自噬在葛根素促成骨细胞分化调节中的作用

目的探讨自噬在葛根素调节h FOB1.19人成骨细胞分化过程中的作用。方法将成骨细胞分为A组(成骨分化诱导组)、B组(A组+10^-8 mol/L葛根素3 h)和C组[B组+5 mmol/L自噬抑制剂3-甲基嘌呤(3-MA) 1 h]。采用碱性磷酸酶(ALP)和茜素红染色检测成骨细胞的分化及钙化;透射电镜检测成骨细胞内自噬小体的改变情况;免疫荧光染色检测自噬微管相关蛋白轻链3-Ⅱ(LC3-Ⅱ)在细胞内的分布情况;Western blotting检测成骨细胞内成骨分化和自噬相关蛋白的表达情况。结果 ALP和茜素红染色显示,葛根素(10-8 mol/L)能够明显促进成骨细胞的分化,加入3-MA后葛根素的促成骨细胞分化作用受到抑制;葛根素促进成骨细胞自噬小体增加,3-MA抑制自噬小体增加;免疫荧光显示葛根素促进LC3-Ⅱ在细胞核表面的聚集,加入3-MA后,这种现象被抑制;葛根素促进成骨分化蛋白矮小相关转录因子2 (RUNX 2)、骨保护素(OPG)和自噬相关蛋白LC3-Ⅱ、BECLIN 1的表达,3-MA抑制LC3-Ⅱ和BECLIN 1的表达。结论葛根素通过自噬调节成骨细...     

大黄素对人类风湿关节炎成纤维样滑膜细胞自噬的影响

目的 基于cGAS/STING信号通路研究大黄素对人类风湿关节炎成纤维样滑膜细胞(MH7A)自噬的潜在作用。方法 采用CCK-8法检测MH7A细胞增殖的结果,并根据细胞存活率筛选出药物的浓度,并加入自噬抑制剂3-MA进一步验证大黄素对自噬的影响;采用MDC法检测MH7A细胞自噬情况;采用蛋白免疫印迹法(Western blot)检测cGAS、STING、p-STING、LC3-I、LC3-II、P62和Beclin-1的蛋白表达水平。结果 MDC染色结果提示,大黄素能够增强MH7A细胞自噬;Western blot结果提示,大黄素可降低MH7A细胞自噬相关蛋白cGAS、STING、p-STING和P62的表达,增加LC3-II和Beclin-1的表达。加入自噬抑制剂3-MA后,MH7A细胞P62蛋白表达升高,LC3-II和Beclin-1蛋白表达降低。结论 大黄素可能通过下调cGAS/STING信号通路加速自噬,抑制MH7A细胞增殖。     

3- 甲基腺嘌呤对 EPCs 超微结构和 LC3-II蛋白表达的影响研究

目的 探索3-甲基腺嘌呤(3-MA)对大鼠骨髓源性内皮祖细胞(EPCs)超微结构和自噬体膜型(LC3-II)蛋白表达的 影响。方法 采用密度梯度法取大鼠骨髓EPCs,体外诱导分化并鉴定;分成对照组和4个3-MA浓度组(分别加入1.25、2.5、5、 10mmol/L 3-MA) 。采用单丹(磺)酰戊二胺(MDC)荧光染色和透射电镜观察 3-MA 给药后 EPCs 自噬的发生。Western blot 检测 3-MA给药后EPCs内LC3-II蛋白表达的变化。结果 MDC荧光染色和透射电镜均观察到5、10mmol/L组EPCs超微结构明显 区别于正常及死亡细胞,是典型的凋亡形态图。Western blot 检测 3-MA 给药 24h 后 EPCs LC3-II 蛋白表达降低;与对照组比较,5、 10mmol/L组LC3-II蛋白表达明显降低(P<0.05)。结论 5、10mmol/L的3-MA对EPCs超微结构和LC3-II蛋白表达均有影响, 推测浓度≥5mmol/L 的 3-MA 能抑制 EPCs 的自噬。     

吴茱萸碱联合自噬抑制剂氯喹抗肝癌作用的研究

目的 研究吴茱萸碱(EVO)联合自噬抑制剂氯喹(CQ)的抗肝癌作用.方法 用CCK8法检测EVO及EVO联合CQ对HepG2细胞活性的影响,Western blot法检测微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)/微管相关蛋白1轻链3-Ⅰ(LC3-Ⅰ)(LC3-Ⅱ/LC3-Ⅰ)、P62、血管内皮生长因子(VEGFA)蛋白表...     

自噬对AngⅡ诱导的心肌成纤维细胞增殖及迁移的影响及机制

目的 观察自噬对血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导的新生大鼠心肌成纤维细胞(cardiac fibroblats,CFs)增殖及迁移的影响,并探讨其作用机制.方法 消化法培养新生大鼠CFs,AngⅡ诱导新生大鼠CFs增殖,MTT比色法、细胞划痕实验分别检测细胞增殖及迁移情况;Western blot法检测微管相关蛋白轻链3(LC3)、beclin-1及DGKξ蛋白的表达;DGKξ shRNA慢病毒感染CFs下调DGKξ蛋白水平,观察其对CFs自噬的影响.结果 AngⅡ10-7 mol·L-1明显促进CFs增殖并增加自噬相关蛋白LC3及beclin-1的表达;自噬阻断剂3-甲基腺嘌呤(3-MA,5 mmoL/L)及氯喹(CQ,5tmol/L)可逆转AngⅡ诱导的CFs增殖和迁移;AngⅡ诱导CFs增殖的同时DGKξ蛋白表达明显下调;DGKξ下调增加AngⅡ诱导的自噬相关蛋白LC3及beclin-1表达.结论 AngⅡ通过激活自噬诱导CFs增殖及迁移,其作用机制可能与DGKξ蛋白下降有关.     

竹节香附素A调控mTOR通路对肠癌HCT116细胞自噬及凋亡的影响

目的 探讨竹节香附素A(RA)抑制HCT116细胞的增殖及其相关机制.方法 利用四甲基偶氮唑蓝(MTT)检测RA对HCT116细胞的增殖抑制;流式细胞术(FCM)检测RA对细胞凋亡的影响;透射电镜观察RA诱导自噬;Western blot检测凋亡、自噬相关蛋白表达.结果 RA能够明显抑制HCT116细胞的增殖,并呈浓度、时间依赖性.透射电镜观察到双层膜自噬体的存在;FCM显示RA能够诱导HCT116细胞凋亡,且随着浓度的增大凋亡率增加.Western blot检测显示,自噬相关蛋白Beclin-1、微管相关蛋白1轻链3(LC3)表达增高;抑制凋亡蛋白Bcl-2表达减少;而促凋亡蛋白Bax,凋亡相关蛋白Cleaved-caspase-3、Cleaved-聚二磷酸腺苷核糖多聚酶(PARP)表达增加;RAPA靶蛋白(mTOR)信号通路中mTOR蛋白表达增加,磷酸化mTOR(p-mTOR)蛋白表达减少.加入mTOR通路抑制剂雷帕霉素(RAPA)后Beclin-1、LC3蛋白表达增加,凋亡率增加,且Cleaved-caspase-3、Cleaved-PARP表达也增加.结论 RA能够抑制肠癌细胞HCT116的增殖;并能诱导细胞自噬和凋亡,其机制可能是通过调控mTOR信号通路实现.     

左旋棉酚通过细胞自噬下调Namalwa细胞中B淋巴细胞刺激因子的表达

目的探讨左旋棉酚[(-)-gossypol]下调Namalwa细胞中B淋巴细胞刺激因子(B lymphocyte stimulator,BLyS)的可能机制。方法采用CCK8、台盼蓝活细胞染色检测左旋棉酚在体外对Namalwa细胞的生长抑制作用,吖啶橙(acridine orange,AO)染色观察经左旋棉酚处理后细胞自噬的形态特征,Western blot检测细胞中BLyS和自噬蛋白微管蛋白轻链3(LC3)的表达情况。结果左旋棉酚对Namalwa细胞有明显的增殖抑制作用,并且具有时间和剂量依赖性;AO染色后细胞内可观察到明显的细胞自噬小体;Western blot结果显示,左旋棉酚可下调Namalwa细胞中BLyS的表达,并上调LC3Ⅱ蛋白的表达;自噬特异性抑制剂3-methyladenine(3-MA)可显著抑制左旋棉酚对BLyS的下调与LC3Ⅱ的上调作用。结论左旋棉酚可能通过诱导细胞自噬来下调B淋巴细胞中BLyS的表达,从而抑制细胞生长。     

苦参碱对食管癌Eca-109细胞自噬的影响及作用机制

目的 探讨苦参碱对食管癌Eca-109细胞自噬的影响及其可能的作用机制。方法 将传代培养的Eca-109细胞分为对照组、低浓度苦参碱组、中浓度苦参碱组和高浓度苦参碱组。对照组细胞加入含体积分数10%胎牛血清的RPMI 1640培养基，低、中、高浓度苦参碱组细胞分别加入终质量浓度为1.0、1.5、2.0 g·L^-1的苦参碱溶液；采用四甲基偶氮唑盐法检测各组细胞的活性，免疫荧光细胞化学染色法检测各组细胞中自噬相关蛋白微管相关蛋白轻链3(LC3)的定位及表达，Western blot法检测各组细胞中多聚蛋白62(P62)、LC3蛋白的相对表达量，实时荧光定量聚合酶链式反应法检测各组细胞中Beclin1 mRNA的相对表达量，透射电子显微镜观察对照组和高浓度苦参碱组Eca-109细胞的超微结构。另取传代培养的Eca-109细胞，将细胞分为对照组、自噬抑制剂组、苦参碱组和自噬抑制剂+苦参碱组。对照组细胞加入5 mL含体积分数10%胎牛血清的RPMI 1640培养基，自噬抑制剂组细胞加入终浓度为7μmol·L^-1的3-甲基腺嘌呤(3-MA)溶液，苦参碱组...     

白藜芦醇通过上调SIRT1/自噬通路改善Aβ25-35对PC12细胞增殖的抑制作用

目的 探讨SIRT1/自噬通路在白藜芦醇改善Aβ25-35诱导的PC12细胞增殖活力下降中的作用.方法 用不同浓度(1、5、10、20 μmol/L)白藜芦醇(resveratrol,RSV)单独或加入5 mmol/L自噬特异抑制剂(3-methyladenine,3-MA)、2μmol/L SIRT1选择性抑制剂(EX527)处理PC12细胞2h后,再加入30μmol/L淀粉样蛋白25-35片段(Amyloid β-protein fragment 25-35,Aβ25-35)处理24 h.采用CCK-8检测细胞增殖活力,电镜检测自噬体,Western blot检测自噬标志蛋白P62、LC3和SIRT1蛋白的表达水平.结果 30 μmol/L Aβ25-35可显著降低PC12细胞的增殖活力,为对照组的45.14％(P＜0.05).RSV预处理可显著改善Aβ25-35诱导的PC12细胞增殖活力下降(P＜0.05),且随浓度增加,保护作用增强.同时,RSV可明显促进自噬体的形成,上调SIRT1、LC3-Ⅱ的表达和降低P62的表达,且随浓度增加,表达变化越显著;而加入3-MA和EX527处理能显著抑制RSV的上述效应.结论 RSV可通过激活SIRT1/自噬通路改善Aβ25-35诱导的神经细胞毒性.     

钙蛋白酶在TOCP诱导人神经母细胞瘤SH-SY5Y细胞自噬中的调节作用

目的探究钙蛋白酶对三邻甲苯基磷酸酯(TOCP)诱导的人神经母细胞瘤SH-SY5Y细胞自噬的调节作用。 方法不同浓度TOCP处理未分化和分化的SH-SY5Y细胞,用荧光分光光度法检测钙蛋白酶活性。抑制钙蛋白酶活性后,通过Western blotting检测TOCP对自噬相关蛋白的影响；抑制钙通道后,用荧光分光光度法检测胞质钙离子浓度对钙蛋白酶活性的影响。 结果TOCP诱导未分化和分化的SH-SY5Y细胞钙蛋白酶活性上升；钙蛋白酶抑制剂可降低TOCP诱导的自噬相关蛋白LC3-II和Beclin1表达。维拉帕米和2-APB阻断钙通道后钙蛋白酶活性降低,维拉帕米而非2-APB可以抑制TOCP诱导的自噬相关蛋白LC3-II表达。 结论TOCP在未分化和分化的SH-SY5Y细胞中诱导的自噬受钙蛋白酶活性的调节。     

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.     

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.     

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.     

Dynamic Changes in Serum Estradiol and Progesterone levels in Patients of Premenstrual Syndrome with Adverse Flow of Liver-qi

Objective:To probe into the influence of changes of ovarian hormones on the pathogenesis of the specific sub-type premenstrual syndrome(PMS)and reveal partial microcosmic mechanisms of adverse flow of liver-qi.Methods:Estradiol(E2)and progesterone(P)levels in serum were determined at different phases of menstrual cycle by radioimmunoassay.Results:In the group of PMS with adverse flow of liver-qi.the secretive peak value Of E2 and P at the follicular phase significantly decreased,and the secretive peak value at the luteal phase did not come into being.Conclusions:Low E2 and P secretive peak at the follicular phase and absence of secretive peak at the luteal phase is one of the microcosmic mechanisms of PMS with adverse flow of liver-qi.One of the pathophysiologic mechanisms of specific sub-type PMS is probably the continuous low level of E2and P.     

Real-time Three-dimensional Echocardiography in Assessment of Left Ventricular and Right Ventricular Volumes

Real-time three-dimensional echocardiography (RT3DE)is a new ultrasound technique that enables dynamic threedimensional visualization and quantification of the heart in real time. Investigation of feasibility and methodology of RT3DE in determining left ventricular (LV) and right ventricular (RV) volumes, RT3DE was performed in 35 normal adults using Philips SONOS 7500 system with a 2-4 MHz matrix array transducer. The 60°×60° "pyramid" volume database was obtained and analyzed on a TomTec echo workstation. Both LV and RV volumes were calculated with four 3DE methods (i.e. apical 2, 4, 8, and 16-plane) through manually tracing ventricular endocardial borders in end diastole and end systole. Stroke volumes were then calculated. LV volume was also measured by 2DE Simpson's rule using GE VIVID 7 ultrasound machine.     

SCREENING FOR A 21-CHROMOSOME ABNORMALITY IN PREIMPLANTED EMBRYOS OF ELDERLY WOMEN

Increasing maternal age is the only etiological factor unequivocally linked to Down's syndrome in humans. The occurrence rate of newborns with Down's syndrome is about 1/220 in women over 35 years old. However, the occurrence rate in embryos fertilized in vitro, of the elder woman is unclear. Using FISH we screened the number of chromosome 21 in preimplanted embryos of 5 elderly women (average age, 38.4 years) to study the feasibility and necessity of screening trisomy 21 in embryos in patients over 35 years old at the in vitro fertilization (IVF) center.