Ox-LDL对PMA诱导的THP-1巨噬细胞自噬的影响

目的探讨氧化型低密度脂蛋白（Ox-LDL）对PMA诱导的THP-1巨噬细胞自噬的影响。方法 PMA诱导THP-1细胞24 h,使其分化为巨噬细胞,再分别用0、10、20、40或80 mg/L的Ox-LDL处理24 h,逆转录聚合酶链反应（RT-PCR）和免疫印迹技术（Western blotting）分别检测Beclin-1以及LC3的mRNA及蛋白表达;细胞免疫荧光检测LC3在细胞内的含量;MDC染色检测自噬囊泡的形成。结果随着Ox-LDL浓度的增加,THP-1源性巨噬细胞Beclin-1 mRNA及蛋白表达显著降低（P〈0.05）;LC3 mRNA表达无明显改变（P〉0.05）,但蛋白水平LC3II/LC3I显著降低（P〈0.001）;免疫荧光结果表明随着Ox-LDL浓度的增加,LC3II含量降低,MDC染色结果显示自噬囊泡随着Ox-LDL浓度的增加而减少。结论 Ox-LDL抑制PMA诱导的THP-1巨噬细胞自噬。     

不同水平自噬对替莫唑胺抑制神经胶质瘤细胞增殖的影响

目的探讨不同水平自噬对替莫唑胺抑制神经胶质瘤细胞增殖的影响。方法使用0（对照）、10、25、50滋g/L的雷帕霉素（RAPA）预处理人胶质瘤细胞（U87细胞）0.5h;再加入200滋mol/L替莫唑胺作用24h,收集U87细胞。采用流式细胞术检测自噬标志物微管相关轻链蛋白3（LC3）-Ⅱ表达量,RT-PCR法检测自噬相关基因Beclin-1mRNA相对表达量,MTT法检测细胞增殖抑制率。结果对照组、10滋g/L组、25滋g/L组和50滋g/L组U87细胞LC3-Ⅱ表达量、Beclin-1mRNA相对表达量及细胞增殖抑制率比较,差异均有统计学意义（均P＜0.05）;经两两比较发现,随着RAPA浓度的增加,LC3-Ⅱ表达量及Beclin-1mRNA相对表达量均逐渐升高（均P＜0.05）,而细胞增殖抑制率先下降后逐步升高（均P＜0.05）。结论不同水平自噬对替莫唑胺抑制神经胶质瘤细胞增殖产生不同的影响,随着自噬水平的提高,替莫唑胺对神经胶质瘤细胞的增殖抑制率先下降后逐渐升高。     

新藤黄酸抑制人肝癌细胞BEL-7402自噬作用研究

目的 观察新藤黄酸（gambogenic acid,GNA）对人肝癌细胞BEL-7402自噬的影响,为GNA抗肿瘤研究提供理论依据。方法 体外培养人肝癌BEL-7402细胞株,采用MTT法检测GNA对人肝癌细胞BEL-7402存活率的影响;倒置显微镜观察给药前后细胞形态变化;单丹磺酰尸胺（monodansylcadaverine,MDC）染色,荧光显微镜观察细胞自噬小体;透射电子显微镜进一步观察自噬小体;Western blot法检测自噬相关蛋白微管相关蛋白1轻链3（microtubule associated protein 1 light chain 3,LC3）和p62、Beclin1的表达水平及加入自噬诱导剂雷帕霉素（rapamycin,Rap）和自噬抑制剂氯喹（chloroquine,CQ）后自噬相关蛋白的表达情况。结果 GNA能显著抑制人肝癌细胞BEL-7402的增殖并呈时效及量效关系;倒置显微镜观察结果显示GNA组细胞随药物浓度增大,细胞悬浮增多,高浓度给药组细胞出现碎片化;荧光显微镜下观察结果显示,随药物浓度的增加,荧光点状聚集增多;透射电子显微镜观察发现,随GNA浓度的增加,细胞内自噬空泡数量增多,与对照组比较,加入自噬诱导剂Rap后,自噬空泡数量显著增多;Western blot法检测结果显示,随GNA浓度的增加,Beclin1、p62、LC3-Ⅱ蛋白表达水平均增加（P<0.05）,LC3-Ⅰ无明显变化,表明BEL-7402细胞自噬前期被激活,后期进程被抑制。结论 GNA能抑制人肝癌细胞BEL-7402的增殖,其机制可能部分是通过抑制自噬后期自噬体与溶酶体的融合,抑制人肝癌BEL-7402细胞的保护性自噬。     

自噬在子宫肌瘤中作用研究

目的 研究子宫肌瘤细胞中自噬的程度,探讨自噬对子宫肌瘤的影响.方法 随机选取2013年7月至2014年6月该院因子宫肌瘤住院的患者20例,取子宫肌瘤组织和同一病例正常子宫平滑肌.其中各取10例组织消化为细胞,利用单丹黄酰尸胺(MDC)染色后检测荧光度,检测自噬囊泡数量.另取10例组织提取蛋白,利用Western blot检测自噬相关蛋白Ⅱ型微管相关蛋白轻链3(LC3-Ⅱ)、Beclin-1和P62表达水平.结果 肌瘤细胞中自噬囊泡荧光度明显低于正常子宫平滑肌细胞(P<0.05),肌瘤细胞中LC3-Ⅱ、Beclin-1和P62蛋白水平也明显低于正常子宫平滑肌细胞(P<0.05).结论 子宫肌瘤细胞中自噬水平明显降低,自噬抑制可能为子宫肌瘤发生的原因之一.     

红景天苷对97H细胞自噬的影响

目的：探讨红景天苷对人高转移性肝癌细胞株97H自噬的影响。方法：97H细胞经红景天苷处理后,采用透射电镜、MDC染色和吖啶橙AO染色观察自噬现象;采用qRT-PCR方法检测自噬相关基因的mRNA水平;用Western blot方法检测自噬相关蛋白的表达。结果：红景天苷可诱导97H细胞发生自噬,且上调Beclin-1基因mRNA水平,下调p62基因mRNA水平（P<0.05）,上调Beclin-1和LC3-Ⅱ/Ⅰ蛋白的表达,下调p62蛋白的表达（P<0.05）。结论：红景天苷可以诱导97H细胞的自噬作用。     

自噬在白藜芦醇调节胶质瘤干细胞辐射敏感性中的作用

目的研究白藜芦醇联合x射线对胶质瘤干细胞辐射抗性的影响。方法采用甲基四唑蓝（MTT）法检测白藜芦醇和X射线对胶质瘤干细胞增殖的抑制作用;光镜下观察胶质瘤干细胞的形态变化;采用Hoechst荧光染色观察凋亡小体;采用单丹磺酞尸胺（MDC）染色和Westernblot分析自噬在x射线诱导胶质瘤干细胞死亡中的作用。结果MTT检测结果显示,与阴性对照组和单独X射线处理组相比,白藜芦醇联合X射线可明显抑制胶质瘤干细胞生长（均P〈0．05）;在加入自噬抑制剂3-MA后,胶质瘤干细胞抑制率下降（P〈0．05）。显微镜下观察联合处理组的细胞形态,细胞不再成球形生长;Hoeehst33258荧光染色结果显示,联合处理组细胞出现凋亡形态;MDC染色结果显示,联合处理组细胞出现大量自噬泡;Westernblot分析结果显示,联合处理组细胞的Bel一2表达减少（P〈0．01）,LC3Ⅱ／LC3I的比值增大（P〈0．01）。结论白藜芦醇可提高胶质瘤干细胞的辐射敏感性,白藜芦醇联合X射线处理可诱导胶质瘤干细胞发生凋亡和自噬;自噬对于提高胶质瘤干细胞的辐射敏感性起到重要作用。     

羟氯喹逆转慢性髓性白血病细胞K562/IMA对伊马替尼耐药的作用机制研究

目的探讨羟氯喹逆转慢性髓性白血病细胞K562/IMA对伊马替尼（IMA）耐药的作用机制。方法体外培养人慢性髓性白血病细胞K562/IMA（对10滋mol/L的IMA耐药）,将细胞分为对照组（Con组）、IMA干预组（IMA组）、羟氯喹+IMA干预组（HCQ+IMA组）,并在含相关药物的培养基中继续培养。采用CCK-8法检测细胞增殖率的变化,AnnexinV-FITC/PI染色后流式细胞术检测细胞凋亡率,Transwell小室实验检测细胞侵袭能力,免疫荧光法染色检测细胞自噬激活标志蛋白LC3Ⅰ/Ⅱ的表达,MDC染色检测细胞自噬酸性囊泡的产生,Westernblot法检测细胞自噬相关蛋白p62、Beclin-1及凋亡相关蛋白Bax、Bcl-2、Caspase-3、Caspase-9的表达。结果IMA组细胞增殖率明显高于HCQ+IMA组（P＜0.05）,细胞凋亡率明显低于HCQ+IMA组（P＜0.05）。HCQ+IMA组细胞侵袭能力明显低于IMA组（P＜0.05）。HCQ+IMA组细胞LC3Ⅰ/Ⅱ蛋白表达下调,酸性囊泡形成水平降低,与IMA组比较差异均有统计学意义（均P＜0.05）。HCQ+IMA组细胞p62蛋白表达水平高于IMA组（P＜0.05）,而Beclin-1蛋白表达水平低于IMA组（P＜0.05）,同时凋亡蛋白Bax、Caspase-3、Caspase-9的表达水平高于IMA组（均P＜0.05）,抑制凋亡蛋白Bcl-2的表达水平低于IMA组（P＜0.05）。结论羟氯喹可以逆转IMA耐药CMLK562细胞对IMA的敏感性,其作用机制或与抑制细胞自噬有关。     

NF-κB对肝癌细胞自噬形成的影响

目的研究抑制核转录因子（NF—κB）对肝癌细胞HepG2自噬的作用及影响。方法常规培养HepG2细胞后分为两组：SN50处理组及空白对照组,然后采用MTT比色法检测细胞生长增殖、Westernblot检测细胞内微管相关蛋白LC3-Ⅱ水平以及MDC染色后,采用荧光显微镜对自噬进行定性观察。结果经SN50处理24、48、72h后,细胞增殖抑制率分别为（20．45±3．70）％、（31．94±4．20）％和（36．44±4．60）％（P〈0．05）,自噬相关蛋白LC3-Ⅱ水平的表达上调．同时荧光染色也提示肝癌细胞自噬活性增强。结论阻断NF—κB可以在-定时间内显著抑制HepG2细胞的增殖,其机制可能是增强了细胞的自噬作用。     

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。     

鹦鹉热衣原体Ⅲ型分泌蛋白SINC通过激活MAPK/ERK信号通路促进宿主细胞自噬

目的 探讨鹦鹉热衣原体（Cps）SINC蛋白对宿主细胞自噬的影响,及MAPK/ERK信号通路在其中的调控作用。方法 用重组的SINC蛋白刺激小鼠单核巨噬细胞（RAW 264.7）,Western blot检测LC3-Ⅱ和Beclin-1的水平及ERK1/2的磷酸化程度,并用间接免疫荧光法检测SINC蛋白刺激后细胞LC3的水平,透射电镜检测自噬特殊结构。用50μmol MEK1/2抑制剂U0126预处理RAW 264.7细胞后1 h,再用不同浓度SINC蛋白刺激不同时间,用间接免疫荧光检测LC3的水平,并用Western blot检测LC3-Ⅱ和Beclin-1的表达水平。结果 Western blot检测结果显示,以2μg/mL SINC蛋白刺激RAW 264.7细胞12 h时,LC3-Ⅱ和Beclin-1的表达水平显著上调,并达到峰值;间接免疫荧光检测发现SINC刺激后可使细胞内LC3荧光斑点数明显增多,透射电镜下可见更多的自噬小体和自噬溶酶体。2μg/mL SINC蛋白刺激RAW 264.7细胞15 min时p-ERK1/2/ERK1/2比值明显上调;加入抑制剂U0126后,LC3...     

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.