悬浮培养HEK-293 N3S细胞生产重组腺病毒Ad-GFP的实验研究

利用5L生物反应器悬浮培养HEK-293 N3S细胞生产携带绿色荧光蛋白基因的重组腺病毒(recombinant adenovirus-green fluorescent protein,Ad-GFP),为规模化生产腺病毒基因药物建立一种稳定可行的生产工艺。复苏的种子细胞进行逐级放大最后接入5L搅拌式生物反应器中,采用含5％胎牛血清（FBS）的DMEM/F12培基灌流培养293 N3S细胞,当细胞密度达到(2～4)×10⁶个/mL时感染Ad-GFP,48h后收获细胞,经两步氯化铯超速离心获得纯化的Ad-GFP。采用紫外分光光度计比色法和高压液相色谱法（HPLC）测定病毒颗粒数和纯度,采用组织培养半数感染剂量（TCID₅₀）法检测腺病毒的感染滴度。连续培养10～12d,细胞密度可达到(2～4)×10⁶6个/mL左右,纯化的Ad-GFP感染滴度和颗粒数分别为1.0×10¹¹IU/mL和1.68×10¹²VP/mL,比活性为6.0%,A₂₆₀A₂₈₀比值为1.33,产品纯度达到99.2%。建立了5L生物反应器悬浮培养293 N3S细胞生产重组腺病毒Ad-GFP的生产工艺,对携带其他基因的重组腺病毒药物生产具有一定的指导意义。     

乳酸脱氢酶基因调控对HEK-293细胞代谢和腺病毒生产的影响

减少乳酸积累一直是哺乳动物细胞生物技术产业的一个目标。体外培养动物细胞时,乳酸积累主要是2种代谢途径作用的综合结果:一方面,葡萄糖在乳酸脱氢酶A(lactate dehydrogenase A,LDHA)的作用下生成乳酸;另一方面,乳酸可通过乳酸脱氢酶B(LDHB)或乳酸脱氢酶C(LDHC)氧化为丙酮酸重新进入三羧酸循环。本研究综合评估了乳酸代谢关键基因调控对人胚胎肾细胞(human embryonic kidney 293 cells,HEK-293)细胞生长、代谢和人腺病毒(human adenovirus,HAdV)生产的影响,有效提高了HEK-293细胞的HAdV生产能力,并为哺乳动物细胞的乳酸代谢工程调控提供了理论基础。通过改造乳酸代谢关键调控基因(敲除ldha基因以及过表达ldhb和ldhc基因),有效改善了HEK-293细胞的物质和能量代谢效率,显著提高了HAdV的生产。与对照细胞相比,3个基因改造均能促进细胞生长,降低乳酸和氨的积累,明显增强细胞的物质和能量代谢效率,显著提高了HEK-293细胞的HAdV生产能力。ldhc基因过表达对HEK-293细胞的生长、代谢和HAdV生产调控最显著,最大细胞密度提高了约38.7%,乳酸对葡萄糖得率和氨对谷氨酰胺得率分别下降了33.8%和63.3%,HAdV滴度提高了至少16倍。此外,相比于对照细胞株,改造细胞株的腺苷三磷酸(adenosine triphosphate,ATP)生成速率、ATP/O_(2)比率、ATP与腺苷二磷酸(adenosine diphosphate,ADP)的比值以及还原型辅酶Ⅰ(nicotinamide adenine dinucleotide,NADH)含量均有不同程度的提高,能量代谢效率明显改善。     

一次性生物反应器悬浮培养HEK293细胞生产Ad-IFNγ的工艺

腺病毒载体是极具发展前景的基因治疗载体之一,为获得一条新型腺病毒规模化生产工艺,研究采用5 L振荡激流式一次性生物反应器悬浮培养HEK293细胞来生产重组腺病毒载体。细胞经种子链逐步扩增后,接种至AP10生物反应器,采用CD293无血清培养基流加培养悬浮HEK293细胞,细胞密度达约2.0×106个/m L时,以30 MOI(Multiplicity of infection)感染重组Ad-IFNγ(Recombinant adenovirus-interferon gamma),48 h后收获细胞,3次冻融裂解离心收获上清病毒粗产品。采用壳蛋白免疫法快速测定粗产品滴度。结果表明,采用振荡激流式一次性生物反应器,流加HEK293细胞悬浮培养6 d,密度可达2.0×106个/m L,Ad-IFNγ粗产量达1.49×1013 IFU(Infectious unit),单细胞包装量达3 800 IFU/cell。采用阴离子交换层析法纯化重组腺病毒,回收率35.9%。建立了利用5 L激流式一次性生物反应器悬浮培养HEK293细胞生产重组腺病毒载体Ad-IFNγ的初步工艺。     

高渗胁迫和灌流培养策略提高HEK 293细胞生产重组腺病毒载体产量

由于各种疾病在全球范围内的肆虐,国际市场对重组腺病毒载体(adenoviral vector,Adv)疫苗的需求量急剧增加,而工艺研究是解决这一问题的有效手段之一。在细胞接毒前施加高渗胁迫可以提高分批培养模式下的Adv产量,新兴的灌流培养也可以显著提高Adv的产量。将高渗胁迫工艺与灌流培养相结合,有望进一步提升高细胞密度生产过程中的Adv产量。本研究利用摇瓶结合拟灌流培养作为生物反应器灌流培养的缩小模型,使用渗透压为300–405 mOsm的培养基研究了高渗胁迫对细胞生长和Adv生产的影响。结果显示,在细胞生长阶段使用370 mOsm的高渗透压培养基,在病毒生产阶段使用300 mOsm的等渗透压培养基的灌流培养工艺有效地提高了Adv的产量。进一步研究发现这可能归因于病毒复制后期HSP70蛋白的表达量增加。将这种工艺放大至生物反应器中,Adv的产量达到3.2×10¹⁰ IFU/mL,是传统灌流培养工艺的3倍。本研究首次将高渗胁迫工艺与灌流培养相结合的策略应用于HEK 293细胞生产Adv,同时揭示了高渗胁迫工艺增产Adv的可能原因,为HEK 293细胞生产其他类型Adv的工艺优化提供了借鉴。     

VK3诱导悬浮培养的胡萝卜细胞凋亡

Menadione (VK3), a quinone that undergoes redox cycles leading to the formation of superoxide radicals, was found to induce cell death in suspension culture of carrot cells. The effect of menadione was in a dose-dependent manner. 100-800 mumol/L menadione caused 10-33 percent cell death. When concentration of menadione reached 1 mmol/L, 100 percent of cell death was observed. DNA cleavage, a hallmark of apoptosis was further studied. DNA ladders were observed in cells treated with 600 and 800 mumol/L menadione but not with lower concentration treatments where only very low percentage of cell death was found. There was no DNA ladders in the cells treated with 1 mmol/L menadion indicating that necrosis may occur. In situ detection of nuclear DNA fragmentation by TUNEL reaction revealed fragmented nuclear DNA in cells treated with 100-800 mumol/L menadion but not in cells treated with 1 mmol/L menadione.     

藏红花细胞悬浮培养动力学研究

基于已经建立的藏红花细胞悬浮培养体系,研究了其悬浮培养动力学.结果表明,悬浮培养时细胞的生长周期约为20 d,在20 d时生物量达到最大,为12.3 g DW/L,但是藏红花素的合成周期大约为28 d,在28 d时藏红花素的含量和产量达到最大,分别为95.8 mg/g和0.92 g/L.藏红花素的积累与细胞的生长之间的关系为半生长偶联型,为其生物反应器放大培养提供了参数等理论依据.     

搅拌式生物反应器悬浮培养水母雪莲细胞的研究

应用 2L通气搅拌式生物反应器一步批式培养水母雪莲细胞。采用倾斜式搅拌桨代替透平桨 ,研究了搅拌转速、通气量和接种量对细胞生长和黄酮合成的影响 ,发现在 75r min、70 0～1000L min和 4.0～ 5.0gDCW L接种量下细胞生长和黄酮合成比较好。经过 12d培养细胞干重达 13.8gDCW L ,黄酮产量 416mg L ,黄酮含量占细胞干重的 30%。水母雪莲细胞生长及黄酮合成的进程表明 ,黄酮积累与细胞生长呈正相关。对细胞聚集体分布的研究发现 ,流变压力使细胞聚集体分裂 ,使反应器中细胞生长受到影响 ,黄酮产量较摇瓶中降低     

重组腺病毒Ad-Mig基因治疗裸鼠肾细胞癌的实验研究

目的:研究腺病毒介导的小鼠Mig基因对BALB/c裸鼠肾细胞癌的抗肿瘤效果,探讨肾细胞癌治疗的新途径.方法:利用786-O肾癌细胞皮下注射BALB/c裸鼠建立肾细胞癌模型,应用携带Mig基因的重组腺病毒(Ad-Mig)直接进行瘤内注射治疗,观察裸鼠皮下肿瘤生长情况和荷瘤裸鼠的生存期;用乳酸脱氢酶(LDH)释放法检测CTL和NK的杀伤活性.结果:Mig基因能显著抑制荷瘤裸鼠皮下肿瘤的生长,并使鼠生存期明显延长,还能显著增强鼠脾细胞NK和CTL杀伤活性.结论:重组腺病毒Ad-Mig基因对鼠肾细胞癌有显著治疗效果.     

石竹细胞悬浮培养研究

石竹细胞继代周期为 7d时 ,悬浮细胞培养系生长最快 ,生长率最高 ,而且培养物中胚性细胞较多 ,并能保持较快的分裂和生长 ,能促进已形成的大细胞团的生长和分化。转代时接种物与新鲜培养基的体积比以1∶2较好 ,悬浮系细胞生长最快 ,生长率最高 ,以 1∶2和 1∶3的高倍稀释接种有利于胚性细胞的形成及产生小的胚性细胞团 ,对悬浮系添加椰乳和水解乳蛋白的混合物 ,可较大幅度地提高悬浮细胞系的生长速率 ,单独添加上述两种物质的效果均不如二者的综合效应好。在 6种不同激素组合中 ,配方 2 (2 ,4 D 1 .5mg/L +NAA0 .5mg/L +6 BA 0 .5mg/L)最好 ,生长率最高。配方 5 (2 ,4 D 1 .5mg/L +NAA 0 .5mg/L +6 BA 1 .0mg/L)其次 ;配方 1 (2 ,4 D 1 .0mg/L +NAA 0 .5mg/L +6 BA 0 .5mg/L)次之。     

悬浮培养CHO 细胞生产重组人促红细胞生成素条件的优化*

目的:通过对贴壁培养CHO细胞筛选驯化,得到高表达的细胞后进行悬浮培养生产重组人促红细胞生成素(rHuEPO)。方法:利用96孔板和24孔板对CHO细胞进行筛选,得到高表达细胞株后进行驯化,使其适合悬浮培养,经过摇瓶扩增后接种到生物反应器中无血清培养,每天监测葡萄糖含量,测rHuEPO表达量。结果:悬浮培养CHO细胞生产rHuEPO,生产周期短,表达量比贴壁培养高出很多,操作方便,减少污染,易于放大,并建立了适合悬浮培养的CHO细胞株,为工业化悬浮培养CHO细胞生产rHuEPO提供了技术基础。结论:经过工艺优化后利用无血清悬浮培养生产促红细胞生成素平均表达量较贴壁培养高,生产周期短,有利于降低生产成本。     

Transient Transfection Factors for High-Level Recombinant Protein Production in Suspension Cultured Mammalian Cells

The efficient transfection of cloned genes into mammalian cells system plays a critical role in the production of large quantities of recombinant proteins (r-proteins). In order to establish a simple and scaleable transient protein production system, we have used a cationic lipid-based transfection reagent-FreeStyle MAX to study transient transfection in serum-free suspension human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. We used quantification of green fluorescent protein (GFP) to monitor transfection efficiency and expression of a cloned human IgG antibody to monitor r-protein production. Parameters including transfection reagent concentration, DNA concentration, the time of complex formation, and the cell density at the time of transfection were analyzed and optimized. About 70% GFP-positive cells and 50-80 mg/l of secreted IgG antibody were obtained in both HEK-293 and CHO cells under optimal conditions. Scale-up of the transfection system to 1 l resulted in similar transfection efficiency and protein production. In addition, we evaluated production of therapeutic proteins such as human erythropoietin and human blood coagulation factor IX in both HEK-293 and CHO cells. Our results showed that the higher quantity of protein production was obtained by using optimal transient transfection conditions in serum-free adapted suspension mammalian cells.     

质谱检测叶酸缺乏人胚肾细胞中低组蛋白H3赖氨酸79甲基化修饰

目的：探讨叶酸（Folic acid,FA）缺乏在培养的人胚肾细胞（HEK-293）中对细胞组蛋白修饰水平的影响。方法：人胚肾细胞分两组培养,一组正常培养,一组无叶酸培养。细胞提取组蛋白后通过高效液相色谱一线性离子阱/静电场轨道阱高分辨质谱（HPLC-LTQ/Orbitrap Ms）检测组蛋白的修饰以比较叶酸缺乏对人胚肾细胞组蛋白修饰的影响。结果：用高分辨质谱方法成功检测到人胚肾细胞的五个组蛋白变体H1,H3,H4,H2a和H2b上的33个组蛋白修饰位点,其中23个修饰位点为uniprot数据库上已经报道的组蛋白修饰位点,而其余10个为未报道修饰位点。通过质谱比较正常和叶酸缺乏组人胚肾细胞修饰谱发现H3K79me1和H3K79me2在叶酸缺乏培养组中检出率较低。进一步用蛋白免疫印迹的方法也证明了在叶酸缺乏的人胚肾细胞中H3K79me1水平低于正常培养组。结论：细胞中叶酸缺乏影响组蛋白甲基化包括H3K79me2和H3K79me1修饰水平,提示细胞外营养因素叶酸水平可影响组蛋白修饰水平从而参与疾病如神经管畸形（Neural tube defect,NTD）的发生。     

质谱检测叶酸缺乏人胚肾细胞中低组蛋白H3赖氨酸79甲基化修饰

目的:探讨叶酸(Folic acid,FA)缺乏在培养的人胚肾细胞(HEK-293)中对细胞组蛋白修饰水平的影响。方法:人胚肾细胞分两组培养,一组正常培养,一组无叶酸培养。细胞提取组蛋白后通过高效液相色谱一线性离子阱/静电场轨道阱高分辨质谱(HPLC-LTQ/Orbitrap Ms)检测组蛋白的修饰以比较叶酸缺乏对人胚肾细胞组蛋白修饰的影响。结果:用高分辨质谱方法成功检测到人胚肾细胞的五个组蛋白变体H1,H3,H4,H2a和H2b上的33个组蛋白修饰位点,其中23个修饰位点为uniprot数据库上已经报道的组蛋白修饰位点,而其余10个为未报道修饰位点。通过质谱比较正常和叶酸缺乏组人胚肾细胞修饰谱发现H3K79me1和H3K79me2在叶酸缺乏培养组中检出率较低。进一步用蛋白免疫印迹的方法也证明了在叶酸缺乏的人胚肾细胞中H3K79me1水平低于正常培养组。结论:细胞中叶酸缺乏影响组蛋白甲基化包括H3K79me2和H3K79me1修饰水平,提示细胞外营养因素叶酸水平可影响组蛋白修饰水平从而参与疾病如神经管畸形(Neural tube defect,NTD)的发生。     

Optimization of HEK-293S cell cultures for the production of adenoviral vectors in bioreactors using on-line OUR measurements

The culture of HEK-293S cells in a stirred tank bioreactor for adenoviral vectors production for gene therapy is studied. Process monitoring using oxygen uptake rate (OUR) was performed. The OUR was determined on-line by the dynamic method, providing good information of the process evolution. OUR enabled cell activity monitoring, facilitating as well the determination of the feeding rate in perfusion cultures and when to infect the culture. Batch cultures were used to validate the monitoring methodology. A cell density of 10 × 10⁵ cell/mL was infected, producing 1.3 × 10⁹ infectious viral particles/mL (IVP/mL).To increase cell density values maintaining cell specific productivity, perfusion cultures, based on tangential flow filtration, were studied. In this case, OUR measurements were used to optimize the dynamic culture medium feeding strategy, addressed to avoid any potential nutrient limitation. Furthermore, the infection protocol was defined in order to optimize the use of the viral inoculum, minimizing the uncontrolled release of particles through the filter unit mesh. All these developments enabled an infection at 78 × 10⁵ cell/mL with the consequent production of 44 × 10⁹ IVP/mL, representing a cell specific productivity 4.3 times higher than for the batch culture.     

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human FcγRIIIa and the rat α2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.     

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.     

抗MRP3单链抗体-sTRAIL融合蛋白诱导U251多形性胶质母细胞瘤凋亡

采用重组PCR技术获得抗多药耐药相关蛋白3(multidrug resistance protein 3, MRP3)的单链抗体(scFv)与人源可溶性肿瘤坏死因子相关凋亡诱导配体(soluble TNF-related apoptosis inducing ligand, sTRAIL)的融合蛋白质的基因编码序列, 利用原核表达载体pMAL-c2,构建含麦芽糖结合蛋白(maltose binding protein, MBP)标签肽的antiMRP3(scFv)-sTRAIL融合蛋白, 经亲和层析柱纯化. 获得纯化的antiMRP3(scFv)-sTRAIL融合蛋白,用MRP3阳性U251多形性胶质母细胞瘤做增殖抑制实验、细胞凋亡诱导实验,结果均显示具有明显的活性, 而MBP无明显作用. 上述结果表明,成功表达了antiMRP3(scFv)-sTRAIL融合蛋白, 该融合蛋白具有诱导U251多形性胶质母细胞瘤细胞凋亡的活性, 为开发靶向性抗肿瘤药物奠定了基础.     

Programmed cell death 6 (PDCD6) inhibits angiogenesis through PI3K/mTOR/p70S6K pathway by interacting of VEGFR-2

Programmed cell death 6 (PDCD6) was originally found as a pro-apoptotic protein, but its molecular mechanism is not well understood. In this study, we have attempted to investigate the effects of PDCD6 on the inhibition of angiogenesis-mediated cell growth as a novel anti-angiogenic protein. Purified recombinant human PDCD6 inhibited cell migration in a concentration-time-dependent manner. We also found that overexpressed PDCD6 suppressed vascular endothelial growth factor (VEGF)-induced proliferation, invasion, and capillary-like structure tube formation in vitro. PDCD6 suppressed phosphorylation of signaling regulators downstream from PI3K, including Akt, mammalian target of rapamycin (mTOR), glycogen synthase kinase-3β(GSK-3β), ribosomal protein S6 kinase (p70S6K), and also decreased cyclin D1 expression. We found binding PDCD6 to VEGFR-2, a key player in the PI3K/mTOR/P70S6K signaling pathway. Taken together, these data suggest that PDCD6 plays a significant role in modulating cellular angiogenesis.