Studies On The Role Of Zebrafish Heme Oxygenase 1 In Hypoxia Adaptation And The Development Of Transgenic Zebrafish

Heme oxygenase 1(HO1)is an enzyme that catalyzes the first and ratelimiting reaction in heme degradation.HO1 has protective effects through anti-inflammatory,anti-apoptosis and anti-oxidant stress in many tissues and cells.The expression of HO1 can be dramatically induced by a variety of stress-associated agents,including heavy metals,UV irradiation and oxidative stress.Our previous studies have found that hypoxia treatment can increase ho1 mRNA level significantly in crucian carp and zebrafish embryos.In this paper we studied the role of zebrafish Ho1 in hypoxia adaptation and the development of transgenic zebrafish.The results are as follows:We detected the expression of ho1 mRNA in ZF4 cells exposed to hypoxia for 24h,48h and 72h using Real-Time PCR.It was found that ho1 mRNA was induced significantly by hypoxia.It suggested that Drhol may play an important role in hypoxia adaption of zebrafish.The expression of Ho1 in ZF4 cells under hypoxia was inhibited by ZnPP ? with different concentrations.The viability of ZF4 cells was analyzed using CCK-8 assay.The result showed that the viability of ZF4 cells under hypoxia was markedly reduced by ZnPP ?in a concentration-dependent manner.Hemin,the inducer of Ho1,could restore the effect.Based on the result,we selected 10 ?M ZnPP ? to perform further study.ZF4 cells were randomly divided into four groups:normoxia group,hypoxia group,ZnPP?+Hypoxia group and ZnPP ?+Hemin+Hypoxia group.The normoxia group was treated with 10?M DMSO under nomoxia.The latter three groups were treated with 10?M DMSO?10 ?M ZnPP ??10?M ZnPP IX+ Hemin respectively under hypoxia.We detected the growth of ZF4 cells under light microscope and found that inhibition of Ho1 expression can increase the death rate of ZF4 cells under hypoxia.These results showed that inducible expression of Ho1 can protect ZF4 cells against cell death under hypoxia.The expression of Ho1 was inhibited by ZnPP ?.The apoptosis of ZF4 cells were analyzed using Hochest staining,DNA ladder assay and Caspase 3 activity detection.The results showed that there was no apoptosis in nomoxia group.Hypoxia induced a few apoptosis in ZF4 cells.Inhibition of Ho1 expression can significantly increase the apoptosis rate of ZF4 cells under hypoxia.Hemin,the inducer of Ho1,could reduce cell apoptosis to a certain extent.These results showed that inducible expression of Ho1 can protect ZF4 cells against apoptosis under hypoxia.Zebrafish gDNA was extracted and used as template to amplify 1765bp Drhol promoter.The Drhol promoter was cloned into pBK-To12 and got recombinant plasmid pDrhol-Pro-GFP.Using crucian carp and zebrafish cDNA,Cahol ORF and hsp70 promoter were also amplified respectively.They were cloned into pT2AL200R and got recombinant plasmid pCahol-hsp-GFP.Plasmids pDrhol-Pro-GFP and pCahol-hsp-GFP were microinjected into one cell zebrafish embryos respectively.The embryos were detected under fluorescence microscope.We found that GFP expressed in many microinjectd embryos.It showed that the two promoters were active.Then,we mixed the two recombinant plasmids with transposase mRNA respectively and injected them into one cell zebrafish embryos to get transgenic zebrafish Tg(Drhol-Pro:GFP)and Tg(Cahol:GFP).After zebrafish Tg(Drho1-Pro:GFP)grew up,gDNA were extracted from the fin and PCR were performed to determine whether Drhol-Pro:GFP inserted into zebrafish gDNA.The result sHowed that we got two FO transgenic zebrafish Tg(Drhol-Pro:GFP).Zebrafish Tg(Cahol:GFP)is too small to get its fin.They will be identified two month later.The construct of the two transgenic zebrafish(Tg(Drho1-Pro:GFP)and Tg(Cahol:GFP)will be useful for further study on the protective role of Ho1 in fish hypoxia adaptation.