Application of circular RNA CircOGDH as acute ischemic stroke treatment target
A technology of ischemic stroke and therapeutic target, which is applied in the biological field, can solve the problem that the application of pathological changes in stroke has not been reported, and achieve the effect of reducing neuronal apoptosis
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Embodiment 1
[0021] Example 1. Screening process and identification of CircOGDH
[0022] In the early stage, the joint analysis of next-generation sequencing data of middle cerebral artery occlusion (middle cerebral artery occlusion, MCAO) mouse brain tissue and MCAO mouse blood chip data was carried out, and real-time fluorescent quantitative PCR (Quantitative Real-time PCR, qPCR) was carried out. The verification screen obtained the circular RNA CircOGDH generated by the transcription of the oxoglutarate dehydrogenase (Oxoglutarate Dehydrogenase, OGDH) gene (Fig. 1.A-C). The stability of CircOGDH was further confirmed by RnaseR enzyme treatment (Figure 1.D), and RNA in situ hybridization (FISH) was used to find that CircOGDH was mainly expressed in the cytoplasm of ischemic penumbra neurons in MCAO mice (Figure 1.E) .
Embodiment 2
[0023] Example 2. Knocking down CircOGDH can reduce the apoptosis of primary neurons caused by ischemia and hypoxia
[0024] 2.1 Transfection of sh_CircOGDH adenovirus can knock down the expression of CircOGDH in primary neurons
[0025] In order to explore the biological function of CircOGDH in the process of hypoxia-induced neuronal apoptosis, we designed the ADV_GFP_shRNA_CircOGDH adenovirus (sh_CircOGDH for short) targeting the reverse splicing sequence of CircOGDH to knock down the expression of CircOGDH in cells ( Figure 2A ). Transfection of ADV_GFP_shRNA_CircOGDH adenovirus or ADV_GFP_shRNA_CircCtrl adenovirus (negative control) in primary neurons, high expression of GFP (green fluorescence) can be observed under a fluorescent microscope, proving that adenoviruses can invade primary neuronal cells ( Figure 2B ). Through RT-qPCR experiments, we found that transfection of sh_CircOGDH adenovirus could significantly reduce the expression of CircOGDH in primary neuronal ...
Embodiment 3
[0029] Example 3. Knocking down CircOGDH reduces neuronal apoptosis in MCAO model mice
[0030] 3.1 Experimental Design and Construction of MCAO Model Mice
[0031] Experimental design such as Figure 4A As shown, use a stereotaxic apparatus to inject sh_CircCtrl or sh_CircOGDH adenovirus into two parts of the left cortex of the mouse, and the specific coordinates are as follows: 1) 0.2 mm behind the Bregma point, 2 mm left, and 1.5 mm deep; 2) 0.2 mm behind the Bregma point, left 3.0mm, 2.0mm deep. On the 6th day after virus injection, MCAO modeling was carried out ( Figure 4A ), 3 hours after modeling, samples were taken for follow-up experiments. Since the adenovirus carries the GFP fluorescent protein, in order to determine whether the virus injection site is accurate and the spread of the virus, we injected the sh_CircCtrl adenovirus into the cortex of MCAO mice and took samples, and made frozen sections of the brain tissue, and then performed immunofluorescence exper...
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