AGR2调控IFITM3的表达促进肝癌细胞的增殖

目的:探讨重组人前梯度同源蛋白2(anterior gradient homolog 2,AGR2)的表达对肝癌细胞增殖能力的影响及其可能的作用机制.方法:通过实时荧光定量PCR、Western blot、免疫组织化学检测40例肝癌患者癌组织和对应癌旁组织中AGR2基因、干扰素诱导跨膜蛋白3(interferon-induced transmembrane protein 3,IFITM3)的表达情况,构建AGR2的过表达质粒pcDNA3.1-AGR2,并将其转染至肝癌细胞中,运用实时荧光定量PCR和蛋白质印迹法分别检测AGR2 mRNA和蛋白的表达,CCK-8(cell counting kit 8)法检测细胞的增殖能力,利用流式细胞技术检测细胞凋亡,蛋白质印迹法检测IFITM3的表达.结果:在原发性肝癌组织中AGR2,IFITM3呈现高表达;成功构建AGR2的过表达质粒pcDNA3.1-AGR2,成功构建稳定高表达AGR2的肝癌HepG2细胞.pcDNA3.1-AGR2转染组HepG2细胞中AGR2 mRNA和蛋白的表达水平明显高于阴性对照组和空白对照组.与阴性对照组比较,pcDNA3.1-AGR2转染组HepG2细胞的体外增殖能力和克隆形成能力明显升高,IFITM3蛋白的表达水平也随之升高.结论:上调AGR2可以增加IFITM3的表达,促进肝癌细胞的增殖.     

丙型肝炎病毒1b基因型核心蛋白对HepG2细胞Bax与Bcl-2基因表达的影响

目的研究丙型肝炎病毒（HCV）1b基因型核心蛋白（C）对HepG2细胞B细胞淋巴瘤-2基因（Bcl-2）与Bcl-2相关X蛋白（Bax）表达的影响,以探索1b型HCV C蛋白与HepG2细胞凋亡的关系。方法利用RT-PCR扩增出HCV-1b-C基因,经双酶切后连接pcDNA3.1（-）,成功构建真核表达载体pcDNA3.1（-）/HCV-1b-C。利用脂质体转染HepG2细胞,RT-PCR及Western Blot检测其mRNA及蛋白的表达,RT-PCR及Western Blot检测转染成功后HCV-1b-C对HepG2细胞Bax与Bcl-2表达的影响,并设转染空质粒组及未处理组作对照。结果成功构建真核表达载体pcDNA3.1（-）/HCV-1b-C;瞬时转染HepG2细胞,成功表达HCV C mRNA及蛋白;转染C基因组的Bax的mRNA及蛋白相对表达量减少,与转染空质粒组及未处理组比较差异均有统计学意义（P〈0.01）;转染C基因组的Bcl-2的mRNA及蛋白相对表达量增多,与转染空质粒组及未处理组比较差异均有统计学意义（P〈0.01）。结论 1b基因型HCV C蛋白转染HepG2细胞会导致Bax表达减少及Bcl-2表达增多,降低Bax/Bcl-2比值,可能是抑制HepG2细胞凋亡的机制之一。     

重组质粒pcDNA3.1/SurP/Trail联合吉西他滨诱导胰腺癌细胞凋亡的实验研究

目的研究重组质粒pc DNA3.1/Sur P/Trail联合吉西他滨诱导胰腺癌细胞凋亡的能力。方法将人胰腺癌细胞株SW1990分为4组,第1组转染重组质粒联合吉西他滨(联合组),第2组单纯转染重组质粒(重组质粒组),第3组单纯加入吉西他滨(吉西他滨组),第4组未加任何处理(空白对照组)。通过Western blotting检测各组细胞中Trail蛋白表达的情况,并用流式细胞仪检测SW1990胰腺癌细胞凋亡率。结果 (1)重组质粒组、联合组均有Trail蛋白表达,吉西他滨组、空白对照组则无Trail蛋白表达;(2)联合组SW1990细胞凋亡率显著高于其他各组(P0.05);重组质粒组和吉西他滨组SW1990细胞凋亡率显著高于空白对照组(P0.05);重组质粒组与吉西他滨组比较,差异无统计学意义(P0.05)。结论重组质粒pc DNA3.1/Sur P/Trail与吉西他滨联可显著提高SW1990胰腺癌细胞的凋亡率。     

Survivin基因在牛蒡子苷元增强人肺癌H460细胞化疗敏感性中的作用

目的 研究Survivin基因在牛蒡子苷元促进肺癌细胞化疗敏感性中的作用.方法 应用RT-PCR技术从H460细胞中扩增全长人Survivin cDNA,并将其克隆至pcDNA3.1(+)表达载体,构建人Survivin基因真核表达质粒.将pcDNA3.1-Survivin重组质粒或空载体分别转染H460细胞,孵育24 h后,应用Western blot法检测Survivin过表达情况;转染细胞与牛蒡子苷元单独或联合顺铂处理,应用Annexin-Ⅴ/PI染色法检测细胞凋亡情况.结果 与未转染和转染空载体质粒的H460细胞相比,转染pcDNA3.1-Survivin重组质粒H460细胞Survivin水平显著增加,提高大约10倍.低剂量顺铂对H460细胞Survivin蛋白表达影响不显著,而牛蒡子苷元可显著抑制Survivin蛋白表达水平.当牛蒡子苷元与顺铂联合作用时,Survivin蛋白表达抑制程度更大.与转染空载体质粒相比,转染Survivin表达重组质粒的细胞对牛蒡子苷元单独或联合顺铂诱导的凋亡具有抗性,细胞凋亡比率显著下降.结论 牛蒡子苷元抑制人肺癌细胞中Survivin基因表达.牛蒡子苷元促人肺癌细胞顺铂敏感性与抑制Survivin通路有关.     

LIGHT、IFN-γ基因转染人肝癌HepG2细胞凋亡及其Caspase-3和survivin表达

目的 观察LIGHT、IFN-γ基因转染人肝癌细胞HepG2的凋亡及其Caspaae-3和survivin的表达变化. 方法 用LIGHT、IFN-γ真核细胞表达质粒pcDNA4C-LIGHT-cDNA、pcDNA4C-IFN-γ-cDNA及其转化的E.coli JM-109感受态大肠杆菌,经质粒提取后,转染人肝癌细胞HepG2,同时设对照组(未转染).分别于转染后12、24、48 h收集HepG2细胞.流式细胞仪检测细胞凋亡和Caspase-3、survivin. 结果 转染后细胞凋亡及Caspase-3表达量在对照组、LIGHT单转组、联合转染组呈逐渐递增趋势,survivin表达量呈递减趋势. 结论 LIGHT转染后能通过调节HepG2细胞Caspase-3及survivin的表达来发挥促细胞凋亡作用,INF-γ能增强LIGHT诱导的HepG2细胞凋亡,且显著上调其Caspase-3的表达.     

丙型肝炎病毒核心区蛋白对 HepG2细胞周期、细胞凋亡和细胞端粒酶活性的影响

目的　研究丙型肝炎病毒核心区 (HCV C)蛋白对肝癌细胞HepG2细胞周期、细胞凋亡和细胞端粒酶活性的影响。方法　首先运用基因重组技术构建含有HCV C基因的真核表达质粒pcDNA3.1( ) ,然后利用脂质体介导将重组真核表达质粒转染HepG2 ,经G4 18筛选获得稳定转染HepG2细胞 (HCV C转染HepG2细胞 ) ,经逆转录 聚合酶链反应技术 (RT PCR)和间接免疫荧光法证实其中有HCV C蛋白表达。然后进行如下实验 :( 1)利用四甲基偶氮唑蓝比色 (MTT)法检测HCV C转染HepG2细胞、空白质粒转染HepG2细胞和未转染HepG2细胞的生长增殖率 ;经流式细胞术(FACS)检测 3组细胞的细胞周期 ;( 2 )经流式细胞术检测细胞凋亡率 ;( 3)经端粒重复扩增 酶联免疫吸附试验 (TRAP ELISA)法检测上述 3组细胞端粒酶活性表达情况。结果　 ( 1)HCV C转染HepG2细胞增殖率显著高于空白质粒转染HepG2和未转染HepG2细胞增殖率 ;HCV C转染HepG2细胞S期所占百分率高于未转染HepG2细胞S期所占百分率 ;( 2 )HCV C转染HepG2细胞凋亡率显著低于无HCV C转染细胞凋亡率 ;( 3)上述 3组细胞端粒酶活性之间差异无显著性。结论　 ( 1)HCV C蛋白具有抑制细胞凋亡的作用 ;( 2 )HCV C蛋白促进HepG2从G0 /1期进入S期 ,从而可能促进细胞生长增殖 ,抑制细胞凋亡 ;( 3)HCV     

HCV核心蛋白与截短型HBV表面抗原中蛋白的协同反式激活功能

目的 探讨HCV核心蛋白与截短型HBV表面抗原中蛋白的协同反式激活作用。方法 构建表达HCV核心蛋白的重组质粒pcDNA3.1(-)-core和表达截短型HBV表面抗原中蛋白的重组质粒pcDNA3.1(-)-Mt；转染HepG2细胞，从转录和翻译水平鉴定病毒基因的瞬时表达；与报告质粒pSV-lacZ共转染HepG2细胞，检测β－半乳糖苷酶（β-gal)表达活性，酶的活性反映了表达的肝炎病毒蛋白对SV40病毒早期启动子（增强子）功能的影响。结果 构建成HCV核心蛋白及截短型HBV表面抗原中蛋白的重组表达载体pcDNA3.1(-)-core、pcDNA3.1(-)-Mt；在HepG2细胞均能瞬时表达相应的病毒蛋白；单独的共转染实验中pcDNA3.1(-)-core、pcDNA3.1(-)-Mt组的β-gal的表达分别是对照的4．6和3．2倍，两种质粒共同转染时酶的表达是对照组的8．4倍；表达质粒对β－gal表达的激活作用呈剂量依赖性。结论 HepG2细胞中表达的HCV核心蛋白和截短型HBV表面抗原中蛋白均具有反式激活SV40早期启动子（增强子）的功能，并且两种蛋白的反式激活功能具有协同特性。本实验有助于解释HCV、HBV感染，尤其是共同感染的致病（癌）机制。     

缺氧诱导因子1α对肝癌细胞HepG2干细胞特性及表阿霉素敏感性的影响

目的探讨缺氧诱导因子(HIF)1α对肝癌细胞HepG2干细胞特性及表阿霉素敏感性的影响。方法以肝癌细胞为研究对象,肝癌细胞HepG2脂质体转染过表达HIF-1α的质粒作为实验组,转染pcDNA3.1空质粒作为对照组,单独HepG2细胞为HepG2组。实时荧光定量PCR检测HIF-1αmRNA表达,Western Blot检测HIF-1α蛋白表达;流式细胞术检测细胞表面CD133表达。不同浓度表阿霉素(0、6.25、12.5、25、50μmol/L)作用3组细胞24 h,MTT法检测细胞活性,流式细胞术检测表阿霉素(50μmol/L)处理后细胞凋亡情况。计量资料多组间比较采用单因素方差分析,进一步两两比较采用t检验。结果相较于HepG2组及对照组,实验组HIF-1αmRNA的表达水平明显升高,差异具有统计学意义(P值均<0.001);Western Blot结果显示实验组HIF-1α蛋白高表达。HepG2组、对照组和实验组细胞的CD133比例分别为0.040%±0.003%、0.030%±0.010%、20.110%±0.600%,实验组的CD133阳性率显著高于HepG2组和对照组(P值均<0.001)。表阿霉素浓度为25、50μmol/L时,HepG2组和对照组的细胞活性明显受到抑制,显著低于实验组(P值均<0.05)。50μmol/L表阿霉素作用48 h后,实验组的细胞凋亡率(67.9%±2.5%)较HepG2组(93.6%±1.5%)和对照组(93.0%±1.2%)明显降低(P值均<0.001)。结论过表达HIF-1α的质粒成功转染至HepG2细胞,HIF-1α可提高肝癌细胞干细胞比例使其对表阿霉素耐药。     

HBC通过NEU1调控肝癌细胞中基因表达的初步研究

目的探讨乙肝病毒核心蛋白(hepatitis B virus C protein,HBC)对唾液酸酶1(neuraminidase 1,NEU1)及相关基因表达的影响。方法通过脂质体将pcDNA3.1和pcDNA3.1-HBC质粒分别转染HepG2细胞和Huh7细胞,采用Quantitative real-time-PCR(q-PCR)及Western blot检测NEU1的表达;PCR扩增NEU1基因并与pcDNA3.1载体质粒连接,构建NEU1过表达质粒pcDNA3.1-NEU1;将pcDNA3.1-NEU1和对照质粒分别转染HepG2细胞,收集细胞,进行转录组测序,获得差异表达基因,用q-PCR对差异表达基因进行验证;将pcDNA3.1-HBC和对照质粒转入肝癌细胞,q-PCR检测HBC对NEU1相关的差异表达基因的影响;在HBC阳性肝癌细胞,利用NEU1的小干扰质粒抑制其表达,q-PCR检测HBC是否通过NEU1调控相关基因的表达。结果与对照组肝癌细胞相比,转染HBC质粒的肝癌细胞NEU1表达显著上调;PCR鉴定pcDNA3.1-NEU1插入片段正确,重组质粒构建成功;转录组测序显示,与对照组比较,过表达NEU1的HepG2细胞有8个基因表达显著不同,其中6种基因表达上调,2种基因表达下调;q-PCR检测转录组测序获得的差异表达基因与转录组测序的结果一致;与对照肝癌细胞相比,NEU1相关的上调差异表达基因在HBC阳性肝癌细胞中高表达,且干扰NEU1表达后以上基因在HBC阳性肝癌细胞中表达下调;与NEU1相关的下调差异表达基因在HBC基因组中低表达,且干扰NEU1表达后相关基因在HBC阳性肝癌细胞中表达上调。结论HBC能够通过NEU1调控肝癌细胞中相关基因的表达。     

乙型肝炎病毒X基因下调miR-192对人肝癌细胞株HepG2凋亡的影响

目的 探讨乙型肝炎病毒X基因(HBx)通过调节人肝癌细胞株HepG2中miR-192的表达而抑制其凋亡的机制.方法 设立3个细胞组:稳定转染HBx基因的HepG2细胞(HepG2/HBx),稳定转染空载体pcDNA3.1的HepG2细胞(HepG2/pcDNA3.1)以及未作转染的HepG2细胞.用流式细胞术分析3个细胞组的凋亡率差异,用Taqman探针荧光定量PCR检测3组细胞中miR-192的表达水平.转染miR-192后,用流式细胞术检测HepG2细胞凋亡率的变化,同时用SYBR Green荧光定量PCR和Western blot检测细胞中p53、PUMA表达的变化.计量资料均数的比较用单因素方差分析.结果 HepG2/HBx细胞的凋亡率为2.37％±0.35％,较HepG2/pcDNA3.1、HepG2细胞(11.46％±0.69％、12.50％±0.66％)明显降低(F=171.722,P＜0.01).miR-192表达在HepG2/HBx细胞中为49.1％±5.9％,较HepG2/pcDNA3.1、HepG2细胞(98.0％±8.9％,100％)也明显下调(F=14.319,P＜ 0.05).转染miR-192后HepG2细胞的凋亡率(15.74％±1.17％)较转染相应阴性对照的HepG2细胞的凋亡率(10.74％±1.15％)显著升高(F=18.415,P＜0.05),同时,p53、PUMA基因在mRNA (953:1.68±0.12比0.90±0.09,F=43.115,P＜0.05 ; PUMA:1.66±0.10比0.98±0.06,F=22.541,P＜0.05)和蛋白质水平(p53:3.07比1,PUMA:2.13比1)的表达均显著上升.结论 miR-192促进HepG2细胞凋亡,HBx通过下调miR-192抑制HepG2细胞凋亡.     

Trousseau Syndrome in a 25-Year-Old Woman with Occult Colon Malignancy,Lynch Syndrome,and Chronic Thromboembolic Pulmonary Hypertension

We present a rare case of thrombosis associated with an occult colon malignancy (Trousseau syndrome) in a 25-year-old woman who also presented with previously unidentified Lynch syndrome and acute-on-chronic thromboembolic pulmonary hypertension. Staged treatment included bilateral pulmonary endarterectomy under deep hypothermic circulatory arrest, followed 11 days later by laparoscopic subtotal colectomy and creation of a primary anastomosis. The patient tolerated both procedures well and recovered normal functional status. Final pathologic analysis of the resected colon mass revealed a pT3N0, stage IIA adenocarcinoma; no adjuvant therapy was administered. At her one-year follow-up visit, the patient was cancer-free, remained on lifelong apixaban anticoagulation, and was undergoing routine monitoring and genetic counseling. This case highlights the need for multidisciplinary management of a patient with severe chronic thromboembolic pulmonary hypertension and a concomitant malignancy.     

Large Lipomatous Hypertrophy of the Interventricular Septum

We present the case of a 58-year-old woman who had large lipomatous hypertrophy of the interventricular septum, a condition that is reported very infrequently. Preoperative cardiac magnetic resonance images revealed an inhomogeneous, infiltrating mass that was suppressed in fat-suppression mode. The extensive mass was causing right ventricular dysfunction, so we excised it through a right ventricular approach. The findings on histologic analysis of the mass were consistent with lipomatous hypertrophy. The patient died of septic shock on the 28th postoperative day. In addition to the patient''s case, we discuss the characteristics and diagnosis of this rare entity.     

Conservative Management of Cardiac Hemangioma for 11 Years

Cardiac hemangiomas are benign tumors with an unpredictable natural history. Surgical resection is the treatment of choice; however, conservative management can be an alternative in some patients.We report a case of a left-sided cardiac hemangioma that we managed conservatively for 11 years without obvious major complications in the patient, an adult woman.     

肝硬变腹水患者钾钠氯及酸碱失衡

目的研究肝硬变腹水患者的钾、钠、氯及酸碱失衡。方法肝硬变腹水患者１５４例，血Ｋ＋、Ｎａ＋、Ｃｌ－测定采用ＥＥＬ公司自动分析仪及火焰光度计。血气及酸碱度测定采用ＩＬ１３０２型自动微机分析仪。结果低血钾者５７例，高血钾者６例，低血钠者８１例，高血钠者１２例，低血氯者３４例，高血氯者８例。２４例血气及酸碱度测定结果显示，以碱中毒者为主。依次为呼碱、呼碱＋代酸、代酸、代碱、呼酸。本组高血钾、低血钠与Ｃｈｉｌｄ分级、ＢＵＮ、Ｃｒ值相关。从本组资料表明，重症肝硬变腹水患者水盐代谢失衡多为医源性所致，且加重原有失衡。高钾血症、急性低钠血症及高钠血症大多如此，多为住院后发生，常可危及生命。结论肝硬变腹水患者的高钾血症、低钠血症和高钠血症大多在肝肾功能低下，不适当的治疗所致，是影响预后的重要因素     

Resolution of Right-Sided Heart Failure Symptoms after Resection of a Primary Ovarian Carcinoid Tumor

Carcinoid tumors are rare neuroendocrine malignancies that typically originate from the gastrointestinal tract. Patients who are diagnosed with carcinoid heart disease generally have poor prognoses because of advanced metastases during staging and few therapeutic options. We present the case of a 61-year-old woman with right-sided heart failure, secondary to carcinoid heart disease caused by a primary ovarian carcinoid tumor. After undergoing surgical resection of the left ovary and fallopian tube, the patient experienced complete resolution of her heart failure symptoms. In addition to the patient''s case, we discuss the diagnosis, nature, and treatment of this rare condition.     

Prompt Recognition of Left Ventricular Free-Wall Rupture Aided by the Use of Contrast Echocardiography

In the modern period of reperfusion, left ventricular free-wall rupture occurs in less than 1% of myocardial infarctions. Typically, acute left ventricular free-wall rupture leads to sudden death from immediate cardiac tamponade. We present the case of a 59-year-old woman who sustained a posterior-wall myocardial infarction and subsequent cardiac arrest with pulseless electrical activity. A bedside transthoracic echocardiogram showed pericardial effusion with cardiac tamponade. Emergency pericardiocentesis yielded 500 mL of blood, and spontaneous circulation returned. Contrast-enhanced echocardiograms revealed inferolateral akinesis and a new, small myocardial slit with systolic extrusion of contrast medium, consistent with left ventricular free-wall rupture. During immediate open-heart surgery, a small hole in an area of necrotic tissue was discovered and repaired. This case highlights the usefulness of bedside contrast-enhanced echocardiography in confirming acute left ventricular free-wall rupture and enabling rapid surgical treatment.     

Cardiac Sarcoidosis Presenting as Constrictive Pericarditis

In patients with cardiac sarcoidosis, the sarcoid granulomas usually involve the myocardium or endocardium. The disease typically presents as heart failure with ventricular arrhythmias, conduction disturbances, or both. Constrictive pericarditis has rarely been described in patients with sarcoidosis: we found only 2 reports of this association.We report the case of a 57-year-old man who presented with clinical and hemodynamic features of constrictive pericarditis, of unclear cause. He was admitted for treatment of recurrent pleural effusion. After a complicated hospital course, he underwent pericardiectomy. His clinical and hemodynamic conditions improved substantially, and he was discharged from the hospital in good condition. The pathologic findings, the patient''s clinical course, and his response to pericardiectomy led to our diagnosis of cardiac sarcoidosis presenting as constrictive pericarditis. In addition to the patient''s case, we discuss the nature and diagnostic challenges of cardiac sarcoidosis. Increased awareness of this disease is necessary for its early detection, appropriate management, and potential cure.     

Benefits and Limitations of Multimodality Imaging in the Diagnosis of a Primary Cardiac Lymphoma

Primary cardiac tumors are far rarer than tumors metastatic to the heart. Angiosarcoma is the primary cardiac neoplasm most frequently detected; lymphomas constitute only 1% of primary cardiac tumors.We present the case of a 55-year-old woman with a recently diagnosed intracardiac mass who was referred to our institution for consideration of urgent orthotopic heart transplantation. Initial images suggested an angiosarcoma; however, a biopsy specimen of the mass was diagnostic for diffuse large B-cell lymphoma. The patient underwent chemotherapy rather than surgery, and she was asymptomatic 34 months later.We use our patient''s case to discuss the benefits and limitations of multiple imaging methods in the evaluation of cardiac masses. Certain features revealed by computed tomography, cardiac magnetic resonance, and positron emission tomography can suggest a diagnosis of angiosarcoma rather than lymphoma. Cardiac magnetic resonance and positron emission tomography enable reliable distinction between benign and malignant tumors; however, the characteristics of different malignant tumors can overlap. Despite the great usefulness of multiple imaging methods for timely diagnosis, defining the extent of spread and the hemodynamic impact, and monitoring responses to treatment, we think that biopsy analysis is still warranted in order to obtain a correct histologic diagnosis in cases of suspected malignant cardiac tumors.     

Acute Coronary Thrombosis and Multiple Coronary Aneurysms in a 22-Year-Old Man with the Human Immunodeficiency Virus

The human immunodeficiency virus (HIV) can cause diverse cardiovascular complications. In HIV patients on antiretroviral therapy, the prevalence of myocardial infarction has steadily increased over the years. Young patients who are naïve to antiretroviral therapy and who experience coronary events are not well represented in the medical literature. We describe the case of a 22-year-old man, infected with HIV for 4 years and never treated with antiretroviral therapy, who emergently presented with a non-ST-segment-elevation myocardial infarction. Coronary angiograms revealed thrombosis and multiple coronary artery aneurysms; however, no areas of atherosclerotic stenosis were apparent. He was successfully treated with coronary stenting, antiplatelet therapy, and anticoagulation. Nine months after the initial presentation, he exhibited excellent exercise capacity, and no ischemia was evident. We discuss the various therapeutic approaches in this case.