Application of CD146 + mesenchymal stem cell subpopulation in preparation of medicine for preventing and treating premature ovarian failure

A technology of premature ovarian failure and mesenchymal stem cells, which is applied in the application field of medicine, can solve the problems such as the lack of therapeutic effect of CD146+ mesenchymal cell subsets on premature ovarian failure

Pending Publication Date: 2022-04-19
北京三有利康细胞科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the specific surface markers of mesenchymal cell subsets in the treatment of premature ovarian failure, let alone the therapeutic effect of CD146+ mesenchymal cell subsets on premature ovarian failure

Method used

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  • Application of CD146 + mesenchymal stem cell subpopulation in preparation of medicine for preventing and treating premature ovarian failure
  • Application of CD146 + mesenchymal stem cell subpopulation in preparation of medicine for preventing and treating premature ovarian failure
  • Application of CD146 + mesenchymal stem cell subpopulation in preparation of medicine for preventing and treating premature ovarian failure

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Preparation and isolation method of CD146+ / - mesenchymal stem cell subpopulation

[0032] 1. Materials and methods:

[0033] 1) Cell culture

[0034] The first passage of human umbilical cord-derived mesenchymal stem cells (UC-MSCs) was provided by the Shandong Provincial Cord Blood Bank (Qingdao, China). Cells were cultured with 90% minimal essential medium (αMEM, BI) and 10% fetal bovine serum (Gibco, Australia) at 37°C, 5% CO 2 After further passage (P), MSCs were harvested with trypsin-edta (Gibco, Australia), counted, and re-seeded into 100 mm cell culture dishes at a density of 20,000 cells / cm2.

[0035] 2) Isolation of CD146+MSCs and CD146-MSCs

[0036] Umbilical cord MSCs were separated into CD146+ / - subsets by MACS (Magnetic Activated Cell Sorting). Cells were incubated with FcR blocking reagent and CD146 microbeads (CD146 Bead Kit; Miltenyi Biotec) before magnetic separation. The LS column was used to sort CD146+ cells, and the CD146- cells in the LS colu...

Embodiment 2

[0047] Construction of premature ovarian failure (POF) model in mice and cell therapy

[0048] 1. In order to establish a premature ovarian failure model, female C57BL / 6 mice aged 6-8 weeks in the experimental group were subcutaneously injected with busulphan (Bu, 30 mg / kg, dissolved in DMSO), and intraperitoneally injected with cyclophosphamide (Cy, 120 mg / kg, dissolved in physiological saline), and the mice in the control group (n=20) were injected with the same amount of DMSO and saline. HE staining results showed that follicle development stopped at the primary follicle stage, resulting in a decrease in antral follicles in POF ovaries. Consistent with the sinus follicle damage, the serum AMH level in the POF group also increased significantly, indicating that the mouse model of premature ovarian failure was successfully established.

[0049] In the POF group (n=20), mice received no cell therapy after chemotherapy. In the CD146+ / - MSCs treatment group (n=20), 1 week afte...

Embodiment 4

[0066] 1. Fertility experiment of premature ovarian failure mouse model after cell therapy

[0067] According to the method described in Example 3, the mouse model of premature ovarian failure was constructed, and two kinds of cell therapy were performed. After the second injection of CD146+ / - MSCs, adult males and females who were confirmed to have fertility were raised in a ratio of 1:2. . The litter size was recorded for each gestation.

[0068] 2. Histological Analysis of Ovaries and Spleen

[0069] At 4w, the ovaries were collected and fixed with 4% formaldehyde. The ovaries were cut into 5 μm sections, stained by ultrasound, and evaluated by light microscope for histology. Count and record as preantral follicles (primitive, primary), preantral follicles (secondary, mature), or atretic follicles. Spleens were processed the same as ovaries for H&E staining. Images were taken under a light microscope to record the histological changes of the spleen.

[0070] 3. Biolum...

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Abstract

The invention provides application of a CD146 + mesenchymal stem cell subset in preparation of a medicine for preventing and treating premature ovarian failure, and belongs to the technical field of biological medicines. The invention provides an application of a CD146 + mesenchymal stem cell subset in preparation of a medicine for preventing and treating premature ovarian failure. The treatment efficiency of the CD146 + MSCs and the treatment efficiency of the CD146-MSCs on a mouse POF model are compared, compared with a model group, the MSCs remarkably improve the premature ovarian failure, and the CD146 + subgroup has a slightly good trend compared with the CD146-MSCs. Meanwhile, the invention also provides an application of the CD146 + mesenchymal stem cell subset in preparation of a medicine for improving the immunoregulation ability of a premature ovarian failure patient. Experiments show that the CD146 + mesenchymal stem cell subgroup can significantly reduce the expression level of chemotactic factors and inflammatory genes and inhibit T cell proliferation, which indicates that the CD146 + subgroup has better immunoregulation ability.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to the application of a CD146+ mesenchymal stem cell subpopulation in the preparation of medicines for preventing and treating premature ovarian failure. Background technique [0002] Mesenchymal stem cells (MSCs) have been used in the treatment of various refractory diseases, including tissue damage diseases and immune diseases. Currently, mesenchymal stem cells have become a new type of treatment in regenerative medicine. The surface of mesenchymal stem cells expresses CD105, CD73, CD44 and CD90, but does not express hematopoietic markers such as CD45, CD34, CD11b, CD19 and HLA-DR. However, MSCs are a heterogeneous subset that express different surface markers and have different biological characteristics. Therefore, isolating and identifying MSC subsets from different sources to evaluate their potential and determine their therapeutic functions for diseas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/28A61P15/08A61P37/02
CPCA61K35/28A61P15/08A61P37/02
Inventor 王立生孙慧燕张林
Owner 北京三有利康细胞科技有限公司
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