Study Of Prediction And Intervention Treatment Measure Of Newborn Hyperbilirubinemia

Objective Severe hyperbilirubinemia can develop acute bilirubin encephalopathy even kemicterus.To reduce the incidence of acute bilirubin encephalopathy even kemicterus,this study was conducted from clinical and laboratory.①To search for one simple and easy clinical parameter to estimate newborn hyperbilirubinemia.In order to investigate relationship of umbilical cord alpha-fetoprotein level and neonatal jaundice,we checked umbilical cord AFP(include premature and term infants),then bilirubin levels were measured on day 3 of life.We also need to found the relationship of AFP serum levels and bilirubin levels after birth.②Neonatal serum bilirubin levels is significantly associated with the activity of UGT1A1.To provide experimental evidence for prevention and gene treatment on newborn hyperbilirubinemia by studing devolping principle of UGT1A1 of rat liver in premature period.③Gene therapy can correct severe neonatal hyperbilirubinemia for long time,so it is a very potential treatment method.To obtain human UDP-glucuronyltransferase 1A1 gene and constructe a recombinant adeno-associated virus vector expressing UGT1A1,to be prepared for appling recombinant adeno-associated virus vector expressing UGT1A1 to correct hyperbilirubinemia caused by UDP-glucuronyl-transferase transferase deficiency.Methods1.Umbilical cord alpha-fetoprotein level and serum bilirubin of premature infant and full-term infant were measured.We also tested the serum alpha-fetoprotein concentration at the first month after birth.①Umbilical cord alpha-fetoprotein level of 38 cases premature infant(gestational age 32 to 33 weesks 8 cases,34 to 35 weeks 13 cases,36 to 37 weeks 17 cases) was measured, serum total bilirubin level of all premature infant was tested on postnatal 3 days.②Umbilical cord alpha-fetoprotein level of 120 full-term newborn and serum total bilirubin level on postnatal 3 days were tested,serum alpha-fetoprotein concentration of 25 full-term newborn with hyperbilirubinemia and 30 full-term newborn without hyperbilirubinemia were measured during the first month after birth.Scatterplot figure was drawed,straight line regression equation was calculated.2.UGT1A1 activity in liver of fetal rat(gestational days 17 to 19,five rats in each group) and postnatal rat(one-day to five-days rat afterbirth,10 rats in each group,5 two-week-old rats afterbirth ) was determined by Matern method,autoradiograms were made.3.The total RNA was extracted from cultured HepG2 cells.The gene sequence coding UGT1A1 was amplified by reverse transcription-polymerase chain reaction(RT-PCR). Constructing clone vector expressing UGT1A1,screening masculine recon and sequencing. The fragment was inserted into pAAV-MCS ultimately,recombinant pAAV-UGT1A1 plasmid was identified by double enzyme restriction digestion method and PCR.The HEK293T cell was cotransfected with pAAV-UGT1A1,pAAV-RC,pHelper simultaneously.The shape changes of HEK293T cell were observed by light microscope, number of cell with green fluorescence was counted with fluorescence microscope,so transfectous efficiency can be got indirectly,and titer of recombinant adeno-associated virus was determined.Results1.Umbilical cord AFP levels of premature infant group and full-tern infant group were correlated respectively with serum total bilirubin levels of postnatal 3 days infant linearly. There was significant difference in serum AFP level during the first month afterbirth between hyperbilirubinemia group and control group(P＜0.01).2.UGT1A1 activity of fetal rat liver with gestational age 17 to 19 days was very low, the UGT1A1 activity of fetal rat liver with gestational age 19 days was only 6.7%of adult values,but the UGT1A1 activity increased markedly from low level to near adult values during 1 to 2 postnatal days,and the UGT1A1 activity of 5-day-old rat afterbirth was 88.1 %of adult values.There was significant difference between four groups(gestational age 17 and 19 days rats,1-day-old rats and mature rats).This regularity was also appeared on autoradiogram.3.The UGT1A1 cDNA was amplified successfully from the total RNA extracted from cultured HepG2 cells by RT-PCR.Positive clone vector expressing UGT1A1 was selected. UGT1A1 was inserted into pAAV-MCS ultimately,Recombinant adeno-associated virus vector expressing UGT1A1 was constructed and expressed successfully in HEK293T cell. Cells cotransfected with pAAV-UGT1A1 appeared shrinking or shedding under light microscope.A lot of cells hotogenesed green fluorescence under fluorescence microscope, transfectous efficiecy was over 70%.Titer of recombinant adeno-associated virus was 10^5.6pFU/ml.Conclusions1.Ambilical cord and serum AFP levels of neonate may suggested as an indicator for the functional maturity of the liver cells.It can provid a new parameter to predict hyperbilirubinemia.The range of umbilical cord AFP levels also correlate with subsequent bilirubin levels.Umbilical cord AFP levels can predict severe hyperbilirubinemia in order to intervent as early as possible,so it maybe spreaded in clinical in future.2.UGT1A1 activity in the liver of premature rat is very low,the activity of UGT1A1 increases quickly and reaches the levels of adult rat after birth.Experimental evidences of activity of UGT1A1 in perinatal and matural period are provided by these results.We will select the suitable duration to conduct gene therapy for newborn hyperbilirubinemia caused by liver UGT1A1 activity deficiency.3.This expriment constructs rAAV expressing the human bilirubin UDP-glucuronyl-transferase gene successfully,establishs the foudation of gene therapy for hyperbiliru-binemia because of bilirubin UDP-glucuronyltransferase deficiency.