凹唇壁蜂的耐寒性及生殖潜力研究

在滞育期和滞育结束后 (成虫出茧之前 ) ,分别测定凹唇壁蜂越冬成虫的耐寒性 ,结果表明 :滞育期成虫的过冷却点和体液冰点分别为 -2 6 4 8℃和 -2 0 91℃ ,解除滞育后分别为 -1 8 4 0℃和-1 1 78℃。自然条件下 ,凹唇壁蜂在我国北方大部分地区可安全越冬。在筑巢前期 ,对雌蜂的生殖系统解剖观察发现 ,卵巢由 6根卵巢管组成 ,卵母细胞总数平均为 55 9± 4 4 6个 ,卵原区在成虫筑巢期不再产生卵母细胞。     

多普勒B超鉴定中国大鲵性别

首次报道了从影像学水平上鉴定中国大鲵性别的研究结果。采用多普勒B超,常规方法直接扫描雌雄中国大鲵腹部,并记录大鲵的精巢或卵巢的形状、内部回声。实验结果表明,雌雄中国大鲵的检测结果存在明显的差畀,在雄性中国大鲵的腹部,可检测到精巢组织;在雌性中国大鲵的腹部,可检测到为卵巢组织和卵泡。初步分析了实验结果,并认为将多普勒B超鉴定法与泄殖孔法鉴定相结合可以作为中国大鲵性别鉴定的可靠方法。     

过表达热休克蛋白70提高CHO细胞表达抗体的能力

通过在中国仓鼠卵巢细胞(CHO)中过表达热休克蛋白70以提高其表达抗体的能力。首先从中国仓鼠基因组DNA中扩取HSP70基因,构建真核表达质粒pcDNA3.1-HSP70,再将重组质粒稳定转染到CHO/dhfr-细胞中,筛选获得稳定的细胞系,运用RT-qPCR检测和Western blot分析HSP70基因的过表达。在过表达HSP70的CHO细胞组和对照细胞组(转染空载体pcDNA3.1的CHO细胞组)中分别转染表达抗-HBs的质粒,应用ELISA检测两组细胞表达抗-HBs的能力。RT-qPCR结果显示实验组CHO细胞中HSP70基因的表达量明显高于对照组细胞;ELISA检测结果表明过表达HSP70的CHO细胞组抗-HBs表达量高于对照组细胞(P<0.05)。研究揭示HSP70能有效促进细胞内分泌性蛋白的表达。     

Phase-dependent nucleotide substitution in protein-coding sequences

It is well known that due to the degeneracy of genetic code, most of the silent substitutions appear in the third codon position, so the mutation frequency of the third codon position is much higher than that of the first two positions. However, it remains unknown whether the directionality of point mutation in three codon positions is similar or not. In this paper, through analyzing 15 sets of orthologous genes, it is revealed that most of the substitution types are significantly different between any two codon positions, especially between the 2nd and the 3rd phases. Furthermore, the average frequencies of each type of substitution calculated from the fifteen sets of orthologous genes are similar to those identified in single nucleotide polymorphisms (SNPs) of human and mouse genome. The present analyses suggest that the nucleotide substitution in protein-coding sequences is not only context-dependent (so called neighboring-nucleotide effects), but also phase-dependent, which is of significance to improving the prevalent nucleotide-evolution models.     

Isotope labeling to determine the dynamics of metabolic response in CHO cell perfusion bioreactors using MALDI‐TOF‐MS

The steady‐state operation of Chinese hamster ovary (CHO) cells in perfusion bioreactors requires the equilibration of reactor dynamics and cell metabolism. Accordingly, in this work we investigate the transient cellular response to changes in its environment and their interactions with the bioreactor hydrodynamics. This is done in a benchtop perfusion bioreactor using MALDI‐TOF MS through isotope labeling of complex intracellular nucleotides (ATP, UTP) and nucleotide sugars (UDP‐Hex, UDP‐HexNAc). By switching to a ¹³C₆ glucose containing feed media during constant operation at 20 × 10⁶ cells and a perfusion rate of 1 reactor volume per day, isotopic steady state was studied. A step change to the ¹³C₆ glucose medium in spin tubes allowed the determination of characteristic times for the intracellular turnover of unlabeled metabolites pools, (≤0.56 days), which were confirmed in the bioreactor. On the other hand, it is shown that the reactor residence time (1 day) and characteristic time for glucose uptake (0.33 days), representative of the bioreactor dynamics, delayed the consumption of ¹³C₆ glucose in the bioreactor and thus the intracellular ¹³C enrichment. The proposed experimental approach allowed the decoupling of bioreactor hydrodynamics and intrinsic dynamics of cell metabolism in response to a change in the cell culture environment. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1630–1639, 2017     

苯酚的生物降解基因组成及其调控机制

苯酚的生物降解受多组分的降解基因控制,形成邻位和间位等不同的代谢途径.结合部分实验结果,介绍了邻位和间位代谢系统的基因组成及其作用机制等方面的研究进展,讨论了调节基因在苯酚降解菌中的作用和不同因素对苯酚降解的影响.     

Control of passive permeability of chinese hamster ovary cells by external and intracellular ATP

External ATP causes passive permeability change in several transformed cells, but not in untransformed cells. We studied the effect of external ATP on the passive permeability of CHO-K1 cells, a transformed clone of Chinese hamster ovary cells. Treatment of the cells with external ATP alone did not produce a permeability change, and this was observed only when a mitochondrial inhibitor, such as rotenone or oligomycin, was present together with ATP. These inhibitors reduced the concentration of intracellular ATP and a permeability change by external ATP was observed when intracellular ATP was decreased more than 70%. This requirement for permeability change of CHO-K1 cells was quite unique, since passive permeability change of other transformed cells so far tested was induced by ATP alone. Treatment of CHO-K1 cells with cyclic AMP analogues increased their sensitivity to external ATP about 2-fold. The roles of external and intracellular ATP in controlling passive permeability are discussed.     

来曲唑联合妈富隆治疗多囊卵巢综合征患者的疗效探讨

目的:探讨来曲唑联合妈富隆治疗多囊卵巢综合征患者的临床疗效。方法:选择2015年12月至2017年12月我院接诊的90例多囊卵巢综合征患者,通过随机数表法分为观察组(n=46)和对照组(n=44)。对照组给予妈富隆治疗,观察组联合来曲唑治疗。比较两组治疗前后血清卵泡刺激素(FSH)、黄体生成素(LH)、睾酮(T)、血清血管内皮生长因子(VEGF)、内皮抑素(ES)、肝细胞生长因子(HGF)、单核细胞趋化蛋白-1(MCP-1)水平的变化和治疗后的临床疗效,并随访半年,记录排卵率和妊娠率。结果:治疗后,两组血清FSH、LH、T、VEGF、ES、HGF、MCP-1水平均较治疗前明显降低(P0.05),且观察组以上指标均明显低于对照组(P0.05);观察组临床总有效率[91.30%vs. 75.00%]、排卵率[78.26%vs. 36.96%]、妊娠率[59.09%vs. 5.91%]均明显高于对照组(P0.05)。结论:来曲唑联合妈富隆治疗多囊卵巢综合征的临床效果显著优于妈富隆单药治疗,其可有效改善患者内分泌,提高妊娠率,可能与降低血清VEGF、ES、HGF、MCP-1的表达相关。     

重组中华仓鼠卵巢细胞培养过程控制的研究进展

中华仓鼠卵巢细胞(Chinese Hamster Ovary Cells,CHO)是科研及生产中用于蛋白表达的常用体系。与大肠杆菌相比,CHO获得高表达的细胞株所需时间更长,蛋白产量更低。因此,大规模培养细胞所需的成本较高,培养条件也不易掌握。但该体系产生的蛋白纯度高,因而被广泛用于工业生产中。本文对CHO细胞的培养方式、pH值测定、渗透压、溶氧及培养液成分的选择等多方面条件进行综述,为优化中华仓鼠卵巢细胞培养的策略及具体方法提供理论依据。     

Functional Divergence of Multiple Duplicated Foxl2 Homeologs and Alleles in a Recurrent Polyploid Fish

Evolutionary fates of duplicated genes have been widely investigated in many polyploid plants and animals, but research is scarce in recurrent polyploids. In this study, we focused on foxl2, a central player in ovary, and elaborated the functional divergence in gibel carp (Carassius gibelio), a recurrent auto-allo-hexaploid fish. First, we identified three divergent foxl2 homeologs (Cgfoxl2a-B, Cgfoxl2b-A, and Cgfoxl2b-B), each of them possessing three highly conserved alleles and revealed their biased retention/loss. Then, their abundant sexual dimorphism and biased expression were uncovered in hypothalamic–pituitary–gonadal axis. Significantly, granulosa cells and three subpopulations of thecal cells were distinguished by cellular localization of CgFoxl2a and CgFoxl2b, and the functional roles and the involved process were traced in folliculogenesis. Finally, we successfully edited multiple foxl2 homeologs and/or alleles by using CRISPR/Cas9. Cgfoxl2a-B deficiency led to ovary development arrest or complete sex reversal, whereas complete disruption of Cgfoxl2b-A and Cgfoxl2b-B resulted in the depletion of germ cells. Taken together, the detailed cellular localization and functional differences indicate that Cgfoxl2a and Cgfoxl2b have subfunctionalized and cooperated to regulate folliculogenesis and gonad differentiation, and Cgfoxl2b has evolved a new function in oogenesis. Therefore, the current study provides a typical case of homeolog/allele diversification, retention/loss, biased expression, and sub-/neofunctionalization in the evolution of duplicated genes driven by polyploidy and subsequent diploidization from the recurrent polyploid fish.