CN111454900A

CN111454900A - Preparation method of bone marrow mesenchymal stem cell exosome

Info

Publication number: CN111454900A
Application number: CN202010383835.5A
Authority: CN
Inventors: 李立; 胡宗强; 张升宁; 赵英鹏
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-05-08
Filing date: 2020-05-08
Publication date: 2020-07-28

Abstract

The invention relates to the technical field of bioengineering, and discloses a preparation method of a bone marrow mesenchymal stem cell exosome, which comprises the following steps: adding a culture medium without exosome serum into bone marrow mesenchymal stem cells, diluting the culture medium into a cell suspension, and inoculating the cell suspension into a cell culture bottle for culture; step two, adding the miR-142-3p inhibitor and the transfection reagent into a cell culture bottle, and then continuing to culture; and step three, collecting the cultured supernatant, and then extracting the exosome by a centrifugation method. The bone marrow mesenchymal stem cell exosome transfected with the miR-142-3p inhibitor has the effect of inhibiting the death of T lymphocyte iron, so that the development of liver cancer is slowed down.

Description

Preparation method of bone marrow mesenchymal stem cell exosome

Technical Field

The invention relates to the technical field of bioengineering, and particularly relates to a preparation method of a bone marrow mesenchymal stem cell exosome.

Background

Liver cancer is a common malignant tumor, which can be divided into primary and secondary types, wherein the primary liver cancer originates from liver epithelium or mesenchymal tissue; secondary or metastatic liver cancer refers to the invasion of malignant tumors of multiple organ origins in the whole body to the liver. The number of new cases and deaths is far greater in developing countries than in developed countries, and more men and women are likely to have more morbidity and mortality worldwide.

Meanwhile, research has proved that Hepatitis B Virus (HBV) is the main pathogen causing liver cancer, and liver cancer caused by HBV infection accounts for about 80% of all liver cancer cases. Although the medical technology is continuously improved, the early diagnosis rate of liver cancer in China is only about 20 percent, and the survival rate of liver cancer patients after 5 years of treatment is less than 10 percent

Exosomes refer to 30-150nm vesicles containing complex RNAs and proteins. Many cells secrete exosomes under both normal and pathological conditions, mainly from multivesicular bodies formed by endolysosomal invaginations in cells, and secreted into the extracellular matrix by fusion of the multivesicular bodies and cell membranes [22 ]. A large number of researches show that exosome is an important regulatory factor for intercellular communication, and has important regulation and control effects on aspects such as body immune response, antigen presentation, cell migration, differentiation, tumor invasion and the like. The exosome has rich sources, is easy to modify, carries substances which are easy to be absorbed by cells, can avoid immunological rejection reaction caused by cell transplantation, is easy to carry out multiple injections, and gradually becomes a very promising treatment means.

Disclosure of Invention

The invention provides a preparation method of a bone marrow mesenchymal stem cell exosome aiming at the problems in the prior art.

In order to solve the technical problem, the invention is solved by the following technical scheme:

the preparation method of the bone marrow mesenchymal stem cell exosome comprises the following steps:

adding a culture medium without exosome serum into bone marrow mesenchymal stem cells, diluting the culture medium into a cell suspension, and inoculating the cell suspension into a cell culture bottle for culture;

step two, adding the miR-142-3p inhibitor and the transfection reagent into a cell culture bottle, and then continuing to culture;

and step three, collecting the cultured supernatant, and then extracting the exosome by a centrifugation method.

Preferably, in the second step, the culture environment is first cultured in a mixed gas of 37 ℃ temperature, 0.5% oxygen volume concentration and 5% CO volume concentration for 24 hours; then, the cells were cultured in a mixed gas of 37 ℃ at a CO volume concentration of 5% and a nitrogen volume concentration of 95% for 48 hours.

Preferably, the culture medium in the first step is low-sugar DMEM culture medium containing 110 mg/L sodium pyruvate, 10-30mM glutamine and 10% fetal bovine serum.

Due to the adoption of the technical scheme, the invention has the remarkable technical effects that:

the bone marrow mesenchymal stem cell exosome transfected with the miR-142-3p inhibitor has the effect of inhibiting the death of T lymphocyte iron, so that the development of liver cancer is slowed down.

Drawings

FIG. 1 is a graph showing the effect of miR-142-3p overexpression on the expression of an iron death marker in T lymphocytes in the present example.

FIG. 2 is a graph showing the effect of miR-142-3p overexpression on Gln transport and xCT-related protein expression level in the present example.

FIG. 3 is a targeting relationship between miR-142-3p and S L C3A2 in the present example

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples.

Example 1

In the second step of this embodiment, the culture environment is first cultured in a mixed gas at 37 ℃ and oxygen volume concentration of 0.5% and CO volume concentration of 5% for 24 hours; then, the cells were cultured in a mixed gas of 37 ℃ at a CO volume concentration of 5% and a nitrogen volume concentration of 95% for 48 hours.

In this example, the medium in step one is low sugar DMEM medium containing sodium pyruvate 110 mg/L, glutamine 10-30mM, and fetal bovine serum 10%.

First, experimental verification means

The exosome prepared by the method has the effect of inhibiting liver cancer, and is verified by the following experiments:

a xenograft mouse model is established by using BA L B/c mice of about 1 week old, after 1 week, 0.1m L HBV infection negative liver cancer cell suspension (containing 5 × 106 cells in logarithmic growth phase) is inoculated to the axilla of a nude mouse subcutaneously, when the tumor volume grows to about 100mm3, exosomes extracted in the implementation is injected into the abdominal cavity, the registration is continuously carried out for 12 days, 8 mice are used as one group, a control group is injected with a mixture of physiological saline and 1% DMSO, the tumor diameter of the mice is measured every week, the mice are killed after 5 weeks according to the V which is the shortest diameter × and the longest diameter × 0.5.5, the tumor size of the mice is measured, and various indexes are detected.

Second, experimental results

(1) Expression conditions of ROS and MDA of over-expression miR-142-3p on T lymphocytes in experimental group and control group

As shown in FIG. 1, the results show that miR-142-3p is overexpressed to significantly up-regulate ROS levels and MDA content in T lymphocytes, compared to a control group.

(2) Influence of miR-142-3p overexpression on Gln transport and xCT related protein expression level in experimental group and control group

As shown in FIG. 2, the RAPI protein lysate was used to extract total proteins from each group of T lymphocytes, using Westernblotting

Detection of Gln transporter-related proteins (S L C38A1 and S L C1A5) and cystine/glutamate antiporter (xCT)

Expression levels of related proteins (S L C7a11 and S L C3a2) and the results showed that over-expression of miR-142-3P significantly upregulated the expression level of S L C3a2 but had no significant effect on the expression levels of S L C38a1, S L C1a5 and S L C7a11 (P >0.05) compared to the control group.

(3) Targeted relationship between over-expressed miR-142-3p and S L C3A2 in experimental group and control group

As shown in FIG. 3, the luciferase activity was detected by using the dual-luciferase reporter gene detection kit, and the result showed that the luciferase activity was significantly reduced (P <0.05) compared with the control group, thereby indicating that miR-142-3P has a direct targeting relationship with S L C3A 2.

Experimental results show that miR-142-3p is overexpressed in HBV infection positive liver cancer exosomes and has influence on tumor infiltration T lymphocyte iron death and liver cancer development, and miR-142-3p regulates T lymphocyte iron death through targeted down-regulation of S L C3A2 so as to promote liver cancer development.

In summary, the above-mentioned embodiments are only preferred embodiments of the present invention, and all equivalent changes and modifications made in the claims of the present invention should be covered by the claims of the present invention.

Claims

1. The preparation method of the bone marrow mesenchymal stem cell exosome is characterized by comprising the following steps:

2. The method for preparing an exosome of mesenchymal stem cells according to claim 1, characterized in that: in the second step, the culture environment is that the culture is firstly carried out for 24 hours in mixed gas with the temperature of 37 ℃, the oxygen volume concentration of 0.5 percent and the CO volume concentration of 5 percent; then, the cells were cultured in a mixed gas of 37 ℃ at a CO volume concentration of 5% and a nitrogen volume concentration of 95% for 48 hours.

3. The method for preparing bone marrow mesenchymal stem cell exosomes according to claim 1, wherein the culture medium in the first step is low-sugar DMEM culture medium containing sodium pyruvate 110 mg/L, glutamine 10-30mM and fetal bovine serum 10%.