Pharmacological Properties Researches Of Oleosin-hEGF Expressed In Safflower Oil Body In Skin Wound Repair

Human epidermal growth factor?hEGF?is widely used in skin wound repair.However,its poor stability and poor transdermal absorption have severely limited its clinical application.Therefore,this subject mainly aims at its shortcoming.The hEGF gene was fused with oleosin to construct the pOTB-oleosin-hEGF expression vector,and the oleosin-hEGF protein was expressed in safflower seeds.The expression of oleosin-hEGF fusion protein can overcome the shortcomings of natural hEGF,enabling it to be stably stored in vitro,which lays a foundation for further development and utilization of hEGF.After T3 generation of hEGF safflower seeds were planted,6-8 pieces of fresh young leaves were grown,0.2g of fresh leaves were picked,and the genome of the leaves was identified by PCR.After the safflower seeds were matured,the fusion was extracted from the seeds by gradient centrifugation.SDS-PAGE and Western blot were used to identify whether the target protein oleosin-hEGF could be successfully expressed in the transgenic safflower seeds.The oleosin-hEGF protein expressed by each line was semi-quantified by Quantity One software.Subsequently,Subsequently,a mice model of in vivo transdermal absorption was prepared,and the transdermal absorption effect of oleosin-hEGF was evaluated by immunohistochemical staining and Western blotting.The activity analysis of oleosin-hEGF to promote the proliferation of NIH/3T3 cells was measured by MTT assay.Then,the rat skin model of mechanically injured was constructed.After different drugs treatment,the pharmacodynamic activity of oleosin-hEGF on rat skin wound repair was evaluated by pathological section,related gene and protein expression.The results of the c experimental studies are as follows:?1?The pure T3 transgenic safflower oil body was obtained by gradient centrifugation.The results of microstructure observation showed that the transgenic safflower oil body was evenly dispersed.The particle size distribution of the transgenic safflower oil body was determined to be between 700 and 1000nm by particle size measurement,which was significantly smaller than that of the wild type safflower oil body?1.5?m-2.5?m?.?2?The T3 generation transgenic safflower genome PCR showed a target band at342bp,which was identical to the plasmid positive control.SDS-PAGE and Western blot were used to detect oleosin-hEGF fusion protein,and the target band appeared at31kDa,which was the same as the theoretical value of the target protein.The hEGF standard protein was used as a positive control,and the oleosin-hEGF protein expressed by each line was semi-quantified by Quantity One software.The results showed that in the T3-17 strain,the maximum amount of target protein per gram of safflower seed was 69.32mg.?3?Cell proliferation efficiency was measured by using the oleosin-hEGF fusion protein expressed from T3-17 transgenic seed oil bodies.The bFGF protein is known to have a better ability to promote cell proliferation,the efficiency of NIH/3T3 cell proliferation is compared between the oleosin-hEGF fusion protein and the bFGF standard protein.The results showed that the oleosin-hEGF fusion protein promoted the proliferation of NIH/3T3 cells,which was was similar to the bFGF standard protein.While the wild-type oil body did not have significant proliferative activity.This indicates that the oleosin-hEGF fusion protein expressed from the oil body of T3-17 transgenic seeds has significant proliferative activity for NIH/3T3cells.?4?In the transdermal absorption experiment of oleosin-hEGF,the results showed that a large amount of positive protein appeared in the subcutaneous tissue of oleosin-hEGF group,and the target protein absorbed through the skin quickly.In the oleosin-hEGF group,the absorption of oleosin-hEGF protein reached the maximum at30 min after administration,while the transdermal rate was relatively slow in the hEGF standard protein-treated group,which began to be absorbed in the skin at 30min after administration.The absorption of hEGF standard protein reached the maximum amount until 60 min after administration,but due to the instability of the protein,its content in mice decreased after 60 min of administration,while the target protein permeation of oleosin-hEGF group was maintained at a high level for 30 min to 60 min.?5?In the skin injury test of mechanically injured rats,after treatment with oleosin-hEGF fusion protein and hEGF protein,wound healing and regeneration of newborn skin can be accelerated.On the 4th,7th and 14th day after treatment,the wound healing rate of oleosin-hEGF fusion protein treatment group was higher than that of hEGF treatment group?P<0.05?.After observing the pathological section of newborn skin,it was found that in the oleosin-hEGF fusion protein-treated group,the regeneration of the connective tissue from the subcutaneous neoplasia,the integrity of the structure of the newborn skin,the amount of collagen deposition,the proliferation of epidermal cells,and the regeneration of new blood vessels were better than blank control group,wild type safflower oil treatment group and hEGF standard protein treatment group.At the same time,total RNA and total protein were extracted from newborn skin tissue,and the expression of endogenous TGF-?1,bFGF and VEGF were detected by qRT-PCR and Western blotting.The results showed that TGF-?₁,bFGF and VEGF all increased from 4d to 7d in the whole process of wound repair,and reached the maximum on the 7th day,then they decreased from 7th to 14th day,and returned to normal level on the 14th day.This indicates that the oleosin-hEGF fusion protein has a better role in promoting wound repair.