| Background and objective:So far?there is little agreement of themost favorable management to PPROM? the time from rupture membranesto delivery is a critical factor to improving neonatal outcomes of PPROM.If the puncture is sealed or healed?the amniotic cavity may be completedagain, gestational age may be prolonged,meanwhile,there was an increasedrate of neonatal surviving and a reduction rate of perinatalmorbidity.Sealing membranes should be a new therapy to PPROM.In thepresent study?we developed the model of PROM in vitro and found themost efficient fibrin clot. Next?we cultured the HAEC on the fibrin clot?HAEC was observed with growth factor (EGF,bFGF,TGF-β1) or no with.Finally? the model of HAEC damaged was performed?we observed therestoration of damaged HAECs with formulated fibrin clot and cell growthfactor.The purpose of this study was to found the experimental testimony tothe use of repairing of PPROM rupture with formulated fibrin clot and cellgrowth factor in clinic. Methods: (1)Amniotic fluid and membranes were obtained from31~36 gestational weeks?we designed a model for studying rupturedmembranes in vitro and various blood components for fibrin sealant, therewere five groups(A,B,C,D,E).Artifical standardized punctures were sealedby various blood components,the termination times of amniotic fluidleakage were counted,fibrin clot and ruptured fetal membranes werevisualized by light microscopy after each experiment.We found the mostefficient components of fibrin clot. (2)The cultivated HAEC on the formulated fibrin clot were examinedby phase contrast microscopy,Giemsa staining and scanning electronmicroscopy.We measured apoptosis of HAECs on the formulated fibrin clotwith growth factor (EGF,bFGF,TGF-β1) by TUNEL. (3)Trepan was drilled the culture sheets coated with HAEC to makequantified models of damaged HAEC,on which the lacks were thencovered with fibrin clot.Subsequently,different concentration of EGF, bFGFand TGF-β1 were added into the sheets respectively.After the predesignedculturing time, the growing and transiting conditions of HAEC wereobserved under inverted microscope after Giemsa staining. Also, theproliferating conditions of HAEC were detected by using5-bromodeoxyuridine(BrdU). Results:(1)The median times for amniotic fluid leakage of A groupwas 19.83 minutes,B group was 3956.00 minutes,C group was 2988.50minutes, D group was 2670.00 minutes, E group was 4200.00 minutes?thecontrol group(amniotic fluid)was 2.99 minutes.The median times foramniotic fluid leakage of B,C,D or E group were significantly longer thanA group(p<0.05).There was amniotic fluid in the injector of E group only,inaddition,the fibrin clot was observed around puncture site,connection wasshown between fibrin clot and membranes. (2)HAECs generate well on the formulated fibrin clot in vitro.HAECs exposure to EGF or bFGF was associated with a lower rate ofapoptosis than control group,however,HAECs exposure to TGF-β1showedopposite effect. (3)In all groups,HAECs could transit toward damaged area on fibrinclot and grow there. The higher transiting speed and the bigger cellnumbers were observed in the EGF and bFGF groups followed by thecontrol group,while the TGF-β1 group showed the relatively poorerresults.Compared with the control group,all of the EGF groups (EGFconcentration ranging from 10ng/ml to 80ng/ml) and bFGF groups (bFGFconcentration ranging from 5ng/ml to 80ng/ml )showed better HAECproliferating effect(P<0.05),especially 20ng/ml EGF group and 40ng/mlbFGF group got the significantly best proliferating results among their owngroups respectively(P<0.05).Besides, the effective inhibiting concentrationof TGF-β1 was 0.8ng/ml～12.8ng/ml(P<0 .05). Conclusion: (1)Fibrin clot that contain blood components in vitromay be efficient for sealing of ruptured membranes, four components thatinvolved cryoprecipitate ,thrombin,aprotinin and cacl2 to formalize fibrinclot were the best components in experiments. (2)HAECs generate on the formulated fibrin clot in vitro. HAECsexposure to EGF or bFGF was decreased apopt...
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