Extraction,Identification And Preservation Of Brain Drived Microparticle And The Preliminary Study In Traumatic Brain Injuries

Objective: microparticle,which size in 0.1-1um in diameter is a kind of extracellular vesicles secreted by activated or apoptosis cell.In recent years,the study found that the microparticles can be mediated some diseases,can be used as biological markers of infection or trauma,and has potential therapeutic value.Moreover,it has been reported confirmed that the microparticles which act as material transport carrier have transfer function as transfer m RNAs,micro RNAs,lipid and protein in intercellular spaces.Thus,the basic and clinical medicine research keep a watchful eye on microparticle.The experiment compares to two methods of extraction brain drived microparticle(BDMP),explores the flow cytometry instrument to detect microparticle concentrations,explores frozen storage conditions effect on the BDMP and tests BDMP drived of clinical TBI patients plasma samples,attempts to:(1)compare BDMP extraction method,lay the foundation for the BDMP separation and purification;(2)establish a set of effective test method for identification microparticle,and carry out BDMP research of large-scale in the future;(3)explicit trend of BDMP change under different temperature conditions,and provide a reference for the preservation of the BDMP;(4)Based on the current study which the BDMP exist in mouse,we further confirmed BDMP also exist in the blood of patients after trauma brain injury(TBI),provides new way of thinking for blood coagulation dysfunction after TBI patients.Methods:(1)Apply flow cytometry technology and RNA quantitative extraction technology test BDMP produced by grinding method and enzymic digestion method respectively,observing different structure of BDMP;(2)Apply flow cytometry technology test fluorescent standard for calculating the difference of within-day and between-day,establish TBI mice model.Apply flow cytometry technology to detect the contents of BDMP;(3)preservation BDMP within environment of 4 ?,20 ?,80 ?,and use flow cytometry technology to detect liquid nitrogen respectively for different period of time;(4)collect blood specimen of TBI patients,use flow cytometry technology,Western blot technology and transmission electron microscope respectively to test the BDMP.Results: 1)The methodological results showed the difference of within-day and between-day were 5.8% and 4.8% respectively.Applied the determination method of flow cytometry,the contents of mouse BDMP in brain tissue in mice after trauma brain injury higher than that of brain tissue in normal mice significantly,with significant difference(P < 0.05);2)Comparing the two extraction methods of BDMP,which are grinding method and digestion method.The concentration of BDMP is greater in grinding method than digestion method.Grinding method of extraction of GFAP positive BDMP content greater than the digestion method,grinding method to extract the Annexin? positive BDMP content is less than the digestion method;Two methods of extracting Annexin ? and GFAP positive BDMP did not differ between the BDMP.Using micro electrophoresis system to detect fluorescence,we accurate analyse nucleic acid samples.Results show that the BDMP contains genetic material RNA,and grinding method to extract BDMP contains RNA concentration is greater than of the digestion method;3)In the different time point under different temperature,we use flow cytometry technique measuring BDMP,after 3days/3 weeks under different temperature,BDMP properties would change,and the effects of different temperature on BDMP show no difference;4)According to the flow cytometry technique,western blot and electron microscope found that BDMP exist in the peripheral blood of brain trauma patients,and the BDMP has unique structure and function.Generally speaking,its size is between 0.1 ~ 1?m in diameter,and its double molecular phosphatide membrane is rich in phosphatidylserine and neuronal specific marker.Conclusion: 1)Detecting the contents of the BDMP,flow cytometry technique which can be used in the routine testing of BDMP has good reliability,high repeatability,and the advantages of simple operation;Brain cells after suffer craniocerebral trauma in mice release more BDMP.2)we use digestion method to extract BDMP,and compare the two extraction methods of BDMP,which are grinding method and digestion method.We found that variety of microparticle concentration is inconsistent.At the same time,we verify BDMP contain genetic material RNA.The concentration of RNA from BDMP is greater in grinding method than digestion method.3)BDMP tests should take fresh specimens of BDMP.Because the nature of the BDMP may changes after cryopreservation,and leads to the result of the experiment unstable.4)Peripheral blood of patients suffer tauma brain injury and healthy people,BDMP has unique structure and function,and GFAP positive and/or Annexin V BDMP levels in the peripheral blood of brain trauma patients more than healthy people.Therefore,we speculate the body after craniocerebral trauma,blood brain barrier broke,and neuron and glial cell release BDMP,so through damaged blood-brain barrier exist in peripheral blood.The relationship between level of BDMP in the peripheral blood of cerebral trauma patients and patient's physique,gender,age,disease severity and the course of the disease has yet to be further research.