金属离子对培养日本对虾肝胰腺细胞的影响

研究了培养基中分别加入Ca^2 、Mg^2 和Zn^2 对培养日本对虾肝胰腺细胞的影响,以测定细胞的RNA／DNA值作为评价指标。当Ca^2 浓度为1g／L时,细胞生长最好;本实验中随Mg^2 浓度的增加,培养的日本对虾肝胰腺细胞的RNA／DNA值也升高;当Zn^2 浓度为80μg／L时,其RNA／DNA值最高;培养基中混合加入Ca^2 和Mg^2 有助于细胞贴壁生长。     

青海湖裸鲤体外肝胰细胞原代与传代培养

采用组织块移植培养技术,分别用DMEM和RPM11640培养基对青海湖裸鲤肝胰组织细胞进行原代培养。培养48h组织块周围有细胞迁出,并形成生长晕。培养一周可形成单层细胞。对原代培养的单层细胞用胰蛋白酶-EDTA消化后,传代培养至第四代。确立青海湖裸鲤肝胰细胞培养条件为：培养基为DMEM,培养温度为27℃,pH值为7．0—7．5,原代培养血清浓度为20％,传代培养的血清浓度为10％,无需通入CO2和添加细胞生长因子。     

中华绒螯蟹白化症、正常肝胰腺组织结构及脂肪酸组成的比较研究

采用气相色谱、组织学及电镜技术分别对中华绒螯蟹雄蟹白化症肝胰腺与正常肝胰腺脂类和脂肪酸组成、显微及超微结构进行了比较研究,结果表明：正常肝胰腺的总脂含量（31.54%肝胰腺湿重）显著高于白化症肝胰腺（19.07%肝胰腺湿重）,两种蟹肝胰腺的脂类组成没有显著差异,但脂肪酸组成存在一定的差异,白化症肝胰腺中C18∶1的含量显著高于正常肝胰腺,中性脂和极性脂HUFA正常蟹肝胰腺含量都高于白化症蟹,但没有显著差异。组织学结果和超微结构观察表明：白化症肝胰腺肝小管的上皮细胞壁结构紊乱,微绒毛及微绒毛黏膜大部分脱落,细胞内仅见有零星的线粒体、脂肪滴分布,R细胞呈现空泡化。     

pH值对中国龙虾消化酶活力的影响

采用酶学分析方法研究了pH对中国龙虾胃蛋白酶、类胰蛋白酶、淀粉酶、纤维素酶和脂肪酶活力的影响。结果表明,在设定的pH范围内,中国龙虾各消化酶的活力均随着pH的升高呈现先升后降的变化趋势。其中,胃、肠、肝胰腺内胃蛋白酶最适pH均为2.2,类胰蛋白酶最适pH分别为8.8-9.2、8.4、8.8,淀粉酶最适pH分别为7.0、7.0、7.4,纤维素酶最适pH分别为4.2、4.2-4.6、5.4,脂肪酶最适pH分别为7.2-7.6、7.2、6.8-7.2。同时测得中国龙虾胃、肠、肝胰腺内的生理pH分别为5.33、6.93、6.60。中国龙虾的消化酶活力存在器官特异性。在最适pH下,胃蛋白酶活力顺序为胃>肠>肝胰腺,类胰蛋白酶、纤维素酶、脂肪酶的活力顺序均为肝胰腺>肠>胃,淀粉酶的活力顺序为肠>肝胰腺>胃。     

肝外胆管组织中干性细胞的研究现状和应用潜能

肝外胆管是肝脏胆管系统的组成部分,包括胆囊、胆总管等组织。近年来研究发现肝外胆管组织中存在一群具有干性特征的细胞。这群细胞在体外可以扩增培养,形成类器官结构,改变培养条件可以使胆管上皮干性细胞分化为具有一定功能的肝细胞、胆管细胞以及胰腺β样细胞等。将分化后的功能细胞分别植入肝脏受损、胆管缺陷、胰腺受损的模型小鼠中,功能细胞可以修复模型小鼠受损的肝脏或胆管,分化后的功能细胞甚至可以在胰腺受损的模型小鼠中分泌胰岛素。该文描述了肝外胆管组织的发育过程,阐明了干细胞的定位分布与具体特征,介绍了肝外胆管组织干细胞治疗疾病的相关应用,为后续研究启发思路并提供参考。     

西洋参细胞大量培养的研究

当西洋参细胞培养在Ms培养液中,KN0₃含量提高一倍而去掉NH₄NO₃。时细胞生长速率和皂甙产量分别比在正常培养液中提高65．1％和166．2％。黑节草寡糖素和人参寡糖素均有利于西洋参细胞的生长和皂甙含量的提高,尤其能增加Rg组皂甙的含量。西洋参细胞悬浮培养以生产皂甙收获的最佳时期为培养25天以上,其合成皂甙的高峰在细胞生长的对数期稍后出现。细胞悬浮培养和发酵培养过程中均未见pH值回升的现象。pH值稳定的发酵培养和pH值任其变化的培养相比,其皂甙含量,生长速率和生物量均要高。最后对细胞的培养方式进行了比较。     

Zn2+对中华绒螯蟹溞状幼体肝胰腺超微结构的影响

研究了不同Zn2+浓度对中华绒螯蟹(Eriocheir sinensis)()状幼体肝胰腺细胞超微结构的影响.当Zn2+浓度超过200 μg/L时,与对照组相比,肝胰腺结构受到了显著的影响.成熟和正在形成的B细胞的空泡中有很多含有金属的电子致密颗粒(EDG,可能为金属蛋白复合体),随着B细胞的成熟和B细胞从肝胰腺管壁上脱落,这些EDG也被释放到管腔中,因此肝胰腺的管腔中常有许多此类颗粒存在.B细胞空泡或管腔中的EDG与环境中Zn 2+浓度高低呈正相关.E细胞质中也出现了很多空泡,与正常E细胞的结构明显不同.R细胞的细胞质常解体形成大的空泡,被破坏的程度较为严重.在Zn2+浓度>1 000 μg /L时,肝胰腺遭到严重破坏,细胞结构几乎不存在.研究结果表明,肝胰腺的B细胞在Zn 2+的解毒方面起重要作用,但当Zn2+浓度超过了肝胰腺B细胞的解毒能力时,就会引起肝胰腺细胞(如R和E细胞)结构异常,甚至破坏整个肝胰腺细胞结构,从而影响幼体的生长,甚至造成死亡.     

Zn[2+]对中华绒螯蟹zao状幼体肝胰腺超微结构的影响

研究了不同Zn[2+]浓度对中华绒螯蟹(Eriocheir sinensis)zao状幼体肝胰腺细胞超微结构的影响。当Z[2+]浓度超过200 μg/L时，与对照组相比，肝胰腺结构受到了显著的影响。成熟和正在形成的B细胞的空泡中有很多含有金属的电子致密颗粒(EDG，可能为金属蛋白复合体)，随着B细胞的成熟和B细胞从肝胰腺管壁上脱落，这些EDG也被释放到管腔中，因此肝胰腺的管腔中常有许多此类颗粒存在。B细胞空泡或管腔中的EDG与环境中Zn[2+]浓度高低呈正相关。E细胞质中也出现了很多空泡，与正常E细胞的结构明显不同。R细胞的细胞质常解体形成大的空泡，被破坏的程度较为严重。在Zn[2+]浓度>1000 μg/L时，肝胰腺遭到严重破坏，细胞结构几乎不存在。研究结果表明，肝胰腺的B细胞在Zn[2+]的解毒方面起重要作用，但当Zn[2+]浓度超过了肝胰腺B细胞的解毒能力时，就会引起肝胰腺细胞(如R和E细胞)结构异常，甚至破坏整个肝胰腺细胞结构，从而影响幼体的生长，甚至造成死亡。     

无包涵体杆状病毒在中国对虾主要器官组织中的分布

研究自然发病和人工感染的中国对虾胃等10种器官组织内杯状病毒的分布情况,结果在病虾的胃、皮下组织、鳃、循环血细胞,肠的上皮和结缔组织细胞中,观察到大量病毒粒子,是该杆状病毒侵染的主要器官组织;在类淋巴、,肝脏、肝胰腺的组织细胞中,观察到中等数量的病毒粒子;在症状典型的病重虾腹神经索,肌肉组织细胞中观察到少量病毒粒子。表明病毒在病虾机体内广泛分布,已形成周身性感染。该病毒主要侵染以上器官的疏松结缔组织细胞、上皮细胞和循环血细胞,其次是神经胶质细胞和心肌细胞。在中国对虾的神经胶质细胞、心肌细胞、循环血细胞以及各主要器官的疏松结缔组织细胞中,目前尚未见到报道存在有杆状病毒粒子。     

Vero细胞培养A/Shanghai/CN02/2013(H7N9)Va流感病毒适宜条件研究

目的:优化Vero细胞培养H7N9流感病毒的培养条件,建立细胞培养病毒高产培养系统。方法:采用不同病毒接种MOI、培养温度、pH值和TPCK胰酶等条件优化培养H7N9流感病毒Vero细胞适应株A/Shanghai/CN02/2013(H7N9)Va的条件,并将其连续传代后扩大至细胞工厂大规模培养。结果:Vero细胞高产H7N9流感病毒培养条件为DMEM/F12和MEM以1∶1混合,并添加0.3 mg/mL牛血清白蛋白、1%谷氨酰胺、0.5%双抗和1.5μg/mL TPCK胰蛋白酶,pH为7.4时,连续传代病毒血凝效价保持在512,扩大至细胞工厂大规模培养病毒产量稳定。结论:建立的Vero细胞培养H7N9流感病毒条件,能够持续稳定高产,并适用于细胞工厂大规模培养,有望用于H7N9流感细胞疫苗的大规模制备。     

Inoculation and growth conditions for high-cell-density expansion of mammalian neural stem cells in suspension bioreactors

Inoculation and growth conditions for the large-scale expansion of mammalian neural stem cells (NSC) have been determined. We examined suspension culture bioreactors of murine NSC, and concluded that the oxygen level should be kept high (20%), and the osmolarity of the medium should be kept low (below 400 mOsm/kg). The pH of the medium was found to have a large effect on cell proliferation, and the best growth characteristics were obtained within an optimum pH range of 7. 1 to 7.5. The inoculation conditions were also seen to have a large effect not only on the growth characteristics, but also on the number of cells that die in the initial stages of the culture. For large expansion of cells, low inoculum levels (10(4) cells/mL) and single-cell suspensions proved superior, whereas, for fast expansion of cells, higher inoculum levels (10(5) cells/mL) and spheroid inoculum forms were preferred. The inoculum temperature of the medium did not have a large effect on growth characteristics, but the pH greatly influenced cell proliferation. Inoculum pH levels should also be kept between 7.1 and 7.5. If these protocols are followed, high multiplication ratios and viabilities can be obtained in a 5-day batch suspension culture bioreactor run. A large number of cells could then be used in animal models for testing of neural drugs and in research and development toward cures for neurodegenerative disorders such as multiple sclerosis (MS) and Huntington's and Parkinson's disease. The results presented here also point the way toward studies on in vitro expansion of human neural stem cells.     

Discovery of a human erythroid differentiation factor (EDF) and its large-scale production

A novel human protein exhibiting erythroid differentiation activity was discovered in the culture fluids of phorbol ester-stimulated human cells. The differentiation assay system involving Friend virus-derived mouse leukemia cells was used. THP-1 cells of myelomonocytic origin were typical producers. 4beta-Phorbol 12-myristate 13-acetate (PMA) was essential for inducible excretion of the erythroid differentiation factor (EDF). The factor was stable toward heat and pH (acidic or alkaline) but lost its activity on pronase treatment, which suggested its proteinous nature. After an optimization of the condition, production of EDF was performed on a 200-L scale for purification of the protein.     

趋磁细菌W M-1 的培养条件优化研究

采用正交设计实验法研究了趋磁细菌WM-1产磁性细胞的培养条件。并利用L16(44)方案,用16个实验完成了4种培养条件、4个水平的优化工作。研究结果表明,培养基的pH值是影响趋磁细菌WM-1产磁性细胞的重要因素,正交实验结果在90％的置信区间可信。在最优的培养条件下,即pH为7．0,氧气的浓度为4％,m酒石酸:m琥珀酸为1:1,NaNO3 100 mg l-1条件下,磁性细胞的浓度提高到6．5×107 cells ml-1,比优化前提高了约8．3％．趋磁细菌WM-1磁滞回线的测量表明,Hc=230 Oe,Ms=0．9 emu／g dry wt．Cells,及Mr／Ms=0．50。     

Characterization of factors influencing the growth of Anabaena variabilis in a bubble column reactor

The combined effect of superficial gas velocity, pH, initial phosphate concentration, and light intensity on cell growth was investigated for the mass production of cyanobacterial cells. The light intensity was manipulated to maintain a specific irradiation rate (q(i)) at a constant level for high cell density culture. The optimum condition for the batch culture was achieved at a superficial gas velocity of 2.0 cm/s, pH 7.0, and an initial phosphate concentration of 55 mg/l when the specific irradiation rate was controlled above 11.5 micromol/s/g dry cell. In this condition, the specific growth rate and cell productivity were 1.47 day(-1) and 0.98 g dry cell/l/day, respectively.     

在CHO-C28细胞大规模培养中提高收获次数及HBsAg的表达

目的：在CHO—C28细胞大规模培养过程中增加收获次数,延长细胞收获周期,提高HBeAg收获量。方法：采用15L转瓶培养CHO—C28细胞,分3组,对比观察不同小牛血清含量及pH值对细胞生长及HBeAg表达的影响,优化最佳培养条件。结果:三组比较,实验1组的收获次数最多,达25次以上,HBsAg的表达量最高,收液量最多：结论：优化了CHO—C28细胞的培养条件.     

Influence of culture conditions on growth and protective antigenicity of Clostridium chauvoei

. The effect of culture conditions on growth and immunogenicity of Clostridium chauvoei were examined. The pH control and partial feeding of the carbon source at high concentrations were beneficial for growth. The biomass yield was significatively improved, however the butanol concentration reached toxic levels hampering further growth. For each experimental condition the immunogenicity of cells was tested. No differences were found with cells obtained at different temperatures, but it decreased significatively with the partial supply of the carbon source and pH control.     

培养条件对头孢霉菌丝体脂肪酸组分的影响

研究了头孢霉(Cephalosporium sp.)菌丝体最大生产力和多不饱和脂肪酸形成积累的条件。菌丝体最适培养条件为：麦芽糖60g/L\,KNO33g/L、起始pH为60、500mL三角瓶装100mL培养基、接种25%、25℃培养10d则菌丝体达到最大干重。多不饱和脂肪酸形成积累的最适条件为：葡萄糖10～20g/L、(NH4)2SO4或NH4Cl 3g/L、培养基起始pH为40、500mL三角瓶装100mL培养基、接种10%～20%、10℃下照光培养。〖JP2〗因此,在整个生产流程中可采用不同条件分段掌握的技术原则。同时提出在多不饱和脂肪酸的形成和积累途径中油酸(18∶1)向亚油酸(18∶2)的转化是关键,为进一步探索最适培养条件和关键酶的调节提供依据。     

基于DOE设计和氨基酸补加策略提高CHO细胞表达抗CD20单克隆抗体*

中国仓鼠卵巢细胞(CHO)流加培养生产单克隆抗体是目前主流培养方式,其中环境参数(pH和温度)和营养成分均影响细胞生长、碳氮源代谢和外源蛋白表达,是培养过程中关键的控制参数。采用实验设计(design of experiment,DOE)方法研究培养参数(温度、pH)对CHO细胞生长和抗CD20抗体表达的影响,建立营养限制型氨基酸流加策略,实现抗CD20抗体的高表达。结果表明,温度是影响蛋白质表达的显著因素,35℃有助于提高细胞密度和目标抗CD20抗体表达,而pH对抗CD20表达影响不显著,且温度和pH无交互作用,经DOE预测分析最佳培养条件是温度35℃和pH7.0。在该最佳培养条件下,在培养后期酪氨酸和半胱氨酸的浓度都低于0.1mmol/L。在培养的第2天通过补加1.5mmol/L酪氨酸和1mmol/L半胱氨酸避免营养限制,抗CD20抗体表达水平提高了24.1%,且对蛋白糖型无影响。     

沼泽红假单胞菌培养条件的初步研究

就沼泽红假单胞菌Ｐ３．９菌株的主要培养条件进行了初步研究。Ｐ３．９菌株在厌氧光照条件下能利用多种类型的有机碳源物质;其细胞生长的最适ｐＨ为６．５—８,最佳光照强度为２０００—３０００Ｌｘ;培养基中添加酵母膏能明显促进细胞生长。在适宜的培养条件下,菌液细胞数可高达４８亿个／ｍｌ。     

Ammonium uptake in carrot cell structures is influenced by pH-dependent cell aggregation

Semicontinuously grown wild carrot ( Daucus carota L.) cells were used in an investigation of the effect of culture medium pH on ammonium uptake in suspension cultures as a first step in exploring the relationship between pH and anthocyanin biosynthesis. In contrast to published data showing decreasing uptake rates with decreasing culture medium pH, ammonium-limited, semicontinuous carrot cell cultures showed a 25% greater ammonium uptake rate at pH 4.5 than at pH 5.5. When cells that had been grown semicontinuously in medium with a pH of 4.5 or 5.5 were grown in batch cultures at pH 4.5, 5.5 or 6.5 the ammonium uptake rates were those of the semicontinuous cultures, indicating that the pH of the batch culture medium had no effect on ammonium uptake rates over 7 days. The cell culture was composed of very small aggregates when it was grown semicontinuously in medium at pH 4.5, but was composed of large aggregates when it was grown semicontinuously in medium at pH 5.5. The aggregation/disaggregation of the cells was pH dependent, as changing the pH of the semicontinuous culture medium altered the extent of the aggregation. We conclude that the change in culture medium pH caused the cells to aggregate or disaggregate which in turn decreased or increased the rate of ammonium uptake from the medium.