RESUMO
BACKGROUNDS: The plasticity of retinal stem cells (RSCs), a type of cells that can differentiate into neuron cells and photoreceptor cells, endows them with potential therapeutic properties that can be applied to regenerative medicine. Gene modification of these stem cells before trans-differentiation and transplantation enhances their survival and increases their therapeutic function. The different ways to effectively deliver gene into RSCs are still discussed. This study aimed to use the acoustic waves to improve the efficacy of gene delivery for RSCs. METHODS: RSCs were obtained from non-fetal human ocular pigmented ciliary margin tissues. The enhanced green fluorescent protein-encoded murine stem cell retroviruses (MSCV) were prepared and used to infect RSCs. Glass chambers containing RSCs, retroviruses, and various concentrations of polybrene (0, 0.8, 2, 4 and 8 µg/mL) were exposed under 20 or 25 Vp-p ultrasonic standing wave fields (USWF) for 5 min. The percentage of green fluorescent protein positive cells in each sample was calculated and compared to test the efficacy of gene transduction. RESULTS: Our results showed that the efficiency of gene transduction by MSCV infection was enhanced following the concentration of polybrene and the energy of USWF. The percentage of green fluorescent protein positive cells was significantly higher in chambers that contained 8 µg/mL of polybrene and was exposed to 20Vp-p of USWF for 5 min. In addition, the percentage increased in chambers contained 2, 4 and 8 µg/mL of polybrene when they were exposed to 25Vp-p of USWF. Comparing to those did not treated with ultrasound, the efficiency of retroviral transduction to RSCs increased 4-fold after exposed to USWF for 5 min. CONCLUSION: We demonstrated the ability of ultrasound standing waves to improve retroviral transduction into RSCs. We believe that this may be applied to the experimental designs of future studies and may have possible therapeutic uses.
Assuntos
Retroviridae/genética , Som , Células-Tronco/metabolismo , Transdução Genética/métodos , Adulto , Idoso , Agregação Celular , Separação Celular , Células Cultivadas , Brometo de Hexadimetrina/farmacologia , Humanos , Lactente , RetinaRESUMO
The high invasiveness and frequent recurrence of lung adenocarcinoma (LAC) are major reasons for treatment failures and poor prognoses. Alterations in microRNAs (miRNAs) expression have been shown in lung cancers. Recent reports have demonstrated that tumors contain a small subpopulation of cancer stem cells (CSCs) that possesses self-renewing capacity and is responsible for tumor malignancy including metastasis, relapse, and chemoradioresistance. However, a miRNAs-based therapeutic approach in LAC-associated CSCs (LAC-CSCs) is still blurred. Using miRNA/mRNA-microarray and Quantitative RT-PCR, we found that the expression of miR145 is negatively correlated with the levels of Oct4/Sox2/Fascin1 in LAC patient specimens, and an Oct4(high)Sox2(high)Fascin1(high)miR145(low) phenotype predicted poor prognosis. We enriched LAC-CSCs by side population sorting or identification of CD133 markers and found that LAC-CSCs exhibited low miR145 and high Oct4/Sox2/Fascin1 expression, CSC-like properties, and chemoradioresistance. To clarify the role of miR145, we used a polyurethane-short branch-polyethylenimine (PU-PEI) as the vehicle to deliver miR145 into LAC-CSCs. PU-PEI-mediated miR145 delivery reduced CSC-like properties, and improved chemoradioresistance in LAC-CSCs by directly targeting Oct4/Sox2/Fascin1. Importantly, the repressive effect of miR145 on tumor metastasis was mediated by inhibiting the epithelial-mesenchymal transdifferentiation (EMT) and metastastic ability, partially by regulating Oct4/Sox2/Fascin1, Tcf4, and Wnt5a. Finally, in vivo study showed that PU-PEI-mediated miR145 delivery to xenograft tumors reduced tumor growth and metastasis, sensitized tumors to chemoradiotherapies, and prolonged the survival times of tumor-bearing mice. Our results demonstrated that miR145 acts as a switch regulating lung CSC-like and EMT properties, and provide insights into the clinical prospect of miR145-based therapies for malignant lung cancers.