大鼠胰岛微血管内皮细胞分离、纯化与培养的改进

目的：建立稳定可靠的大鼠胰岛微血管内皮细胞分离培养方法。方法：分离纯化大鼠胰岛后进行内皮细胞选择性培养，使用UEA-1包被的免疫磁珠对胰岛微血管内皮细胞进行纯化。免疫荧光法检测经典的内皮细胞标志物第Ⅷ因子相关抗原（vWF）和CD34的表达和吞噬Dil标记的乙酰化低密度脂蛋白（Dil-Ac-LDL）能力。结果：胰岛培养4-5 d后，可见内皮细胞从贴壁的胰岛内爬出，通过UEA-1包被的免疫磁珠筛选出胰岛微血管内皮细胞接种后24 h细胞开始贴壁。该细胞具有单层生长、接触抑制的特性。大鼠胰岛微血管内皮细胞表达vWF和CD34，可摄取Dil-Ac-LDL。结论：本研究方法是一种较为高效的分离、纯化和培养大鼠胰岛微血管内皮细胞的方法。     

成年大鼠胰岛细胞的体外培养

目的： 建立一种高效、实用的成年大鼠胰岛细胞分离、纯化和体外培养的方法。方法：采用胰管内逆行灌注胶原酶溶液消化,密度梯度离心纯化胰岛,显微镜下手挑法捡出全部胰岛。分离后胰岛置胶原中培养，利用RT-PCR检测胰岛细胞特异基因的表达。结果：分离的胰岛细胞呈圆形或椭圆形，双硫腙染色呈猩红色，在胶原中培养能保持形态完整，表达胰岛细胞特异性基因PDX-1和insulin。 结论：本胰岛分离技术已趋于完善,获得的胰岛纯度高，培养效果好。     

Wistar大鼠胰岛细胞体外分离、纯化及鉴定

目的 探索Wistar大鼠胰岛分离纯化的最佳条件.方法 采用肝胰管内灌注胶原酶消化分离胰岛及Ficoll 400密度梯度离心纯化.纯化后的胰岛经组织学染色、电镜及放射免疫法鉴定其特异性和活力.结果 组织学染色显示纯化后胰岛的活力和纯度分别在95%和85%以上.电镜显示纯化后的胰岛形态完整,包膜清晰,分泌颗粒丰富.放射免疫结果表明,低糖组和高糖组分泌胰岛素的浓度有显著差异,证明胰岛功能良好.结论 肝胰管内灌注胶原酶消化法是一种好的消化方法.影响胰岛收获量的因素很多,例如胰腺的充分扩张,胶原酶的浓度和活性以及消化的时间等.     

组织块法培养SD大鼠的成骨细胞及鉴定

背景：获得足够量高纯度的成骨细胞比较困难,因此掌握简单而快速提取成骨细胞并进行培养鉴定势在必行。 目的：应用组织块法对SD大鼠成骨细胞的体外培养与鉴定。 方法：取新生(<24 h)SD大鼠颅骨,去除周围多余的组织,将剔净颅骨剪成1 mm3大小的碎块,分别用常规方法和组织块法(改良)方法进行成骨细胞培养,从形态学、碱性磷酸酶和茜苏红染色等方法鉴定。 结果与结论：利用改良方法培养的细胞具有典型的成骨细胞形态特征,碱性磷酸酶呈阳性染色,茜素红染色后有矿化结节的形成。组织块法培养的成骨细胞具有典型的成骨细胞成分单一、细胞培养时间短、数量、纯度、密度均一致,从而的得到稳定的成骨细胞系,为进行体外实验建立了良好的平台。     

六黄合剂对胰岛素抵抗大鼠胰岛β细胞的保护作用

目的探讨中药复方六黄合剂对胰岛素抵抗大鼠胰岛β细胞的影响。方法动物随机分为空白对照组、模型对照组、六黄合剂组,每组8只。应用地塞米松致大鼠胰岛素抵抗模型,给予六黄合剂干预,检测空腹血糖(FBG),放射免疫分析法检测血清胰岛素(FINS)水平,化学比色法检测胰腺丙二醛(MDA)含量、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)的活性,透射电镜观察胰岛β细胞超微结构的变化。结果与模型对照组相比,六黄合剂组FINS水平和胰岛素抵抗指数(HOMA-IR)明显下降(p0.01),胰腺MDA含量下降(p0.01),SOD活性升高(p0.05)、GSH-Px活性升高(p0.01)。电镜观察IR大鼠胰岛β细胞可见凋亡早期改变,六黄合剂组β细胞超微结构的病理改变明显减轻。结论中药复方六黄合剂对β细胞的保护作用可能与减轻IR大鼠胰腺的氧化应激和增强抗氧化能力相关。     

原代分离的大鼠胰岛细胞对葡萄糖刺激胰岛素分泌的反应性研究

目的:研究原代分离的大鼠胰岛对葡萄糖刺激的胰岛素分泌反应性。方法:胶原酶原位灌注法分离大鼠胰岛,在含0.5%BSA、5.5或11.1mmol/L葡萄糖的培养基中培养不同时间后,用含0.2%BSA、3.3mmol/L葡萄糖的KRB缓冲液预培养胰岛30min,分别换入含不同浓度葡萄糖KRB缓冲液,培养1h,收集上清,RIA法测定胰岛素浓度。结果:大鼠胰岛过夜培养后,在基础(3.3mmol/L)和高浓度(16.7mmol/L)葡萄糖条件下胰岛素分泌量分别为(12.4±3.2)和(45.2±4.2)μU/ml/10islets/h;5.5mmol/L和11.1mmol/L葡萄糖浓度下培养12h和20h后,胰岛对葡萄糖的反应性均明显高于16.7mmol/L和22.5mmol/L葡萄糖组(P<0.05);体外培养5d后,对高糖的反应性为(4.28±0.67)倍。结论:原代分离的大鼠胰岛可在(1～5)d内保持对葡萄糖的反应性。     

胚胎干细胞向胰岛细胞诱导分化的研究

胚胎干细胞(embryonic stem cell，ESC)因具有全能分化潜能而有望成为组织工程和细胞治疗中的重要种子细胞来源。如能将其诱导分化为能分泌胰岛素的细胞，将有助于解决胰岛素绝对或相对缺乏引起的糖尿病代谢失调状态。目前已有研究诱导分化出能分泌胰岛素的细胞，有的将之应用于糖尿病鼠模型并取得了可喜的效果。本对这一领域的研究现状进行综述。     

大鼠细小动脉平滑肌细胞分离培养的新方法

目的：探讨大鼠细小动脉平滑肌细胞分离培养的新方法。方法：取大鼠肺分支动脉,先切成小块进行胶原酶的消化,约 ８ ｈ,而后用含２０％小牛血清的 ＤＥＭＥ培养基贴块培养。结果：培养 ２４ ｈ,可见有大量细胞游出贴瓶底生长,７２ｈ已融合成片,呈典型的“谷峰”样长势。尚有少量内皮细胞,通过消化传代除去。取传第三代细胞进行抗α－ａｃｔｉｎ免疫组织化学染色,大量饮泡,基膜下有密斑,密体。免疫组化鉴定培养细胞纯度为９６％。结论：应用消化贴块法培养的大鼠平滑肌细胞,方法简单,结果可靠,具有应用价值。     

大鼠脊髓祖细胞的分离与体外培养

采用bFGF、EGF及神经元无血清培养基分离培养大鼠脊髓祖细胞，并对脊髓祖细胞进行了分化和电生理实验。脊髓祖细胞可增生分化成神经元和胶质细胞，并具有电流特性。有希望应用于脊髓创伤的治疗。     

一种新生SD大鼠皮质源性神经元的培养方法

BACKGROUND: Primary culture in vitro of neurons plays an important role in the development, regeneration, signal transduction mechanisms, neuropharmacology and gene expressions of the nervous system. OBJECTIVE: To establish a simple method for primary culture of high-purity cortical neurons in neonatal Sprague-Dawley rats. METHODS: Cortical tissues were acquired from neonatal Sprague-Dawley rats born 1 day. In traditional experimental group, the whole cortex was removed; in improved experimental group, the cortical tissues, 2-3 mm thick on the brain surface were removed. Single cell suspensions were prepared after papain digestion and centrifugation and were then seeded onto 24-well culture plates containing neuron solutions for primary culture (1×105 per well). Cells were identified by neuronal specific markers MAP-2 and Tuj1 after 3-day culture. The number of neurons and neurite length were observed under inverted phase contrast microscope and recorded at 6, 24, 48 and 72 hours, 5 and 7 days of culture, resprctively. RESULTS AND CONCLUSION:The cultured cells expressing MAP-2 and Tuj1 were neurons that could be used in the following experiments. The purity of neurons in the improved experimental group was 92% at 3 days, while only 51% in the traditional experimental group. Cells in both two groups had attached to the wall presenting with small processes at 6 hours, and a simple neural network formed at the 3rd day until dense neural networks could be found at the 5th day. To conclude, our culture method herein is simple and convenient, and can be used to produce neurons with high purity, which will be helpful for the experimental studies on cortical neurons from Sprague-Dawley rats. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Preparation and culture of isolated islets and dissociated islet cells from rodent pancreas

Summary Procedures for preparing isolated islets and completely dissociated single islet cells free from the surrounding acinar tissue are described. The mixture of Ficoll and Conray solution (Ficoll-Conray solution) employed for the collection of islets is less viscous and toxic than the plain Ficoll solution and is also applicable for the separation of cells other than islets. Dissociation of islet cells by Dispase results in a satisfactory yield of single islet cells with viability greater than 95%. The islets and dispersed islet cells can be maintained in culture for several months with no deterioration of hormone-producing ability.     

Cultures of human fetal pancreatic islet cells

Cultures of islets cells were obtained from the cadaveric pancreas of 16–25-week human fetuses. During culture two waves of mitotic activity were observed. An increase in the insulin concentration in the culture medium took place after a wave of mitotic activity.Department of Artificial Organs and Assisted Circulation, Laboratory of Pathomorphology, Scientific-Research Institute of Transplantation and Artificial Organs, Ministry of Health of the USSR. Laboratory of Biological Standardization of Hormones, Institute of Experimental Endocrinology and Hormone Chemistry, Academy of Medical Sciences of the USSR. Maternity Home No. 26, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 8, pp. 202–204, August, 1979.     

Insulin-forming activity of monolayer cultures of bovine fetal pancreatic islet cells

The insulin concentration in the growth medium of primary monolayer cultures of bovine fetal pancreatic islet cells grown in the presence of a normal and increased (300 mg%) glucose content, was determined by a radioimmunologic method. A high glucose concentration led to increased secretion of insulin. The results of the cytological study showed definite correlation between mitotic activity of the cells of the culture and the intensity of insulin secretion into the medium.Laboratory of Biological Standardization of Hormones, Institute of Experimental Endocrinology and Hormone Chemistry, Academy of Medical Sciences of the USSR, Moscow. Laboratory of Pathomorphology, Institute of Transplantation of Organs and Tissues, Ministry of Health of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. A. Yudaev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 8, pp. 235–238, August, 1978.     

大鼠大脑皮层神经干细胞培养方法比较

目的:比较不同培养基对大脑皮层神经干细胞增殖的影响,以寻找培养神经干细胞的有效方法。方法:体外分离生后24 h SD大鼠大脑皮层细胞,使用DMEM/F12+B27+bFGF、Neurobasal+B27+bFGF和Neuro-basal+B27三种培养条件,分别用悬浮培养法和贴壁培养法对细胞进行培养。免疫细胞化学技术分别以Nestin、β-TubIII和GFAP标记神经干细胞、神经元和星形胶质细胞。结果:在悬浮培养情况下,三种培养条件均诱导所培养的细胞形成神经球,经染色鉴定神经球中的细胞均表达Nestin;其中DMEM/F12+B27+bFGF培养的神经干细胞增殖速度最快(P<0.01),Neurobasal+B27培养的神经干细胞增殖效率最高(P<0.01)。在贴壁培养情况下,经染色鉴定,部分细胞表达β-TubⅢ或GFAP。结论:大脑皮层神经干细胞不适合贴壁培养,DMEM/F12+B27+bFGF条件适合大脑皮层神经干细胞体外增殖。     

微囊化胰岛细胞移植治疗糖尿病的实验研究

目的观察微囊化大鼠胰岛细胞移植治疗糖尿病小鼠的疗效。方法将糖尿病小鼠随机分为三组：生理盐水注入组、未囊化胰岛细胞移植组和微囊化胰岛细胞移植组。将生理盐水、纯化大鼠胰岛细胞和囊化大鼠胰岛细胞分别移植于三组糖尿病小鼠腹腔。结果分离的胰岛细胞对刺激反应良好，微囊化和未囊化胰岛细胞移植后均可纠正糖尿病小鼠的高血糖状态，但微囊化胰岛细胞移植组可维持正常血糖水平更长时间。结论微囊化胰岛细胞移植治疗糖尿病小鼠具有良好的效果，微囊具有较好的免疫隔离作用。     

Isolation and primary culture of rat renal cortical epithelial cells

Summary Methods are described for the isolation of rat renal cortical epithelial cells by a collagenase and hyaluronidase perfusion, followed by pressing fragments of renal cortex through an 80-mesh screen and further enzymatic dissociation in vitro. Primary monolayer cultures are derived from the resulting suspension of tubular fragments and cells. Fibroblast and endothelial cell overgrowth is suppressed by the use of medium lacking arginine and containingd-valine in place ofl-valine. Further separation of fibroblasts from epithelial cells is achieved by a technique that takes advantage of the differential rate of attachment of the two cell types. The presence of glomerular cells in the cultures is diminished by a complete medium change 48 h after plating the cells.     

构建OGDH基因敲除型SD大鼠模型

目的构建酮戊二酸脱氢酶(OGDH)基因敲除SD大鼠模型。方法设计、构建TALEN重组子,用荧光素酶活性法筛选出最有效重组子,显微注射法将质粒打入受精卵后测序鉴定。对同一时间点的野生型和基因敲除型大鼠分别进行饮食习惯、活动状态、毛发颜色及质量检测,Western Blot检测两者肝组织OGDH蛋白表达。结果成功构建了靶向OGDH基因的TALEN质粒,并筛选了最有效的TALEN重组子(OGDH-T2),成功培育出杂合子18只,成功率为22.5%,未发现OGDH-KO8纯合子的出生。质量检测显示OGDH-KO8大鼠的质量明显高于野生型,且在第10周时最为显著,增幅率达50.2%(P0.001),KO组大鼠的OGDH蛋白表达水平显著降低,下降率为50.6%(P0.01)。结论利用TALEN技术成功构建了OGDH基因敲除型SD大鼠模型。     

大鼠骨髓源性内皮祖细胞的分离培养与鉴定

背景：内皮祖细胞因其分离与培养的方法各不相同,在实验中难以重复。 目的：探讨大量获取骨髓源性内皮祖细胞分离与培养的方法。 方法：通过密度梯度离心法从4周龄SD大鼠骨髓中分离单个核细胞,使用EGM-2 MV培养基进行诱导培养,采用形态学特征观察、摄取Dil-Ac-LDL与结合FITC-UEA-1实验、免疫荧光化学鉴定其表面抗原CD133与VEGFR2等方法对其进行鉴定,并通过管腔形成实验观察形成管腔的能力。 结果与结论：①形态学观察：分离的骨髓单个核细胞经诱导培养后,在生长的早期(8 d左右)、晚期(15 d左右)其细胞形态有一定差异,早期以纺锤形、三角形、圆形细胞多见,晚期以圆形、短梭形细胞多见。②摄取Dil-Ac-LDL与结合FITC-UEA-1实验：显示8,21 d的细胞均为阳性。③免疫荧光化学染色：8 d的细胞表达CD133、VEGFR2。④管腔形成实验：在Matrigel基质上15 h左右能够生成血管样结构。结果表明：利用密度梯度离心法分离大鼠骨髓单个核细胞后以EGM-2 MV进行诱导培养,经过鉴定证明获得的细胞符合内皮祖细胞的特征。这种方法能够简单、快速、可靠、大量地获取内皮祖细胞。     

新生大鼠离体海马神经元原代培养方法及鉴定

目的:建立一种稳定的生后大鼠离体海马神经元的培养方法。方法:无菌环境下将出生24 h内SD大鼠断头取脑分离海马,经消化后差速贴壁,采用无血清培养基进行培养,倒置显微镜下观察不同时间段细胞生长形态变化:采用Hoechst33258与NeuN抗体双染方法鉴定神经元。结果:采用差速贴壁后,使用无血清培养基培养的神经元生长良好,纯度较高,12 d时经鉴定神经元纯度可以达到92%以上。结论:差速离心法后可以采用无血清培养神经元,培养的神经元具有纯度高,结果稳定的优点,该方法为以后进行相关的研究奠定了基础。