CHO细胞高密度流加工艺参数优化

[目的]优化细胞接种密度、培养基、细胞培养温度等参数,提高生产细胞株PCSK9蛋白表达滴度。[方法]试验分四步进行,(1)探讨几款市售培养基优化组合后对CHO细胞株蛋白表达滴度的影响;(2)探讨10.0×10⁶、15.0×10⁶、50.0×10⁶ cells/mL高密度接种流加过程培养基、降温时间等对CHO细胞蛋白滴度的影响;(3)在(2)实验数据基础上继续优化,通过更换培养基继续探讨高密度流加工艺的可行性;(4)在反应器对实验数据进行工艺验证。[结果](1)CHO细胞PCSK9蛋白滴度提升至2.6 g/L,蛋白滴度成倍增长;(2)15.0×10⁶ cells/mL接种,30.0×10⁶ cells/mL降温至34℃,继续优化培养基1^#、2^#配比,蛋白滴度提升至4.5 g/L,反应器工艺验证,细胞生长状态稳定,蛋白滴度突破3.8 g/L;(3)继续筛选市售培养基,成功实现80.0×10⁶ cells/mL高密度流...     

不同饵料密度和盐度下褶皱臂尾轮虫的生活史特征

为探索低盐驯化褶皱臂尾轮虫规模化培育的可行性,本实验以小球藻(Chlorella vulgaris)为饵料,在饵料密度1.0×10⁴、1.0×10⁵、1.0×10⁶、1.0×10⁷和1.0×10⁸ cells·mL^-1下,采用单个体培养法,研究了盐度23及盐度6的褶皱臂尾轮虫(Brachionus plicatilis)的生活史特征。结果表明：随着饵料密度的升高,两组盐度轮虫的生殖期、平均寿命、产卵量、生命期望、净生殖率、世代时间和种群内禀增长率均呈现出先升高再降低(P<0.05);盐度6的轮虫在饵料密度1.0×10⁶、1.0×10⁷和1.0×10⁸ cells·mL^-1下的生殖前期显著小于饵料密度1.0×10⁴和1.0×10⁵ cells·mL^-1下的生殖前期(P<0.05);盐度23的轮虫...     

用去掉免疫球蛋白IgG的腹水提高体外培养的单克隆抗体产量(英文)

发展了一种在实验室大幅度提高抗体产量的方法,即采用添加2%的去免疫球蛋白IgG的腹水于无血清培养基中的方法,培养杂交瘤细胞,获得了高达3.55 ×10⁶/ml的细胞密度,纯化后获得了135μg/ml的单克隆抗体产量,比通常的用无血清培养的单位体积培养液抗体产量高4倍。对于体外生产单克隆抗体而言,这种方法经济并且易于推广,它的成功是抗体产量提高的一个巨大进步。     

四川西北部亚高山云杉天然林生态系统碳密度、净生产量和碳贮量的初步研究

该文利用野外实际调查数据对四川西北部亚高山云杉(Picea asperata)天然林碳密度、净生产量、碳贮量及其分布进行了分析,结果表明,在调查区域,云杉天然林分平均生物量为230.37×10³ kg·hm^-2,其中乔木层为212.77×10³ kg·hm^-2,占林分生物量的92.30%。云杉天然林生态系统各组分的平均碳密度为树干57.85%,树皮47.12%,树枝51.22%,树叶48.27%和树根52.39%,灌木层平均碳密度49.91%,草本层平均碳密度46.34%,地被层平均碳密度43.21%,枯落物层平均碳密度39.44%,土壤碳密度平均值为1.41%,随土层深度增加各层次土壤碳密度逐渐减少。云杉林平均生态系统总碳贮量为273.79×10³ kg·hm^-2,其中乔木层109.30×10³ kg·hm^-2,占云杉林生态系统总碳贮量的39.92%,灌木层5.69×10³ kg·hm^-2,占2.08%,草本层1.26×10³ kg·hm^-2,占0.46%,地被物层0.60×10³ kg·hm^-2,占0.22%,枯落物层0.83×10³ kg·hm^-2,占0.30%,林内土壤(0~100 cm)碳贮量为156.11×10³ kg·hm^-2,占57.01%。云杉林的碳库分布序列为土壤(0~100 cm)>乔木层>灌木层>草本层>枯落物层>地被物层。云杉天然林分平均净生产总量为6 838.5 kg·hm^-2·a^-1,碳素年总净固量平均为3 584.98 kg·hm^-2·a^-1,其中乔木层净生产量为4 676 kg·hm^-2·a^-1,占林分总量的68.38%,碳素年平均固定量2 552.99 kg·hm^-2·a^-1,占林分总量的71.21%。     

巨桉人工林地土壤微生物类群的生态分布规律

研究了四川省洪雅县巨桉人工林地土壤微生物数量及其类群分布规律.结果表明,洪雅县巨桉人工林地土壤微生物数量具有明显的季节变化,各样地均以秋季最多,达到17.42×10⁶CFU·g^-1,春季次之,夏季最少.土壤微生物主要集中在0～20cm的土层,随着土层的加深,微生物数量迅速减少;在0～60cm土层,同一类群微生物数量变动范围较大,其中好气性细菌0.31×10⁶～14.39×10⁶CFU·g^-1,放线菌0.06×10⁶～0.79×10⁶CFU·g^-1,真菌0.02×10⁶～0.07×10⁶CFU·g^-1,厌氧细菌0.07×10⁶～3.22×10⁶CFU·g^-1.巨桉人工林地微生物以细菌为主要类群,占微生物总数的92.83%以上,其次是放线菌和真菌,其微生物组成结构合理.与青冈次生林和农耕地相比,巨桉人工林地微生物数量远远高于青冈次生林地,有利于土壤微生物生长.细菌生理类群的Simpson多样性指数和Shannon-Wiener多样性指数分别达到0.773和1.896.     

灌水量与密度互作对冬小麦籽粒产量和水分利用效率的影响

为明确协同提高冬小麦产量和水分利用效率的适宜灌水量和种植密度,选用大穗型品种‘泰农18’(T18)和中穗型品种‘山农22’(S22)为试验材料,设置4个灌溉水平(不灌水、每次灌水45、60、75 mm)和4个种植密度,其中泰农18选用135×10⁴、270×10⁴、405×10⁴、540×10⁴ 株·hm^-2,山农22选用90×10⁴、180×10⁴、270×10⁴、360×10⁴株·hm^-2,研究了籽粒产量、麦田耗水特性和水分利用效率对灌水量和密度互作效应的响应。结果表明: 籽粒产量、总耗水量、土壤贮水消耗量和水分利用效率均受到灌溉水平、种植密度及两者互作效应的显著影响。每次灌水量为45 mm,泰农18种植密度为405×10⁴株·hm^-2、山农22种植密度为270×10⁴株·hm^-2时,两品种籽粒产量均达到最高,拔节后棵间蒸发量占阶段农田总耗水量的比例最小,1 m以下土壤水消耗比例、水分利用效率高。种植密度与灌溉量合理组合,有利于降低水分无效损耗,提高水分利用效率。     

虫生真菌LL-01鉴定及对两种检疫性粉蚧的致病性

【目的】为丰富南洋臀纹粉蚧Planococcus lilacinus和石蒜绵粉蚧Phenacoccus solani的生防菌资源。【方法】本研究从感病南洋臀纹粉蚧上分离生防菌,采用基因序列分析方法进行种类鉴定,并在室内优化其培养条件,评估其对这2种检疫性粉蚧的致病性。【结果】分离得到1株编号为LL-01的虫生真菌,经r DNA-ITS、18S r DNA和Nad1序列分析确定为蜡蚧轮枝菌;该菌的生长和产孢最适的温度为26℃,光周期为6L︰18D,碳源为果糖,氮源为干酪素,此条件下培养10 d的蜡蚧轮枝菌菌落直径和产孢量分别可达4.66 cm和3.16×10⁸孢子/cm8孢子/cm²;其侵染不同虫龄南洋臀纹粉蚧和石蒜绵粉蚧10 d后的LC_(50)分别为9.80×102;其侵染不同虫龄南洋臀纹粉蚧和石蒜绵粉蚧10 d后的LC_(50)分别为9.80×10⁴-9.17×104-9.17×10⁵孢子/m L和5.00×105孢子/m L和5.00×10⁴-5.30×104-5.30×10⁵孢子/m L。浓度为1.00×105孢子/m L。浓度为1.00×10⁸孢子/m L的蜡蚧轮枝菌侵染南洋臀纹粉蚧和石蒜绵粉蚧后第10天,其累计致死率分别为84.09%-97.62%和89.89%-98.85%,LT_(50)分别为3.70-5.84 d和3.48-5.14 d;在侵染第5天时,蛋白酶和几丁质酶活性分别达峰值19.44 U/m L和15.01 U/m L,脂肪酶活性在侵染第6天时达到峰值7.68 U/m L。【结论】蜡蚧轮枝菌LL-01生长速度快、产孢量高,对这2种检疫性粉蚧的致病性强。     

肝素促进脐血巨核祖细胞体外扩增

目的 :探索肝素在脐带血CD34⁺ 细胞定向扩增巨核祖细胞中的作用。方法采用免疫磁珠法 (MACS)分选CD34⁺ 细胞 ,在TPO ,IL 1 1的扩增体系中加入肝素 ,巨核祖细胞集落分析 (CFU MK)测定巨核祖细胞扩增倍数 ,流式细胞仪检测巨核祖细胞分化过程中的特异性标记 (CD34⁺ ,CD41a⁺ ,CD61⁺ ,CD34⁺ CD41a⁺ ,CD41a⁺ CD61 ⁺ ) ,巨核细胞特异性抗体 (CD41a)免疫组化染色和透射电镜观察鉴定巨核细胞形态及超微结构 ,血小板体外活化实验及NOD/SCID小鼠异种体内移植实验评价扩增的巨核祖细胞的功能。结果 :TPO( 5 0ng/ml)与IL-1 1 ( 5 0ng/ml)双因子联合应用 ,7天巨核祖细胞克隆扩增倍数为 83 1 7± 39 41倍 ,1 0天为 2 0 5 0 6± 74 2 6倍 ,流式细胞仪分析显示 7天CD34⁺ CD41a+ 细胞扩增 1 0 5 1± 4 79倍 ,0天加入肝素后 ,7天巨核祖细胞克隆扩增倍数为 1 0 8 2 5± 32 67倍 ,1 0天为 333 0 6± 2 7 5 4倍 ,7天CD34⁺ CD41a⁺ 细胞扩增到 2 9 93± 6 39倍 ,为无肝素组的 2.85倍 ,与双因子组相比有统计学差异 (P <0.0 1 ) ,肝素在第 5天及第 7天加入没有增加巨核祖细胞扩增效果。经全身照射预处理的NOD/SCID小鼠静脉输注扩增第 7天的巨核细胞 (TPO、IL-11、肝素联合 ) ,可明显加速其血小板及白细胞计数的恢复并提高生存率 ;同时 ,体外血小板活化实验证实扩增的巨核细胞在体外可产生血小板 ,有正常巨核细胞功能。结论 :TPO、IL-11组合的扩增体系中加入肝素可进一步改善脐带血巨核祖细胞的扩增效果 ,优化体外扩增体系。     

不同藻密度下Cd2+浓度对萼花臂尾轮虫生命表统计学参数的影响

为了比较不同食物密度下污染物浓度对受试生物的慢性毒性,筛选出以轮虫为受试生物对水环境中Cd污染进行监测的敏感指标,研究了在不同斜生栅藻密度(1.0×10⁶、3.0×10⁶和5.0 ×10⁶ cells·ml^-1)下不同浓度(2.5、5.0、10.0、20.0和40.0 μg·L^-1)Cd²⁺对萼花臂尾轮虫生命表统计学参数的影响.结果表明：(25±1) ℃下Cd²⁺对轮虫的24 h LC₅₀为37.7 μg·L^-1.与各藻密度下的对照组相比,当藻密度为1.0×10⁶ cells·ml^-1时,20.0和40.0 μg·L^-1的Cd²⁺显著延长了轮虫的世代时间,5.0 μg·L^-1的Cd²⁺显著提高了轮虫的后代混交率;当藻密度为3.0×10⁶ cells·ml^-1时,除了5.0 μg·L^-1外,其他浓度的Cd²⁺显著降低了轮虫的后代混交率;而当藻密度为5.0×10⁶ cells·ml^-1时,Cd²⁺浓度对轮虫的所有生命表统计学参数均无显著影（P>0.05）.藻密度对轮虫的世代时间、生命期望、净生殖率和后代混交率均有显著影响（P<0.05）,Cd²⁺浓度对轮虫的世代时间和后代混交率有显著影响（P<0.05）,两者交互作用对后代混交率有极显著影响（P<0.01）.轮虫的世代时间和后代混交率是在1.0×10.6和3.0×10.6 cells·ml^-1食物密度下对Cd²⁺污染比较敏感的参数,其中后代混交率最敏感.     

不同藻密度下Cd2+浓度对萼花臂尾轮虫生命表统计学参数的影响

为了比较不同食物密度下污染物浓度对受试生物的慢性毒性,筛选出以轮虫为受试生物对水环境中Cd污染进行监测的敏感指标,研究了在不同斜生栅藻密度(1.0×10⁶、3.0×10⁶和5.0 ×10⁶ cells·ml^-1)下不同浓度(2.5、5.0、10.0、20.0和40.0 μg·L^-1)Cd²⁺对萼花臂尾轮虫生命表统计学参数的影响.结果表明：(25±1) ℃下Cd²⁺对轮虫的24 h LC₅₀为37.7 μg·L^-1.与各藻密度下的对照组相比,当藻密度为1.0×10⁶ cells·ml^-1时,20.0和40.0 μg·L^-1的Cd²⁺显著延长了轮虫的世代时间,5.0 μg·L^-1的Cd²⁺显著提高了轮虫的后代混交率;当藻密度为3.0×10⁶ cells·ml^-1时,除了5.0 μg·L^-1外,其他浓度的Cd²⁺显著降低了轮虫的后代混交率;而当藻密度为5.0×10⁶ cells·ml^-1时,Cd²⁺浓度对轮虫的所有生命表统计学参数均无显著影（P>0.05）.藻密度对轮虫的世代时间、生命期望、净生殖率和后代混交率均有显著影响（P<0.05）,Cd²⁺浓度对轮虫的世代时间和后代混交率有显著影响（P<0.05）,两者交互作用对后代混交率有极显著影响（P<0.01）.轮虫的世代时间和后代混交率是在1.0×10.6和3.0×10.6 cells·ml^-1食物密度下对Cd²⁺污染比较敏感的参数,其中后代混交率最敏感.     

阿霉素通过Wnt/β-catenin信号通路抑制口腔鳞癌干细胞迁移和侵袭

目的:探讨阿霉素对口腔鳞癌干细胞迁移、侵袭、凋亡的影响及其可能的机制。方法:体外培养人口腔鳞癌细胞系SCC25,通过流式细胞术分选CD44^-和CD44⁺细胞,RT-PCR检测CD44^-和CD44⁺细胞的Oct4、CD133、CD44和GAPDH的m RNA表达;检测和比较CD44^-和CD44⁺细胞的克隆形成能力。CD44+细胞用阿霉素或β-catenin抑制剂LF3进行处理,分别使用Transwell和细胞划痕检测细胞侵袭和迁移能力,一步法TUNEL检测细胞凋亡水平,WB检测β-catenin和TCF-4的蛋白表达。结果:流式细胞术成功分离CD44^-和CD44⁺细胞,RT-PCR检测CD44+细胞高表达Oct4、CD133和CD44 m RNA,CD44-细胞弱表达Oct4m RNA,不表达CD133和CD44 m RNA;CD44⁺细胞的克隆形成能力显示显著强于CD44^-细胞(P<0.05)。阿霉素显著降低了CD44⁺细胞的侵袭能力和迁移能力(P<0.05),显著提高了CD44+细胞的凋亡率(P<0.05);阿霉素显著降低了CD44+细胞β-catenin和TCF-4的蛋白表达(P<0.05),LF3对β-catenin和TCF-4蛋白表达的影响与阿霉素比较无显著差异(P>0.05)。结论:阿霉素可能通过抑制Wnt/β-catenin信号通路降低口腔鳞癌干细胞迁移、侵袭能力,促进细胞凋亡。     

Influence of culture medium composition, dissolved oxygen concentration and harvesting time on the production of Serratia entomophila, a microbial control agent of the New Zealand grass grub

The bacterium Serratia entomophila (Enterobacteriaceae) has been developed as a commercially available biopesticide for control of the pasture pest Costelytra zealandica. The influence of culture medium composition, dissolved oxygen (DO) concentration and harvesting time were investigated in order to optimise the production of S. entomophila. In batch fermentations, highest yields were achieved using sucrose (40 g L^-1) as the carbon source, followed closely by fructose and molasses. The effect of yeast extract (YE), marmite and bakery yeast as cell growth enhancers was also examined in both batch and fed-batch mode. Culture medium containing 20 g L^-1 of YE (fed-batch) produced the highest cell density. No significant effect on cell yield was detected when cultures were supplemented with bakery yeast or marmite. The DO concentration influenced biomass production: a 5-fold increase in cell density was achieved when the concentration of DO was maintained in the range of 20-50% (5.7×10¹⁰ CFUs mL^-1) in comparison with 1% (1.2×10¹⁰ CFUs mL^-1). In cultures maintained at 1 and 20% DO concentration, cells harvested from the exponential growth phase survived for less than 2 weeks when stored at 4°C. In contrast, high cell survival (85-100%) was achieved when cells were harvested after they had entered the stationary growth phase. Recommendations are provided for the production of robust, high cell density cultures of S. entomophila.     

Process intensification in fed-batch production bioreactors using non-perfusion seed cultures

ABSTRACT

Although process intensification by continuous operation has been successfully applied in the chemical industry, the biopharmaceutical industry primarily uses fed-batch, rather than continuous or perfusion methods, to produce stable monoclonal antibodies (mAbs) from Chinese hamster ovary (CHO) cells. Conventional fed-batch bioreactors may start with an inoculation viable cell density (VCD) of ~0.5 × 10⁶ cells/mL. Increasing the inoculation VCD in the fed-batch production bioreactor (referred to as N stage bioreactor) to 2–10 × 10⁶ cells/mL by introducing perfusion operation or process intensification at the seed step (N-1 step) prior to the production bioreactor has recently been used because it increases manufacturing output by shortening cell culture production duration. In this study, we report that increasing the inoculation VCD significantly improved the final titer in fed-batch production within the same 14-day duration for 3 mAbs produced by 3 CHO GS cell lines. We also report that other non-perfusion methods at the N-1 step using either fed batch or batch mode with enriched culture medium can similarly achieve high N-1 final VCD of 22–34 × 10⁶ cells/mL. These non-perfusion N-1 seeds supported inoculation of subsequent production fed-batch production bioreactors at increased inoculation VCD of 3–6 × 10⁶ cells/mL, where these achieved titer and product quality attributes comparable to those inoculated using the perfusion N-1 seeds demonstrated in both 5-L bioreactors, as well as scaled up to 500-L and 1000-L N-stage bioreactors. To operate the N-1 step using batch mode, enrichment of the basal medium was critical at both the N-1 and subsequent intensified fed-batch production steps. The non-perfusion N-1 methodologies reported here are much simpler alternatives in operation for process development, process characterization, and large-scale commercial manufacturing compared to perfusion N-1 seeds that require perfusion equipment, as well as preparation and storage vessels to accommodate large volumes of perfusion media. Although only 3 stable mAbs produced by CHO cell cultures are used in this study, the basic principles of the non-perfusion N-1 seed strategies for shortening seed train and production culture duration or improving titer should be applicable to other protein production by different mammalian cells and other hosts at any scale biologics facilities.     

球形棕囊藻游离单细胞的密度与囊体形成的关系研究

球形棕囊藻(Phaeocystis globosa Scherffel)主要以囊体形态形成赤潮,由单细胞向囊体形态的转变是赤潮爆发的关键.本研究推测囊体形成的前提是游离单细胞达到一定密度阈值,当密度低于该阈值时,囊体无法形成.基于此,本文探究了不同条件(温度、营养充气搅动、摄食压力、初始密度)下囊体形成时游离单细胞的密...     

中国仓鼠卵巢工程细胞无血清流加培养关键工艺参数的考察

主要考察流加培养基中不同营养成分、流加起始时间及初始接种密度对11G-S细胞无血清流加培养的影响。在研究中以悬浮适应的表达尿激酶原 (Pro-urokinase,Pro-UK) CHO工程细胞系11G-S为研究对象,在100 mL的摇瓶中无血清悬浮流加培养11G-S细胞,同时以活细胞密度、细胞活力及Pro-UK活性为评价依据。结果表明在培养基中氨基酸、无血清添加成分及无机盐对促进细胞生长、细胞活力维持及蛋白表达起着较为重要的作用;且流加起始时间为72 h及初始接种密度为3×105~4×105 cells/     

A high-yielding, generic fed-batch cell culture process for production of recombinant antibodies

A fed-batch cell culture process was developed that has general applicability to all evaluated Sp2/0 (n = 8) and NS0 (n = 1) antibody-producing cell lines. The two key elements of this generic process were a protein-free concentrated feed medium, and a robust, metabolically responsive feeding strategy based on the off-line measurement of glucose. The fed-batch process was shown to perform equivalently at the 15 L development scale and 750 L manufacturing scale. Compared to batch cultures, the fed-batch process yielded a 4. 3 fold increase in the average integral of viable cell concentration and a 1.7 fold increase in average specific antibody production rate, equivalent to a 7.6 fold increase in average final antibody concentration. The highest producing cell line reached a peak viable cell concentration of 1.0 x 10(7) cell mL(-1) and a final antibody concentration of 750 mg L(-1) in a 10 day process. For all lines evaluated, reducing bioreactor pH set point from 7.2 to 7.0 resulted in an additional 2.4 fold increase in average final antibody concentration. The optimized fed-batch process consistently yielded a volumetric productivity exceeding 50 mg L(-1) day(-1). This generic, high-yielding fed-batch process significantly decreased development time, and increased manufacturing efficiency, thereby facilitating the clinical evaluation of numerous recombinant antibodies.     

Vero细胞微载体不同培养方法的评价

对三种不同培养方法进行细胞生长速度、密度、营养及代谢产物浓度的比较分析,以优化和筛选最佳培养条件与方式。用同体积生物反应罐,基本培养条件相同,采用批培养、再循环培养、灌流培养三种方式进行了Vero细胞微载体（CytodexI）的周期培养。三种培养方法均达到预期效果,最终细胞密度分别为每毫升2.09×10     

Low-glutamine fed-batch cultures of 293-HEK serum-free suspension cells for adenovirus production

Recent developments in gene therapy using adenoviral (Ad) vectors have fueled renewed interest in the 293 human embryonic kidney cell line traditionally used to produce these vectors. Low-glutamine fed-batch cultures of serum-free, suspension cells in a 5-L bioreactor were conducted. Our aim was to tighten the control on glutamine metabolism and hence reduce ammonia and lactate accumulation. Online direct measurement of glutamine was effected via a continuous cell-exclusion system that allows for aseptic, cell-free sampling of the culture broth. A feedback control algorithm was used to maintain the glutamine concentration at a level as low as 0.1 mM with a concentrated glucose-free feed medium. This was tested in two media: a commercial formulation (SFM II) and a chemically defined DMEM/F12 formulation. The fed-batch and batch cultures were started at the same glucose concentration, and it was not controlled at any point in the fed-batch cultures. In all cases, fed-batch cultures with double the cell density and extended viable culture time compared to the batch cultures were achieved. An infection study on the high density fed-batch culture using adenovirus-green fluorescent protein (Ad-GFP) construct was also done to ascertain the production capacity of the culture. Virus titers from the infected fed-batch culture showed that there is an approximately 10-fold improvement over a batch infection culture. The results have shown that the control of glutamine at low levels in cultures is sufficient to yield significant improvements in both cell densities and viral production. The applicability of this fed-batch system to cultures in different media and also infected cultures suggests its potential for application to generic mammalian cell cultures.     

HIV/HCV共感染者抗逆转录病毒治疗后免疫恢复情况

由于具有相同的传播途径,人类免疫缺陷病毒(Human immunodeficiency virus,HIV)和丙型肝炎病毒(Hepatitis C virus,HCV)共感染非常普遍,但是关于合并感染的程度,两种病毒之间的相互关系,在艾滋病抗逆转录病毒治疗(Antiretroviral therapy,ART)前后,HCV合并感染对HIV患者免疫细胞恢复的影响仍不明确。为了通过分析CD4⁺和CD8⁺T淋巴细胞数的变化,以了解辽宁省HIV/HCV共感染者ART后免疫恢复的情况,本研究从辽宁省艾滋病抗病毒治疗数据库中筛选符合要求的HIV感染者和HIV/HCV共感染者,收集感染者基本人口学资料及HCV抗体检测结果、HIV/HCV共感染途径等资料。采用t检验或卡方检验进行组间比较,采用Kaplan-Meier乘积极限法绘制生存分析函数图。结果显示,本研究共纳入HIV感染者12742人,HIV/HCV共感染者340人。HIV感染者和HIV/HCV共感染者的不同人口学特征均差异显著(P<0.001)。HIV感染和HIV/HCV共感染者ART治疗后CD4⁺细胞数和CD4⁺/CD8⁺比值显著升高(P<0.05),CD8⁺细胞数比ART前显著下降(P<0.05)。HIV/HCV共感染者随着ART时长,CD4⁺T淋巴细胞数恢复情况始终显著低于HIV感染者(P<0.05)。生存分析曲线表明,HCV/HIV共感染者从艾滋病诊断开始随着ART的治疗CD4⁺细胞恢复情况显著低于HIV感染者,Log-Rank检验统计量为4.483(P=0.034)。本研究揭示,HCV感染对ART患者CD4⁺和CD8⁺T淋巴细胞的恢复有影响。ART后HIV/HCV共感染者中CD4⁺T淋巴细胞计数的改善低于HIV单一感染者,并且单一感染患者对ART的反应比合并感染患者更好。因此,建议在启动ART之前,对每个感染HIV的患者进行HCV抗体筛查。     

High Yield of Human Monoclonal Antibody Produced by Stably Transfected Drosophila Schneider 2 Cells in Perfusion Culture Using Wave Bioreactor

Since it was first introduced in late 1990s Wave bioreactor has been used for protein production by mammalian and insect cell lines. However, using Wave bioreactor to produce human monoclonal antibody by stable Drosophila Schneider 2 (S2) cell transfectants has not been reported before. In this study, S2 cells were co-transfected with an inducible vector expressing human monoclonal antibody heavy and light chains, respectively, specific for hemagglutinin (HA) of H5N1 influenza virus. Stable S2 transfectant clone was selected by limiting dilution assay. Stable S2 transfectant clone that produce the highest amount of human monoclonal antibody was inoculated into two 2-l disposable cellbags, where cell growth and antibody production were compared between batch and perfusion cultures using Wave bioreactor. Here, we report that maximum viable cell density reached 1.06?×?10(7) cells/ml in batch culture; whereas 1.04?×?10(8)?cells/ml was achieved in perfusion culture. The maximum volumetric antibody productivity in batch culture was 52?mg/l/day; while perfusion culture yielded 1,437?mg/l/day. As a result, the total antibody production was 201?mg in batch culture and 8,212?mg in perfusion culture. The antibody produced by both cultures displays full neutralizing activity. Thus, our results provide strong support for using Wave bioreactor in perfusion culture for a large-scale production of human monoclonal antibody by stable S2 cell transfectants.