In vitro and in vivo investigation of a novel two-phase delivery system of 2-methoxyestradiol liposomes hydrogel

For improving effectiveness of conventional chemotherapy of subcutaneous tumor, we selected 2-methoxyestradiol (2-ME) as a model drug, local injectable PLGA-PEG-PLGA copolymer thermosensitive hydrogel loading 2-ME liposomes instead of free 2-ME as a novel two-phase drug delivery system was developed, which avoid rapid clearance of liposomes follwing systemic administration. This new transport system was characterized in vitro and in vivo including rheological behavior, thermo-sensitiveness, stability, released character and intratumoral delivery. The PLGA-PEG-PLGA copolymer solution exhibited still reversible thermosensitive property and better syringeability after incorporated 2-ME liposomes. The 2-ME liposomes were demonstrated stable in the hydrogel by five methods such as scanning electron microscopy (SEM), fluorescent labeling, opalescence, particle size and ultrafiltration methods. Results showed that intact liposomes could be released from the hydrogel and following zero-order model, and sustained release one–two months in vitro and in vivo. In vivo release data demonstrating that 2-ME liposomes could be transported to tumor site, improved therapeutic efficacy and bioavailability of 2-ME liposomes in subcutaneous tumor chemotherapy.     

一体化的光声-荧光显微镜用于活体肿瘤成像

报道了一种利用单一波长激发的同时产生光声和荧光信号的显微成像系统,本成像系统具有超高的成像分辨率(<6μm)。借助外源的造影剂在近红外的吸收特性,利用光声-荧光显微成像系统对活体肿瘤进行光声/荧光成像。实验结果表明,光声-荧光显微镜在早期肿瘤的成像和检测等方面具有潜在的应用价值。因此,通过研究和选择适当的双模态造影剂,该系统在不同病理模型中可以提供更准确的组织信息及生理参数。     

肺癌患者外周血中循环肿瘤细胞的快速分离与体外培养

循环肿瘤细胞(circulating tumor cells, CTCs)是从肿瘤病灶脱落并进入外周血液循环的处于游离状态的肿瘤细胞,代表了肿瘤病灶的分子特征,可用于对肿瘤的“液体活检”。但外周血中CTCs数目极为稀少,使得后续针对CTCs的分子与功能分析面临巨大挑战。鉴于此,本文建立了一种基于微流控芯片和免疫磁珠的能够快速从肺癌患者的外周血中分离CTCs的方法。该方法直接针对全血进行一步分离,可避免血液样本预处理及富集等过程对细胞造成的损伤,从而有效地保护CTCs的活性(>90%)。分离得到的CTCs可富集在小体积中(80 μL),实现高密度的细胞培养,完成体外扩增,扩增后的CTCs可以被进一步冻存、复苏及再次增殖培养,表明已经对患者血液中的CTCs成功建系。本文进一步对CTCs进行了基因突变(EGFR、KRAS、PIK3CA、TP53和BRAF)检测及荧光标记葡萄糖类似物(2-NBDG)摄取的功能分析,证明CTCs存在较大异质性。本研究成功实现了对外周血中稀少的CTCs进行体外培养,并对CTCs进行了基因、蛋白、功能等各个层面的分析,这对于肿瘤精准医疗具有重要的临床意义。     

The potential of flow cytometry in the study of Bacillus cereus

Flow cytometry (FCM) is a rapid method allowing the acquisition of multiparametric data from thousands of individual cells within a sample. As well as measuring the intrinsic light scattering properties of cells, a plethora of fluorescent dyes may be employed to yield information on macromolecule content, surface antigens present or physiological status. Despite FCM's indispensability within other fields e.g. immunology, it is underutilized within microbiological research. In this review, a strong case is presented for the potential of FCM in the study of Gram-positive spore-former, Bacillus cereus . Previous reports where FCM was successfully used in the study of B. cereus are reviewed along with relevant studies involving other members of the genus. Under headings reflecting common research themes associated with B. cereus , specific instances where FCM has generated novel data, providing a unique insight into the organism, are discussed. Further applications are posited, based on the authors' own research with FCM and B. cereus and work extant in the broader field of microbial cytometry. The authors conclude that, while the expense of equipment and reagents is an undeniable disadvantage, FCM is a technique capable of generating significantly novel data and allows the design and execution of experiments that are not possible with any other technique.     

肝细胞癌循环肿瘤细胞标志物及检测方法研究进展

肝细胞癌(hepatocellular carcinoma, HCC)在中国是一种高发病率和高死亡率的恶性肿瘤。肿瘤切除、肝移植是治疗该病最有效的手段,但术后的高复发率和高转移率是影响患者预后的重要因素。外周血循环肿瘤细胞(circulating tumor cells, CTCs)是导致肝细胞癌术后复发和转移的必要因子。综述了CTCs的标记物——磷脂酰肌醇蛋白聚糖-3、转铁蛋白受体、甲胎蛋白、α-L岩藻糖苷酶、上皮细胞粘附因子、高尔基蛋白73和异常凝血酶原等,以及利用这些标记物检测CTCs的特异性和灵敏度,以期为肝细胞癌转移的早期检测、术后的复发、预后评估和选择治疗方案等提供依据。     

Cell surface antigens on rat neural progenitors and characterization of the CD3 (+)/CD3 (-) cell populations

While the hematopoietic lineage has been extensively studied using cluster of differentiation (CD) antibodies, very few data are available on the extracellular epitopes expressed by rat neural progenitors (rNPC) and their derivatives. In the present study, we used flow cytometry to screen 47 cell surface antigens, initially known as immune markers. The quantitative analyses were performed on rat neurospheres and compared with primary cultures of astroglial cells or cerebellar neurons. Several antigens such as CD80 or CD86 were clearly undetectable while others, like CD26 or CD161, showed a weak expression. Interestingly, 10% and 15% of the cells were immunopositive for CD172a and CD200, two immunoglobulin superfamily members preferentially expressed by glial or neuronal cells, respectively. Over 40% of the cells were immunopositive for CD3, CD71, or MHCI. The biological significance of the latter markers in rNPC remains to be determined but analyses of the CD3(-)/CD3(+) populations isolated by magnetic cell separation revealed differences in their cell fate. Indeed, CD3(+) cells did not establish neurospheres and differentiated mostly into GFAP(+) cells while CD3(-) cells were able to generate neurospheres upon mitogen treatment and gave rise to GFAP(+), A2B5(+), Tuj-1(+), and RIP(+) cells under differentiating conditions. In contrast, CD71(-)/CD71(+) cells did not show any significant difference in their proliferating and differentiating potentials. Finally, it is worth noting that an subpopulation of cells in rat neurospheres exhibit an immunoreactivity against anti-CD25 (IL2 receptor) and anti-CD62L (L-selectin) antibodies. The results reveal particular surface antigen profiles, giving new perspectives on the properties of rat brain-derived cells.     

流式核酸单分子计数方法研究进展

研发精确灵敏的核酸定量技术,提升对微量样本的测量能力,在当前抗击新冠疫情的背景下尤其重要。流式核酸单分子计数是一种基于“计数”技术的定量测量方法,该方法无需聚合酶链式反应而对荧光标记的靶分子直接定量,测量结果直接溯源到国际基本单位。作为潜在的基准计量方法,它是当前核酸定量测量技术的重要补充,可广泛应用于临床诊断、转基因标识、食品药品残留检测等多个领域。对流式核酸单分子计数方法的原理、检测体系的硬件组成以及数据分析模型进行了综述,并展望了该方法对构建生物计量标准体系的重要意义。     

基于L5178Y细胞体外Pig-a基因突变试验方法的建立与初步探索

利用小鼠淋巴瘤细胞L5178Y tk+/--3.7.2C和阴性化合物Glc、NaCl及阳性化合物EMS、ENU、4-NQO、B(a)P建立并验证基于哺乳动物细胞的体外Pig-a基因突变检测方法。计算细胞相对倍增速率评价细胞毒性,抗体标记突变型细胞确定流式检测模板,L5178Y细胞经EMS处理后第4、8、12、16、20天检测Pig-a基因突变频率,确定最大突变频率发生时间点,免疫荧光技术检测CD90蛋白在细胞中的定位情况,采用PCR方法进行突变位点分析。结果表明(:1)Glc、NaCl、EMS、ENU、B(a)P、4-NQO所设浓度组RPD均大于50%。(2)Pig-a基因突变频率在给药后第8天出现峰值。Glc和NaCl致Pig-a基因突变频率均小于200×10-6,各浓度组与溶剂对照组间不存在显著性差异(P>0.05),EMS、ENU、B(a)P、4-NQO均可引起Pig-a基因突变频率增加,且与溶剂对照组相比存在显著性差异。(3)免疫荧光成像显示突变型细胞表面无CD90蛋白,野生型细胞正常表达CD45,CD90蛋白。(4)基因突变位点检测显示存在G→C、A→C、C→T三种突变类型。基于小鼠淋巴瘤L5178Y细胞分别在有无S9代谢活化条件下成功建立体外Pig-a基因突变的遗传毒性检测方法,旨为化合物体外遗传毒性评价或药物研发早期遗传毒性筛选提供新方法。     

Two new C3H mouse ascites tumor cell lines capable of proliferation in vivo and in suspension culture: Morphological,karyological, kinetic,and immunological properties

Summary The establishment of mouse tumor cell lines capable of proliferating in vivo and in suspension culture was undertaken. The MM-46 tumor line, initiated from primary mammary carcinoma arising in a C3H/He mouse, was maintained for over 100 generations in the peritoneal cavities of syngenic mice. At the 50th generation of the tumor suspension, cultures were initiated. The established cell lines, designated TMT-1 and TMT-2, were characterized in vitro and in vivo. The morphological finding indicated that TMT-1 and TMT-2 cells from mice closely resembled the MM-46 tumor cells. The oncogenic potential of the cultured cells was comparable to that of the original ascites tumor. The population doubling time of TMT-1 and TMT-2 cell lines was about 12 hr in mice, whereas the population doubling time of both cell lines lengthened to 20 hr in suspension culture. The increase of doubling time in culture was due to the prolongation of the G₁ period. The cell lines, TMT-1 and TMT-2, whether from culture or mice, possessed colony forming ability in soft agar medium. The colony forming ability of the cells decreased gradually through in vivo passages but it recovered upon recultivation of the cells from mouse to culture. Chromosome analysis and cytotoxicity test by anti-MM antiserum indicated that TMT-1 and TMT-2 cell lines closely resembled and had been derived from MM-46 tumor line. Therefore, it is possible to assay cell survival in vitro after in vivo experiments on these cells.