Objective To study the effects of peroxisome proliferators-activated receptor (PPAR) γ on the growth of human hepatocellular carcinoma cells and explore the roles of phosphatase and tensin homologue deleted on chromosome ten (PTEN) and phospho-Akt in this process. Methods SMMC-7721 cells were treated with 15-d-PGJ2 or pioglitazone, which were two kinds of PPARγ ligands, at different concentrations. The viability of SMMC-7721 cells was evaluated by MTT assay. The cell cycle was analyzed by flow cytometry. PTEN mRNA level was determined by RT-PCR. The protein expressions of PTEN and pAkt were measured by Western blot analysis. Results It was demonstrated through MTT assay that both 15-d-PGJ2 and pioglitazone had an inhibitory effect on the growth of SMMC-7721 cells in a time- and dose- dependent manner. According to flow cytometry detection, more cells were arrested in G0/G1 phase. Increased expression of PTEN mRNA was detected in 15-d-PGJ2 or pioglitazone-treated cells through RT-PCR. Increased expression of PTEN protein and decreased expression of pAkt were confirmed by Western blot analysis. Conclusion The ligands of PPARγ could inhibit SMMC-7721 cells proliferation in a time- and dose- dependent manner. The upregulation of PTEN may be involved in the underlying mechanism.
Objective To assess the efficacy of a kind of new material lipid magnetic particle for isolation and detection of lung cancer circulating tumor cells (CTCs). Methods Immune lipid magnetic particles were prepared with reverse evaporation method and they were assembled into kits with EpCAM and EGFR antibody respectively. Their efficacy were evaluated by detecting A549 cells in group A (A549 cells mixed in phosphated buffer solution) and group B (A549 cells mixed in blood from healthy volunteers). Lung cancer CTCs of hospitalized patients were also detected with both immune magnetic particals. Then the detecting efficacy was compared between EpCAM immune lipid magnetic particles and the conventional CellsearchTM system. Results The immune lipid magnetic particles had high capture efficiency for CTCs isolation and identification. The median of EpCAM immune lipid magnetic particles method in detecting A549 cells in group A was 92%, and EGFR was 90%. The median of EpCAM immune lipid magnetic particles method in detecting A549 cells in group B was 85%, and EGFR was 81%. In 13 patients with lung cancer, CTCs can be detected with both immune lipid magnetic particles methods and both medians were 5; In negative control, the medians of both methods were 0 (P＜0.05). EpCAM immune lipid magnetic particles method can detect more CTCs than conventional CellsearchTM system in 3 lung cancer patients. Conclusions Immune lipid magnetic particles have good efficacy for lung cancer CTCs detection and has promising clinical application value. The EpCAM immune lipid magnetic particles have equal efficiency in detecting lung cancer CTCs with EGFR. There is a trend that EpCAM immune lipid magnetic particles is superior to the conventional CellsearchTM system.
ObjectiveTo establish a method of air-liquid interface culture and ciliary beat frequency measurement of mouse tracheal-bronchial epithelial cells to simulate the physiological function of airway epithelium.MethodsBALB/c mouse tracheal-bronchial epithelial cells were obtained by digestion with 1 mg/mL protease in cold temperature overnight, and the digestion time was optimized to ensure the quantity and viability of the obtained cells. After removing fibroblasts by differential velocity adhesion method, the cells were cultured into collagen coated Transwell inserts. Proliferating phase and air-liquid interface culture were promoted with different culture media.ResultsCell numbers obtained by cold protease overnight digestion for 12 h, 14 h and 16 h were (1.78±0.33)×105, (1.93±0.26)×105 and (2.01±0.28)×105, respectively. Cell viability by trypan blue staining were (96.86±0.25)%, (94.73±1.63)% and (86.87±5.95)%, respectively. Cells were 100% confluent in Transwell chamber after 1-week proliferation, and the ciliary beat frequency was observed under microscope after 2 - 3 weeks of air-liquid interface culture. The cilia structure was confirmed by hematoxylin-eosin staining, electron microscopy and immunofluorescence. Ciliary beat frequency of the cells obtained by this method was consistent with that of mouse trachea in vivo, which further demonstrated its capacity in simulating the physiological function of airway epithelium. ConclusionsThe separation and air-liquid interface culture system as well as the ciliary beat frequency measurement method established in this experiment is simple, stable, efficient and reliable, which establishes a substantial foundation for exploring the pathogenesis and treatment mechanism of airway diseases. It can also provide reference for the culture of epithelium in the airway of other species and/or other organs.