol group and AstragalosideⅣgroup. The result demonstrate : the concentration of AstragalosideⅣhas an effect on synthesis of cllogan.This thesis prepares little mouse's immunity and inhibits and compounds the animal model of the skin wound at first, and carry on experiment research on this basis. The result demonstrate : It accords with the empty cold wound surface model that the immunity inhibits and compounds the skin wound model. Oral medication can accelerate the healing of wound,it's mechanism is to improve little mouse's healthy tendency and immunity. External application of APS can also accelerate the healing of wound, mechanism maybe improve the physical and chemistry status and osmotic pressure of wound and generate the level local inflammatory factor and the count of MΦcell. Astragali many candies can be promoted into hyperplasia and collagenic formation of fibrous cells within the range of certain density, the ones that strengthened the wound ground substance are synthetic in order to promote the wound to heal.