AIM: To develop a novel gene-delivery therapeutic based on CRISPR/Cas9 genome editing technology capable of specifically targeting and knocking out the VEGFA gene, thereby achieving sustained suppression of VEGFA expression in retinal pigment epithelial(RPE)cells and providing a new strategy for gene therapy in retinal neovascular diseases.METHODS:Single guide RNAs targeting the human VEGFA gene for knockout were designed, and corresponding recombinant plasmids were constructed. A novel polymer(PTEE)was used to encapsulate the plasmids to prepare a PTEE-loaded anti-VEGFA plasmid(PLAP)gene delivery system. PTEE materials at concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 μg/μL were co-incubated with ARPE-19 cells, and the biocompatibility of PTEE was evaluated using the cell counting kit-8(CCK-8)assay. Recombinant plasmids expressing green fluorescent protein(GFP)were constructed. Lipofectamine 3000 and jetOPTIMUS®DNA transfection reagents were used as control groups, and PTEE nanomaterials were used as the experimental group to encapsulate the plasmids. When the cell confluence reached 80%, the formulations were transfected into ARPE-19 and 293T cells. GFP expression was observed under light microscopy, and the transfection efficiencies of each group were compared. ARPE-19 cells were induced under hypoxia, and PLAP was transfected into the cells. The expression level of VEGFA was detected by enzyme-linked immunosorbent assay(ELISA)to evaluate the efficacy of this novel gene delivery system.RESULTS: After co-incubation of ARPE-19 cells with different concentrations of PTEE for 24 h and 48 h, no significant effect on cell viability was observed in any group. The transfection efficiency of PLAP in ARPE-19 cells was higher than that in the Lipo3000 and jetOPTIMUS groups, with statistically significant differences(P<0.01). Hypoxia for 6 h significantly induced the upregulation of VEGFA mRNA expression in ARPE-19 cells, and under hypoxic conditions, the PTEE group exhibited a significant inhibitory effect on VEGFA expression(P<0.01).CONCLUSION:PLAP exhibits favorable biocompatibility and prominent VEGFA inhibitory effects in vitro, making it a potential candidate drug for gene therapy of retinal neovascular diseases.

The rehabilitation and nursing experience of a patient with recurrent coronary vasospasm and emergency scrotal hematoma in a short time after cardiac radiofrequency ablation was summarized.Key nursing points include:identification and emergency care of coronary vasospasm after cardiac radiofrequency ablation;enhanced psychological care and targeted guidance for smoking cessation management;implementation of scrotal hematoma nursing plan with integrated Chinese and westem medicine;development of individualized follow-up and continuous rehabilitation nursing,help to back to society.Through careful and professional treatment and nursing after 27 d,the patient's left ventricular ejection fraction(LVEF)recovered from 39％to 57％,and the patient was discharged smoothly.After discharge,the patient underwent continuous rehabilitation care,and the scrotal hematoma subsided significantly in 1 month after discharge,and the activity endurance increased.

The rehabilitation and nursing experience of a patient with recurrent coronary vasospasm and emergency scrotal hematoma in a short time after cardiac radiofrequency ablation was summarized.Key nursing points include:identification and emergency care of coronary vasospasm after cardiac radiofrequency ablation;enhanced psychological care and targeted guidance for smoking cessation management;implementation of scrotal hematoma nursing plan with integrated Chinese and westem medicine;development of individualized follow-up and continuous rehabilitation nursing,help to back to society.Through careful and professional treatment and nursing after 27 d,the patient's left ventricular ejection fraction(LVEF)recovered from 39％to 57％,and the patient was discharged smoothly.After discharge,the patient underwent continuous rehabilitation care,and the scrotal hematoma subsided significantly in 1 month after discharge,and the activity endurance increased.

In recent years, the theory of cancer stem cells has provided a new perspective on the treatment of cancers including breast cancer. The accurate sorting of breast cancer stem cells is critical. The sorting procedure is consist of 4 steps: isolation of side population, serum-free suspension culture,determination of specific cell surface markers and of the activity of aldehyde dehydrogenase 1 ( ALDH1 ) enzymatic through the ALDEFLUOR assay. Some studies choose drug resistance as an additional method for sorting cancer stem cells because of the enrichment of cancer stem cells after chemotherapy . The controversy about the sorting outcome of breast cancer stem cell mainly focus on molecular markers like CD44+ CD24- and ALDH1+ . The problem needed to be settled is to identify which sorting method and markers are appropriate.

Objective:The purpose of this study is to investigate the expression of tumor stem cell marker aldehyde dehydrogE-nase 1 (ALDH1) in breast cancer and its clinical significance. Methods:The expression of ALDH1 protein was examined by immunohistochemical staining in 92 breast cancer tissues. The correlation analysis and diseasE-free survival analysis of patients was evaluated based on the clinical follow-up data. Results:Expression of ALDH1 protein had a significant correlation with progesterone receptor (PR) and cerb-B2 (P<0.05), but had no significant correlation with age, tumor size, clinical staging, and lymph node metastasis (P>0.05). The 2-year diseasE-free survival rate of AlDH1-positive patients was lower than that of ALDH1-negative patients (P<0.05). ALDH1-positive patients, who received CEF regimen chemotherapy and hormone therapy, had lower 2-year diseasE-free survival rate than that of ALDH1-negative patients (P<0.05). Conclusion:The decreased diseasE-free survival rate of ALDH1-positive patients is related with drug resistance. ALDH1 could be used as an independent factor for predicting the prognosis of breast cancer.