Method for modifying lemon exosome

A modification method, exosome technology, applied in the field of DNA recombination

Active Publication Date: 2018-06-22
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Indian scholar Stefania Raimondo and others found that lemon-derived exosomes can inhibit the proliferation of chronic myeloid leukemia cells, accumulate in the tumor site of chronic myelogenous leukemia subcutaneous tumor model mice, and inhibit the growth of their tumors (see Raimondo, S ., et al., Citrus limon-derived nanovesicles inhibit cancer cell proliferation and suppress CML xenograft growth by inducing TRAIL-mediated cell death. Oncotarget, 2015.6(23):p.19514-27.), which shows that lemon-derived exosomes can Inhibiting the growth of tumor cells has a certain therapeutic effect, but for glioma, it must have the ability to cross the blood-brain barrier to inhibit the growth of tumor cells

Method used

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  • Method for modifying lemon exosome
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  • Method for modifying lemon exosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 (preparation of PEG-cRGD-lemon exosomes)

[0019] 1. Extract lemon exosomes

[0020] Purchase fresh lemons, peel and squeeze juice, dilute the juice with PBS buffer, and centrifuge at a differential speed (centrifuge at 500g for 10min, centrifuge at 2000g for 20min, centrifuge at 5000g for 30min, centrifuge at 10000g for 1h to get the supernatant, and centrifuge the supernatant at 100000g for 2h to get the precipitate) to obtain the outer Exosome precipitation, PBS buffer resuspension, sucrose density gradient centrifugation (8%, 30%, 45%, 60% sucrose were dissolved in 20mM Tris.Cl respectively) to purify exosomes, dynamic light scattering and transmission electron microscopy Identify the particle size distribution and morphology characterization of exosomes, such as figure 1 shown. The BCA protein quantification method was used to measure the protein content in exosomes, and the obtained exosomes were stored at -80°C for later use.

[0021] 2. (Modified le...

Embodiment 2

[0027] Embodiment 2 (drug effect test)

[0028] (1) The effect on the growth of glioma cells

[0029] In the logarithmic growth phase of the cells, the cell suspensions of human glioma cells U87 and U251 were inoculated in 96-well plates, with about 3500 cells per well, and the 96-well plates were placed at 37°C and 5% CO 2 Cultivate in the incubator for 12 hours, set up 5 exosome concentration gradients for dosing, the highest concentration is 20 μg / ml, and serially dilute until the concentration is 0, and set 5 duplicate wells for each concentration. After adding the medicine, the 96-well plate Placed at 37°C, 5% CO 2 Cultivate for 48h, then add MTT to continue to cultivate for 4h, microplate reader detects the absorbance value (OD value) (detection wavelength 490nm), calculates the relative survival rate of cells under each drug concentration respectively, the result is as follows image 3 shown. Relative cell viability (%)=(OD 实验组 mean / OD 对照组 mean) x 100%.

[0030] Dep...

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Abstract

The invention relates to a method for modifying lemon exosome. The method comprises the following steps that (1) according to the mass ratio of NHS-PEG-VS:cRGD=5:1, NHS-PEG-VS and cRGD are taken to bedissolved in DMF, then according to the ratio between the mass sum of NHS-PEG-VS and cRGD and the mass volume ratio of TEA=6 mg:1 microliter, TEA is added, reaction at a room temperature is carried out for 2 hours, the obtained reaction product is washed with ether for 3 times, standing and precipitating are carried out, sediment is taken for vacuum drying to obtain cRGD-PEG-VS; (2) according tothe mass volume ratio of cRGD-PEG-VS: lemon exosome=1 mg : 500 microliters, the cRGD-PEG-VS is taken and mixed with the lemon exosome with the concentration of 500 micrograms / milliliter, the added sodium carbonate buffer solution is adjusted to a PH value of 9.0, then according to the molar ratio of cRGD:EDC:NHS=1:1:1 in step (1), EDC and NHS are added, the reaction is carried out for a night, andPEG-cRGD- lemon exosome is obtained. According to the method, the obtained PEG-cRGD- lemon exosome can be used in preparing drugs for treating brain glioma.

Description

technical field [0001] The present invention relates to DNA recombination, in particular to plant-derived exosomes and their modification. Background technique [0002] Glioma is the most common malignant tumor of the central nervous system, accounting for 46.6% of primary malignant brain tumors. 5.5%. Glioma has the characteristics of highly diffuse and infiltrative growth, and there is no obvious boundary with the surrounding normal brain tissue. It is difficult to completely remove the surgery and easy to relapse after surgery. The current standard treatment for glioma is in the Radiation therapy and (or) drug chemotherapy are supplemented on the basis of maximal total surgical resection, but the overall prognosis has not been significantly improved, and the clinical efficacy is not ideal. Due to the existence of the blood-brain barrier, there are few drugs used in the treatment of glioma, so it is urgent to develop more new drugs that can cross the blood-brain barrier ...

Claims

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Application Information

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IPC IPC(8): A61K36/752A61K47/60A61K47/64A61P35/00
CPCA61K36/752
Inventor 王莺肖倩
Owner SOUTHERN MEDICAL UNIVERSITY
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