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Method of specifically inducing microglial cell selective polarization with MSX3 gene and application of same

A technology of microglia and MSX3, applied in the fields of genetic engineering and medicine, can solve the problems that there is no research on MSX3, no effective means for microglia, and unclear regulatory mechanism

Active Publication Date: 2015-08-12
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, little is known about the phagocytic polarization mechanism of microglia, the in situ colonization of the central nervous system, and the regulation mechanism of the transcriptional mechanism of M2 polarization is still unclear. There is no effective means, and there is no method that can change the polarization state of microglia from M1 and anchor in the M2 phenotype
There are currently no relevant studies of MSX3 in microglia or disease

Method used

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  • Method of specifically inducing microglial cell selective polarization with MSX3 gene and application of same
  • Method of specifically inducing microglial cell selective polarization with MSX3 gene and application of same
  • Method of specifically inducing microglial cell selective polarization with MSX3 gene and application of same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1: Construction of lentiviruses to overexpress MSX3-GFP and interfere with MSX3i-GFP, and infect mice and human microglia.

[0088] 1. Purification and preparation of microglial cells

[0089] Preparation of mouse microglia by primary culture. Human microglia were from SCIENCELL.

[0090] 2. Preparation of microglial total RNA

[0091] The total RNA of human epidermal cells was extracted by the conventional guanidine isothiocyanate method with the total RNA extraction kit of Shanghai Sangon Bioengineering Co., Ltd. Methods as below:

[0092] Take a 3.5cm-diameter petri dish of microglia growing in a single layer, discard the medium directly, add 1ml of TRIzol to dissolve the cells, and remove the cell lysate with a pipette after the cells are fully dissolved. The cell lysed samples were incubated at 15-30°C for 5 minutes to completely decompose the ribosomes. Add 0.2ml of chloroform to every 1ml of TRIzol, close the cap of the sample tube, shake the tube by...

Embodiment 2

[0130] Embodiment 2: cell experiment (in vitro experiment)

[0131] Using biological experimental methods such as immunocytochemistry, the changes in cell morphology, target gene expression, cell viability, axon growth, and myelination were analyzed from various aspects such as cell morphology and myelin-related protein expression (MBP).

[0132] The specific method is as follows:

[0133] 1) Detection of OPC differentiation and neurite outgrowth by immunocytochemistry

[0134] (1) The MSX3 interference type and overexpression type virus obtained in step 4 (7) of Example 1 were transfected into microglial cells, the medium was changed after 24 hours, and cultured for 3 days, the conditional supernatant was collected and mixed with OPC (Oligodendrocyte precursor cell , oligodendrocyte precursor cells) (10 000 cells / well seeded in 24-well plates) or cortical and hippocampal neurons (10 000 cells / well seeded in 24-well plates) were co-cultured for 120 hours. At each time point,...

Embodiment 3

[0144] Example 3: In Vivo Cell Transplantation Test

[0145] The mice used in the experiment were female C57BL / 6 strain, SPF grade. Weight about 20 ~ 25g, 8 weeks old, purchased from Shanghai Experimental Animal Center. A total of 40 animals were divided into 5 groups of virus transfection groups: Ctrl group (control interference virus transfection for 72 hours), MSX3i+IL-4 group (MSX3 interference virus transfection for 72 hours and IL-4 stimulation for 24 hours), Ctrl+IL -4 groups (contrast interference virus transfection for 72 hours and IL-4 stimulation for 24 hours), MSX3OE group (MSX3 overexpression virus transfection for 72 hours), CtrlOE group (contrast overexpression virus transfection for 72 hours). Extract the MG obtained in Step 4 of Example 1 with a 10 μl syringe Ctrli , MG Ctrli+IL-4 , MG MSX3i+IL-4 , MG CtrlOE and MG MSX3OE The cells were centrifuged to prepare 2 μl of cell suspension, and transplanted into the lateral ventricle of mice on the 8th day afte...

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Abstract

The invention belongs to the field of gene engineering and medicine and particularly relates to a method of specifically inducing microglial cells selective polarization with MSX3 gene and an application of the method in preparation of a drug for treating multiple sclerosis. The method includes following steps: (1) according to the structure of the MSX3 gene, preparing an MSX3 cDNA sequence, constructing an MSX3-over-expressed eukaryotic expression vector and constructing MSX3-over-expressed (MSX3-GFP) lentivirus; and (2) transfecting the MSX3-over-expressed lentivirus to mice and human microglial cells. A research result proves that the MSX3-over-expressed microglial cells have same M2 phenotype, so that diseases progress of EAE can be effectively alleviated.

Description

technical field [0001] The invention belongs to the field of genetic engineering and medicine, in particular to a microglial cell highly expressing homeobox gene MSX3 (msh-like homeobox-3) and its ability to promote remyelination in demyelinating diseases such as multiple sclerosis Applications. Background technique [0002] Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system. Most patients start with relapsing-remitting disease, and then develop into irreversible secondary progressive disease (Trapp, B.D., and Nave, K.A. 2008. Multiple sclerosis: an immune or neurodegenerative disorder? Annu Rev Neurosci 31:247-269.). The restorative remyelination of the body only occurs in the early stage of MS. At present, it is believed that the loss of late remyelination is probably an important reason for the development of MS from relapsing-remitting to irreversible secondary progressive (Scolding, N., Franklin, R., Stevens, S., Heldin, C.H.,...

Claims

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Application Information

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IPC IPC(8): A61K48/00C12N15/867C12N15/12C12N5/10A61P25/00A61P37/02
Inventor 何成曹莉俞仲望
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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