ABSTRACT
Objective:To investigate the effects of down-regulation of Krüppel-like factor 5 (KLF5) on biological functions of rat intestinal epithelial cells IEC-6 in response to ionizing radiation.Methods:Rat intestinal epithelial IEC-6 cells were irradiated with 0, 2, 4, 8, 12, 16 Gy of X-rays and 3 h later the expression of KLF5 in IEC-6 cells was detected by Western blot. IEC-6 cells were irradiated to 12 Gy and 0, 0.5, 1, 2, 3, 5, 7 and 24 h later the expression of KLF5 in IEC-6 cells was detected by Western blot. shRNA sequences targeting rat KLF5 gene were designed, synthesized and inserted into the lentiviral vector. The recombinant lentiviral vectors were packaged in human embryonic kidney 293T cells, and the lentivirus titers were determined. IEC-6 cells were infected with the recombinant lentivirus, and the transfection efficiency was observed under fluorescence microscope. Real-time PCR and Western blot were used to detect the expressions of KLF5 mRNA and protein in the transfected cells 72 h post transfection. The consequent experiments included four groups: negative control group, shKLF5 group, radiation group and radiation + shKLF5 group. The cell viability was observed in KLF5 silencing cells irradiated with 8 Gy by using CCK-8 assay. The cell cycle distribution and apoptosis were detected in KLF5 silencing cells irradiated with 8 Gy by flow cytometry. Immunofluorescence staining was applied to visualize the γ-H2AX foci in nucleus of KLF5 silencing cells irradiated with 2 Gy.Results:The expression of KLF5 increased with the different doses. The expression of KLF5 increased first, then decreased and peaked at 5 h post-irradiation with 12 Gy. KLF5 shRNA lentiviral vectors were successfully constructed. The mRNA and protein level of KLF5 were down-regulated in recombinant lentivirus transfected IEC-6 cells 72 h after transfection. Knockdown of KLF5 markedly induced G 2/M phase arrest ( t=11.56, P<0.05), proliferation inhibition, more apoptosis rate [radiation group: (7.42±0.49)%, radiation + shKLF5 group: (12.49±0.63)%, t=10.98, P<0.05], and more γ-H2AX foci in nucleus post-irradiation than negative control ( t=22.07, 23.89, 11.24, 59.97, 20.85, P<0.05). Conclusions:The KLF5 knockdown intestinal epithelial cell line was successfully established. The down-regulation of KLF5 expression could induce cell arrest at G 2/M, suppress the proliferation of irradiated cells and improve the cell apoptosis, enhance DNA double strand breaks and prolong DNA damage repair.
ABSTRACT
Objective:To observe the extent of brain edema caused by severe cut injury and the protective role of umbilical cord mesenchymal stem cells (UC-MSCs).Methods:A total of 90 female C57L mice were selected and the models of severe cut injury were prepared with surgical blade. According to the random number table, the animals were divided into control group (20 mice), cut group (20 mice), interleukin-6 antibody (IL-6-AB) before cut group (administered IL-6-AB at 18 hours before cut, 15 mice), IL-6-AB after cut group (administered IL-6-AB at 1 hour after cut, 15 mice) and UC-MSCs group (20 mice). The extent of brain edema was detected, the level of IL-6 in brain tissue by ELISA method and the expression of aquaporin-4 (AQP-4) by Western blot assay.Results:Brain water content test showed brain edema in cut group was (81.5±1.8)%, significantly higher than (77.1±2.4)% in control group ( P<0.05). Compared with cut group, brain edema in UC-MSCs group [(76.8±2.4)%] and IL-6-AB before cut group [(76.2±2.9)%] were significantly decreased ( P<0.05), while that in IL-6-AB after cut group [(82.4±1.7)%] was little decreased ( P>0.05). ELISA showed the level of IL-6 in cut group was significantly increased in mouse brain [(16.6±1.3)pg/ml], when compared with control group [(10.3±0.3)pg/ml] ( P<0.01). Compared with cut group, the levels of IL-6 in UC-MSCs group [(10.7±0.6)pg/ml] and IL-6-AB before cut group [(10.1±0.4)pg/ml] were significantly decreased ( P<0.01), while that in IL-6-AB after cut group [(14.9±1.2)pg/ml] was little decreased in mouse brain ( P>0.05). Western blot assay showed that compared with control group (1.0±0.1), the expression of AQP-4 in cut group (2.4±0.5) was significantly increased in mouse brain ( P<0.01). Compared with cut group, the expression of AQP-4 in UC-MSCs group (1.2±0.3) and IL-6-AB before cut group (1.0±0.1) were significantly decreased ( P<0.01), while that in IL-6-AB after cut group (2.3±0.3) was little decreased in mouse brain ( P>0.05). Conclusions:Severe cut injury can increase brain water content and eventually lead to brain edema through upregulating the levels of IL-6 and AQP-4 protein in the brain. Moreover, UC-MSCs effectively prevent the formation of brain edema by inhibiting the above effects.