大鼠骨髓间充质干细胞的分离、培养和鉴定

目的 探讨体外分离和培养大鼠骨髓间充质干细胞(MSCs)的生物学特性,对其表型和生长曲线进行初步鉴定.方法 采用全骨髓培养法提取培养MSCs,绘制原代及三代细胞生长曲线,利用免疫组化法检测表面标志物CD34、CD44;流式细胞分析法检测表面标志物CD90.结果 成功培养出MSCs,免疫组化CD44阳性表达,CD34阴性表达;流式细胞术CD90阳性表达.结论 该实验分离培养的细胞群与MSCs的生物学特性相吻合,说明MSCs易于体外分离、培养和扩增.     

小鼠骨髓间充质干细胞的分离、培养新方法

目的探讨一种新的小鼠骨髓间充质干细胞的分离、培养方法。方法分离小鼠3 d乳鼠双后肢,剔除肌肉筋膜组织,剪碎后接种于含10%胎牛血清的α-MEM中,观察细胞形态学变化,采用CCK8检测细胞增殖能力,绘制细胞生长曲线,采用流式细胞技术鉴定细胞表面抗原,并进行多向分化潜能鉴定。结果原代分离培养的小鼠骨髓间充质干细胞呈长梭形,从骨片周围爬出,传代后,细胞形态一致,生长良好;绘制的细胞生长曲线呈"S"型,流式细胞表型鉴定结果显示,培养的细胞高表达CD90、CD29,低表达CD11b、CD45,成脂油红O染色和成骨茜素红染色均呈阳性。结论采用小鼠3 d乳鼠骨片法能够成功培养出骨髓间充质干细胞,骨髓间充质干细胞可作为肝脏组织工程的种子细胞。     

大鼠骨髓间充质干细胞的分离培养及生物学特性

用密度梯度离心结合贴壁培养法分离纯化大鼠骨髓间充质干细胞(MSCs),体外培养传代扩增,观察细胞生长特性,对细胞进行免疫组化染色鉴定,电镜观察细胞形态。动态观察示培养细胞具有不断增殖的干细胞特性,并保持细胞活性。证实密度梯度离心结合贴壁法以及消化传代能有效分离纯化和扩增骨髓MSCs,培养的细胞具有骨髓MSCs的基本表型和特性。本研究可为临床进行细胞移植奠定基础。     

一种改良大鼠骨髓间充质干细胞培养方法

目的建立大鼠骨髓间充质干细胞(BMSCs)的分离、改良培养、纯化方法,并进行细胞形态学观察、表面标志物鉴定及多向分化能力检测。方法通过改良全骨髓贴壁法对4周龄SD雄性大鼠脱颈处死,无菌条件下分离出骨髓进行原代培养、消化传代培养及纯化。对BMSCs进行形态学观察,收获第四代BMSCs进行流式细胞仪检测其细胞表面标记物CD90、CD29、CD34、CD45的表达率及向成脂方向诱导分化。结果 BMSCs的原代培养形态学观察可见骨髓细胞接种于培养皿后,细胞呈圆型,大小不一,悬浮于培养液中。24 h后部分细胞开始贴壁,呈圆形、梭形或多角形。通过换液去除未贴壁的杂质细胞,可见短梭形、星形细胞分散贴壁生长,四五天可见放射状排列的细胞集落,伸出长短不一、粗细不均的突起,梭形细胞为主,胞浆丰富,胞核大、核仁清晰。7~8 d细胞呈集落生长,融合80%~90%,呈漩涡状,同向排列,9~10 d细胞排列紧密,逐渐融合成片。传代培养可见消化传代后,传代细胞24 h完全贴壁生长。细胞形态均一,呈梭形生长,细胞生长旺盛。四至五天可传代1次。可稳定连续传代7代以上,细胞形态及生长速度未见明显变化。BMSCs表面标记物的表达通过流式细胞仪检测结果显示,培养的第4代大鼠BMSCs均一表达CD90,CD29,阳性率分别为96.9%,96.6%;而CD34,CD45,呈阴性,阳性率分别为0.395%,7.56%。BMSCs加入成脂诱导剂后18 d,诱导而成的脂肪细胞累积脂质,脂滴变大,合并呈串珠状,经油红O染色呈鲜红色。结论与传统全骨髓贴壁法相比,改良后的全骨髓贴壁法操作步骤简单,降低离心对细胞的损害,减少了污染机会,节省经费,且分离的BMSCs细胞活性高,可大量分离、纯化、扩增,所获细胞具有间充质干细胞的一般生物学特性,经诱导培养后具有多向分化潜能。可为组织器官缺损性疾病、恶性肿瘤等的治疗和组织工程提供充足的种子细胞来源,具有重要的现实意义。     

人骨髓间充质干细胞体外培养及其生物学特性分析

目的探讨人骨髓间充质干细胞（MSC）的体外分离培养方法,并分析其生物学特性。方法无菌条件下抽取健康志愿者骨髓,采用密度梯度离心法分离骨髓单个核细胞,培养在含10%胎牛血清的DMEM培养基中,用贴壁筛选法纯化获得MSC。相差显微镜下观察细胞形态学变化,细胞计数法绘制生长曲线,流式细胞仪检测细胞表面抗原及细胞周期。结果原代和传代培养的细胞呈梭形,具有较强的生长增殖能力。细胞表面CD90、CD105表达阳性,CD34、CD11b阴性。第1、3、5代MSC生长曲线呈S形,均经历潜伏期、对数生长期和平台期。第3代MSC中,G0/G1期细胞约占77.42%,S＋G2/M期细胞约占22.58%。结论采用密度梯度离心结合贴壁筛选培养法可获得纯度较高的MSC,且该细胞增殖活性较强。密度梯度离心结合贴壁筛选培养法是一种简单、有效的分离纯化MSC的方法。     

Bcl-2基因修饰的骨髓间充质干细胞治疗急性肝衰竭的初步研究

目的 构建Bcl-2基因修饰慢病毒质粒,建立稳定表达Bcl-2基因的骨髓间充质干细胞系,探讨Bcl-2基因修饰的骨髓闸充质干细胞治疗ALF的可行性。方法 PCR合成Bcl-2基因片段并插入GV287空载体后扩增进行慢病毒包装,使用Bcl-2慢病毒液转染大鼠骨髓间充质干细胞,使用单纯未处理骨髓间充质干细胞,移植急性肝损伤模型大鼠,观察移植后大鼠存活情况及其肝脏病理改变。结果 得到纯化的骨髓间充质干细胞细胞株;成功分离Bcl-2基因DNA片段并构建Bcl-2基因重组慢病毒质粒,并成功转染到骨髓间充质干细胞中,经荧光显微镜检测GFP荧光及免疫印迹蛋白检测,转染后细胞可高表达Bcl-2基因,转染后细胞状态较好,可进行移植实验;模型鼠经过尾静脉移植骨髓间充质于细胞后,共聚焦显微镜下观察肝脏冰冻切片病理,骨髓间充质干细胞,可以定植到损伤肝脏组织。结论 初步证实,骨髓间充质干细胞能够在急性肝损伤大鼠肝脏归巢,并且能发挥一定程度治疗作用。     

血管内皮生长因子对骨髓间充质干细胞分离培养的诱导分化作用

目的　结合骨髓间充质干细胞与血管内皮生长因子（VEGF）的优点,探讨VEGF对其体外诱导分化的作用。为缺血性疾病的治疗提供新思路。方法　成人新鲜骨髓,Percoll梯度分离法培养,单克隆培养法分离传代扩增骨髓基质细胞（MSCs）。在大肠杆菌DH5α中扩增和提取,纯化、克隆pcDNA3.0-VEGF165质粒。脂质体法转染MSCs。流式细胞术检测诱导前后MSCs免疫表型变化。一抗VEGF和CD31（1∶50）,FITC标记的VEGF二抗和cy3标记的CD31二抗与细胞共同孵育,甘油封片,在激光扫描共聚焦显微镜下观察细胞染色情况。结果　转染前表达CD44（75.86%）、CD31（5.63%）。转染后CD44表达明显下调（40.18%）,CD31则明显上调（56.20%）。FITC标记后VEGF抗体使细胞显现绿色荧光,cy3标记的CD31抗体使细胞显红色荧光。结论　转染VEGF后骨髓间充质干细胞向内皮细胞分化且有分泌VEGF的功能,为干细胞植入及血管再生起到指导作用。     

大鼠骨髓间充质干细胞的分离培养纯化与鉴定

目的:探讨骨髓间充质干细胞(BMSCs)体外获取及培养增殖的方法并鉴定。方法:用密度梯度离心法结合贴壁培养法分离、纯化培养大鼠BMSCs,测定生长曲线,流式细胞仪鉴定,免疫细胞化学检测。结果:密度梯度离心结合贴壁培养法能有效分离纯化大鼠BMSCs,P2、4、6代细胞生长曲线基本一致,增值能力强,呈均一成纤维样细胞,P3代以后细胞均一地表达细胞表面抗原CD44、CD90,不表达CD34,弱表达CD11b/c,细胞表达Connexin43。结论:本实验建立了一种体外稳定培养扩增大鼠BMSCs的方法,培养的细胞成分单一,适用于对BMSCs进一步的应用研究。     

人骨髓间充质干细胞体外长期传代培养及恶性转化趋势实验研究

目的 探讨人骨髓间充质干细胞体外长期传代培养、扩增及其生物学特性,观察其恶性转化趋势的可能性.方法 采用密度梯度离心差速贴壁法对人骨髓来源的间充质干细胞进行分离纯化,并进行体外长期传代,观察细胞形态及生长方式;鉴定细胞分化能力;相关癌标志P53、Ki67的表达与成瘤性检测.结果 经过长期体外培养的人间充质干细胞,至36代时,细胞逐渐老化;成骨分化诱导能力丧失;相关癌标志表达阴性;无成瘤性.结论 人骨髓间充质干细胞在体外可以有限传代,无显著恶性转化趋势.     

大鼠羊膜上皮细胞的生物学特性

目的 探讨建立大鼠羊膜上皮细胞分离、培养及鉴定方法及观察其生物学特性.方法 从妊娠14～ 16 d的SD大鼠胎盘剥离羊膜,用胰蛋白酶消化法分离大鼠羊膜上皮细胞,采用免疫荧光细胞化学和流式细胞仪分析细胞表型特征;使用含有10％胎牛血清(FBS)和100 U/ml青霉素-链霉素(PS)溶液的DMEM/F-12的培养基进行细胞培养.结果 每次采用胰蛋白酶消化法从1只孕鼠分离的上皮细胞数为(1～6) ×105/ml,足够达到贴壁培养浓度.所分离的羊膜上皮细胞表达神经前体细胞特异性蛋白nestin和骨髓间充质干细胞表面标记蛋白CD29和CD44,但是未成熟神经元标记蛋白β-Ⅲ-tublin、星形胶质细胞标记物胶质纤维酸性蛋白(GFAP)以及造血干细胞表面标记蛋白CD34表达阴性.在完全培养基培养条件下,羊膜上皮细胞生长增殖较快,原代培养1 w后即可传代.结论 建立了大鼠羊膜上皮细胞分离、培养及鉴定的方法,大鼠羊膜上皮细胞具有神经前体细胞的特异标志和间充质干细胞的某些表型特征.     

妊娠时间与肺结核复发的相关性研究及诊治对策

目的 分析肺结核史患者妊娠时间和肺结核复发间相关性.方法 选取我院收治的有肺结核史的妊娠妇女576例作为研究对象,对其妊娠前肺结核治疗、治愈后妊娠时间、妊娠后复发肺结核等进行分析,总结有肺结核史育龄女性的妊娠时间和肺结核复发之间的关系.结果 肺结核治愈后不同时间段妊娠者的结核复发率比较,差异具有显著性（P＜0.05）,停药后间隔时间越久妊娠,肺结核复发的几率越小.结论 加强孕期痰菌检查,及早发现复发肺结核,提高母婴安全.     

我国骨关节结核诊治的回顾与展望

骨关节结核是危害人们健康的严重感染性疾病,近95％由他处结核病继发而来.罹患骨关节结核疾病后几乎均将致残,严重影响人们的健康、工作和生活.建国以来在党和国家的关心和支持下,骨关节结核的诊治水平取得了长足进步.时至今日,由于多种原因,学科发展和被重视程度受到一定的制约,同整个医疗行业的发展不相适应.回顾过去,展望未来,我们需要重新审视骨关节结核的诊治方法,努力推进骨关节结核诊疗技术的科学发展.     

Effect of phosphorylation of MAPK and Stat3 and expression of c-fos and c-jun proteins on hepatocarcinogenesis and their clinical significance

AIM To study the effect of phosphorylation ofMAPK and Stat3 and the expression of c-fos andc-jun proteins on hepatocellular carcinogenesisand their clinical significance.METHODS SP immunohistochemistry was usedto detect the expression of p42/44~(MAPK), p-Stat3,c-fos and c-jun proteins in 55 hepatocellularcarcinomas (HCC) and their surrounding livertissues.RESULTS The positive rates and expressionlevels of p42/44~(MAPK), p-Stat3, c-fos and c-junproteins in HCCs were significantly higher thanthose in pericarcinomatous liver tissues (PCLT).A positive correlation was observed between theexpression of p42/44~(MAPK) and c-fos proteins, andbetween p-Stat3 and c-jun, but there was nosignificant correlation between P42/44~(MAPK) and p-Stat3 in HCCs and their surrounding livertissues.CONCLUSION The abnormalities of Ras/Raf/MAPK and JAKs/ Stat3 cascade reaction maycontribute to malignant transformation ofhepatocytes. Hepatocytes which are positive forp42/ 44~(MAPK), c-fos or c-jun proteins may bepotential malignant pre-cancerous cells.Activation of MAPK and Stat3 proteins may be anearly event in hepatocellular carcinogenesis.     

Overweight and Obesity and Progression of ADPKD

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Effect of phosphorylation of MAPK and Stat3 and expression of c-fas and c-jun proteins on hepatocarcinogenesis and their clinical significance

AIM To study the effect of phosphorylation ofMAPK and Stat3 and the expression of c-fos andc-jun proteins on hepatocellular carcinogenesisand their clinical significance.METHODS SP immunohistochemistry was usedto detect the expression of p42/44MAPK, p-Stat3,c-fos and c-jun proteins in 55 hepatocellularcarcinomas (HCC) and their surrounding livertissues.RESULTS The positive rates and expressionlevels of p42/44MAPK, p-Stat3, c-fos and c-junproteins in HCCs were significantly higher thanthose in pericarcinomatous liver tissues (PCLT).A positive correlation was observed between theexpression of p42/44MAPK and c-fos proteins, andbetween p-Stat3 and c-jun, but there was nosignificant correlation between p42/44MAPK and p-Stat3 in HCCs and their surrounding livertissues.CONCLUSION The abnormalities of Ras/Rat/MAPK and JAKs/ Stat3 cascade reaction maycontribute to malignant transformation ofhepatocytes. Hepatocytes which are positive forp42/ 44MAPK, c-fos or c-jun proteins may bepotential malignant pre-cancerous cells.Activation of MAPK and Stat3 proteins may be anearly event in hepatocellular carcinogenesis.     

Replication and Inhibitors of Enteroviruses and Parechoviruses

The Enterovirus (EV) and Parechovirus genera of the picornavirus family include many important human pathogens, including poliovirus, rhinovirus, EV-A71, EV-D68, and human parechoviruses (HPeV). They cause a wide variety of diseases, ranging from a simple common cold to life-threatening diseases such as encephalitis and myocarditis. At the moment, no antiviral therapy is available against these viruses and it is not feasible to develop vaccines against all EVs and HPeVs due to the great number of serotypes. Therefore, a lot of effort is being invested in the development of antiviral drugs. Both viral proteins and host proteins essential for virus replication can be used as targets for virus inhibitors. As such, a good understanding of the complex process of virus replication is pivotal in the design of antiviral strategies goes hand in hand with a good understanding of the complex process of virus replication. In this review, we will give an overview of the current state of knowledge of EV and HPeV replication and how this can be inhibited by small-molecule inhibitors.     

Effects of sex and time of day on metabolism and excretion of corticosterone in urine and feces of mice

Non-invasive techniques to monitor stress hormones in small animals like mice offer several advantages and are highly demanded in laboratory as well as in field research. Since knowledge about the species-specific metabolism and excretion of glucocorticoids is essential to develop such a technique, we conducted radiometabolism studies in mice (Mus musculus f. domesticus, strain C57BL/6J). Each mouse was injected intraperitoneally with 740 kBq of 3H-labelled corticosterone and all voided urine and fecal samples were collected for five days. In a first experiment 16 animals (eight of each sex) received the injection at 9 a.m., while eight mice (four of each sex) were injected at 9 p.m. in a second experiment. In both experiments radioactive metabolites were recovered predominantly in the feces, although males excreted significantly higher proportions via the feces (about 73%) than females (about 53%). Peak radioactivity in the urine was detected within about 2h after injection, while in the feces peak concentrations were observed later (depending on the time of injection: about 10h postinjection in experiment 1 and about 4h postinjection in experiment 2, thus proving an effect of the time of day). The number and relative abundance of fecal [3H]corticosterone metabolites was determined by high performance liquid chromatography (HPLC). The HPLC separations revealed that corticosterone was extensively metabolized mainly to more polar substances. Regarding the types of metabolites formed, significant differences were found between males and females, but not between the experiments. Additionally, the immunoreactivity of these metabolites was assessed by screening the HPLC fractions with four enzyme immunoassays (EIA). However, only a newly established EIA for 5alpha-pregnane-3beta,11beta,21-triol-20-one (measuring corticosterone metabolites with a 5alpha-3beta,11beta-diol structure) detected several peaks of radioactive metabolites with high intensity in both sexes, while the other EIAs showed only minor immunoreactivity. Thus, our study for the first time provides substantial information about metabolism and excretion of corticosterone in urine and feces of mice and is the first demonstrating a significant impact of the animals' sex and the time of day. Based on these data it should be possible to monitor adrenocortical activity non-invasively in this species by measuring fecal corticosterone metabolites with the newly developed EIA. Since mice are extensively used in research world-wide, this could open new perspectives in various fields from ecology to behavioral endocrinology.     

心电图、心电向量图与冠状动脉造影结果对比分析

目的:通过分析心电图(Electrocardiogram,ECG)和心电向量图(Vectorcardiogram,VCG)的改变与冠脉造影（CAG）结果进行对比,探讨ECG、VCG在冠状动脉病变中的诊断价值。方法: 选择2008年1月~2009年12月临床拟诊断为冠心病患者108例,行常规ECG、VCG检查,并于1周内进行CAG,对检查结果依据各自的诊断标准进行判定,以CAG为标准诊断法,利用四格表法,计算相关评价真实性的指标并进行比较。结果: ①VCG检测的灵敏度、特异度、准确度显著高于ECG(P<0.05,P<0．01)。②ECG、VCG阳性率与冠脉病变支数组间比较:在单支病变、双支病变中,VCG阳性率明显高于ECG(P<0.05),左主干或三支病变无统计学意义;组内比较:ECG组左主干或三支病变组较单支病变、双支病变阳性率高(P<0.05,P<0.01);VCG组左主干或三支病变组较单支病变阳性率高(P<0.05);与双支病变阳性率比较无统计学意义;③ECG、VCG阳性率与冠脉病变程度组间比较:冠脉病变狭窄50%～69%的VCG阳性率明显高于ECG (P<0.05),其他两组阳性率比较无统计学意义;组内比较:ECG组冠脉病变狭窄≥90%较50%～69%、70%～89%的阳性率高(P<0.05,P<0.01); VCG组狭窄≥90%较50%～69%阳性率高（P<0.01),其他无统计学意义。结论: VCG对冠心病检测价值显著高于ECG。     

Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence

Here we report the structural characterization of the product formed from the reaction between hydroethidine (HE) and superoxide (O(2)(.-)). By using mass spectral and NMR techniques, the chemical structure of this product was determined as 2-hydroxyethidium (2-OH-E(+)). By using an authentic standard, we developed an HPLC approach to detect and quantitate the reaction product of HE and O(2)(.-) formed in bovine aortic endothelial cells after treatment with menadione or antimycin A to induce intracellular reactive oxygen species. Concomitantly, we used a spin trap, 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO), to detect and identify the structure of reactive oxygen species formed. BMPO trapped the O(2)(.-) that formed extracellularly and was detected as the BMPO-OH adduct during use of the EPR technique. BMPO, being cell-permeable, inhibited the intracellular formation of 2-OH-E(+). However, the intracellular BMPO spin adduct was not detected. The definitive characterization of the reaction product of O(2)(.-) with HE described here forms the basis of an unambiguous assay for intracellular detection and quantitation of O(2)(.-). Analysis of the fluorescence characteristics of ethidium (E(+)) and 2-OH-E(+) strongly suggests that the currently available fluorescence methodology is not suitable for quantitating intracellular O(2)(.-). We conclude that the HPLC/fluorescence assay using HE as a probe is more suitable [corrected] for detecting intracellular O(2)(.-).     

荣宝和氯硝柳胺灭螺效果比较及成本分析

目的 评价新型灭螺药物荣宝杀灭钉螺的效果,探讨其推广应用价值.方法 按目前推荐的荣宝灭螺剂量,喷洒法为30 g/m2,浸杀法为50 g/m3;氯硝柳胺喷洒法和浸杀法分别采用2 g/m2和2 g/m3杀螺剂量,分别在室内和现场进行灭螺试验,观察两种药物的灭螺效果并初步分析评估其成本.结果 在现场气温22～30℃条件下,荣宝50 g/m3浸杀3、5、7 d后,螺袋内钉螺校正死亡率均达到100.0%,与氯硝柳胺2 g/m3灭螺效果相似;荣宝30 g/m2剂量喷洒3、5、7、15 d后,钉螺校正死亡率分别为54.5%、58.0%、69.0%、79.1%,氯硝柳胺喷洒组钉螺校正死亡率分别为61.0%、69.4%、76.7%、77.9%.在室温18℃条件下,荣宝以30 g/m2喷洒3、5、7、15 d后,钉螺校正死亡率分别为72.9%、87.2%、91.5%、76.1%;而相应2 g/m2氯硝柳胺喷洒后的钉螺校正死亡率分别为81.3%、95.7%、97.9%、80.4%.同样完成1000 m2的喷洒灭螺任务,荣宝所需灭螺药物和人力资费成本比氯硝柳胺多支出0.114元/m2;完成72 m3的浸杀灭螺任务,荣宝所需灭螺药物和人力资费成本比氯硝柳胺多支出0.127元/m3.50 g/m3荣宝浸杀灭螺剂量,对成鱼(＞250 g)的活力不会造成影响,但对鱼类幼苗仍具较强毒性.结论 荣宝与氯硝柳胺灭螺效果相似,由于其成本较高,氯硝柳胺仍然是目前首选灭螺药物,但荣宝的鱼类毒性低,可作为氯硝柳胺之外有益的补充灭螺药物.