人脐血间充质干细胞的培养条件比较

目的摸索人脐血间充质干细胞（mesenchymal stem cell,MSC）的培养条件。方法根据不同采血量、首次换液时间、胎龄、不同培养基对样本分组，比较不同培养条件对脐血中的间充质干细胞原代生长的影响，以流式细胞仪对培养出的间充质干细胞进行细胞表面标志检测。结果在相同条件下，取10ml的脐血能较大程度培养出间充质干细胞；观察首次换液时间在培养后96h较为合适，延长换液时间有利于数量不占优势的单核细胞充分贴壁：早产胎儿的脐血培养出的间充质干细胞成功率较高；胎牛血清的质和量决定了培养成功与否。培养出的间充质干细胞不表达造血细胞系的标志（CD34、CD45、CD14）及内皮细胞的标志（CD106），强表达CD29、CD44、CD13。结论样本量、首次换液时间、胎龄、培养基的质和量对MSCs的成活、生长有关键作用。     

差速贴壁法纯化分离GFP转基因小鼠骨髓间充质干细胞

目的运用差速贴壁法分离纯化绿色荧光蛋白（GFP）转基因小鼠骨髓问充质干细胞（BMSCs）。方法分离GFP转基因小鼠骨髓细胞直接种植在培养瓶中,分别于2h、4h、6h、8h、10h、12h、14h、16h、9．4h和48h进行首次全量换液,培养3d后按细胞类型对贴壁细胞分别计数,并用CD44、CD45、CD54进行免疫细胞化学染色。另外,选4h、8h、9．4h后换液的细胞进行传代培养,传至第5代,计算扩增倍数,用CD44、CD45、CD54进行免疫细胞化学染色。结果随着首次换液时间的延长,贴壁细胞密度增多,BMSCs的比例减少。首次换液时间在接种后2h的细胞纯度高,但细胞密度低;24h后的细胞密度高而纯度低;8h后的细胞密度大具有较高的纯度。传代后的GFP转基因小鼠BMSCs能稳定表达GFP。结论差速贴壁法能有效分离纯化GFP转基因小鼠BMSCs,首次换液时间在接种后8～10h时的传代细胞纯度高,具有扩增能力,可作为组织工程和基因治疗研究的种子细胞。     

人脐带血间充质干细胞移植对急性心肌梗死大鼠心功能的影响

目的研究脐带血来源的间充质干细胞(mesenchymal stem cells,MSCs)体外分离、纯化及培养的条件,以建立稳定的体外培养体系,满足实验和临床的需要;探讨经尾静脉移植脐血间充质干细胞对心肌梗死大鼠心功能的影响。方法无菌条件下采集健康育龄产妇正常分娩胎儿的脐带血42份,脐血随机分成3组:FBS(胎牛血清)未包被组、FBS包被组和mesencult培养基组。FBS未包被组和FBS包被组均使用含10%FBS的LG-DMEM培养基、mesencult培养基组使用专用来培养干细胞的mesencult培养基,与其添加物配合使用。3组均用Ficoll淋巴细胞分离液,密度梯度法分离脐血单个核细胞(MNC),将单个核细胞按密度1×108/ml接种于T25培养瓶中,置37℃饱和湿度含5%CO2孵育箱培养,72 h后全量换液,去除未贴壁细胞,以后均7 d换液1次。待细胞长到80%铺满瓶底时结束原代培养。按1∶1进行消化传代。观察不同培养条件对脐血MSCs生长的影响。取P2代细胞用流式细胞仪检测细胞表面CD 29、CD 34、CD45、CD 105标志。将36只SD大鼠随机分成MSCs移植组、假手术组和心肌梗死植组各12只,结扎左冠状动脉前降支制备大鼠心肌梗死模型。心肌梗死后1周,经尾静脉注射脐血MSCs,4周后测定大鼠血流动力学。结果 42份脐血中单个核细胞原代培养,其中仅8份传代培养成功,而其中mesencult条件培养基有5份培养成功,明显高于FBS包被组(3份)和FBS未包被组(0份)。流式细胞仪检测第2代的脐血MSCs结果显示,P2代MSCs不表达或极弱表达CD 34,CD45造血细胞标志,稳定地高表达CD 29、CD 105间充质细胞相关的表面抗原标记。这与骨髓MSCs的表面抗原标志相一致。移植后4周,经血流动力学检测,与心梗组比较,MSCs移植组左室心功能明显改善(P0.05)。结论将脐血单个核细胞以高密度(1×108cells/ml)接种在mesencult培养基中可以在体外成功培养出较纯化的脐血MSCs,其培养成功率较高。脐血MSCs的免疫表型符合间充质干细胞特征。脐血MSCs移植能促进心梗大鼠心功能恢复。     

人脐血间充质干细胞体外分离培养与扩增实验研究

目的探讨脐血来源的人间充质干细胞（HMSCs）在体外分离培养与扩增的可行性。方法在无菌条件下收集正常足月胎儿的脐带血,经复合枸橼酸钠抗凝,用相对密度为1．077g／L的Ficoll淋巴细胞分离液分离脐血的单个核细胞,以30％胎牛血清进行培养和扩增,用流式细胞仪检测MSCs的表面标志。结果来源于人脐血的单个核细胞接种于特定培养基后,可产生贴壁细胞,主要表现为破骨样细胞和间充质样细胞,经传几代后,可得纯化扩增的人脐血MSCs。流式细胞仪检测结果显示,人脐血MSCs不表达GD14、CD19,强表达CD105、CD44。结论来源于人脐血的MSCs在体外可以分离培养、扩增,为MSCs的进一步研究奠定基础。     

脐血间充质干细胞肌源性诱导分化的研究

目的研究脐血间充质干细胞(MSC)体外的采集、培养和扩增,探讨MSC肌源性分化的条件和能力.方法选择杂种妊娠犬11只,采集适量脐带血,常规分离后获得单核细胞,分别接种于不同的培养液中进行培养和体外传代扩增.免疫组织化学检测脐血MSC表面抗原的表达;5-氮杂胞嘧啶核苷(5-aza)诱导处理脐血MSC,观察细胞形态结构的变化及肌源性标记物的表达.结果犬脐血MSC在IMDM中生长良好,增殖迅速,每传一代细胞扩增2 ～ 3倍,传至7代后细胞可扩增1.5×102 ～ 2.0×103倍;脐血中包含破骨细胞样和纤维母细胞样贴壁细胞,后者CD29和CD71表达阳性,CD11a、CD11b和CD34表达阴性;脐血MSC在10 μmol/L的5-aza诱导3周后体积延长,与邻近细胞形成连接,并表达肌节性肌动蛋白和结蛋白,与阳性对照结果相似.结论脐血MSC是存在于脐血中的一类不同于造血干细胞的原始细胞,在体外可大量扩增,在诱导剂的作用下向肌源性定向分化.     

血清量及首次换液时间对荧光小鼠骨髓基质细胞体外增殖的影响

目的研究不同量的血清及首次换液时间对荧光小鼠骨髓基质细胞(BMSCs)体外增殖的影响。方法转基因荧光小鼠BMSCs取材后种植于24孔培养板中,分别用含10%、20%、30%胎牛血清的DMEM/F12混合培养基常规培养。于4h、6h、8h、10h、12h、24h、48h、72h首次全量换液,观察其生长情况,用CD44、CD54、CD45抗体对10%、20%胎牛血清传代培养至第5代的细胞进行免疫细胞化学染色。结果BMSCs在含10%、20%胎牛血清的DMEM/F12中生长较好,原代培养的细胞其贴壁细胞数随首次换液时间的延长而增多,但纯度下降。首次换液时间相同而培养液血清含量不同的两组细胞免疫组织化学染色的阳性率差别无显著性(P>0.05),CD44和CD54抗体反应的阳性率随着首次换液时间的延长而逐渐降低,CD45抗体反应的阳性率则随首次换液时间的延长而逐渐升高。结论贴壁分离纯化对转基因荧光小鼠的BMSCs体外培养有效,其体外培养适宜的血清体积分数为0.1～0.2,首次换液时间以8h时为佳。     

人骨髓间质干细胞分离和培养扩增方法的研究

目的 :探索分离、纯化及培养成人骨髓间质干细胞 (hMSC)的最佳方法。方法 :采用Ficoll Paque淋巴细胞分离液和全血接种两种方法分离成人MSC ,筛选体外培养hMSC适合的培养基和适宜的血清含量 ,流式细胞仪检测hMSC表面抗原表达。结果 :经Ficoll Paque淋巴细胞分离液分离后 ,接种于 φ =10 0mL/L胎牛血清的L DMEM培养液 ,细胞贴壁良好 ,增殖速度快 ,hMSC在体外扩增 5代可获得 1× 10 8细胞。全血接种分离hMSC ,细胞贴壁慢、少 ,生长情况欠佳。除L DMEM ,其他类型的培养基均不适合hMSC的培养扩增。流式细胞仪检测结果显示CD2 9、CD44、CD10 5、CD166表达阳性 ,CD14、CD3 3、CD3 4、CD3 8、CD45、CD11a为阴性。结论 :用Ficoll Paque淋巴细胞分离液分离骨髓单核细胞 ,接种于 φ =10 0mL/LFCS的L DMEM培养液 ,分离培养扩增hMSC的效果最好     

脐血间充质干细胞肌源性诱导分化的研究

目的研究脐血间充质干细胞 (MSC)体外的采集、培养和扩增 ,探讨MSC肌源性分化的条件和能力。方法选择杂种妊娠犬 11只 ,采集适量脐带血 ,常规分离后获得单核细胞 ,分别接种于不同的培养液中进行培养和体外传代扩增。免疫组织化学检测脐血MSC表面抗原的表达 ;5 -氮杂胞嘧啶核苷 (5 aza)诱导处理脐血MSC ,观察细胞形态结构的变化及肌源性标记物的表达。结果犬脐血MSC在IMDM中生长良好 ,增殖迅速 ,每传一代细胞扩增 2 ～3倍 ,传至 7代后细胞可扩增 1.5× 10 2～2 .0× 10 3 倍 ;脐血中包含破骨细胞样和纤维母细胞样贴壁细胞 ,后者CD2 9和CD71表达阳性 ,CD11a、CD11b和CD34表达阴性 ;脐血MSC在 10μmol/L的 5 aza诱导 3周后体积延长 ,与邻近细胞形成连接 ,并表达肌节性肌动蛋白和结蛋白 ,与阳性对照结果相似。 结论脐血MSC是存在于脐血中的一类不同于造血干细胞的原始细胞 ,在体外可大量扩增 ,在诱导剂的作用下向肌源性定向分化     

体外分离大鼠骨髓间充质干细胞方法研究

目的确定分离骨髓间充质干细胞 (mesenchymalstemcells,MSCs)过程中的各种条件对获得MSCs群的影响。方法应用不同的分离液、离心温度、速度、培养液及不同品种胎牛血清等条件分离培养细胞并进行对比分析。结果使用 2种分离液 ,在相同离散密度时 ,得到的细胞群相同 ;相同离散密度的 2种分离液 ,不同离心温度、速度及时间 ,如果培养条件相同 ,获得的细胞群也相同 ;用含国产或进口胎牛血清的培养基进行培养所获细胞有明显的不同 ;改良的最小必需培养基 (Dulbecco′sminimumessentialmedium ,DMEM )中添加F12与不添加F12获得的细胞克隆群基本相同。结论无论使用那种分离液 ,只要其离散密度在 10 73g/L均能获得较纯的MSCs;在确定温度、时间、速度等条件下 ,确定基础培养基、选择合适的胎牛血清是取得实验成功的关键。     

人脐血内皮祖细胞体外扩增、鉴定和生物学活性

目的 建立一种稳定的体外分离培养和鉴定人脐血内皮祖细胞(endothelial progenitor cells,EPCs)的方法 ,并探讨利于EPCs增殖的最佳接种浓度.方法 采用密度梯度离心法分离得到单个核细胞,制成1×106/ml、1.5×106/ml、2 × 106/ml、2.5×106/ml、3 × 106/ml、3.5×106/ml、4×106/ml 7个密度接种于铺被人纤维连接蛋白的培养瓶,比较各接种密度的贴壁细胞数和细胞集落形成情况,并采用激光共聚焦显微镜、流式细胞仪和免疫荧光鉴定EPCs.结果 不同接种密度得到的贴壁细胞数和细胞集落数有差别,在2.5×106/ml密度接种时得到的最佳贴壁细胞数和细胞集落数,与其他密度比较差异有统计学意义(P<0.05);双染色阳性细胞为正在分化EPCs;流式细胞仪检测内皮祖细胞对CD34、AC133、VEGFR2的阳性表达率分别为(1.67±0.35)%、(12.63±9.70)%、(66.81±6.63)%;免疫荧光检测内皮祖细胞一致性表达CD34、AC133、VEGFR2;黏附能力和迁移能力检测结果 分别为(33.7±1.6)、(29.2±1.2).结论 采用Ficoll密度梯度离心法结合贴壁筛选法可以较好地分离人脐血EPCs,以2.5×106/ml的密度接种培养可获得良好的细胞分化和增殖.     

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.