Expression Of Cytohrome P450 MRNA In Experimental Miniature Pigs Quantitated With Real-Time Reverse Transcriptions Polymerase Chain Reaction

According to cloning standards and screening TaqMan PCR system, quantitativereal-time RT-PCR method suitable for studying expression revels of CYPs mRNA wasestablished and used to evaluate the feasibility of experimental miniature pigs' livers usingas model of security estimate of drug by quantitation expression livers of CYP3A29 mRNAin livers of 4-6 months old Bama miniature pigs, Guizhou miniature pigs and Rongchangpigs. Then, CYP3A29 mRNA in livers of 0, 40, 120, 180 and 720 days old experimentalminiature pigs seclected according to screening 3 breeds of pigs were quantitated, and theages during which the expression levels of CYP3A29 mRNA in experimental miniaturepigs'livers are close to that of CYP3A4 in human livers. Tissue distribution of CYP1A1,2A19, 2D25, 2E1 and 3A29 mRNA expression, and the effect of rifampicin on CYP3A29mRNA of livers were studied with these screening experimental miniature pigs. Thefollowing was concluded after comparing these results to that of human counterpartdocumented:1. Real-time RT-PCR method for quantitation expression levels of CYPs mRNA inpigs was established. Total volume of PCR was 10μL, including 1×Buffer,5.5 mmol/LMg²⁺,0.8 mmol/L dNTP mix,0.3μmol/L each primer,0.2 U/μL rTaq,0.1μmol/L probeand 1μL template. PCR was conducted by 94℃5 min→40 cycles(94℃15 s→60℃45s→read panel). This method had good properties of broad rang of quantitation, highefficiency of amplification and good repetition of determination.2. The average expression levels of CYP3A29 mRNA in livers of 4-6 months old ofBama miniature pigs, Guizhou miniature pigs and Rongchong pigs were 35.36±14.69, 21.48±11.87 and 38.87±20.11, respectively. These values were close to that of humanlivers' CYP3A4 that was the conterpart of pig's CYP3A29 in human body. This resultindicated transcriptionally that 4-6 months old of Bama miniature pigs, Guizhou miniaturepigs and Rongchong pigs were all suitable for experimental model of security estimate ofdrug metabolismed by human CYP3A4.3. liver had the highest level of CYP3A29 mRNA than the other tissues in Bamaminiature pigs. There were remarkable increase of CYP3A29 mRNA levels in Bamaminiature pigs' livers from 1-40 days and 40-120 days. The average expression levels ofCYP3A29 mRNA in livers of 120 days(sexual mature) and 180 days(bodily mature) old ofBama miniature pigs were 16.7243908±8.17834582 and 29.76705722±18.73595974,respectively. These values were close to that of human livers' CYP3A4. These resultsindicated transcriptionally that sexual mature and bodily mature of Bama miniature pigswere all suitable for animal model of security estimate of drug metabolismed by humanCYP3A4.4. liver had the highest level of CYP1A1, 2A19, 2D25 and 2E1 mRNA than the othertissues in bodily mature of Bama miniature pigs. The average expression levels were13.24145753±9.166365249, 134.5902182±68.02425634, 33.43790049±43.12612521 and162.783024±208.8879079, respectively. These values were quite large different from thatof human livers' CYP1A2, 2A6, 2D6 and 2E1 that was the counterparts of pig's CYP1A1,2A19, 2D25 and 2E1 in human body. These results indicated transcriptionally that bodilymature of Bama miniature pigs were not idea animal models of security estimate of drugsmetabolismed by human CYP1A2, 2A6, 2D6 or 2E1.5. The average expression levels of CYP3A29 mRNA in livers of bodily mature ofBama miniature pigs icreased remarkablely treated with rifampicin, and the value was 7.71times of that of untreated bodily mature of Bama miniature pigs. Expression levels ofCYP1A2, 2A6, 2D6 and 2E1 mRNA were all not affected by treatition of rifampicin. Thisresult indicated induciablely that bodily mature of Bama miniature pigs were all suitablefor experimental model of security estimate of drug metabolismed by human CYP3A4.