MiR-21通过PTEN/PI3K/AKT信号通路抑制胰腺癌细胞系生长侵袭能力的体外研究

目的 探讨MiR-21对胰腺癌细胞生长和侵袭的影响.方法 采用脂质体Lipofectamine 2 000将MiR-21 inhibitor和MiR-21 inhibitor阴性对照转染至胰腺癌细胞ASPC-1,qRT-PCR检测转染效果,细胞免疫荧光、Western blot检测转染后目的蛋白的表达,Transwell、划痕实验检测细胞迁移侵袭能力,MTT法检测细胞的增殖活性,流式细胞术检测细胞周期分布的改变.结果 成功构建稳定下调MiR-21的胰腺癌细胞亚系ASPC-1-MiR-21,荧光定量PCR结果显示MiR-21表达量明显下调,抑制MiR-21表达后ASPC细胞的侵袭和增殖能力均受到明显抑制;抑制MiR-21能明显增强PTEN蛋白和减低p-AKT蛋白表达水平,对AKT蛋白水平无明显影响.结论 抑制MiR-21表达可能通过调控PTEN/AKT通路抑制胰腺癌细胞AS-PC-1增殖和侵袭.     

MiR-29b下调肝肿瘤细胞Mcl-1的表达并诱导其发生凋亡

目的:探讨miR-29b(microRNA-29b)的作用靶点及其对肝肿瘤细胞系Huh7的凋亡诱导作用。方法:荧光定量PCR检测正常肝细胞系LO2及肝癌细胞系Huh7的miR-29b和Mcl-1的表达水平。通过生物信息学分析预测Mcl-1基因是否受microRNA调控。将miR-29b模拟物通过Lipofectamine 2000顺时转染Huh7细胞,检测miR-29b对Mcl-1表达水平的影响。MTT法检测miR-29b模拟物转染对Huh7细胞增殖的影响;Annexin V/PI染色法检测miR-29b对Huh7细胞凋亡的影响。结果：肝肿瘤细胞相比于正常肝细胞高表达Mcl-1低表达miR-29b,在肝癌细胞中转染miR-29b可使Mcl-1的表达水平下降,转染miR-29b可抑制Huh7细胞的增殖并诱导其发生凋亡。结论：MiR-29b下调Huh7细胞Mcl-1蛋白的表达并诱导其发生凋亡。     

慢病毒介导的TSPY1对肝癌SMMC7721和Huh7细胞系增殖的影响

目的：观察转染睾丸特异性蛋白1（ TSPY1）慢病毒表达载体对肝癌细胞SMMC7721和Huh7细胞系增殖能力的影响。方法构建特异性的TSPY1慢病毒过表达载体稳定转染至SMMC7721和Huh7肝癌细胞系,采用荧光定量PCR和蛋白印迹技术检测TSPY1 mRNA和蛋白的表达情况,Cell Counting Kit-8（CCK-8）实验检测转染后细胞的增殖情况。结果在转染过表达TSPY1慢病毒的SMMC7721和 Huh7细胞中, TSPY1 mRNA和蛋白的表达均明显上调（ P＜0.01）。 CCK-8细胞增殖实验发现,过表达TSPY1组SMMC7721和Huh7细胞的增殖能力比对照组增强。结论 TSPY1慢病毒表达载体可明显上调TSPY1 mRNA和蛋白表达,并能够促进肝癌SMMC7721和Huh7细胞的增殖。     

miR-202通过降低肝癌细胞ROCK1表达抑制其迁移和侵袭

目的：研究微小RNA-202（miR-202）对肝癌细胞迁移和侵袭能力的影响,并探讨其可能的分子机制。方法：通过实时定量PCR检测Hep3B、Huh7及HCCLM3人肝癌细胞和LO2人正常肝细胞中miR-202的表达水平。以miR-202表达水平最低的HCCLM3细胞为转染对象,转染miR-202模拟物或空白对照miRNA,以miR-202表达水平最高的Hep3B细胞为转染对象,转染miR-202抑制物或阴性对照miRNA,并利用实时定量PCR验证转染效率。采用Transwell迁移和侵袭实验检测过表达和敲低miR-202对肝癌细胞迁移和侵袭能力的影响。生物信息学网站检索并应用荧光素酶报告基因实验验证miR-202的靶基因。实时定量PCR和Western blot检测miR-202靶基因的表达水平。结果：与正常肝细胞LO2相比,miR-202在肝癌细胞的表达水平显著降低;HCCLM3细胞转染miR-202模拟物后,细胞miR-202的表达水平显著增加;过表达miR-202后,HCCLM3细胞的迁移和侵袭能力显著降低;Hep3B细胞转染miR-202抑制物后,细胞miR-202的表达水平显著降低;敲低miR-202后,Hep3B细胞的迁移和侵袭能力显著增强;生物信息学检索及荧光素酶报告基因证明ROCK1基因为miR-202的靶基因。过表达miR-202可明显降低肝癌细胞中ROCK1基因的表达,而敲低miR-202可明显增加肝癌细胞中ROCK1基因的表达。结论：miR-202在肝癌细胞中低表达,miR-202通过抑制ROCK1的表达抑制肝癌细胞的迁移和侵袭。     

LINC00665通过let-7i/HMGA1促进肝癌细胞增殖与侵袭的机制研究

目的 探讨LINC00665通过let-7i/HMGA1对肝癌细胞增殖与侵袭能力的影响。方法 通过qRT-PCR分析LINC00665在肝癌组织中的表达情况。选择人肝癌细胞Hep3B和Huh7细胞，分别将其分为siRNA-NC组、siRNA-LINC00665组、let-7i mimics-NC组、let-7i mimics组进行转染；采用qRT-PCR和Western blot检测各组的HMGA1 mRNA和蛋白水平；CCK8法检测各组细胞的增殖活力；划痕实验检测各组细胞的划痕愈合率；Transwell实验检测各组细胞的侵袭数量。结果 LINC00665在肝癌组织中的表达量显著高于正常组织。hep3B和Huh7细胞转染48 h后，与si-NC组相比，si-LINC00665组细胞的增殖、迁移和侵袭能力下降，HMGA1的mRNA和蛋白水平下降，let-7i的表达水平升高；与let-7i mimics-NC组相比，let-7i mimics组细胞的增殖、迁移和侵袭能力下降，HMGA1的mRNA和蛋白水平下降。结论 LINC00665通过let-7i/HMGA1途径，在肝癌进展中调控肿瘤增...     

长期索拉菲尼暴露促进Huh7肝癌细胞上皮间质变的实验研究

目的探讨索拉菲尼耐药肝癌细胞发生上皮间质变(EMT)及侵袭、转移能力增强的机制。方法通过药物连续诱导人肝癌细胞株Huh7建立索拉菲尼耐药肝癌细胞株Huh7R,显微镜下观察细胞形态学变化;CCK-8细胞增殖试验检测Huh7R细胞增殖能力;荧光定量PCR检测ABC家族耐药基因ABCB1、ABCC1、ABCG2及EMT相关调控锌指蛋白转录因子Snail、Slug基因表达水平;Westernblot检测耐药蛋白ABCG2,EMT标记分子E-cadherin、Vimentin及N-cadherin,转录因子Snail、Slug蛋白表达水平;Transwell小室侵袭试验检测Huh7R细胞迁移、侵袭能力。结果Huh7R细胞呈细长形,伴纤维状突起,形态学上符合EMT改变;CCK-8细胞增殖试验显示在索拉菲尼作用下,Huh7R细胞生存率高于Huh7细胞;荧光定量PCR结果显示,Huh7R细胞ABCB1、ABCC1、ABCG2及Snail、Slug基因表达水平均高于Huh7细胞（均P＜0.05）;Westernblot显示,Huh7R细胞上皮标记蛋白E-cadherin表达水平低于Huh7细胞（P＜0.05）,而间质标记蛋白Vimenti、N-cadherin及Snail、Slug蛋白表达水平均高于Huh7细胞（均P＜0.05）;Transwell小室侵袭试验显示Huh7R细胞迁移、侵袭能力均较Huh7细胞增强。结论长期低剂量索拉菲尼暴露会促进肝癌细胞Snail和Slug表达,诱导肿瘤细胞发生EMT,增强其侵袭、转移能力。     

miR-30c减轻肝细胞肝癌的侵袭与转移

目的:观察miR-30c/snail在肝细胞肝癌(hepatocellular carcinoma,HCC)中的表达并探讨其在肝癌侵袭中的作用?方法:定量PCR检测肝癌及肝癌细胞系中miR-30c/snail的mRNA表达水平,通过免疫组织化学和Western blot分别检测肝癌及肝癌细胞系中snail的蛋白表达水平,并在肝癌细胞株中转染miR-30c模拟物或抑制物,分析其对肝癌细胞侵袭的作用?结果:与无脉管转移组相比,miR-30c在有脉管转移组的HCC组织中表达明显下调,同时在高转移肝癌细胞株MHCC-97H中也有相似的结果?转染miR-30c模拟物可减少snail的表达,并减少MHCC-97H细胞的侵袭性,然而,转染miR-30c抑制剂则增加snail的蛋白水平,增加HepG2细胞的侵袭力?结论:上调miR-30c的表达通过直接抑制snail,可减弱HCC的侵袭性?一种新的针对miR-30c的治疗策略可能有利于伴侵袭转移的HCC患者?     

WWP2沉默对肝癌细胞系黏附、侵袭和迁移的影响

目的 探讨WWP的E3泛素连接酶2(ww domain-containing protein 2,WWP2)沉默对肝癌细胞系黏附、侵袭和迁移的影响作用。方法 通过RNA沉默技术降低肝癌细胞系的WWP2水平,用Transwell法检测细胞迁移和侵袭,CCK-8法检测细胞的增殖情况,Western blot法检测caspase7、caspase8、Bcl-2、Bax、PTEN、p-Akt及Akt的蛋白水平。结果 沉默WWP2后,肝癌细胞BEL-7404和Huh7的WWP2mRNA 和蛋白的表达水平明显下降(P＜0.001),BEL-7404细胞和Huh7细胞的凋亡相关标志物caspase7、caspase8及Bax的蛋白水平表达都显著升高(P＜0.01)、PTEN的蛋白水平升高(P＜0.01),Bcl-2、p-Akt的蛋白水平显著下降(P＜0.01),肝癌细胞BEL-7404和Huh7的细胞活力、黏附能力、侵袭能力和迁移能力都明显减弱(P＜0.001)。结论 沉默WWP2可抑制肝癌BEL-7404细胞和Huh7细胞增殖、黏附、侵袭和迁移,并促进细胞凋亡,其作用机制可能与PTEN/Akt信号通路相关。     

lncRNAENST00000484679促进肝癌索拉菲尼耐药细胞迁移侵袭的机制研究

目的探讨差异表达的长链非编码RNA（lncRNA）对肝癌索拉菲尼耐药细胞迁移、侵袭的影响。方法采用ln-cRNA芯片微阵列分析肝癌索拉菲尼耐药细胞Huh7SR和亲本细胞Huh7中的lncRNA,筛选出差异表达lncRNA;荧光定量PCR法进一步验证差异表达的lncRNA。将转染后的肝癌索拉菲尼耐药细胞Huh7SR分为ENST00000484679-小干扰RNA（siRNA）组和阴性对照（siNC）组。ENST00000484679-siRNA组加入lncRNAENST00000484679-siRNA转染,siNC组加入无关序列NC-siRNA转染。Transwell实验观察肝癌索拉菲尼耐药细胞株迁移侵袭能力的变化;Westernblot法检测肝癌索拉菲尼耐药细胞株的上皮-间质转化（EMT）标记蛋白钙黏蛋白N（N-cadherin）、钙黏蛋白E（E-cadherin）、波形蛋白（Vimentin）表达水平。结果芯片筛选出lncRNAENST00000484679在肝癌索拉菲尼耐药细胞株中高表达,荧光定量PCR进一步验证了Huh7SR细胞ENST00000484679mRNA表达水平高于Huh7细胞（P＜0.01）。ENST00000484679-siRNA组ENST00000484679mRNA表达水平低于siNC组,迁移细胞数和侵袭细胞数均低于siNC组,N-cadherin、Vimentin蛋白表达水平均低于siNC组,E-cadherin蛋白表达水平高于siNC组,差异均有统计学意义（均P＜0.05）。结论lncRNAENST00000484679促进肝癌索拉菲尼耐药细胞的迁移和侵袭能力可能与EMT有关。     

银椴苷通过调控 miR-218/NEDD9轴影响肝癌细胞 增殖、迁移、侵袭和凋亡

摘要:目的 探讨银椴苷对肝癌细胞生物学行为的影响和分子机制。方法 采用不同浓度(0.1、0.2、0.4、0.8、1.6 mmol/L)的银椴苷分别处理肝癌细胞 Huh748h,通过四甲基偶氮唑蓝(MTT)法、流式细胞术、Transwell实验检测细胞 活力、凋亡率以及迁移、侵袭能力。实时荧光定量 PCR(qRT-PCR)检测 miR-218和神经元细胞表达发育下调基因 9 (NEDD9)mRNA 的表达水平,蛋白质印迹(Westernblot)检测 NEDD9 蛋白表达水平。双荧光素酶报告基因实验和 Westernblot验证 miR-218和 NEDD9的靶向调控关系。将 miR-218抑制物(anti-miR-218)转染 Huh7细胞,检测干扰 miR-218表达联合银椴苷处理对 Huh7细胞活力、凋亡、迁移和侵袭的影响。结果 与空白组比较,银椴苷处理组 Huh7 细胞活力、迁移和侵袭数、NEDD9mRNA 和蛋白表达显著降低,凋亡率、miR-218表达显著升高(均 P<0.05)。miR-218 可与 NEDD9直接结合。过表达 miR-218后 NEDD9表达水平降低(均 P<0.05),干扰 miR-218后 NEDD9表达水平升 高(均P<0.05)。干扰 miR-218表达可逆转银椴苷对 Huh7细胞增殖、迁移侵袭、凋亡的影响(均 P<0.05)。结论 银 椴苷可诱导肝癌细胞凋亡,抑制其增殖、迁移和侵袭,其机制可能是通过上调 miR-218/NEDD9轴实现的。     

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…     

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.     

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.     

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.     

猪肺磷脂注射液联合经鼻持续气道正压通气对呼吸衰竭早产儿的临床疗效及肌酸激酶同工酶活性的影响

目的 探讨猪肺磷脂注射液联合经鼻持续气道正压通气(NCPAP)对呼吸衰竭早产儿的临床疗效及肌酸激酶同工酶活性(CK-MB)的影响.方法 选取呼吸衰竭早产儿80例,分为观察组和对照组各40例.对照组采用NCPAP给氧治疗,观察组给予NCPAP给氧联合猪肺磷脂气管内给药.观察两组患儿治疗前及治疗12h、24 h后PaO2、PaCO2、血氧饱和度(SaO2)、pH的变化情况,检测治疗前及治疗5d后血清CK-MB水平;评估两组患儿的临床治疗效果.结果 两组患儿PaO2、PaCO2、SaO2、pH比较,差异均有统计学意义(P＜0.05),其中观察组治疗后的PaO2、SaO2、pH均高于对照组,PaCO2则低于对照组.两组的PaO2、SaO2、pH均随观察时间延长而升高(P＜0.05),PaCO2均随观察时间的延长而降低(P＜0.05).观察组治疗有效率为87.5％,显著高于对照组的70.0％ (P ＜0.05).治疗5d后两组患儿血清CK-MB水平均较前降低(P＜0.05),且观察组明显低于对照组(P＜0.05).结论 猪肺磷脂注射液气管内给药联合NCPAP可以显著降低呼吸衰竭早产儿CK-MB的含量,提高治疗有效率,起到很好的呼吸循环支持作用.