生产抗Tim3单抗CHO细胞N-1灌流高密度培养工艺研究

通过开发高表达量、高效率重组人源化靶向Tim3的单克隆抗体细胞培养工艺,来显著降低Tim3抗体生产成本,提升药品市场可及性。本研究对CHO细胞N-1种子灌流培养工艺和生产抗Tim3单抗CHO细胞高密度培养工艺进行小试工艺开发,实现了高效细胞培养抗体生产。结论:基于交替切向流动过滤(ATF)的灌注过程,N-1种子细胞密度可达53×10^(6)cells/mL;接种至终反应器后进行超高接种密度(uHSD)的补料分批生产,细胞跳过适应期直接进行对数增殖,2 d后细胞便进入稳定期进行蛋白高效表达。对比常规补料分批式生产,细胞周期由14 d缩短为8 d,产品表达量由3.51 g/L提升至5.68 g/L,产品关键质量参数与原始工艺高度类似,生产效率提升3倍左右。研究初步表明,采用对CHO细胞N-1种子灌流培养工艺对抗Tim3单抗CHO细胞高密度培养工艺进行小试工艺开发,实现了高效细胞培养抗体生产。

High dose glargine alters the expression profiles of microRNAs in pancreatic cancer cells

AIM: To investigate the effect of high dose glargine on the expression profiles of microRNAs in human pancreatic cancer cells. METHODS: Real-time polymerase chain reaction array (RT-PCR) was applied to investigate miRNAs differentially expressed in Sw1990 cells treated with or without 100 IU/L glargine. Stem-loop RT-PCR was used to confirm the results of the array assay in Sw1990 and Panc-1 cells. The effects of miR-95 on cell growth, apoptosis, invasion and migration abilities were respectively examined by CCK8 assay, apoptosis assay, Matrigel invasion and migration assay in Sw1990 and Panc-1 cells. Nude mice xenograft models with Sw1990 cells were built to investigate pancreatic cancer growth in vivo after transfection by the lentivirus pGLV3-GFP-miR-95. RESULTS: Ten miRNAs were significantly up-regulated and 2 miRNAs down-regulated in glargine treated Sw1990 cells when compared with non-treated cells (2.48-fold changes on average, P < 0.01). miR-95, miR-134 and miR-34c-3p are the top three miRNAs regulated by glargine (3.65-fold, 2.67-fold and 2.60-fold changes respectively, P < 0.01) in Sw1990 cells. Stem-loop RT-PCR confirmed that high dose glargine up-regulated the expression of miR-95 and miR-134 in both Sw1990 and Panc-1 cells. The most obvious change is the apparent increase of miR-95. Forced expression of miR-95 significantly increased cell proliferation (Sw1990: 2.510 ± 0.129 vs 2.305 ± 0.187, P < 0.05; Panc-1: 2.439 ± 0.211 vs 2.264 ± 0.117, P < 0.05), invasion (Sw1990: 67.90 ± 12.33 vs 47.30 ± 5.89, P < 0.01; Panc-1: 37.80 ± 8.93 vs 30.20 ± 5.14, P < 0.01), migration (Sw1990: 101 ± 6.00 vs 51.20 ± 8.34, P < 0.01; Panc-1: 91.80 ± 9.22 vs 81.50 ± 7.47, P < 0.01) and inhibited cell apoptosis (Sw1990: 22.05% ± 1.92% vs 40.32% ± 1.93%, P < 0.05; Panc-1: 20.17% ± 0.85% vs 45.60% ± 1.43%, P < 0.05) when compared with paired negative controls, whereas knockdown of miR-95 obtained the opposite effect. Nude mice xenograft models confirmed that miR-95 promoted the growth of pancreatic cancer in vivo when compared with negative control (tumor volume: 373.82 ± 23.67 mLvs 219.69 ± 17.82 mL,P < 0.05). CONCLUSION: These observations suggested that mod- ulation of miRNA expression may be an important mechanism underlying the biological effects of glargine.

Entanglement Fidelity as a Measure of Preservation of Entanglement in Local Noisy Process

A new formula of entanglement fidelity has been introduced, which can serve as a measure of the preservation of entanglement between two initially entangled subsystems exposed to local noisy environments. For a simple model we derive analytic expressions of concurrence and entanglement fidelity and draw the relationship between them. We find that such entanglement fidelity exhibits the behavior similar to that of the concurrence in quantum evolutions.

Terrain-Aided Navigation Considering Map Error

Terrain referenced navigation estimates an aircraft navigation status by utilizing a radar altimeter measuring a distance between the aircraft and terrain elevation. Accurate digital elevation map is essential to estimate the aircraft states correctly. However, the elevation map cannot represent the real terrain perfectly and there exists map error between the estimated and the true maps. In this paper, an influence of the map error on measurement equation is analyzed and a technique to incorporate the error in the filter is proposed. The map error is divided into two sources, accuracy error and resolution error. The effectiveness of the suggested technique is verified by simulation results. The method modifies a sensor noise covariance only so there is no additional computational burden from the conventional filter.

Mesenchymal Stem Cells Express Low Levels of Cardiomyogenic Genes and Show Limited Plasticity towards Cardiomyogenic Phenotype

Mesenchymal stem cell differentiation towards osteogenic, chondrogenic and adipogenic lineages have been extensively described and reproduced in the literature. In contrast, cardiomyogenic differentiation still remains largely controversial. In this study the authors aim to shed new light into this unclear phenomenon and test whether BMMSC （bone marrow mesenchymal stem cells） and ATMSC （adipose tissue derived mesenchymal stem cells） are able to differentiate into functional cardiomyocytes, investigating two differentiation protocols. AT and BMMSC behaved differently when cultured in differentiation media and presented lower levels of proliferation and alkaline phosphatase production, expression of cardiomyocyte-specific transcription factors such as GATA-4, Nkx2-5 and proteins such as ct and 13 Myosin Heavy Chains. Furthermore, MSC started to express higher levels of Connexin-43 and c~ sarcomeric actinin protein. Unfortunately, though, MSC did not present cardiomyocyte-like electrophysiological properties. In order to analyze a possible explanation for such limited plasticity, the authors decided to address the issue using a quantitative approach. Gene expression was quantified by Real time PCR, and, for the first time, the authors show that a possible explanation for limited plasticity of MSC is that even though differentiated cells presented differential gene expression, the levels of key cardiomyogenic genes did not reach expression levels presented by adult cardiomyocytes, nor were maintained along differentiation, reaching peaks at 4 days of stimulation, and decaying thereafter.

High-throughput sequencing-based genome-wide identification of micro RNAs expressed in developing cotton seeds

Micro RNAs(mi RNAs) have been shown to play critical regulatory roles in gene expression in cotton. Although a large number of mi RNAs have been identified in cotton fibers, the functions of mi RNAs in seed development remain unexplored. In this study, a small RNA library was constructed from cotton seeds sampled at 15 days post-anthesis(DPA) and was subjected to high-throughput sequencing. A total of 95 known mi RNAs were detected to be expressed in cotton seeds. The expression pattern of these identified mi RNAs was profiled and 48 known mi RNAs were differentially expressed between cotton seeds and fibers at 15 DPA. In addition, 23 novel mi RNA candidates were identified in 15-DPA seeds. Putative targets for 21 novel and 87 known mi RNAs were successfully predicted and 900 expressed sequence tag(EST) sequences were proposed to be candidate target genes, which are involved in various metabolic and biological processes, suggesting a complex regulatory network in developing cotton seeds. Furthermore, mi RNA-mediated cleavage of three important transcripts in vivo was validated by RLM-5′ RACE. This study is the first to show the regulatory network of mi RNAs that are involved in developing cotton seeds and provides a foundation for future studies on the specific functions of these mi RNAs in seed development.