Acta Pharmacologica Sinica 2005 Jul; 26 (7): 845-850; doi: 10.1111/j.1745-7254.2005.00138.x

Aim: To construct pEGFP-N3 recombinant vectors carrying adrenomedullin (AM) or fragments of the AM gene, and to express AM or fragments of AM from the pEGFP-N3 recombinant vectors (pEGFP-N3-AM1-2 and pEGFP-N3-AM1-3) and study their biological properties on cultured rat renal mesangial cells (RMC).

Methods: Total RNA of rat kidney was obtained using TriZol reagent. The cDNA was synthesized by reverse transcriptase using oligo-deoxythymidine as primer. The fragments of AM gene were then amplified by polymerase chain reaction (PCR) with specific upstream and downstream oligonucleotides. The PCR products were digested with EcoRI and BamHI and subcloned into the plasmid pEGFP-N3. Facilitated by cationic liposomes, RMC were transfected with pEGFP-N3-AM1-2 or pEGFP-N3-AM1-3. After 24 h, green fluorescent protein (GFP) fluorescent images were examined with a fluorescence microscope. After 48 h, the proliferation of RMC was detected using the MTT assay, and the mRNA expression of transforming growth factor-β1 (TGF-β1) was measured by semiquantitative PCR.

Results: DNA sequence reports verified that pEGFP-N3-AM1-2, which carried the full length AM gene translation fragment (preproadrenomedullin preproAM_1-185), and pEGFP-N3-AM1-3, which carried the translation fragment of preproAM [without adrenotensin (ADT, preproAM_150-185)], were constructed successfully. After 24 h, green fluorescence was observed in RMC into which either pEGFP-N3-AM1-2 or pEGFP-N3-AM1-3 was transfected, while in the control cells no fluorescence was observed. Either pEGFP-N3-AM1-2 or pEGFP-N3-AM1-3 delivery inhibited the proliferation of RMC (P<0.01) and decreased the mRNA transcription level of TGF-β1 in RMC (P<0.05). However, no significant difference was observed between the effects of pEGFP-N3-AM1-2 and pEGFP-N3-AM1-3.

Conclusion: pEGFP-N3-AM1-2 and pEGFP-N3-AM1-3 were constructed successfully and were functionally expressed in RMC. Expressing the fragment of AM without ADT has similar inhibitory biological effects on RMS proliferation and TGF-β1 transcription with full length preproAM.

Keywords: adrenomedullin; transfection; glomerular mesangium; transforming growth factor-β1