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Mouse ovarian stem cell transplanting and tracking method

A stem cell and quality stem cell technology, applied in the field of mouse ovarian stem cell transplantation and tracking, can solve the problem of not being able to reflect the survival of transplanted cells as a whole, and achieve the effects of prolonging cell survival time, simple method and high sensitivity

Active Publication Date: 2019-09-20
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods either require sophisticated instruments, or can only reflect a certain section and cannot reflect the survival of transplanted cells as a whole, which has certain limitations.

Method used

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  • Mouse ovarian stem cell transplanting and tracking method
  • Mouse ovarian stem cell transplanting and tracking method
  • Mouse ovarian stem cell transplanting and tracking method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Design of tracking primers for human UCMSCs in mouse ovary

[0049] The DNA sequences of the human and mouse GAPDH genes were downloaded from the NCBI website, and the two sequences were compared using the Clustalw website, and primers were designed for different positions in the two sequences. Primer design was completed using primer 5 software.

[0050] The designed primer sequences are:

[0051]

[0052]

[0053] This pair of primers was attached figure 1 Detection, hGAPDH primers can only amplify the GAPDH gene sequence of human UCMSCs but cannot amplify the mouse gene sequence, and the same mGapdh primer can only amplify the DNA sequence of mouse tissue but cannot amplify the gene sequence in human UCMSCs, These two pairs of primers can be used for cell tracking after non-syngeneic transplantation in humans and mice.

Embodiment 2

[0054] Embodiment 2: UCMSCs mouse ovary orthotopic injection was performed using a Hamilton microinjector, specifically including the following steps:

[0055] S1. Prepare cells: Discard the medium, rinse with PBS three times, digest the cells with 0.25% trypsin, and stop the digestion with complete medium. Pipette into a single cell suspension, centrifuge at 1000rpm for 5min, discard the supernatant, resuspend the cells in 5ml of medium, take 10μl of cells for counting, adjust the cells to an appropriate density with PBS solution, and place the cells on ice when ready.

[0056] S2. The mice were anesthetized with 4% chloral hydrate, the back hair was cut off, and after disinfection, a 1 cm longitudinal incision was made on both sides of the spine, and the skin, fascia, and muscle were cut layer by layer, and the fat pad was gently clamped with curved forceps , exposing both ovaries.

[0057] S3. Slowly draw 10 μl of the cell suspension into the bilateral ovaries along the lo...

Embodiment 3

[0059] Embodiment 3: Utilize TIANGEN kit to extract mouse ovary total DNA, mainly including the following steps:

[0060] S1. The ovaries were collected at each time point in liquid nitrogen, and the DNA was extracted immediately. Add 50 μl of PBS to the ovary and grind it with an electric grinder until no large pieces of tissue can be seen.

[0061] S2. Extract the total DNA according to the instructions of the kit, and dissolve it with TE solution. After mixing, use a Nanogdrop ultra-micro spectrophotometer to detect the DNA concentration and purity, adjust to a final concentration of 8-10 ng / μl with TE solution, and store the extracted DNA at -20°C.

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PUM

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Abstract

The invention provides a mouse ovarian stem cell transplanting and tracking method. Survival rate of transplanted cells is prolonged by applying sodium hyaluronate gel; aiming at limitation of existing cell tracking methods, a primer is designed according to human and mouse GAPDH gene nonhomologous sequences, and PCR (polymerase chain reaction) technology is applied for transplanted cell in-vivo tracking; a simple, convenient and sensitive method for tracking retention of ovarian local stem cells is provided, thereby having great actual application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a mouse ovarian stem cell transplantation and tracking method. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Stem cells have great development potential and clinical application value in the fields of disease treatment and tissue repair. In recent years, stem cells have become one of the important directions in the field of life sciences and have attracted extensive attention. When tissue cells are inflamed or damaged, stem cells can be used as seed cells to differentiate into desired cells, or improve the local microenvironment through a paracrine method, so as to b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686C12N15/11
CPCC12Q1/686C12Q2565/125
Inventor 赵世斗焦文林秦莹莹陈子江颜磊刘冉燕涛
Owner SHANDONG UNIV
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