阅读说明：本技术 一种促进脐带间充质干细胞体外增殖并促进其分化的培养基 (Culture medium for promoting in-vitro proliferation and differentiation of umbilical cord mesenchymal stem cells ) 是由 朱阳晨 赵玲 于 2020-05-24 设计创作，主要内容包括：本发明公开了一种促进脐带间充质干细胞体外增殖并促进其分化的培养基。本发明发现,L-组氨酸衍生物5具有体外促进hUC-MSCs增值的作用,并可以诱导其成骨分化。因此,L-组氨酸衍生物5可以用于体外制备hUC-MSCs种子细胞并促进其成骨分化,具有制备成促进hUC-MSCs体外增殖并诱导其成骨分化的培养基的前景。(The invention discloses a culture medium for promoting in-vitro proliferation and differentiation of umbilical cord mesenchymal stem cells. The invention discovers that the L-histidine derivative 5 has the function of promoting the proliferation of the hUC-MSCs in vitro and can induce the osteogenic differentiation of the hUC-MSCs. Therefore, the L-histidine derivative 5 can be used for preparing hUC-MSCs seed cells in vitro and promoting osteogenic differentiation of the hUC-MSCs seed cells, and has the prospect of preparing a culture medium for promoting the hUC-MSCs to proliferate in vitro and inducing the osteogenic differentiation of the hUC-MSCs.)

1. The application of the L-histidine derivative with the following chemical structure in promoting the in vitro proliferation of the umbilical cord mesenchymal stem cells and inducing the osteogenic differentiation of the umbilical cord mesenchymal stem cells.

2. A culture medium for promoting the in vitro proliferation and inducing the osteogenic differentiation of umbilical cord mesenchymal stem cells, which contains an L-histidine derivative with the following chemical structure.

Technical Field

The invention belongs to the field of stem cells, relates to in-vitro proliferation and induced differentiation of umbilical cord mesenchymal stem cells, and particularly relates to a culture medium for promoting in-vitro proliferation and differentiation of umbilical cord mesenchymal stem cells.

Background

Stem cell engineering refers to the manipulation of stem cells in vitro, including in vitro proliferation, directed induction, transverse differentiation, gene modification, tissue formation, and the like. The scientific value lies in the attractive application prospect, and the treatment of diseases by using the cell technology is the development direction of future medicine. At present, the main contents of stem cell engineering research are ES cell isolation culture, directed induced differentiation, gene manipulation, embryo engineering, nuclear transplantation, etc., and the aim is to deeply research the differentiation mechanism of pluripotent stem cells, establish in vitro three-dimensional culture conditions, construct different tissues and organs, and develop novel biomaterials which can be applied in vivo.

Human umbilical cord mesenchymal stem cells (hUC-MSCs) are a class of mesenchymal stem cells with multipotential differentiation capacity and self-renewal capacity. The umbilical cord has the advantages of easily-obtained materials, difficulty in pollution, no relation to moral, ethical and legal problems and the like, and is widely used for extracting the hUC-MSCs. Moreover, researches find that the in vitro amplification and multidirectional differentiation capacity of the hUC-MSCs is stronger than that of mesenchymal stem cells from other tissues, and the hUC-MSCs are very suitable for being used as seed cells in stem cell engineering.

The problem to be solved in the first place for stem cell research and application is how to rapidly obtain a large number of stem cell seed cells in vitro; the second problem to be solved is if the differentiation of the seed cells into the target cells is induced.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a culture medium for promoting the in-vitro proliferation and differentiation of umbilical cord mesenchymal stem cells.

The technical scheme of the invention is as follows:

the application of the L-histidine derivative with the following chemical structure in promoting the in vitro proliferation of the umbilical cord mesenchymal stem cells and inducing the osteogenic differentiation of the umbilical cord mesenchymal stem cells.

A culture medium for promoting the in vitro proliferation and inducing the osteogenic differentiation of umbilical cord mesenchymal stem cells, which contains an L-histidine derivative with the following chemical structure.

The beneficial technical effects are as follows:

the invention discovers that the L-histidine derivative 5 has the function of promoting the proliferation of the hUC-MSCs in vitro and can induce the osteogenic differentiation of the hUC-MSCs. Therefore, the L-histidine derivative 5 can be used for preparing hUC-MSCs seed cells in vitro and promoting osteogenic differentiation of the hUC-MSCs seed cells, and has the prospect of preparing a culture medium for promoting the hUC-MSCs to proliferate in vitro and inducing the osteogenic differentiation of the hUC-MSCs.

Drawings

FIG. 1 is a chemical structural formula of L-histidine derivative 5;

FIG. 2 is a diagram of phenotypic flow assays for hUC-MSCs;

FIG. 3 shows the proliferation promoting rate of hUC-MSCs by different concentrations of L-histidine derivative 5;

FIG. 4 shows alizarin red staining results.

Detailed Description

The following examples are intended to illustrate the essence of the present invention, but should not be construed as limiting the scope of the present invention.

First, test materials

DMEM/F12 medium and fetal bovine serum were purchased from Gibco.

Glutamine and diabody were purchased from Nanjing Senega Biotech, Inc.

The PBS buffer solution is prepared according to the formula, stored at 4 ℃ and used up within 24 h.

Collagenase type iv and pancreatin were purchased from shanghai diligent kang biotechnology limited, and used as described herein.

The chemical structural formula of the L-histidine derivative 5 is shown in figure 1, and the purity is not lower than 98%.

Second, test method

1. Extraction culture and identification of hUC-MSCs

The hUC-MSCs used are the same as those in patents 2020104333269 and 2020104333714, and the preparation and identification methods are as follows:

collecting umbilical cord of newborn produced by normal full-term caesarean section at about 12cm (storing umbilical cord in PBS buffer solution containing 1% double antibody at 4 deg.C, extracting stem cells within 6h, culturing), washing with PBS buffer solution containing 1% double antibody to remove umbilical artery and vein and umbilical cord adventitia, and shearing to about 1mm³Placing the tissue blocks in a constant temperature shaking instrument at 37 deg.C, adding collagenase type IV and pancreatin, digesting for 60min and 30min respectively to extract cells, resuspending the cells in DMEM/F12 medium containing 20% FBS, 25mmol/L glutamine and 1% double antibody, and adding 1.0 × 10⁶and/mL, inoculating the cells into a cell culture bottle, changing the culture medium after culturing for 4 days, changing the culture medium for 1 time after 2-3 days, and carrying out passage when 80% of the cells are fused. The 5 th generation cells were used for the experiment.

Taking 5 th generation hUC-MSCs, carrying out pancreatin digestion, fully and uniformly blowing to prepare single cell suspension, and adding CD34-PE, CD45-PE, CD73-PE, CD90-PE and CD 105-FITC. Incubating for 30min at room temperature in a dark place, fixing paraformaldehyde, and detecting by a flow cytometer.

2. In vitro proliferation of hUC-MSCs

2.1 grouping

Low concentration drug group: culture with DMEM/F12 medium containing 10. mu. M L-histidine derivative 5(DMSO Co-solvent), 20% FBS, 25mmol/L glutamine and 1% double antibody;

high concentration drug group: culturing with DMEM/F12 medium containing 20 μ M L-histidine derivative 5(DMSO Co-dissolved), 20% FBS, 25mmol/L glutamine and 1% double antibody;

control group: DMEM/F12 medium containing 20% FBS, 25mmol/L glutamine and 1% double antibody was used for culture, and DMSO solvent with the same volume as that of the low-concentration and high-concentration drug groups was added to the medium.

2.2 measurement of cell proliferation Activity by MTT method

Taking 5 th generation hUC-MSCs, digesting with pancreatin, fully and uniformly blowing, and preparing into single cell suspension with each well being 4 × 10⁴The cells were plated in 96-well plates at 5% CO₂Culturing in an incubator at 37 ℃; after 24h, replacing the corresponding culture medium according to the grouping, continuously culturing for 48h, adding 20 mu L of MTT solution (5mg/mL) into each hole, continuously culturing for 4h, discarding the supernatant, adding 150 mu L of LDMSO into each hole, oscillating the shaker at low speed for 10min to fully dissolve the crystal, measuring the absorbance value (OD) of each hole at 490nm wavelength by using an enzyme-linked immunosorbent detector, and calculating the proliferation promoting rate of the L-histidine derivative 5 to the hUC-MSCs according to a formula:

proliferation promoting rate (%) - (drug OD 490-control OD490 value) ÷ control OD490 value × 100%.

Each set of 5 replicates and the test was run in 3 replicates.

3. Alizarin red staining determination of hUC-MSCs osteogenic differentiation

Taking 5 th generation hUC-MSCs, digesting with pancreatin, fully and uniformly blowing, and preparing into single cell suspension with each well being 5 × 10⁴Inoculum at 24-well plates at concentration/mL in 5% CO₂Culturing in an incubator at 37 ℃; after 24h, the control group and the high concentration drug group (abbreviated as drug) in the above groups are selectedGroup) medium was changed and the culture was continued, and the medium was changed 1 time every 3 days. After 18d, alizarin red staining is adopted to observe the formation of mineralized nodules in the culture plate.

4. Data analysis

In SPSS 17.0, data are presented as mean. + -. SD and tested for t, with P < 0.05 indicating that the difference is statistically significant.

Third, test results

1. Extraction culture and identification results of hUC-MSCs

Phenotypic identification of hUC-MSCs flow results As shown in Table 1 and FIG. 2, CD34-PE and CD45-PE negative expression, CD73-PE, CD90-PE and CD105-FITC positive expression, consistent with the phenotypic characteristics of hUC-MSCs.

TABLE 1 phenotypic identification results of hUC-MSCs

	Expression rate
		CD34-PE	0.35％
CD45-PE	0.47％
		CD73-PE	95.2％
CD90-PE	96.8％
		CD105-FITC	98.5％

2. Proliferation promoting rate of hUC-MSCs by L-histidine derivative 5

The proliferation promoting rate of the L-histidine derivative 5 on the hUC-MSCs is shown in Table 2 and FIG. 3, and it can be seen that the L-histidine derivative 5 has the function of promoting the in vitro proliferation of the hUC-MSCs and has a dose effect.

TABLE 2 proliferation-promoting Rate of L-histidine derivative 5 on hUC-MSCs

	Proliferation promoting rate
		L-histidine derivative 5 (10. mu.M)	(118.2±6.9)％
L-histidine derivative 5 (20. mu.M)	(205.5±8.4)％

3. Osteogenesis inducing effect of L-histidine derivative 5 on hUC-MSCs

The alizarin red staining result is shown in fig. 4, and no obvious bone mineralization nodules are seen in the control group and obvious bone mineralization nodules are seen in the drug group. The alizarin red staining method is a method for reflecting the osteogenic differentiation degree by measuring the bone mineralization nodule degree, and the higher the number of the bone mineralization nodules is, the higher the osteogenic differentiation degree is.

The above test results show that the L-histidine derivative 5 has the effect of promoting the proliferation of hUC-MSCs in vitro and can induce the osteogenic differentiation of the hUC-MSCs. Therefore, the L-histidine derivative 5 can be used for preparing hUC-MSCs seed cells in vitro and promoting osteogenic differentiation of the hUC-MSCs seed cells, and has the prospect of preparing a culture medium for promoting the hUC-MSCs to proliferate in vitro and inducing the osteogenic differentiation of the hUC-MSCs.