Cu2+对离体培养猪腺垂体细胞GH分泌的影响

目的：研究铜离子（Cu2＋）对离体培养猪腺垂体细胞生长激素（GH）分泌的影响。方法：试验选取32-35日龄仔猪腺垂体细胞,于10%小牛血清的Dulbecco MEM培养液（DMEM）中离体培养48 h。之后用含不同浓度Cu2＋（0 mg/L、0.025 mg/L、0.1 mg/L、0.4 mg/L、1.6 mg/L）的无小牛血清DMEM培养液培养36 h,于12 h、24 h、36 h收集细胞培养液,用Linco公司的试剂盒及放射免疫法测定培养液中GH浓度。结果：铜离子离体培养腺垂体细胞24 h,0.025 mg/L、0.1 mg/L、0.4 mg/L组培养液中GH浓度均高于0 mg/L组,其中0.1 mg/L组与0 mg/L组差异显著（P〈0.05）。结论：铜离子可促进离体培养猪腺垂体细胞分泌GH。     

17β雌二醇对谷氨酸钠诱导增殖的新生大鼠大脑皮质星形胶质细胞细胞周期的影响

目的探讨17β雌二醇对谷氨酸钠诱导增殖的星形胶质细胞细胞周期的影响.方法将细胞周期同步化处理的培养星形胶质细胞(1)加入不同浓度(0、10、100、1000 nmol/L)的17β雌二醇;(2)加入浓度100nmol/L的17β雌二醇分别作用24、48和72h;(3)加入浓度100 nmol/L17β雌二醇和1mmol/L谷氨酸钠分别作用24、48和72h;采用流式细胞术观察星形胶质细胞周期的变化.结果 (1)100、1000nmol/L 17β雌二醇促进星形胶质细胞增殖效果明显;(2)100 nmol/L17β雌二醇具有强化1mmol/L谷氨酸钠促进星形胶质细胞增殖的作用,效果一直延续到72h.结论雌激素具有促进星形胶质细胞增殖的作用,与谷氨酸钠共同作用,有一定的协同效应.     

甲状腺素T4对体外高温培养奶牛乳腺上皮细胞生长和凋亡的影响

以体外培养的奶牛乳腺上皮细胞为模型,采用台盼蓝染色绘制生长曲线,细胞流式检测细胞凋亡,以正常培养温度(38℃)为对照,研究体外高温培养条件下(42℃),添加不同浓度(0.01、01和1mol/L)甲状腺素(thyroxine,T4)对细胞生长和凋亡的影响.结果表明,不同浓度的T4在38℃有促进奶牛乳腺上皮细胞生长的趋势,但是变化不显著(P>0.05),而T4对缓解高温所造成的乳腺上皮细胞的生长抑制作用也不显著(P>0.05);不同的T4都能够极显著缓解42℃培养1 h和3 h的奶牛乳腺上皮细胞的凋亡(P<0.01),并且对缓解42℃培养3 h的细胞凋亡效果更加明显,但是对42℃培养5 h和8 h的细胞,仅1 μmol/L的T4能够极显著缓解其凋亡(P<0.01).结果提示,T4对高温造成的奶牛乳腺上皮细胞的生长抑制没有明显的缓解作用,但能缓解高温诱导的奶牛乳腺上皮细胞凋亡.     

二甲双胍对人脐带间充质干细胞形态、增殖、表面标志及细胞周期的影响

目的探讨不同浓度二甲双胍（METF）对人脐带间充质干细胞（hUC-MSC）形态、增殖、表面标志及细胞周期的影响。 方法取健康足月新生儿脐带在体外分离出hUC-MSC进行传代培养,至第3代（流式细胞仪分析）对细胞进行鉴定,取第6代处于对数生长期的hUC-MSC （相对老化）,将对照组与不同浓度METF （0.1,1,5,10,20?mmol/L）干预的细胞进行比较,观察不同浓度METF干预对细胞的形态、增殖率（MTT法分别于24、48、72?h检测）、及细胞表面标志和细胞周期的影响,采用One-Way ANOVA,及LSD-t检验进行统计学分析。 结果（1）METF为0.1?mmol/L、1?mmol/L,细胞形态无显著改变,当药物浓度为5?~?20?mmol/?L时,随着药物浓度增加、培养时间延长,细胞形态改变越显著。（2）METF为0.1?mmol/L（24?h：101.28±0.98,24?h：104.06±1.76,24?h：101.51±0.67）促进hUC-MSC增殖,药物浓度为1?~ 10?mmol/L在培养初期可增加间充质干细胞的增殖率,随着培养时间的延长,细胞的增殖逐渐被抑制。METF为20?mmol/L（24?h：86.64±0.66,48?h：58.38±2.52,72?h：17.75±1.35）抑制细胞增殖,抑制作用随着时间延长而增强（P?< 0.05）。（3）当METF浓度为5,10,20?mmol/L时,随着药物浓度的增加,CD105的表达逐渐减弱（F?= 17.539,P?< 0.05）。METF未对CD44、CD90产生影响。（4）METF为0.1?mmol/L时降低G0/G1期的比例（64.16±1.20,P?< 0.05）,促进间充质干细胞的增殖,随着药物浓度的增加,细胞增殖逐渐被抑制。 结论METF浓度在0.1mmol/?L促进hUC-MSC增殖,而在浓度5 ~ 20?mmol/L时抑制人脐带间充质干细胞的增殖及表面标志CD105的表达,不同浓度的METF均未对CD44、CD90的表达产生影响。     

INS-1E细胞葡萄糖毒性模型的建立

目的:建立胰岛细胞系INS-1E细胞的葡萄糖毒性模型。方法:将INS-1E细胞分别在不同葡萄糖浓度(5.5 mmol/L、16.7mmol/L、25 mmol/L、30 mmol/L)的1640完全培养基中培养不同时间(48 h、72 h、96 h、120 h),分别在不同时间点取细胞进行细胞功能检测,实时荧光定量PCR法检测胰岛素m RNA的表达,ELISA检测葡萄糖刺激的胰岛素的分泌。结果:与对照组相比,高糖浓度(5.5 mmol/L、16.7 mmol/L、25 mmol/L、30 mmol/L)培养基中培养48 h后,INS-1E细胞的胰岛素合成和分泌的功能均增加(P均0.05),随着培养基中葡萄糖浓度的升高以及培养时间的延长,INS-1E细胞胰岛素合成及分泌的功能逐渐下降,当在葡萄糖浓度为30 mmol/L的培养基中培养120 h后,胰岛素m RNA合成及葡萄糖刺激的胰岛素分泌均显著降低(P均0.01)。结论:INS-1E细胞在30 m M的葡萄糖中培养120 h形成稳定的葡萄糖毒性模型。     

MS-275联合平阳霉素对Tca-8113细胞生长和凋亡的影响

目的:观察组蛋白去乙酰化酶抑制剂(HDACIs)MS-275联合抗生素类化疗药物平阳霉素(PYM)对口腔鳞状癌细胞Tca-8113的生长及凋亡的影响。方法:以体外培养的口腔鳞状细胞癌Tca-8113细胞为研究对象,应用MTT法检测不同浓度(0、1、2、4、8μmol/L)MS-275、(0、0.05、0.1、0.2、0.4μmol/L)平阳霉素(PYM)单独和联合用药对Tca-8113细胞增殖活性的影响;Annexin-V-FITC/PI双染流式细胞术定量检测细胞的凋亡情况。结果:不同浓度(1、2、4、8μmol/L)MS-275和(0.05、0.1、0.2、0.4μmol/L)PYM均可显著抑制Tca-8113细胞的增殖,并促进其凋亡,且呈浓度依赖性(P0.05)。4μmol/L MS-275和0.2μmol/L PYM联合应用时,其抑制Tca-8113细胞增殖和促进其凋亡的作用均显著高于单独应用MS-275或PYM(P0.05)。结论:MS-275能有效增强口腔鳞状细胞癌Tca-8113细胞对平阳霉素(PYM)的敏感性,体外实验中呈现较好的抗肿瘤效应。     

五味子甲素对大鼠成骨细胞增殖分化的影响

目的　探讨五味子甲素体外对大鼠成骨细胞增殖与分化的影响。方法　用改良的组织块法分离培养新生大鼠颅骨成骨细胞 ,五味子甲素以不同浓度加入细胞培养体系 ,作用不同时间后 ,用MTT法检测成骨细胞的增殖情况 ;用对硝基苯二钠基质动力学法 (PNPP)测定细胞内碱性磷酸酶的活性 ,用改良的Lowry法测蛋白含量。 结果　五味子甲素在 0 75× 10 -5mol/L及 0 75× 10 -9mol/L浓度范围内 2 4h ,72h ,0 75× 10 -4 ～ 0 75× 10 -7mol/L 48h促进成骨细胞增殖 ,在 0 75× 10 -5～ 0 75× 10 -6mol/L浓度范围内 48h提高成骨细胞内碱性磷酸酶的活性。结论　五味子甲素体外能促进成骨细胞的增殖与分化。     

促甲基化因子对西索米星发酵的影响*

研究发现,发酵培养基中添加2·0~3·0g/L蛋氨酸或7·5~10·0mg/L氯化钴可明显促进西索米星的合成。蛋氨酸的添加时机和添加方式对西索米星产物合成的作用明显不同。在产物合成中前期(30~48h)添加蛋氨酸的效果最佳。当发酵液中蛋氨酸初始浓度为0·656g/L时,与在产物合成初期一次性添加相比,1·5g/L蛋氨酸在产物合成初期、中期和后期均分成3次添加的效果更优,当发酵至91h结束时,发酵液中西索米星浓度可达0·70g/L。     

牛αS1-酪蛋白5＇调控序列驱动人α-LA基因与LacZ基因在牛乳腺上皮细胞中的表达

将牛αS1-酪蛋白5＇调控序列约1．2kb的片段,连接到含SV40启动子调控下β-半乳糖苷酶基因（LacZ）的PSV载体上,做为启动子,在其后连接0．76kb的人α-乳白蛋白基因（α-LA）,构建真核表达载体αS1-LA-psv．采用组织块接种法,培养奶牛乳腺上皮细胞,经传代纯化后,对细胞接种存活率、群体倍增时间、生长曲线、形态学等生物学性状进行检测,用免疫荧光细胞染色法对培养的奶牛乳腺上皮细胞进行角蛋白18鉴定,结果表明,成功建立奶牛乳腺上皮细胞系,细胞传至20代以上时仍保持旺盛的增殖活力．将构建的真核表达载体αS1-LA-psv转染奶牛乳腺上皮细胞,培养24～120h均检测到了β-半乳糖苷酶的表达;培养72h检测到细胞中人α-乳白蛋白的表达,表达量约为0．64g／L．实验结果表明,建立的奶牛乳腺上皮细胞系具有外源基因表达活性,得到的牛αS1-酪蛋白5＇调控序列能作为启动子指导外源基因的表达,构建的真核表达载体能在体外培养的牛乳腺上皮细胞中同时表达人α-乳白蛋白和β-半乳糖苷酶．     

纳米硒对肉鸡肝细胞中细胞谷胱甘肽过氧化物酶活性的影响

以亚硒酸钠和蛋氨酸硒为对照,研究了纳米单质硒(纳米硒)对肉鸡肝细胞中细胞谷胱甘肽过氧化物酶(cGPx)活性的影响。每种硒源分别以0.01、0.05、0.10、0.30、0.50、1.0μmol/L6个硒添加浓度培养肉鸡肝细胞,测定培养后0、24、48、72、96h肉鸡肝细胞cGPx活性。结果显示:亚硒酸钠添加浓度(以硒计)在0.01￣0.10μmol/L、蛋氨酸硒和纳米硒添加浓度(以硒计)在0.01￣0.30μmol/L,cGPx活性随着硒添加浓度的增加而增加;亚硒酸钠添加浓度在0.10￣1.0μmol/L、蛋氨酸硒添加浓度在0.30￣1.0μmol/L,cGPx活性随着硒添加浓度的增加而下降,而纳米硒添加浓度在0.30￣1.0μmol/L,cGPx活性始终保持在高峰平台。结果表明,3种硒源的剂量-效应关系曲线中的最适剂量范围宽度依次为:纳米硒>蛋氨酸硒>亚硒酸钠。     

l-threonine production by polyauxotrophic mutants ofArthrobacter globiformis

A number of methionine, methionine+lysine-, and (methionine+lysine+isoleucine)-auxotrophic mutants producing threonine have been isolated from a glutamate-producing strain ofArthrobacter globiformis by a three-step mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine. The best double mutant ML24 requiring methionine and lysine for growth produced 3.2 gl-threonine per L in the synthetic Alföldi medium (200 mmol/L) glucose, 80 mmol/L ammonium nitrate) supplemented with 5 μg biotin per L and optimum (0.5 mmol/L) methionine and lysine concentrations.     

In vitro development of bovine embryos in Buffalo rat liver- or bovine oviduct-conditioned medium

A culture system for bovine embryos was developed using Buffalo rat liver cell (BRL) line-conditioned medium without serum. Zygotes, obtained by in vitro maturation and fertilization of oocytes, were cultured either in unconditioned medium (TCM 199 or DMEM/F12) or in the same medium conditioned by bovine oviduct or BRL cells. No serum was added during conditioning or during embryo culture. The DMEM/F12 medium was superior to TCM 199 for development of bovine embryos to the 5 to 8-cell stage: on average between 50 and 57% of the embryos reached this stage after 2 d of culture in DMEM/F12 or in conditioned medium, while 36% reached this stage in TCM 199. Further development to the blastocyst stage was enhanced by conditioning. The highest percentage of blastocysts was achieved in DMEM/F12 medium conditioned with BRL cells (30%). The yield of blastocysts was similar in TCM 199 and in DMEM/F12 media conditioned with bovine oviduct cells (22 versus 20%), but after conditioning with BRL cells, DMEM/F12 medium yielded a higher percentage of blastocysts than TCM 199 (30 versus 18%). This might be explained by the fact that viability of BRL cells was better in DMEM/F12 medium than in TCM 199 when serum was omitted. Blastocysts produced in BRL-conditioned medium had a higher number of cells than blastocysts obtained in bovine oviduct-conditioned medium, and their transfer to recipients led to pregnancies and birth of calves. In conclusion, culture of bovine embryos in DMEM/F12 medium conditioned with BRL cells without serum led to the development of good-quality blastocysts and is thus a promising method for producing embryos for the study of potential embryotrophic factors. The use of rat liver cell lines guarantees against bovine viruses and allows for better production of embryos.     

芦笋抗S—（2—氨乙基）—L—半胱氨酸（AEC）变异体的筛选

S-(2-氨乙基)-L-半胱氨酸(AEC)可抑制芦笋愈伤组织的生长,此抑制作用可被赖氨酸或甲硫氨酸部分解除。用0.5mmol/L的AEC进行筛选,得到抗性愈伤组织AR10并再生植株。AR10愈伤组织经一年多的继代培养,在离开选择剂组培继代两代后仍保持对AEC的抗性。抗性系愈伤组织还表现出对2mmol/L的半胱氨酸具交叉抗性,对1mmol/L的赖氨酸加苏氨酸表现部分交叉抗性。AR10再生植株一部分保持对AEC的抗性,而一部分则无抗性。对抗性愈伤组织及其再生植株的氨基酸分析表明,愈伤组织内游离赖氨酸、苏氨酸、甲硫氨酸都有增加,而在再生植株内却发现半胱氨酸和赖氨酸的特异性增加,分别是对照植株的5.4和4.6倍。     

小鼠精原干细胞在三种培养基中的生长行为

目的：建立小鼠精原干细胞（SSCs）的体外长期培养体系。方法：用分别添加了等量的胶质细胞源神经营养因子（GDNF）、可溶性GFRα1和hFGF的DMEM／F12、KSR和StemPro-34 SFM三种无血清培养基和MEF饲养层分别培养经差异贴壁分选富集的小鼠SSCs,通过形态观察、标志基因的RT-PCR和免疫细胞化学分析检测其SSCs本原。结果：DMEM／F12与KSR可支持小鼠SSCs在体外存活6-7d,而StemPro-34 SFM能能维持SSCs体外增值一个月。结论：StemPro-34 SFM支持小鼠SSCs的体外增殖。     

利用高通量生物反应器优化一种CHO细胞无血清培养基

研究以DMEM/F12(1:1 V/V)培养基为基础,添加不同添加剂优化一种适宜CHO DG44细胞生长的廉价培养基。以细胞密度和细胞活率为主要指标,对DMEM/F12(1:1 V/V)培养基进行了优化。通过正交试验和单因素试验筛选出了CHO DG44细胞生长的最佳培养基。正交试验结果表明添加8mg/L Insulin、10mg/L Transferrin、12mM Glutamine、9mg/L Ethanolamine、9mg/L Sodium selenite、0.5×Lipids、0.5×Vitamin,对细胞生长有较好促进作用,细胞密度从0.6×106 cells/mL上升到1.8×106 cells/mL。在此基础上添加2.5g/L Malt Peptone和2.5g/L YeastExtract可使细胞密度达到2.65×106 cells/mL,基本上达到商业培养基的培养效果,而成本降低了约60%。     

Effects of stem cell factor and granulocyte macrophage-colony stimulating factor on survival of porcine type A spermatogonia cultured in KSOM.

Spermatogenesis is initiated with the divisions of the type A spermatogonial stem cells; however, the regulation of this stem cell population remains unknown. In order to obtain a better understanding of the biology of these cells, type A spermatogonia were isolated from 80-day-old pig testes by sedimentation velocity at unit gravity. The cells were cultured for up to 120 h in Dulbecco's modified Eagle's medium/Ham's F-12 medium (DMEM/F12) or a potassium-rich medium derived by the simplex optimization method (KSOM). At the end of the 120-h culture period, 30-50% of the spermatogonia were viable in KSOM, whereas in DMEM/F12 very few cells survived. Using KSOM as the culture medium, the effects of stem cell factor (SCF) and granulocyte macrophage-colony stimulating factor (GM-CSF) were studied. SCF significantly enhanced the percentage of cell survival at 100 ng/ml but not at lower concentrations. In comparison, GM-CSF promoted survival at relatively low concentrations (0.01, 0.1, and 1 ng/ml). At a higher dose (10 ng/ml), a significant reduction in percentage of cell survival was observed. The combination of SCF with GM-CSF had no significant effect on the percentage survival of type A spermatogonial cells. These data indicate that SCF and GM-CSF play a role in the regulation of survival and/or proliferation of type A spermatogonia.     

胎鼠中脑多巴胺细胞混合培养方法探索

目的 探索一种新的胚胎大鼠腹侧中脑黑质细胞的混合培养方法,以获得具有高比例多巴胺神经元的原代细胞体系,有利于在体外条件下进行帕金森病(Parkinson's disease,PD)的发病机制和防治的研究.方法 分离E15 (Embryonic Day 15)SD胎鼠中脑黑质区域组织,分散为单细胞后,分别用DMEM/F12+ 10％FBS含血清培养基和Neurobasal+ N1无血清培养基设置不同组别进行培养,通过免疫组织化学方法检测在不同培养条件下细胞的生长状态及多巴胺能神经元的比例.结果 DMEM/F12+ 10％ FBS与Neurobasal+ N1先后一周交替换液的的培养体系中获得的TH阳性神经元占神经元的比例可达31％左右,明显高于单独使用DMEM/F12+ 10％ FBS培养组(约10％).结论 DMEM/F12+ 10％ FBS与Neurobasal +N1先后一周交替换液是一种黑质神经元和胶质细胞混合培养并能获得高比例多巴胺神经元的有效培养方法.     

Amino acid-rich medium (Leibovitz L-15) enhances and prolongs proliferation of primary cultured rat hepatocytes in the absence of serum.

Multiple rounds of cell division were induced in primary cultured rat hepatocytes in serum-free, modified L-15 medium supplemented with 20 mM NaHCO3 and 10 ng/ml EGF in a 5% CO2/95% air incubator. A 150% increase in cell number and DNA content was observed between day 1 and day 5. The time course of DNA synthesis of hepatocytes cultured in L-15 medium differed from that in DMEM/F12 medium in that there were four peaks of 3H-thymidine incorporation in the L-15 medium, at 60 h, 82 h, 96 h, and 120 h, but only one peak at 48 h in modified DMEM/F12 medium. Labeling studies of the hepatocytes indicated that more than 60% of the cells were stained with antibromodeoxyuridine (BrdU) antibody in the periods of 48-72 h and 72-96 h after plating at densities between 1.5 x 10(5) and 6.0 x 10(5) cells per 35-mm dish. Even at a density of 9.0 x 10(5) cells/dish, about 40% of the cell nuclei were stained with BrdU in the periods of 48-72 h and 72-96 h. In addition, about 20% of the hepatocytes in culture initiated a second round of the cell cycle between 48 and 96 h in culture. Proliferating cells, which were mononucleate with a little cytoplasm, appeared in small clusters or colonies in the culture from day 4. These proliferating cells produced albumin. The addition of essential amino acids to the DMEM/F12 medium enhanced the DNA synthesis of hepatocytes, thus indicating that the higher level of amino acids in L-15 medium may be an important factor in its enhanced ability to support the proliferation of primary cultured rat hepatocytes.     

Human epidermis reconstructed on synthetic membrane: Influence of experimental conditions on terminal differentiation

Summary Cell suspensions of human keratinocytes seeded onto cell culture inserts may undergo terminal differentiation in the absence of fibroblasts. Among the parameters that control these morphogenic events, exposure to air and the composition of the culture medium were investigated. In the latter case, three media were considered DMEM:Ham’s F12, MCDB 153, and keratinocyte SFM medium at equivalent calcium (1.5 mM) and fetal calf serum (5%) concentrations. Immunochemical methods and transmission electron microscopy show that cells cultured in DMEM:Ham’s F12 medium, and then raised at the air-liquid interface, form a basal layer plus suprabasal cell layers corresponding to thestratum spinosum, stratum granulosum, andstratum corneum. The suprabasal keratinocyte layers show morphologies that resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-filaggrin granules. When the cultures are kept submerged, the keratinocytes show occasional keratohyalin granules and are connected by fewer desmosomes. Additionally, no properstratum corneum is formed. In keratinocyte SFM medium and MCDB 153, cultures raised at the air-liquid interface are not able to form an epithelium of normal architecture and do not express terminal differentiation markers. Differentiation is initiated, however, since desmosomes and bundles of keratin filaments appear; on the other hand, filaggrin is not expressed even after 28 d in culture. Membrane-bound transglutaminase is expressed throughout the entire suprabasal compartment in MCDB153 and DMEM:Ham’s F12 media but never appears in keratinocyte SFM medium. These studies show the relative independence of epidermal differentiation program to the composition (including the calcium concentration) of the media contacting the dermis and filling the extracellular space. Conversely, differentiation appears to depend on elements of basal medium and/or components synthesized by keratinocytes under the influence of the culture medium.     

l-lysine production by S-2-aminoethyl-l-cysteine-resistant mutants ofArthrobacter globiformis

Using mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, a number of homoserine auxotrophs have been isolated from a glutamate-producing Arthrobacter globiformis excreting L-lysine in good amounts. For further improvement, mutants resistant to the lysine analog S-(2-aminoethyl)-L-cysteine have been isolated from homoserine auxotrophs. For the three potent mutants tested, White's medium was found to be the best. Glucose, ammonium nitrate and biotin were found to be optimum at 280 mmol/L, 40 mmol/L and 22 nmol/L, respectively. With optimal glucose, ammonium nitrate and biotin, the strain AECrVI yielded 36 g lysine per L in flask culture.