| Part One Expression of hepatocyte transporters and FXR protein in infant with cholestatic hepatitisObjective : To analyze the expression of multiple drug resistance 3 (MDR3), bile salt export pump(BSEP), multidrug resistant protein 2 (MRP2) and farnesoid X receptor(FXR) in liver tissue of infant with cholestatic hepatitis and to understand the clinical significance. Methods: Clinic datas were collected in 22 infants with cholestasis hepatitis from January 2000 to October 2009, liver biopsy was performed in all patients, normal liver tissue was obtained from 10 living donors of liver trans- plantation. The protein expresses of MDR3, BSEP, MRP2 and FXR were exa- mined by immunohistochemistry. The protein expression levels were quantita- tively analyzed by Leica Qwin software. Statistical correlations between the pro- tein expression levels and indices of blood testing were calculated.Results: The normal liver showed canalicular membrane straining for MDR3, BSEP and MRP2 and nuclear localization straining for FXR in hepato- cytes. Compared with the normal liver, strong canalicular membrane staining for MDR3, weak canalicular membrane staining for MRP2 and weak nuclear localization straining FXR were observed in patient liver, BSEP was unchanged in patient. Optical density value of MDR3 expression in patient and normal liver were 0.13±0.02 and 0.11±0.03 respectively, MDR3 protein levels were signi- ficantly increased in patient liver (t=-2.239, P<0. 05). Optical density value of BSEP expression in patient and normal liver were 0.08±0.02 and 0.07±0.01 respectively, BSEP protein levels were not significantly altered (t=-1.625, P=0.114). Optical density value of MRP2 expression in patient and normal liver were 0.11±0.05 and 0.17±0.04 respectively, MRP2 protein levels were signi- ficantly decreased in patient liver (t=3.418, P=0.002). Optical density value of FXR expression in patient and normal liver were 0.09±0.02 and 0.15±0.06 respectively, FXR protein levels were significantly decreased in patient liver (t=7.676, P<0. 001). Serum levels of ALT were negative correlated with MRP2 protein levels (r=-0.488, P<0.05). Serum levels of TB, DB,γ-GT and ALT were not correlated with MDR3 and FXR protein levels (P>0.05).Conclusion: In infant with cholestatic hepatitis, the protein expression of MRP2 and FXR were decreased, MDR3 protein levels were increased, BSEP protein levels were unchanged compared with normal control. MDR3 protein levels were upregulation which may represent an adaptive response aimed at the enhanced phosphatidylcholine excretion to normally protects the biliary epithe- lium from bile salt toxicity. Downregulation of MRP2 may decrease the bile flow and potentiate the hepatocellular toxicity by enabling the accumulation of bile acids, which may aggravate the cholestasis. Downregulation of MRP2 was probably controlled by downregulation of FXR. These results suggest that the altered expressions of the hepatocyte transporters may play a role in the patho- genesis of the infant with cholestatic hepatitis. Part Two Effect of urosodeoxycholic acid on ABCB4 and FXR mRNA levels in infant with cholestatic hepatitisObjective : Analysis of the expression of ABCB4 and FXRmRNA and efficacy of UDCA in infant with cholestatic hepatitis, and to identify the diffe- rence of ABCB4 and FXRmRNA levels before and after UDCA treatment.Methods: Clinic datas were collected in 58 infants with cholestatic hepatitis from July 2008 to July 2010. Patients were randomly divided into two groups, 30 patients received Glutathione and ademetionine served as conven- tional treatment group, 28 patients received a combination of Glutathione, ade- metionine and UDCA served as UDCA group. Normal controls were 30 healthy infants. Total RNAS were extracted from peripheral blood , and were reverse transcripted to cDNA. The mRNA levels were determined by SYBR Green I realtime fluorescent quantitative reverse transcription polymerase chain reaction, and analysis method were performed to detect the mRNA expression of ABCB4 and FXR gene. Relative expression of mRNA in two groups were compared by 2-△△Ct method of relative quantification. Expressed correlations between the gene expression levels and indices of blood testing were calculated.Results: Serum levels of TB, DB andγ-GT were significantly decreased after UDCA treatment in UDCA group patients(P<0.05). The decreased levels of serum TB and DB were higher in UDCA group than Conventional treatment group(P<0.05). Compared with normal controls, FXR mRNA levels were not significantly changed(P>0.05), ABCB4mRNA levels were increased 4.53-fold in infant with cholestatic hepatitis(P<0.05). After UDCA treatment, ABCB4 mRNA levels were increased 2.04-fold(P<0.05), while FXRmRNA levels were not altered in UDCA group(P>0.05). Serum levels of TB, DB,γ-GT and ALT were not correlated with the ABCB4mRNA levels(P>0.05). Conclusion: UDCA exerted beneficial effects in infant with cholestatic hepatitis which improved clinic and liver function. UDCA may up-regulate ABCB4mRNA. In infant with cholestatic hepatitis, ABCB4 mRNA levels were not changed, it could not be demonstrated that FXR up-regulate ABCB4 mRNA. Part Three Analysis of ABCB4 gene mutations in infant with cholestatic hepatitisObjective : The purpose of this study was to investigate whether ABCB4 gene mutations are associated with infant with cholestatic hepatitis and to deter- mine whether they can be a new genetic biomarker for predicting the prognosis of cholestatic hepatitis and characterized the genotypes with respect to clinic features.Methods: Genomic DNA were obtained from peripheral blood of 100 patients and 149 healthy subjects. Sequencing of ABCB4 gene spanned all 28 exons and comprised 100-300bp of the flanking intronic region around each exon in 100 patients, genomic variants detected in patients were measured in 149 health controls. The genotype distributions and allele frequencies were compared between patients and controls, and the association of genotypes with clinic features were studied.Results: A total of 5 variant sites were found in patients, they were single nucleotide substitutions, 4 of which were in Hardy-Weinberg equilibrium. In health controls, the most common genotype of rs2302387, rs1202283, rs8187- 791 and rs2230028 were G/G, G/A, C/C and A/A respectively. The genotype distributions and allele frequencies of rs8187791 were not significantly differ- ences with other peoples in all regions. The genotype distributions and allele frequencies of rs2302387, rs1202283 and rs2230028 were not significantly differences with Han Chinese in Beijing and Japanese, and were significantly differences with European and African. The distributions of four SNPs genotype and alleles did not differ between patients and controls. Comparision of liver funcion in patient was not significantly differences in 4 SNPs genotype. There were no significant differences in haplotype distribution between patients and controls.Conclusion: ABCB4 mutations were not responsible for infant with cho- lestatic hepatitis, and they could not be a genetic biomarker for predicting the prognosis. As we knew, this was the first study about rs 2302387, rs1202283,rs8187791 and rs2230028 genotype distributions in healthy infant in Nanning region. The distribution of rs8187791 genotype was similar in all regions. Rs23- 02387, rs1202283 and rs2230028 genotype distributions were similar with Han Chinese in Beijing and Japanese, but were significant differences with European and African. |
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