大黄素通过PPARγ/NF κB信号通路抑制LPS诱导的RAW2647细胞炎症反应

【摘要】 目的 研究大黄素对LPS诱导的炎症反应的抑制作用及其相关的信号通路。方法 将小鼠巨噬细胞RAW2647细胞分为6组,即Control组、LPS组、大黄素组(Emodin组)、LPS+大黄素组(LPS+Emodin组)、LPS+大黄素组+siRNA PPARγ (LPS+Emodin+siRNA PPARγ组)、LPS+大黄素组+siRNA scrambled (LPS+Emodin+siRNA scrambled组)。使用ELISA测定TNF α水平,qPCR和western blot测定ICAM 1、MCP 1和PPARγ mRNA和蛋白水平,并使用western blot测定NF κB p65磷酸化水平。结果 与Control组相比较,LPS干预6小时后RAW2647细胞TNF α、ICAM 1和MCP 1的表达以及NF κB p65磷酸化水平均明显升高,PPARγ表达水平明显下降,差异均有统计学意义(P均<005);与LPS+Emodin组相比较,LPS组TNF α、ICAM 1和MCP 1的表达以及NF κB p65磷酸化水平升高更明显,PPARγ表达水平下降更明显,差异均有统计学意义(P均<005);同时siRNA PPARγ可以有效阻断Emodin的作用,差异均有统计学意义(P均<005);且LPS+Emodin组和LPS+Emodin+siRNA scrambled组间各分子表达及磷酸化水平未见明显差别,差异均无明显统计学差异(P>005)。结论〓大黄素可能通过PPARγ/NF κB信号通路抑制LPS诱导的RAW2647细胞炎症反应,为大黄素应用于支气管哮喘及ARDS等炎症相关性疾病的临床治疗奠定了基础。     

肺腺癌细胞A549 hnRNP B1基因RNA干扰的体外研究

目的 体外研究特异的小干扰RNA(siRNA)对肺腺癌细胞A549 hnRNP B1 基因表达及生长的抑制作用. 方法设计和构建由H1启动子驱动产生的特异的hnRNP B1 siRNA 表达质粒,转染至A549细胞,荧光定量PCR法及Western blot检测hnRNP B1基因的表达,MTT法检测该表达质粒转染后对A549细胞生长的影响.结果 针对hnRNP B1 基因构建的重组质粒具有明显的干扰效果,受特异hnRNP B1 siRNA 干扰的A549细胞hnRNP B1 mRNA及蛋白表达下调,细胞生长受到抑制.结论 应用RNA干扰技术可阻止hnRNP B1基因的表达,有望成为肿瘤基因治疗新的研究方向.     

hnRNP B1 siRNA对肺腺癌细胞A549 p53表达的影响

目的 探讨核内不均一核糖核蛋白(hnRNP)B1调节肺腺癌细胞A549抑癌基因p53表达的作用.方法 采用前期研究已构建并通过体外实验证实具有较好干扰效果的两对hnRNP B1特异性的小干扰RNA(siRNA)真核细胞表达载体(重组质粒A和D)转染的人肺腺癌细胞株A549作为实验组,对照组为转染空质粒的A549细胞和未转染任何质粒的A549细胞.各组细胞均用10 μmol/L顺铂作用24 h收集细胞.实时荧光定量RT-PCR检测p53 mRNA的表达,Western blot检测P53及磷酸化P53蛋白表达的变化.结果 各组细胞p53 mRNA和蛋白表达量无明显差异,说明hnRNP B1特异性siRNA对A549细胞p53基因mRNA和蛋白表达无影响.转染hnRNP B1 siRNA细胞磷酸化P53蛋白表达率分别为0.87±0.06、0.75±0.06,与空质粒转染组(0.30±0.08) 和未转染组(0.21±0.04)相比明显增高(P<0.01).结论 hnRNP B1 siRNA可上调肺腺癌细胞A549 P53活性,参与肺癌的发生发展.     

STAT3 siRNA对肺腺癌A549细胞STAT3及其下游基因表达的影响

目的：观察RNA干扰沉默STAT3基因对人肺腺癌A549细胞STAT3、CyclinD1、Bcl-2、BAX、Caspase-9蛋白表达的影响及其相关性及对细胞生长的影响,探讨肺癌基因治疗的新途径。方法：采用化学合成法,合成针对STAT3基因不同位点设计的3条小干扰RNA（small interfering RNA,siRNA）序列（siRNA1-3）和1条带有荧光标记的应用STAT3-siRNA转染处理人肺腺癌细胞系A549,转染16h后荧光显微镜观察转染效率,RT-PCR检测STAT3基因表达的变化,蛋白免疫印迹法检测转染后A549细胞STAT3,CyclinD1,BAX,Bcl-2,Caspase-9蛋白表达,AM-BLUE检测A549细胞生长情况。结果：经RNA干扰能有效减低A549细胞STAT3mRNA表达水平（P〈0.05）,转染后A549细胞STAT3、CyclinD1、Bcl-2蛋白的表达水平均显著降低（P〈0.05）,而BAX、Caspase-9蛋白的表达水平升高（P〈0.05）。STAT3蛋白表达分别与CyclinD1、Bcl-2蛋白表达呈正相关（r=0.854,0.910,P〈0.05）;与BAX蛋白表达程负相关性（r=-0.848,P〈0.05）;与Caspase-9蛋白表达程无相关性（r=-0.787,P〉0.05）。RNA干扰法沉默STAT3基因后,A549细胞增殖受到明显抑制。结论：STAT3-siRNA可有效抑制STAT3、CyclinD1、Bcl-2在人肺腺癌A549细胞中的表达,升高BAX、Caspase-9在人肺腺癌A549细胞中的表达,抑制A549细胞生长。     

应用荧光定量PCR和Western—blot检测RNA干扰肺腺癌A549细胞STAT3基因表达水平的实验研究

目的：研究RNAi技术对人肺腺癌A549细胞系中STAT3基因的阻断效应,探讨STAT3在肺癌中的重要作用,为肺癌的基因治疗寻找新的靶点。方法：将化学合成siRNA用脂质体介导法转染肺腺癌A549细胞,应用荧光定量PCR法检测RNA干扰后STAT3 mRNA表达量,得出抑制率。应用Western-blot检测RNA干扰后对STAT3蛋白表达的影响。结果：成功将siRNA转染肺腺癌A549细胞,荧光定量PCR结果显示,在转染24h、48h、72h后,各干扰组与阴性组比较STAT3基因表达量明显下调（P〈0.05）,Western blot显示转染前后STAT3蛋白的表达水平明显受到抑制,干扰组与阴性组空白组相比具有显著差异（P〈0.05）,内参基因在各组的表达量无明显差异。结论：将合成的STAT3 siRNA能有效抑制STAT3蛋白的表达、降低STAT3 mRNA水平,对STAT3有高效和特异的沉默作用,以STAT3为靶点的RNA干扰技术可望成为肺癌基因治疗的新策略。     

LPS刺激对人结肠癌SW480、肺癌A549细胞HMGB1以及HBD-1表达的影响

目的观察脂多糖（LPS）刺激对SW480、A549细胞HMGB1、HBD-1表达的影响。方法以浓度为5、10、50和100ng／μl LPS刺激SW480、A549细胞24h后,提取细胞RNA,并用RT—PCR法检测HMGB1以及HBD-1基因mRNA的表达水平。结果SW480以及A549细胞,HMGB1以及HBD-1均有较高的表达。在SW480细胞中,不同的LPS刺激浓度下,5ng／μl LPS即可上调HMGB1基因mRNA的表达,但其表达量并不随LPS浓度的增高而有所增加;而同样刺激条件下HBD-1基因mRNA表达水平未见明显变化（P〉0．05）。在A549细胞中,不同的LPS刺激浓度下HMGB1以及HBD-1基因mRNA表达量未见明显变化（P〉0．05）。结论HMGB1及HBD-1固有表达于肠道及呼吸道上皮细胞。LPS能上调SW480细胞HMGBl的表达,其表达量无浓度依赖性,但LPS不能上调HBD-1的表达。在A549细胞,LPS不能诱导HMGB1以及HBD-1的表达。     

SCNN1B在非小细胞肺癌组织中的表达及其调节肺癌对顺铂的敏感性

【摘要】 目的 探讨SCNN1B在非小细胞肺癌(NSCLC)组织中的表达及其调节肺癌对顺铂的敏感性。 方法 选取本院2017年12月~2019年12月收治的130例NSCLC患者,RT PCR法检测其肺癌组织中SCNN1B mRNA水平;另设A549细胞组、A549/DDP细胞组、siRNA-SCNN1B组、siRNA-NC组,其中siRNA-SCNN1B组、siRNA-NC组分别转染SCNN1B过表达质粒及空载质粒（阴性对照）,RT-PCR法检测各组SCNN1B mRNA相对表达量,验证SCNN1B转染效果;并检测各组半数抑制浓度（IC50）、细胞凋亡、细胞周期,Western blot法检测耐药相关蛋白表达。 结果 低分化NSCLC患者SCNN1B mRNA低表达率明显高于中高分化患者,TNM分期Ⅳ期患者中SCNN1B mRNA低表达率明显高于Ⅲ期（P＜0.05）;A549/DDP细胞组SCNN1B mRNA水平显著低于A549细胞组（P＜0.05）;与A549/DDP细胞组比较,siRNA-SCNN1B组SCNN1B mRNA水平明显上升（P＜0.05）;siRNA-SCNN1B组IC50明显低于siRNA-NC组,逆转系数下降（P＜0.05）;与A549细胞组比较,A549/DDP细胞组细胞凋亡率明显下降,细胞周期G0/G1期比例明显上升（P＜0.05）,siRNA SCNN1B组细胞凋亡率则较A549/DDP细胞组明显上升,细胞周期G0/G1期比例较A549/DDP细胞组明显下降（P＜0.05）;与A549细胞组比较,A549/DDP细胞组Bcl-2、生存素、周期蛋白D1(CyclinD1)、表皮生长因子受体(EGFR)、多药耐药相关蛋白-1(MRP1)、肺耐药相关蛋白（LRP）水平明显上升（P＜0.05）;siRNA-SCNN1B组Bcl-2、生存素、Cyclin D1、EGFR、MPR1、LRP水平则较A549/DDP细胞组明显下降（P＜0.05）。结论 SCNN1B与NSCLC患者肺癌组织分化程度、肿瘤分期密切相关,靶向上调SCNN1B可下调Bcl-2、生存素、Cyclin D1、EGFR、MPR1、LRP等蛋白表达。     

RNA干扰Grp78蛋白对肺癌细胞A549的作用研究

目的 探讨采用RNA干扰Grp78蛋白表达,观察对肺癌细胞A549的作用.方法 通过转染Grp78的siRNA质粒到肺癌细胞A549并进行表达,观察A549细胞的Grp78蛋白的表达及细胞的侵袭和转移能力的变化.结果 RNA干扰肺癌细胞A549的Grp78的表达,能够显著地抑制癌细胞的黏附、伸展,以及MMP-2,MMP-9蛋白的表达,甚至诱导细胞凋亡.结论 siRNA特异性下调Grp78蛋白,可以抑制肺癌细胞A549的侵袭和转移,甚至导致细胞凋亡.     

全氟化碳对脂多糖诱导肺泡上皮细胞炎性反应及单免疫球蛋白白介素1相关蛋白表达的影响

【摘要】 目的 探讨全氟化碳（PFC）对脂多糖（LPS）诱导人肺泡上皮A549细胞炎性反应及单免疫球蛋白白介素 1受体相关蛋白（SIGIRR）表达的影响。方法 实验分为正常细胞组、全氟化碳+脂多糖（PFC+LPS）组、全氟化碳（PFC）组及脂多糖（LPS）组。各组细胞经相应处理24 h后,用酶联免疫吸附法（ELISA）检测培养上清中的TNF α、IL 6和IL 10含量,用蛋白免疫印迹法（Western blot）检测核转录因子 κB （NF κB）和SIGIRR的表达。结果 ELISA结果显示,LPS组TNF α、IL 6水平较正常细胞组升高（P＜005）;PFC+LPS组TNF α、IL 6水平较LPS组下降（P＜005）;与正常细胞组相比,PFC组IL 10水平升高（P＜005）,PFC+LPS组IL 10水平较LPS组升高（P＜005）。Western blot 结果显示,与正常细胞组相比,LPS组核内磷酸化NF κB p65表达上调（P＜005）,PFC+LPS组核内磷酸化NF κB p65表达水平较LPS组降低（P＜005）;与正常细胞组相比,LPS组SIGIRR表达水平下调（P＜005）,PFC+LPS组SIGIRR表达水平较LPS组上调（P＜005）。PFC本身对TNF α、IL 6含量及NF κB p65表达无影响,但PFC可促进A549细胞释放IL 10及上调SIGIRR表达（P＜005）。结论 PFC可减轻A549细胞对LPS诱导的炎性反应,促进抗炎因子IL 10分泌及上调负调控分子SIGIRR表达是可能的分子机制。     

天麻素通过调节miRNA-221/PI3K/Akt通路对LPS诱导的肺泡上皮细胞损伤的影响

目的 探索天麻素(gastrodin,GAS)及磷脂酰肌醇3-激酶/蛋白激酶B (PI3K/Akt)通路在脂多糖(LPS诱导的肺泡上皮细胞损伤中的作用及可能机制。方法 将人肺泡上皮细胞A549分为正常对照组、LPS组、GAS组、miR-221 siRNA组、control siRNA组,采用RT-qPCR实验检测miRNA-221 mRNA的表达水平;ELISA实验检测细胞培养液中炎性因子的含量;MTT实验检测细胞活力;Western blot实验检测细胞中磷脂酰肌醇3-激酶(PI3K)、磷酸化蛋白激酶B (p-Akt)、蛋白激酶B (Akt)、Bcl-2相关X蛋白(Bax)、B细胞淋巴瘤基因-2 (Bcl-2)及半胱氨酸/天冬氨酸蛋白酶9 (Caspase9)蛋白的表达情况。结果 与LPS组比较,GAS组中GAS可明显降低LPS诱导的A549细胞中miRNA-221 m RNA的表达,升高p-Akt蛋白的表达,差异均具有统计学意义(P<0.05);与LPS组比较,miRNA-221 siRNA组抑制miRNA-221的表达后,可促进LPS诱导的A549细胞中PI3K、p-Akt...     

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.     

安心颗粒对急诊经皮冠状动脉介入术后生活质量影响的研究

目的：评价使用安心颗粒对急诊经皮冠状动脉介入术（PPCI）术后生活质量的影响.方法：将160例接受PPCI的急性ST段抬高型心肌梗死患者随机分为安心颗粒组（术前顿服安心颗粒8.8g,术后安心颗粒4.4 g/次,每日2次）和对照组（仅接受基础药物治疗）.所有患者均服用阿司匹林、氯吡格雷和阿托伐他汀.分别在入院时、出院前1d、出院后180 d时,应用心肌梗死多维度量表（MIDAS）、中文版SF-36评价量表对患者生活质量评分.并观察术后30 d以内的出血并发症、血小板减少症发生情况.结果：入院时和出院前1d,两组患者的心肌梗死MIDAS、SF-36量表评分比较无差异（P＞0.05）;出院后180 d时,与对照组比较,安心颗粒组MIDAS、SF-36评分明显减低（P＜0.05）;组内与入院时比较,两组出院前1d、出院后180 d时,MIDAS、SF-36评分均降低（P＜0.05）.两组患者在随访期间均无大量出血、少量出血、重度和极重度血小板减少症发生,安心颗粒组有4例、对照组有7例发生不明显出血（P＞0.05）.两组发生轻度血小板减少症的患者数比较无差异（P＞0.05）.结论：PPCI使用安心颗粒,能改善急性ST段抬高型心肌梗死患者的生活质量,且不增加出血风险.     

The comparative studies of the influences of Urapidil and Nicardipine on sino-atrial node function, atrio-ventricular node function and hemodynamics

Objective:To investigate the influences of urapidil and nicardipine on rabbit sinus function,atrio-ventricular node function and hemodynamics.Methods:Thirty-two Angora's rabbits were selected and randomly divided into four groups.U1 group:urapidil 0.25 mg/kg;U2 group:urapidil 0.5 mg/kg;N1 group:nicardipine 10 μg/kg;N2 group:nicardipine 20 μg/kg.All these medicine were administrated within 30 seconds.Measurements were taken before and after the administration of urapidil or nicardipine for the following data:mean blood pressure(MAP),heart rate(HR),sino-atrial conduction time(SACT),maximal sinoatrial recovery time(SNRTmax)corrected sinus node recovery time(CSNRT),index of sinus node recovery time(SNRTI),Wenckebach A-V conduction frequency (WB),and P-R interval.Results:Significant MAP and HR changes were identified in all of the four groups before and after administration of both urapidil and nicardipine.No significant changes could be found in the rest of the parameters.Intergroup analysis showed that SACT and CSNRT of N1 and N2 groups were shorter than those of the U2 group(P＜0.01);the MAP decreased(P＜0.01)and the HR increased drastically(P＜0.01).Conclusions:Neither urapidil(0.25 mg/kg,0.5 mg/kg)nor nicardipine(10μg/kg,20μg/kg)has any significant influence on rabbit sinus function or rabbit atrio-ventricular node function.Nicardipine could be a better choice than urapidil for parafunctional sinus node patients.     

Expression of OPGmRNA and ODFmRNA in the patients of hip fracture due to osteoporosis

Objective：To investigate the gene expression of osteoprotegerin（OPG） and osteoclast differentiation factor（ODF） in the bone tissue of patients with hip fracture due to osteoporosis. Methods：OPGmRNA and ODFmRNA in the bone tissue in 50 cases of osteoporosis sufferers（over 50 years old） with hip fracture（Observer Group） and 30 cases of hip facture sufferers with no osteoporosis（Control group） were analyzed with the Semi-Quantitative RT-PCR method. Results：The mRNA expressed of ODF, OPG were both high in the patients with hip fracture. In the control group, the expression of OPG mRNA was observed, while the expression of ODF mRNA was very slight. Conclusion：Aged patients contained all signals including OPG, ODF that are essential for inducing osteoclastogenesis and promoting bone resorption.     

The clinicopathological and immuohistochemical analysis of solid-pseudopapillary tumor of the pancreas： report of 9 cases

Objective：To investigate the clinical features, pathological characteristics and immunophenotype of solid-pseudopapillary tumor of the pancreas（SPTP）. Methods：Nine surgically treated cases of SPTP were retrospectively reviewed. Hematoxylin and Eosin（HE） staining and immunohistochemical staining were used to analyze all cases, and the general clinical data was collected. Results：Six patients were asymptomatic except for a palpable mass. Two patients complained of vague-epigastric pain. One patient appeared jaundice. The tumor was encapsulated and solid tissues alternately with cystic tissues. Histologically, the histological structure of solid portion was pseudopapillary with a fibrovascular core. Tumor cells were uniform and medium-sized which were arranged in sheets ets or nests or pseudopapillary patterns. Immunohistochemical studies demonstrated that SPTP proved positive in vimentin（9/9 cases）, AAT（9/9 cases）, NSE（9/9 cases）, ACT（7/9 cases）, CK20（2/9 cases）, CgA（1/9 cases）, S-100（3/gcases）, PR（4/gcases）, Syn（3/9 cases） and CD56（5/9cases）, negative in CEA and ER. Conclusion：SPTP is a tumor predominantly occurring in young women frequently without special symptoms. This tumor has various characteristical histological patterns with different immunophenotype.     

Electro-Acupuncture Combined with the Trigger Point Needle-Embedding for Treatment of Primary Trigeminal Neuralgia in 31 Cases

In recent years, the author of this essay has applied electro-acupuncture combined with the trigger point needle-embedding for treatment of primary trigeminal neuralgia in 31 cases, yielding satis- factory results as reported in the following.     

Investigation of Matrix Metalloproteinase -1, 2 and tissue inhibitor of matrix metalloproteinases-1 in Endometriosis

Objective： To explore the role of matrix metalloproteinase-1,2 （MMP-1, MMP-2） and tissue inhibitor of matrix metalloproteinases-1 （TIMP-1） in endometriosis. Methods： The eutopic and ectopic endometria from 40 subjects suffering from endometriosis and regular.endometria from 40 subjects （excluding endometriosis） were collected and examined by in situ hybridization technology and western blot assay. Results： Both expressions of MMP-1 and -2 were stronger in ectopic endometrium and eutopic endometrium than in normal endometrium. On the contrary, the expression of TIMP-1 in ectopic endometrium and eutopic endometrium was lower. The differences were significant （P 〈 0.01 ）. Moreover, there was no relationship among the expressions of MMP-1, 2 and TIMP-1 in ectopic endometrium. Conclusion： The expressions of MMP-1, 2 and TIMP-1 lose balance and lack of periodic changes in ectopic endometrium , which explains the biological invasive behavior of endometriosis. It was suggested-that regulating the balance between the MMPs and TIMP-1 should be an ideal therapeutic target to endometriosis.     

Shi Da-zhuo (史大卓) —An outstanding cardiologist of integrative medicine

Prof. SHI Da-zhuo, Ph.D., male, was born on March 20, 1960. Prof. SHI entered the Ph.D. program in 1990 at the China Academy of Chinese Medical Sciences under the supervision of Prof. CHEN Ke-ji, majoring in the treatment of cardiovascular diseases. After receiving his Ph.D. degree in 1993, Prof. SHI started working at the Cardiovascular Center in Xiyuan Hospital affiliated to China Academy of Chinese Medical sciences.     

Persist in and Carry Forward the Integrative Medical Research——Speech at the 6th National General Congress of Chinese Association of Integrative Medicine by Academician CHEN Zhu,Minister of Ministry of Health

The 6th National General Congress of Chinese Association of Integrative Medicine （CALM） was convened at 19-20, April 2008 in Beijing. Academician CHEN Zhu, the minister of Ministry of Health indicated at the congress that the integration of Chinese and Western medicine is very well in keeping with the situation of our country and the general rule of development in medical science; and as a good integration of Chinese medicine and Western medicine, it is mutually beneficial and advantageous to both of them. Seeing the creativity shown in integrative medical investigation in theoretic and methodological sides, we should and must persist in and develop it.