Study On The Purification And Characterization Of Fibrinolytic Active Protein Of American Cockroach

Periplaneta Americana L.is the scientific name of the Chinese medicine cockroach,also known as lady sweet,negative plate,tea vermina,etc.Among medicinal insects,cockroach has a wide range of species,and is rich in trace elements,proteins,essential amino acids,peptides and other nutrients.Ancient Chinese medicine has recorded that it has the functions of promoting blood circulation and removing blood stasis,benefiting water and swelling,and detoxifying.It also has a significant pharmacological effect in the treatment of antihypertensive,vascular sclerosis,hepatitis,scalding burn,etc.,but traditional medicine uses whole insect,and little is known about the specific medicinal ingredients and pharmacological mechanism.In this paper,a fibrinolytic active protein component was extracted from cockroach.Firstly,purifing and testing its activity.Then,the specific effects of anti-thrombotic and fibrinolytic enhancement in vivo and in vitro were studied,and the safety was determined.Tumor treatment has always been a hot topic.With the awakening of traditional medicine,the study of traditional Chinese medicine on tumor has attracted much attention.Based on this demand,this paper also studied the effect of fibrinolytic protein on the growth,proliferation,death and apoptosis of tumor cells.In this paper,segmented crude proteins were extracted by tissue homogenate extraction,and then separated into a monomer Fibrinolyric Proteins（FP）from the Americana cockroach,by means of deep ammonium sulfate salting out and anion exchange.Showing that the purity of the protein was a single component and its molecular weight was about 16 k D.In order to further understand the physicochemical properties of FP,the effects of different physicochemical properties on the active protein were also investigated.The results of single factor experiment showed that the active component was the most stable at 4℃ and 37℃,and the activity was lost at 65℃ or above.Metal K⁺,Na⁺ and EDTA had basically no effect on the active component,Ba²⁺andβME had slight inhibitory effect on the active component,and Fe³⁺almost completely inhibited the activity of FP.The optimal p H of the active protein is 7.In addition,considering the possibility of future clinical application,safety assessment also was carried out on the extraction of FP experiment.Establishing a kind of healthy mouse model to inject the target protein,prove it has no acute toxicity,will not cause hemolytic reaction,and there is no obvious bleeding phenomenon.In vivo and in vitro anti-thrombotic experiments in mice have proved its anti-thrombotic effect.Thrombus is closely related to tumor.This paper preliminarily studied the anti-cancer effect of the protein,through the cultivation of OVCAR-3,SCG-7901 in vitro,showed that different concentration gradient of fibrinolytic activity of the protein function in the same cell inhibitory effect is different,the result also showed its inhibitory effect on OVCAR-3 is better,to SCG-7901 cell inhibitory effect is not obvious,target protein are selective for the proliferation inhibition.The fluorescent staining of OVCAR-3 cells showed that the protein could regulate the process of cancer cell death or apoptosis.The research results provide a theoretical basis for the nuclear targeted diagnosis and treatment of thrombotic diseases and tumors.However,it remains to be further studied whether the fibrinolytic protein contained in it has the function of directly damaging tumor cells and the mechanism of specific inhibition,as well as its mechanism of promoting angiogenesis.