The Application Research Of Early Embryos Activity Crud Extracton Mouse Tissue Wound Repair

This research aims to verify whether the early chick embryo cell actives can be used for clinical application in terms of skin damage repair and substitute the mammalian embryonic stem cells.In the experiment,firstly,the breeding eggs will be hatched,then the extracted early chick embryo cell activity can be prepared as salves which apply to animal wound test.Through homology alignment,this experiment concludes that chicken and mice have a high degree of homology in protein active ingredient about wound healing.The significance of this experiment is to evaluate the clinical effect of embryo stem cell activity and provide new ideas and references for clinical application.1.Separate early chicken embryo cells and extract active substanceTo determine the optimum incubation time and conditions,this study adopts fresh eggs at 37℃,humidity of 55% in an incubator,and adopts machine spoon method to select hatch eggs for 0h,24 h,48h,72 h to isolate primary cells.This research uses the Wright-Giemsa staining fluid to observe the extracts under the microscope.The results show that the forms of early chick embryo cells are rounded,spherical,the cell is small,but nuclear is much larger.Moreover,the best time to separate eggs’ cell is at 72 h,and cell collection rate is the highest.In order to determine how to extract the chicken embryo primary cells,at first,the chicken embryo primary cells have been frozen and thawed three times in selected liquid nitrogen under the circumstances of-80 refrigerator and ℃-20 refri℃ gerator,second the cells are melted at 37 constant ℃temperature water bath.By comparing and combining references found that there is no otherness in liquid nitrogen and-80 ℃refrigerator,the rate of cell lysed at 87%,85%;while there is difference as opposed to-20 is 70%.Considering operation and ℃cost,this test selected-80℃as freezing temperature.2.The determination of ointment baseIn order to explore the prescription and preparation technology of O/W type ointment.By appearance,cold resistant,heat resistant test,centrifuge test and inspect p H as indicator,also select suitable emulsifier,then determine the best prescription through orthogonal experiment.Optimum preparation is to take stearate 10 g,white petrolatum 5g,lanolin 1.5g,borneol 1.5g,liquid paraffin 3g as the oil phase,and heat to 80℃,then Weigh glycerol 5g,Tween-805 g,triethanolamine 0.7g,ethyl paraben 0.06 g,add distilled water to adjust oil and water two phases to 100 g,and heat to 85℃.After the prescription completely dissolved,the oil phase will be slowly added to the aqueous phase,stirring constantly until completely emulsified,when the substrate temperature drops to about 40 ℃,add 10 g extracts to matrix.Finally,irritation experiment can be tested on live animal broken skin models with painted ointment,the result shows that this ointment has no irritation for skin and can be used in animal clinical trials.3.Establish an animal trauma modelFor the sake of studying the early embryo cell actives ointment effect on wound healing,Kunming mice burn,knife cut,and eye injury model have been constructed,and the early chick embryo cell active ointment smears on the wound surface.The results show that: in the burn model,apply early chick embryo cell active ointment to heal wounds faster than erythromycin ointment group and the control group,in the 12 days,the area of the wound smear early chick embryo cell active paste group was 0.62cm2;wound healing was 60.16%;the control group of the wound area was 1.13cm2;wound healing rate was 26.50%;and the difference was extremely significant(p <0.01).While in the knife wounds model,apply early chick embryo cell paste active group compared with erythromycin ointment group and the control group,its wound heal faster in the former.At 7 days,the wound length is 1.10 cm by applying homemade ointment group,there is no distinct differences of the erythromycin ointment group which wound length is 1.35 cm.By comparison the control group wounds was 1.58 cm,and the difference was significant(p <0.05).In the eye injury model,dropping early chick embryo cell active extract to the eye could repair,inhibit inflammation,and promote hair growth.In this test,by using different modes of drug administration,such as subcutaneous injection,eye drop and smear,to explore the effects of early chick embryo cell activity on mouse body,measuring body temperature of mice,the temperature fluctuations are within 0.5 ℃,also fevers,allergies and other immune rejection phenomena do not appear.In consequence,although early chick embryo cell active is xenobiotics,a big race difference exists with the mouse,still by the protein sequence alignment,b FGF homology up to 90%,EGF and SCF is 53%,VEGF is 78%,IGF to 76%,TGF-β is 56%,PDGF is 80%,the cells of the active ingredient has a high degree of homology,and could promote wound healing effectively,meanwhile it can be applied to clinical animal experiments.