CN113817776A - Application of GBP2 in regulating and controlling mesenchymal stem cell osteogenic differentiation - Google Patents

Abstract

The invention discloses a preparation for regulating and controlling mesenchymal stem cell osteogenic differentiation, which takes a GBP2 inhibitor or an accelerant as an active ingredient; and discloses application of the preparation in preparing a product for regulating and controlling mesenchymal stem cell osteogenic differentiation. The invention researches the biological characteristics of hUC-MSC stably overexpressing GBP2 by constructing the hUC-MSC, and finds that GBP2 is compared with normal MSC^highThe osteogenic differentiation function of hUC-MSC is influenced, which shows that GBP2 can effectively regulate the osteogenic component of hUC-MSCThe discovery of the function of the differentiation of hUC-MSC can help to understand the osteogenic differentiation of hUC-MSC more clearly, and the function of guiding the clinical treatment of osteoporosis is achieved.

Description

Application of GBP2 in regulating and controlling mesenchymal stem cell osteogenic differentiation

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of GBP2 in regulation and control of mesenchymal stem cell osteogenic differentiation.

Background

Mesenchymal Stem Cells (MSCs) are adult stem cells derived from the mesoderm with a variety of differentiation potentials and self-renewal capabilities. The MSC source is very wide, can be separated from various tissues such as umbilical cord, bone marrow, fat, placenta and the like, and can be differentiated into fat, osteogenic cells and chondroblastic cells under different culture conditions.

MSCs are common progenitor cells of adipocytes and osteoblasts, and can regulate the balance of adipogenic and osteogenic differentiation, maintaining bone homeostasis. Research has shown that the change of proliferation and differentiation of mesenchymal stem cells (increased adipogenic differentiation, decreased osteogenic differentiation) is one of the main causes of osteoporosis. Therefore, revealing the molecular mechanism of differentiation of mesenchymal stem cells into osteoblasts will help to further understand mesenchymal stem cells and may provide a new approach for the treatment of osteoporosis.

Disclosure of Invention

The invention aims to provide a preparation for regulating and controlling mesenchymal stem cell osteogenic differentiation and application thereof.

In order to achieve the purpose, the specific technical scheme of the invention is as follows:

the invention utilizes the lentivirus overexpressed by GBP2 to construct the mesenchymal stem cell GBP2 stably overexpressing GBP2^highhUC-MSC, the biological characteristics of which are researched, and GBP2 is found^highThe osteogenic differentiation capacity of the hUC-MSC is obviously higher than that of normal MSC. Confirmation of alkaline phosphatase (ALP) staining, GBP2^highThe staining area of hUC-MSC was significantly more than normal MSC. The q-PCR method proves that the expressions of the osteogenesis related genes ALP and OPN are obviously increased. The above shows that GBP2 can affect human umbilical cord-derived MSOsteogenic differentiation of C.

Based on this, the invention provides a preparation for regulating and controlling osteogenic differentiation of mesenchymal stem cells, which comprises a GBP2 inhibitor or promoter as an active ingredient.

Preferably, the mesenchymal stem cells comprise human umbilical cord-derived mesenchymal stem cells.

In the present invention, GBP2 is a member of the gamma interferon (IFN-. gamma.) induced Guanylate Binding Protein (GBP) family, which encodes a protein that is a GTPase that hydrolyzes GTP to GDP. GBP2 is mainly present in the golgi membrane and can be secreted into the extracellular domain as a secretory protein.

Further, the GBP2 promoter includes agents that promote GBP2 expression or activity function.

Further, the reagent for promoting GBP2 expression is an expression vector containing GBP2 gene.

Further, the vector includes a lentiviral vector.

In a second aspect, the invention provides an application of the preparation in preparing a product for regulating and controlling the osteogenic differentiation of the mesenchymal stem cells.

Preferably, the preparation promotes the osteoblast differentiation ability of the MSC by allowing the osteoblast differentiation associated marker to be significantly up-regulated by overexpressing the GBP2 gene.

Preferably, the osteoblast differentiation-related markers include an early marker ALP and a late marker OPN for osteoblast differentiation maturation.

In a third aspect, the invention provides the use of the preparation in the preparation of a medicament for the treatment of osteoporosis.

In a fourth aspect, the present invention provides a method for promoting osteogenic differentiation of mesenchymal stem cells, comprising the steps of:

1) obtaining mesenchymal stem cells and culturing;

2) constructing an overexpression GBP2 vector;

3) the constructed overexpression GBP2 vector was transfected into mesenchymal stem cells and then cultured.

The transfection comprises the following specific steps: inoculating hUC-MSC with good growth state into a 6-pore plate, changing cell liquid and adding lentivirus (MOI is 10) and/or infection enhancing liquid for transfection when the cell adherence state is good; preferably, the infection enhancing fluid comprises infection enhancing fluid a (hittra).

The methods for constructing vectors overexpressing GBP2 are well known to those skilled in the art.

Based on the technical scheme, the invention has the following beneficial effects: the invention researches the biological characteristics of hUC-MSC stably overexpressing GBP2 by constructing the hUC-MSC, and finds that GBP2 is compared with normal MSC^highThe morphology and phenotype of the hUC-MSC are not changed, but the osteogenic differentiation function is influenced, which shows that GBP2 can effectively regulate the osteogenic differentiation function of the hUC-MSC, and the discovery helps to more clearly understand the osteogenic differentiation of the hUC-MSC and plays a guiding role in clinically treating osteoporosis.

Drawings

FIG. 1A: observing the cell morphology of the hUC-MSC under an inverted microscope; FIGS. 1B, C: cell morphology after Giemsa staining, wherein panel B is observed under 4-fold microscope, and panel C is observed under 10-fold microscope; FIG. 1D: detecting the result by flow cytometry; FIGS. 1E, F: the result of fat-forming differentiation, namely a self-differentiation group (E) and an induction group (F) of the fat-forming of the hUC-MSC are detected by oil red O staining; FIGS. 1G, H: ALP staining to detect the results of osteogenic differentiation, hUC-MSC osteogenic self-differentiation group (G) and induction group (H); FIG. 1I, J: q-PCR detects the expressions of adipogenic differentiation related genes PPAR-gamma, ADI (I) and osteogenic induction related genes ALP and OPN (J). Note, self-resolved group (Control), Induced group (Induced).

FIGS. 2A-C: the fluorescence microscope is used for observing the expression of GFP after the transfection of the lentiviral vector (FIG. 2A: no enhancement solution is added, FIG. 2B: A enhancement solution is added, FIG. 2C: P enhancement solution is added), and the GFP is observed under a green fluorescence microscope to be green and to be green, which indicates the success of the transfection; fig. 2D, E: detecting the transfection efficiency (GFP channel expression efficiency) of the lentivirus and counting results by flow cytometry; FIG. 2F: q-PCR detects the expression of the mRNA level of GBP2 gene.

FIGS. 3A, B: giemsa detects the morphology of GBP2 overexpressed MSC, wherein a picture A shows the morphology of GBP2 overexpressed MSC observed under a microscope, and a picture B shows the morphology observed under the microscope after Giemsa staining; FIG. 3C: qPCR assayMeasuring the degree of overexpression of GBP 2; FIG. 3D: flow cytometry detection of GBP2 overexpressing MSC phenotype; fig. 3E, F: oil red O to GBP2^highStaining the self-differentiation group (E) and the induction group (F) of the hUC-MSC lipidization and observing under a microscope; FIG. 3G, H ALP vs GBP2^highStaining of the hUC-MSC osteogenic self-differentiation group (G) and the osteogenic induction group (H); FIG. 3I, J: q-PCR detection of expression of adipogenic differentiation marker genes ADI, PPAR- γ (I) osteogenic differentiation marker genes OPN, ALP (J).

ALP staining of NC groups and GBP2^highAlkaline phosphatase activity of the hUC-MSC group; figure 4e. imagej analysis of NC induction groups and GBP2^highALP staining area in MSC-induced group.

FIG. 5A, B.q-PCR detection of NC-inducible and GBP2^highThe hUC-MSC induces the relative expression of the bone marker genes ALP and OPN in the group.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

All materials, reagents and the like in the following examples are commercially available unless otherwise specified.

Example 1 culture and identification of human umbilical cord-derived MSCs

Firstly, separating and obtaining primary human umbilical cord mesenchymal stem cells (hUC-MSC) from human umbilical cord tissues according to a conventional method, culturing the hUC-MSC, and then inoculating the hUC-MSC into a complete culture medium to culture the P1 generation hUC-MSC. Frozen for subsequent experiments. Taking out a frozen hUC-MSC, rapidly thawing in 37 deg.C warm water, adding 10% FBS-containing alpha-MEM, resuspending at 1000rpm, centrifuging, discarding supernatant, suspending cells with alpha-MEM, inoculating to T75 cell culture flask, placing in 5% CO₂37 ℃ incubator. Passages were then performed every 2 days with 0.25% trypsin digestion. Cell morphology was observed by inverted microscopy (FIG. 1A) and Giemsa staining (FIG. 1B, C), showing that hUC-MSCs were long spindle-shaped under adherent conditions,and grows in a vortex shape. Taking partial cells to incubate flow antibodies (APC-CD14, APC-CD34, APC-CD45, PE-CD73, PE-CD90 and PE-CD105) in a dark state to detect the expression of the hUC-MSC surface antigen, and finding that hUC-MSC surface markers CD73, CD90 and CD105 are highly expressed (the expression of the hUC-MSC surface markers CD73, CD90 and CD 105: (the expression of the hUC-MSC surface markers is high)>95%), low expression of CD14, CD34 and CD 45: (<2%) (fig. 1D).

Taking the other part of cells to perform adipogenic osteogenesis induced differentiation, wherein the adipogenic differentiation induced system is as follows: dexamethasone 1X 10^{- 6}mol/l，IBMX5×10^-4mol/l, insulin 1X 10^-5ng/l and indometacin 5X 10^-4mol/l; the osteogenic induced differentiation system is as follows: dexamethasone 1X 10^-7mol/l, beta-glycerophosphate 1X 10^-2mol/l and vitamin C phosphate 5X 10^-5mol/l or more were purchased from Sigma, USA. And (3) replacing liquid for adipogenic differentiation and osteogenic differentiation cells every two days, culturing for about two weeks, carrying out oil red O staining on the adipogenic differentiation cells, and detecting the expression of key transcription factors PPAR-gamma and ADI of adipogenic differentiation by q-PCR. Alkaline phosphatase is used for staining osteoblast differentiated cells, and q-PCR is used for detecting the expression of the osteogenic key transcription factors OPN and ALP.

Oil red O staining showed no lipid droplets from the differentiation group and distinct red lipid droplets in the induction group (fig. 1E, F); ALP staining showed a significant increase in induced group alkaline phosphatase activity (fig. 1G, F); q-PCR detection showed that the expression of the induced constitutive osteogenesis related genes was significantly higher than that of the self-differentiation group (FIG. 1I, J).

Example 2 construction of hUC-MSCs stably overexpressing GBP2

The conventional method is utilized to construct a lentivirus overexpression vector, the GV492 vector and BamHI/AgeI enzyme digestion are adopted and purchased from Shanghai Jikai GeneChemicals Co., Ltd, the enhancement solution belongs to a reagent matched with the lentivirus vector, and the experiment is operated according to a reagent instruction.

The hUC-MSC with good growth state is inoculated into a 6-well plate and divided into 4 groups: MSC group (not transfected by lentivirus overexpressing GBP 2), separate lentivirus overexpressing GBP2 transfection group (V), V + P infection enhancing fluid group, V + A infection enhancing fluid group, each group being provided with 3 multiple wells to inoculate cells in an amount of 1X 10⁵Hole, the next day, when the adherence state of the cells is good, the cells are changed and the slow virus is added respectivelyMOI 10, infection-enhancing fluid P (HiTransP) (REVG005, Kjekjelda gene) and infection-enhancing fluid A (HiTransA) (REVG005, Kjekjelda gene) were transfected. After incubation at 37 ℃ for 12h 1ml complete medium was added, the complete medium was replaced for 24h, the third day of transfection was performed and Puromycin (Puromycin) (2. mu.g/ml) was added, and transfected cells were selected. When the cells grew over the 6-well plate, the cells were passaged to T75 culture bottles for further culture, and the transfection efficiency, i.e., GFP fluorescence expression, was observed under a fluorescence microscope after culture for a certain period of time (FIGS. 2A-C); GFP expression was detected by flow cytometry on harvested cells, indicating that the enhancement effect of A enhancing fluid was superior to that of P enhancing fluid (FIG. 2D, E). When the q-PCR is used for detecting the GBP2 gene expression, both the A enhancing solution and the P enhancing solution can obviously enhance the GBP2 expression (2F). Based on the results, an A enhancing fluid with higher transfection efficiency was selected as the optimal infection condition.

In conclusion, it was found that the transfection efficiency of infection enhancing fluid A treatment group was significantly higher than that of transfection enhancing fluid P. The primers for obtaining the target gene fragment are shown in table 1, and the primers for constructing and identifying the recombinant plasmid are shown in table 2.

TABLE 1 acquisition primers for target Gene fragments

Note: P1/P2 contains exchange pairing base, enzyme cutting site and containsSequence of the 5' end portion of the target geneUsed for PCR to adjust the target gene.

TABLE 2 identification primers for recombinant plasmid construction

Example 3 study of GBP2 on the osteogenic differentiation of MSC

The overexpression vector constructed above was transfected into hUC-MSC under the same conditions as in example 2. The experiment was divided into 3 groups: MSC group (untransfected vector group), NC group (control group of vectors transfected with disordered fragments), GBP2^highMSC group (group transfected with GBP2 vector). GBP2 overexpression MSC morphology was observed using a microscope (fig. 3A) and Giemsa staining (fig. 3B).GBP2 was detected by flow cytometry to overexpress the hUC-MSC phenotype (fig. 3D). It was demonstrated that transfection of hUC-MSCs overexpressing GBP2 did not affect cell morphology and phenotype. The q-PCR was used to detect the degree of overexpression of GBP2, indicating that lentivirus-transfected hUC-MSC GBP2 was significantly overexpressed (FIG. 3C).

Normal hUC-MSC and GBP2^highRespectively inoculating hUC-MSC to 6-well plates, respectively setting as a self-differentiation group and an induction group, respectively adding an inducer to the induction group to perform osteogenesis induction for adipogenesis, respectively adding a complete culture medium (90% alpha-MEM culture medium + 10% FBS) to the self-differentiation group, changing the culture medium every two days, and performing oil red O staining and alkaline phosphatase (ALP) staining after 14 days.

The alkaline phosphatase (ALP) staining method comprises the following steps: washing with PBS buffer for 1 time; then 200 mul of fixing solution (40 mul of citric acid, 2ml of deionized water and 3ml of acetone) are added into each hole, the fixing solution is removed after 30 seconds of fixing, 200 mul of staining solution is added, the staining solution is removed after 30 minutes of room temperature treatment, a small amount of PBS buffer solution is added for washing for 2 times, and finally 1ml of PBS buffer solution is added into each hole for observation under a microscope.

The oil red O dyeing method comprises the following steps: removing the culture medium, and adding 1% neutral formaldehyde for fixation for 20 min; washing with PBS for 1 time, dyeing with oil red O, and keeping out of the sun at room temperature for 30 min; PBS was washed 1 time, 1ml of PBS was added to each well, and the shape, size and distribution density of lipid droplets were observed under an inverted microscope.

The oil red O is found to stain the self-differentiation and adipogenesis induction groups, and GBP2^highNo lipid droplets from the differentiation group of hUC-MSC, GBP2^highThe hUC-MSC-induced group showed a distinct red lipid droplet (FIG. 3E, F). ALP staining of the autodifferentiation and osteogenesis inducible groups was found, in contrast to GBP2^highGBP2 comparing hUC-MSC self-differentiation group^highThe hUC-MSCs induced a significant increase in group alkaline phosphatase activity (FIG. 3G, H).

q-PCR respectively detects the expression of marker genes ADI and PPAR-gamma of adipogenic differentiation and osteogenic differentiation marker genes OPN and ALP of the autogenic differentiation and the induction group, and the results show that: in the Induction group, GBP2^highThe expression of the hUC-MSC adipogenic and osteogenic related gene is increased, which indicates that GBP2^highThe hUC-MSCs can differentiate into adipocytes and osteoblasts (FIG. 3I, J).

ALP staining for NC groups and GBP2^highAlkaline phosphatase Activity of the hUC-MSC group, indicating GBP2^highThe activity of hUC-MSC alkaline phosphatase is obviously higher than that of NC group (FIGS. 4A-D); ImageJ analysis of ALP staining area, GBP2^highALP staining area was significantly larger in the MSC-induced group than in the NC-induced group (FIG. 4E), indicating GBP2^highThe osteogenic differentiation capacity of MSC is obviously higher than that of NC group.

q-PCR analysis of NC Induction groups and GBP2^highRelative expression of bone marker genes ALP and OPN in hUC-MSC induction group, and the result shows that GBP2^highThe expression of ALP and OPN in the hUC-MSC induction group is obviously higher than that in the NC induction group (FIG. 5A, B), which shows that GBP2^highThe hUC-MSC can promote the osteogenic differentiation of the MSC.

The sequences of the primers used for detecting the expression of ALP, OPN, ADI, PPAR-gamma are shown in Table 3:

TABLE 3 primer sequences

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

SEQUENCE LISTING

<110> military medical research institute of military science institute of people's liberation force of China

Application of <120> GBP2 in regulating and controlling mesenchymal stem cell osteogenic differentiation

<130> P210241

<160> 14

<170> PatentIn version 3.5

<210> 1

<211> 48

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

aggtcgactc tagaggatcc cgccaccatg gctccagaga tcaacttg 48

<210> 2

<211> 43

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

tccttgtagt ccataccgag tatgttacat attggctcca atg 43

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

taatgatcct cggttgtgca 20

<210> 4

<211> 22

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

ccttatagtc cttatcatcg tc 22

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

tcaagatcat cagcaatgcc 20

<210> 6

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

cgataccaaa gttgtcatgg a 21

<210> 7

<211> 22

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

ttccattcac aagaacagat cc 22

<210> 8

<211> 22

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

ctttgattgc actttggtac tc 22

<210> 9

<211> 19

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

tcacccaagc aacaaagtc 19

<210> 10

<211> 19

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

aaagaccaac cagatgcag 19

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 11

cagaagaagg acaaactggg 20

<210> 12

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 12

attgtatgtc ttggacagag c 21

<210> 13

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 13

ccataccagt taaacaggct g 21

<210> 14

<211> 19

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 14

tcagggttta gccatgtgg 19

Claims (10)

1. A preparation for regulating osteogenic differentiation of mesenchymal stem cells, which comprises a GBP2 inhibitor or promoter as an active ingredient.

2. The preparation of claim 1, wherein the mesenchymal stem cells comprise human umbilical cord-derived mesenchymal stem cells.

3. The formulation of claim 1 or 2, wherein the GBP2 promoter comprises an agent that promotes GBP2 expression or active function.

4. The formulation of claim 3, wherein the agent that promotes the expression of GBP2 comprises an expression vector comprising the GBP2 gene, preferably wherein the vector is a lentiviral vector.

5. Use of the preparation of any one of claims 1 to 4 for preparing a product for regulating osteogenic differentiation of mesenchymal stem cells.

6. The use of claim 5, wherein said preparation promotes the osteoblast differentiation capacity of MSCs by overexpressing GBP2 gene, thereby significantly up-regulating the expression of osteoblast differentiation-related markers.

7. The use according to claim 6, wherein said osteoblast differentiation related markers comprise the early marker ALP and the late marker OPN for osteoblast differentiation maturation.

8. Use of a formulation according to any one of claims 1 to 4 in the manufacture of a medicament for the treatment of osteoporosis.

9. A method for promoting osteogenic differentiation of mesenchymal stem cells, comprising the steps of:

1) obtaining mesenchymal stem cells, and carrying out subculture;

2) constructing an overexpression GBP2 vector;

3) the constructed overexpression GBP2 vector was transfected into mesenchymal stem cells and then cultured.

10. The method according to claim 9, characterized in that the transfection comprises the specific steps of: inoculating hUC-MSC with good growth state into 6-well plate, changing cell liquid and adding slow virus (MOI 10) and/or infection enhancing liquid for transfection when cell adherence state is good.

Images