粒径对微胶囊成膜及微囊化细胞生长代谢的影响

目的:考察粒径对微胶囊强度及微囊化细胞生长代谢的影响,为制备性能优良的生物微胶囊提供实验依据。方法:制备不同粒径的凝胶珠,测定其在相同成膜条件下的球磨强度,进而用台盘蓝拒染法测定微囊化细胞的增殖及活率。结果:小粒径的微胶囊具有更厚的微囊膜及更高的球磨强度,另外小粒径微胶囊培养细胞能够获得更多的细胞数(350μm,570μm和900μm微囊内的细胞数量分别为:5.67×107、4.71×107和3.89×107/mL microcapsule,P<0.05)及更高的细胞活率(350μm、570μm和900μm微胶囊的细胞活率分别为:83.70%、67.64%和75.73%,P<0.05)。结论:粒径能影响微胶囊的强度及微囊化细胞的生长、代谢。     

miRNA-21种子序列反义核酸及抗多发性骨髓瘤应用

成熟microRNA的种子序列通过与其靶mRNA的3'-UTR区完全互补结合,发挥负性调节作用.针对miR-21的种子序列设计并合成8个碱基的微小反义核酸(tiny anti-miR-21,t-antimiR-21),研究t-antimiR-21对多发性骨髓瘤的抑制效应.利用LipofectamineTM2000转染多发性骨髓瘤RPMI-8266细胞系,激光共聚焦显微镜检测荧光标记的反义寡核苷酸的细胞内定位,流式细胞仪检测转染效率;四甲基偶氮唑蓝(MTT)法测细胞增殖抑制率;台盼蓝拒染法测活细胞数;Annexin V/PI双染流式细胞仪检测细胞凋亡率.结果显示:t-antimiR-21主要定位于细胞质,与antimiR-21具有相同的转染效率,但是t-antimiR-21转染后降解较快.t-antimiR-21显著抑制细胞增殖和促进细胞凋亡,最佳作用浓度为0.4μmol/L,最佳作用时间为48 h.结果表明t-antimiR-21可用于血液系统相关肿瘤的实验治疗,miRNA-21可作为多发性骨髓瘤基因治疗的潜在靶点.     

人毛乳头细胞组织化学研究

毛乳头细胞是一种高度特殊化的成纤维细胞。本文通过对体外培养的毛乳头细胞进行组织化学染色研究发现,它对阿新蓝、甲苯胺蓝和PAS染色均呈阳性,并对甲苯胺蓝显异染性．与原位时的细胞染色结果相同,表明在体外培养下．毛乳头细胞合成和分泌酸性、中性粘多糖的能力仍能维持较长时间;在细胞聚集区和多层化细胞团中有丰富的细胞外基质,阿新蓝和PAS染色呈强阳性,说明细胞外基质的存在与毛乳头细胞的聚集有很大关系;另外毛囊真皮鞘细胞对阿新蓝、甲苯胺蓝染色呈阳性反应．无甲苯胺蓝的异染性,PAS染色阴性,而真皮成纤维细胞这些染色均阴性,说明它与毛乳头细胞关系密切。     

原位复染鉴别细胞的凋亡与坏死

用10μg／ml去甲斑蝥素作用于人红白血病K562细胞24h,诱导其发生凋亡。采用台盼蓝染液染色,再瑞特-吉姆萨染液原位复染的方法,验证了凋亡细胞与坏死细胞质膜通透性不同的说法。利用此方法可以在普通光学显微镜下把一张涂片上的凋亡细胞、坏死细胞及活细胞区别开来。该实验也表明,常规用台盼蓝染液拒染细胞数来统计活细胞数的方法是值得商榷的。     

不同频率聚焦超声与血卟啉对人体肿瘤细胞的协同作用

用 1 .4,2 .1 4,2 .1 6MHZ 频率的聚焦超声及超声结合血卟啉的方式分别对体外培养的人红白血病细胞K₅₆₂ 、人黑色素瘤细胞LiBr及人胃癌细胞进行照射 ,杀伤效果用以盼蓝染色 ,MTT和FDA法进行测试 .结果表明 ,同一细胞对不同频率超声有不同敏感性 ;声照条件相同时 ,不同细胞杀伤不同 .超声结合血卟啉照射会大大增加细胞死亡 ,同时未观察到细胞对声频的敏感性 .细胞杀伤测试表明台盼蓝法与MTT法有较好的一致性     

小鼠Kupffer细胞分离及细胞培养

目的 采用在体胶原酶灌注、不连续密度梯度离心、选择性贴壁3步法分离Kupffer细胞（Kupffer cells,KCs）,探讨其在分离小鼠KCs的应用及其对KCs生物活性的影响.方法 根据原位灌注和梯度离心方法不同随机分为4组：无胶原酶原位灌注＋3层梯度离心组（A）、无胶原酶原位灌注＋双层梯度离心组（B）、胶原酶原位灌注＋3层梯度离心组（C）和胶原酶原位灌注＋双层梯度离心组（D）.采用F4/80（BM8）免疫染色及吞墨实验判断细胞纯度和功能、台盼蓝拒染实验判断细胞的活力,探讨不同方法KCs分离的效果及细胞活性.结果 刚分离的KCs细胞近似圆形,接种l h后收获细胞纯度较高,但细胞得率相对较低.培养4 h后KCs得率相对较高,培养28 d仍能存活.免疫荧光可显示分离的为KCs,台盼蓝染色显示各组细胞的活力均在90 %左右,在体胶原酶灌注和双层梯度离心可以增加KCs的得率,双层梯度离心法可以增加分离KCs的纯度.结论 在体胶原酶灌注对提高KCs得率较为重要,在体胶原酶灌注、不连续密度梯度离心、选择性贴壁3步法分离小鼠KCs的的方法简便、高效、稳定,培养的KCs具有良好的细胞生物学性状.     

白屈菜红碱对宫颈癌细胞的抑制作用研究

目的:研究白屈菜红碱(chelerythrine,CHE)对宫颈癌细胞(Hela)的增殖抑制作用,为CHE在预防和治疗宫颈癌方面提供实验依据.方法:不同浓度CHE作用于体外培养的Hela细胞后,采用台盼蓝拒染法和噻唑蓝(MTT)法研究白屈莱红碱时宫颈癌(Hela)细胞增殖能力的影响,流式细胞仪(FCM)检测细胞周期变化.结果:细胞接种密度越小、药物作用时间越长、药物浓度越大,CHE对Hela细胞的增殖抑制率越大,过大的细胞接种密度以及过低的药物浓度均不会引起明显的细胞死亡;CHE作用后的Hela细胞周期分布有明显变化,G1期细胞减少,S期和G2/M期细胞明显增多.结论:CHE在体外能明显抑制Hela细胞生长,其机理可能与CHE阻滞细胞周期、诱导细胞凋亡有关.     

人颈椎间盘髓核细胞的体外培养和鉴定

目的：建立人颈椎间盘髓核细胞体外培养体系,并对其细胞表型进行鉴定。方法：采用酶消化法分离人颈椎间盘髓核细胞,进行单层培养,倒置相差显微镜观察细胞生长和形态,流式细胞仪测定细胞周期和凋亡率,并行甲苯胺蓝、Ⅱ型胶原及CK8免疫组化染色对其细胞表型进行鉴定。结果：原代髓核细胞凋亡率6.1±1.4%,S期细胞比例7.3±0.5%。贴壁后形态为多角形或短楔形,传代后生长加速。细胞呈甲苯胺蓝异染性;Ⅱ型胶原免疫组化染色阳性;只有少量椭圆形大细胞CK8免疫组化染色阳性。结论：成功建立人颈椎间盘髓核细胞体外培养模型,并证实成年后髓核内仍有少量细胞保持脊索细胞表型。     

人颈椎间盘髓核细胞的体外培养和鉴定

目的:建立人颈椎间盘髓核细胞体外培养体系,并对其细胞表型进行鉴定。方法:采用酶消化法分离人颈椎间盘髓核细胞,进行单层培养,倒置相差显微镜观察细胞生长和形态,流式细胞仪测定细胞周期和凋亡率,并行甲苯胺蓝、Ⅱ型胶原及CK8免疫组化染色对其细胞表型进行鉴定。结果:原代髓核细胞凋亡率6.1±1.4%,S期细胞比例7.3±0.5%。贴壁后形态为多角形或短楔形,传代后生长加速。细胞呈甲苯胺蓝异染性;Ⅱ型胶原免疫组化染色阳性;只有少量椭圆形大细胞CK8免疫组化染色阳性。结论:成功建立人颈椎间盘髓核细胞体外培养模型,并证实成年后髓核内仍有少量细胞保持脊索细胞表型。     

细胞计数板在荧光显微镜观察B细胞吞噬现象的应用探讨

目的:基于细胞计数板建立一种简单、快速使用免疫荧光显微镜观察B淋巴细胞吞噬卡介苗(BCG)现象的新方法,对即将进行流式细胞检测的样品进行质控,提高流式细胞术检测吞噬率的稳定性,同时为流式细胞仪检测吞噬率提供镜下依据。方法:B细胞与FITC标记的BCG共培养24 h后,PE anti-human CD19抗体直接标记细胞膜,应用细胞计数板在荧光显微镜下观察B细胞吞噬现象,流式细胞仪检测吞噬率。结果:应用细胞计数板在荧光镜下可观察到B细胞与BCG的荧光标记及B细胞与BCG共标记现象,证实B细胞可吞噬BCG,流式细胞仪检测结果显示吞噬率为13.9%。结论:应用细胞计数板在荧光镜下可观察B细胞吞噬现象,且操作简便快速,能对流式细胞检测的样品进行质控,并提供镜下依据。     

Discrete roles for histone acetylation in human T helper 1 cell-specific gene expression

To better understand the control of T helper (TH) 1-expressed genes, we compared and contrasted acetylation and expression for three key genes, IFNG, TBET, and IL18RAP and found them to be distinctly regulated. The TBET and the IFNG genes, but not the IL18RAP gene, showed preferential acetylation of histones H3 and H4 during TH1 differentiation. Analysis of acetylation of specific histone residues revealed that H3(Lys-9), H4(Lys-8), and H4(Lys-12) were preferentially modified in TH1 cells, suggesting a possible contribution of acetylation of these residues for induction of these genes. On the other hand, the acetylation of IL18RAP gene occurred both in TH1 and TH2 cells the similar kinetics and on the same with residues, demonstrating that selective histone acetylation was not universally the case for all TH1-expressed genes. Histone H3 acetylation of IFNG and TBET genes occurred with different kinetics, however, and was distinctively regulated by cytokines. Interleukin (IL)-12 and IL-18 enhanced the histone acetylation of the IFNG gene. By contrast, histone acetylation of the TBET gene was markedly suppressed by IL-4, whereas IL-12 and IL-18 had only modest effects suggesting that histone acetylation during TH1 differentiation is a process that is regulated by various factors at multiple levels. By treating Th2 cells with a histone deacetylase inhibitor, we restored histone acetylation of the IFNG and TBET genes, but it did not fully restore their expression in TH2 cells, again suggesting that histone acetylation explains one but not all the aspects of TH1-specific gene expression.     

人胚胎角膜上皮细胞系的建立和生物学特性

为构建角膜基础研究的稳定载体和操作平台,通过合法渠道获得人胚胎,并对角膜上皮细胞进行了分离培养。经反复传代,初步建立了人胚胎角膜上皮细胞系,并对其生物学特性进行了研究。结果显示,培养细胞具有较典型的上皮细胞特征。细胞生长较快,最快时两天可传代一次。K19和PCNA在各代胚胎角膜细胞均有表达,K19的表达部位位于胞浆,而PCNA的表达部位位于胞核。处于细胞周期中S期的细胞比例大约为11%￣23%。染色体核型分析表明各代细胞染色体的数目和形态相似。因此,人胚胎角膜上皮细胞适合于建立相对稳定的细胞系。培养细胞具有分化上的幼稚性和较强且稳定的增殖能力,细胞生长状态良好,而且该细胞系的遗传特征较为稳定。     

The PIWI‐Interacting RNA Molecular Pathway: Insights From Cultured Silkworm Germline Cells

The PIWI‐interacting RNA (piRNA) pathway, one of the major eukaryotic small RNA silencing pathways, is a genome surveillance system that silences selfish genes in animal gonads. piRNAs guide PIWI protein to target genes through Watson–Crick RNA–RNA base‐parings. Loss of piRNA function causes genome instability, inducing failure in gametogenesis and infertility. Studies using fruit flies and mice as key experimental models have resulted in tremendous progress in understanding the mechanism underlying the piRNA pathway. Recent work using cultured silkworm germline cells has also expanded our knowledge of piRNA biogenesis in particular, since these silkworm cells are the only cells of germline origin that can be cultured. In this review, we describe elucidation of the piRNA pathway using cultured silkworm cells as an experimental model by focusing on recent work in biochemistry and structural biology. Earlier studies that made important contributions to the field are also described.     

Embryonic undifferentiated cells show scattering activity on a surface coated with immobilized E-cadherin

Rearrangement of cell-cell adhesion is a critical event in embryonic development and tissue formation. We investigated the regulatory function of E-cadherin, a key adhesion protein, in the developmental process by using E-cadherin/IgG Fc fusion protein as an adhesion matrix in cell culture. F9 embryonal carcinoma cells usually form colonies when cultured on gelatin or fibronectin matrices. However, F9 cells cultured on the E-cadherin/IgG Fc fusion protein matrix formed a scattered distribution, with a different cytoskeletal organization and E-cadherin-rich protrusions that were regulated by Rac1 activity. The same scattering activity was observed in P19 embryonal carcinoma cells. In contrast, three types of differentiated cells, NMuMG mammary gland cells, MDCK kidney epithelial cells, and mouse primary isolated hepatocytes, did not show the scattering activity observed in F9 and P19 cells. These results suggest that migratory behavior on an E-cadherin-immobilized surface is only observed in embryonic cells, and that the regulatory mechanisms underlying E-cadherin-mediated cell adhesion vary with the state of differentiation.     

Generation of cloned goats (Capra hircus) from transfected foetal fibroblast cells, the effect of donor cell cycle.

The neomycin-resistant gene (neo(r)) is probably the most commonly used selectable marker gene in gene targeting and gene transfection research. In this study, the neo(r) gene construct was introduced into in vitro cultured goat foetal fibroblast cells (IV-5), and the cells were selected with 900 microg/ml G418. The G418-resistant colonies were analysed by neo-specific PCR, karyotyping and anti-intermediate filament proteins antibody (anti-vimentin) staining. Cell cycle analysis of the neo(r) positive foetal fibroblast cell colony (IV-5.1) cultured in a variety of cell cycle-arresting medium indicated that 74.2% of cells cultured in serum-deprived medium for 3 days and 71.7% of cells grown to confluence were at G0/G1 stage of cell cycle, respectively, in comparison to 61.6% of cells in normal culture (cycling) medium. Nocodazole treatment for 17 hr in vitro culture could increase the number of cells at G2/M stage of cell cycle from 20.3% (in cycling medium) to 39.7%. In total, one early pregnancy was observed by B ultra-sound scanning in a surrogate transferred with cloned embryos from IV-5.1 cells at M stage (cells were cultured in nocodazole medium). Seven cloned goats, including two that miscarried at a late stage, were derived from the IV-5.1 cell clone cultured in starved medium (G0). Indeed, one surrogate receiving three blastocysts reconstituted from the starved donor cells, gave birth to three live cloned goats, all of which are healthy and doing well. PCR, Southern blot and G418 resistance in vitro of fibroblast cells from cloned goats confirmed that all cloned goats are positive for neo(r) transgene. This study demonstrates that a foreign gene, such as the neo-resistant gene, can be introduced into goat foetal fibroblast cells, and that the resulting transgenic cells are capable of being cloned to produce 100% transgenic animals.     

全反式维甲酸体外诱导P19细胞向心肌方向分化

目的探讨不同浓度全反式维甲酸(all-trans retinoic acid,atRA)诱导P19细胞向心肌分化的效力。方法细胞分成P19细胞组,2nm/L atRA诱导组,5nm/L atRA诱导组,8nm/L atRA诱导组。各组细胞经过诱导、聚集培养、聚集体贴壁培养10天后,RT-PCR检测GATA-4,α-肌球蛋白重链(α-myosin heavychain,α-MHC)的mRNA表达,免疫荧光双标检测α-sarcomeric actin和cTnT蛋白共表达,Western blot检测cTnT的蛋白表达。结果 atRA可诱导聚集P19细胞表达GA-TA-4、a-MHC mRNA;α-sarcomeric actin和cTnT的表达和共表达增加;5nm/L atRA组,8nm/L atRA组GATA-4、a-MHCmRNA的表达量显著高于P19细胞组;5nm/L atRA组,8nm/L atRA组两种蛋白的表达和共表达量显著高于P19细胞组,以5nm/L atRA组最高,显著高于其它组。结论 atRA可诱导聚集P19细胞向心肌分化,其中5nm/L atRA组效果最好。     

A new human cholangiocellular carcinoma cell line (HuCC-T1) producing carbohydrate antigen 19/9 in serum-free medium

Summary A human cholangiocellular carcinoma cell line, HuCC-T1, was established in vitro from the malignant cells of ascites of a 56-yr-old patient. Histologic findings of the primary liver tumor revealed a moderately differentiated adenocarcinoma. Tumor cells from the ascites have been cultured with RPMI 1640 medium containing 0.2% lactalbumin hydrolysate and the cultured cells grew as monolayers with a population doubling time of 74 h during exponential growth at Passage 25. They had an epithelial-like morphology and were positive for mucine staining. Ultrastructural studies revealed the presence of microvilli on the cell surface and poorly developed organelles in the cytoplasm. The HuCC-T1 cell was tumorigenic in nude mice. The number of chromosomes in HuCC-T1 ranged from 61 to 80. These human cholangiocellular carcinoma cells in serum-free medium secreted several tumor markers, including carbohydrate antigen 19/9, carbohydrate antigen 125, carcinoembryonic antigen, and tissue polypeptide antigen. The carbohydrate antigen 19/9 secretion level of HuCC-T1 cells cultured in PRMI 1640 medium with 1% fetal bovine serum was sixfold higher than that with 0.2% lactalbumin hydrolysate. These findings suggest that HuCC-T1 will provide useful information to clarify the mechanism of tumor marker secretion and tumor cell growth in the human cholangiocellular carcinoma.     

Cultured proliferating rat mammary epithelial cells

Summary Normal epithelial cells from the rat mammary gland proliferated in culture when plated with lethally irradiated cells of the LA7 rat mammary tumor line. Proliferation of the normal rat cells occured as the LA7 cells slowly died from the radiation. By labeling the cultures with³H-thymidine it was determined that most of the proliferating rat cells were those adjacent to the LA7 feeder cells. The epithelial cells from the primary culture proliferated after subsequent passages if the cells were plated at each subculture with newly irradiated LA7 cells. If the cells were plated at a ratio of ∼1:8 rat:LA7 a confluent layer of normal rat cells covered the plastic substrate after 6 to 7 wk. The cells have so far been carried up through Passage 7, which amounted to ∼19 doublings in cell number, and still proliferate vigorously. The growth medium for this culture system was Dulbecco’s modified Eagle’s medium:Ham’s F12 1:1 supplemented with fetal bovine serum, insulin, and antibiotics. The presence in the cells of keratin, desmosomes, and cell junctions attested to their epithelial origin. The cultures were composed of cells with diploid or near diploid chromosome numbers. Samples of the cultured cells were implanted into the cleared fat pads of nude mice. Most of the implants from Passage 2 formed normal mammary ductal structures, but the incidence of outgrowths decreased significantly with later passages until no out-growths resulted from the implantation of cells from Passage 5. The one unusual, feeder-independent cell line that arose from a primary culture seemed to be immortal in culture, contained a hyperdiploid chromosome complement, and formed abnormal structures when implanted into cleared fat pads. This work was supported by the Veterans Administration, Washington, DC, and by CA grant 05388 from the U.S. Public Health Service, Washington, DC.     

Penaeid (Penaeus japonicus) lymphoid cells replicate by cell division in vitro

Penaeid cell culture has gained much attention as a potential model to facilitate researches on the characterization of the virus and to develop more sophisticated and improved diagnostic procedures for use in the aquaculture industry. However, to date, cell division processes of cultured penaeid cells have not been found, which is suggested as one of the reasons that block the establishment of the continuous penaeid cell lines. We reported here the cell division processes of cultured lymphoid cells of Penaeus japonicus. The culture medium used was based on M199 and was modified by supplementing saline components. Cultures were incubated at 25 degrees C, and 5% CO2 was supplemented. In primary cultured lymphoid cells, dividing cells in different shapes were found. Cell division processes of 12 dividing lymphoid cells were tracked. After cell division, their daughter cells turned into fibroblast-like or epithelioid cells. These results proved that the culture conditions used were suitable for lymphoid cells of I japonicus to proliferate in vitro and that cultured lymphoid cells still had the ability to carry out cell division. These findings would give light to the establishment of continuous penaeid cell lines and would also provide us with the knowledge of cell division processes of the penaeid.