The Effect Of TF Gene Silencing On HUVECs With Placental Abruption

Background:Placental abruption (PA) is the severe complication in the late trimester ofpregnancy. PA which increases the incidence and the mortality of the newborns also isone of the important reasons for the death of the fetus and the newborns with lowbody weight, for example the small for gestational age infant. The internal morbilityof the PA was reported 0.06%～2.1% by yangjianbo and so on. The mortality which is20%～35% in these newborns whose mother has the PA is 15 times than that of thehealth.The Wad Medani hospital in SUDAN reported the incidence of PA is 6.5% andthe mortality of the fetal death and death in one month after born is 20.2%.Salihureported that the incidence of PA of the single birth, twins and triplets respective is6.2/1000,12.2/1000and 15.6/1000 in the 15488000 newborns who was born in theUSA during 1995-1998. Along with the augment of the newborns, the incidence of PAis increasing. In these newborns whose mother has PA, the proportion of the bloodclotting disorder is very high. However, in the severe PA and other pregnantcomplication, the high clotting state can result in the placental thrombus, affect theclotting function and life-safe of the fetus, also is the latent and dangerouspathological change to the newborns. There are exogenous pathway and endogenous pathway in the blood clottingprocess. Recent research shows that the exogenous pathway plays an important rolein the course of the pathological blood clotting. However, the tissue factor, as theinitiator of the exogenous pathway, coupled with many diseases including theplacental hemorrhage, has many intimate correlations with the blood clotting of thenewborns.There are more tissue factor antigens in the mother's amniotic fluid, placenta,deciduas and myometrium than the blood plasma. Some proteins and polypeptidescan pass placental barrier with the exocytosis and zendocytosis manners. Thesefuctions are determined to the special receptors on the syncytial trophoblast ofplacentas. When crossing the cell interspace of the placenta barrier before the normaldelivery,some hormone-like proteic substance, e.g peptidaseα, can be degraded bythe enzyme in the barrier, and will not have any physiological effect. Recentresearches showed that the mechanism was related with the trophoderm of themother-surface and the thick villus of the baby-surface of the placenta. Accordingly,some giant molecules were permitted crossing placenta, such as IgG.The major pathological change of PA is the breakage of the helicine arteries ofthe uterus. At the same time, the other peripheral tissue may be injured. Kuczynskireported that there are more TF in the placenta and myometrium than that of theblood plasma, but the level of TFPI seems to be very low. These illustrated that theexpression of the TF increased in the situation of the PA. Many tissue factors,released from the placental villus and decidua in the dissection, may cross theplacenta, come into the blood circulation of the fetus and effect the clotting system ofthe newboms. On the other hand, the injured tissues may active the clotting system,induce the clotting disorder in the fetus and newborns. Seriously, because of theaugment of the thrombin, many fibrinogens tum into fibrins, lead to DIC. Our prophase researches also discovered that a part of the newboms in hospital whosemother had PA were in the high clotting state, but most of the newboms of thesevere lying-in mother had already in the low clotting or low fibrinogens state whenthey came into our hospital. Because mothers had a rather integrity coagulationalmechanism, more substance which active the clotting system can induce the DIC.However, the regulatory mechanism of the newborns is not so good, only a littlesubstance can result in DIC. If these cases do not be deal with in time, it would bedangerous to the fetus and newboms, lead to fetal death or DIC, finally the death ofthe newborns. Although there are so many treatments for the high-risk newboms, thegeneral treatment at present is using the fibrinogen passively, the platelets and otherblood anticoagulation factor. It was hypothesis that the priming of the clotting processmay be prevented by transient silencing the TF of the exogenous pathway, decreasingthe depletion of the blood coagulation factor and blood platelets, preventing andcuring the impact of the placental hemorrhage to the clotting function of thenewborns.As having rich sources, the Human Umbilical Vein Endothelial Cell (HUVEC)has a lot of advantages for disease research. And it has become a good research toolin many diseases, mostly focusing on the hemorrhage and clotting disease,histological engineering vascular prosthesis, artherosclerosis and so on.A hypothesis is proposed that when curing the blood clotting disease, it has animportant significance to transiently shut the TF expression in the exogenouspathway. Through this way, we can interrupt the priming of the clotting process,decrease the dissipation of the blood coagulation factor and blood platelet and stopthe impact of the placental hemorrhage on the clotting function of the newboms.RNA interference (RNAi) is a technique which has an extensive applicationalprospect in the gene function study and gene therapy. It can down regulate the expression of the aim genes. In recent years, there have been many progressesmainly aiming at inducing the silence of specificity target genes, interfering signalconduction pathway, inhibition AIDS and hepatitis viral genes and overespression ofthe oncogenes and cancer associated gene It can also keeps silent and resting stateof these viruses. So, it may become a new treatment method for these kind diseases.To prevent the start of blood clotting and interfere the pathologic process, it wassupposed what the expression of TF transiently was shut off with the RNAitechnology and the effect of PA on the blood fuction of newborns.On the base of the primary culture of HUVECs, we constructed a interferevector of TF. Utilizing RNAi developed in recent years, we transfected the vector,pENTR^TM/U6-siRNA/TF, into HUVECs of normal and placental abruptionrespectively. The expression of TF gene was silenced in our experiment whichconfirmed by RT-PCR and immunofluorescence staining. This might found the basesof the gene treatment of the disease.Material and Methods:1,According to the manual of the kit, the levels of TF and TFPI of the blood ofmothers, cords and newborns with placental abruption were detected with Elisa.Then the values of TFPI/TF were calculated.2,Culture in vitro and identification of Human Umbilical Vein EndothelialCells(HUVECs)Natal umbilical cord about 10 cm are cut by asepsis scissors, with the blood ofcord blood vessel squeezed.PBS washes out the bloodstain on the outside andterminator of umbilical cord,then 20-40ml PBS washes the lumen of umbilical veintill the effluent fluid become colorless or the color of light washed meat water, with5-10ml 0.1%collagenaseâ…¡pouting into the umbilical vein, incubating in 37℃anddigesting 12minutes,we collect the digest fluid,20ml PBS wash the umbilical vein again, finally we add some fetal bovine serum to stop the digestion. Next, thecollected fluid was centrifugated in common temperature for 1000r/min×10min,abandon the supematant, add the maximal medium to the collected cell sediment,blow the cell group again and again until it become single cells, inoculate them into25cm² culture flask, cultured in 37℃, 5%CO₂.24 hours later, when we observe thecells, we find that most cells which have become fusiform or triangle grow extendlyin adherence way. We change the medium, at the same time, add epidermal growthfactor (EGF) into the medium. Culture cells for another 48-72hours till cells almostoutgrow the culture flask, then we assess related antigens in immunofluorescencestaining, with cells used in experiment are second or third generation.3,Construction and identification of TF gene vector by RNAiWe designed specific TF genotype sequence T12 and T6, which afforded by thefree on-line software come from Invitrogen Co., the Web addresshttps://rnaidesigner.invitrogen.com/maiexpress/.First, we composed shRNAsequence and then ds oligo double strand. After annealed, ds oligo double strand wasconnect to pENTRTM/U6 cohesive end of BLOCK-iTTM U6 RNAi Entry, Vector,which was transduced into competent cells. The cells were multiplied,meanwhile the vectors were transcribed also. The specific DNA was extracted fromthe cells and then sequenced. According to the sequence, we chose the correctsequence T12 as the interference vector and T6, which was wrong sequence as thenegative control group for the further experiment。4,The study of bioimmunofunction in TF-silenced HUVECs of placental abruptionThere are two groups in the experiment, the normal group and the pathologicalgroup. Three different treatments are established in each group.(1)the blank;(2) thefalse-intervention;(3) the TFgene silencing triel. There are three samples in achtreatments. After these treatments, we can observe the changes of mRNA expression of the HUVECs before and after the gene silencing and the changes of theimmunofluorescence of the TF protein level to discuss the changes in the biologicalfunction.(1) Transfection: Inoculate 5×10⁴/ml HUVECs to each pore of the 12-pore plate,then add 0.5ug plasmid DNA into each pore when the cells have grown to 60%-80%of the pore, transfect the constructive carrier to the HUVECs, cultivate them for 48hours in 37℃(2)RT-PCR detection: Dislodge the supematant of each pore, use two-stepsRT-PCR to detect the mRAN. The first reactive condition is 42℃20min—95℃5min—5℃5min; human tissue factor cDNA sequence (NM-001993), design a coupleprimers, the sequence of the upstream is 5'-CCGGCACAGCCTTTAACAACCT-3';the sequence of the downstream is 5'-CGTTTGCTCTCGATTCCATGTG-3'.choosethe humanβ-actin gene (CF62602) as the inter-reference, amplify theβ-actin gene, itsupstream sequence is 5'-TGGCACCACACCTTCTACAATG-3'; its downstreamsequence is 5'-CCGTGGTGGTGAAGCTGTAGC-3'. The PCR reactive condition is:94℃5min—32×(94℃30s, 50℃30s, 72℃30s)—72℃5min. Electrophorete theamplifying production in the 1.5% agarose gel, image it with Bio-rad gelatinoussystem, scan and analyse the results of the zone-gray scale by Gel-Pro Analyzersoftware.use the densitive ratios of the TF and the amplifying zone of the beta-actinto represent the relative intension of TF.(3) The immunofluorescence of the TF expression:put the germ-free glasses(1×1cm) into the 12-pore plate, innoculate HUVECs (5×10⁴/ml) to the glasses, thantransfect the constructive carrier to HUVECs in the above way,dislodge thesupematant, soak in PBS, dry in the room temperature, use paraform to fix,cultivatewith rabbit-anti -human TF Ab (the concentration 1:200)in wit box, clean 3 timeswith PBS, add FITC labeled goat-anti-rabbit-Ab (the concentration 1:400),cultivate for 2 hours, than observe in the microscope.Results:1,The TF level of blood with PA was significantlly higher than the normals', (P＜0.05) and the same result founded in cord. But the TF level of blood with PA wassignificantlly lowerr than the normals', (P＜0.05) and the same result founded incord.2,From the fetal umbilical cords in normal control group and placental abruptiongroup, HUVECs are successfully cultured. And the morphology of the cells hasbeen assessed. The cells in early period are small multiple angles or globular. 2-3days later, they become flat multiple angles jointing each other, arranging inunilayer slabstone-like way, with distinct boundaries, abundant andochylema andclear nuclears which are round or ellipse. There are many caryocinesia phases,1-2 nucleoli, microaggregates inside the cells. They mixed together after 3-4days.Till 7-10days the cells body becomes multiple angles and joints each other.Weassessâ…§factor related antigens in immunofluorescence to confirm they areHUVECs. When cells passage 3-4 generations they would reach the experimentdemand.2,After the plasmid competences were amplificated, plasmid DNA obtained fromthe competences were sequenced to show that the pENTRTM/U6-TF-shRNAwas the positive clone. The result of RT-PCR demonstrated that the level of TFmRNA of HUVECs transfected by pENTRTM/U6-TF-shRNA significantlydecreased.3,The pENTRTM/U6-TF-shRNA, which was sequenced a correct result, wastransfected to the HUVECs of the normal and pathological group. PT-PCR outcomes was scanned and analyzed by the zonal gray scale with Gel-ProAnalyzer software.There were significantly differences between the normal andpathological group, (F=23.44, P＜0.001), the same results among themanagement. (F=33.63, P=0.01) Before management, there was a significantdifference for the levels of TF mRNA between normal and pathological froup,(P=0.023) and the same result for the second management. (P=0.01) But therewas no significantly difference between the normal and pathological froup afterRNAi. (P=0.516) There were significantly differences among the threemanagements in the themselves groups of normal and pathological onesrespectively. (F=19.30, P=0.002, F=27.66, P=0.001)Conclusion:1,The TF levels of parturients with PA, their cord blood and newborns weresignificantly higher than the normals'. Their TFPI and TFPI/TF levels weresignificantly lower than normals'. The procoagulant activitities were enhancedin puerperants and newboms with PA. And the antiprocoagulant activities weredecreased in puerperants and newborns with PA.. This situation leads to thehypercoagulable state in newboms.2,Because of their abundant resources, the HUVEC is a good research objectfor experiments. A lot of HUVECs can be cultured from fetal umbilical vein.The HUVECs of our cultured were confirmed by morohology andimmunofluorescence.3,The vector of targeting TF gene for RNAi was successfully constructed.The targeting sites of T12 was successfully cloned. The vectors were transfectedinto HUVECs and played the biological function. And they silenced theexpression of TF mRNA. 4,The level of TF mRNA of HUVECs in the placental abruption group washigher than the one in normal group. (P=0.023) It may be one reason of thehypercoagulation state in the mothers and bewborns5,pENTRTM/U6-TF-shRNA could significantly inhibit the expression of TFmRNA of HUVECs in both the normal and the pathological group. It maybecome the novel method.