A Study On The New Functions Of Extracellular Vesicles And Their Correlation With Preeclampsia

ObjectiveExtracellular vesicles(EVs)are one of the most popular research subjects that have received much attention in recent years.However,there are still many unknowns in their functional studies.They exist in biological fluids and participate in various physiological and pathological processes.Preeclampsia(PE)is a pregnancy specific syndrome,and its pathogenesis is not fully understood.Poor invasion of trophoblast cells is the main pathophysiological change of PE.Therefore,we made an in-depth exploration of the new functions of extracellular vesicles and investigated the effect of extracellular vesicles on poor invasion of PE trophoblast cells.MethodI.Screening of extracellular vesicle culture conditions in acellular state1.Selection of cultivation conditions(1)Selection of cultures-plasma ratio:Mix the same culture medium with plasma in different proportions.Extracellular vesicles were separated and lysed after 72 hours of culture,and vesicles protein content was extracted and detected.The optimal ratio was the result with the highest amount of vesicles protein.(2)Selection of medium:Three kinds of medium(whole blood cell medium,1640 medium and amniotic cell medium)were used to culture extracellular vesicles.The culture medium and plasma were mixed according to the above optimal ratio(2:1),and the extracellular vesicles were separated and lysed after 72 h of culture.The RNA and DNA contents of the vesicles were extracted and detected.The medium with the highest RNA and DNA content was the best.(3)Selection of culture time:Using the best culture medium screened above,mix the culture medium with plasma in a ratio of 2:1,the culture suspension was harvested at 0h,12h,24h,48h and 72h.Extracellular vesicles were separated and collected.The vesicles concentration was detected by Nanoparticle tracking analysis(NTA).The culture time with the highest vesicle growth efficiency was the best.2.Separation and detection of extracellular vesicles and their nucleic acid and protein(1)Extracellular vesicles isolation:Extracellular vesicles were extracted from plasma using an Exosupur kit(Echo Biotech,Beijing,China)according to the size exclusion chromatography(SEC)principle,and the distilled suspension was finally concentrated using a 10 kDa Amicon?millipore(Merck,Germany).(2)Extracellular vesicle protein extraction and detection:the extracellular vesicle suspension was lysed to obtain total vesicle protein.Pierce BCA protein assay kit(Thermo Scientific,USA)was used to calculate the protein content of the sample through the BSA standard protein solution curve.TM(3)Extracellular vesicle nucleic acid extraction and detection:total DNA and RNA of vesicles were extracted using a DNA isolation kit(Rengen Biosciences Co.)and Trizol reagent(Invitrogen),respectively.Nucleic acid quality and OD 260/280 values of samples were measured using a NanoDrop ND-1000 instrument,and nucleic acid integrity was assessed by standard denatured gel electrophoresis.(4)NTA detection of extracellular vesicle concentration:Laser light source is used to irradiate nanoparticle suspension,detect the scattered light of nanoparticle,and calculate the nanoparticle concentration by counting the number of scattered particles.A video with a duration of 60 seconds was shot at a frame rate of 30 frames per second,and the particle motion was analyzed using NTA software(ZetaView 8.02.28).II.Explore new functions of extracellular vesicles1.Study on extracellular vesicle proliferation(1)Fresh plasma was treated with 0.2μm filter membrane for sterilization,and culture suspensions were collected at 0h and 72h by adding twice the volume of medium under the above culture conditions.Extracellular vesicles of culture suspension were separated and purified,and particle concentration was detected by NTA.The proliferative capacity of extracellular vesicles was interpreted by particle number,size,and movement.(2)The morphology and expression of signature protein were detected by Transmission electron microscopy(TEM)and WB test.2.Study on extracellular vesicle function(1)Study on vesicle migration and invasion function:xCELLigence RTCA(ACEA Biosciences)instrument was used to detect the migration and invasion function of extracellular vesicles.The vesicle suspension was added to RTCA microplate 16(ACEA Biosciences)and incubated in the instrument for 24h.During this period,automatic monitoring was performed for 10 seconds every 15 minutes,and the data was expressed as cell index(CI),which was used to calculate the migration and invasion ability of extracellular vesicles.(2)Prediction of half-life of extracellular vesicles:Normal human plasma was filtered and sterilized with 0.2μm filter membrane,and incubated at 37℃ incubator for 30 days.Cultures were collected once a day to separate and purify extracellular vesicles and detect vesicle RNA and protein content.The half-life of a vesicle is assumed to be the time when the amount of RNA and protein in the vesicle is reduced to 50%.3.Study on the correlation between extracellular vesicles and diseases(1)The plasma of 6 healthy pregnant women and 6 PE pregnant women was collected,the age and gestational age of two groups were matched.4ml of pregnant women’s whole blood was collected,and the separated plasma was treated with 0.2μm filter membrane for debacteria.The plasma was added into the medium at the rate of 1:2 for 0h and 72h.Cultures are collected,separated and purified to obtain four groups of extracellular vesicles.The correlation between the extracellular vesicles and PE was analyzed and compared by NTA technology.(2)The trophoblast cells were mixed with 4 groups of vesicles for 12 h respectively,and the migration and invasion function of trophoblast cells were detected by xCELLigence RTCA(ACEA Biosciences)instrument,to explore the effect of PE plasma vesicles on the poor invasion of PE trophoblast cells in pregnant women.Result1.Culture conditions of plasma extracellular vesicles(1)The results of vesicle protein detection showed that the protein concentration of vesicles extracted after culture medium and plasma were significantly increased compared with that before culture(P<0.0001).After culture,the protein content of vesicles in the three groups also increased with the increase of the proportion of medium.In order to improve the efficiency of subsequent vesicle extraction,2:1 mixing ratio of culture-plasma was selected as the culture condition for subsequent studies.(2)The results of vesicle DNA and RNA detection showed that the RNA and DNA of plasma extracellular vesicles were significantly increased after using the three kinds of culture medium compared with that before culture(P<0.05).The increase amplitude of vesicle nucleic acid cultured in three medium was different,and the concentration of vesicle nucleic acid cultured in amniotic fluid cell medium was the most significant.In view of the above results,we selected amniotic fluid cell medium with a 2:1 ratio mixed with plasma as the culture condition for subsequent studies.(3)The results of NTA showed that the concentration of vesicles decreased slightly within 12 hours of culture.With the extension of culture time,the concentration of extracellular vesicles began to increase gradually after 24 hours.The proliferation rate of vesicle concentration was significantly increased after 72 hours(P=0.0064).In order to improve the efficiency of the follow-up experiment,72 hours of culture was selected as the culture condition for the follow-up study.2.Extracellular vesicles have the ability to proliferate offspring vesiclesNTA results showed that extracellular vesicle concentration increased significantly after culture(logarithmic analysis:11.80±0.61 vs.10.91±0.54,P=0.0035).The volume of neonatal extracellular vesicles was significantly larger than that of maternal extracellular vesicles(136.87±7.17 vs.92.98±7.71,P=0.0004).The main peak of the particle size of newborn vesicles was different(115.15±6.45 vs.76.97±7.98,P=0.0004).Screenshots from the NTA video also show a denser distribution of newborn vesicles.3.The morphology and marker expression of newborn extracellular vesicles were the same as that of maternal extracellular vesiclesTEM results showed that the newborn vesicles were also typical small vesicles with membrane structure,consistent with the morphological characteristics of the mother’ s extracellular vesicles,with clear cup-holder shape.Compared with before culture,vesicle volume increased after culture.The results of WB experiment showed that the extracellular vesicles isolated before and after culture had the expression of the signature proteins CD9,HSP70 and TSG101,and the non-signature protein calnexin was not detected.4.New extracellular vesicles showed stronger migration and invasion abilityThe results showed that extracellular vesicles had migration and invasion ability.Compared with pre-culture,the invasion ability of newborn vesicles was significantly increased(0.51±0.11 vs.0.30±0.051,P=0.027).5.The half-life of extracellular vesicles in vitro is about 14 daysAccording to the results of daily vesicle RNA and protein content detection,the amount of RNA and protein in extracellular vesicles decreased by 50%in 10-20 days after isolation compared with 0 h.The experimental results of 10-20 days showed that the extracellular vesicles survived for about 14 days in vitro.6.Both plasma vesicles and neonatal vesicles in PE patients can inhibit trophoblast migration and invasion(1)Compared with healthy pregnant women,the plasma extracellular vesicle concentration of PE pregnant women was slightly decreased,but there was no significant difference.The proliferation of vesicles in pregnant women with PE after culture was lower than that in healthy pregnant women(logarithmic analysis:11.80±0.61 vs.11.39±0.25,P=0.20).After culture,the average particle size and main peak of particle size of vesicles in the two groups were significantly increased compared with that before culture(P<0.01),and the increase of vesicles in PE group was significantly lower than that in healthy group(P<0.01).(2)The trophoblast function test results after co-incubation with extracellular vesicles showed that,compared with NC group,the migration and invasion ability of trophoblast cells co-incubated with PE vesicles were decreased(P>0.05).Compared with PE maternal vesicles,PE neonatal vesicles(PE-72h group)showed stronger inhibition on trophoblast invasion function(0.70±0.15 vs.0.23±0.05,P=0.0017).Conclusion1.After cultured under appropriate conditions in vitro,the extracellular vesicles that left the mother cells would produce new extracellular vesicles of the offspring generation,which had the ability to self-proliferate.Both maternal and progeny extracellular vesicles have independent migration and invasion functions.2.There were differences in the number and function of plasma extracellular vesicles between healthy and PE patients.Plasma vesicles in PE patients can inhibit trophoblast migration and invasion.Our study suggests that extracellular vesicles have a previously unrecognized ability to produce functional progeny,and their effects on trophoblast cells may be related to the mechanism of the occurrence and development of PE.