Dispase Ⅱ酶对人角膜缘干细胞培养结果的影响

目的 分析Dispase Ⅱ酶对人角膜缘干细胞培养的影响。方法 将110 例人角膜随机分为A、B 2组,在组织块培养过程中分别用和不用Dispase Ⅱ酶处理组织块,观察并分析2种处理方法培养时的细胞生长速度及培养成功率。结果 2 组细胞生长的最早及最晚时间均为(4.00±0.45)d和(11.00±0.27)d,培养成功率分别为93.54%±3.12%和98.51%±1.44%,不使用DispaseⅡ酶处理组比使用DispaseⅡ酶处理组不但培养成功率高,而且从培养至见到细胞生长时间早,统计学上有非常显著的差异(P<0 01,P<0 001)。结论 在人角膜缘干细胞的组织块培养中,不用Dispase Ⅱ酶处理可以得到更快的细胞生长速度,更高的培养成功率。

细胞培皿构形对毫米波辐照剂量的影响

对细胞层次上进行的毫米波生物学效应实验中使用的培皿内的毫米波辐照剂量进行了分析.以8mm波长的毫米波为参考,通过时域有限差分法计算了两种典型构形的培皿中辐照到细胞上的毫米波功率密度和细胞对毫米波的比吸收率的数据,并对结果进行了定量表示和定性分析.计算和分析结果表明,培皿构形对辐照到细胞上的毫米波功率密度和细胞对毫米波的比吸收率在培皿内分布的作用十分显著,对实验结果会造成较大影响,在相关实验中必须考虑.

动物无血清细胞培养基的研究现状及进展

动物细胞培养基是细胞赖以体外生长、繁殖、分化的重要因素。随着哺乳动物细胞培养规模的扩大和生物药物需求的增长,基于细胞及产品特性的无血清培养基的研制已成为细胞工程领域的重要课题。本文主要对无血清细胞培养基及其常用添加成分、无血清细胞培养基在生物科学领域的应用、近年来的研究进展等进行论述。

大丽轮枝菌寡糖素诱导的棉花组培细胞的过氧化物酶表达分析

将已在察氏液体培养基中培养 3 d的强毒性 (V44 )或弱毒性 (V 6 4)大丽轮枝菌接种到新鲜的相同培养基中 ,加入棉花黄萎病感病品种 (S)或抗病品种 (R)的悬浮培养细胞 (或其细胞匀浆 )。培养 4d后收获真菌孢子 ,用于致萎力测定 ,收获菌丝体细胞壁用于制备真菌寡糖素 (H,来自 V44或 L,来自 V6 4) ,然后用该寡糖素诱导悬浮培养的棉花细胞 (S或 R)。棉花细胞的过氧化物酶 (POD)同工酶结果表明 :(1)所有寡糖素均诱导棉花细胞产生新的过氧化物酶 ;(2 )感病品种豫棉 6号的活组培细胞 S(或其细胞匀浆 )显著影响真菌 ,用生长过程中接触该活细胞 (或其细胞匀浆 )的真菌所制备的寡糖素再诱导该活细胞 (即 S→ L (H )→ S)时 ,棉花细胞在 36 h的 POD同工酶谱与其对照 ,即 L(H)→ S比较 ,酶带增减变化显著。致萎力测定结果表明感病品种的活细胞提高了病原毒性和致病力 ;(3)耐病 (豫棉 8号 )或抗病品种 (中棉 12号 )的活组培细胞 R(或其细胞匀浆 )对真菌的影响不同于感病品种。 R→ L(H )→ R在 36 h的 POD同工酶谱与 L (H)→ R比 ,酶带没有增减变化。但是在 H→ R或 H→ S中于 9h和 2 4h出现的酶带在 R→ H→ R或 R→ H→ S中分别提前到 6 h和 9h,致萎力测定结果表明抗病品种细胞对病原毒性影响很小。

烧伤皮肤再生的核心技术——原位培植干细胞复制皮肤

20 0 1年第二期中国烧伤创疡杂志以科普讲座的形式刊登了三篇烧伤皮肤再生医学的基础科研和临床应用的内容 ,受到广大读者的欢迎。应大家的要求 ,本刊再次刊登烧伤皮肤原位干细胞培植技术相关的内容五讲 。

人胚胎干细胞向肌腱细胞分化方法的专家共识

胚胎干细胞具有无限增殖能力，可能为组织工程提供肌腱细胞或肌腱祖细胞的来源。为规范人胚胎干细胞来源的肌腱细胞的分化方法，提高人肌腱细胞的纯度，保证分化方法的可重复性，使其更系统、规范和有效地应用于临床和生物安全性评价体系中，科技部国际合作专项有关专家制定了《人胚胎干细胞向肌腱细胞分化方法共识》。共识建议通过两步法将人胚胎干细胞诱导为肌腱细胞：首先利用不同材料表面将人胚胎干细胞诱导为成体间充质干细胞，然后采用高密度种植形成无支架组织工程肌腱，并利用静态或动态力学刺激在体内外环境下将成体间充质干细胞诱导成肌腱细胞。由此建立的体外组织工程肌腱可作为检测评价模型。对小分子化合物、医用材料和药物进行肌腱相关的毒理学系统分析和安全性评价。

异种血清对猪血管内皮细胞适应模型的影响

目的:观察胎牛血清对PAEC适应模型的影响。方法:以猪自体血清原代培养PAEC并传代,接种于96孔板后分别以5%正常人血清(NHS组)、胎牛血清(FCS组)和补体灭活的胎牛血清(FCS+C组)诱导6d,加入25%正常人血清,以MTT比色法检测细胞的存活率,并以免疫组化的方法观察各组HO-1的表达情况。结果:NHS组和FCS+C组细胞存活率均升高,同对照组相比差异显著,两组间亦有差异,HO-1表达均增加。FCS组与对照组无差异。结论:质量不佳的胎牛血清或补体灭活不充分的胎牛血清会干扰PAEC适应模型的诱导。

兔脂肪干细胞的体外成脂成骨诱导及BrdU标记情况的初步研究

目的探索兔脂肪间充质干细胞体外分离培养的方法及生物学特性,并探讨5-溴脱氧尿嘧啶核苷(BrdU)标记脂肪间充质干细胞的效果。方法取兔颈后脂肪采用胶原酶消化方法分离培养干细胞,诱导培养基定向成脂、成骨细胞诱导及组织化学鉴定。采用BrdU法标记干细胞,免疫组化法鉴定其体外标记情况。结果兔脂肪组织中可以分离培养出生长旺盛的干细胞,定向诱导后表现出成脂,成骨细胞的特性。BrdU可标记干细胞的核,体外标记率达95%。结论兔脂肪干细胞易于体外分离培养,具有较强的自我更新能力及多向分化的潜能。BrdU对脂肪干细胞的标记效果满意,操作简单易行。

二甲双胍对炎症状态下人视网膜血管内皮细胞生物学行为的保护作用及其机制

背景研究表明炎性过程参与糖尿病视网膜病变（DR）的发生和发展，而炎症作用的靶细胞为视网膜血管内皮细胞（RVECs）。二甲双胍是临床上常用的降血糖药物，近年来表明其可发挥保护心血管、预防肿瘤和保护血－脑屏障等多重生物学效应，但其对炎症状态诱导的人视网膜血管系统异常是否具有防护作用尚不清楚。目的观察二甲双胍对肿瘤坏死因子-α（TNF-α）刺激下人视网膜血管内皮细胞（RVECs）增生、移行以及分泌单核细胞趋化蛋白-1（MCP-1）和白细胞介素-8（IL-8）的影响，探讨二甲双胍对炎症环境中人RVECs生物学行为的保护作用。方法对原代RVECs进行培养和传代并分为正常对照组、5 mmol/L二甲双胍组、TNF-α（2.5 ng/ml）组、TNF-α＋不同浓度（5、10、20、40 mmol/L）二甲双胍组，分别按照分组方法在培养液中添加相应药物处理24 h。分别于处理前及处理后24 h采用Image-Pro Plus Software 7.0软件计数各组细胞数目；采用MTS法检测各组RVECs的吸光度（A490）值以评价细胞的代谢活力；采用Transwell小室法检测各组迁移细胞数；采用ELISA法检测各组细胞上清液中MCP-1和IL-8质量浓度，并对各组间检测结果进行比较。结果正常对照组、5 mmol/L二甲双胍组、TNF-α组、TNF-α＋5 mmol/L二甲双胍组、TNF-α＋10 mmol/L二甲双胍组、TNF-α＋20 mmol/L二甲双胍组和TNF-α＋40 mmol/L二甲双胍组细胞数目总体比较差异有统计学意义（F＝189.31，P〈0.01）；各组细胞代谢活力（A490值）分别为0.32±0.02、0.32±0.03、0.97±0.02、0.90±0.05、0.76±0.15、0.74±0.05和0.41±0.03；各组细胞移行数目分别为（1 214±49）、（1 200±45）、（1 648±43）、（1 309±48）、（1 279±73）、（961±60）和（942±106）/视野；各组细胞上清液中MCP-1质量浓度分别为（0.385±0.050）、（0.362±0.060）、（2.285±0.200）、（1.131±0.180）、（0.622±0.120）、（0.537±0.090）和（0.492±0.130）μg/ml，IL-8质量浓度分别为（0.385±0.080）、（0.390±0.120）、（1.123±0.130）、（0.899±0.180）、（0.680±0.060）、（0.417±0.090）和（0.335±0.100）μg/ml，组间A490值、移行细胞数、MCP-1质量浓度和IL-8质量浓度的总体比较差异均有统计学意义差异（F＝73.31、103.89、150.92、268.32，均P〈0.01），TNF-α组细胞数目、A490值、移行细胞数、MCP-1质量浓度和IL-8质量浓度均明显高于正常对照组，TNF-α＋10 mmol/L二甲双胍组、TNF-α＋20 mmol/L二甲双胍组、TNF-α＋40 mmol/L二甲双胍组细胞数目、A490值、移行细胞数、MCP-1质量浓度和IL-8质量浓度均明显低于TNF-α组，差异均有统计学意义（均P〈0.05）。结论二甲双胍对TNF-α诱导的人RVECs增生、移行及分泌MCP-1、IL-8的能力均有抑制作用，从而可能对炎症环境中的RVECs发挥保护作用。

Chemical Constituents of the Suspension Cell Cultures of Maytenus hookeri

Suspension cell cultures of Maytenus hookeri Loos. (Celastraceae) in SH media were established from the calli induced from the leaves and young steins of M. hookeri on MS media with the supplement of 2 mg/L 2,4-D and 0.1 mg/L KIN (kinetin). Ethyl acetate extract of the cultures showed inhibitory activities against Penicillium avellaneum UC-4376 which was sensitive to maytansinoids. Exhaustive isolation of natural products from a large scale of suspension cell cultures did not yield maytansine instead of affording nine compounds including one novel triterpenoid, named 2, 3-diacetoxyl maytenusone (1), and eight known ones including squalene (2), beta-sitosterol (3), 2', 3', 4-triacetyl-sitoindoside I (4), salaspermic acid (5), maytenonic acid (6), 2alpha-hydroxy-maytenonic acid (7), 6, 11,12-trihydroxy-8, 11, 13-abietrien-7-one (8) and 11, 12-dihydroxy-8, 11, 13-abietatrien-7-one (9) elucidated on the basis of 1D and 2D NMR data. The H-1-NMR and C-13-NMR assignments were made for 1, 5, 6 and 7, while the C-13-NMR assignments for 5 and 6 were revised. The chemical results suggested that the suspension cell cultures of M. hookeri did not produce maytansinoids under the reported experiment conditions.

氮水平和形态配比对巴西橡胶树花药苗生长及氮代谢、光合作用的影响

采用砂培方法研究了不同供氮水平及NH4+/NO3-配比对巴西橡胶树花药苗生长、氮代谢及光合作用相关指标的影响。结果表明,随供氮水平的提高,橡胶花药苗茎秆、叶、地上部及单株总干重、植株各部位氮含量和氮积累量、叶绿素含量均相应升高,其中粗根、茎秆、叶片含氮量、单株吸氮量及叶绿素含量各处理间差异均达显著水平;株高、茎粗、细根干重、叶片光合速率先增后减,且以8 mmol/L氮水平时最大;低氮水平下细根硝酸还原酶(NR)活性较高。不同NH4+/NO3-配比试验中,纯NH4+营养下小苗各部位氮含量、氮积累量最高(除细根和叶柄氮的积累量);NH4+/NO3-为75/25时,小苗株高、茎粗、叶柄、叶片、植株地上部及株干重最大,NH4+/NO3-为50/50时,小苗细根干重、茎秆干重、叶绿素含量、光合速率最大,且显著大于纯NO3-营养处理;而NH4+/NO3->50/50时,处理间差异不显著(除细根和茎秆N含量);纯NO3-营养下小苗细根NR活性最高,但生长最弱。结果说明橡胶花药苗不适宜于纯NO3-环境,而适宜于纯NH4+及NH4+/NO3-混合营养,其适宜水平为8 mmol/L和NH4+/NO3->50/50。

Cell Growth and Ajmalicine Accumulation in a Full Habituated Catharanthus roseus Cell Line C_ (20)hi

A full habituated cell line C_ 20hi was screened from 2,4_D dependent line (C_ 20D) of Catharanthus roseus (L.) G. Don. The investigation involved the cell growth, ajmalicine production and enzyme activity related to indole alkaloid biosynthesis in both cell lines. These results indicated that C_ 20hi cells grew faster than C_ 20D cells, and average ajmalicine content in C_ 20hi cells was 18.4 times more than that in C_ 20D when cultured in the production medium. In the growth medium, average ajmalicine content in C_ 20hi cells was 31.9 times more than that in C_ 20D cells, while the cell growth has no obvious difference. The comparison of enzyme activities in C_ 20hi and C_ 20D cells indicated that tryptophan decarboxylase (TDC), strictosidine synthase (SSS) and geraniol_10_dehydrogenase (G10H) activities have no close relation to ajmalicine accumulation, although the activities of these enzymes were higher when cells were cultured in the production medium than in the growth medium. The C_ 20hi cells are relatively stable in five years of culture.

Productions of Taxol and Related Taxanes by Cell Suspension Cultures of Taxus yunnanensis

A high taxol yield cell line of Taxus yunnanensis Cheng et L. K. Fu keeps a high taxol_producing level after successive subcultures for more than eight years. In this study, eight taxanes were isolated from the suspension cell cultures of this cell line. Based on NMR and MS analyses, and comparison with literature data and standards, their structures were determined to be 2α,5α,10β_triacetoxy_14β_propionyloxy_4(20),11_taxadiene (1), 2α,5α,10β_triacetoxy_14β_(2′_methyl)_butyryloxy_4(20),11_taxadiene (2), 2α,5α,10β_14β_tetra_acetoxy_4 (20),11_taxadiene (3, taxuyunnanine C), 2α,5α,10β_triacetoxy_14β_(2′_methyl_3′_hydroxy)_butyryloxy_4(20),11_taxadiene (4, yunnanxane) and its 3′_epimer (5), baccatin Ⅳ (6), baccatin Ⅲ (7) and taxol (8), respectively. Among those compounds, 3, 5, 6 and 7 were reported to be isolated from the suspension cell cultures of T. yunnanensis for the first time. TLC and HPLC analyses indicated that the chemical constituents of the culture solution were similar to those of cultured cells. Moreover, the highest taxol content of this cell line reached 0.3% and the cell line could be applied for a large_scale culture.

Callus Induction and Cell Suspension Culture of Eggplant(Solanum melongena L.)

[Objective] The aim of this article is to establish a cell suspension culture system for eggplant. [Method] Using orthogonal design, appropriate media were screened for callus induction, subculture and cell suspension culture. [Result] The appropriate medium for callus induction was MS supplemented with 0.2 mg/L 6-BA and 0.2 mg/L NAA; the appropriate subculture medium was MS supplemented with0.2 mg/L 6-BA, 0.2 mg/L NAA, and 0.1 mg/L KT; the suitable medium for cell suspension culture was liquid MS medium supplemented with 0.4 mg/L NAA and 0.2mg/L. Cell growth curve was similar to ＂S＂ shape in the above suspension medium.The cell growth included four phases, the initial phase（1-3 d）, the logarithmic phase（3-7 d）, the steady phase（7-8 d）, and the decline phase（8-11 d）. With the increasing culture time, the number of suspension cells increased, and it reached the maximum value at the 7thd, about 3.8 ×105cells/ml. Then the number of cells began to decline rapidly. The cell vigor was the highest at the initial phase. Suspension cells grew best in the liquid MS medium supplemented with NAA（0.4 mg/L）and 6-BA（0.2 mg/L）. The mitotic index reached the maximum, about 4.1% in the above medium, which suggested that this medium was suitable for cell suspension culture of eggplant. [Conclusion] Cell suspension culture system of eggplant provides a significant method for eggplant biotechnology. Genetic transformation and mutants screening can be carried out with this system.

Vascular Channel Formation by Osteosarcoma Cells in Vitro: Vasculogenic Mimicry

Objective: To observe whether there is evidence for vascular channel formation by osteosarcoma cellsin vitro and to illustrate mechanism of vasculogenic mimicry in osteosarcoma.Methods: Osteosarcoma cell lines (U-2OS) were tested for their ability to form tubular networks in three-dimensional culture containing type I collagen. The structures of the tubular networks were observed under a phase contrast microscope and an electron microscope.Results: Observation under light microscopy and electron microscopy showed that high aggressive osteosarcoma cells line (U-2OS) formed networks containing channels when grown in three-dimensional culture containing type I collagen in the absence of endothelial cells or fibroblasts.Conclusion: These observations strongly suggest that aggressive osteosarcoma cells may generate vascular channels that facilitate tumor perfusion independent of tumor angiogenesis and have the ability of vasculogenic mimicry. Key words osteosarcoma cells line - vasculogenesis mimicry - angiogenesis - 3-dimensional cultures This study was supported in part by the National Natural Sciences Foundation of China (No. 30271314).

Single Polar Compound Bioelectret Material and Its Influence on the Cell Growth

To explore the influence of compound bioelectret material′s dielectric property on the cell growth,several kinds of compound bioelectret materials of collagen/chitosan were developed Their TSDC(thermally stimulated depolarization current)spectra were analyzed,and the compound bioelectret collagen/chitosan whose t α and I α were 37℃ and 2×10 -9 A respectively at polarized state was selected The cell culture study showed that the compound bioelectret material could promote normal cell growth when singly negatively polarized,and could inhibit cancer cell growth when singly positively polarized It proves that the rational designation of compound bioelectret has a broad application for clinical medicine

Study on the Browning in Cell Suspension Culture of Taxus cuspidata

[Objective] This study aimed to investigate the browning of T. cuspidata cells in suspension culture and provide the guidance for the cell suspension culture of T. cuspidata. [Method] T. cuspidata callus was used as experimental materials, to explore the effect of different medium, N/P ratio, pH, shaking speed, illumination time and light intensity and other factors on browning of T. cuspidata cells in suspension culture. [Result] Non-browning callus was transferred to 2MB5 medium （pH 7.0） for illumination culture at 22℃ under light intensity of 1 500 lx with shaking speed of 90 r/min for 24 h. Results showed that the cell browning was significantly inhibited. [Conclusion] This study laid the foundation for cell suspension culture of T. cuspidata and had important significance to the large-scale industrial production of paclitaxel.

A Study of Rabbit Lens Epithelial Cells Survival and Growth on the Rabbit Capsular Bag in Vitro

Objective: To study the proliferation, migration and metaplasm of residual rabbit lens epithelial cells (LECs) after extracapsular cataract extraction(ECCE)based on the rabbit capsular bag model in vitro. Methods: Sham cataract surgery, including anterior capsulorhexis, nucleus hydroexpression and aspiration of lens fibers, was performed on 20 rabbit lens. The capsular bags were isolated and pinned to sterile non-toxic silicone rings on petri dishes. The capsular bags were incubated with Eagle's minimum essential medium (DMEM) supplemented with 10% fetal calf serum (FCS) and monitored for 3 weeks by phase-contrast microscopy, after which light microscopy was performed on them.Results: After a latent period of 2-3 d, outgrowth was observed across the posterior capsule. Growth proceeded rapidly so that the posterior capsule was totally covered by a confluent monolayer of cell at 6-8 day. Capsular wrinkles became increasingly apparent as time progressed, causing a marked rise in light scatter. An increase in capsular tension also came.Conclusion: This model exhibits many of the in vito characteristics of the lens capsule after extracapsular surgery and may prove useful in further elucidating the cellular mechanisms of posterior capsule opacification and developing strategies for inhibiting cell growth with this system.

Primary Culture of Bovine Mammary Epithelial Cells

[ Objective] To investigate the feasibility of the primary culture of bovine mammary epithelial cells in biochemical incubator. [ Method] In vitro, bovine mammary epithelial cells were isolated and cultured by the tissue explant method in order to investigate the optimal culture conditions. The morphology observation and identification of the cultured cells were performed by inverted microscope observation, Giemsa staining and cytokeratin immunohistochemistry. [ Result] Observed with inverted microscope, most of the bovine mammary epithelial cells were polygonal and displayed typical slabstone-like appearance. As it can be seen from cell staining results, the cell body was big and the nucleus was stained dark blue and was round or oval in shape, with clearly visible nucleoli, generally 2 -4 nucleoli. The tissue-specific expression of cytokeratin 14 and cytokeratin 18 genes in mammary epithelial cells was identified by cytokeratin immunohistochemistry. [ Conclusion] Primary bovine mammary epithelial cells were successfully cultured in biochemical incubator.