Characterization and identification of primarily cultured astrocytes and microglia / 生物工程学报

The aim of this study was to investigate the growth characteristics of primarily cultured astrocytes and microglia of different generations and then optimize the method for obtaining primary astrocytes and microglia effectively. Primarily cultured microglia were isolated and purified from the cortices of neonatal mice. The proliferation curve of mixed glia cells was measured by Cell Counting Kit-8 (CCK-8) assay, the proportion of astrocytes and microglia was detected by flow cytometry, and the polarization of the two types of glia cells was identified by immunofluorescence staining. Cell growth results showed that the mixed glia cells of P0 and P1 generation had the best proliferative activity; 97.3% of the high purity microglia could be obtained by mechanical shaking at 170 r/min for 30 min, and there was no significant difference in the morphology of ionized calcium-binding adapter molecule 1 (Iba-1) positive microglia and the proportion of M1 and M2 phenotype among the P0, P1 and P2 generations of microglia isolated by the above methods. Moreover, 95.7 % of the high purity astrocytes could be obtained by astrocyte cell surface antigen-2 (ACSA-2) magnetic beads separation, and there was no significant difference in the morphology of glial fibrillary acidic protein (GFAP) positive astrocyte and the proportion of A1 and A2 phenotype among the P0, P1 and P2 generations of astrocyte isolated by the above methods. Taken together, this study observed the growth characteristics of primarily cultured microglia and astrocyte in vitro, and then proved the best generations for purifying microglia and astrocytes. Finally, we optimized the methods of obtaining microglia and astrocyte, and verified that continuous culture within 2 generations will not affect the functional phenotypes of glia cells. These results provide technical support for studying the molecular mechanism of inflammation-associated diseases in nervous system.

Visible light emitted quantum dots targeting EGFR in triple-negative breast cancer / 中华核医学与分子影像杂志

Objective:To explore the feasibility and conditions of in vitro and in vivo imaging of triple-negative breast cancer using visible light emitted quantum dots(QDs) as the carrier to target epidermal growth factor receptor (EGFR). Methods:The water-soluble QDs reacted with Cetuximab to synthesize the probe QD-Cetuximab. The morphology, particle size, stability and luminescence properties of the probe were examined. Human breast cancer cells MDA-MB-468 (EGFR+ ) and MDA-MB-453 (EGFR-) were cultured. Cytotoxicity assays, in vitro imaging and fluorescence intensity quantification were performed after cells incubation with QD-Cetuximab and QDs. Eight MDA-MB-468 tumor-bearing mice models were constructed, 100 μl QD-Cetuximab and QDs were injected through the tail vein. In vivo imaging and probe distribution were obtained at different time points. Independent-sample t test was used to analyze the data. Results:QD-Cetuximab had a particle size of (40.34±2.44) nm detected by transmission electron microscope (TEM), a hydrated particle size of (57.85±4.69) nm detected by dynamic light scattering (DLS), and a stable structure. When the concentration of QD-Cetuximab was ≤50 nmol/L, the relative survival rate of cells was more than 90%, and when the concentration exceeded 100 nmol/L, the relative survival rate of cells was reduced to (72.52±4.91)% ( P<0.05). The red fluorescence of MDA-MB-468 incubated with QD-Cetuximab was stronger than that of MDA-MB-468 incubated with QDs and MDA-MB-453 incubated with QD-Cetuximab or QDs. The confocal fluorescent intensity quantitative determination showed that the ratio of QD-Cetuximab group/QDs group was 5.1 (863.36/169.97). Flow cytometry showed that the uptake of QD-Cetuximab and QDs by MDA-MB-468 increased with incremental incubating concentration, and the former was more significantly( t values: 12.25-38.11, all P<0.05). When the incubating concentration was 25, 50, 100, and 200 nmol/L, the quantitative average fluorescent intensity ratio of QD-Cetuximab group/QDs group was 5.4, 6.9, 7.4 and 6.2, respectively. The QD-Cetuximab and QDs probes mainly accumulated in the liver in vivo. The fluorescence emitted by tumor was not obvious under the high fluorescence of liver as a background. However, the fluorescence was visible in the isolated tumor tissue, and the quantitative fluorescence intensity of experimental group and control group were (2.46±0.60)×10 4 and (1.29±0.05)×10 4, respectively ( t=3.392, P=0.015). Conclusions:Cetuximab can increase the targeting ability of QDs and promote cell uptake. Although the isolated tumor imaging results are acceptable, further modification of QDs should be considered to reduce the liver uptake and improving in vivo fluorescence imaging efficiency.