Application of N-acetylglucosamine as marker in preparation of reagent for diagnosing ischemic cerebral apoplexy
An acetylglucosamine and ischemic stroke technology, applied in the field of medical diagnosis, can solve problems such as the influence of metal implants on examination, and achieve the effects of reducing the volume of cerebral infarction, high accuracy and specificity, and improving the therapeutic effect
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Embodiment 1
[0035] Targeted Metabolomics Validation:
[0036] 1. Preparation of middle cerebral artery occlusion / reperfusion (MCAO / R) mouse model
[0037] Mice were intraperitoneally injected with 1% sodium pentobarbital, anesthetized, fixed in the supine position, and incised in the midline of the neck. The right common carotid artery, external carotid artery and internal carotid artery were separated and exposed, a ligature was reserved at the proximal end of the common carotid artery, the external carotid artery was ligated at the distal end, and the distal end of the external carotid artery was freed. A section of the internal carotid artery was freed along the internal carotid artery to the deep part, and a ligature was reserved at the proximal end of the external carotid artery. The common carotid artery at the proximal end and the internal carotid artery at the distal end were clamped with a small non-invasive vascular clip, and a small opening was cut with micro scissors at the d...
Embodiment 2
[0053] Metabolite functional verification:
[0054] 1. TTC staining
[0055] After modeling MCAO / R, the brain tissue of each group of mice was taken out, placed in a -70°C refrigerator until the brain tissue was completely frozen, taken out on the ice box, and the frozen brain tissue was cut into 5 coronal brain slices with a thickness of about 2 mm. , quickly put the cut brain slices into the 2% TTC solution, paying attention to placing the brain slices in one direction. Subsequently, incubate in a 37°C oven for 30 min in the dark. Placed on a black cardboard to take pictures and transferred to a computer, Image J image analysis software was used to measure the volume of cerebral infarction. Calculated as follows:
[0056] Percentage of infarct volume = right hemisphere infarction volume / total brain volume × 100%;
[0057] The larger the infarct volume, the more severe the stroke. Under normal circumstances, the volume of cerebral infarction caused by MCAO / R is 30%-40%; ...
Embodiment 3
[0065] Functional validation of metabolite-regulated enzymes
[0066] 1. Experimental reagents and equipment
[0067] N-acetylglucosamine kinase (NAGK) overexpressed adeno-associated virus (AAV-NAGK) and empty adenovirus (AAV-control) were provided by Shanghai Jiman Biotechnology Co., Ltd. and tested: GPAAV-CMV-M_Nagk-EF1- The titer of ZsGreen1-WPRE was 1.21E+13VG / mL; microsyringe (1 μL); brain localizer (Stoelting, USA).
[0068] 2. Synthetic primers
[0069] carrier information such as figure 1 ; Design PCR amplification fragment primers, and introduce the homologous sequence at the end of the linearized cloning vector at the 5' end of the primer, so that the 5' and 3' most end sequences of the amplification product are completely consistent with the sequences at both ends of the linearized cloning vector, respectively. The sequence is as follows:
[0070]
[0071] 3. In situ injection of adeno-associated virus into the brain
[0072] After the mice were anesthetized...
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