前列腺基质细胞的原代培养方法

目的:建立一种操作简单、成功率高、重复性好的前列腺增生组织原代基质细胞(PSC)培养方法。方法:采用胶原酶消化法、组织块贴壁法和胰酶消化组织块贴壁法,从70岁及以上男性的良性前列腺增生组织中分离培养PSC,通过显微镜观察比较PSC的数量、形态、培养周期,用免疫荧光染色法鉴定PSC的纯度。结果:胶原酶消化法得到的贴壁细胞少,细胞体积较小且形态无法铺展,增殖能力较弱;组织块贴壁法培养72h后细胞会从组织边缘缓慢爬出,生长周期长;胰酶消化组织块贴壁法,细胞培养7d后基本融合,折光性强,细胞多呈长梭形,通过免疫荧光染色鉴定,基质细胞纯度在95%以上。结论:利用胰酶消化组织块贴壁法建立了一种易行、高效且重复性好的前列腺增生组织基质细胞培养方法。     

小鼠胃 Cajal 间质细胞分离与培养实验方法探讨

目的:尝试优化体外培养Balb/c小鼠胃Cajal间质细胞(interstitial cells of Cajal,ICC)的实验方法,为深入探索该细胞的生理病理作用机制提供基础。方法:无菌条件下取出小鼠胃组织,使用酶解法消化分离细胞,将细胞悬液接种于含有SCF(干细胞因子)的M199培养基中培养,并进行传代。倒置显微镜下观察不同时间段细胞生长状态,采用ICC特异性标志物c-Kit(酪氨酸激酶受体)进行免疫荧光鉴定。结果:细胞培养24 h后基本已贴壁,呈梭形或三角形,有短突起;72 h后细胞胞体变大,突起伸长;5 d后,细胞之间通过突起彼此相互连接,开始形成网状结构;传代后细胞依然保持其固有特征。免疫荧光鉴定可见细胞c-Kit抗体荧光染色阳性。结论:使用酶解法成功分离细胞,细胞数量较多但不增殖,传代后可见细胞纯度较好,稳定培养3周以上后细胞形态逐渐发生变化并开始凋亡。     

大鼠附睾上皮细胞的原代培养及纯化鉴定

目的建立大鼠附睾上皮细胞原代培养及纯化方法。方法利用酶消化法和组织块法对大鼠附睾上皮细胞进行原代培养,然后用胰酶两步消化法进一步纯化附睾上皮细胞,最后分别利用免疫荧光和免疫组织化学染色对原代培养的细胞及相关蛋白表达情况进行鉴定。结果酶消化法较组织块法得到的附睾上皮细胞纯度高,免疫荧光染色结果证明所得附睾上皮细胞主要是主细胞,免疫组织化学结果证明培养的附睾上皮细胞中有雄激素受体和雌激素受体α的表达。结论利用酶消化法对大鼠附睾上皮细胞进行体外培养,方法简单易行,成功率高。     

树鼩原代皮肤成纤维细胞的分离培养技术

建立稳定的树鼩(Tupaiabelangeri)皮肤成纤维细胞的体外培养体系,可为有关此类细胞的实验和疾病树鼩细胞模型提供技术支持。取树鼩大腿内侧皮肤用组织块贴壁法和胶原酶Ⅰ消化法分离皮肤细胞,胰蛋白酶差别消化法纯化细胞;用MEM(10%FBS)完全培养基和含低血清生长添加物(LSGS)的培养基培养细胞;免疫荧光和蛋白印迹法鉴定细胞,并测定细胞的生长、冻存和复苏特性。经树鼩皮肤细胞分离效果比较,胶原酶消化法比组织块贴壁法更适合用于树鼩原代皮肤细胞分离;对分离及冻存复苏后细胞生长状况观察比较发现,添加了LSGS的MEM培养基更利于细胞存活、生长;细胞形态观察、免疫荧光和蛋白印迹检测鉴定所分离的细胞为树鼩皮肤成纤维细胞。成功建立了树鼩原代皮肤细胞的分离、纯化方法,并优化了该细胞的培养条件。     

大鼠膀胱Cajal间质细胞的分离、培养和鉴定

目的:探索大鼠膀胱Cajal间质细胞(ICC)的分离和培养方法,为进一步研究其在膀胱中的作用提供条件.方法:取大鼠的膀胱组织,采用Ⅱ型胶原酶酶解法分离细胞,将细胞悬液接种于含50ng/ml SCF、15%(v/v)FBS的DMEM培养基中,进行培养.用c-kit特异性杭体标记细胞,免疫荧光鉴定ICC细胞.结果:培养8小时后的ICC贴壁良好,并保持其固有特征:两个长的突起,多个短的侧突.胞体小,核大,c-kit抗体荧光染色阳性.结论:酶解法分离大鼠膀胱ICC并培养成功.     

成年小鼠原代皮肤成纤维细胞的分离培养及其生物学特性

目的:建立一种周期短、成本低的成年小鼠原代皮肤成纤维细胞分离培养方法,并探索其生物学特性。方法:取8~12周龄BALB/c小鼠背部、尾尖、耳部皮肤,配制2种血清的细胞培养液,采用组织块贴壁法、酶消化法、酶消化组织块贴壁法进行原代皮肤成纤维细胞的培养,通过显微镜观察比较原代细胞的数量、形态、培养周期及纯度;通过免疫荧光、CCK-8、UVB辐照、流式细胞术进行生物学特性鉴别。结果:背部皮肤组织块贴壁使用Gibco胎牛血清培养7 d无细胞游出,CLARK特级胎牛血清细胞游出较多。背部皮肤经酶消化法得到细胞贴壁少;经组织块贴壁法细胞生长慢,培养周期长;酶消化组织块贴壁法细胞游出速度快、数量多、呈长梭形。尾尖取材量少,得到细胞少;耳部皮肤取材方便,但细胞纯度低。CCK8增殖曲线呈S型;相较于对照组,UVB辐照后细胞凋亡率增高17%。结论:CLARK特级胎牛血清、背部皮肤取材、酶消化组织块贴壁法是培养成年小鼠原代皮肤成纤维细胞最优的方案,可增加细胞得量、缩短培养周期,降低成本。     

大鼠主动脉内皮细胞和平滑肌细胞的原代培养及生物学特性比较

目的培养大鼠主动脉平滑肌细胞和内皮细胞,细胞纯化与鉴定,比较生物学特性的差异。方法采用血管环贴壁法培养动脉内皮细胞,组织块贴壁法培养动脉平滑肌细胞,并采用有限稀释法挑选内皮细胞单克隆,免疫细胞荧光鉴定二者的特异性标志,相差显微镜观察二者单个细胞及细胞群体在形态上的差异性,CCK-8试剂盒检测细胞的增殖,比较二者对胰酶消化,粘附,冻存后复苏的情况。结果血管环贴壁法成功培养血管内皮细胞,组织块培养法成功培养出血管平滑肌细胞,内皮细胞能够形成单克隆集落,培养的细胞均表达相应的特异性标志,内皮细胞增殖速度和平滑肌细胞有差异,内皮细胞对胰酶的耐受性较差,内皮细胞粘附所需时间短,对冻存后的耐受性较好。结论组织块贴壁法适合内皮细胞和平滑肌细胞的培养,有限稀释法能够纯化原代培养的内皮细胞,大鼠主动脉平滑肌细胞和内皮细胞在细胞形态、增殖、粘附、对胰酶的反应、冻存后复苏均存在差异。     

树鼩原代小胶质细胞的分离培养、纯化与鉴定

本文旨在建立树鼩(Tupaia belangeri)小胶质细胞原代培养及纯化的方法,为利用新型实验动物树鼩进行相关研究工作提供实验材料。将新生树鼩大脑皮质机械分离,皮质组织块用胰蛋白酶消化后制成细胞悬液;培养9～10 d后,分别采用直立手拍法、温和胰酶消化法以及恒温振荡法分离纯化树鼩小胶质细胞,通过差速贴壁进一步纯化。荧光显微镜下,利用小胶质细胞的特异性标记物CD11b抗体进行鉴定。结果显示,小胶质细胞分离培养第3天时呈静息状态,表现为梭形、杆状、分支状等不规则形态。细胞免疫荧光CD11b呈阳性。不同纯化方法细胞免疫荧光并计数显示,直立手拍法所获得的细胞产量明显高于恒温振荡法(P 0.05),细胞阳性率( 96%)明显高于温和胰酶消化法( 90%,P 0.05)。直立手拍法可获得产量及纯度高的树鼩原代小胶质细胞。     

一种同时分离培养大鼠肝细胞及肝枯否细胞技术的建立

目的:建立简便、经济的同步分离培养肝细胞及kupffer细胞的方法.方法:采用肝脏原位灌洗结合离体胶原酶灌注消化的方法获得总细胞悬液,差速离心分离肝细胞及肝非实质细胞,经多次低速离心可分离肝细胞,经percoll密度梯度离心以及选择性贴壁法得到纯化的kupffer细胞.台盼蓝染色鉴定细胞活力.使用倒置相差显微镜、HE染色、PAS染色及白蛋白免疫组织化学染色对培养肝细胞的形态及功能进行检测.使用光学显微镜、荧光显微镜及CD68免疫荧光染色鉴定分离的kupffer细胞.结果:体外成功的同步分离培养了肝细胞及kupffer细胞,肝细胞产率为1.37± 0.53× 108/大鼠,kupffer得率为3.45± 0.41×106/g肝脏.细胞存活率及纯度都可达90％.肝细胞培养24h后呈典型肝细胞形态,7天后仍具有糖原合成和白蛋白合成能力.贴壁后的kupffer细胞呈典型的星型或三角形,且其标志分子CD68免疫荧光染色阳性.结论:应用改良的原位灌注方法可以很好的同时分离具有活性及功能的肝细胞和kupffer细胞.     

大鼠少突胶质前体细胞(OPCs)纯化培养及糖氧剥夺(OGD)模型的建立

目的建立大鼠脑少突胶质前体细胞(oligodendrocyte precursor cells,OPCs)分离纯化培养及糖氧剥夺(oxygen glucose deprivation,OGD)模型。方法出生3d内的SD大鼠乳鼠取脑,经胰蛋白酶消化法培养混合胶质细胞,混合培养10d后,震摇及差速贴壁法分离纯化OPCs,纯化培养3d后鉴定、诱导分化OPCs为少突胶质细胞(oligodendrocyte,OL)及进一步OGD干预。免疫荧光法鉴定OPCs纯度及分化为OL的能力;MTT法检测OGD(37℃,1%O2,5%CO2)干预0.5h、1h、2h及4h时细胞活力改变,Edu染色检测细胞增殖情况。结果免疫荧光显示纯化培养的OPCs 95%以上表达NG2+A2B5,且可分化为MBP阳性的OL。OGD 2h时,MTT显示细胞活力明显下降,Ed U染色阳性率明显降低。结论震摇及差速贴壁法可获得高纯度的OPCs,且细胞具有分化为OL的能力。2h可作为OPCs OGD模型缺血缺氧损伤合适时间。     

Long-term culture of adult rat hepatocyte spheroids

Hepatocytes from adult rats were cultured on poly-HEMA-coated surface to form spheroids in hormonally defined media as previously shown with newborn rat hepatocytes. Spheroidal aggregates of adult rat hepatocytes were morphologically similar to those of newborn rat hepatocytes and could also form a monolayer of uniform liver parenchyma-like cells when transferred on collagen-coated surfaces even after 2 months of culture. Under these culture conditions, albumin and transferrin secreted in vitro by adult rat hepatocyte spheroids were detectable by immunoprecipitation method at least until 2 months of culture. The production of proteins by hepatocyte spheroids could be regulated in vitro by IL-6: the secretion of alpha 2-macroglobulin was increased and the secretion of albumin was decreased in the presence of this cytokine. In addition, cytochrome P450 IA1 was strongly induced by methylcholanthrene in adult rat hepatocyte spheroids, and the induction remained relatively constant up to 22 days of culture. These cells were also able to metabolize lidocaine to monoethylglycinexylidine when measured up to 14 days of culture, showing the presence of a relatively high level of P450 IIIA2. The UDP-glucuronyltransferase activity, specific for bilirubin conjugation, decreased to 18% of the initial value after 2 weeks of culture. This work showed that adult rat hepatocytes in long-term spheroid culture kept differentiated functions, providing a new model for the in vitro study of hepatocyte functions and complementing that of newborn rat hepatocytes using the same system.     

胚胎小肠Cajal细胞的发育研究

目的研究人胚胎小肠cajal细胞的发育变化规律。方法采用全层铺片结合切片的免疫细胞化学技术。结果Cajal细胞呈酪氨酸激酶受体(Kit)和波形蛋白(vinlentin)免疫反应阳性。在胚胎发育早期,cajal细胞较少,为单层,稀疏分布于肌间神经丛周围,细胞为梭形,可见两个短而小的突起,未见分支;随着胎龄的增加,Cajal细胞数量增多,胞体增大,突起伸长,并出现分支。此时,肌间神经丛周围的Cajal细胞出现两层,其长轴彼此垂直,分别平行于环行肌和纵行肌。与此同时环行肌层内亦可见少许Cajal细胞;出生前,肌间神经丛部位的Cajal细胞接近成熟,两层细胞的突起进一步增多、伸长,彼此间形成与成人相似的完整的细胞网络。此时深肌丛附近亦可见少量Cajal细胞。结论人的小肠Cajal细胞发育有一定的时间顺序,即肌间神经丛周围最先出现,肌内次之,深肌丛较晚,出生前肌间神经丛周围的Cajal细胞已经接近成熟。这种发育演变若发生异常,可能导致某些胃肠动力障碍性疾病。     

Dose-dependent and time-limited proliferation of cultured murine interstitial cells of Cajal in response to stem cell factor

Interstitial cells of Cajal (ICCs) play a role as pacemakers for gastrointestinal movement. Although some in vivo experiments showed that the c-kit receptor tyrosine kinase (KIT) and its ligand, stem cell factor (SCF), might be required for the development of murine ICCs near birth, in vitro experiments would be useful to clarify the role of SCF-KIT system for the development of ICCs. We attempted to establish a culture system in order to investigate the proliferation of ICCs. Murine gastrointestinal cells from embryos or neonates were cultured with SCF and stained with anti-KIT antibody and/or alcian-blue. The numbers of KIT+ cells a n d alcian-blue+ cells we re counted, and the number of KIT+.alcian-blue- cells, which represent ICCs was calculated. Clusters containing KIT+ cells were formed in culture. The number of KIT+.alcian-blue- cells from day-18 post coitum embryos increased in response to SCF up to a concentration of 50 ng/ml or for 8 days. The number of cells from day-2 post-partum neonates increased for 4 days, and then remained constant in the presence of SCF. In contrast, the number of cells from day-6 post-partum neonates did not increase and remained constant, even in the presence of SCF. ICCs showed a dose-dependent and time-limited proliferation in response to SCF in the in vitro culture system used here in.     

高糖环境对豚鼠膀胱ICCs细胞形态及电生理的影响

目的：研究高糖环境下豚鼠膀胱Cajal间质细胞（interstitial cells of Cajal,ICCs）形态及细胞内钙离子荧光的改变,探讨糖尿病膀胱（diabetic cystopathy,DCP）的发病机制。方法：采用酶消化法原代培养,激光共聚焦技术观察5、10、15mol/L葡萄糖浓度下培养24、72h的ICCs,每浓度/时间取30个细胞测量其长度,再从中随机选10个细胞,以200ms/张图片的速度进行扫描,获得每个时间点细胞内钙离子荧光强度值。结果：糖浓度增高培养时间延长,ICCs内钙离子荧光值升高（P=0.00）,只有15mol/L/24h降低（P=0.00）;ICCs细胞长度缩短（P=0.00）,只有10mol/L/72h细胞长度增长（P=0.00）。结论：高糖环境对豚鼠膀胱ICCs形态学及电生理学造成显著影响,这种改变可能是造成DCP发病的重要原因之一。     

The lack of genotoxicity of sodium fluoride in a battery of cellular tests

In a comprehensive assessment of genotoxicity, sodium fluoride was evaluated in a battery of cellular tests providing different genetic end points and biotransformation capabilities. The tests included the following: rat hepatocyte primary culture/DNA repair assay, Salmonella typhimurium histidine locus reversion assay, adult rat liver epithelial cell/hypoxanthine guanine phosphoribosyl transferase mutation assay, and sister chromatid exchange in two target cell types, human peripheral blood lymphocytes and Chinese hamster ovary cells. Negative findings were made in all assays, indicating that sodium fluoride is not genotoxic in these assays.Abbreviations ARL adult rat liver epithelial cell - CHO Chinese hamster ovary cell - HGPRT hypoxanthineguanine phosphoribosyl transferase - HPBL human peripheral blood lymphocyte - HPC hepatocyte primary culture - SCE sister chromatid exchange     

A Comparison of the Polypeptide Pattern Between a Brain Primary Culture and Fractions of Bulk-Isolated Glial Cells

Abstract: This paper reports on the electrophoretic protein/polypeptide pattern of a rat brain primary culture. For comparison, the polypeptide pattern of neuronal and glial enriched fractions from adult rat brain and cerebral hemispheres from newborn and adult rat have been analysed. Water-soluble and SDS-extractable polypeptide fractions appeared and/or increased in amount in the cultures until confluency. The polypeptide pattern of the cultures most resembled that of the glial cell fractions, showing some of this fraction's specificity. Removal of fetal calf serum and addition of 0.1 mM dibutyryl cyclic adenosine monophosphate (dB-cAMP) produced few changes in the electrophoretic pattern. The study thus provides evidence in favour of the astroglial nature of the brain primary culture. It also shows that the cells undergo some maturation in the culture.     

Organotypic tissue culture of adult rodent retina followed by particle-mediated acute gene transfer in vitro

Background

Organotypic tissue culture of adult rodent retina with an acute gene transfer that enables the efficient introduction of variable transgenes would greatly facilitate studies into retinas of adult rodents as animal models. However, it has been a difficult challenge to culture adult rodent retina. The purpose of this present study was to develop organotypic tissue culture of adult rodent retina followed by particle-mediated acute gene transfer in vitro.

Methodology/Principal Findings

We established an interphase organotypic tissue culture for adult rat retinas (>P35 of age) which was optimized from that used for adult rabbit retinas. We implemented three optimizations: a greater volume of Ames'' medium (>26 mL) per retina, a higher speed (constant 55 rpm) of agitation by rotary shaker, and a greater concentration (10%) of horse serum in the medium. We also successfully applied this method to adult mouse retina (>P35 of age). The organotypic tissue culture allowed us to keep adult rodent retina morphologically and structurally intact for at least 4 days. However, mouse retinas showed less viability after 4-day culture. Electrophysiologically, ganglion cells in cultured rat retina were able to generate action potentials, but exhibited less reliable light responses. After transfection of EGFP plasmids by particle-mediated acute gene transfer, we observed EGFP-expressing retinal ganglion cells as early as 1 day of culture. We also introduced polarized-targeting fusion proteins such as PSD95-GFP and melanopsin-EYFP (hOPN4-EYFP) into rat retinal ganglion cells. These fusion proteins were successfully transferred into appropriate locations on individual retinal neurons.

Conclusions/Significance

This organotypic culture method is largely applicable to rat retinas, but it can be also applied to mouse retinas with a caveat regarding cell viability. This method is quite flexible for use in acute gene transfection in adult rodent retina, replacing molecular biological bioassays that used to be conducted in isolated cultured cells.     

Loss of interstitial cells of Cajal after pulsating electromagnetic field (PEMF) in gastrointestinal tract of the rats.

Exposure to the magnetic field has remarkably increased lately due to fast urbanization and widely available magnetic field in diagnosis and treatment. However, biological effects of the magnetic field are not well recognized. The myoelectric activity recorded from the gastrointestinal and urinary systems is generated by specialized electrically active cells called interstitial cells of Cajal (ICCs). Thus it seems rational that ICC have significant vulnerability to physical factors like an electromagnetic field. The aim of this study was to evaluate the influence of pulsating electromagnetic field (PEMF) (frequency 10 kHz, 30ms, 300 muT burst, with frequency 1Hz) on ICCs density in the rat gastrointestinal tract. Rats were divided into two groups (n=32). The first group was exposed to PEMF continuously for 1, 2, 3, and 4 weeks (n = 16), and the second group (n=16) served as a control. Tissue samples of the rat stomach, duodenum and proximal colon were fixed and paraffin embedded. The tangential sections of 5 microm thickness were stained immunohistochemically with anti-c-Kit (sc-168) antibody and visualized finally by DAB as chromogen (brown end product). C-Kit positive branched ICC-like cells were detected under the light microscope, distinguished from the c-kit-negative non-branched smooth muscle cells and from the c-kit positive but non-branched mast cells and quantitatively analyzed by MultiScan computer program. Apoptosis detection was performed with rabbit anti-Bax polyclonal antibody (Calbiochem, Germany) and LSAB 2 visualization system. The surface of c-Kit immunopositive cells decreased after exposure to PEMF in each part of the gastrointestinal tract. Reduced density of ICCs was related to exposure time. The most sensitive to PEMF were ICCs in the fundus of the stomach and in the duodenum, less sensitive were ICCs in the colon and pacemaker areas of the stomach. No marked changes in ICC density in the pyloric part of the stomach were observed. We demonstrate that the PEMF induced apoptosis dependent decrease in ICC expression.     

Biochemical evidence for cellular dedifferentiation in adult rat cardiac muscle cells in culture: expression of myosin isozymes

Myosin isozyme pattern in adult rat cardiac ventricular muscle cells in long-term culture was investigated. The myosin isozymes profile of cultured cardiac myocytes underwent a change in a serum-containing medium from two weeks onward, showing an embryonic rat ventricular myosin isozymes pattern that contained predominant isozyme V3. When adult cardiac myocytes were grown in a serum-containing medium supplemented with T4, these cells contained a predominant V1 band whose electrophoretic mobility and Ca2+-ATPase activity were comparable to those of the adult rat ventricle in vivo. This study has demonstrated that the adult cardiac ventricular muscle cells in long-term culture contain a predominant myosin isozyme V3 unlike their counterparts in vivo. Supplemented T4 modulated the embryonic type isozyme V3 to the adult type V1.     

Activities of enzymes of phospholipid and fatty acid synthesis in fetal and adult rat type II pneumocytes

Although differentiated fetal and adult type II pneumocytes are ultrastructurally similar, it is not known whether there are metabolic differences between them. We measured the activities of selected enzymes of phospholipid and fatty acid synthesis in fetal and adult rat type II cells, in late gestation fetal rat lung explants and in intact lung from rat fetuses of comparable gestational age. The activity of 1-acylglycerophosphocholine acyltransferase was significantly greater in adult type II cells than in fetal type II cells, fetal explants or intact fetal lung. The activity of CDP diacylglycerol:glycerol-3-phosphate 3-phosphatidyltransferase was similar in fetal and adult type II cells, but significantly lower in explants and intact fetal lung. There was a significant positive correlation between the percentage of alveolar epithelial cells in the cultures and tissue studied and CDP diacylglycerol:glycerol-3-phosphate 3-phosphatidyltransferase activity. This suggests that the previously reported correlation between phosphatidylglycerol synthesis and the percentage of alveolar epithelial cells in various lung culture systems may be related to the activity of this enzyme. Phosphatidylglycerol synthesis and CDP diacylglycerol:glycerol-3-phosphate 3-phosphatidyltransferase activity may be metabolic markers of type II cells, whereas the acyltransferase activity may be an indicator of type II cell maturation.