?.large Scale Purification Of High Purity ?2-macroglobulin And The Study Of Its Important Biological Functions ?.study On The Different Atherosclerosis Susceptibilities Of Six Strains Inbred Mice And The Identification Of Rcn2 As An Atherosclerosis Susc

?2-Macroglobulin(?2-M)is a large molecular weight glycoprotein and belongs to alpha macroglobulin superfamily.It's present mainly in palsma.?2-M consists of four subunits,known as tetramer.Each subunit of ?2-M has five functional areas which determine its characteristics and biological functions.?2-M can participate in physiological and pathological activities widely,of which cleaning both endogenous and exogenesis proteases as a broad spectrum inhibitor of protease is primarily known.Studies indicated that ?2-M has anti-radiation function,and presents a good therapeutic effect on pneumonitis,cystitis,and other mucocutaneous caused by radiation.Moreover,?2-M can bind several cytokines,and regulate their biological activities.Studies indicated that ?2-M has anti-tumor function,and involves in coagulation regulation.?2-M can also be used as a biomarker for the diagnosis and prognosis of some diseases.Further study on biological characteristics and preparation of ?2-M can contribute to its application in clinical treatment.However,human plasma,the source for ?2-M purification,is not sufficient especially in developing countries.Currently human plasma is mainly applied to manufacture immunoglobulin,human serum albumin(HSA),and coagulation factors.It is unrealistic and costly to produce ?2-M with plasma.Cohn Fraction IV(Cohn F IV)is usually discarded as a byproduct of Cohn fractionation process through ethanol precipitation,but it contains abundant ?2-M.Therefore,Cohn Fraction IV could be a very cost-effective source for the preparation of plasma derivatives.Based on the background described above studies were divided into four parts.(1)Purification of ?2-M from Cohn F IV on the laboratory scaleActivity detection of ?2-M is essential during the purification process.?2-M is a kind of proteinase inhibitors,and could react with proteinase to form ??2-M–proteinase complex?.Different from other proteinase inhibitors,?2-M won't damage the reactive site.The entrapped proteinase is not inactivated but only sterically hindered from the access to the macromolecular substrates.BAPNA,a small molecular substrate,could get access to the reactive site of entrapped proteinase.After the reaction between entrapped proteinase and BAPNA,absorption at 410 nm was detected with the microplate reader.Several critical parameters including the amount of trypsin,BAPNA,and soybean trypsin inhibitor(SBTI)in this assay should be optimized.Then standard curve of ?2-M activity also need to be established.According to this standard curve,the ?2-M activity of sample could be detected.This kind of chromogenic substrate assay provides a convenient method to detect ?2-M activity.Several studies have described the utilization of Cohn Fraction IV for the preparation of PC,Tf,or AAT,but no studies have been reported on the preparation of ?2-M from Cohn Fraction IV,in spite of its high concentration in the discarded paste.Previous reports have described the purification of ?2-M by means of gel filtration,immunoadsorbent chromatography,and Cibacron Blue sepharose chromatography,often in combination.In this study,we identified the composition of Cohn F IV by two-dimensional electrophoresis.Salting-out method,CaptoTM Core 700 chromatography media,Aerosil 380,and PlanovaTM 35 N were tested for ?2-M purification.Immobilized zinc affinity chromatography and gel filtration were applied for ?2-M purification for the reason that ?2-M is a zinc-binding protein,and has a huge molecular weight.The final protein,with a purity of approximately 98% and a yield of nearly 48%,was obtained from Cohn Fraction IV after additional one step blue sepharose affinity chromatography.The final product was identified by LC-ESI-MS/MS.The effects of temperature,p H,and methylamine on ?2-M activity were evaluated to avoid activity loss during preparation and preservation.The results suggested that ?2-M activity could be readily inactivated at temperatures above 50°C,at pH levels above 9.0 or below 4.0,or in the presence of methylamine.(2)Large scale purification of high purity ?2-M from Cohn F IVSeveral publications reported the purification of ?2-M.However,the following drawbacks may limit their further applications: 1.All of the previously described protocols used human plasma as the starting material for purifying ?2-M,but these processes are difficult to be fitted into current plasma fractionation processes without major changes.2.The described methods rarely simultaneously achieved high purity and recovery.Immunoadsorption is an effective method to purify ?2-M,but it is too expensive to be applied in the large scale production.3.Few reported isolation processes for ?2-M are suitable for large-scale production.Though the rivanol method was a good solution for scaled-up preparation,Chinese government has forbidden the use of rivanol in plasma derivatives in China since 1990 s.4.Most other published procedures are only applicable to plasma obtained from subjects with Hp type1-1,which represent only 14.5% of all plasma.Here we report a high-purity process for producing ?2-M from Cohn F IV based on a salting-out method,immobilized zinc affinity chromatography,and ultrafiltration.The LCESI-MS/MS analysis and a comparison of the amino acid composition demonstrated that the final product was ?2-M.To the best of our knowledge,this is the first report to use Cohn Fraction IV as a starting material for the isolation of ?2-M.Our process enhances plasma utilization and is applicable to all plasma materials regardless of Hp type.After three batches purification,280 vials ?2-M(mg/vial)were obtained.The final protein with an average purity of 94.9%,specific activity of 26.7 ?g/mg,and yield of 20.5%,was achieved.We established new quality standard for ?2-M.Some core indicators including purity and specific activity were reset.The Chelating Sepharose Fast Flow we used in the immobilized zinc affinity chromatography process will facilitate pilot-scale preparation because of its low cost,fast linear flow rate,and high resistance to pressure.(3)Optimization of virus inactivation methods during the process of ?2-M purificationThe use of human plasma products derived from un-safe plasma has previously been associated with the transmission of human immunodeficiency virus type 1(HIV-1),hepatitis B virus(HBV),and hepatitis C virus(HCV).The use of purified plasma products still has a risk to transmit blood-borne infectious agents,though the development of virus inactivation and/or removal technologies has greatly improved the safety.Pasteurization,dry-heat treatment,irradiation,S/D,and chromatography were used to inactivate a wide range of lipidand non lipid-enveloped viruses during the purification process of plasma proteins.In this study,viruses with a range of physicochemical properties were chosen to test the ability of pasteurization,dry-heat treatment,irradiation,S/D,and chromatography to inactivate or eliminate viruses.The stabilizers during the process of pasteurization,dry-heat treatment,and irradiation were optimized.According to these virus inactivation results,pasteurization and dry-heat can be concluded to be two effective steps for elimination of both lipid-and non lipid-enveloped viruses without stabilizers.Complete virus inactivation was achieved after 25 kGy irradiation with almost 40% ?2-M activity lose.S/D is effective to inactivate lipid-enveloped viruses,and chromatography also has the ability of removing virus to a certain extent.It is desirable to incorporate at least two distinct effective virus inactivation steps which complement each other for plasma-derived medicines.Therefore,two of pasteurization,dryheat treatment,and S/D need to be incorporated for futher application.This study may lay a foundation for the selection of virus inactivation treatment in the industrial preparation of ?2-M.(4)Study on the important biological functions of ?2-MIt is reported that ?2-M presents an effective treatment for radiation induced ulcer of skin and mucosa,radiological pneumonia,radiological cystitis,keratitis,and corneal ulcer.In this study L-929 cells were chosen because of theirs abundant functional receptor of ?2-M.The influence of ?2-M on the proliferation,reactive oxygen species production,and apoptosis of L-929 cells after being irradiated were investigated.?2-M was used to treat mice after being irradiated.The survival and weight changes were assessed.Influence of ?2-M on storage of red cell was also investigated by measuring routine blood rest,crystal osmotic pressure,free hemoglobin,and analyzing blood gas.?2-M significantly improved the proliferation of L-929 cells,and reduced the reactive oxygen species production and apoptosis of cells.However,better influence was not observed on the weight recovery and survival of irradiated mice.Hemolysis was observed after being treated by ?2-M.Therefore,?2-M didn't show good effect on the storage of red cells.Atherosclerosis is the underlying cause of ischemic heart disease,stroke,peripheral artery disease,and chronic kidney disease.Ischemic heart disease and stroke are the first and second leading causes of mortality respectively in the world.Mortality from coronary artery heart disease has declined in the past three decades due to effective prevention programs,lipid-lowering statin and angioplasty therapies,but this disease continues to claim more lives than any other disease.The prevalence of atherosclerotic cardiovascular disease in developing countries is increasing so rapidly that the worldwide disease burden may have overtaken that of other major causes of death,including cancer,chronic obstructive pulmonary disease and diabetes.Currently atherosclerosis is treated from using a combination of statin,niacin,aspirin,a ?-receptor blocker or angiotensin-converting enzyme inhibitor that alleviates hyperlipidemia and hypertension and prevents thrombotic complications,to angioplasty/stenting and coronary artery bypass surgery.The introduction of intravascular stent placement to prevent constrictive vascular remodeling has been successful to an extent in reducing the incidence of restenosis after vascular surgery,but the late restenosis of the artery still occurs in 30% to 50% of the patients within 3 to 6 months of the procedure,and the risk of developing a major acute cardiovascular event in these patients remains high.Neointimal hyperplasia is the main pathology process of atherosclerosis and in-stent restenosis.One possible mechanism suggested that the neointima is derived from abnormal proliferation and migration of smooth muscle cells from the tunica media to the intima.The activation of smooth muscle cells is cytokine release as a consequence of mechanically induced endothelial denudation.However,although vascular surgery invariably results in endothelial denudation,not all patients undergoing these surgical procedures suffer restenosis.Thus,some other factor(s)may be important in determining susceptibility to vascular reocclusion after surgery.A possible genetic influence on vascular stenosis has been suggested by several recent studies.However,whether the development and severity of the neointimal hyperplasia phenotype are also influenced by genetic factors has not been explored in the apolipoprotein E-deficient mouse strains.Whether the gene Rcn2,a novel regulator of cytokine expression,is an atherosclerosis susceptibility gene remains unknown.Both potassium chloride and gender play an important role on the heart disease.Direct evidence about the role of them on the pathogenesis process of atherosclerosisis is lacking.On the basis of the background described above studies were conducted as follows.(1)Study on the different atherosclerosis susceptibility of six mouse strains with apolipoprotein E-deficiencyVascular remodeling of carotid artery is an important predictive phenotype for many human cardiovascular diseases,such as atherosclerosis and hypertension.The model of arterial remodeling based on ligation of left common carotid artery near carotid bifurcation has been used frequently to study research about vascular remodeling.In the present study this kind of arterial remodeling model was applied to six inbred strains of mice.Wide differences regarding to neointimal formation,lumen area increase,and medial hypertrophy were observed.Apart from C3 H,the ligated carotid arteries of other five strains mice presented positive remodeling with varying degrees increase of lumen area,B6(171%),BAL SM F1(63%),SWR(38%),BALB(15%),and SMJ(12%),compared with right lumen.The left medial area of all strains increased significantly,B6(209%),SWR(170%),BALB(79%),and SMJ(68%),compared with right one except for C3 H and BAL SM F1.B6 revealed the most dramatically increase in medial area of left carotid compared with other stains.Cells in left carotid medial area were significantly more than right ones in four strains,B6,SWR,BAL SM F1,BALB,suggesting that hyperplasia was one main contribution to medial area enlargement.SWR and B6 mice were susceptible to ligation-induced neointima formation,but BALB,SMJ and C3 H mice were pretty resistant.Cellular composition in advanced plaques of SWR strain was determined by immunostaining.Anti-?-smooth muscle actin and MOMA-2 antibody were used to identify smooth muscle cells and macrophage respectively.Neointimal lesions consist primarily of smooth muscle cells near the fabric cap and no macrophage was observed.Because of the transformation from a quiescent contractile phenotype into a synthetic phenotype,a great number of smooth muscle cells did not react well with anti-?-smooth muscle actin antibody,which is a specific antibody of contractile smooth muscle cell.B6 and C3 H were chose to study the proliferation and migration of smooth muscle cell.The smooth muscle cell derived from C3 H grew and migrated significantly slowly than B6.(2)Identification of Rcn2 as an atherosclerosis susceptibility geneLigation of left common carotid artery was used to study the role of gene Rcn2 in the pathogenesis process of atherosclerosis.Gene Rcn2 was knocked out based on B6 apo E-/-mice,and B6 apo E-/-mice were used as control.Western and chow diet were applied in this study.No neointimal lesion was observed in Rcn2-/-mice fed with chow diet,while large lesions were found in B6 apo E-/-mice.Both B6 apo E-/-and Rcn2-/-mice fed with western diet developed huge lesions in the vessels.The medial area of ligated arteries in B6 apo E-/-mice increased significantly compared with contralateral ones,while the medial area of ligated arteries in Rcn2-/-mice decreased.B6 apo E-/-mice presented positive remodeling with increase of lumen area in both diet groups compared with contralateral lumen,while the lumen area of ligated arteries in Rcn2-/-mice decreased,and the differences between B6 apo E-/-and Rcn2-/-mice are significant in both diet groups.Cellular composition in neointimal lesions of B6 apo E-/-and Rcn2-/-mice were analyzed by immunostaining.Neointimal lesions of B6 apo E-/-mice consist primarily of smooth muscle cells and a few focally accumulated macrophages in chow diet group.A few focally accumulated macrophages were observed in the lesion of B6 apo E-/-mice fed with western diet,while no smooth muscle cells were observed because of the transformation from contractile phenotype into synthetic phenotype.The neointimal lesion of Rcn2-/-mice fed with western diet was full of macrophages,which was totally different from B6 apo E-/-mice.The endothelial cells derived from Rcn2-/-mice migrated significantly slowly than the ones from B6 apo E-/-mice.Knocking out Rcn2 resulted in slower proliferation of endothelial cells compared with control,but there is no statistical significance.Rcn2 should be a promising candidate atherosclerosis susceptibility gene because of the aobve results.(3)Role of potassium chloride and sexuality on the pathogenesis process of atherosclerosisGender and potassium chloride are considered to be two important factors in the heart disease development.A significant inverse correlation of serum Rcn2 concentrations with potassium chloride concentration was observed in our lab.In this study carotid ligation was conducted for male and female B6 apo E-/-mice to investigate possible difference of atherosclerosis susceptibility between different genders.The influence of potassium chloride on the proliferation and migration of endothelial cells derived from B6 apo E-/-and Rcn2-/-mice were also assessed.Though small differences were found between male and female B6 apo E-/-mice in neointimal lesion,difference of medial area between ligated and contralateral arteries,and ratio between neointimal lesion and medial area of ligated artery,no statistic significance was observed.The left carotid arteries of male mice presented positive remodeling with more than 70% increase of lumen area compared with right lumen,while negative vascular remodeling was observed in female mice.Potassium chloride did not show significant influence on the proliferation and migration of endothelia cells isolated from the aortas of B6 apo E-/-and Rcn2-/-.One possible explanation suggested that the effect of potassium chloride on cells is a chronic process.