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Objective To knockout the exon51 of DMD gene in HEK293T cells using the CRISPR/Cas9 system. Methods Design the target sequences of sgRNA and clone them into plasmid PX459 respectively; transfer these plasmids into HEK293T cell and extract the total genome DNA; test the activity of sgRNAs with surveyor assay, choose the most efficient one in each end;construct plasmid PX459-2sgRNA and transfer it into HEK293T cells;check whether the exon51 has been knocked known with PCR and T vector sequencing. Results 50% of HEK293T cells' DMD gene exon51 were knocked out,showing a high gene editing efficiency. Conclusions We successfully establish a platform to target knockout the exon51 of DMD gene and provide an important experimental basis for the treatment of DMD and other genetic diseases.
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BACKGROUND: Umbilical cord mesenchymal stem cells (UC-MSCs) are a group of cells that have self-renewal, highly proliferative and multidrug differentiation potential. The properties of UC-MSCs and their tumor tropism make them an ideal tool for glioma cell therapy. These cells can act by paracrine or as a delivery system for genes and drugs. It has been demonstrated that UC-MSCs can inhibit the growth of glioma and improve the survival after transplantation into the brain. OBJECTIVE: To summarize the molecular mechanisms and safety of UC-MSCs in the treatment of glioma and to provide a useful reference for further research. METHODS: We searched the PubMed and CNKI databases from 2000 to 2017 with the English terms of "glioma; umbilical cord mesenchymal stem cells" and the Chinese terms of "glioma; umbilical cord mesenchymal stem cells; safety; molecular mechanism". Based on the inclusion and exclusion criteria, 55 articles were finally reserved for review. RESULTS AND CONCLUSION: UC-MSCs have obvious effect on treating glioma. These cells can treat glioma through homing mechanism and paracrine mechanism as gene carrier and co-culture. Moreover, UC-MSCs have certain safety in the treatment of glioma.
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Objective To investigate the effects of regular and irregular mandatory treadmill running (TR) on learning and memory abilities,stress response,survival and maturation of newborn neurons in dentate area of C57 mice. Methods Forty-eight male C57BL/6 mice were randomly divided into regular running group (n=16,definite time and quantity), irregular running group (n=16,definite time and different quantities) and sedentary group (n=16).Injection of bromodeoxyuridine (BrdU,50 mg/kg) was given intraperitoneally to mice from the 1st d to the 7th d of exercise,and then,these mice were subjected to a 4-wk TR training course or sedentary exercise.Double irnmunofluorescence labeling was employed to observe the effect of TR on survival and maturation of newborn neurons. Mice were trained on the Morris water maze to test the abilities of learning and memory. Corticosterone (CORT).level in the venous blood was measured with a radioimmunoassay kit. Results As compared with that in mice of the irregular running group and sedentary group,significant decrease of latency was noted in mice of the regular running group on the 1st, 3rd, 4th and 5th d of place navigation test (P＜0.05). As compared with that in mice of the sedentary group, significant decrease of latency was noted in mice of the irregular running group and regular running group on the 2nd d of place navigation test (P＜0.05),while no significant difference of that was noted between mice of the irregular running group and regular running group (P＞0.05).The platform was removed for a 60-s probe test 4 h after the last trial to test the recall ability, and the times of staying in the target zone in each group were regular running group＞irregular running group＞control group, and significant difference was noted between each 2 groups (P＜0.05). The numbers of double immunofluorescence labeled cells in the dentate gyrus were counted as regular running group＞irregular running group＞control group, and significant difference was noted between each 2 groups (P＜0.05). The serum CORT level was measured as regular running group＜irregular running group＜control group, and significant difference was noted between each 2 groups (P＜0.05). Conclusion Mandatory treadmill running could improve the learning and memory abilities,which may be related to the increment of survival and maturation of neural precursor cells,and decreased level of serum CORT; and the effect of regular mandatory treadmill running is better than that of irregular mandatory treadmill running.
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This study is to investigate the protective effects of the SB203580 against radiation induced mortality and intestinal injury of mice. A total of 67 male C57BL/6 mice (20.0-22.0 g) were matched according to body weight and randomly assigned to one of three groups: control, total body irradiation exposure (IR, 7.2 Gy) only, and IR (7.2 Gy) + SB203580 (15 mg x kg(-1)). 30 days survival rate was observed in the experiment. In intestinal injury experiment, the expression levels of caspase-3, Ki67, p53 and p-p38 were assayed in the mice intestine crypts. The results showed that the 30 days survival rate was 100% (control), 0 (IR) and 40% (IR+ SB203580), separately. Compared to the IR groups, the positive cells of caspase-3, p53 and p-p38 in crypt cells decreased 33.00%, 21.78% and 34.63%, respectively. The rate of positive cells of Ki67 increased 37.96%. Significant difference was found between all of them (P < 0.01). SB203580 potently protected against radiation-induced lethal and intestinal injury in mice, and it may be a potential radio protector.
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Animales , Masculino , Ratones , Apoptosis , Efectos de la Radiación , Caspasa 3 , Metabolismo , Inhibidores Enzimáticos , Farmacología , Imidazoles , Farmacología , Intestinos , Metabolismo , Patología , Antígeno Ki-67 , Metabolismo , Ratones Endogámicos C57BL , Piridinas , Farmacología , Traumatismos Experimentales por Radiación , Metabolismo , Mortalidad , Patología , Protectores contra Radiación , Farmacología , Distribución Aleatoria , Proteína p53 Supresora de Tumor , Metabolismo , Irradiación Corporal Total , Proteínas Quinasas p38 Activadas por Mitógenos , MetabolismoRESUMEN
OBJECTIVE@#To compare the behavioral improvement to find the best transplantation approach for treating brain injury through transplanting amniotic-derived mesenchymal stem cells into brain injured rats in different ways.@*METHODS@#Eighty brain injured Wista rats were randomly divided into a control group with brain injury alone (n=20) and a treatment group(n=60) which were further evenly divided into Group A (transplanted through the vena caudalis), Group B (transplanted through the ventriculus cerebri lateralis), and Group C (transplanted through the injured brain area). Each group was transplanted with amniotic-derived esenchymal stem cells, and their therapeutic efficacy would be evaluated through the neurological severity score (NSS).@*RESULTS@#Compared with other groups, the behaviors of Group C had markedly improved. There was statistically significant difference in the 2 groups (P0.05).@*CONCLUSION@#Transplanting the amniotic-derived mesenchymal stem cells into the injured brain area may be effective for brain injury in rats.