Similar effects on rat renal mesangial cells by expressing different fragments of adrenomedullin gene in vitro.
OBJECTIVE
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-beta1 (TGF-beta1) 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 preproAM1-185), and pEGFP-N3-AM1-3, which carried the translation fragment of preproAM [without adrenotensin (ADT, preproAM150-185)], 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-beta1 in RMC (P<0.05). However, no significant difference was observed between the effects of pEGFP-N3-AM1-2 and pEGFP-N3-AM1-3.


CONCLUSIONS
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-beta1 transcription with full length preproAM.
Chen
Shao-Wei
SW
Department of Physiology and Pathophysiology, Shanghai Medical College, Fudan University, Shanghai 200032, China.
Wang
Xiao-Fang
XF
Shao
Ying
Y
Xue
Hong
H
Zhou
Li
L
Yao
Tai
T
Lu
Li-Min
LM
D053607:Adrenomedullin; D000818:Animals; D049109:Cell Proliferation; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D050527:Mesangial Cells / Q000166:cytology / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D053773:Transforming Growth Factor beta1
D010446:Peptide Fragments; D010455:Peptides; D011498:Protein Precursors; D011994:Recombinant Proteins; C508962:Tgfb1 protein, rat; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C112034:preproadrenomedullin; D053607:Adrenomedullin