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。     

原代大鼠肝细胞分离方法与冻存条件的优化

目的 :优化原代大鼠肝细胞 (PRH)分离方法与冻存条件。方法 :建立低浓度胶原酶原位循环灌流法分离肝细胞 ;分别采用含 2 %与l0 %DMSO的冻存液于 - 70℃或液氮中冻存PRH ,并比较各组冻存后PRH活力。结果 :1.该分离方法肝细胞产量为 1.5 84± 0 .5 2 5× 10 8/10 0g大鼠体重 ,存活率达 95 .2± 2 .9% ,肝细胞纯度 >95 % ,胶原酶用量减少为 4 .5mg/10 0 g大鼠体重。 2 .含 2 %DMSO冻存液于 - 70℃组与液氮组PRH存活率均可达 90 %～ 95 % ;ALB冻存 14d - 70℃组PRH明显高于液氮组与新鲜分离组 ,LDH值于冻存 7d的 - 70℃组与液氮组明显低于新鲜分离组 ,其它各组间均无显著差异。结论 :低浓度胶原酶原位循环灌流法为高效的PRH分离技术 ;含 2 %DMSO冻存液于 - 70℃可作为原代肝细胞冻存的理想条件。     

人端粒酶逆转录酶诱导的猪肝细胞初步鉴定

目的观察应用四步灌流法分离后原代猪肝细胞的数量和活力,初步鉴定人端粒酶逆转录酶(hTERT)诱导的猪肝细胞的生物学特性。方法四步灌流法分离12只猪肝脏组织中的猪肝细胞,采用锥虫蓝拒染法测定其肝实质细胞数量和活力;将含hTERT真核表达的载体转染到原代猪肝细胞,经G418硫酸盐筛选,获得耐药性的猪肝细胞克隆并传代3代。观察原代猪肝细胞和hTERT诱导后的猪肝细胞的形态,检测两种猪肝细胞不同时期培养上清液中白蛋白、尿素氮的浓度。结果每只肝脏的肝细胞产量约为2.0×1010个,具有活力的肝细胞所占百分比为(95.5±3.2)%。原代猪肝细胞和hTERT诱导的猪肝细胞培养上清中分泌白蛋白和尿素氮在前3天无统计学差异(P>0.05),第4、5天差异有统计学意义(P<0.05)。结论四步灌流法分离获取的猪肝细胞产量高、功能活性好。hTERT诱导的猪肝细胞具有正常肝细胞的基本功能,可作为生物人工肝及细胞移植等的理想的细胞源。     

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     

大鼠肝细胞的分离及BRL细胞培养上清对原代肝细胞增殖的影响

目的 :研究大鼠肝细胞分离的方法及正常大鼠肝细胞来源的BRL细胞培养上清对原代大鼠肝细胞贴壁和增殖的影响。方法 :采用体外胶原酶 2步灌注法分离大鼠肝细胞 ,台盼兰染色法计算细胞数及细胞活率。以四甲基偶氮唑蓝 (MTT)法检测不同浓度的BRL细胞培养上清及含体积分数 15 %胎牛血清的普通培养液对肝细胞贴壁和增殖的影响。结果 :平均每只大鼠可获 2× 10 8个肝细胞 ,平均活力 94 .3%。用普通培养液培养肝细胞 ,肝细胞贴壁率低 ;BRL细胞培养上清能明显促进肝细胞的贴壁和生长 ,且有量效关系 (P <0 .0 5 )。结论 :用本法分离的大鼠肝细胞有较高的获取率和活力 ,BRL细胞培养上清能促进正常大鼠肝细胞的贴壁及增殖     

胶原酶灌注法制备小鼠肝细胞

目的:建立体外培养小鼠肝细胞的实验体系。方法:采用两步原位胶原酶灌注法分离小鼠肝细胞,在RPM I1640培养液中培养。结果:用胶原酶灌注法分离的小鼠肝细胞活率可达90%以上,产量平均为2.8×107,完全满足实验要求。肝细胞在RPM I1640培养液中2 h即可贴壁,48 h内细胞贴壁充分,伸展良好,1周后死细胞增多。结论:胶原酶灌注法成功制备出小鼠肝细胞。     

Ⅱ型胶原酶加压灌流提高成年大鼠心肌细胞分离效率

目的 探讨效率更高的成熟心肌细胞分离方法.方法 采用Ⅱ型胶原酶升主动脉逆行灌流法分离成年大鼠心肌细胞.对照组采用Langendorff装置灌流;实验组在Langendorff装置基础上加压匀速灌流.在倒置显微镜下观察细胞形态,并计算杆状细胞比率及产量;通过台盼蓝染色和局部场刺激评价心肌细胞活性.结果 分离即刻,实验组杆状细胞比率显著高于对照组[(90.3±4.4)%vs.(53.4±5.2)%,P<0.01];实验组活细胞产量也显著高于对照组[(3.6±0.7)x 10<''7>vs.(1.9±0.6)×10<''7>,P<0.01].复钙过程中,实验组发生自发性收缩的细胞比率及变圆细胞比率均明显低于对照组[(10.4±2.1)%vs(18.9±4.5)%,P<0.01;(5.3±1.3)%vs.(8.6±1.7)%,P<0.05].实验组台盼蓝染色阴性的杆状细胞比率和局部场刺激条件下收缩细胞比率明显高于对照组[(95.3±8.2)%vs.(90.6±9.8)%,P<0.05;(92.2±7.6)%vs.(85±6.4)%,P<0.01].结论 Ⅱ型胶原酶加压灌流可获得高产量、高活性的心肌细胞,提高了成熟心肌细胞的分离效率.     

高活力原代小鼠肝细胞的分离与纯化

目的建立一种稳定的能获得高活力和高纯度原代小鼠肝细胞的分离和纯化方法。方法对传统的两步原位胶原酶灌注法进行4个方面的优化,主要包括选择逆向灌流、将持续灌注法改为间断灌注后夹闭门静脉法、严格控制胶原酶消化时间和将分离的肝细胞悬液进行低速Percoll单密度离心纯化。分离后的肝细胞进行体外培养。肝细胞活力和得率用台盼蓝染色法检测。结果新鲜分离的小鼠原代肝细胞活力能稳定达到87%±3%,平均活细胞总数为8×105每克小鼠体重,绝大部分细胞在体外培养2h后贴壁生长。结论优化后的肝细胞分离方法更为稳定、有效,分离到的肝细胞具有高活力的优点。     

长爪沙鼠肝细胞体外分离培养体系的建立

目的：建立长爪沙鼠原代肝细胞分离培养体系。方法：以雄性长爪沙鼠肝细胞为供体,采用组织消化法和Seglen两步灌流法分离肝细胞,以台盼蓝染色检测细胞得率和活率,PAS法鉴定肝细胞,倒置显微镜观察肝细胞形态变化,并使用含有多种细胞因子的培养基维持培养。结果：组织消化法和Seglen两步灌流法平均每只长爪沙鼠肝脏分离获得肝细胞分别为(1.33?.34)?07个、(3.97?.15)?07个,细胞活率分别为(29.4?.05)%、(80.3?.56)%,这两种方法在细胞得率及活率方面存在显著差异。肝细胞内因有大量的糖原颗粒,经PAS法染色后被染成红色。结果表明肝细胞在贴壁后72h内,肝细胞形态发生显著变化。结论：采用胶原酶经肝门静脉灌流分离肝细胞是一种高效获得肝细胞的方法。各种细胞因子有利于维持肝细胞在体外的生长分化,长爪沙鼠原代肝细胞分离培养体系的建立将为肝脏相关疾病研究和防治药物的开发提供技术支持。     

分离活体小鼠肝细胞的改良灌流法

Berry等首先建立了生理条件下用胶原酶灌流肝脏分散肝细胞，获得了成功，后来经过Seglen等发展，使这种胶原酶灌流技术更加完善。虽然Seglen灌流法细胞产量大、存活率高．但需特殊灌流装置、胶原酶耗费多、操作难度大，不易掌握。为建立一种便于各实验室开展简便、廉价的分离肝细胞方法，我们设计了分离小鼠肝细胞改良灌流法。经初步应用，获得了良好效果。     

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…     

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.     

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.     

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.     

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.     

Acupuncture Treatment of Vomiting

CASE HISTORY A female patient, 46 years old, head of the foreign affairs department of a certain university in Beijing, paid her first visit on October 9, 2006, with the chief complaint of vomiting for one month. She got vomiting after meals in early September. Before that, she had discomfortable sensation in the stomach due to angry with others, but she didn't pay much attention. Later, it developed into vomiting after eating. After the vomiting, the discomfort would be relieved, but with slight hypodynamia. She was once diagnosed as having 'neurogenic vomiting'. Having taken some western and Chinese drugs, the above symptoms were a little bit improved, but she would have nausea upon eating and with regurgitation. Because of the fear for vomiting, she did not dare to have food intake, with body weight reduction of 6 kilos in one month.     

Qiu Baoguo''''s Experience in Treating Consumptive Syndromes after Radio-Chemotherapies

Radiotherapy and chemotherapy are the important modern medical therapies for malignant tumors,yet they can also bring about serious local and systemic toxic side reactions so to decrease the patient;'s life quality,manifested by a series of consumptive symptoms.Having engaged in the combined work of Chinese and western medicine for nearly 50 years,the research fellow Qiu Baoguo in Henan Provincial Academy of TCM has developed his unique views on the TCM study of consumptive syndromes.The author of this essay had once the fortune tO follow Dr.Qiu in clinic,and specially would like to introduce in the following Dr.Qiu's experience in treating consumptive syndromes after radio-chemotherapies for patients with malignant tumor.     

Insomnia Due to Deficiency of Both the Heart and Spleen Treated by Acupuncture-Moxibustion and Chinese Tuina

OBJECTIVE: To observe therapeutic effects of the comprehensive therapy of acupuncture-moxibustion and Chinese Tuina for treatment of insomnia due to deficiency of both the heart and spleen. METHODS: 92 cases were divided randomly into the treatment group (treated by acupuncture-moxibustion and Chinese Tuina) and the control group (treated by acupuncture-moxibustion). RESULTS: The therapeutic effect of the treatment group was obviously superior to that of the control group (the CHI2 test showed P < 0.01). CONCLUSIONS: The comprehensive therapy of acupuncture-moxibustion and Chinese Tuina can give marked therapeutic effects for treatment of insomnia due to deficiency of both the heart and spleen.     

Survey and practice of reporting quality of randomized controlled clinical trials on traditional Chinese medicine

Evidence obtained from randomized controlled trials （RCTs） has been generally accepted as the gold standard in the evaluation of clinical effectiveness. Readers need to understand the trial design, implementation, results, analysis and interpretation, so as to fully Jnderstand the results of RCTs. Thus, the investigators of RCTs have to report these items in a complete, accurate and clear manner. Since 1998, we have conducted several evaluations on the reporting quality of RCTs published in Chinese journals on traditional Chinese medicine （TCM） and results have shown that there is an urgent need for higher quality RCTs on TCM.