Chen Shao-Wei, Wang Xiao-Fang, Shao Ying, Xue Hong, Zhou Li, Yao Tai, Lu Li-Min
Department of Physiology and Pathophysiology, Shanghai Medical College, Fudan University, Shanghai 200032, China.
Acta Pharmacol Sin. 2005 Jul;26(7):845-50. doi: 10.1111/j.1745-7254.2005.00138.x.
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).
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.
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.
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.
构建携带肾上腺髓质素（AM）或AM基因片段的pEGFP-N3重组载体，从pEGFP-N3重组载体（pEGFP-N3-AM1-2和pEGFP-N3-AM1-3）表达AM或AM片段，并研究它们对培养的大鼠肾系膜细胞（RMC）的生物学特性。
使用TriZol试剂获得大鼠肾脏的总RNA。以寡聚脱氧胸苷为引物，通过逆转录酶合成cDNA。然后用特异性上下游寡核苷酸通过聚合酶链反应（PCR）扩增AM基因片段。PCR产物用EcoRI和BamHI消化，并亚克隆到质粒pEGFP-N3中。在阳离子脂质体的辅助下，用pEGFP-N3-AM1-2或pEGFP-N3-AM1-3转染RMC。24小时后，用荧光显微镜检查绿色荧光蛋白（GFP）荧光图像。48小时后，用MTT法检测RMC的增殖，并用半定量PCR法检测转化生长因子-β1（TGF-β1）的mRNA表达。
DNA序列报告证实，成功构建了携带全长AM基因翻译片段（前肾上腺髓质素前体AM1-185）的pEGFP-N3-AM1-2和携带前体AM翻译片段[无肾上腺紧张素（ADT，前体AM150-185）]的pEGFP-N3-AM1-3。24小时后，在转染了pEGFP-N3-AM1-2或pEGFP-N3-AM1-3的RMC中观察到绿色荧光，而在对照细胞中未观察到荧光。pEGFP-N3-AM1-2或pEGFP-N3-AM1-3的递送均抑制了RMC的增殖（P<0.01），并降低了RMC中TGF-β1的mRNA转录水平（P<0.05）。然而，pEGFP-N3-AM1-2和pEGFP-N3-AM1-3的作用之间未观察到显著差异。
成功构建了pEGFP-N3-AM1-2和pEGFP-N3-AM1-3，并在RMC中功能性表达。表达无ADT的AM片段对RMS增殖和TGF-β1转录具有与全长前体AM相似的抑制生物学作用。
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