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Mouse fetal liver stromal cell construction method and application thereof in hemopoiesis assistance

A stromal cell and construction method technology, applied in the field of mouse fetal liver stromal cell construction method and its application in hematopoietic assistance, to improve the hematopoietic microenvironment, promote proliferation and rapid expansion, and promote recovery

Inactive Publication Date: 2019-03-08
SHANGHAI CHILDRENS HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, research at home and abroad focuses on using FL stromal cells as feeder cells to induce other types of cells to differentiate into HSCs and expand them in large quantities. Function: Whether its hematopoietic auxiliary function in vivo is different from that in vitro, and whether it can promote the niche, proliferation and differentiation of exogenous HSC in the recipient remains to be explored

Method used

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  • Mouse fetal liver stromal cell construction method and application thereof in hemopoiesis assistance
  • Mouse fetal liver stromal cell construction method and application thereof in hemopoiesis assistance
  • Mouse fetal liver stromal cell construction method and application thereof in hemopoiesis assistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Stromal cell acquisition and in vitro culture, surface marker expression detection

[0028] Pregnant mice were sacrificed by cervical dislocation, FL was taken out under aseptic conditions and blown into a cell suspension, passed through a 30 μm filter to prepare a single cell suspension, and FL mononuclear cells were separated with lymphocyte separation medium, according to 0.5*10 6 -2*10 6 / cm 2 The density was inoculated on a petri dish, and the culture medium was high-glucose DMEM+15% fetal bovine serum (v / v)+1% penicillin / streptomycin (v / v)+0.1mM β-mercaptoethanol, placed at 37°C, 5%CO 2 Cultivate in a concentration incubator. Change the medium for the first time 24 hours after inoculation, and change the medium every 2-3 days thereafter. When the cell confluence reaches about 80%, subculture according to 1:2. BM stromal cells were obtained from mouse tibia and femur, and the culture method was the same as that of FL stromal cells except that the cult...

Embodiment 2

[0031] Example 2 Transplantation of FL stromal cells into the bone marrow cavity

[0032] BM LK cells were isolated by two C57BL / 6 CD45.1 / CD45.2 The female mice were sacrificed by cervical dislocation, the tibia and femur were taken out under aseptic conditions, the BM cells were washed out, passed through a 30 μm filter to prepare a single cell suspension, and then the BM mononuclear cells were separated by lymphocyte separation medium, and then sorted by magnetic beads Kit operation process to separate Lin - c-Kit + Cells were counted and placed at 4°C for later use.

[0033] FL stromal cells and MEF cell suspensions were prepared by culturing passage 4 (P4) E13.5FL stromal cells and P4MEFs, digested with trypsin, centrifuged, resuspended in PBS, counted, and placed at 4°C for later use.

[0034] Cell bone marrow transplantation: 20 C57BL / 6 CD45.2 / CD45.2 Mice (all received 9.0Gy the day before 60COγ-ray radiation) were divided into 4 groups, 5 rats in each group, respec...

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Abstract

The invention discloses a mouse fetal liver stromal cell construction method and application thereof in hemopoiesis assistance. The method herein includes acquiring fetal liver stromal cells, performing in-vitro culture, isolating bone marrow LK cells and performing cell intramedullary transplanting. The fetal liver stromal cells can promote recovery of PLT (platelets) and RBC (red blood cells) inearly stage of hemopoiesis reconstruction and promote HSC (hepatic stellate cells) proliferation and quick amplification, which is important to improving host hemopoiesis microenvironment in HSC transplantation and improving hemopoiesis reconstruction efficiency.

Description

technical field [0001] The invention belongs to the technical field of hematopoietic cell construction, and in particular relates to a mouse fetal liver stromal cell construction method and its application in hematopoietic assistance. Background technique [0002] Stromal cells were first isolated from guinea pig BM by Friedenstein A et al. Different from hematopoietic cells, they are a type of fibroblast-like cells that can adhere to the walls of plastic vessels. Differentiate from adipocytes, so stromal cells are gradually called mesenchymal stem cells (MSCs). However, due to the lack of a unified standard for the identification of mouse MSCs, the MSCs established by different researchers at home and abroad vary greatly. These differences are mainly reflected in the expression of surface markers. It is generally believed that MSC does not express the blood line marker CD45, but some researchers have isolated CD45 + mouse BM MSCs. CD34, as a marker of hematopoietic cells...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/073
CPCC12N5/0603C12N5/067
Inventor 曾凡一黄淑帧薛燕王沄汉
Owner SHANGHAI CHILDRENS HOSPITAL
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