新生大鼠原代心肌细胞分离方法的改进

目的对传统的原代乳鼠心肌细胞分离方法进行改良,获得活性好、纯度高、反应性好的原代乳鼠心肌细胞。方法利用断头开胸取心法取新生24 h内的Wistar大鼠心肌组织,无菌剪碎后用0.25%不含EDTA胰酶混合II型胶原酶消化法,采用自制消化组合装置分离消化细胞。逐日在倒置相差显微镜下观察细胞状态。采用心肌细胞特异性蛋白α-横纹肌肌动蛋白(α-actin)单克隆抗体对培养的心肌细胞进行免疫荧光鉴定。另外使用异丙肾上腺素和去甲肾上腺素作用于细胞,观察细胞反应情况。结果培养后24 h即可观察到部分细胞搏动,可初步确定为心肌细胞;48 h后细胞逐渐展开,连续观察20 d,发现心肌细胞在3~14 d期间搏动频率与形态无明显变化。对比观察发现断头开胸取心法获取的原代心肌细胞中红细胞数量明显减少。免疫荧光鉴定发现,本方法获得的心肌细胞纯度高达95%。此外,分离的原代乳鼠心肌细胞对异丙肾上腺素和去甲肾上腺素反应灵敏。结论断头开胸取心法配以混合酶液消化心脏组织分得的原代乳鼠心肌细胞反应性好、纯度高、有活力,可满足大多数实验的要求。     

成年大鼠肝细胞的分离及培养

用含有0.5mmol/L EGTA的Hanks液100ml灌流大鼠肝脏,然后用100U/ml胶原酶溶液100ml循环灌流10min,再用100ml的0.5%胰酶灌流。分离的肝细胞用台盼蓝排斥试验显示87%～89%活率,产率可达4.7×10~8,具有~3H-TdR掺入活性。原代培养3d出现新增殖的肝细胞,20d增殖约5倍,可进行连续传代培养。     

大鼠胰岛细胞原代培养方法比较研究

目的：比较减碎消化法和原位灌注消化法在大鼠胰岛细胞原代培养中的优缺点,探讨一种简单易行的大鼠胰岛细胞培养方法。方法：雄性SD大鼠30只随机分成减碎组（CC组,n=15）,胰腺剪为小组织块后,Ⅴ型胶原酶逐级消化分离;原位灌注组（YY组,n=15）,采用胆总管逆行灌注Ⅴ型胶原酶分离胰岛。双硫腙（DTZ）染色检测胰岛纯度,葡萄糖刺激胰岛素释放试验检测胰岛功能。结果：2组获得的胰岛对于葡萄糖刺激均具有良好的分泌活性（P〈0.01）;YY组比CC组获得的胰岛数量多（P〈0.01）,形态较好,等量胰岛的胰岛素释放量亦多（P〈0.05）。结论：胆总管原位灌注Ⅴ型胶原酶消化可以获取数量多,活性和纯度较好的胰岛。     

大鼠肺泡Ⅱ型上皮细胞的分离、纯化、原代培养及鉴定

目的建立大鼠肺泡Ⅱ型上皮细胞（alveolar typeⅡepithelial cell,AT-Ⅱ）分离、纯化、原代培养及鉴定方法。方法取Wistar大鼠肺脏,采用胰蛋白酶消化法分离AT-Ⅱ,通过差速离心法、红细胞裂解法、贴壁选择法、免疫黏附选择法纯化AT-Ⅱ,倒置相差显微镜下观察细胞形态,鞣酸染色法进行细胞鉴定。结果与结论AT-Ⅱ呈单个或岛状生长,细胞呈圆形或椭圆形,细胞浆内有大量的反差明显的细胞小颗粒,细胞核明显。鞣酸染色法鉴定阳性。通过改进的酶消化法获得的AT-Ⅱ生长状态良好,纯度高,符合体外实验要求。     

幼龄大鼠骨骼肌卫星细胞原代培养的实验研究

目的:为改进大鼠骨骼肌卫星细胞的体外培养方法,获取高纯度的肌卫星细胞。方法:选用幼龄SD大鼠,通过两步消化法和简易的两次差速贴壁法得到高纯度的肌卫星细胞。所得细胞通过免疫组织化学方法加以鉴定。结果:骨骼肌卫星细胞纯化率高,细胞生长良好,后期增殖较快。结论:本实验成功建立了骨骼肌卫星细胞的纯化和鉴定方法,可用于骨骼肌细胞增殖和细胞移植的相关研究。     

小鼠肾小球分离方法比较及原代足细胞鉴定

 

目的 通过比较小鼠肾小球分离技术,明确小鼠原代足细胞培养的最优方法。方法 分别采用差异过筛法和免疫磁珠法分离C57/BL6J小鼠肾小球,用Ⅳ型胶原酶消化去除包曼囊,用相差显微镜观察肾小球纯度和形态结构,采用免疫荧光染色和聚合酶链反应法(polymerase chain reaction,PCR)鉴定足细胞的标志蛋白Podocin的分布和表达。结果 差异过筛法获得肾小球纯度达90.2%±1.6%,一只C57/BL6J小鼠可以得到(10 421±2421)个肾小球;视野中肾小球结构完整,可见肾小管碎片。免疫磁珠法获得的肾小球纯度达96.7%±1.2%,一只C57/BL6J小鼠可以得到(16 112±2651)个肾小球;视野中肾小球结构完整,基本无肾小管。免疫磁珠法获得肾小球纯度和数目均高于差异过筛法,差异均有统计学意义(P＜0.05)。7～10 d可见肾小球周围大量多边形细胞爬出,呈铺路石样外观,符合足细胞特征;免疫荧光染色和PCR法均证实有足细胞特异性蛋白Podocin表达。结论 C57/BL6J小鼠肾小球的分离,免疫磁珠法明显优于差异过筛法,可以更简便、高效地培养出原代足细胞。

     

新生大鼠皮层神经元的培养与鉴定

目的 建立一种新生大鼠大脑皮层神经元原代培养方法.方法 体外原代培养新生大鼠大脑皮质神经元,应用免疫细胞化学法鉴定神经元.结果 用神经基础培养基(Neurobasal-A)+B27培养的神经元纯度可达70%,生长状态较佳.结论 该培养技术是大鼠皮层神经元体外培养的一种较理想的方法.     

逆转录病毒介导的对原代培养成年大鼠成肌细胞基因转染的研究

目的　探索成年大鼠成肌细胞的原代培养方法以及逆转录病毒对其的基因转染。方法　取成年SD大鼠的胸大肌 ,利用组织块法进行培养 ,并对培养细胞进行形态学研究和免疫细胞化学鉴定。以逆转录病毒为载体 ,将绿色荧光蛋白基因导入成年大鼠成肌细胞 ,利用倒置荧光显微镜及流式细胞仪对转染细胞进行观察。结果　采用组织块培养法 ,2周后成肌细胞的密度可达 10 7/ml,免疫细胞化学分析显示 90 %以上的细胞呈骨骼肌特异的myogenin抗体染色阳性。荧光显微镜和流式细胞仪检测发现eGFP基因可在成年大鼠成肌细胞中有效地表达 ,其转染效率约为 12 82 %。结论　利用组织块法成功培养成年大鼠原代成肌细胞 ,该方法实用性强 ,所得成肌细胞纯度高 ;逆转录病毒可介导成肌细胞的基因转导 ,为深入研究心肌梗死瘢痕中转基因成肌细胞自体移植奠定了基础。     

重力因素对体外培养心肌细胞的影响作用

目的探讨重力因素对心肌细胞的影响。方法通过建立的乳鼠心肌细胞长期动态培养模型,利用回转器模拟失重效应。结果模拟失重可抑制心肌细胞功能,导致心肌细胞收缩力减弱,搏动频率减慢,搏动节律异常,自回转的第３～７天,其心肌收缩力分别减弱２６．１％,３１．８％,５７．５％,６７．４％和８０．６％（Ｐ＜０．０５）;其搏动频率分别减少２２．４％,４８．７％,７６．６％,８９．２％,９６．４％（Ｐ＜０．０５）;从回转的第４天起,心肌细胞团出现搏动节律异常现象,最终发展为９０．４％的心肌细胞团停止搏动。回转停止后,部分心肌细胞的搏动功能可逐步自行恢复,自回转停止的第３～７天,其恢复搏动的心肌细胞团分别为０．９５％,５．９％,１６．０％,２３．９％和３８．７％（Ｐ＜０．０５）;而离心超重则可使心肌细胞收缩力明显增强,离心２～８天,其收缩力分别增加１１．２％,４０．４％,８０．２％,７７．６％,６９．０％,７５．６％（Ｐ＜０．０５）,并维持正常搏动频率和搏动节律正常。结论重力因素能直接作用并影响体外培养的心肌细胞功能,提示失重或模拟失重引起心脏功能的退行性变化可能与重力因素对心肌细胞的直接影响作用有关。     

新生大鼠胰岛细胞的体外单层培养

胰岛细胞体外培养中成纤维细胞的过度生长是影响胰岛细胞贴壁率的主要因素，曾经采用多种方法以限制成纤维细胞的生长，有的效果不理想，有的方法较复杂。作者采用接种后两次转板的方法，使成纤维细胞明显减少，胰岛细胞贴壁成活率提高，且方法简便。１材料与方法１．１胰...     

DHPLC快速诊断中国人静止型-α4.2地中海贫血

目的 开发基于断裂点序列信息的-α^4.2缺失检测技术。方法对中国人-α^4.2基因断裂点区域及其正常同源片断的进行测序获得可用于基因诊断的信息，设计RCR／DHPLC方法快速检测-α^4.2基因。结果 序列同源性分析表明：1个包含4个单核苷酸位点的单体型存在于所有的10个中国人-α^4.2基因中，采用盲法检测了40个样本的基因型以验证DHPLC方法的可靠性，检测结果和Gap—PCR的完全一致。结论 DHPLC是一种快速、敏感和可靠的-α^4.2基因检测技术。     

小鼠骨髓干细胞的分离培养鉴定及诱导分化的研究

目的分离培养鉴定小鼠的骨髓间充质干细胞,观察体外培养生长特性,并在特定条件下诱导分化,探讨其成脂成骨分化能力。方法采用全骨髓培养法培养小鼠骨髓间充质干细胞,观察干细胞的形态和生长特性,用流式细胞仪对其细胞表型CD29、CD90、CD34、CD44、CD31、CD45进行鉴定,成脂成骨诱导分化能力鉴定。结果原代分离的细胞培养24 h,干细胞开始贴壁,胞体呈圆形,其他血细胞悬浮。培养3 d,贴壁细胞逐渐变梭形;培养第4 d,细胞开始分裂;随着细胞数目的增加,逐渐形成漩涡状排列,细胞形态呈梭形、三角形、多边形、不规则形;培养第10 d,贴壁细胞长满瓶底的80%,按1∶2传代。选择第3代骨髓干细胞分析显示,CD29、CD44、CD90阳性表达,CD31、CD34、CD45阴性表达。细胞成脂成骨诱导后染色鉴定呈阳性。结论采用全骨髓法培养可获得生长状态良好,增殖能力强的骨髓间充质干细胞,方法简便、实用。     

A simple and rapid method of preparing schematic anatomic drawings using modern computer technology

Function and usage of the graphic-software "MacDraw" is described and illustrated by drawing a lumbar vertebrae and pathologic clefts in a layer technique.     

The approximate planimetric method: a simple,rapid and reliable method for estimation of lesion size in acute ischemic stroke

Magnetic resonance imaging is increasingly used in stroke trials for early diagnosis and follow-up of lesion size. Since volumetric measurement remains a laborious and time-consuming task, a rapid and reliable method for the assessment of lesion size has been developed and validated in diffusion weighted imaging (DWI) and fluid attenuated inversion recovery (FLAIR) sequences. These were serially obtained in 40 patients less than 8 h after the onset of symptoms of a middle cerebral artery territory stroke (day 1), as well as on days 3 and 18. For each of 16 (DWI) or 20 (FLAIR) transverse sections obtained on each occasion, lesion size was estimated as a percentage of the total hemisphere. Percentage values from all sections were summed up and expressed as arbitrary units. Results obtained using this approximate planimetric method (APM) were compared with those from a standard volumetric approach. Lesion volumes as determined by both methods were highly correlated (DWI: r = 0.966, FLAIR: r = 0.979, p < 0.001). To conclude, the APM is simple, rapid and reliable for the estimation of lesion size in acute ischemic stroke. It can be recommended for broader application in clinical trials.     

A simple and rapid radioimmunoassay of triiodothyronine in unextracted serum.

A simple, accurate, and rapid procedure for radioimmunoassay (RIA) of triiodothyronine (T3) is described. Serum is denatured by sodium trichloroacetate and the T3 released is allowed to react with 125I-T3-labeled T3 anti-serum. The displaced 125I-T3 is rapidly taken up by an anionic-resin sponge, and this uptake is linearly related to serum T3 concentration. The entire assay procedure was completed in the same tube in about 1 hr. The T3 antibody was specific, and T4-to-T3 conversion in the assay did not occur. Recovery of exogenously added T3 was virtually complete. The mean T3 concentration in 92 euthyroid subjects (142 ng/100 ml) is comparable to the values described in a number of radioimmunoassays for T3.     

A rapid and simple Sep Pak method for purification of radioiodinated IQNP, a high affinity ligand for the muscarinic receptor

A simplified procedure for the purification of 1-azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (IQNP) stereoisomers utilizing a silica Sep Pak (SSP) is described. Iodine-131-E- and iodine-125-Z-(R,R)-IQNP were isolated after SSP purification in 80% and 75% radiochemical yields, respectively. The biodistribution of iodine-131-E-/iodine-125-Z-(R,R)-IQNP, purified either by SSP or high performance liquid chromatography (HPLC), was evaluated in female rats and demonstrated no significant differences in the uptake in various organs and cerebral regions. The utilization of SSP thus affords a simple and rapid method for the purification of IQNP for use in a variety of animal studies.     

A simple method for domestic animal identification in Argentina using PCR-RFLP analysis of cytochrome b gene

We developed a simple, quick assay in order to discriminate forensic samples among human, and common domestic and livestock species of the Pampean region, Argentina. A mitochondrial cytochrome b fragment amplified with universal primers was separately digested with three restriction enzymes (AluI, HaeIII, and HinfI) and the resulting fragments were resolved through electrophoresis in polyacrylamide gels. This PCR-RFLP method allowed us to identify the target species and worked on degraded samples. The assay was successfully applied in livestock robbery cases in Argentine, and may be useful when attempting a first assessment as to the specific status of a forensic evidence.