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Mesenchymal stem cell for inhibiting tumor growth and preparation method and application

A technology of mesenchymal stem cells and tumors, applied in the field of mesenchymal stem cells and preparations that inhibit tumor growth, can solve problems such as limitations

Active Publication Date: 2018-10-30
北京华奥玄德生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of MSCs in the field of tumor therapy has been greatly limited, because there is still a lot of controversy about the relationship between MSCs and tumors, and its tumor-promoting and tumor-suppressing effects have been confirmed by experiments.

Method used

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  • Mesenchymal stem cell for inhibiting tumor growth and preparation method and application
  • Mesenchymal stem cell for inhibiting tumor growth and preparation method and application
  • Mesenchymal stem cell for inhibiting tumor growth and preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Isolation and identification of human umbilical cord mesenchymal stem cells

[0036] 1. Isolation and culture of human umbilical cord mesenchymal stem cells (MSCs)

[0037] Mesenchymal stem cells (MSCs) were isolated by the umbilical cord tissue slice method, and a total of 16 MSCs from isolated umbilical cords were isolated. The specific method is as follows:

[0038] (1) Put the isolated umbilical cord from normal delivery into PBS buffer solution containing 200 U / mL penicillin and 200 U / mL streptomycin. In order to ensure the viability of the umbilical cord tissue, the fresh umbilical cord should be separated within 6 hours.

[0039] (2) Flush the residual blood in the umbilical vein and umbilical artery with a 20mL syringe, and cut the umbilical cord tissue into 1mm pieces with tissue scissors 3 Then filter the obtained small pieces of umbilical cord tissue with a 200-mesh filter, collect the umbilical cord tissue pieces on the 200-mesh filter, remove the ...

Embodiment 2

[0050] Example 2: Screening of TLR5-positive MSCs

[0051] 1. Detection of TLR5mRNA expression level in MSC

[0052] For the MSCs of 16 umbilical cords isolated and cultured in Example 1, the mRNAs of different batches of MSCs were extracted with TRIZOL lysate according to the instructions, reverse-transcribed into cDNAs according to the instructions of the kit, and the expression level of TLR5 was detected by q-PCR method. The primer sequence in 1 was used to amplify TLR5 and β-actin, and the expression level of TLR5 mRNA in each batch of MSCs was detected and calculated. turn out( Figure 4 ), the MSC isolated from No. 16 umbilical cord expressed a relatively high level of TLR5.

[0053] Table 1 TLR5 and β-actin primer sequences

[0054]

[0055] 2. Sorting TLR5-positive MSCs by flow cytometry

[0056] The No. 16 umbilical cord P2 generation MSCs were used for amplification, and about 1×10 MSCs in good growth state were collected. 8 (P4 generation), washed twice with...

Embodiment 3

[0058] Example 3: Inhibitory effect of TLR5-positive MSCs on tumors

[0059] 1. Collection of MSC-related conditioned culture supernatant

[0060] (1) Inoculate the obtained MSCs and TLR5+MSCs in 18cm culture dishes, about 5×10 4 Cells were cultured in a cell constant temperature incubator, wherein two groups of culture dishes were set for TLR5+MSCs.

[0061] (2) When the cells adhere to the wall and grow to a confluency of 50%-60%, add the agonist CBLB502 (Salmonella typhimurium flagellin derivative) (final concentration 100ng / mL) to one of the TLR5+MSC culture dishes, Continue to culture in a constant temperature incubator at 37°C for the preparation of MSC, TLR5+MSC and TLR5+MSC+CBLB 502 conditioned culture supernatants respectively.

[0062] (3) Induce in a constant temperature incubator at 37°C for 48 hours, suck off the complete medium in the three groups of culture dishes, replace it with serum-free DMEM medium, 20 mL per dish, continue to incubate for 24 hours and co...

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Abstract

The invention discloses a mesenchymal stem cell for inhibiting tumor growth and a preparation method and an application, and relates to the technical field of the medicine. The provided mesenchymal stem cell for inhibiting the tumor growth is a TLR5 positive mesenchymal stem cell. The preparation method for the TLR5 positive mesenchymal stem cell is to separate TLR5 high-expression positive mesenchymal stem cells by using a flow-type or overexpress TLR5 on the mesenchymal stem cell. The invention further provides a cultural supernatant containing a serum-free medium and the TLR5 positive mesenchymal stem cell, and further provides applications of the TLR5 positive mesenchymal stem cell and the cultural supernatant for preparing an anti-tumor drug. The cultural supernatant in TLR5+MSC conditions has the remarkable inhibiting effects to the in-vitro proliferation, the migration and the clonality of a tumor cell. The TLR5-activated MSC is capable of remarkably improving the in-vitro killing activity of a NK cell. The TLR5-activated MSC is capable of inhibiting the tumor growth in Lewis lung cancer mice.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a mesenchymal stem cell for inhibiting tumor growth, a preparation method and application thereof. Background technique [0002] Tumor is one of the major diseases leading to death in the world. The tumor microenvironment plays an important role in the occurrence, development, invasion and metastasis of tumors. Mesenchymal stem cells (MSCs) can participate in the immune regulation of the tumor microenvironment in vivo, and have a close relationship with tumors. The microenvironment is closely related. On the one hand, tumor tissue secretes a large number of inflammatory factors and chemokines during the growth process, recruiting MSCs to homing to the tumor site; on the other hand, active substances such as IDO, PGE2, TGF-β1, and MMP-9 secreted by MSCs It can act on tumor cells and immune cells to affect the growth of tumors. MSC has the function of repairing the b...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C12N5/10A61K35/28A61P35/00
CPCA61K35/28A61P35/00C12N5/0665C12N2510/00
Inventor 段海峰薛冰华刘金肖秀孝秦亚茹于婷婷
Owner 北京华奥玄德生物医药科技有限公司
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