Transfection of 12/15-lipoxygenase effectively alleviates inflammatory responses during experimental acute pancreatitis

BACKGROUND Acute pancreatitis(AP),the initially triggered inflammatory process in the pancreas,can be life-threatening.It has been reported that 15-lipoxygenase may promote the removal of damaged intracellular components,maintain intracellular homeostasis,and promote apoptosis by upregulating the activity of caspases.Despite an increased understanding of the lipoxygenase pathway in inflammation and immune diseases,the role of the Alox15 gene product in modulating the inflammatory changes during AP is not well defined.AIM To investigate the effect of Alox15 expression in cerulein-induced AP in rats.METHODS Model rats were transfected with Alox15 by injecting a recombinant lentivirus vector encoding Alox15 into the left gastric artery before inducing AP.The expression of Alox15 was then assessed at the mRNA and protein levels.RESULTS Our in vivo results showed that serum amylase activity and pancreatic tissue water content were significantly reduced in Alox15-transfected rats.Further,the mRNA expression levels of tumor necrosis factor alpha,interleukin(IL)-1β,IL-6,and monocyte chemoattractant protein-1,as well as the protein expression of nuclear factor kappa B in pancreatic tissue were reduced.Additionally,we observed an upregulation of cleaved caspase-3 that implies an induction of apoptosis in pancreatic cells.The transfection of Alox15 resulted in a lower number of autophagic vacuoles in AP.CONCLUSION Our findings demonstrate a regulatory role of Alox15 in apoptosis and autophagy,making it a potential therapeutic target for AP.

Involvement of eicosanoids in the pathogenesis of pancreatic cancer: the roles of cyclooxygenase-2 and 5-lipoxygenase

The interplay between inflammation and cancer progression is a growing area of research. A combination of clinical, epidemiological, and basic science investigations indicate that there is a relationship between inflammatory changes in the pancreas and neoplastic progression. Diets high in ω-6 polyunsaturated fatty acids provide increased substrate for arachidonic acid metabolism by cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) to form eicosanoids. These eicosanoids directly contribute to pancreatic cancer cell proliferation. Both COX-2 and 5-LOX are upregulated in multiple cancer types, including pancreatic cancer. In vitro studies using pancreatic cancer cell lines have demonstrated upregulation of COX-2 and 5-LOX at both the mRNA and protein levels. When COX-2 and 5-LOX are blocked via a variety of mechanisms, cancer cell proliferation is abrogated both in vitro and in vivo. The mechanism of COX-2 has been shown to include effects on apoptosis as well as angiogenesis. 5-LOX has been implicated in apoptosis. The use of COX-2 and 5-LOX inhibitors in clinical studies in patients with pancreatic cancer has been limited. Patient enrollment has been restricted to those with advanced disease which makes evaluation of these drugs as chemopreventive agents difficult. COX-2 and 5-LOX expression have been shown to be present during the early neoplastic changes of pancreatic cancer, well before progression to invasive disease. This indicates that the ideal role for these interventions is early in the disease process as preventive agents, perhaps in patients with chronic pancreatitis or hereditary pancreatitis.

Expression of 5-Lipoxygenase in human colorectal cancer

AIM: To evaluate the 5-lipoxygenases (Loxs) expression level in human colorectal cancer specimens in order to determine its clinicopathologic significance in human tumorigenesis. METHODS: The relative quantity of 5-Lox mRNA in paired 91 colorectal tumor and adjacent normal mucosa samples was determined by real time quantitative PCR. Additionally, the expression of 5-Lox and cyclooxygenase (Cox)-2 proteins was also examined using immunohistochemical staining methods. RESULTS: There was a marked increase in 5-Lox mRNA levels in the tumor compared with paired normal mucosa samples (P < 0.0001). Sixty six (72.5%) tumors showed high 5-Lox mRNA levels. The positivity rate of 5-Lox and Cox-2 protein expression was 68.7% and 79.1% respectively. There was a significant association between tumoral 5-Lox mRNA level and tumor size (Rho = 0.392, P = 0.0002), depth or vessel invasion. CONCLUSION: These results suggest that 5-Lox is up-regulated in colorectal cancer and that inhibition of its expression might be valuable in the prevention and treatment of colorectal cancer.

QSAR modeling of benzoquinone derivatives as 5-lipoxygenase inhibitors

The inhibitors of 5-LOX control the overproduction of pro-inflammatory mediators known as leukotrienes(LTs)and thus have therapeutic relevance in the treatment of various diseases like asthma,rheumatoid arthritis,inflammatory bowel disease and certain types of cancers.This has increased the search for efficient therapeutic agents for protein 5-LOX and this process is now primarily based on QSAR.In this study,we have developed four different quantitative structure and 5-LOX inhibition activity relationship models of benzoquinone derivative by exploiting CoMFA,RF,SVM,and MLR chemometric methods.Performance of the QSAR models was measured by using cross-validation technique as well as through the external test set prediction.RF model outperforms all other models.SVM and MLR models failed due to the poor performance of the external test set prediction.CoMFA model,which shows relatively good performance was used to explore the essential structural regions where the modification was necessary to design a novel scaffold with improved activity.Moreover,molecular docking of all the derivatives to the binding site of 5-LOX was done to show their binding mode and to identify critical interacting residues inside the active site of 5-LOX.The docking result confirms the stability and rationality of the CoMFA model.

Variants of the arachidonate 5-lipoxygenase-activating protein (ALOX5AP) gene and risk of ischemic stroke in Han Chinese of eastern China

Variants of the arachidonate 5-1ipoxygenase-activating protein （ALOX5AP） gene have been suggested to play an important role in the pathogenesis of atherosclerosis and ischemic stroke. This study was aimed to explore the association of ALOX5AP variants with ischemic stroke risk in Han Chinese of eastern China. A total of 690 ischemic stroke cases and 767 controls were recruited. The subjects were further subtyped according to the Trial of Org 10172 in Acute Stroke Treatment （TOAST） criteria. On the basis of that, two polymorphisms of the ALOX5AP gene （rs10507391 and rs12429692） were determined by TaqMan genotyping assay. In addition, plasma leukotriene B4 （LTB4） levels were analyzed in these subjects. There was no evidence of association between the two variants of ALOX5AP and the risk of ischemic stroke or its TOAST-subtypes. Haplotype analysis and stratification analysis according to sex, age, body mass index, hypertension, and diabetes also showed negative association. Analysis of LTB4 levels in a subset of cases and controls revealed that LTB4 levels were significantly higher in ischemic stroke cases than in the controls （70.06± 14.75 ng/L vs 57.34±10.93 ng/L; P = 0.000） and carriers of the T allele of the rs10507391 variant were associated with higher plasma LTB4 levels （P = 0.000）. The present study suggests there is no association of the two polymorphisms in the ALOX5AP gene with ischemic stroke risk in Han Chinese of eastern China.

15-Lipoxygenase inhibition of Commelina benghalensis,Tradescantia fluminensis,Tradescantia zebrina

Objective:To evaluate the 15-lipoxygenase inhibitory activity of the methanol leaf extracts of Commelina benghalensis,Tradescantia fluminensis(T.fluminensis)and Tradescantia zebrina.Method:The inhibitory activity was evaluated using a spectrophotometric assay by observing the increase in absorbance at 234 nm due to the formation of the product 13-hydroperoxyoctadecadienoic acid.The extracts were also tested for the presence of terpenoids,saponins,tannins,flavonoids,steroids,phenolic compounds,alkaloids and cardiac glycosides.Results:All the extracts inhibited the action of 15-lipoxygenase at a concentration of 0.2μg/mL.T.fluminensis and Tradescantia zebrina exhibited higher than 50%inhibition with T.fluminensis at 87.2%.T.fluminensis was partitioned with ethyl acetate and hexane and their IC_(50)values were determined at 8.72μg/mL and 98.04μg/mL,respectively.Conclusions:T.fluminensis is a potentially good source of 15-lipoxygenase inhibitors.

5-Lipoxygenase and cysteinyl leukotriene receptors in neuroinflammation and neuronal injury

Brian ischemic injury and central neurodegenerative diseases as leading contributors to disability and death have become a majorclinical and public health concern worldwide.Neuroinflammation plays a pivotal role in the pathological progression of cerebral ischemia and neurodegenerative diseases including Parkinson disease(PD).Therefore,it is important to find effective therapeutic targets to attenuate inflammation and delay the progression of brain injury.Cysteinyl leukotrienes(CysLTs) are potent inflammatory mediators synthesized from arachidonic acid by 5-lipoxygenase(5-LOX) in the central nervous system.Two distinct G-protein-coupled receptors,CysLT1 R and CysLT2 R,mediate most of the known CysLTs biological responses.Accumulating evidence has demonstrated that postischemic inflammation and neuronal loss are mediated by 5-LOX and CysLTRs fol owing focal cerebral ischemia.We recently reported that the expression of 5-LOX,CysLT1R and inflammatory vascular cell adhesion molecule-1(VCAM-1) was upregulated in the hippocampus of rats with transient global cerebral ischemia,which was closely associated with delayed neuronal death in the hippocampal CA1 area.5-LOX inhibitor zileuton,CysLT1R antagonist ONO-1078 and montelukast dose-dependently reduced hippocampal CA1 neuronal death and inhibited the increased expression of 5-LOX and VCAM-1.In vitro ischemia-like injury in 5-LOXtransfected PC12 cells,oxygen-glucose deprivation(OGD) induced cell death mediated by5-LOX via ROS/P38 MAPK pathway.The nonselective 5-LOX inhibitor caffeic acid inhibited OGDstimulated activation of 5-LOX and ROS/P38 MAPK signaling and improved neuronal survival.In PD model,high concentrations of rotenone caused directly PC12 neurotoxicity,which was modulated by 5-LOX and abolished by suppression of 5-LOX.It is well known that microglia is major modulators of inflammatory response after brain injury.Overactivated microglia and production of proinflammatory cytokine IL-1β,IL-6 and TNF-α contribute to the neuroinflammation and brain injury.5-LOX,CysLT1R and CysLT2R are involved in microglial activation and resultant neurotoxic responses.It has been found that low concentrations of rotenone can activate 5-LOX and CysLT1R on microglial cells to enhance microglial inflammation and microglia-dependent neuronal death in vitro.5-LOX inhibitor zileuton and CysLT1R antagonist montelukast protected neurons from microglia-dependent rotenone neurotoxicity.Furthermore,lipopolysaccharide(LPS)induced microglial activation and microglial neurotoxicity mediated by CysLT2R in vitro.Both pharmacological blockade(CysLT2R antagonist HAMI3379) and RNA interference(specific short hairpin RNA) of CysLT2 R significantly attenuated LPS-triggered microglial inflammation and subsequent neuronal death.Collectively,the present results indicate the role of 5-LOX and CysLTRs in neuroinflammation and brain injury.Modulation of 5-LOX and CysLTRs may be potential therapeutic approaches for inflammation-related brain disorders such as cerebral ischemia and PD.However,further research is needed to clarify the mechanisms underlying the regulation of neuinflammatory processes by 5-LOX and CysLTRs.

The effects of 5-lipoxygenase inhibitor and cysteinyl leukotriene receptor 1 antagonist on 1-methyl-4-phenylpyridine-induced neurotoxicity

OBJECTIVE Previously we demonstrated the neuroprotective effect of 5-lipoxygenase(5-LOX)inhibitor as well as cysteinyl leukotriene receptor 1(Cys LT1)antagoniston rotenone-induced microglial activation and neuronal death.In this study,we determined the effects of 5-LOX inhibitor zileuton and Cys LT1 antagonist montelukast on neurotoxicity induced by 1-methyl-4-phenylpyridine(MPP+)in an in vitro model of Parkinson disease(PD).METHODS The neurotoxicity of MPP+,a neurotoxin relevant to PD,on the PC12 cells was measured by MTT assay,lactate dehydrogenase(LDH)release and double fluorescence staining with Hoechst/propidiumiodide(PI).The protective effects of 5-LOX inhibitor zileuton and Cys LT1 antagonist montelukast were investigated by the above methods.RESULTS We found that exposure of PC12 cells to MPP+led to a reduced cell viability and an increased level of LDH in a concentration-dependent manner.Pretreatment with zileuton and montelukast significantly attenuated viability loss and LDH release in MPP+-treated PC12 cells.Furthermore,MPP+increasednecrotic cell death in PC12 cells.Administration of montelukast significantly decreased MPP+-induced cell necrosis in PC12 cells.CONCLUSION The 5-LOX inhibitor zileuton and Cys LT1 antagonist montelukast have a neuroprotective effects on MPP+-induced neurotoxicity in PC12 cells.The 5-LOX inhibitor and Cys LT1 antagonist might raise a possibility as potential therapeutic agent for PD and other inflammation-related the central nervous system disorders.

5-lipoxygenase expression in a brain damage model induced by chronic oral administration of aluminum

A preliminary study has found that the 5-lipoxygenase inhibitor, caffeic acid, has a marked protective effect on acute brain injury induced by intracerebroventricular microinjection of aluminum. In this experiment, chronic brain injury and neuronal degeneration model was established in rats by chronic oral administration of aluminum, and then intervened using caffeic acid. Results showed that caffeic acid can downregulate chronic aluminum overload-induced 5-1ipoxygenase mRNA and protein expression, and repair the aluminum overload-induced hippocampal neuronal damage and spatial orientation impairment. It is suggested that direct intervention of 5-lipoxygenase expression has a neuroprotective role in the degeneration induced by chronic aluminum overload brain injury model.

A Facile Total Synthesis of 2-Geranyl-2',3,4',4-tetrahydroxydihydrochalcone with Highly 5-Lipoxygenase Inhibiting Activity

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C1q/TNF-related protein 5 promotes atherogenesis by enhancing transcytosis and oxidative modification of low-density lipoprotein through increasing 12/15-lipoxygenase

Objective Increased transcytosis of low-density lipoprotein (LDL)across the endothelium and oxidation of LDL deposited within the subendothelial space are crucial early events in atherogenesis. C1q/TNF-related protein (CTRP) 5 is a novel secreted glycoprotein and its biological functions are largely undefined.

Further evidence for the roles of 5-lipoxygenase and cysteinyl leukotriene receptors in cerebral ischemic injury

Arachidonic acid-metabolizing enzyme 5-lipoxygenase (5-LOX) produces pro-inflammatory mediators:leukotrienes (including cysteinyl leukotrienes, CysLTs). 5-LOX and CysLTs are involved in the pathophysiological process after brain injury, and the actions of CysLTs are mediated via activation of their receptors, CysLT1 and CysLT2. We have recently reported the expressions of 5-LOX, CysLT1 and CysLT2 in human brains with traumatic injury and tumors, and short-term neuroprotective effects of Cys-LT1 antagonists in stroke models of rats and mice.

Expression manner of 5-lipoxygenase in human traumatic brain injury and astrocytoma

5-lipoxygenase (5-LO) is a key enzyme of arachidonic acid metabolism. The metabolites, leuklotrienes, are important mediators in asthma, inflammatory and allergic disorders. The activation of 5-LO includes the gathering of 5-LO,adhering onto the membranes of nuclei and entrance to the nuclei. In the central nervous system, 5-LO is widely

Biological Evaluation of Lysionotin:a Novel Inhibitor of 5-Lipoxygenase for Anti-glioma

Objective:To explore the potential mechanism of lysionotin in treating glioma.Methods:First,target prediction based on Bernoulli Naïve Bayes profiling and pathway enrichment was used to predict the biological activity of lysionotin.The binding between 5-lipoxygenase(5-LO)and lysionotin was detected by surface plasmon resonance(SPR)and molecular docking,and the inhibitory effects of lysionotin on 5-LO and proliferation of glioma were determined using enzyme inhibition assay in vitro and cell viability analysis,respectively.Furthermore,the pharmaceutical effect of lysionotin was explored by cell survival rate analysis and liquid chromatography with tandem mass spectrometry(LC-MS/MS).The protein expression,intracellular calcium ion concentration and cytoskeleton detection were revealed by Western blot,flow cytometry and fluorescence labeling,respectively.Results:Target prediction and pathway enrichment revealed that lysionotin inhibited 5-LO,a key enzyme involved in the arachidonic acid metabolism pathway,to inhibit the proliferation of glioma.Molecular docking results demonstrated that 5-LO can be binding to lysionotin through hydrogen bonds,forming bonds with His600,Gln557,Asn554,and His372.SPR analysis further confirmed the interaction between 5-LO and lysionotin.Furthermore,enzyme inhibition assay in vitro and cell survival rate analysis revealed that 50%inhibition concentration of lysionotin and the median effective concentration of lysionotin were 90 and 16.58µmol/L,respectively,and the results of LC-MS/MS showed that lysionotin inhibited the production of 5S-hydroperoxy-eicosatetraenoic acid(P<0.05),and moreover,the LC-MS/MS results indicated that lysionotin can enter glioma cells well(P<0.01)and inhibit their proliferation.Western blot analysis demonstrated that lysionotin can inhibit the expression of 5-LO(P<0.05)and downstream leukotriene B4 receptor(P<0.01).In addition,the results showed that lysionotin affected intracellular calcium ion concentration by inhibiting 5-LO to affect the cytoskeleton,as determined by flow cytometry and fluorescence labeling.Conclusion:Lysionotin binds to 5-LO could suppress glioma by inhibiting arachiodonic acid metabolism pathway.

Butyrolactones,inhibitors of 5-lipoxygenase from fungal metabolites

In our ongoing search for new inhibitors of 5-lipoxygenase（5-LOX） from microbial resources,Aspergillus F06Z-509 was found to produce active components.Three active compounds named F06Z-509-A,B and C were obtained and identified as butyrolactoneⅡ,ⅠandⅢby NMR and MS data analyses.They showed inhibitory activity against 5-LOX with IC50 of 21.43, 22.51 and 11.83μg/mL,respectively.Butyrolactones are shown to be inhibitors of 5-LOX for the first time.

Short-term intensive atorvastatin therapy improves endothelial function partly via attenuating perivascular adipose tissue inflammation through 5-lipoxygenase pathway in hyperlipidemic rabbits

Background Atherosclerosis is a kind of disease with multiple risk factors,of which hyperlipidemia is a major classical risk factor resulting in its pathogenesis and development.The aim of this study was to determine the effects of short-term intensive atorvastatin （IA） therapy on vascular endothelial function and explore the possible mechanisms that may help to explain the clinical benefits from short-term intensive statin therapy.Methods After exposure to high-fat diet （HFD） for 8 weeks,the animals were,respectively,treated with IA or low-dose atorvastatin （LA） for 5 days.Blood lipids,C-reactive protein （CRP）,tumor necrosis factor-α （TNF-α）,interleukin-6 （IL-6）,nitric oxide （NO）,endothelin-1 （ET-1）,and endothelium-dependent vasorelaxation function were,respectively,measured.mRNA and protein expression of CRP,TNF-α,IL-6,macrophage chemoattractant protein-1 （MCP-1）,and 5-lipoxygenase （5-LO） were also evaluated in pericarotid adipose tissue （PCAT） and cultured adipocytes.Results HFD increased serum inflammatory factor levels; induced significant hyperlipidemia and endothelial dysfunction,including imbalance between NO and ET-1; enhanced inflammatory factors and 5-LO expression; and promoted macrophage infiltration into adipose tissue.Five-day IA therapy could significantly decrease serum inflammatory factor levels and their expression in PCAT; restore the balance between NO and ET-1; and improve endothelial function and macrophage infiltration without significant changes in blood lipids.However,all of the above were not observed in LA therapy.In vitro experiment found that lipopolysaccharide （LPS） enhanced the expression of inflammatory factors and 5-LO in cultured adipocytes,which could be attenuated by short-time （6 hours） treatment of high-dose （5 pmol/L） but not low-dose （0.5 μmol/L） atorvastatin.In addition,inhibiting 5-LO by Cinnamyl-3,4-dihydroxy-α-cyanocinnamate （CDC,a potent and direct 5-LO inhibitor） could significantly downregulate the above-mentioned gene expression in LPS-treated adipocytes.Conclusion Short-term IA therapy could significantly ameliorate endothelial dysfunction induced by HFD,which may be partly due to attenuating inflammation of PCAT through inhibiting 5-LO pathway.

Structural optimization and biological evaluation of 1,5-disubstituted pyrazole-3-carboxamines as potent inhibitors of human 5-lipoxygenase

Human 5-lipoxygenase (5-LOX) is a well-validated drug target and its inhibitors are potential drugs for treating leukotriene-related disorders. Our previous work on structural optimization of the hit compound 2 from our in-house collection identified two lead compounds, 3a and 3b, exhibiting a potent inhibitory profile against 5-LOX with IC50 values less than 1 mu mol/L in cell-based assays. Here, we further optimized these compounds to prepare a class of novel pyrazole derivatives by opening the fused ring system. Several new compounds exhibited more potent inhibitory activity than the lead compounds against 5-LOX. In particular, compound 4e not only suppressed lipopolysaccharide-induced inflammation in brain inflammatory cells and protected neurons from oxidative toxicity, but also significantly decreased infarct damage in a mouse model of cerebral ischemia. Molecular docking analysis further confirmed the consistency of our theoretical results and experimental data. In conclusion, the excellent in vitro and in vivo inhibitory activities of these compounds against 5-LOX suggested that these novel chemical structures have a promising therapeutic potential to treat leukotriene-related disorders. (C) 2016 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.

白藜芦醇激活细胞外信号调节激酶5信号蛋白促进小鼠MC3T3-E1细胞增殖

背景:细胞外信号调节激酶5信号蛋白对生物体的存活不可或缺,白藜芦醇能通过多种途径促进成骨细胞增殖,但其是否能通过细胞外信号调节激酶5信号蛋白调控成骨细胞功能还需进一步验证。目的:探究细胞外信号调节激酶5对MC3T3-E1细胞增殖以及相关分泌蛋白的调控作用,进一步验证白藜芦醇通过激活细胞外信号调节激酶5完成上述过程。方法:小鼠MC3T3-E1前成骨细胞分别用完全培养基、XMD8-92(细胞外信号调节激酶5抑制剂)、表皮生长因子(细胞外信号调节激酶5激活剂)和白藜芦醇单独干预及XMD8-92+表皮生长因子、白藜芦醇+XMD8-92干预后,通过Western blot检测各组细胞内细胞外信号调节激酶5、磷酸化细胞外信号调节激酶5蛋白,增殖相关蛋白Cyclin D1、CDK4、PCNA,以及成骨细胞分泌蛋白骨保护素、核因子κB受体活化因子配体的表达情况,使用细胞免疫荧光染色检测各组细胞外信号调节激酶5、骨保护素和核因子κB受体活化因子配体荧光强度,使用EdU染色检测各组细胞增殖情况。白藜芦醇干预MC3T3-E1细胞的适宜浓度及时间由细胞形态学观察和CCK-8实验确定。结果与结论:①细胞外信号调节激酶5信号蛋白的激活能有效促进MC3T3-E1细胞增殖、上调骨保护素/核因子κB受体活化因子配体比值;②白藜芦醇干预MC3T3-E1细胞的适宜浓度及时间为5μmol/L,24 h;③白藜芦醇可以激活细胞外信号调节激酶5信号蛋白,进而促进成骨细胞增殖,并上调骨保护素/核因子κB受体活化因子配体比值;④研究结果表明,白藜芦醇可以通过激活细胞外信号调节激酶5信号蛋白促进MC3T3-E1细胞增殖,并通过激活细胞外信号调节激酶5信号蛋白上调骨保护素/核因子κB受体活化因子配体比值。

基于改进YOLOv5的红花目标检测算法研究

为实现农业非结构环境下采摘机器人对红花的准确识别,提出了一种基于改进YOLOv5的红花目标检测算法。将CBAM注意力机制嵌入到YOLOv5网络,提高了小尺寸目标物在高层次特征中的表现力;建立一种Alpha-IoU目标位置损失函数对原损失函数GIOU存在的梯度消失问题进行改进,提高了被遮挡红花的预测率,并通过在目标检测网络中增加分割检测模块,提高宽和高小于最低像素的小目标物检测精度,利用图像扩增数据集对改进后的YOLOv5算法进行训练,再分别与改进前后YOLOv5网络和Faster R-CNN网络在不同红花品种、不同自然光照情况、不同天气条件和不同遮挡情况下进行对比。试验结果表明:改进后的YOLOv5算法P值、R值分别为90.45%和0.90,对非结构环境下盛开期的未采摘红花mAP值达到94.48%,在不同影响因素下都可以准确识别出红花且置信度较高,可为红花采摘机器人自动化作业中的红花识别提供技术支持。

过表达溶质载体家族1成员5和敲低慢病毒载体构建及稳定转染RAW264.7细胞株

背景:溶质载体家族1成员5(solute carrier family 1 member 5,SLC1A5)在多种疾病中发挥了潜在作用,但确切作用机制尚不清楚。构建稳定的SLC1A5过表达和敲低细胞模型可为深入研究SLC1A5在疾病中的确切作用机制以及发现潜在治疗靶点提供有力的实验工具。目的:构建小鼠SLC1A5过表达和敲低的慢病毒载体,以建立稳定转染的RAW264.7细胞株,为深入探讨SLC1A5在炎症中的作用提供实验基础。方法:根据SLC1A5基因序列设计合成引物并使用聚合酶链反应扩增该基因片段。将目的基因定向接入经Age I/Nhe I酶切的载体质粒GV492中构建重组慢病毒质粒,对阳性克隆进一步筛选后测序比对结果;pHelper1.0质粒载体、pHelper2.0质粒载体、目的质粒载体与293T细胞共同培养并转染,获得慢病毒原液进行包装和滴度测定;在此基础上,通过体外培养RAW264.7细胞,确定嘌呤霉素工作质量浓度;不同滴度的慢病毒分别与RAW264.7细胞共同培养,根据荧光强度确定转染效率;用嘌呤霉素挑选出稳定转染细胞,实时荧光定量聚合酶链反应和蛋白免疫印迹方法检测稳定转染细胞株的SLC1A5基因和蛋白表达水平。结果与结论:(1)测序序列与目的序列一致提示重组慢病毒载体构建成功;(2)过表达SLC1A5慢病毒的滴度为1×10~9 TU/mL,敲低SLC1A5慢病毒的滴度为3×10~9 TU/mL;(3)确定RAW264.7细胞嘌呤霉素工作质量浓度为3μg/mL;(4)过表达/敲低SLC1A5慢病毒转染RAW264.7细胞的最佳条件皆为HiTransG P转染增强液且感染复数值等于50;(5)过表达SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量明显上调,而敲低SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量显著下调。结果表明,成功构建了小鼠SLC1A5过表达和敲低的慢病毒载体并获得稳定转染的RAW264.7细胞株。