Downregulation of cold-inducible RNA-binding protein activates mitogen-activated protein kinases and impairs spermatoRenic function in mouse testes

Cold-inducible RNA-binding protein （CIRP） is an RNA-binding protein that is expressed in normal testes and downregulated after heat stress caused by cryptorchidism, varicocele or environmental temperatures. The purpose of this study was to investigate the functions of CIRP in the testes. We employed RNAi technique to knock down the expression of CIRP in the testes, and performed haematoxylin and eosin staining to evaluate morphological changes following knockdown. Germ cell apoptosis was examined by terminal deoxynucleotidal transferase-mediated dUTP nick end labelling （TUNEL） assay, and mitogen-activated protein kinase （MAPK） signalling pathways were investigated by Western blotting to determine the possible mechanism of apoptosis. We found that using siRNA is a feasible and reliable method for knocking down gene expression in the testes. Compared to controls, the mean seminiferous tubule diameter （MSTD） and the thickness of the germ cell layers decreased following siRNA treatment, whereas the percentage of apoptotic seminiferous tubules increased. The p44/p42, p38 and SAPK/JNK MAPK pathways were activated after downregulation of CIRP. In conclusion, we discovered that downregulation of CIRP resulted in increased germ cell apoptosis, possibly viathe activation of the p44/p42, p38 and SAPK/JNK MAPK pathways.

Neuroprotective effects of cold-inducible RNA-binding protein during mild hypothermia on traumatic brain injury

Cold-inducible RNA-binding protein（CIRP）, a key regulatory protein, could be facilitated by mild hypothermia in the brain, heart and liver. This study observed the effects of mild hypothermia at 31 ± 0.5℃ on traumatic brain injury in rats. Results demonstrated that mild hypothermia suppressed apoptosis in the cortex, hippocampus and hypothalamus, facilitated CIRP m RNA and protein expression in these regions, especially in the hypothalamus. The anti-apoptotic effect of mild hypothermia disappeared after CIRP silencing. There was no correlation between mitogen-activated extracellular signal-regulated kinase activation and CIRP silencing. CIRP silencing inhibited extracellular signal-regulated kinase-1/2 activation. These indicate that CIRP inhibits apoptosis by affecting extracellular signal-regulated kinase-1/2 activation, and exerts a neuroprotective effect during mild hypothermia for traumatic brain injury.

Artificial cold exposure induced stroke in renovascular hypertensive rats and its association with cold-inducible RNA binding protein mRNA expression in brain tissue and blood pressure

BACKGROUND： High incidence of stroke at interchange period of autumn and winter was demonstrated by epidemiological survey, and the specific causes should be further investigated. OBJECTIVE： To investigate the influence of artificial cold exposure on the incidence of stroke in renovascular hypertensive rats （RHR）, and analyze the association with blood pressure and cold-inducible RNA binding protein （CIRP） mRNA expression in brain tissue. DESIGN： A completely randomized grouping design, a randomized control animal trial. SETTINGS： Lab of Neurology, the First Affiliated Hospital of Sun Yat-sen University; Department of Chemistry, Open laboratory of Chemical Biology, Institute of Molecular Technology for Drug Discovery and Synthesis, University of Hong Kong. MATERIALS： Male SD rats （n=460）, weighing 80 - 100 g were obtained from Guangdong Province Health Animal Unit. A modified RXZ-300A intelligent artificial climate cabinet （Ningbo Jiangnan Instrument Co. ,Ltd., China）. METHODS： The experiment were processed in the Lab of Neurology, the First Affiliated Hospital of Sun Yat-sen University and the Open Laboratory of Chemical Biology, Institute of Molecular Technology for Drug Discovery and Synthesis, University of Hong Kong from October 2004 to November 2005. Rats （n = 400） were operated to establish 2-kidney 2-clip RHR model as described previously. The sham-operated rats （n =60） served as normotensive controls. Eight weeks later, 300 of RHR were randomly selected according to their systolic blood pressure （SBP） and divided into 3 sub-groups （n =100 per group）： mild hypertensive group （SBP of 160 - 200 mm Hg）, moderate hypertensive group （SBP of 200 - 220 mm Hg） and severe hypertensive group （SBP 〉 220 mm Hg）. Each group was further divided into two groups （n =50） under ACE and non-ACE. Normal sham-operated SD rats （n =60）, SBP 〈 140 mm Hg, were randomly divided into two groups： Sham-operated control group （n =30） under ACE and non-ACE. To establish the ACE and non-ACE treatment, rats were housed individually in artificial climate cabinet, and ACE was designed as three cycles of 12-hour light of 22℃ （7 ： 00 - 19 ： 00） and 12-hour dark of 4℃（19 ： 00 - 7 ： 00）. The non-ACE group was kept at 22℃ throughout the experiment. MAIN OUTCOME MEASURES： Blood Pressure changes were measured and stroke symptom were observed; Expression of the CIRP were examined by reverse transcription-polymerase chain reaction. RESULTS： Finally 360 rats were involved in the analysis of results. ①Incidence of stroke： The incidence of stroke in 2k2c RHR was significantly higher after a three-day intermittent （12-hour） ACE （29.3%） as compared with that in non-ACE （17.3%） （P 〈 0.05）. Furthermore, the severe hypertensive 2k2c RHR （BP 〉 220 mm Hg） was found to have much higher incidence of stroke （66%, 33/50） than the mild （8%, 4/50） and moderate （18%） hypertensive 2k2c RHR. ②CIRP mRNA in brain tissue： ACE treatment stimulated the mRNA expression of CIRP in non-stroke 2k2c RHR but not in stroke 2k2c RHR （P 〈 0.05）. CONCLUSION： High blood pressure and low expression of CIRP are associated with ACE induced stroke.

Molecular Cloning, Expression Pattern, and 3D Structural Prediction of the Cold Inducible RNA- Binding Protein(CIRP) in Japanese Flounder(Paralichthys olivaceus)

Cold-inducible RNA-binding protein(CIRP) is a kind of RNA binding proteins that plays important roles in many physiological processes. The CIRP has been widely studied in mammals and amphibians since it was first cloned from mammals. On the contrary, there are little reports in teleosts. In this study, the Po CIRP gene of the Japanese flounder was cloned and sequenced. The genomic sequence consists of seven exons and six introns. The putative Po CIRP protein of flounder was 198 amino acid residues long containing the RNA recognition motif(RRM). Phylogenetic analysis showed that the flounder Po CIRP is highly conserved with other teleost CIRPs. The 5' flanking sequence was cloned by genome walking and many transcription factor binding sites were identified. There is a Cp Gs region located in promoter and exon I region and the methylation state is low. Quantitative real-time PCR analysis uncovered that Po CIRP gene was widely expressed in adult tissues with the highest expression level in the ovary. The m RNA of the Po CIRP was maternally deposited and the expression level of the gene was regulated up during the gastrula and neurula stages. In order to gain the information how the protein interacts with m RNA, we performed the modeling of the 3D structure of the flounder Po CIRP. The results showed a cleft existing the surface of the molecular. Taken together, the results indicate that the CIRP is a multifunctional molecular in teleosts and the findings about the structure provide valuable information for understanding the basis of this protein's function.

Genes for RNA-binding proteins involved in neuralspecific functions and diseases are downregulated in Rubinstein-Taybi iNeurons

Taking advantage of the fast-growing knowledge of RNA-binding proteins(RBPs)we review the signature of downregulated genes for RBPs in the transcriptome of induced pluripotent stem cell neurons(iNeurons)modelling the neurodevelopmental Rubinstein Taybi Syndrome(RSTS)caused by mutations in the genes encoding CBP/p300 acetyltransferases.We discuss top and functionally connected downregulated genes sorted to“RNA processing”and“Ribonucleoprotein complex biogenesis”Gene Ontology clusters.The first set of downregulated RBPs includes members of hnRNHP(A1,A2B1,D,G,H2-H1,MAGOHB,PAPBC),core subunits of U small nuclear ribonucleoproteins and Serine-Arginine splicing regulators families,acting in precursor messenger RNA alternative splicing and processing.Consistent with literature findings on reduced transcript levels of serine/arginine repetitive matrix 4(SRRM4)protein,the main regulator of the neural-specific microexons splicing program upon depletion of Ep300 and Crebbp in mouse neurons,RSTS iNeurons show downregulated genes for proteins impacting this network.We link downregulated genes to neurological disorders including the new HNRNPH1-related intellectual disability syndrome with clinical overlap to RSTS.The set of downregulated genes for Ribosome biogenesis includes several components of ribosomal subunits and nucleolar proteins,such NOP58 and fibrillarin that form complexes with snoRNAs with a central role in guiding post-transcriptional modifications needed for rRNA maturation.These nucleolar proteins are“dual”players as fibrillarin is also required for epigenetic regulation of ribosomal genes and conversely NOP58-associated snoRNA levels are under the control of NOP58 interactor BMAL1,a transcriptional regulator of the circadian rhythm.Additional downregulated genes for“dual specificity”RBPs such as RUVBL1 and METTL1 highlight the links between chromatin and the RBP-ome and the contribution of perturbations in their cross-talk to RSTS.We underline the hub position of CBP/p300 in chromatin regulation,the impact of its defect on neurons’post-transcriptional regulation of gene expression and the potential use of epidrugs in therapeutics of RBP-caused neurodevelopmental disorders.

BH3-only protein Bim is involved in myocardial injury induced by co-stress of ischemia and cold stress in rats

Objectives To investigate the effect of co-exposure of myocardial ischemia and cold stress on myocardial injury in rats and the relative mechanism.Methods Myocardial ischemia model was established by ligation of left coronary artery.SD rats were randomly allocated to 4 groups; sham+normal temperature(S group),sham+cold stress(SC group),myocardial ischemia+ normal temperature(Ⅰgroup), myocardial ischemia+cold stress(IC group).On the condition of 26℃,SC and IC groups were keeped in a 4℃artificial chamber for 8h(8;00-16:00) for 4 consecu- tive days.Car diac function was assessed by echocardiography;pathological change was analyzed by HE staining;myocardial infarct size was determined by TTC staining;Bim,Caspase-3 expression in myocardium was determined by western blotting.Results It was demonstrated that co-exposure of myocardial ischemia and cold stress could significantly make the cardiac muscle in abnormal shape,increase the infarct size and the expression of Bim and Caspase-3.Conclusions Co-exposure of myocardial ischemia and cold stress may aggravate the cardiac injury,pro- apoptosis protein Bim is involved.

Expression of a Carrot 36 kD Antifreeze Protein Gene Improves ColdStress Tolerance in Transgenic Tobacco

Antifreeze proteins （AFPs） enable organisms to survive under cold conditions, and have great potential in improving cold tolerance of cold-sensitive plants, In order to determine whether expression of the carrot 36 kD antifreeze protein gene confers improved cold-resistant properties to plant tissues, we tried to obtain transgenic tobacco plants which expressed the antifreeze protein. Cold, salt, and drought induced promoter Prd29A was cloned using PCR from Arabidopsis. Two plant expression vectors based on pBI121 were constructed with CaMV35S：AFP and Prd29A：AFP. Tobacco plantlets were transformed by Agrobacterium-medicated transformation. PCR and Southern blotting demonstrated that the carrot 36 kD afp gene was successfully integrated into the genomes of transformed plantlets. The expression of the afp gene in transgenic plants led to improved tolerance to cold stress. However, the use of the strong constitutive 35S cauliflower mosaic virus （CaMV） promoter to drive expression of afp also resulted in growth retardation under normal growing conditions. In contrast, the expression of afp driven by the stress-inducible Prd29A promoter from Arabidopsis gave rise to minimal effects on plant growth while providing an increased tolerance to cold stress condition （2℃）. The results demonstrated the prospect of using Prd29A-AFP transgenic plants in cold-stressed conditions that will in turn benefit agriculture.

血清eCIRP、suPAR预测脓毒症致急性呼吸窘迫综合征患者预后的价值分析

目的探讨血清细胞外冷诱导RNA结合蛋白(eCIRP)、可溶性尿激酶纤溶酶原激活物受体(suPAR)预测脓毒症致急性呼吸窘迫综合征(ARDS)患者预后的价值。方法选取2019年1月—2023年6月上海交通大学医学院附属第九人民医院急诊科收治的脓毒症致ARDS患者84例(ARDS组),按照1∶1比例选取单纯脓毒症患者84例(非ARDS组),根据预后将脓毒症致ARDS患者分为死亡亚组(37例)和存活亚组(47例)。采用酶联免疫吸附法检测血清eCIRP、suPAR水平。通过多因素Logistic回归和受试者工作特征(ROC)曲线分析脓毒症致ARDS患者死亡的因素及血清eCIRP、suPAR水平预测价值。结果与非ARDS组比较,ARDS组血清eCIRP、suPAR水平升高(t/P=14.330/<0.001、10.632/<0.001);84例脓毒症致ARDS患者90 d死亡率为44.05%(37/84);死亡亚组患者血清eCIRP、suPAR、脓毒性休克比例、机械通气时间≥3 d比例、序贯器官衰竭评估(SOFA)评分、降钙素原、血乳酸均高于存活亚组(χ^(2)/t/P=13.805/<0.001、5.229/<0.001、10.932/0.001、4.334/0.037、4.850/<0.001、7.592/<0.001、5.926/<0.001);SOFA评分高、血乳酸高及血清eCIRP、suPAR高为脓毒症致ARDS患者死亡的独立危险因素[OR(95%CI)=1.523(1.123~2.067)、2.558(1.123~5.824)、1.094(1.017~1.178)、1.365(1.117~1.670)]。血清eCIRP、suPAR及二者联合预测脓毒症致ARDS患者死亡的AUC分别为0.787、0.779、0.871,二者联合的AUC大于血清eCIRP、suPAR水平的单独预测(Z/P=2.005/0.045、2.205/0.028)。结论血清eCIRP、suPAR水平升高与脓毒症致ARDS患者预后不良有关,且二者联合预测的价值较高。

急性脑梗死患者血清miR-106a-5p、NINJ1、CIRP水平及其临床意义

目的探讨急性脑梗死(ACI)患者血清微小核糖核酸-106a-5p(miR-106a-5p)、神经损伤诱导蛋白1(NINJ1)、冷诱导RNA结合蛋白(CIRP)的水平及其临床意义。方法选择2022年1月至2023年6月该院神经内科收治的151例ACI患者作为ACI组,另选择同期65例体检健康者作为对照组,根据静脉溶栓后90 d预后情况将ACI患者分为预后不良组和预后良好组。采用实时荧光定量聚合酶链反应检测血清miR-106a-5p水平,通过酶联免疫吸附试验检测NINJ1、CIRP水平。采用多因素Logistic回归分析影响ACI患者预后的因素,采用受试者工作特征(ROC)曲线分析血清miR-106a-5p、NINJ1、CIRP水平对ACI患者预后的预测价值。结果与对照组比较,ACI组血清miR-106a-5p、NINJ1、CIRP水平明显升高,差异均有统计学意义(P<0.05)。随访90 d,151例ACI患者预后不良发生率为35.10%。与预后良好组比较,预后不良组年龄明显增大,美国国立卫生研究院卒中量表(NIHSS)评分及血清miR-106a-5p、NINJ1、CIRP水平明显升高,差异均有统计学意义(P<0.05)。年龄增加、NIHSS评分升高和血清miR-106a-5p、NINJ1、CIRP水平升高均为ACI患者预后不良的独立危险因素(P<0.05)。血清miR-106a-5p、NINJ1、CIRP水平联合检测预测ACI患者预后不良的曲线下面积为0.937,大于血清miR-106a-5p、NINJ1、CIRP水平单独检测预测的0.777、0.773、0.778(P<0.05)。结论ACI患者血清miR-106a-5p、NINJ1、CIRP水平升高是预后不良的独立危险因素,血清miR-106a-5p、NINJ1、CIRP水平联合检测对ACI患者预后不良有较高的预测价值。

马铃薯淀粉酶StBAM9互作蛋白的鉴定及其互作机制分析

本实验室前期研究表明β-淀粉酶9(StBAM9)在马铃薯抗低温糖化中具有重要作用,但其并无β-淀粉酶活性。为了研究StBAM9在马铃薯抗低温糖化中的功能机制,我们构建了低温贮藏后块茎cDNA酵母双杂交文库,并以StBAM9蛋白为诱饵,对其进行了互作蛋白的筛选和分析,结果显示分别以全长StBAM9和截去转运肽的StBAM9为诱饵筛选到的候选互作蛋白中有12个是共有的。酵母双杂交结果显示有4个蛋白(StDUF842、StTPR01660、StTPR22129和StTPR45174)与StBAM9互作。进一步通过谷胱甘肽-S-转移酶融合蛋白沉降技术验证表明,其中的2个蛋白StTPR01660和StTPR4517与StBAM9互作。双分子荧光互补结果显示只有StTPR01660与StBAM9互作于淀粉粒上,而StTPR01660自身定位于细胞质。因此,我们推测StBAM9可能通过从细胞质中招募StTPR01660到淀粉粒上发挥淀粉降解的功能。

冷诱导RNA结合蛋白在大鼠低体温性急性肺损伤中的表达变化

背景海上遇难者落水后常死于海水长时间浸泡后的低体温及相关并发症,肺是低体温损伤的重要器官之一,但目前针对低体温造成急性肺损伤(acute lung injury,ALI)的研究相对匮乏。目的探讨冷诱导RNA结合蛋白(coldinducible RNA-binding protein,CIRP)在低体温性ALI大鼠模型中的变化及其可能的作用机制。方法将40只雄性成年SD大鼠随机分为0 h组(0 h,8只)和实验组(32只)。实验组分别用低温海水浸泡12 h、16 h、20 h、24 h(每组8只),构建低体温性ALI动物模型。ELISA检测白细胞介素-6(interleukin-6,IL-6)、IL-1β、细胞外CIRP的含量,对肺组织进行HE染色、TUNEL染色和CIRP免疫组织化学染色,qRT-PCR检测CIRP mRNA的表达。结果与0 h组相比,实验组出现不同程度肺损伤,病理切片可见肺泡壁增厚或结构破坏,肺泡腔内出血,伴中性粒细胞和淋巴细胞浸润等;16 h、20 h、24 h实验组血清中IL-6的表达显著增加,20 h、24 h实验组血清中IL-1β表达显著增加,差异均有统计学意义(P<0.01);各实验组大鼠血清中CIRP表达增加均有统计学差异(12 h、16 h、20 h组,P<0.05;24 h组,P<0.01)。TUNEL结果显示,各实验组的大鼠肺组织出现不同程度的细胞凋亡现象(P<0.01);通过炎症因子的表达水平、肺组织病理改变以及细胞凋亡情况选择低温海水浸泡24 h为最佳构建模型时间,免疫组织化学染色和qRT-PCR观察到24 h组肺组织内CIRP的表达增加。结论肺组织和外周血清的CIRP在低体温性ALI大鼠模型中随时间延长而表达增高,提示其可能与低体温致ALI相关,同时与炎症因子IL-1β、IL-6水平呈现一致性趋势变化,因此推测CIRP可能通过促进炎症因子释放发挥作用,本研究初步证实了CIRP在肺组织区域和整体与低体温ALI的相关性。

动物冷应激相关蛋白和分子的最新研究进展

恒定的体温是维持生命活动的重要基础,恒温动物需要内源性产热源。寒冷条件下动物体温降低的直接后果是细胞内整体水平上蛋白质的合成被抑制,但冷诱导RNA结合蛋白(cold-inducible RNA binding protein,CIRP)、RNA结合基序蛋白3(RNA-binding motif protein 3,RBM3)、解偶联蛋白(uncoupling protein,UCP)、热休克蛋白(heat shock proteins,HSPs)以及少数细胞因子的表达却增加,从而确保细胞在寒冷环境中的内稳态和生存。对冷应激条件下动物体内被激发的蛋白及相关分子的最新研究进展进行综述,以期为冷应激蛋白分子领域的深入研究提供借鉴。

体外循环术中血液灌流降低主动脉夹层术后急性肾损伤发生风险

目的探究急性主动脉夹层体外循环术中应用血液灌流器对患者术后急性肾损伤(AKI)发生风险的影响。方法纳入本院2022年7月至2022年10月被诊断为急性A型主动脉夹层并行孙氏手术治疗的患者,回顾性收集其基线资料、手术情况、各项临床生化数据及预后结果进行分析。结果共有57名患者被纳入研究,其中23名患者接受了血液灌流治疗。血液灌流组的AKI发生率显著低于无血液灌流组(43.5%vs.85.3%,P=0.001),特别是AKI 1~2级的发生(34.8%vs.61.8%,P=0.046)。且血液灌流组术后7 d肌酐最大值[121(82,218)μmol/L vs.174(112,220)μmol/L,P=0.216]和胱抑素C最大值[1.63(1.34,2.85)mg/L vs.2.58(1.68,4.23)mg/L,P<0.05]均低于无血液灌流组。血液灌流组术后3 d降钙素原最大值较无血液灌流组有下降[10.72(7.28,27.00)g/L vs.6.50(2.60,11.0)g/L,P=0.011]。血液灌流组血清中冷诱导RNA结合蛋白的浓度较无血液灌流组未显示出差异[987(664,1335)vs.993(741,1451)ng/L,P=0.733]。结论急性主动脉夹层手术术中联合血液灌流可减少术后AKI发生风险,尤其是1级和2级AKI的风险。

TREM-1与CIRP在胃癌组织中的表达及其对胃癌预后的评估价值

目的探讨髓样细胞触发受体-1(TREM-1)与冷诱导RNA结合蛋白(CIRP)在胃癌组织中的表达及其对胃癌预后的评估价值。方法选取2016年9月至2019年9月该院收治的108例胃癌患者作为研究对象,收集胃癌患者的癌组织标本作为胃癌组,对应的癌旁组织标本作为癌旁组。采用免疫组织化学法检测胃癌组及癌旁组TREM-1、CIRP表达情况;采用χ^(2)检验比较不同临床病理特征胃癌患者TREM-1、CIRP表达情况;采用多因素Cox回归分析胃癌患者预后的危险因素。结果胃癌组TREM-1阳性表达率(78.70%)高于癌旁组(37.96%),差异有统计学意义(χ^(2)=36.876,P<0.05);胃癌组CIRP阳性表达率(73.15%)高于癌旁组(43.52%),差异有统计学意义(χ^(2)=19.505,P<0.05)。低分化、肿瘤最大径>5 cm、临床分期为Ⅲ~Ⅳ期、有淋巴结转移的胃癌患者TREM-1、CIRP阳性表达率均高于中/高分化、肿瘤最大径≤5 cm、临床分期为Ⅰ~Ⅱ期、无淋巴结转移的胃癌患者,差异均有统计学意义(P<0.05)。TREM-1阳性表达患者3年生存率为41.18%,低于TREM-1阴性表达患者的91.30%,差异有统计学意义(P<0.05);CIRP阳性表达患者3年生存率为36.71%,低于CIRP阴性表达患者的93.10%,差异有统计学意义(P<0.05)。低分化、临床分期为Ⅲ~Ⅳ期、有淋巴结转移的胃癌患者3年生存率均低于中/高分化、临床分期为Ⅰ~Ⅱ期、无淋巴结转移的胃癌患者,差异均有统计学意义(P<0.05)。多因素Cox回归分析结果显示,临床分期为Ⅲ~Ⅳ期(HR=2.425,95%CI:1.591~3.697)、有淋巴结转移(HR=2.646,95%CI:1.709~4.096)、TREM-1阳性表达(HR=3.343,95%CI:2.081~5.373)、CIRP阳性表达(HR=2.898,95%CI:1.843~4.557)是胃癌患者生存的危险因素(P<0.05)。结论TREM-1、CIRP在胃癌患者中均呈高表达,且与患者临床病理特征及预后密切相关,能够作为胃癌预后评估的生物学标志物,有望为胃癌提供新的治疗靶点。

CIRBP对小鼠血管平滑肌细胞增殖及迁移的调控及机制研究

目的:探讨冷诱导RNA结合蛋白(CIRBP)对小鼠血管平滑肌细胞(VSMCs)增殖及迁移过程的调控及机制。方法:将雄性C57BL/6J小鼠分为假手术对照组、假手术干扰组、颈总动脉损伤对照组和颈总动脉损伤干扰组,每组5只。造模后按照组别分别用阴性对照腺病毒(AD-NC)和沉默CIRBP腺病毒(AD-CIRBPI)转染,28 d后观察CIRBP表达及血管内膜的增生情况。将小鼠VSMCs分为对照组和腺病毒沉默组,分别用AD-NC和AD-CIRBPI转染细胞48 h,然后加入激活雷帕霉素靶蛋白复合物1(mTORC1)活性的胰岛素。通过RT-qPCR检测CIRBP的mRNA表达,Western blot检测CIRBP、磷酸化核糖体蛋白S6(p-S6Ser235/236)和磷酸化4E结合蛋白1(p-4EBP1Thr37/46)蛋白水平变化,Ki67免疫荧光染色和CCK-8实验检测细胞增殖,划痕实验检测细胞迁移,HE染色检测颈动脉内膜增生程度。结果:沉默CIRBP后,VSMCs的活力下降,Ki67阳性细胞比率降低,细胞迁移速度减慢,同时mTORC1活性下降。加入胰岛素恢复mTORC1活性后,细胞活力、Ki67阳性细胞率和细胞迁移速度下降幅度减弱。损伤小鼠颈总动脉内皮后CIRBP表达增加,体内干扰小鼠CIRBP表达后,小鼠血管内皮损伤后内膜增生程度减轻。结论:CIRBP通过mTORC1途径加强小鼠VSMCs增殖及迁移,促进小鼠血管损伤后血管内膜增生。

鲤与生长性状相关的EST-SSRs标记筛选

以大头鲤(Cyprinus pellegrini Tchang)(母本)与荷包红鲤(Cyprinus carpio wuyuanensis)抗寒品系(父本)杂交产生的F1作为亲本,以其自交F2作为分离群体,结合"拟测交"策略,用EST-SSRs标记对其中92个个体进行基因型检测。利用Windows Map Manager2.0的标记回归法对符合拟测交分离的70个EST-SSRs标记进行单标记回归分析,检测出8个与鲤体长、体高、体厚性状相关的标记。对同一标记不同基因型个体间生长性状进行显著性比较,通过t检验,找到与生长性状相关的基因型。将上述8个EST序列与GenBank数据库进行BLAST比对,有3条EST序列与蛋白库中已知功能的序列高度同源。其中HLJE225与编码斑马鱼(Denio rerio)冷诱导基因RNA结合蛋白的基因同源性水平高达97%,HLJE222与编码斑马鱼DAZ相关蛋白1基因同源性水平也高达97%,HLJE599与编码斑点叉尾(Ictalurus punctatus)核糖体蛋白L6基因的同源水平为87%。本研究鉴定的与鲤生长性状相关的功能基因及有利用价值的基因型,为鲤重要生长性状的QTL(数量性状基因座)定位和分子标记辅助育种提供了一定的依据。

低温诱导蛋白及其与植物的耐寒性研究进展

低温诱导蛋白是植物在温度逆境条件下诱导产生的一系列蛋白,以抗冻蛋白、脱水蛋白、热激蛋白和热稳定蛋白较多,而且低温诱导蛋白质一旦在体内形成,植物体就会尽快地适应外界环境,表现出较强的抗逆性.本文对几种主要的低温诱导蛋白——抗冻蛋白、脱水蛋白、热激蛋白和热稳定蛋白的特性及其与植物耐寒性的关系研究进行综述,以期为进一步阐明植物耐寒的分子机制以及提高植物耐寒力研究提供新的思路.

大黄鱼冷诱导结合蛋白(CIRP)基因cDNA克隆及低温胁迫对其时空表达的影响

为研究冷诱导结合蛋白(CIRP)在大黄鱼低温适应过程中的作用,采用同源克隆技术获得了大黄鱼CIRP基因,并通过实时荧光定量PCR(qRT-PCR)技术检测了低温胁迫下大黄鱼各组织中CIRP基因的表达变化。结果显示,大黄鱼CIRP基因cDNA序列全长1211 bp,推测编码一个由182个氨基酸组成、分子量为19 ku的蛋白。序列比对显示硬骨鱼类CIRP基因序列较为保守,特别是N端的RNA识别基序(RRM)高度保守。系统进化树分析显示大黄鱼CIRP与银鳕相似性最高(81.5%),所有硬骨鱼类聚为一个大支。慢性低温胁迫中(12℃缓降至6℃),皮肤和肌肉中CIRP基因的表达量呈随温度降低而升高的趋势,6℃时表达量分别为12℃时的11.56倍和9.03倍;鳃、脑和心脏中CIRP基因表达量均呈先显著上升后显著下降的趋势,其峰值均出现在8℃时,表达量分别为12℃时的5.07、7.69和2.83倍;肠、肾脏、肝脏和脾脏中的表达量随温度降低而下降,6℃时的表达量最低,与12℃时相比降幅分别为80.0%、65.6%、91.2%、55.6%。急性低温胁迫(由12℃骤降至8℃并胁迫4 h)条件下,鳃、皮肤、脑、肝脏、心脏中CIRP基因表达量均呈先升高后降低的变化,反映了机体对低温胁迫的应激、适应过程;肌肉和肠中的表达量则始终显著低于胁迫前。慢性和急性低温胁迫中各组织CIRP表达量变化的差异提示,大黄鱼机体在低温胁迫响应和适应中有着多种调节机制。研究表明,CIRP基因参与了大黄鱼应对低温胁迫的过程。

植物低温诱导蛋白和诱导基因研究新进展

现就近年来植物低温诱导蛋白和低温诱导基因的研究进展进行了概述 。

民猪CIRP基因的克隆与冷诱导研究

采用生物信息学结合RT-PCR的方法,克隆民猪的冷诱导RNA结合蛋白(Cold inducible RNA-binding protein,CIRP)基因,获得3个变异体序列。猪CIRP变异体1基因cDNA长1 278 bp(GenBank登录号:HQ908794),编码172个氨基酸;变异体2基因cDNA长1 653 bp(GenBank登录号:HQ908795),编码182个氨基酸;变异体3基因cDNA长1 765 bp(Genbank登录号:HQ908796),编码144个氨基酸。CIRP变异体2与变异体1相比,在其编码区的第501碱基处,出现了一段375 bp的插入片段,该插入片段的第46～48个碱基为TAG,使翻译提前终止;变异体3与变异体2相比,在其编码区的第428碱基处,出现一段115 bp插入片段,该插入片段的第5～7个碱基为TGA,使翻译提前终止。3种转录变异体的核苷酸序列同源性为86.45%,氨基酸序列同源性为87%。冷诱导后,CIRP基因在肌肉中的表达水平出现显著升高(P<0.05)。