CN109966500B

CN109966500B - Wolfberry polysaccharide-containing protective solution and application method thereof in improving pathological microenvironment of mesenchymal stem cells in vivo

Info

Publication number: CN109966500B
Application number: CN201910319863.8A
Authority: CN
Inventors: 朱永朝
Original assignee: General Hospital of Ningxia Medical University
Current assignee: General Hospital of Ningxia Medical University
Priority date: 2019-04-19
Filing date: 2019-04-19
Publication date: 2022-12-30
Anticipated expiration: 2039-04-19
Also published as: CN109966500A

Abstract

A protective solution containing lycium barbarum polysaccharide and an application method thereof in improving a complex pathological microenvironment in vivo of mesenchymal stem cells. The method adopts the separation, culture and identification of mesenchymal stem cells; identifying the use efficacy of the protective solution containing the lycium barbarum polysaccharide, and identifying the components and the proportion of the protective solution; the method comprises the steps of protecting the action mechanism of the liquid, and the like, and replaces the existing methods of gene editing or chemical drug treatment and the like by using the lycium barbarum polysaccharide which is a natural medicine extract as a main component, namely: the mesenchymal stem cells are intervened by using pure natural substances, so that the mesenchymal stem cells are better adapted to complex pathological microenvironment in vivo, and the survival capability and the treatment potential are improved. The protective solution can remarkably promote the survival rate of the mesenchymal stem cells in a pathological microenvironment in vivo and improve the treatment effect.

Description

Wolfberry polysaccharide-containing protective solution and application method thereof in improving pathological microenvironment of mesenchymal stem cells in vivo

Technical Field

The invention relates to a new application of a wolfberry extract in the field of biomedicine, and more particularly relates to a wolfberry polysaccharide-containing protective solution and an application method thereof in improving a complex pathological microenvironment in vivo of mesenchymal stem cells.

Background

Mesenchymal Stem Cells (MSCs) are a multipotent precursor cell of a wide source, derived from the mesoderm in the early embryonic development stage, with the potential to self-renew and differentiate into all cell types of the three germ layers. Based on self-renewal, multidirectional differentiation potential and immunoregulation function, the MSCs have incomparable treatment advantages in the aspects of tissue repair, immunoregulation and the like compared with the traditional treatment means. Clinical researches prove that the MSCs show subversive treatment effects in the treatment of various diseases such as diabetes, cardiovascular diseases, autoimmune diseases, mental diseases, liver failure, cancers and the like, and have strong clinical transformation potential.

Although MSCs show strong therapeutic effects in basic and clinical studies, after MSCs enter a patient, severe pathological microenvironments created by elements such as blood oxygen deficiency, free radicals, inflammation and active amino acids cause the risk of death and functional changes of MSCs in a plurality of days after transplantation, and the therapeutic effect is seriously influenced. Therefore, a method is sought, factors for limiting the curative effect of the MSCs are broken through, the viability of the MSCs in a pathological microenvironment is improved, various biological functions of the MSCs are maintained, and a possible regulation mechanism and clinical significance are explored, so that the key scientific problem to be solved is urgently needed.

At present, in order to improve the survival rate, the proliferation capacity, the differentiation potential and the paracrine level of the MSCs in a pathological microenvironment in vivo, the method is mainly realized by methods of gene modification, bioactive molecule pretreatment, culture environment pretreatment and the like. However, in the prior art, on one hand, gene editing or chemical medicine intervention is performed on cells, normal physiological and metabolic activities of the cells are affected to different degrees, the original biological characteristics of the cells and the malignant transformation potential of the cells to tumor cells are possibly changed, unknown safety problems exist, and clinical transformation of mesenchymal stem cells is seriously affected; on the other hand, the intervention effect has limitation, namely the mesenchymal stem cells can not be provided with the capability of comprehensively coping with the complicated pathological microenvironment in vivo only aiming at the condition of one pathological microenvironment in vivo.

Therefore, there is an urgent need in the art to develop a safe and effective protective solution and suitable for enhancing the resistance of mesenchymal stem cells to the in vivo pathological microenvironment.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a lycium barbarum polysaccharide-containing protective solution and application thereof in improving the complex pathological microenvironment of mesenchymal stem cells in vivo.

Lycium Barbarum Polysaccharide (LBP) is a water-soluble polysaccharide extracted from Lycium barbarum, a plant of multi-branched shrub of Solanaceae, ningxia. Research has shown that LBP plays an important biological role in antioxidation, immunomodulation, anti-aging, promotion of recovery from radiation and chemotherapy, etc. Has potential therapeutic effects on diabetes, cranial nerve injury repair, arthritis, and tumor.

The lycium barbarum polysaccharide belongs to natural plant extracts, and has strong safety and broad-spectrum biological functions through long-term application and research. The invention fully proves that the lycium barbarum polysaccharide is used as a main component, plays an important biological function in improving the adaptation of the mesenchymal stem cells to complex and harsh pathological microenvironment in vivo, and has incomparable advantages in the aspects of safety, effectiveness and the like compared with the prior art.

The technical scheme of the invention is as follows: a protective solution containing lycium barbarum polysaccharide and an application method thereof in improving the pathological microenvironment of mesenchymal stem cells in vivo.

The protective solution containing the lycium barbarum polysaccharide comprises the following components in percentage by weight: 20-100g of lycium barbarum polysaccharide, 20-50g of human serum albumin, 200-250mg of vitamin C, 80-100mg of glutathione and 1L of phosphate buffer solution with pH =7.2, and the components are mixed and stirred uniformly according to the required requirements to obtain the preparation.

The invention has the following remarkable effects:

the invention uses the natural medicine extract medlar polysaccharide as the main component to replace the prior methods of gene editing or chemical medicine treatment, and the like, namely: the mesenchymal stem cells are intervened by using pure natural substances, so that the mesenchymal stem cells are better adapted to complex pathological microenvironment in vivo, and the survival capability and the treatment potential are improved. The preparation can remarkably promote the survival rate of mesenchymal stem cells in vivo pathological microenvironment and improve treatment effect. The concrete is characterized in that:

1. the lycium barbarum polysaccharide belongs to a plant for both food and medicine, and the safety of the lycium barbarum polysaccharide is fully determined through a large amount of long-term research and application;

2. the invention does not involve the intervention of genetic modification, chemical induction of signal pathways and other abnormal physiological means of cells;

3. in-vitro experiments prove that the protective solution has the effects of improving the proliferation capacity of mesenchymal stem cells and inhibiting cell aging for the first time;

4. the protective solution has the biological function of improving the resistance of the mesenchymal stem cells to complex pathological microenvironment in vivo, and is firstly verified by animal experiments;

5. the application of the protective solution in mesenchymal stem cells is easy to operate and good in timeliness.

Drawings

FIG. 1 is a morphological observation diagram of human placenta-derived mesenchymal stem cells cultured in a serum-free manner according to the present invention;

FIG. 2 is a schematic diagram of flow cytometry for detecting serum-free culture hfPMSC surface markers;

FIG. 3 is a schematic diagram of the analysis of differentiation potential of serum-free cultured human placental fetal-derived MSCs according to the present invention;

FIG. 4 is a schematic view of the protective solution of the present invention promoting human placenta-derived mesenchymal stem cells to inhibit pulmonary fibrosis;

FIG. 5 is a schematic view of the effect of the protective solution of the present invention on enhancing the inhibition of collagen deposition in a mouse pulmonary fibrosis model by PMSCs;

FIG. 6 is a schematic diagram of the protective solution of the present invention for improving the survival rate of PMSCs in a pulmonary fibrosis mouse model;

FIG. 7 is a schematic diagram of the protective solution of the present invention for improving the proliferation capacity of human placental mesenchymal stem cells;

fig. 8 is a schematic diagram of the protective solution of the present invention for enhancing the viability of human placental mesenchymal stem cells in a pathological microenvironment of pulmonary fibrosis;

fig. 9 is a schematic diagram of the protective solution of the present invention significantly inhibiting apoptosis of human placental mesenchymal stem cells in a pathologic microenvironment of pulmonary fibrosis;

fig. 10 is a schematic diagram of the protective solution of the present invention increasing the autophagy of fmscs in a pulmonary fibrosis mouse model;

FIG. 11 is a schematic diagram illustrating the protective solution of the present invention up-regulating the occurrence of autophagy of PMSCs in a pathological microenvironment of pulmonary fibrosis;

FIG. 12 is a schematic diagram of the protective liquid up-regulating the Nrf2/ARE signal pathways of PMSCs in a pulmonary fibrosis pathological microenvironment.

Detailed Description

In order to make the technical scheme of the invention clearer and easier to understand, the following description refers to the accompanying drawings to further explain the embodiments of the invention in detail.

The protective solution containing the lycium barbarum polysaccharide comprises the following components in percentage by weight: 20-100g of lycium barbarum polysaccharide, 20-50g of human serum albumin, 200-250mg of vitamin C, 80-100mg of glutathione and 1L of phosphate buffer solution with pH =7.2, and the components are mixed, stirred uniformly and filtered according to the required requirements to obtain the preparation.

The application method of the protective solution containing the lycium barbarum polysaccharide in improving the complex pathological microenvironment in vivo of the mesenchymal stem cells comprises the following specific steps:

as shown in FIG. 1, placenta tissue of healthy puerpera of term is collected, and the tissue with a thickness of about 0.5cm on the placenta side is cut into pieces of 1mm 3, and after thoroughly rinsing with HBSS, 1g/L of Collagenase A and 1. Mu.g/mL of DNase I are added, and digested in water bath at 37 ℃ for 2.0h. The cells in the supernatant were collected and transferred to serum-free medium (STEMCELL Technologies) and cultured at 37 ℃ in a 5% carbon dioxide incubator. After 24h, the solution is changed for the first time, and when 80-90% of cells are fused, the cells are passaged according to the proportion of 1: 3. The 3 rd generation human placenta-derived mesenchymal stem cells cultured by the serum-free culture medium have uniform shape, adhere to the wall, grow in a fusiform vortex manner and conform to the typical shape (40 x) of the mesenchymal stem cells.

As shown in fig. 2, mesenchymal stem cells were prepared into a single cell suspension, and to flow cell tubes to which 1 × 10 6 cells were added, fluorophore-labeled mabs were added, respectively, according to the instructions: igG2a-FITC, igG1-PE, CD14-FITC, CD34-FITC, CD45-FITC, CD73-PE, CD90-FITC, CD105-PE, HLA-DR-FITC, and isotype control antibody to a final volume of 100 μ L; keeping away from light, standing at room temperature for 20min, rinsing with PBS, and detecting by flow cytometry. Serum-free cultured hfPMSC are CD73, CD90 and CD105 positive cells, while hematopoietic marker molecules CD14, CD34, CD45 and HLA-DR are negative, with surface marker molecules typical of mesenchymal stem cells.

As shown in FIG. 3, mesenchymal stem cells were seeded at 1X 10/well in 6-well plates, and when the cells were grown and reached a confluence of about 80%, the original medium was discarded, and adipocyte-inducing solution (high-glucose DMEM medium containing 10% FBS 1nmol/L dexamethasone 0.2mmol/L indomethacin 0.5 mmol/L3-isobutyl-1-methylxanthine and 10mg/L insulin) and osteoblast-inducing solution (high-glucose DMEM medium containing 10% FBS0.1nmol/L dexamethasone 10 mmol/L-sodium glycerophosphate and 0.5mmol/L ascorbic acid) were added, respectively, and the solution was changed 2 times per week in a cell culture chamber saturated with 5% CO2 at 37 ℃ for 2 to 3 weeks, stained with oil red O and rubicin, respectively, and observed under an inverted microscope.

After induction, the human placental fetal side-derived cells can respectively form lipid droplets (A) which are colored bright red and calcium nodule deposits (B) which are colored red, and the separated cells are prompted to have multidirectional differentiation potential and accord with the characteristics of mesenchymal stem cells.

As shown in fig. 4, the protective solution promotes human placenta-derived mesenchymal stem cells to inhibit pulmonary fibrosis. Selecting 2 nd generation normal growth mesenchymal stem cells, digesting with trypsin, and inoculating 6000-8000 cells/cm 2 in a culture dish. When the cells grow to 60-70% confluence, adding a protective solution into the cell culture solution according to the dilution ratio of 1.

The diagram is as follows: HE staining analysis of treatment effects of human placenta-derived mesenchymal stem cells and protective solution-treated mesenchymal stem cells after bleomycin-treated mice for 21d, and normal bleomycin-treated mice for 21d were used as control groups (40 ×); B. ashcroft score analysis of fibrosis (n = 5) (. P < 0.05). The result shows that the protective solution can obviously improve the function of the mesenchymal stem cells in improving the pulmonary fibrosis.

In the figure: control represents normal mice; bleo indicates bleomycin treatment; PMSCs represent human placenta-derived mesenchymal stem cells; fPMSCs represent human placental derived MSCs after treatment with the protective solution.

As shown in fig. 5, the protective solution increased the inhibitory effect of PMSCs on collagen deposition in the mouse pulmonary fibrosis model. A. Carrying out masson staining analysis on the influence of the human placenta-derived mesenchymal stem cells after the bleomycin-treated mice are treated for 21d and the influence of the mesenchymal stem cells after the bleomycin-treated mice are treated for collagen deposition, and respectively taking normal mice and bleomycin-treated mice as a control group (400 x); B. quantitative analysis of lung collagen content (n = 5) (. P < 0.05). The result shows that the protective solution obviously promotes the inhibition effect of the mesenchymal stem cells on the generation of collagen in pathological tissues.

Note that Control is normal mice; bleo: treating the bleomycin; PMSCs: human placenta-derived mesenchymal stem cells; PMSCs LBP: and (4) treating the human placenta-derived mesenchymal stem cells by using the protective solution.

As shown in fig. 6, a mouse pulmonary fibrosis model was prepared using bleomycin, and alveolar lavage fluid (BALF) was collected on day 7 after molding. The trypsin-digested mesenchymal stem cells were seeded at 2000 cells/well in a 96-well plate while maintaining the ratio of 1: adding protective solution and BALF according to the proportion of 100. The proliferation of cells after 72h of culture was examined by CCK8, and cells without protective solution were used as a control group. The result shows that the protective solution obviously improves the proliferation capacity of the mesenchymal stem cells in a pathological microenvironment created by the pulmonary alveolar lavage fluid of the pulmonary fibrosis model mouse.

In the figure, a. Normal PMSCs; B. and (4) pretreating the PMSCs by using a protective solution. PMSCs are treated by Dil Stain, MSCs after being pretreated by normal MSCs and protective solution for 24 hours are injected into bleomycin to prepare a 3 rd pulmonary fibrosis mouse model, after 5 days, lung tissues are taken to prepare a frozen section, and the existence condition (red mark) of mesenchymal stem cells in the lung tissues is observed by a fluorescence microscope after DAPI staining and mounting (40 x). PMSCs are human placenta-derived mesenchymal stem cells. The result shows that the mesenchymal stem cells treated by the protective solution still have the capability of improving the survival capacity of the mesenchymal stem cells in a pathological model animal body.

As shown in fig. 7, 2 nd generation of normally grown mesenchymal stem cells were selected, trypsinized, and seeded into 96-well plates at 1000 cells/well while maintaining the ratio of 1: the protective solution is added according to the proportion of 100. The proliferation of 24h, 48h, 72h, 96h and 120h cells was tested by CCK8, and cells without protective solution were used as control group. The result shows that the protective solution can better maintain the long-term culture of the mesenchymal stem cells in vitro and keep higher proliferation capacity.

As shown in fig. 8, the schematic diagram of the protective solution for enhancing the viability of human placental mesenchymal stem cells in the pathological microenvironment of pulmonary fibrosis is that a mouse pulmonary fibrosis model is prepared by using bleomycin, and alveolar lavage fluid (BALF) is collected at 7 days after molding. The trypsinized mesenchymal stem cells were seeded into 96-well plates at 2000 cells/well in a 1: adding protective solution and BALF according to the proportion of 100. The proliferation of cells after 72h of culture was measured by CCK8, and cells without protective solution were used as a control group. The result shows that the survival rate of the human placental mesenchymal stem cells in pulmonary fibrosis model mouse alveolar lavage can be remarkably improved by adding the protective solution.

As shown in fig. 9, the protective solution significantly inhibits apoptosis of human placental mesenchymal stem cells in the pathological microenvironment of pulmonary fibrosis. Inoculating the mesenchymal stem cells after trypsinization into a 35mm culture dish, and treating the cells by using a protective solution and BALF when the cells grow to 60-70%. The following are shown in the figure: a mouse pulmonary fibrosis model was prepared using Bleomycin and Alveolar Lavage Fluid (BALF) was collected at day 7 after molding. And (3) planting the human placenta mesenchymal stem cells in a 35mm culture dish, and when the cell fusion degree reaches 60-70%, performing the steps of 1: the protective solution and/or BALF is added in a proportion of 100. Using the Dead Cell Apoptosis Kit with annexin V Alexa Fluor ^TM 488&A Propidium Iodide (PI) kit detects the apoptosis condition of cells after 24h of culture, and the cells without protective solution are used as a control group. The result shows that pulmonary fibrosis model mouse alveolar lavage can induce human placental mesenchymal stem cells to undergo apoptosis, but the protective solution can obviously inhibit apoptosis of the cells.

As shown in fig. 10, the protective solution enhanced the occurrence of autophagy of fmscs in a mouse model of pulmonary fibrosis. In the figure: A. normal PMSCs; B. and (4) pretreating the PMSCs by using a protective solution. PMSCs are infected by mRFP-GFP-LC3 adenovirus, normal PMSCs and PMSCs after being pretreated by protective solution for 24 hours are injected into bleomycin to prepare a 7 th-day pulmonary fibrosis mouse model, lung tissues are taken after 24 hours to prepare a frozen section, and the autophagy occurrence and degree (punctate aggregation) of the PMSCs are observed by a fluorescence microscope after DAPI staining and sealing (400 x). The results show that the protective solution can significantly up-regulate the occurrence of autophagy in PMSCs in a pulmonary fibrosis mouse model.

As shown in fig. 11, the protective solution up-regulates the occurrence of PMSCs autophagy in the pathological microenvironment of pulmonary fibrosis. The method comprises the steps of performing intraperitoneal injection of 10g/L pentobarbital sodium for anesthesia, preparing a pulmonary fibrosis model by injecting 50 mu L bleomycin (1 g/L) into a trachea, infecting mesenchymal stem cells by mRFP-GFP-LC3 adenovirus, performing tail vein injection of normal and protective solution for pretreatment of human placenta-derived MSCs for 24 hours on the 3 rd day after model building, respectively preparing lung frozen sections 24 hours after cell injection, and observing the condition of punctate aggregation in mesenchymal stem cells in lung tissues under a fluorescent confocal microscope. In the figure: A. PMSCs are pretreated by protective solution for 24h, alveolar lavage fluid is added to a pulmonary fibrosis mouse model on day 7 to continue culturing for 24h, and expression levels of PMSCs autophagy marker molecules Beclin1 and LC3I/II are detected by Western blotting; B. expression of Beclin1 and LC3-II in a was quantified (. < 0.05). The result shows that the protective solution can up-regulate the autophagy of PMSCs in a pulmonary fibrosis microenvironment in vitro simulation, and the protective solution is prompted to achieve the effect of improving the PMSCs to deal with the pulmonary fibrosis pathological microenvironment by regulating and controlling the autophagy.

As shown in fig. 12, the protective solution of the present invention up-regulates Nrf2/ARE signal pathways of PMSCs in a pulmonary fibrosis pathological microenvironment.

In the figure: A. PMSCs are pretreated by the protective solution for 24h, alveolar lavage fluid on the 7 th day of a pulmonary fibrosis mouse model is added to continue to be cultured for 24h, and the expression levels of Nrf2 and HO-1 in the PMSCs are detected by Western blotting; B. expression of Nrf2 and HO-1 in a was quantified (. Sp. < 0.05). The result shows that the protective solution can up-regulate the expression of PMSCs Nrf2 and HO-1 in a pulmonary fibrosis pathological microenvironment, and the Nrf2 signal path plays an important regulation role in influencing the biological functions of PMSCs by the protective solution.

The invention uses the natural medicine extract lycium barbarum polysaccharide as the main component of the protective solution, replaces the existing methods of gene editing or chemical medicine treatment and the like, and intervenes the mesenchymal stem cells by using pure natural substances, so that the mesenchymal stem cells are better adapted to the complex pathological microenvironment in vivo, and the viability and the treatment potential are improved.

Claims

1. The protection solution containing the lycium barbarum polysaccharide is characterized by comprising the following components in parts by weight: 20-100g of lycium barbarum polysaccharide, 20-50g of human serum albumin, 200-250mg of vitamin C, 80-100mg of glutathione and 1L of phosphate buffer solution with pH =7.2, and the components are mixed, stirred uniformly and filtered according to the required requirements to obtain the preparation.