大鼠原代培养肝细胞的生物学特性研究

目的 :观察培养的原代大鼠肝细胞形态学和功能的动态变化 ,探讨其短期培养内的最佳功能状态。方法 :采用Seglen胶原酶二步灌流法分离肝细胞。光镜、透射电镜和免疫组化方法观察细胞形态学改变 ;并收集不同时期培养上清监测生化指标的改变。结果 :平均每只大鼠肝获取 1.2 1× 10 8个肝细胞 ,平均活力为 93.6 %。光镜下见肝细胞生长良好 ,可存活 3周 ,电镜下可见丰富的细胞器 ,细胞连接及胆小管 ,细胞角蛋白 18强阳性表达。肝细胞功能检测白蛋白分泌及尿素合成功能在第 3,4天时较好。结论 :肝细胞的形态和功能具有一致性 ,原代肝细胞在培养第 3,4天功能较好 ,可能为肝细胞移植和生物人工肝应用的最佳时机。     

微载体黏附培养SD大鼠肝细胞的生物学特性

目的:探讨微载体黏附培养SD大鼠肝细胞的生物学特性. 方法:对胶原酶消化法分离获取的SD大鼠肝细胞行微载体黏附培养,在倒置显微镜及扫描电镜下观察肝细胞形态变化,采用CL-800全自动生化分析仪检测不同时间培养上清中白蛋白、乳酸脱氢酶(LDH)和尿素的含量. 结果:微载体黏附培养肝细胞的正常形态及白蛋白、尿素合成功能可维持1 wk以上,LDH漏出量、白蛋白及尿素水平1 wk内呈波动性变化,在培养第3日白蛋白及尿素水平最高、LDH漏出量最低. 结论:微载体培养可提供高活性、高密度生长的肝细胞,可望为肝细胞移植、生物型人工肝提供较理想的细胞材料;微载体培养肝细胞在培养第3日功能最佳,可能为应用于肝细胞移植、生物型人工肝的最佳时间.     

大鼠肝细胞分离、原代培养及生物学特性研究

目的 :探讨体外原代培养大鼠肝细胞的分离方法、原代培养肝细胞的生物学特性。　方法 :以SD大鼠作肝细胞供者 ,采用胶原酶消化法分离获取肝细胞 ,并进行原代培养 ;以锥虫蓝染色法测细胞活力 ,在倒置显微镜下观察肝细胞形态变化 ,采用Beckman全自动生化分析仪检测各培养体系不同时间培养上清中Albumin、LDH、Urea的含量。　结果 :胶原酶消化法所获取的肝细胞形态完整、贴壁良好、活性高、功能强。LDH漏出量、白蛋白分泌及尿素合成功能在 1周内出现波动性变化 ,第 3天时LDH漏出量最低 ,白蛋白分泌及尿素合成功能较好。　结论 :胶原酶消化法为一较好的肝细胞分离方法 ,分离的肝细胞在培养第 3天功能最佳     

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.     

永生化胚胎肝细胞系的构建及其生物学特性

目的:构建永生化肝细胞系并对其生物学特性及某些功能进行研究. 方法:利用SV40T基因和真核表达载体pcDNA3.1经脂质体转染至体外分离培养的人胚胎肝细胞,使其永生化,进一步鉴定其形态学特征和生物学功能. 结果:经G418 700～300 mg/L筛选,40 d后获得一株阳性克隆. 形态学观察发现,该细胞具有原代培养肝细胞的大多数典型特征. 永生化胚胎肝细胞克隆形成率为31.2%,染色体核型分析表明细胞核型无明显异常,软琼脂集落形成试验表明细胞在软琼脂中不能生长,免疫组化证明SV40T基因已整合入细胞,而且该细胞具有合成白蛋白的功能. 结论:新建胚胎肝细胞系具有与原代肝细胞类似的形态特征及生物学功能,可以成为生物人工肝及肝细胞移植研究中的理想细胞材料.     

SV40LT抗原介导的人源性肝细胞系的构建

目的：建立人源性永生化肝细胞系，为生物人工肝和肝细胞移植等提供合适的细胞源。方法：利用SV40L Tag基因和真核表达载体pcDNA3．1(-)经脂质体转染至来源于25岁男性脑死亡供肝的原代培养细胞，使其永生化，进一步鉴定其形态学特征和生物学功能。结果：经G418 700～300μg／ml筛选，42天后获一抗G418永生化肝细胞系。该细胞系呈单层贴壁、上皮细胞样形态生长。体外培养可无限传代，具有分泌ALB、ALT、AST及LDH的功能。超微结构观察发现，转染肝细胞具有原代培养肝细胞的大多数典型特征，如较大的细胞核、胞浆内含有丰富的糖原颗粒、大量的线粒体和粗面内质网等。经RT—PCR和Westernb lot检测，转染肝细胞的ALB mRNA阳性和细胞色素P450 2E1阳性。结论：新建人源性肝细胞系与原代培养肝细胞具有类似的形态学特征和生物学功能。     

原代培养大鼠肝细胞的基因转染

目的：研究原代培养大鼠肝细胞的基因转染的方法．方法：采用胶原酶灌注法获取原代培养大鼠肝细胞．利用脂质体转染法将含有绿色荧光蛋白(GFF)和Neo基因的真核细胞表达载体(pEGFP-N3)转染原代培养大鼠肝细胞．用荧光显微镜观察和Neo基因原位杂交染色方法检测肝细胞内基因表达情况．结果：获取的原代大鼠肝细胞活细胞率达95％,荧光显微镜下观察可见转染基因的细胞可发出绿色荧光,原位杂交显示有Neo基因的表达．结论：pEGFP-N3基因可转入大鼠肝细胞并获得表达,可用于标记原代培养的大鼠肝细胞,有利于研究肝细胞移植后移植的肝细胞在体内的分布及功能．     

28℃条件下短期培养新生广西巴马小型猪肝细胞的初步观察

目的对28℃条件下短期培养猪肝细胞的可行性进行初步评价.方法将刚分离的新生广西巴马小型猪肝细胞采用28℃条件下的短期培养1～3 d后,恢复37℃继续培养,观察升温前后肝细胞的形态及LDH、AST的释放量,检测28℃短期培养后肝细胞的氨清除率和尿素合成量.结果在28℃短期培养的肝细胞,用倒置相差显微镜观察到典型的形态特征,细胞未见明显增殖,恢复37℃继续培养后,肝细胞迅速增殖并维持典型的肝细胞形态;经28℃短期培养后的肝细胞和未经28℃短期培养的肝细胞均保持较强的尿素合成和氨清除能力,但在28℃培养2 d和3 d后的肝细胞尿素合成量较在28℃培养1 d后的肝细胞尿素合成量显著降低(P＜0.01);培养期内LDH、AST漏出量较少.结论28℃短期培养能较好地维持猪肝细胞的形态学特征和功能,有望成为一种有效的保存和运输生物人工肝细胞材料的方法.     

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.     

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.     

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.     

TCM Treatment of Ankylosing Spondylitis by Tonifying the Kidney and Strengthening the Governor Vessel

Ankylosing spondylitis is a chronic and progressive disorder with inflammation mainly involving the central axis joints. It mainly affects the cervical spine and the lumbosacral area, with the pathogenesis closely related to the kidney and the Governor Vessel (GV). TCM holds that the syndrome is deficiency in origin and excess in superficiality, which is due to insufficiency of the kidney, deficiency of GV, and blocking of the channels with the invasion of exogenous evil, leading to poor circulation of qi and blood and malnutrition of the bones, muscles and joints. The TCM method of tonifying the kidney and strengthening GV to regulate circulation of qi and blood and check the arthralgia pain should be adopted, with the Kidney-Tonifying and GV Strengthening Decoction (益肾强督汤) prescribed.     

IMPROVING P-gp EXPRESSION IN HUMAN MONONUCLEAR CELLS IN VITRO TRANSFECTED BY MULTIDRUG RESISTANCE-1 mRNA

CHEMOTHERAPY playsa greatrolein the treat- ment of malignanttumors,especiallyingynecolo- gicalones.But inanticancerchemotherapy,leuko-cytopeniaisfrequentlytheprimarydose-limitingsideeffect factor.Moreover,cancersarefrequentlychemoresistantbe-causeof overexpressionof P-glycoprotein(P-gp), which isencodedby multidrugresistancegene (MDR1 ) and detectableinup to50% ofhuman cancersand renderscellsresistancetoanticancerdrugs.The safetyand potentialtherapeuticbenefitof mdr1 gene transferredto h…