14,15-环氧化二十碳三烯酸对棕榈酸盐诱导的H9c2心肌细胞凋亡的影响及其机制

目的:观察14,15-环氧化二十碳三烯酸(14,15-EET)对棕榈酸盐(Palmitate)诱导的H9c2大鼠心肌细胞凋亡的影响及其机制。方法:传代培养大鼠心肌细胞H9c2,分为正常组、溶媒组、Palmitate组、Palmitate+14,15-EET组,噻唑蓝比色法(MTT)检测细胞存活率、流式细胞仪检测细胞凋亡以及Western blot检测Bax,Bcl-2蛋白表达。结果:Palmitate的刺激可显著降低H9c2细胞的存活率并呈剂量依赖效应,14,15-EET呈剂量依赖性增加不同浓度Palmitate刺激后H9c2细胞存活率;Palmitate可诱导H9c2细胞凋亡,14,15-EET的作用显著抑制了Palmitate诱导的H9c2细胞的凋亡,联用胞外信号调节蛋白激酶(ERK1/2)及蛋白激酶B(PKB或AKT)信号通路抑制剂显著抑制了14,15-EET的抗凋亡作用。Palmitate的诱导使Bax表达增加,Bcl-2表达减少;14,15-EET可下调Bax,上调Bcl-2的表达,联用ERK1/2及AKT信号通路抑制剂能显著抑制14,15-EET对Palmitate诱导后H9c2细胞Bax表达水平的下调及Bcl-2表达水平上调的作用(P均<0.05)。结论:Plamitate可诱导H9c2细胞凋亡,14,15-EET可通过下调Bax,上调Bcl-2的表达抑制Palmitate诱导的H9c2心肌细胞凋亡作用。这些作用可由ERK1/2和AKT信号通路介导。     

miR-21-5p靶向TGF-β1调节大鼠心肌细胞增殖和凋亡

目的 探讨miR-21-5p通过调控转化生长因子-β₁(transforming growth factor-β₁,TGF-β₁)对大鼠心肌细胞H9c2增殖和凋亡的影响。方法 抑制或过表达TGF-β₁后,通过RT-qPCR检测H9c2细胞中miR-21-5p的表达水平,双荧光素酶报告基因实验验证miR-21-5p和TGF-β₁的靶向关系,CCK-8及Annexin V-FITC/PI检测H9c2细胞增殖和凋亡,Western blotting检测H9c2细胞中TGF-β₁的表达水平。结果 miR-21-5p表达抑制后使H9c2细胞增殖受到抑制并诱导其凋亡(P＜0.05)。miR-21-5p靶向负调控TGF-β₁的表达水平(P＜0.05)。过表达TGF-β₁显著抑制H9c2细胞增殖并诱导其凋亡(P＜0.05)。结论 miR-21-5p通过靶向下调TGF-β₁的表达水平,促进大鼠H9c2心肌细胞增殖和抑制细胞凋亡。     

TGF-β2对心肌细胞H9C2转录因子表达的影响及其组蛋白H3乙酰化调控机制

目的 探讨转化生长因子-β2(transforming growth factor beta2,TGF-β2)对心肌细胞H9C2转录因子Mef2c和GATA4的影响及其与组蛋白H3乙酰化的关系.方法 比较TGF-β2对细胞生长的影响.利用Real-time PCR检测心肌细胞的心脏相关转录因子Mef2c和GATA4mRNA的相对表达量,筛选合适的TGF-β2干预浓度.采用Western blot检测心肌细胞乙酰化组蛋白H3(acetylated histone H3,acH3)的表达差异.染色质免疫共沉淀(chromatin immunoprecipitation,ChIP) Real-time PCR检测Mef2c和GATA4启动子区与acH3结合DNA的相对表达水平.比色分析法检测TGF-β2对心肌细胞的总体组蛋白乙酰化酶(HATs)活性的影响.结果 ①TGF-β2干预组相对于对照组心肌细胞的增殖能力增强(P＜0.05).②心肌细胞的心脏相关转录因子Mef2c和GATA4mRNA的相对表达量随着TGF-β2浓度的升高呈现先升高后降低的趋势,在5 μg/L的浓度时均达到最高,Mef2c mRNA相对表达量是对照组的1.8倍,GATA4mRNA是对照组的2.3倍(P＜0.05).③TGF-β2干预72 h后acH3蛋白表达量是对照组的4倍(P＜0.05),HATs活性是对照组的1.34倍(P＜0.05).④TGF-β2干预组Mef2c和GATA4启动子区组蛋白H3的乙酰化水平分别是对照组的2.56和2.16倍(P＜0.05).结论 5μg/L TGF-β2可以促进心肌细胞H9C2的分裂与增殖,并上调心脏相关转录因子Mef2c和GATA4的表达,组蛋白H3乙酰化水平的升高可能是Mef2c和GATA4表达上调的机制之一.     

七氟醚后处理通过调节FTO/KCNJ2对缺氧/复氧所致心肌细胞损伤的保护作用

摘要:目的 探讨七氟醚后处理对缺氧/复氧(hypoxia/reoxygenation,H/R)诱导的心肌细胞损伤的影响及机制。方 法 从基因表达综合数据库(Gene Expression Omnibus,GEO)获取心肌缺血再灌注损伤(myocardial ischemia reperfusion injury,MIRI)相关数据集 GSE160516,将差异表达基因 KCNJ2纳入本研究。采用 qRT-PCR 检测 KCNJ2在 H/R 和七氟醚处理的心肌细胞中的表达。RIP实验验证 KCNJ2和 FTO 的结合关系。分别采用 MTT 法和流式细胞术检测 细胞活力和凋亡率,使用乳酸脱氢酶(LDH)检测试剂盒测定细胞的 LDH 释放量。结果 七氟醚后处理可抑制 H/R 诱 导的心肌细胞损伤。相对于 Control组,H/R 心肌细胞中 KNCJ2表达下调,而七氟醚可提高 H/R 心肌细胞中 KCNJ2 的表达(均P<0.01)。相对于 H/R组,七氟醚组细胞增殖活力提高、LDH 释放水平和凋亡率下降,但七氟醚对 H/R 心 肌细胞的保护作用能够被敲减 KCNJ2部分逆转(均P<0.05)。FTO 能够通过抑制 KCNJ2的 m6A 修饰进而增强 KCNJ2的表达;七氟醚可上调 FTO 的表达进而影响 KCNJ2的稳定性和表达水平;敲减 FTO 能够抑制七氟醚对 H/R 心肌 细胞的保护作用,而该作用可被 KCNJ2过表达部分挽救。结论 七氟醚后处理通过上调 FTO/KCNJ2的表达对 H/R 诱导的心肌细胞损伤起保护作用。     

阿托伐他汀对心肌肥大的抑制作用及FoxO1表达的影响

目的探讨阿托伐他汀(atorvastatin)对血管紧张素Ⅱ(AngⅡ)诱导的肥大心肌细胞的抑制作用及对转录因子FoxO1表达的影响。方法将H9C2细胞分为3组,对照组:未给予任何干预;心肌肥大组:给予AngⅡ(10-7 mol/L)刺激细胞;阿托伐他汀组:预先给予atorvastatin(10-5 mol/L),30min后AngⅡ(10-7 mol/L)刺激细胞。western-blotand real time PCR检测H9C2细胞FoxO1蛋白及mRNA含量,脑钠肽(brain natriuretic pepide,BNP)作为判断心肌肥大的指标。结果 AngⅡ诱导心肌细胞肥大后FoxO1表达较正常心肌细胞明显降低(P<0.05),而给予阿托伐他汀干预的肥大心肌细胞其FoxO1表达较肥大心肌明显升高(P<0.05)。结论阿托伐他汀可能通过上调FoxO1表达,从而抑制心肌肥大。     

FGF-2上调Survivin蛋白的表达拮抗心肌细胞凋亡

目的探讨成纤维细胞生长因子-2(FGF-2)拮抗心肌细胞凋亡的作用机制。方法以H9c2大鼠心肌细胞作为实验对象,用无血清培养基同步化培养24小时,心肌细胞随机分为正常组(Control)、缺氧/复氧(H/R)组和H/R+FGF-2组。采用CCK-8比色法检测心肌细胞存活率,Hoechst染色和流式细胞术检测心肌细胞凋亡。采用Western blot检测生存素(Survivin)蛋白的表达。结果与正常组相比,H/R明显抑制心肌细胞活力,诱导心肌细胞凋亡(均P<0.01);与H/R组相比,FGF-2显著改善H/R心肌细胞活力,拮抗H/R心肌细胞凋亡,同时上调Survivin蛋白的表达(均P<0.05)。结论 FGF-2拮抗心肌细胞凋亡的作用与其上调Survivin蛋白表达有关。     

TGF-β1诱导大鼠心肌细胞肥大中钙调素依赖蛋白激酶Ⅱ信号途径研究

目的：探讨TGF-β1诱导的大鼠心肌细胞肥大中钙调素依赖蛋白激酶Ⅱ信号途径的信号表达.方法：建立TGF-β1诱导的体外大鼠心肌细胞肥大模型.PI染色标记细胞双链RNA法检测心肌细胞RNA表达量,间接检测心肌细胞肥大.实时荧光定量PCR检测心肌细胞肥大相关基因肌球蛋白重链β亚型（β-MHC）的表达.Western blotting检测心肌细胞中p-CaMKⅡ蛋白表达.结果：TGF-β1可诱导体外培养心肌细胞肥大基因的表达,β-MHC的表达明显高于对照组（P＜0.01）.PI染色TGF-β11组PI含量明显增高（P＜0.01）,TGF-β13 μg/L组心肌细胞内PI含量最高（80.57±3.56）;与对照组相比,TGF-β1可明显上调的表达p-CaMKⅡ（108.83±12.15 vs 10.6±1.35,P＜0.01）,并于TGF-β1诱导2h表达达峰.结论：TGF-β1可诱导心肌细胞肥大的形成,此过程中CaMKⅡ蛋白表达明显增加.     

丙烯醛对缺氧复氧H9c2心肌细胞损伤的影响及机制初探

目的研究丙烯醛对缺氧复氧H9c2心肌细胞损伤的影响,并探讨其促凋亡作用机理。方法建立H9c2心肌细胞缺氧复氧(H/R)损伤模型,分为正常对照组、丙烯醛组(ACR)、缺氧复氧组(H/R)、丙烯醛+缺氧复氧组(ACR+H/R)。用10μmol/L丙烯醛预处理H9c2心肌细胞30min后2h缺氧16h复氧,采用MTT法检测细胞存活率,4′,6-二脒基-2-苯基吲哚(DAPI)荧光染色检测细胞核凋亡的形态学改变,流式细胞术检测细胞凋亡,Western blot检测凋亡相关蛋白cytochrome c(cyt c)、caspase 9和caspase 3的表达水平。结果与H/R组相比,ACR+H/R组H9c2细胞存活率下降、凋亡增加(P<0.05),cyt c的线粒体蛋白表达水平下调、细胞浆蛋白表达水平上调,caspase 9和caspase 3蛋白表达水平下调并出现明显裂解蛋白。结论作为一种来源于人类生活环境的心血管毒物,丙烯醛能加剧缺氧复氧H9c2心肌细胞的损伤,可能与cyt c由线粒体释放到细胞浆、激活caspase级联反应导致线粒体功能损伤有关。     

FGF-1 对TGF-β1 诱导的H9c2 心肌细胞凋亡与Caspase-3，Caspase-8 和Caspase-9 表达的关系研究

目的 观察酸性成纤维细胞生长因子（FGF-1）对转化生长因子-β1（TGF-β1）诱导的H9c2心肌细胞凋亡作用及对半胱氨酸蛋白酶-3（Caspase-3）,半胱氨酸蛋白酶-8（Caspase-8）和半胱氨酸蛋白酶-9（Caspase-9）表达的影响,并探讨可能机制。方法 体外培养H9c2 细胞,随机分为5 组：模型组,肝素（Heparin）组、FGF-1 组、FGF-1/Heparin 组和正常对照组。采用3-（4,5- 二甲基噻唑-2）-2,5-二苯基四氮唑溴盐（MTT）比色分析检测H9c2 细胞的存活率,TUNEL 染色检测H9c2 细胞凋亡指数和Western blot 分析检测H9c2 细胞Caspase-3,Caspase-8 和Caspase-9 的表达。结果 模型组H9c2 细胞存活率与正常对照组比较,差异有统计学意义（P <0.05）,模型组降低;FGF-1 组和FGF-1/Heparin 组H9c2 细胞存活率与模型组比较,差异有统计学意义（P <0.05）,FGF-1 组和FGF-1/Heparin 组均升高。模型组H9c2 细胞凋亡指数,Caspase-3、Caspase-8 和Caspase-9 的表达与正常对照组比较,差异有统计学意义（P <0.05）,模型组升高;FGF-1 组和FGF-1/Heparin 组H9c2 细胞凋亡指数,Caspase-3、Caspase-8 和Caspase-9 的表达与模型组比较,差异有统计学意义（P <0.05）,FGF-1 组和FGF-1/Heparin 组降低。结论 FGF-1 对TGF-β1诱导的H9c2 心肌细胞凋亡具有抑制作用,其机制可能与抑制Caspase-3、Caspase-8 和Caspase-9 的表达有关。     

BRD4基因对TGF-β1诱导的心肌细胞凋亡和p38MAPK信号通路的影响

目的：探讨溴结构域蛋白4（BRD4）基因对转化生长因子β1（TGF-β1）诱导的心肌细胞凋亡的影响,阐明其可能的作用机制。方法：将H9c2细胞分为空白对照组、si-CN组（转染阴性对照siRNA）和si-BRD4组（转染特异性siRNA）,采用Western blotting法检测各组细胞中BRD4蛋白表达水平。大鼠心肌H9c2细胞随机分为对照组、TGF-β1组（20μg·L^-1TGF-β1处理细胞24 h）、si-NC+TGF-β1组（转染阴性对照的siRNA后,使用TGF-β1处理细胞24 h）和si-BRD4+TGF-β1组（转染BRD4特异性siRNA后,使用TGF-β1处理细胞24h）。MTT法检测各组细胞增殖活性,Annexin Ⅴ-FITC/PI双染法检测细胞凋亡率,Western blotting法检测各组细胞中BRD4、B淋巴细胞瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、活化的含半胱氨酸的天冬氨酸蛋白水解酶3（Cleaved caspase3）、P38和磷酸化P38（p-P38）蛋白表达水平。结果：与空白对照组比较,si-BRD4组H9c2细胞中BRD4蛋白表达水平明显降低（P<0.01）。与对照组比较,TGF-β1组细胞增殖活性明显降低（P<0.05）,细胞凋亡率明显升高（P<0.05）,Bcl-2蛋白表达水平明显降低（P<0.01）,Bax、Cleaved caspase3和p-P38蛋白表达水平均明显升高（P<0.01）;与TGF-β1组比较,si-BRD4+TGF-β1组细胞增殖活性明显升高（P<0.05）,细胞凋亡率明显降低（P<0.05）,Bcl-2蛋白表达水平明显升高（P<0.05）,Bax、Cleaved caspase3和p-P38蛋白表达水平均明显降低（P<0.05）。结论：抑制BRD4基因表达可减弱TGF-β1诱导的心肌细胞凋亡,其机制可能与抑制P38丝裂原活化蛋白激酶（MAPK）信号通路有关。     

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…     

Acupuncture Treatment of Vomiting

CASE HISTORY A female patient, 46 years old, head of the foreign affairs department of a certain university in Beijing, paid her first visit on October 9, 2006, with the chief complaint of vomiting for one month. She got vomiting after meals in early September. Before that, she had discomfortable sensation in the stomach due to angry with others, but she didn't pay much attention. Later, it developed into vomiting after eating. After the vomiting, the discomfort would be relieved, but with slight hypodynamia. She was once diagnosed as having 'neurogenic vomiting'. Having taken some western and Chinese drugs, the above symptoms were a little bit improved, but she would have nausea upon eating and with regurgitation. Because of the fear for vomiting, she did not dare to have food intake, with body weight reduction of 6 kilos in one month.     

Qiu Baoguo''''s Experience in Treating Consumptive Syndromes after Radio-Chemotherapies

Radiotherapy and chemotherapy are the important modern medical therapies for malignant tumors,yet they can also bring about serious local and systemic toxic side reactions so to decrease the patient;'s life quality,manifested by a series of consumptive symptoms.Having engaged in the combined work of Chinese and western medicine for nearly 50 years,the research fellow Qiu Baoguo in Henan Provincial Academy of TCM has developed his unique views on the TCM study of consumptive syndromes.The author of this essay had once the fortune tO follow Dr.Qiu in clinic,and specially would like to introduce in the following Dr.Qiu's experience in treating consumptive syndromes after radio-chemotherapies for patients with malignant tumor.     

Insomnia Due to Deficiency of Both the Heart and Spleen Treated by Acupuncture-Moxibustion and Chinese Tuina

OBJECTIVE: To observe therapeutic effects of the comprehensive therapy of acupuncture-moxibustion and Chinese Tuina for treatment of insomnia due to deficiency of both the heart and spleen. METHODS: 92 cases were divided randomly into the treatment group (treated by acupuncture-moxibustion and Chinese Tuina) and the control group (treated by acupuncture-moxibustion). RESULTS: The therapeutic effect of the treatment group was obviously superior to that of the control group (the CHI2 test showed P < 0.01). CONCLUSIONS: The comprehensive therapy of acupuncture-moxibustion and Chinese Tuina can give marked therapeutic effects for treatment of insomnia due to deficiency of both the heart and spleen.     

Analysis of CK-19,PDX-1,Nestin,Ngn3 in different donor islet purity in rats

Objective: To investigate if there are the CK-19, PDX-1, Nestin, Ngn3 positive cells in the donor islets of different purity in rats. Methods: Thirty male adult SD rats were randomly divided into 3 groups. Islets were isolated using digestion by ductal injection of collagenase. Group Ⅰ (n=10): Separating cell preparations were not purified, Group Ⅱ(n=10): Islet sediment was purified with 25% Ficoll400 ,Group Ⅲ (n=10): Islet sediment was purified with 25% and 11% Ficoll-400. The levels of protein of CK-19, PDX-1, Nestin and Ngn3 were detected by immunohistochemistry and the mRNA of CK-19, PDX-1, Nestin, Ngn3 was amplified by RT-PCR. Results: After two different purification methods applied, three islet preparations of different purities were obtained. The difference of islet purity was significant among various groups (P＜0.05). Compared with group Ⅱ and group Ⅲ,the protein and mRNA of CK-19, PDX-1, Nestin,Ngn3 were both higher in group Ⅰ; group Ⅲ was poorly expressed. Conclusions: The three different islet purity donor islet have different CK-19, PDX-1, Nestin, Ngn3 positive cells within them, indicating that there are some islet stem cells in the purified donor islet.     

Dr.Zhang Ren''''s Experience in the Acupuncture Treatment of Different Diseases with the Same Therapeutic Principle

Dr.Zhang Ren,the chief physician,is the chairman of Shanghai Acupuncture and Moxibustion Association.Having been engaged in medicine for about 40 years,he is experienced in treating various intractable diseases.In his long years of clinical practice,he advocates taking the TCM differentiation as the basis to seek for the acupuncture method for treatment of modern intractable diseases.The author of this essay had the fortune to follow Dr.Zhang in study.The following is a summary of Dr.Zhang's experience in the acupuncture treatment for different intractable diseases with the same therapeutic principle.     

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.     

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.