浅低温体外循环手术前后基因表达研究中内参基因的选择

目的筛选浅低温体外循环（cardiopulmonary bypass,CPB）手术前后基因表达变化研究的内参基因。方法通过阅读文献选择基因表达研究中常用的11个管家基因（housekeeping genes,HKGs）,采用Beacon Designer软件设计PCR引物;收集4例浅低温CPB辅助手术患者CPB前、体温最低点、CPB末以及术后1d、3d、7d的静脉血24份,提取总RNA,采用SYBR Green I实时荧光定量RT—PCR法检测各样品中11个HKGs的循环阈值;对所得循环阈值进行转换,采用geNorm程序对候选基因的表达稳定性和所需内参基因数量进行分析。结果11个HKGs稳定性排序为：UBC〈RPLP0〈PGK1〈18S—rRNA〈PPIA〈GAPDH〈ACTB〈TBP〈B2M〈EEF1A1／HPRT1;该研究需要的最佳内参基因数量为3个,即EEF1A1、HPRT1和B2M。结论确定了浅低温CPB手术前后11个HKGs中表达稳定性高的EEF1A1、HPRT1和B2M等3个基因为该条件下基因表达研究的内参基因。     

利用优化的显微技术观察和定位Merm1/Wbscr22在人肺癌细胞NCI-H1299中的表达

目的 优化细胞免疫荧光染色技术结合激光扫描共聚焦显微镜技术的实验步骤,观察并定位Merm 1/Wbscr22在人肺癌细胞NCI-H1299中的表达,以期对后续深入研究Merm1/Wbscr22的分子功能提供技术支持.方法 采用Western blot 及细胞免疫荧光染色结合激光扫描共聚焦显微镜技术观察Merm1/Wbscr22在NCI-H1299细胞的表达与定位,改进并优化免疫荧光染色实验步骤.结果 Merml/Wbscr22蛋白在NCI-H1299细胞内过量表达,并定位于细胞核内.细胞免疫染色步骤显著地影响Texas Red-鬼笔环肽的非特异性染色及F-actin、Merm1/Wbscr22、细胞核DNA染色荧光的强度.结论 结合细胞免疫荧光染色技术与激光扫描共聚焦显微镜技术可以清楚地观察Merm1/Wbscr22在人肺癌细胞NCI-H1299的表达与定位,该技术的最优实验步骤是细胞固定、透膜、封闭、Texas Red-鬼笔环肽标记F-actin、一抗结合、二抗结合、DAPI标记细胞核DNA,最后共聚焦显微镜成像,这样既能减少Texas Red-鬼笔环肽的非特异性染色,各组分荧光强度又足够清晰利于观察.     

不同月经阶段子宫内膜内参基因的稳定性分析

目的筛选子宫内膜不同时期基因表达研究中稳定的内参基因。方法招募月经周期规律的20～40岁育龄期妇女,分别于黄体期（11人）和增殖期（9人）抽吸入选者的子宫内膜,比较这两个阶段候选内参基因ATP5B、B2M、CYC1、RPL13A、SDHA、TOP1、YWHAZ、18SrRNA、ACTB、GAPDH的稳定性,选择最稳定的基因作为合适的内参基因。结果CYC1和RPL13A是增殖期内膜最稳定的内参基因,而YWHAZ和ACTB则是黄体期内膜最合适的内参基因。结论确定不同时期子宫内膜的内参基因为研究其基因表达和相关功能奠定了基础。     

小鼠基因转录表达分析中内参基因的优选

目的 建立小鼠基因转录表达分析中内参基因的选择方法.方法 以C57BL/6J和C3H/HeJ两个品系3个不同组织及2个不同发育阶段为研究对象,应用反转录实时定量PCR技术,评价GAPDH(glyceraldehyde-3-phosphate dehydrogenase)、HPRTl(hypoxanthine phosphoribosyl transferase)、B2M(β2-microglobulin)、PPIA(peptidylprolyl isomerase A)、ACTB(Actin-beta)和18S rRNA(18S ribosomal RNA)等6个看家基因在下丘脑、垂体与卵巢中mRNA水平的表达稳定性.结果 GeNorm统计分析表明,GAPDH和HPRT1表达最为稳定,PPIA等次之,B2M在不同组织和发育阶段中都几乎无表达.结论 成功筛选到GAPDH和HPRT1两个稳定表达的看家基因,证实了小鼠基因表达转录分析中内参基因选择的必要性和可行性.     

穿心莲实时定量PCR分析中内参基因的选择

[目的]选择合适的内参基因用于穿心莲实时定量PCR分析中的校正.[方法]以穿心莲根、茎、幼叶、花、萌发种子和成熟叶片为实验材料,应用实时定量PCR技术分析18S rRNA、甘油醛-3-磷酸脱氢酶(GAPDH)、肌动蛋白(Actin)和泛素连接酶(UBC)4个常用内参基因在穿心莲不同组织中的表达水平,利用GeNorm和NormFinder软件进行数据分析.[结果]4个候选内参基因在穿心莲不同组织中表达的稳定性各异,其中UBC的表达水平最为稳定.[结论]可以选择UBC作为内参基因,用于穿心莲不同组织中基因表达水平的校正.     

乙肝相关肝癌组织实时定量RT-PCR分析中内参基因的选择

目的 实时定量RT-PCR已被广泛应用于基因的表达分析.选择合适的内参基因可以消除不同标本在RNA的产量、质量以及逆转录效率上可能存在的差别,从而获得目标基因特异性表达的真正差异.方法 本研究应用实时定量PCR技术,研究了HMBS、C-TBP、HPRT、UBC和18s rRNA 5个内参基因在男性乙肝相关肝癌组织中的表达情况.结果 结果 表明,这5个内参基因表达存在差异.结论 经过geNorm程序统计学分析,确定了HMBS和C-TBP两个看家基因适用于校正目标基因的表达量,为研究男性乙肝相关肝癌组织中目标基因的表达奠定基础.     

肝癌实时定量RT-PCR中适宜内参基因的选择

目的 探计肝癌(HCC)组织及细胞系实时定量RT-PCR最佳的内参基因选择.方法 通过实时定量RT-PCR,检测HMBS、CTBP1、HPRT1、UBC、B2M、SDHA、RPLI3A、EIF4A1、TUBB、YWHAZ、ACTB、GUSB、GAPDH、18 s rRNA和28 s rRNA 15个内参基因在70例HCC、癌旁肝组织及阿法替尼作用后4个HCC细胞系的表达情况.并使用NormFinder及geNorm软件分析最优化的内参基因.结果 15个内参基因表达存在差异,Cq值在8～29.2间.GAPDH在HCC及癌旁肝组织表达差异有显著性(P＜0.05).结论 NormFinder及geNorm软件均显示HMBS+UBC组合为肝组织的最佳内参,HMBS+ RPLI3A组合为体外肝癌细胞系的最适宜内参.     

阳春砂实时荧光定量PCR内参基因的筛选

【目的】筛选稳定表达的内参基因,用于阳春砂不同发育时期的果皮和种子团中基因表达量实时荧光定量PCR(qRTPCR)检测的校正。【方法】以阳春砂3个不同发育时期的果实(分为果皮和种子团)为材料,根据高通量测序得到的转录组和表达谱数据,选择5个表达稳定的常用内参基因β-actin、EF-1α、GAPDH、PGK和TUA作为候选基因,并利用qPCR技术检测它们在不同样品中表达水平的变化,使用GeNorm和NormFinder软件对基因的稳定性进行分析。【结果】5个内参基因在不同发育时期的果皮和种子团中的表达稳定性有明显的差异,其中GeNorm分析得到的内参基因的稳定顺序为:EF-1α=TUAPGKGAPDHβ-actin,NormFinder的分析结果中稳定性最好的是EF-1α,其次为TUA,其他3个内参基因稳定性的排列顺序与GeNorm软件的结果一致。【结论】可选用EF-1α和TUA作为阳春砂果实发育过程中基因表达量差异分析的双内参基因。     

实时荧光定量PCR和RT-PCR检测宫颈癌细胞株HPV-16 E6和E7基因的实验研究

目的 探讨实时荧光定量PCR和RT-PCR检测HPV-16 E6和E7基因的实验方法.方法 将制备的含HPV-16 E6和E7基因的质粒作为标准品,建立实时荧光定量PCR和RT-PCR方法,分别对3种宫颈癌细胞株进行HPV-16 E6和E7基因的DNA和RNA拷贝数的定量检测.结果 建立的实时荧光定量PCR标准曲线相关系数大于0.99,PCR效率在90%以上.HPV-16 E6和E7基因的DNA和RNA拷贝数在宫颈癌细胞株 CaSki、SiHa和HeLa中从高到低依次是CaSki＞SiHa＞HeLa.结论 用实时荧光定量PCR和RT-PCR方法研究HPV-16 E6和E7基因的DNA和RNA含量结果可靠,为进一步研究两个基因与宫颈病变的关系奠定了基础.     

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…     

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.     

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.     

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.