Bitter gourd exosome, and extraction method and application of same
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An extraction method and exosome technology, applied in the field of medicine, to achieve the effects of high impurity removal rate, blood-brain barrier protection, and reduced penetration
Active Publication Date: 2020-06-02
XUZHOU MEDICAL UNIV
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Whether exosomes can be extracted from bitter melon and use
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Embodiment 1
[0029] Example 1: Extraction method of bitter melon exosomes
[0030] See attached figure 1 For the extraction process shown, take 500g of fresh Yunnan mountain wild bitter gourd, remove the seeds and wash three times, each time for 1-3min, and dry after washing;
[0031] After drying, squeeze bitter gourd juice with a fresh juicer, about 200ml, and centrifuge the bitter gourd juice continuously at 4°C, 1000g, 10min; 3000g, 20min; 10000g, 40min; after centrifugation, take the supernatant and discard the precipitate;
[0032] Ultracentrifuge the obtained supernatant, centrifuge at 4°C, 150,000g for 90min in an ultra-fast refrigerated centrifuge, discard the supernatant after centrifugation, take the precipitate, and suspend the precipitate in 1-2ml of PBS buffer;
[0033] The suspension was filtered with a 0.22um filter membrane, and the filtrate was centrifuged again in an ultracentrifuge at 4°C and 150,000g for 90min. After centrifugation, the precipitate was collected, the ...
Embodiment 2
[0037] Example 2: Brain protection of bitter gourd exosomes
[0038] 1. Experimental animals
[0039] Male Sprague-Dawley (SD) rats, weighing 250±20g, clean grade, provided by the Experimental Animal Center of Xuzhou Medical University, experimental animal production license: SCXK (Shanghai) 2018-0006.
[0040] 2. Main reagents
[0041] Bitter melon exosomes: extracted according to the method described in the examples; TTC (2,3,5-triphenyltetrazolium chloride; Shanghai Sangong), Evans blue (EB; Beijing Yinuokai Technology Co., Ltd.), Trichloroacetic acid (Beijing Yinuokai Technology Co., Ltd.), CD54 / ICAM-1 Antibody#4915 (CST), TSG101 Antibody (proteintech), CD63 Antibody (proteintech)
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Abstract
The invention discloses a bitter gourd exosome, and an extraction method and application of same. Extraction comprises the following steps: taking fresh wild bitter gourds of Yunnan, removing seeds, and cleaning and drying the bitter gourds; obtaining bitter gourd juice by squeezing, centrifuging the bitter gourd juice continuously, taking the supernatant after centrifugation and discarding the sediment; conducting ultra-centrifugation of the obtained supernatant, discarding the supernatant, taking the sediment and suspending the sediment in a phosphoric acid buffer salt solution of 1-2mL; andfiltering the suspension with a 0.22 mum filtering film, centrifuging the filtrate in an ultra-centrifugation refrigeration machine for centrifugation again, discarding the supernatant, taking the sediment and suspending the sediment in the phosphoric acid buffer salt solution of 1-2mL. The extracted bitter gourd exosome can be used for preparing medicines of ischemic cerebral apoplexy. The extraction method disclosed by the invention is convenient and effective, the extraction purity is high, and the extracted bitter gourd exosome can protect nerve injuries after cerebral apoplexy.
Description
technical field [0001] The invention relates to the field of medicine, in particular to a bitter melon exosome, an extraction method and application thereof. Background technique [0002] Ischemic stroke is characterized by high morbidity, disability and mortality. Cerebral stroke includes hemorrhagic stroke and ischemic stroke, of which ischemic stroke accounts for about 87%. Ischemic stroke is mainly caused by cerebral blood vessel blockage, decreased blood flow and insufficient blood supply to the brain. The clinical method of treating cerebral apoplexy is to use t-PA for thrombolysis, but t-PA has a strict treatment time window of 4.5 hours. Exceeding the treatment time window will cause hemorrhagic conversion (more serious damage). Safe and effective therapeutic drugs. [0003] Exosomes are extracellular vesicles released by cells into the environment with a particle size of 30-200nm, which are butterfly-shaped and composed of specific proteins, lipids and nucleic aci...
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