乳猪肝细胞的分离、培养及功能检测

目的为构建生物人工肝进行肝细胞的准备.方法采用胶原酶半原位灌流法分离单个乳猪肝细胞,并对其活力及单层和聚集培养后的白蛋白、尿素合成功能进行检测.结果采用本方法从每头乳猪中分离到的单个肝细胞数为(3.1±1.5)×1010,活性超过95%.在加入激素和生长因子的培养基中单层培养时,肝细胞功能良好,可维持2周左右.而在未加入激素和生长因子的培养中肝细胞虽能存活1周,但功能于24 h后即丧失.球形聚集培养可实现肝细胞的大量培养,且生物学活性较单层培养显著提高.结论采用胶原酶半原位灌注法所得单个乳猪肝细胞基本能满足构建生物人工肝对肝细胞数量的要求.聚集培养接种密度大,细胞生物学活性高,可用于构建生物人工肝.     

用于生物人工肝的乳猪肝细胞球形聚集培养

采用胶原酶门静脉灌注法分离乳猪肝细胞 ,旋转振荡法进行乳猪肝细胞的球形聚集培养 ,旨在探索一种新颖的肝细胞培养方法用于生物人工肝。台盼蓝拒染法判定细胞活力 ,同时分别采用光镜、透射电镜和扫描电镜进行培养肝细胞的形态学检测。结果显示 :肝细胞的产量平均为 ( 4 2～ 5 0 )× 1 0 6个 /g肝组织 ,即时存活率达 ( 90± 5 ) % ;旋转振荡法进行乳猪肝细胞的球形聚集培养 ,2 4 h后绝大多数细胞形成了球形聚集体 ,细胞超微结构正常且保持了较高活性。旋转振荡法是一种简便、经济的球形聚集培养肝细胞的方法     

乳猪肝细胞的生物学特性观察

目的 从细胞数量和细胞功能上探讨乳猪肝细胞用于生物人工肝的可行性.方法 采用RPMI1640培养液37 ℃培养分离的乳猪肝细胞,观察乳猪肝细胞生长和增殖特征,检测乳猪肝细胞白蛋白合成、尿素合成、氨清除量、细胞色素P450 (cytochrome P450,CYP)和葡萄糖醛酸基转移酶(UDP-glucuronyl transferases,UGT)活性.结果 在7 d的培养过程中,乳猪肝细胞呈典型的多角形,至少可增殖7倍以上,细胞生长曲线无明显潜伏期;乳猪肝细胞白蛋白和尿素合成量分别在130 ng·10-6cells·h-1和150 μmol·10-6cells·h-1以上,氨清除量和UGT活性在第3天较第1天显著下降(P＜0.05),其后基本维持稳定,CYP活性逐渐下降.结论 体外分离培养的乳猪肝细胞具有较强的增殖能力、合成功能和生物转化功能,能够为生物人工肝提供大量高活性的肝细胞.     

三明治构型大鼠原代肝细胞长期培养及生物学特性的研究

目的观察三明治构型大鼠原代肝细胞长期培养的形态学变化,并对其功能进行测定。方法采用改良原位2步法门静脉胶原酶灌注分离单肝细胞,台盼蓝拒染实验观察细胞活力,利用三明治培养构型培养成年大鼠原代肝细胞,倒置显微镜下连续观察肝细胞的形态学变化,定期收集培养细胞上清液,检测培养肝细胞的分泌及合成功能,并与单层胶原培养肝细胞比较。结果平均每个鼠肝可获取2×108~3×108个肝细胞,活率在(93±3)%;体外肝细胞培养第3天,清蛋白分泌功能、尿素合成能力恢复到最佳状态。三明治构型培养的肝细胞清蛋白分泌功能、尿素合成能力在培养的14d内始终维持较高的水平,并形成肝索样结构,伴胆小管网络形成,肝细胞形态维持达28d以上。结论三明治构型肝细胞培养体系更接近于肝细胞体内生长环境,肝细胞可在较长时间内保持良好的形态结构和功能,三明治构型不仅可以应用于肝细胞的基础研究,而且为肝细胞移植和生物人工肝治疗肝衰竭奠定了一定的基础。     

猪肝细胞在聚砜膜生物界面生长的初步研究

目的初步了解中国实验乳猪肝细胞在聚砜膜生物界面生长的情况,探索聚砜膜作为生物人工肝中空纤维反应器膜材料的可行性.方法将分离的新生实验小型猪肝细胞接种到聚砜膜和聚苯乙烯界面,进行形态学观察及生物学功能测定,观察培养猪肝细胞的酶漏出量.结果乳猪肝细胞在聚砜膜界面培养呈球型黏附、铺展,形成多细胞聚集体.与聚苯乙烯组相比较,聚砜膜组的尿素合成和蛋白分泌能力更强,氨转化率也明显提高,LDH和AST的漏出量无显著差异.结论聚砜膜是良好的乳猪肝细胞生长的生物界面,可以作为生物人工肝中空纤维反应器的膜材料.     

猪肝细胞的分离与原代培养

目的：建立稳定的猪肝细胞的分离与原代培养体系,满足生物人工肝体外支持系统对肝细胞的需求。 方法:应用改良Seglen法进行肝脏原位胶原酶灌流分离猪肝细胞,进行平面培养,动态观察细胞生长过程中的形态结构变化。 结果：平均每只猪可获得2×10¹⁰个肝细胞,细胞活率达到92.5%。在平面培养过程中维持了正常肝细胞的形态。 结论：原位胶原酶灌流法是获得大量高活率的猪肝细胞的首选方法,猪肝细胞原代分离培养是目前解决组合型生物人工肝脏（HBLSS）应用中细胞来源问题的主要途径。     

Ficoll及Percoll分离、纯化金黄地鼠肝细胞方法比较研究

目的建立经济有效的原代地鼠肝细胞分离纯化方法,并比较Ficoll及Percoll两种分离液分离出的肝细胞纯度及活性的差异。方法对传统的两步原位胶原酶灌流法进行简化,行肝脏原位灌流,离体后Ⅳ胶原酶进行消化;采用Ficoll及Percoll两种细胞分离液进行分离、纯化;纯化后于37℃5%CO2培养箱内孵育培养,0.4%台盼蓝拒染实验检测肝细胞的存活率。结果 Ficoll及Per-coll分离的地鼠肝细胞总数分别为（1.46±0.91）cells.g-1肝组织、（1.79±0.89）cells.g-1肝组织,即时分离的肝细胞活力和纯度相近,都大于95%,但后期二者差异较大,Percoll分离得的肝细胞活率较高,且细胞活性下降较缓慢。结论通过简化的门静脉原位灌流法和低浓度胶原酶离体消化,Per-coll或Ficoll分离液纯化,即时分离的肝细胞可保持良好的功能和活力,二者无显著差别,但从细胞培养的要求出发,Percoll优于Ficoll分离液。     

荧光探针Fura—2／AM测定大鼠肝细胞胞内游离钙离子浓度

采用胶原酶原位灌注法分离纯化了大鼠肝细胞，并用荧光探针Ｆｕｒａ／２／ＡＭ测定了细胞内游离钙离浓度度。结果显示，胶原酶原位灌注法可分离获得高纯度，高活率的单个大肝肝细胞，适用用荧光探针Ｆｕｒａ－２／ＡＭ测定胞内游离钙离子浓度；测得新分离大鼠肝细胞静息状态下的细胞内游离子浓度为１７４．５±５７．３ｎｍｏｌ／Ｌ。     

大量分离乳猪肝细胞方法的实验观察

目的:通过两种不同方法大量分离乳猪肝细胞,观察分离肝细胞的活性和功能,探讨猪肝细胞的分离方法。方法:本实验以肝上下腔静脉作为体外乳肝细胞分离的灌注口,用倒置相差显微镜、血细胞计数板计数,台盼蓝拒染试验判断细胞活力,并用自动生化仪进行肝细胞功能的检测。结果:实验组分离的每克肝肝细胞数和肝细胞活性明显高于对照组,两者差异有统计学意义(P<0.05);实验组AST、ALT、LDH以及TBIL浓度低于对照组,但差异无统计学意义(P>0.05)。结论:本实验改进了胶原酶体外两步灌流法分离乳猪肝细胞,为肝细胞的大量分离提供了一定的理论和实验依据。     

双层胶原培养肝细胞

体外培养的肝细胞有多种临床用途,其中将肝细胞置入特殊的生物反应器,构建生物人工肝支持系统有重要的临床价值.但生物人工肝用于临床的前提是必须有大量的健康肝细胞.因双层胶原更接近肝细胞体内环境,故我们于1999年10月至2000年3月,采用双层胶原培养肝细胞,并比较其与传统的单层胶原培养蛋白分泌功能的变化.     

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.