| Objective Construct the stable expression toll-like receptor4(TLR4), myeloiddifferentiation protein-2(MD2) and leukocyte differentiation antigen14(CD14)genes of cytology model in vitro, using genetic engineering and molecular biologymethod. Provide a simple and feasible laboratory technology and method for pyrogentest of medical injection drugs.Method1. Clone target sequence：using the pcDNA.3.1-TLR4-YFP plasmid as template,obtain the TLR4DNA by PCR. Connect recycled PCR products into pMD19-T clonevector. Extract the total RNA of human peripheral blood mononuclear cells and obtainthe MD2and CD14DNA by RT-PCR. Connect recycled PCR products into pMD19-Tclone vector respectively.2. Constuct eukaryotic expression vector：pMD19-T-TLR4clone and pCAG-GFPexpression vector were double digested by KpnI and SmaI simultaneously, andrecovered the fragments. Then the two DNA fragments were ligated, to constructpCAG-GFP-TLR4expression plasmid; pMD19-T-MD2plasmid and pCAG-GFPexpression vector were double digested by KpnI and EcoRI simultaneously, andrecovered the fragments. Then the two DNA fragments were ligated, to constructpCAG-GFP-MD2expression plasmid; pMD19-T-CD14plasmid and pCAG-GFPexpression vector were double digested by KpnI and EcoRI simultaneously, andrecovered the fragments. Then the two DNA fragments were ligated, to constructpCAG-GFP-CD14expression plasmid. 3. Plasmid transfect and expression detection: transfect the successfully builtpCAG-GFP-TLR4, pCAG-GFP-MD2and pCAG-GFP-CD14expression plasmidsinto PC-3M, HIT cell line by electrical transfection methods. After the cell lines weretransfected48h, observe the GFP expression in the cell lines by fluorescencemicroscope.Results1. Successfully obtained DNA of human TLR4, MD2and CD14by PCR andconstructed the pMD19-T-TLR4, pMD19-T-MD2and pMD19-T-CD14plasmids.Restriction enzyme digestion, sequencing and BLAST identified the sequence wascorrect.2. Successfully constructed pCAG-GFP-TLR4, pCAG-GFP-MD2andpCAG-GFP-CD14plasmids. Restriction enzyme digestion, sequencing and BLASTidentified the sequence was correct.3. The pCAG-GFP-TLR4, pCAG-GFP-MD2and pCAG-GFP-CD14plasmids weretransfected into PC-3M, HIT cell lines by electrical transfection methods. Aftertransfect48h, the GFP expressed, indicating the plasmids were successfully expressedin vitro culture conditions.ConclusionSuccessfully constructed expression vectors of pyrogen reactions related gene, iehuman TLR4, MD2and CD14gene eukaryotic expression vectors pCAG-GFP-TLR4,pCAG-GFP-MD2and pCAG-GFP-CD14. Transfected them into PC-3M, HIT cellsand expressed successfully which made a good preparation for building pyrogendetection cell model. Objectives Explore the protective effect of new triterpenoids on cisplatin(DDP)-induced acute kidney Injury in rats and study its mechanism.Methods1. Protective effect of new triterpenoids on cisplatin-induced acute kidney Injury inrats.①The model of acute renal injury in rats was induced by a single intraperitonealinjection of DDP at the dose of6mg/kg.②The situation of food and drink, color, and weight changes during theadministration were observed in rats, and after modeling five days, the animalswere sacrificed, the blood were collected.③After rats were sacrificed, collect the double part of kidney, weighted, andcalculated the renal rate.④Kidney were dehydrated and embedded in paraffin,then sectioned at athickness of4μm for PASM staining to observe pathological changings.⑤The Blood urea nitrogen and Serum creatinine contents were detected.2. The mechanism of new triterpenoids protection effect against cisplatin-inducedacute kidney Injury in ratsRenal tissue was collected to measure the effect of new triterpenoids onmalondialdehyde(MDA)levels, glutathione peroxidase (GSH-PX) activities andsuperoxide dismutase(SOD) activities.Result1. Protective effect of new triterpenoids on cisplatin-induced acute kidney Injury in rats.Compare with NS group, the body weight of the rats in DDP model groupcontinually decreased, the renal rate increased, pathological changings were apparent.DDP significantly increased the serum levels of BUN and Scr(P<0.01).However,the four kinds of new triterpenoids all can effectively relieve cisplatin inducednephrotoxicity.BUN levels were significantly decreased in H0801treatment group(P<0.05) compare with model group; And Scr levels in serum were significantlydecreased in each treatment group(P<0.01).2. The mechanism of new triterpenoids protection effect against cisplatin-inducedacute kidney Injury in rats.Compare with NS group, the MDA level in renal tissue were significantlyincreased in DDP model group(P<0.01).The GSH-PX and SOD activities wereinhibited at the same time. However, the activity of SOD and GSH-PX weresignificant increased in H0801and H0806treatment groups(P<0.01,P<0.01).Conclusions the new triterpenoids has a protective effect of cisplatin-induced acutekidney injury in rats. Its mechanism may be related to antioxidative stress capability. |
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