CN108486046B

CN108486046B - Stem cell preparation for resisting hypoxia injury, preparation method thereof and application thereof in preparation of medicine for treating acute myocardial infarction

Info

Publication number: CN108486046B
Application number: CN201810137026.9A
Authority: CN
Inventors: 陈维倩; 沈晗; 崔光浩; 沈振亚
Original assignee: Suzhou University
Current assignee: Cangkun Biotechnology (Shanghai) Co.,Ltd.
Priority date: 2018-02-09
Filing date: 2018-02-09
Publication date: 2020-09-25
Anticipated expiration: 2038-02-09
Also published as: CN108486046A

Abstract

The invention discloses an anti-hypoxia injury stem cell preparation, a preparation method thereof and application thereof in preparing a medicament for treating acute myocardial infarction. The Fstl1 modified MSCs provided by the invention have excellent anti-hypoxia injury capability, can improve the transplantation survival rate, and can effectively improve the cardiac function after infarction through direct myocardial injection.

Description

Stem cell preparation for resisting hypoxia injury, preparation method thereof and application thereof in preparation of medicine for treating acute myocardial infarction

Technical Field

The invention belongs to the cell medicine technology, and particularly relates to an anti-hypoxia injury stem cell preparation, a preparation method thereof and application thereof in preparation of a medicine for treating acute myocardial infarction.

Background

Myocardial infarction is a cardiovascular disease seriously harming human health, and the incidence of ischemic myocardial infarction is continuously increased along with the continuous improvement of living standard of Chinese people. Ischemic myocardial infarction can lead to myocardial cell necrosis and scarring, which in turn affects cardiac function. Most of the current medicines or apparatus treatments can only relieve symptoms, but can not reverse the damage of heart tissues. Although heart transplantation can completely improve the heart condition, the heart transplantation is difficult to be widely applied clinically due to factors such as donor source scarcity, immunological rejection, expensive treatment cost and the like.

The stem cell transplantation for treating the myocardial infarction is expected to become an important treatment means for treating the myocardial infarction due to various advantages. Mesenchymal Stem Cells (MSCs) are a primary adult stem cell with abundant and easily available sources, and are mainly present in tissues such as bone marrow, fat, umbilical cord, placenta, amniotic fluid, and the like. In recent years, MSCs are widely used as seed cells for cell transplantation in the field of regenerative medicine due to their excellent tissue repair ability. Several studies have demonstrated that MSCs function primarily on paracrine secretion. The bottleneck problem which plagues stem cell transplantation at present is that the survival rate of stem cell transplantation is low, so how to improve the survival rate of stem cells to the maximum extent is a research hotspot in the field. The local poor hypoxic microenvironment of myocardial infarction is the main reason for the low survival rate of transplanted stem cells, so the improvement of the anti-hypoxic damage capability of stem cells is the future direction of effort.

Follistatin-like protein 1 (Follistatin like 1, Fstl 1), originally cloned from the mouse osteoblast line MC3T3-E1, is a secreted extracellular glycoprotein that is not involved in extracellular matrix architecture, but regulates cellular physiological activities at extracellular level by altering the cellular microenvironment. Fstl1 is an important endogenous active factor for maintaining cardiac homeostasis and pathological remodeling, and is widely expressed in the heart. As a marker of left ventricular remodeling in chronic contractile heart failure, Fstl1 can reduce pathological cardiac hypertrophy from pressure overload; however, it is unclear whether Fstl1 can improve the ability of stem cells to resist hypoxic damage, thereby improving the retention of transplanted cells.

Disclosure of Invention

The invention discloses an anti-hypoxia injury stem cell preparation, a preparation method thereof and application thereof in preparing a medicament for treating acute myocardial infarction.

The invention adopts the following technical scheme:

a preparation method of a stem cell preparation for resisting hypoxia injury comprises the following steps of mixing stem cells with Fstl1 overexpression lentivirus liquid in a culture medium, and changing the liquid after 10-15 hours to obtain the stem cell preparation for resisting hypoxia injury.

The invention also discloses a preparation method of the medicine for preventing or treating myocardial infarction, which comprises the following steps of mixing stem cells and Fstl1 overexpression lentivirus liquid in a culture medium, and changing the liquid after 10-15 hours to obtain a stem cell preparation for resisting hypoxia injury; mixing the stem cell preparation with anoxia damage resisting agent and dispersing medium, such as buffer solution and physiological saline, to obtain medicine for preventing or treating myocardial infarction; the specific administration mode can adopt direct myocardial injection or menstrual injection of various routes, and the like.

Preferably, the stem cells are mixed with Fstl1 overexpression lentiviral fluid in the presence of polybrene (polybrene) at a final concentration of 8 μ g/ml in culture medium to increase the efficiency of viral infection.

In the technical scheme, the stem cells are mouse bone marrow mesenchymal stem cells; the amount of Fstl 1-overexpressing lentivirus fluid was calculated as multiplicity of infection (MOI) = 10. In the Fstl1 overexpression lentivirus solution, the titer of the Fstl1 overexpression lentivirus is 10⁷～10⁸TU/ml; the culture medium is generally DMEM/F12 medium.

Preferably, male C57BL/6J mice of 2-3 weeks old are selected, the mice are killed by cervical dislocation, and the mice are soaked in 75% alcohol for 10 min; soaking the two lower limbs of the mouse in a DMEM/F12 serum-free basic culture medium under an aseptic condition, and removing muscles on the surfaces of thighbones and shinbones; sucking DMEM/F12 serum-free basal medium added with the cyan/streptomycin by using a 1ml syringe and flushing a bone marrow cavity; centrifuging after all bone marrow is flushed out, removing supernatant, adding a fresh special culture medium for mouse bone marrow-derived MSCs (mesenchymal stem cells) to repeatedly blow and beat to obtain a single cell suspension, and inoculating the cell suspension into a culture dish; after 72h, changing the liquid and removing the non-adherent cells, then changing the liquid every two days, and carrying out passage when the colonies in the culture dish are mutually fused and the cell density reaches about 70%, wherein the passage is marked as P1 generation; after MSCs are transmitted to P5 generation, mixing stem cells with Fstl1 overexpression lentivirus solution in a culture medium to prepare a stem cell preparation for resisting hypoxia injury; preferably according to 3X 10⁵/cm²The cell suspension was seeded in a petri dish.

Preferably, the stem cells and Fstl1 overexpression lentivirus solution are mixed in a culture medium, and the solution is changed after 12 hours to obtain the stem cell preparation for resisting hypoxia injury; more preferably, the medium is replaced and cultured for a period of time to obtain more antioxidant damaged stem cells, and the culturing time can be 72 hours or more.

The invention also discloses the stem cell preparation for resisting the hypoxic injury, which is prepared according to the preparation method of the stem cell preparation for resisting the hypoxic injury; the application of the Fstl1 overexpression lentivirus in improving the stem cell transplantation survival rate or the application of the Fstl1 overexpression lentivirus in preparing an anti-hypoxia injury stem cell preparation.

The stem cell preparation for resisting the hypoxic injury judges the cell infection efficiency by observing mCherry fluorescence under an inverted fluorescence microscope, and can find that most cells display strong mCherry fluorescence, thereby prompting the success of lentivirus infection.

As a primary adult stem cell with abundant and easily obtained sources, MSCs are widely used in the field of regenerative medicine as seed cells for cell transplantation due to their excellent tissue repair ability. However, the existing MSCs have the problem of weak ability of obviously resisting hypoxia injury, so that the actual stem cell repair effect is far lower than that of the theoretical design. The invention can obviously improve the anti-hypoxia injury capability of the stem cells after the stem cells are transformed by using Fstl1, thereby improving the transplantation survival rate of the stem cells, and the invention also discloses the application of Fstl1 in improving the transplantation survival rate of the stem cells.

The prior art researches the effect of Fstl1 on myocardial infarction, but no document relates to the reconstruction of stem cells by Fstl1, and no report about the effect of the Fstl1 on the reconstruction of the stem cells is reported; the Fstl1 modified MSCs provided by the invention have excellent anti-hypoxia injury capability and can improve the transplantation survival rate, so that the invention also discloses the application of Fstl1 in preparing an anti-hypoxia injury stem cell preparation; the invention also discloses application of the stem cell preparation for resisting the hypoxia injury in preparing a stem cell system for resisting the hypoxia injury or improving the stem cell transplantation survival rate. The invention also discloses application of the stem cell preparation for resisting hypoxia injury in preparing medicaments or health-care products for preventing or treating myocardial infarction. The stem cell preparation for resisting hypoxia injury has the capability of resisting a local severe hypoxia microenvironment caused by myocardial infarction, and can become an important means for regulating and controlling the steady state and pathological remodeling of the heart.

Drawings

FIG. 1 is a graph showing morphological characterization of mouse bone marrow-derived MSCs;

FIG. 2 is a flow chart of mouse bone marrow-derived MSCs;

FIG. 3 is a graph showing that persistent hypoxic stimulation caused a significant decrease in the expression of Fstl1 in MSCs;

FIG. 4 shows that both MSCs-mCherry and MSCs-Fstl1 strongly express mCherry fluorescence;

FIG. 5 is a flow chart of the identification of MSCs-mCherry and MSCs-Fstl 1;

FIG. 6 shows MSCs-Fstl1 highly expressed and highly secreted Fstl 1;

FIG. 7 shows that MSCs-Fstl1 are more effective against hypoxia-induced apoptosis;

FIG. 8 shows that MSCs-Fstl1 have a higher cell proliferation capacity in an anoxic environment;

FIG. 9 shows that the MSCs-Fstl1 have better cell viability in both normoxic and hypoxic environments;

FIG. 10 is a M-shaped sonogram showing that MSCs-Fstl1 cell transplantation significantly improved myocardial function after myocardial infarction;

FIG. 11 shows that MSCs-Fstl1 cell transplantation significantly promoted EF, FS, LVID, d and LVPW, d indicates recovery;

FIG. 12 is a schematic representation of the reduction in myocardial infarct size following MSCs-Fstl1 cell transplantation (Masson staining);

FIG. 13 shows that MSCs-Fstl1 cell transplantation effectively reduced myocardial infarct area (statistical results);

FIG. 14 is a graphical representation of mCherry autofluorescence and immunofluorescence co-localization to assess engraftment of transplanted cells in hypoxic myocardium;

FIG. 15 is a DiI marker of transplanted cells to assess their residence in hypoxic myocardium.

Detailed Description

The following examples are presented only to assist those skilled in the art in a more complete understanding of the present invention, and are not intended to limit the invention in any way. In the present invention, the liquid exchange means that the virus-infected liquid is 100% replaced by a normal complete cell culture medium.

Example preparation and Performance testing of an anti-hypoxic injury Stem cell preparation

The main materials and sources used were as follows:

c57BL/6J mice (center for research on showa new drug, this experiment was approved by the ethical committee of the university of suzhou); DMEM/F12 medium (Gibco, USA); mouse bone marrow-derived MSCs-dedicated medium (seiko organism, china); trypsin (Sigma, usa); clean bench (antai, china); carbon dioxide incubator (Thermo, usa); bench top centrifuge (Thermo, usa); flow cytometry (Millipore, usa); inverted fluorescence microscopy (ZEISS, germany); nanodrop2000 ultramicro spectrophotometer (Thermo, usa); real-time fluorescent quantitative PCR instrument (ABI, usa); a full-band multifunctional microplate reader (BIO-TEK, USA); reverse transcription kit (Takara, japan); FITC-CD29 antibody (Biolegend, USA); APC-CD44 antibody (Biolegend, USA); FITC-CD90 antibody (Biolegend, USA); PE-CD45 antibody (Biolegend, USA); APC-CD117 antibody (Biolegend, USA); fstl1 ELISA detection kit (R & D, usa); Click-iT Plus EdU Alexa Fluor 647 flow assay kit (Life, USA); annexin V-FITC kit (BD, USA); CCK-8 kit (Dojindo, Japan); fstl1 overexpression lentivirus (LV-Fstl 1) and control (LV-mCherry) (Kjecqal, China)

1.1 obtaining mouse bone marrow-derived MSCs

Selecting 2-3 weeks old male C57BL/6J mouse, killing the mouse by cervical dislocation method, soaking the mouse in 75% alcohol for 10min for disinfection; soaking the two lower limbs of the mouse in a DMEM/F12 serum-free basic culture medium under an aseptic condition, and removing the muscles on the surfaces of the femur and the tibia by using scissors and tweezers; sucking DMEM/F12 serum-free basal medium added with the cyan/streptomycin by using a 1ml syringe and slowly flushing a bone marrow cavity; centrifuging after all bone marrow is flushed out, removing supernatant, adding fresh mouse bone marrow-derived MSCs special culture medium, repeatedly blowing to obtain single cell suspension, and repeating the steps according to 3X 10⁵/cm²Inoculating the cell suspension into a culture dish at the density of (1); after 72h, changing the liquid and removing the non-adherent cells, and then changing the liquid every two days until the colonies in the culture dish are mutually concentratedPassages were performed when confluent and cell density reached around 70%, at which time the marker was P1. FIG. 1 shows that MSCs from P6 generation have uniform distribution and uniform morphology, and are shown as fibroblast-like or flat shape, and a few are fusiform with protrusions of different lengths and thicknesses.

1.2 flow cytometry identification of mouse bone marrow-derived MSCs

When the degree of cell confluence reaches 80%, conventional pancreatin digestion is carried out to adjust the cell concentration to 5X 10⁶cells/ml; respectively adding FITC-CD29 antibody, APC-CD44 antibody, FITC-CD90 antibody, PE-CD45 antibody and APC-CD117 antibody, and incubating at 4 deg.C for 30 min; PBS washed and flow cytometry to detect cell surface markers. FIG. 2 is a graph showing the results of flow cytometry, and mouse bone marrow-derived MSCs expressed CD29, CD44, and CD90, but did not express CD45 and CD 117.

1.3 sustained hypoxic stimulation caused a significant decrease in the expression of Fstl1 in MSCs

Continuous hypoxia stimulation (1% O) was performed when the MSCs confluency reached 70%₂) Cells were harvested at 0h, 24h and 48h respectively and total RNA, inverted and qRT-PCR extracted. Each sample was prepared in 4 duplicate wells, and the reaction system was 10. mu.l with GAPDH as internal control. The primer sequences used were as follows: fstl1: 5-TTATGATGGGCACTGCAA-3 and 5-ACTGCCTTTAGAGAACCAG-3; GAPDH 5-TGCCCAGAACATCATCCCT-3 and 5-GGTCCTCAGTGTAGCCCAAG-3. Figure 3 shows that sustained hypoxic stimulation caused significant down-regulation of Fstl1 expression in MSCs, suggesting that Fstl1 may have an important role in protection from hypoxic injury. # ##P<0.001（0h vs 24h）；***P<0.001（0h vs 48h）。

1.4 preparation of anti-hypoxic injury Stem cells

In the step 1, when the confluence degree of the MSCs reaches 50%, converting the number of virus particles according to multiplicity of infection (MOI) = 10; respectively sucking Fstl1 overexpression lentivirus (LV-Fstl 1) liquid (Gickae company) and corresponding no-load control virus liquid (LV-mCherry), and adding polybrene (8 mu g/ml) to increase the virus infection efficiency; 37 ℃ and 5% CO₂Infecting the incubator overnight; after 12h, the virus solution is replaced and a normal culture medium is added; observing mCherry fluorescence under an inverted fluorescence microscope after 72hThe cell infection efficiency is judged by strength, the Fstl1 high expression and secretion conditions are detected by qRT-PCR and ELISA, and MSCs are identified by flow assay to indicate a mark. The stem cell preparation for resisting the hypoxia injury is called MSCs-Fstl1, and the corresponding control is called MSCs-mCherry.

FIG. 4 is a fluorescence image of MSCs-mCherry and MSCs-Fstl1, wherein strong mCherry fluorescence can be seen in the MSCs-mCherry and MSCs-Fstl1 group cells, and typical MSCs appearance forms are still kept, thus proving that the lentivirus infection is successful; FIG. 5 is a graph showing the results of flow cytometry detection of MSCs-mCherry and MSCs-Fstl1, wherein both MSCs-mCherry and MSCs-Fstl1 express CD29 and CD44, and do not express CD45 and CD 117.

1.5 qRT-PCR identification of Fstl1 transcript levels in MSCs-Fstl1 cells

Total RNAs were extracted conventionally from MSCs-mCherry and MSCs-Fstl1 and the transcript level was determined as in example 1.3 for Fstl 1. As shown in FIG. 6A, the level of Fstl1 transcript in MSCs-Fstl1 cells increased 9.18-fold over the control MSCs-mCherry groupP<0.001。

1.6 ELISA identification of Fstl1 secretion levels in MSCs-Fstl1 cells

A96-well microplate coated with the Fstl1 capture antibody is prepared into a solid phase carrier, a specimen (MSCs-mCherry or MSCs-Fstl1 supernatant) or a standard substance, a biotinylated Fstl1 detection antibody and HRP-labeled avidin are sequentially added, and the mixture is thoroughly washed and developed by a substrate. The shade of color was positively correlated with the Fstl1 content of the sample. The absorbance (OD value) was measured at a wavelength of 450nm with a microplate reader, and the Fstl1 concentration in the sample was calculated from the standard curve.

FIG. 6B shows that the supernatant of MSCs-Fstl1 cells had a Fstl1 concentration 12.44-fold higher than the control, demonstrating that MSCs-Fstl1 could achieve high secretion of Fstl1P<0.001。

1.7 Annexin V marker detection of the ability of MSCs-Fstl1 to resist hypoxia-induced apoptosis

Mixing MSCs-mCherry and MSCs-Fstl1 at 8X 10⁴Culturing in 12-well plate, replacing with fresh culture medium after overnight adherence, and continuously hypoxia stimulating (1% O)₂) 48h, digesting with 0.25% pancreatin without EDTA and collecting the cells, washing 2 times with PBS, adding 100. mu.l Annexin V labeling buffer to resuspend the cells, concentrating the cellsDegree of about 10⁶One per ml. Adding 5 μ l annexin V-FITC, mixing, reacting at room temperature in dark place for 15min, and detecting with flow cytometer.

The Annexin V staining experiment of FIG. 7 shows that under hypoxic stimulation, the proportion of Annexin V + cells in the MSCs-Fstl1 group was significantly lower than that in the MSCs-mCherry group.

1.8 EdU labeling detection of proliferation potency of MSCs-Fstl1 in hypoxic environment

Inoculating MSCs-mCherry and MSCs-Fstl1 into 12-well plate at a certain density, culturing, and performing continuous hypoxia stimulation (1% O) after adherence₂) 48h, adding EdU (10. mu.M), incubating at 37 ℃ for 1h, trypsinizing and collecting cells, washing 2 times with PBS, fixing, performing fluorescence infiltration reaction, and detecting by a flow cytometer.

The EdU infiltration experiment results in FIG. 8 show that the proliferation potency of the MSCs-Fstl1 group was higher than that of the MSCs-mCherry group under both normoxic and hypoxic conditions.

1.9 CCK-8 experiment to detect the cell viability of MSCs-Fstl1 in an anoxic environment

MSCs-mCherry and MSCs-Fstl1 were plated at density in 96-well plates. After the wall is attached, continuous hypoxia stimulation (1% O) is carried out₂) And (3) replacing 100 mul of fresh culture medium and 10 mul of CCK-8 reaction solution in each hole for 48 hours, and measuring the light absorption value at 450nm by using an enzyme-labeling instrument every 0.5 hour between 0.5 and 2 hours.

The results of the CCK-8 experiment in FIG. 9 show that the cell viability of MSCs-Fstl1 under normoxic and hypoxic conditions is 1.24 times and 2.19 times that of MSCs-mCherry, respectivelyP<0.05，**P<0.01，***P<0.001。

Example II hypoxia-damage-resistant Stem cell preparation is effective in improving cardiac function after myocardial infarction and has better retention effect

The main materials and sources used were as follows:

c57BL/6J mice (center for research on showa new drug, this experiment was approved by the ethical committee of the university of suzhou); small animal ventilators (alcoett biology, shanghai); surgical instruments (sixty-six vision, suzhou); suture (golden ring medical, shanghai); small animal ultrasound imaging system (Visual sonic Vevo 2100); inverted fluorescence microscopy (ZEISS, germany); masson staining kit (Sigma, usa); mCherry antibody (Abcam, usa); FITC-labeled goat anti-rabbit IgG secondary antibody (senta cruz, usa); CM-DiI (Invitrogen, USA)

2.1 establishment of mouse myocardial infarction model

About 25g of C57BL/6J male mice are selected as experimental objects, and a left anterior descending coronary artery (LAD) ligation method is adopted to prepare the myocardial infarction model. After the abdominal cavity injection anesthesia, the patient is intubated through an oral trachea, and then connected with an air respirator, the breathing frequency is 110 times/min, the tidal volume is 3ml, and the respiratory suction ratio is 1: 1.3. In the right lateral position, the lateral skin of the left chest is incised through the left longitudinal incision, the pectoralis major is peeled off, the chest is opened through the third and fourth intercostal transverse incisions, the heart is exposed, and the pericardium is torn open through forceps. The left coronary artery was visualized by a surgical microscope. And ligating the LAD together with a small amount of myocardial tissue at the position of about 1-2 mm of the lower edge of the left auricle, wherein the depth of the needle insertion is about 1mm, and the width is controlled within 3 mm. Closing the chest layer by layer. The sham group (sham) passed only under the LAD without knots, the rest of the model group; after ligation, the part from the ligation position to the apex of the heart becomes white by naked eyes, and after 7 days, the left ventricle tissue is taken for cardiac tissue staining, so that obvious fibrosis can be seen, and the successful establishment of the myocardial infarction model is proved.

2.2 myocardial injection of anti-hypoxic injury Stem cell preparation

Mixing the stem cell preparation MSCs-Fstl1 with hypoxia injury resistance and a dispersion medium PBS to obtain the medicine for preventing or treating myocardial infarction. After LAD ligation according to the method of step 1, two sites, i.e., the lower left and the lower right, near the ligation site were selected for injection of drugs, and the amount of MSCs per mouse was 5X 10⁵Mu.l/20. mu.l, 10. mu.l per site, PBS as a negative control. The appropriate angle is selected to avoid injection into the left ventricular cavity. A slight lightening of the myocardial color indicates that the solution has entered the infarcted ventricular wall.

2.3 Heart ultrasonic testing of myocardial function behind myocardial infarction

7 days mouse anesthesia after myocardial infarction (method same as before), left lateral decubitus after depilation, placing the probe of the heart ultrasonic diagnostic apparatus on the anterior wall of the heart, taking a left ventricular two-dimensional short axis view at the level of papillary muscle, simultaneously recording M-type scanning, measuring left ventricular Ejection Fraction (EF), Fractional Shortening (FS), left ventricular end diastolic inner diameter (LVID; d) and left ventricular end diastolic wall thickness (LVPW; d) continuously for 3 cardiac cycles.

Referring to the attached drawings 10 and 11, after 7 days of myocardial infarction/transplantation, the heart function of the mice injected with PBS alone completely accords with the typical ultrasonic characteristics of the heart after myocardial infarction, EF and FS are obviously reduced, LVID is obviously reduced, d is obviously increased, and the reconstruction of the ventricle after myocardial infarction is obvious. Each index of the cardiac function of the MSCs-mCherry group is obviously superior to that of the PBS group which is injected singly. The MSCs-Fstl1 group had the best effect of improving cardiac function after myocardial infarction, and was significantly different from the other groups. *P<0.05，**P<0.01，***P<0.001。

2.4 Masson staining to assess myocardial infarction area

Mice were sacrificed 7 days post myocardial infarction and left ventricular tissue was taken for routine tissue sectioning and Masson staining. The areas of the parts were observed and photographed by a normal optical microscope and analyzed by Image analysis software Image J. The myocardial infarction area is calculated according to the following formula:

myocardial infarction area (%) = actual myocardial infarction area/actual heart cross-sectional area;

referring to fig. 12 and fig. 13, the infarct area of each group 7 days after myocardial infarction was observed by Masson staining, and the infarct areas of the MSCs-mCherry group and the MSCs-Fstl1 group were found to be 74.75% and 52.06% of those of the PBS group, respectively; the myocardial infarction area of the MSCs-Fstl1 group is the lowest and is 69.64 percent of that of the MSCs-mCherry group; *P<0.05. Figure 12 shows a representative picture of each set of samples with a 1mm scale.

2.5 mCherry immunofluorescence assessment of MSCs-Fstl1 residency

Mice were sacrificed 1 day after cell transplantation and myocardial infarction, left ventricular tissue was frozen and sectioned, mCherry immunofluorescence staining was performed according to the conventional procedure, FITC-labeled goat anti-rabbit IgG secondary antibody was added, and encapsulated with DAPI-containing anti-fluorescence attenuation encapsulating agent, and mCherry self signal (red), FITC signal (green) and DAPI signal (blue) were simultaneously observed and photographed with a fluorescence microscope.

Referring to fig. 14, the fluorescence of each group was observed under a fluorescence microscope, and the cell retention of the MSCs-Fstl1 group was found to be significantly higher than that of the MSCs-mCherry control group. Photographs show representative pictures from each set of samples, with a 50 μm ruler.

2.6 CM-DiI-labeled MSCs-Fstl1 to assess its residence in hypoxic hearts

Digesting and collecting MSCs-mCherry and MSCs-Fstl1, carrying out heavy suspension on each group of MSCs by CM-DiI dyeing working solution (1 mu g/ml), carrying out incubation for 5min at 37 ℃, carrying out incubation for 15min at 4 ℃, washing for 2 times by PBS, and carrying out myocardial infarction operation and local myocardial injection. The mice were sacrificed 3 days after transplantation, tissues near the injection site were routinely sectioned, and CM-DiI signals were observed in the local cell transplantation area by fluorescence microscopy.

Referring to FIG. 15, the CM-DiI fluorescence of each group was observed directly under a fluorescence microscope, and the CM-DiI signal region of the MSCs-Fstl1 group was found to be significantly higher than that of the MSCs-mCherry control group, indicating that the MSCs-Fstl1 retention in hypoxic myocardium was superior to that of the MSCs-mChery, with a scale of 200 μm.

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

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Claims

Use of Fstl1 in the preparation of a formulation for improving stem cell transplant survival, wherein said stem cells are mouse bone marrow mesenchymal stem cells.
Use of Fstl 1-overexpressing lentivirus for the preparation of a formulation for improving the survival rate of stem cell transplantation, wherein said stem cells are mouse bone marrow mesenchymal stem cells.
3. The application of the stem cell preparation for resisting hypoxia injury in preparing the stem cells with high transplantation survival rate is characterized in that the stem cells are mouse bone marrow mesenchymal stem cells, and the preparation method of the stem cell preparation for resisting hypoxia injury comprises the following steps: mixing the stem cells with Fstl1 overexpression lentivirus solution in a culture medium, and changing the solution after 10-15 hours to obtain an anti-hypoxia injury stem cell preparation; calculating the dosage of Fstl1 overexpression lentivirus fluid according to MOI = 10; selecting 2-3 weeks old male C57BL/6J mice, killing the mice by cervical dislocation, and soaking the mice in 75% alcohol for disinfection; soaking the two lower limbs of the mouse in a DMEM/F12 serum-free basic culture medium under an aseptic condition, and removing muscles on the surfaces of thighbones and shinbones; sucking DMEM/F12 serum-free basal medium added with the cyan/streptomycin by using a syringe and flushing a bone marrow cavity; centrifuging after all bone marrow is flushed out, removing supernatant, adding a fresh special culture medium for mouse bone marrow-derived MSCs (mesenchymal stem cells) to repeatedly blow and beat to obtain a single cell suspension, and inoculating the cell suspension into a culture dish; after 72h, changing the liquid and removing the non-adherent cells, changing the liquid every two days, and carrying out passage when the colonies in the culture dish are mutually fused and the cell density reaches 70%, wherein the passage is marked as P1 generation; after MSCs passed to P5 passages, stem cells were mixed with Fstl 1-overexpressing lentiviral fluid in culture medium to prepare a stem cell preparation resistant to hypoxic injury.
4. Use according to claim 3, wherein the stem cells are mixed with Fstl 1-overexpressing lentiviral fluid in culture medium in the presence of polybrene.
5. The use according to claim 3, wherein the stem cells are mixed with Fstl 1-overexpressing lentiviral fluid in DMEM/F12 medium; the solution was changed after 12 hours.