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Method for enhancing proliferative capacity of hUCMSCs and application of method

A proliferative ability, plkd-cmv-g&pr-u6-shrna-snora7a technology, applied in biochemical equipment and methods, using vectors to introduce foreign genetic material, cells modified by introducing foreign genetic material, etc., can solve the problem that hUCMSCs have not yet developed. Proliferation ability and other issues, to achieve the effect of enhanced cell proliferation ability, high proliferation ability, and good pluripotency

Active Publication Date: 2016-04-06
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

[0009] At present, there is no research and application of snoRA7A to maintain and enhance the proliferation ability of hUCMSCs

Method used

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  • Method for enhancing proliferative capacity of hUCMSCs and application of method
  • Method for enhancing proliferative capacity of hUCMSCs and application of method
  • Method for enhancing proliferative capacity of hUCMSCs and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Construction of eukaryotic expression vector pLKD-CMV-G&PR-U6-snoRA7A, infecting hUCMSCs (HUCMSCs).

[0045] 1. hUCMSCs isolation and culture

[0046] Human umbilical cords were obtained from the Obstetrics and Gynecology Department of Changhai Hospital. hUCMSCs were obtained from primary culture. As the number of passages increases, the morphology and proliferation rate of hUCMSCs will change. In order to ensure the accuracy and reliability of the experiment, all of our in vitro experiments select the second-generation hUCMSCs as the research objects.

[0047] 2. Preparation of human genomic DNA

[0048] Use Promega Genomic DNA purification kit, the method is as follows:

[0049] [1]. Collect hUCMSCs into a clean 1.5ml OD tube;

[0050] [2]. Add 600 microliters of nuclear lysate, repeatedly pipette with a pipette gun to lyse the tissue until the visible tissue pieces disappear, and let it stand at 65°C for 20 minutes;

[0051] [3]. Add 3 microliters o...

Embodiment 2

[0092] Example 2: Cell experiment

[0093] Using fluorescence microscope to take photos of CCK8 cell doubling time and other biological experimental methods to analyze the changes of cell morphology and cell proliferation ability.

[0094] The specific method is as follows:

[0095] 1) Comparison of cell growth status

[0096] The same passages infected with snoRA7A were visualized with an inverted microscope oe - hUCMSCs, control group CON-hUCMSCs, snoRA7A oe - hUCMSCs can better maintain the fibroblast-like growth morphology, and the cell density is higher after the same passage time. Such as figure 1 shown.

[0097] 2) Real-time quantitative PCR (Real-timePCR) to detect the expression of snoRA7A after transfection

[0098] ① Extract the total DNA at different time points after transfection, and reverse transcribe it into cDNA.

[0099] ② Design primers to detect the expression level of snoRA7A. GAPDH was used as an internal reference for detection.

[0100] The pri...

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Abstract

The invention relates to the technical field of biology, in particular to new application of snoRA7A and a method for enhancing the proliferative capacity of hUCMSCs under an invitro culture condition. According to the method for enhancing the proliferative capacity of the hUCMSCs, expression plasmids of the snoRA7A are successfully constructed, the plasmids are used for transfecting the hUCMSCs, hUCMSCs achieving high expression of the snoRA7A are obtained, and compared with original hUCMSCs, the obtained hUCMSCs are higher in proliferative capacity and better in pluripotency. A larger number of cells with a better functional state can be obtained under the invitro culture condition, and the problem that biological activity of the proliferative capacity and the like of the hUCMSCs is decreased along with increase of the passage number in the invitro culture process is solved. By means of the method for enhancing the proliferative capacity of the hUCMSCs, a new idea is provided for maintaining and enhancing of the cell proliferative capacity of the hUCMSCs in invitro culture and passage process, and a wider prospect is provided for clinical application of the hUCMSCs.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for enhancing the proliferation ability of hUCMSCs and its application. Background technique [0002] Mesenchymal stem cells (mesenchymal stem cells, MSCs) are important members of the stem cell family, derived from the mesoderm and ectoderm in the early stages of development. MSCs have the characteristics of self-replication, massive proliferation, multi-directional differentiation potential, hematopoietic support and immune regulation. At present, MSCs are mainly extracted from fat, bone marrow, umbilical cord, and umbilical cord blood. Human umbilical cord mesenchymal stem cells (hUCMSCs) are a kind of MSCs that exist in the umbilical cord. Controversy, the number of cells and other advantages. [0003] hUCMSCs are of great significance in the application of clinical disease research. For example, hUCMSCs can be induced to differentiate into the cells we need, and tran...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10
CPCC12N15/113C12N15/85C12N2310/10C12N2330/51
Inventor 张燕徐辰刘厚奇王越严冰浩张浩顾道兰
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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