| Bone morphogenetic proteins ( BMPs) are secreted signaling molecules belonging to the transforming growth factor ( TGF - β) superfamily of growth factors. The name BMPs were first given to three proteins purified from a deminer-alize bovine bone preparation that induced ectopic cartilage and eddochondral bone when implanted in experimental animals. More than 30 BMPs have been i-dentified to date, such as BMP2, BMP4, BMP7, BMPS A and so on.Recent experiments have shown that BMPs regulate biological processes as diverse as cell proliferation, apoptosis, differentiation, cell - fate determination and porphogenesis. Moreover, the vertebrate BMPs are involved in the development of nearly all organs and tissues, including the nervous system, urinary system, reproductive system, liver, lung as well as in critical steps in the establishment of the basic embryonic body plan. BMP4 is a important member in BMPs subfamily, it starts to be expressed as early as 6. 5dpc, At 7. 5dpc, BMP4 becomes widely expressed in the embryo. As development proceeds, BMP4 transcripts become detectable in many tissues and regulate the morphogenesis and maintenance of multiple tissues and organs. BMP4 performs its signal transduction by binding with BMP receptors. The ligand first binds to the type II receptor, which then recruits and phosphorylates a type I receptor. The activated type I receptor subsequently phosphorylates the intracellular target molecules , among which are the SMAD family signal transducers. Base on that, we established the ischemia/reperfusion model and investigated the expression ofBMP4 in normal adult mouse kidney and its effect on ischemia/reperfusion injury. Our experiment consisted of four parts as followings; #1Expression of bone morphogenetic protein - 4 mRNA in normal adult mouse kidney and its change in ischemia/reperfusion kidney. #2 The histological change of ischemia/ reper-fusion kidney of the mice with BMP4 knockout and its mechanism. #3 The effect of ischemia/reperfusion injury on renal function of mouse with BMP4 knockout.Materials1. Animals; 190 male wildtype mice were provided by experimental animal center of Southwestern Medical Center in USA.2. main Apparatus; MJ PCR machine, 5417C centrifuge, Heating pad, 0-lympus fluorescent microscopy, Stable temperature water bath, Microclip, auto-clip, EC - 105 electrophoretic machine, UV autocamera, electrobalance, FB warmer, liquid nitrogen tank, Milli - Q filter machine, Vacuum oven, Auto-warming paraffin tank, Stainless forceps, Stainless knife, RM - 2045 section machine, MT-360 vortex machine, Multifunction blood analyse machine.3. Main Reagent; PFA, PBS buffer, NaOH, Ethanol, Xylenes, DEPC water , Proteinase K, EDTA, Triethanolamine, Tris - HC1, Polyvinyl pyrrolidone, DTP, Dextran Sulfate, Deionized formamide, yeast tRNA, 35S-UTP, Rnasin, Hematoxylin, Eosin, Kodak emusion, TRIZol, random primer, First - Strand buffer, SDS, DNA taq polymerase, β - mercaptoethanol, SSC, BMP4 primer, BMPR primer.Methods1. Expression of bone morphogenetic protein 4 mRNA in normal adult mouse kidney and its change in ischemia/reperfusion kidney; Male wildtype mice were randomly divided into two groups (n = 10 in each group). Anesthesia was induced by injection of avertine, the left renal artery was identified. Then the kidneys were removed immediately in one group, and the left renal arterywas occluded using microclip for 60 min in another group. Then kidneys were removed at 24 h after reperfusion. A half of kidneys were fixed in 4% paraform-aldehyde, the others were stocked in liquid nitrogen. To observe the expression of BMP4mRNA in normal adult mouse kidney and its change in ischemia/reper-fusion kidney by in situ hybridization and RT - PCR.2. The histological change of ischemia/ reperfusion kidney of the mice with BMP4 knockout and its mechanism: Three groups mice with different genotype were selected by PCR. I group: wildtype (BMP4/BMPR: +/+ ; + / + ) ,H group: BMP4 knockout ( BMP4/BMPR.- +/ - ; +/ + ) , III gr...
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