共伴侣蛋白FKBP51在高脂诱导肥胖中的作用

目的 通过FKBP51基因敲除小鼠模型和细胞脂肪分化模型,探究FKBP51在高脂诱导肥胖中的机制。 方法 4周龄雄性野生型和FKBP51基因敲除小鼠以普通或高脂饲料单只单笼喂养6周,每周记录小鼠体重和饮食量,应用MM-100代谢笼系统,监测各组小鼠24 h内氧气消耗量、二氧化碳产生量、呼吸交换率和产热量的变化,对上述小鼠肝脏组织进行油红O染色,同时检测肝脏和脂肪组织代谢相关基因的表达情况。同时对取自野生型和FKBP51基因敲除小鼠的永生化成纤维细胞(MEF)进行脂肪诱导分化,观察FKBP51缺失对脂肪分化的影响。 结果 高脂饮食诱导后,两种基因型小鼠饮食量无明显变化,但与野生型小鼠相比,FKBP51基因敲除小鼠体重明显减轻,肝脏中脂滴减少。FKBP51基因敲除小鼠在基础和高脂诱导条件下,氧气消耗量、二氧化碳产生量、呼吸交换率以及产热量均高于野生型小鼠,肝脏中糖异生相关酶PEPCK、G6Pase等表达上调,脂肪中能量代谢相关基因UCP-1等表达上调。此外,FKBP51基因缺失的MEF诱导脂肪分化,细胞内脂滴明显少于野生型MEF细胞。 结论 FKBP51基因在脂肪合成和能量代谢方面起着重要作用,因此FKBP51基因缺失小鼠能够抵制高脂诱导的肥胖。     

Fkbp51基因敲除对小鼠肝脏转录组基因可变剪接的影响

目的通过分析Fkbp51基因敲除(knock out,KO)与野生型(wild type,WT)小鼠肝脏表达谱,研究Fkbp51基因敲除对肝脏组织基因可变剪接的影响。方法利用二代测序对Fkbp51 KO与WT小鼠肝脏进行表达谱测序,用Top Hat对RNA测序结果进行可变剪接分析,筛选出KO与WT小鼠肝组织中差异的内含子保留(intron retetion,RI)和外显子跳跃(exon skipping,SE)。通过在线工具DAVID对这些差异可变剪接体进行基因功能(gene ontology,GO)和代谢通路(kyoto encyclopedia of genes and genomes,KEGG)富集分析,同时用NCBI基因数据库对这些基因进行注释。结果 (1)Fkbp51缺失可导致小鼠肝脏mRNA可变剪接发生变化;(2)Fkbp51基因敲除造成小鼠肝脏mRNA可变剪接表达量的变化;(3)通过GO与KEGG分析,我们发现这些发生差异可变剪切的基因主要与脂肪相关衍生物的代谢、免疫、胆汁酸分泌等通路相关。(4)与差异内含子保留相关的基因主要与肌动蛋白细胞骨架调控,氨基酸及其衍生物代谢相关。结论 Fkbp51基因敲除能够改变基因组中mRNA的可变剪切,进而影响小鼠肝脏的代谢功能。     

三磷腺苷合酶抑制因子1基因敲除对小鼠巨噬细胞线粒体功能和三磷腺苷水平的影响

目的探讨三磷腺苷合酶抑制因子1(ATPIF1)基因敲除对小鼠巨噬细胞内三磷腺苷(ATP)水平及线粒体功能的影响。方法选取5只野生型C57BL/6小鼠为WT组,5只ATPIF1基因敲除小鼠为KO组。处死小鼠后,提取小鼠骨髓细胞进行培养,并诱导其分化成熟为骨髓来源巨噬细胞(BMDM)。在未加脂多糖(LPS)的基础状态及LPS刺激2、4 h后收集细胞,应用ATP检测试剂盒检测细胞内ATP水平,应用细胞能量代谢分析仪检测细胞氧消耗率,采用实时荧光定量聚合酶链反应检测细胞内三羧酸循环中关键酶丙酮酸激酶(PKm)、异柠檬酸脱氢酶(IDH)、柠檬酸合酶(CS)、α-酮戊二酸脱氢酶(OGDC) mRNA相对表达量。结果与WT组比较,在基础状态下KO组小鼠BMDM内ATP水平、基础氧耗量、用于ATP合成的氧耗量、最大氧耗量及PKm、IDH、CS、OGDC mRNA相对表达量显著增加(P <0. 05,P <0. 01); LPS刺激2、4 h后,KO组小鼠BMDM内ATP水平、基础氧耗量、用于ATP合成的氧耗量和最大氧耗量及CS、OGDC mRNA相对表达量均显著增加(P <0. 05)。结论 ATPIF1基因敲除可增加小鼠BMDM内ATP水平,并提高三羧酸循环相关酶mRNA表达和线粒体氧化磷酸化水平。     

OPN诱导小鼠MSCs向表皮细胞分化促进创面愈合的研究

目的 以骨桥蛋白(osteopontin,OPN)基因敲除小鼠及野生型小鼠为研究对象,探讨骨髓间充质干细胞(mesenchymal stem cells,MSCs)分化为表皮细胞的潜能及骨桥蛋白在分化过程中的作用.方法 体外实验:将分离培养的野生型(WT)和基因敲除型(KO)新生鼠(鼠龄1d)MSCs鉴定纯度后分别设为WT组和KO组,取其第3代分别在表皮转化培养体系中孵育,通过镜下观察、流式细胞术、免疫荧光染色、Western blot等方法,比较两组表皮细胞标志物(CK14)表达水平的差异.动物实验:实验分4组,WT组小鼠创周注射GFP-MSCs (WT/MSCs)组,WT小鼠创周注射PBS(WT/PBS)组,KO小鼠创周注射GFP-MSCs (KO/MSCs)组,KO小鼠创周注射PBS(KO/PBS)组,每组各8只雄鼠,鼠龄6～8周,体质量20 ～25 g,按分组将分离培养的GFP-MSCs和PBS注射到创周,观察创面愈合及GFP-MSCs向表皮细胞分化的情况.结果 分离培养的MSCs呈梭形或成纤维细胞样生长,流式细胞术检测MSCs的纯度超过91％;诱导分化第12、17天的两组细胞检测发现,WT组和KO组的细胞均表达CK14;第17天免疫荧光结果示WT组CK14的表达阳性率(0.936 3±0.009 5)明显高于KO组(0.647 5±0.023 1),(P＜0.01);Western blot检测结果也显示KO组的CK14表达量(0.3894±0.016 8)明显低于WT组(0.614 6±0.015 3),(P＜0.01).动物实验WT/PBS组创面愈合速度快于KO/PBS组(P＜0.05),WT/MSCs组的创面愈合速度快于WT/PBS组(P＜0.05),注射的GFP-MSCs向表皮细胞分化的能力WT/MSCs组强于KO/MSCs组(P＜0.05).结论 OPN在体内、体外均可诱导MSCs分化为表皮细胞,进而促进创面愈合.     

科技动态

Dat1基因敲除小鼠的行为学研究多巴胺(DA)运输体基因(Dat1)编码重吸收多巴胺能神经递质的蛋白质,起终止多巴胺能神经递质活动的功能。为了研究KO小鼠行为异常的行为和生化机制,Vladimir MPogorelov等人研究了野生型(WT)、Dat1杂合型(HZ)、和Dat1基因敲除(KO)3种雄性小鼠(3~5月龄)的行为学试验,这3种小鼠均来自C57BL/6J和129 Sv/J杂交Dat1小鼠,杂交15代。KO小鼠中Dat1表达消失,HZ小鼠有50%的Dat1表达。跟WT对照组相比,KO小鼠细胞外的DA增加2~5倍。尽管KO小鼠的旷野活动能力跟WT和HZ相似,但能力较之为强。HZ小鼠紧张度低,能…     

mGluR5拮抗剂对FMR1基因敲除小鼠树突棘的影响及其机制探讨

目的:探讨代谢性谷氨酸受体5(mGluR5)拮抗剂2-甲基-6-苯基乙炔嘧啶(MPEP)对脆性X综合征树突棘发育的影响及其机制.方法:新生FMR1基因敲除(KO)小鼠腹腔注射MPEP 1次、1周,以同龄KO鼠和野生鼠(WT)注射生理盐水对照.免疫印迹法检测微管相关蛋白1B(MAP1B)含量改变,并以单片Golgi染色方法观察皮质感觉区第Ⅴ层锥体神经元树突棘长度和密度.结果:KO小鼠树突棘长度和密度都显著高于WT小鼠,MAP1B含量也明显增高. MPEP干预1周MAP1B表达显著减少,树突棘长度、密度明显降低. MPEP 1次干预明显减少MAP1B,对树突棘无影响.结论:脆性X综合征树突棘的发育异常与mGluR5信号的过度激活有关,MAP1B的过表达可能是主要环节.     

成纤维细胞生长因子21参与调控非诺贝特对肝脏脂质代谢的改善作用

目的：探讨成纤维细胞生长因子21（FGF21）介导非诺贝特对小鼠肝脏脂质代谢的调控。方法：将实验小鼠分为4组,均予高脂饮食喂养：野生型对照组（WT+HFD+CTL）、野生型非诺贝特治疗组（WT+HFD+FF）、FGF21基因敲除对照组（FGF21 KO+HFD+CTL）、FGF21基因敲除非诺贝特治疗组（FGF21KO+HFD+FF）。ELISA法检测小鼠血清的FGF21及Adiponectin含量;HE与油红O染色分别用于检测肝脏组织形态学变化和脂质堆积情况;TG和TC检测试剂盒用于检测血清TG及TC的变化;qRT-PCR检测PPARα、FGF21、肝脏胆固醇调节元件结合蛋白Srebp-2、脂质氧化、分解、转运等代谢调节基因（Acaca、Acacb ABCG5、ABCG8、Cyp7a1）、PPARγ、脂联素的mRNA表达。结果：非诺贝特处理4周能显著上调小鼠肝脏PPARα和FGF21表达水平,抑制小鼠体质量增加,下调血清异常血脂水平,减轻肝脏脂质沉积状况（P ＜0.05）。非诺贝特引发的保护作用在FGF21基因缺失后出现一定程度的减弱。HE和油红O染色结果显示,敲除FGF21基因减弱非诺贝特对高脂饮食喂养小鼠肝脏脂质堆积的改善作用。在分子机制方面,非诺贝特处理能显著上调脂质氧化、分解、转运等代谢调节基因（Acacb、ABCG5、Cyp7a1等）的表达,下调Srebp-2的表达（P ＜0.05）,然而,FGF21基因缺失时,这些效应被显著抑制（P ＜0.05）。同时,FGF21基因敲除可显著抑制非诺贝特诱导的PPARγ和Adiponectin的mRNA表达上调及血清水平的Adiponectin含量升高现象（P ＜0.05）。结论：FGF21介导非诺贝特发挥抗脂肪肝病变的生物学效应。     

miR-125a-5p基因敲除C57BL/6J小鼠的构建

目的：构建miR-125a-5p基因敲除小鼠并进行基因鉴定,并探讨基因敲除小鼠体内miR-125a-5p对靶基因Foxp3的调控。方法：利用CRISPR/Cas9基因编辑技术,以C57BL/6J小鼠为背景,对miR-125a-5p基因位点进行基因敲除,培育纯合miR-125a-5p基因敲除小鼠。采用裂解法提取鼠尾组织基因组DNA,进行PCR反应检测亲代纯合miR-125a-5p基因敲除小鼠基因型,并选用雌、雄均为纯合子的基因敲除鼠作为亲代种鼠进行配种繁殖。通过全自动蛋白表达分析系统检测小鼠脾脏组织Foxp3的蛋白表达。结果：成功构建miR-125a-5p基因敲除小鼠,琼脂糖凝胶电泳显示PCR产物为240 bp或0 bp单条带的为纯合型基因敲除（KO）小鼠,鉴定获得668 bp或428 bp单条带的为野生型（WT）小鼠,与预期的目的基因片段分子量大小一致,成功鉴定了miR-125a-5p基因敲除小鼠的基因型。选用雌、雄均为纯合子的基因敲除鼠进行配种繁育,获得了一批miR-125a-5p敲除纯合子基因型小鼠。与WT小鼠相比,miR-125a-5p基因敲除小鼠的脾脏组织中Foxp3表达降低（...     

基于CRISPR/Cas9技术PCSK9基因敲除小鼠模型的构建及表型验证

目的 基于CRISPR/Cas9技术构建前枯草杆菌蛋白原转换酶9(Preprotein convertase subtilisin/kexin type9,PCSK9)基因敲除小鼠模型并对其表型进行初步验证。方法 利用非同源末端连接(NHEJ)修复引入突变的方式,针对PCSK9基因靶序列设计单链向导RNA(sgRNA),将sgRNA和Cas9 mRNA显微注射到C57BL/6J小鼠受精卵,获得F0代敲除小鼠,通过繁育及基因型鉴定获得PCSK9基因敲除(PCSK9 KO)纯合子小鼠。采用PCR方法对PCSK9 KO小鼠进行基因鉴定,Western Blot检测肝组织PCSK9及低密度脂蛋白受体(LDL-R)蛋白的表达,同时检测心、脑、肾、脂肪组织中的PCSK9蛋白表达水平,q-PCR检测PCSK9的mRNA表达水平。采用酶法检测血清中总胆固醇(TC)及甘油三酯(TG)水平。结果 PCR产物凝胶电泳以及PCSK9蛋白及mRNA表达结果表明成功获得全身性PCSK9基因敲除小鼠,肝组织LDL-R(3.42±0.56)蛋白表达显著增高。与WT组相比,PCSK9小鼠血清TC(90.75±17.81...     

ABCA6缺失对生酮饮食喂养的小鼠代谢的影响

目的：检测ATP转运蛋白6(ATP binding cassette transporter A6,ABCA6)基因缺失对生酮饮食喂养的小鼠代谢的影响。方法：给予小鼠生酮饮食(ketogenic diet,KD)或普通饮食(normal diet,ND)2周,通过Western blot检测肝脏ABCA6的表达水平;给予ABCA6敲除(KO)小鼠或对照(WT)小鼠KD 或ND 2周,检测小鼠的体重、血糖、血清甘油三酯(TG)、胆固醇(TCH)、游离脂肪酸(FFA)、酮体(β-HB)和转氨酶(AST、ALT)等指标,测定肝脏TG和TCH含量,并通过real-time PCR 检测代谢相关基因的表达水平。结果：生酮饮食诱导小鼠肝脏ABCA6的表达;在普通饮食条件下,ABCA6 KO小鼠与对照小鼠在体重、血糖及血清TG、TCH、FFA、β-HB等代谢相关指标、肝脂质含量和代谢相关基因表达水平等方面无明显差别;在生酮饮食条件下,和WT小鼠相比,ABCA6 KO小鼠的血糖、FFA和TCH降低,肝脏TG蓄积更显著,肝脏FAS、DGAT2、PCK1和HMGCS2 mRNA显著增加。结论：ABCA6缺失引起小鼠对生酮饮食的代谢障碍,ABCA6可能参与肝脏的脂质代谢过程。     

Value of D-Dimers in patients with acute aortic dissection

Objective: To evaluatel the value of D-dimers in patients with acute aortic dissection (AAD). Methods: This study consisted of 16 patients with AAD and 27 non-AAD patients. Serum D-dimets were measured by Sta-Liatest D-DI immunoturbidimetric assay. Results: D-dimer level was higher (P ＜ 0.001) in patients with AAD(7.91 ± 5.52 μg/ml) than that in non- AAD group(1.57±1.24 μg/ml). D-dimer was positive (＞0.4 μg/ml) in all patients with AAD and in 10 control group patients (37%). Among patients with acute AAD, D-dimers tended to be higher in Stanford A than in Stanford B (8.67 ± 4.31 μg/ml vs. 3.24±1.27 μg/ml, P ＜0.01). D-dimer values tended to be higher in more extended disease(3.84 ± 1.65 μg/ml, 8.57 ± 3.58 μg/ml and 11.87 ± 5.69 μg/ml in thoracic aorta, thoracic and abdominal aorta, thoracic and abdominal aorta and iliacal arteries, respectively, P ＜ 0.05 for both 8.57 ± 3.58 and 11.87 ± 5.69 vs. 3.84 ± 1.65 ). Including the control group into the analysis, we found a sensitivity of 100%, a negative predictive value of 100%, and a specificity of 66% and a positive predictive value of 64% for D-dimer in diagnosis of AAD in our patients with suspected AAD. Conclusion: D-dimer was elevated in patients with AAD. A negative D-dimer test result could be useful in excluding AAD.     

Research of combined liver-kidney transplantation model in rats

Objective： To set up a simple and reliable rat model of combined liver-kidney transplantation. Methods： SD rats served as both donors and recipients. 4℃ sodium lactate Ringer＇s was infused from portal veins to donated livers,and from abdominal aorta to donated kidneys, respectively. Anastomosis of the portal vein and the inferior vena cava （IVC） inferior to the right kidney between the graft and the recipient was performed by a double cuff method, then the superior hepatic vena cava with suture. A patch of donated renal artery was anastomosed to the recipient abdominal aorta. The urethra and bile duct were reconstructed with a simple inside bracket. Results： Among 65 cases of combined liver-kidney transplantation, the success rate in the late 40 cases was 77.5%. The function of the grafted liver and kidney remained normal. Conclusion： This rat model of combined liver-kidney transplantation can be established in common laboratory conditions with high success rate and meet the needs of renal transplantation experiment.     

APPLICATION OF LORNOXICAM TO PATIENT-CONTROLLED ANALGESIA IN PATIENTS UNDERGOING ABDOMINAL SURGERIES

FOR anesthesiologis s ,treatingpostoperativepainhas alwaysbeen a problem.Althoughopioidshave been provedtobe effective,theirsideeffectscouldnotbeignored.With thedevelopmentofscienceand pharmacology,many drugs with aspectsof satisfactoryanalgesicefficacyand couldbe welltoleratedby patientshave been developed.And lornoxicamisone of them, which isa non-steroidalanti-inflammatorydrug (NSAID ), with analgesic, anti-infl-ammatory,andantipyreticproperties.Itseliminationhalf-time(3 to 5 hours) isle…     

BLOOD PRESSURE CHANGE WITH AGE IN SALT-SENSITIVE TEENAGERS

Objective To observe blood pressure change with age in salt-sensitive teenagers whose salt sensitivity were determined by repeated testing.Methods Salt sensitivity was determined through intravenous infusion of normal saline combined with volume-depletion by oral diuretic furosemide in 55 teenagers. After five years, salt sensitivity was re-examined and subject blood pressure was followed up. Blood pressure changes in salt-sensitive teenagers were compared to that of non-salt sensitive teenagers over five years.Results After 5 years, the repetition rate of salt sensitivity determined by intravenous saline loading is 92.7%. In teenagers with salt sensitivity on the baseline, both the systolic blood pressure increments and increment rates were much higher than non-salt sensitive teenagers (12.7±12.1 mmHg vs. 2.8±5.2 mmHg, P＜ 0.01; 12.2%± 12.0% vs. 2.5% ±4.4%, P＜ 0.001,respectively). There was a similar trend for diastolic blood pressure (8.4 ± 6.4 mmHg vs. 3.7 ± 6.4 mmHg, P = 0.052; 13.2% ±10.6 % vs. 6.8%± 10.1%, P = 0.053, respectively).Conclusions Salt sensitivity determined by intravenous saline loading showed good reproducibility. Blood pressure increments with age were much higher in salt-sensitive teenagers than non-salt sensitive teenagers, especially in terms of systolic blood pressure.     

Development on Adjustable Calibration Marker for Shock Wave Focus

Shock wave lithotripsy （SWL） is a treatment of choice for upper urinary stones. However, this procedure is inappropriate for obese patients because the focus is often unable to reach the target owing to the limited focal distance in shock wave source. Although treating such patients in a blast path may increase the application length of shock wave source, it＇s difficult to find this path on the lithotripter monitor. For this reason, we invented an adjustable calibration marker in order to set an effective focus in the shock wave hath.     

Study on the TCM Pathogenesis of Angiopathic Complications of Type II Diabetes

Excess production of reactive oxygen species(ROS)of mitochondrion mediated by hyperglycemia is the common pathogenesis of angiopathic complications of diabetes.TCM holds that the damp from the dysfunction of spleen.kidney and liver is the causative factor of complications of diabetes.This is similar to the mechanism of Ros resulting in angiopathic complications of diabetes.When the angiopathic complications of type II diabetes mellitus(T2DM)are difierentiated as caused by turbid damp in TCM can be explained as ROS.Since the obstruction of pathogenic damp in channels and collaterals is said to be the main pathogenesis,the treating principle should be dissolving the damp to remove the obstruction.     

Leptin, Ghrelin and TNF-α before and after Hypo-caloric Traditional Chinese Diet and Auricular Acupuncture

INTRODUCTION Obesity is a complex emergent problem, which can be possibly solved not only by the diet but also by the life style and promotion of a constant physical exercise. 1, 2 No doubt careful attentions must be given to the nutritional condition of obese people, the dietary habits, the somatic build (i.e. distribution of fat mass) and the organic functions linked to formation of the fat mass. All the parameters should be constantly monitored before, during and after a diet treatment. 3, 4, 5     

Relationship between Dysglycemia and Carotid Atherosclerosis in Tibetan Population

People with dysglycemia are at high risk for atherosclerotic diseases. This study aims at investigating the atherosclerotic vascular damage in dysglycemia and its metabolic origin in Tibetan population.     

Acupuncture Combined with Spinal Tui Na for Treatment of Primary Dysmenorrhea in 30 Cases

Objective: To observe the therapeutic effects in acupunture treatment of primary dysmenorrhea combined with spinal Tui Na, and study its mechanism. Methods: Thirty cases of the treatment group were treated by acupuncture combined with spinal Tui Na, and thirty cases in the control group were treated by routine acupuncture. Results: The total effective rate was 93.3% in the treatment group, and 73.3% in the control group, with a significant difference between the two groups (P<0.05). Conclusions: Acupuncture combined with spinal Tui Na has good prospects for treatment of primary dysmenorrhea.     

Luo Lingjie's Experience in Treating Chronic Nephropathy

In treating chronic nephropathy,Luo Lingjie,a chief physician,pays attention to regulating the balance between yin and yang,treating infection if present,and removing pathogenic factors.He prescribes gentle drugs and uses carefully strongly warming-tonifying ones,emphasizes the importance of persuading the patient to persist in treatment with medication and nurse one's health for recuperation,and is good at combined use of TCM and western medicine therapy and brings the merits of various therapies into full play,with obvious theraoeutic effects.