CN111206018A - Preparation method of mesenchymal stem cells for treating acute liver failure - Google Patents
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Abstract
The invention discloses a preparation method of mesenchymal stem cells for treating acute liver failure, wherein the mesenchymal stem cells with higher purity are successfully separated and cultured by using a full bone marrow wall-attaching method, and are subjected to IL-1 β pretreatment, the IL-1 β pretreatment obviously enhances the expression of CXCR4 of the mesenchymal stem cells during treatment, and further improves the homing capacity of the mesenchymal stem cells to damaged livers, so that the improvement effects of the mesenchymal stem cells on the survival rate, the liver function, the liver necrosis and the hepatocyte apoptosis of rats with the acute liver failure are obviously improved, and the liver reconstruction is further promoted.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method of mesenchymal stem cells for treating acute liver failure.
Background
Acute Liver Failure (ALF) is a clinical syndrome characterized by liver function decompensation, blood coagulation dysfunction, and the like, which is caused by necrosis or dysfunction of a large number of hepatocytes in a short period of time. Although the survival rate of the acute liver failure is obviously improved along with the development of medical diagnosis and treatment technology, the death rate of the acute liver failure is still as high as 40 to 62.2 percent.
Orthotopic liver transplantation is the most effective treatment method for acute liver failure at present, but the wide application of the orthotopic liver transplantation in clinic is severely limited due to donor deficiency, immune rejection and the like. Liver cell transplantation can rapidly play roles of synthesis, detoxification, bilirubin excretion promotion and the like, can partially solve the problem of liver donor shortage, but is difficult to realize clinical breakthrough due to low transplantation survival rate, selective proliferation of transplanted cells and the like. Therefore, the search for a new treatment scheme for acute liver failure is a problem that needs to be solved urgently in the society at present, and the research progress of stem cells in clinical application in recent years provides a new hope for the treatment of acute liver failure.
Mesenchymal Stem Cells (MSCs) are adult stem cells characterized by continuous self-renewal, multipotential differentiation, and low immunogenicity, and have anti-inflammatory, anti-apoptotic, cell proliferation-promoting, and immunoregulatory properties, and thus are widely used in the research and treatment of various diseases including acute liver failure.
In an acute liver failure model caused by acetaminophen, the intravenous application of the mesenchymal stem cells can effectively improve the survival rate of a model mouse, improve the liver function, improve the death of liver cells, promote the liver regeneration and enhance the oxidation resistance of the liver. In addition, the good curative effect of mesenchymal stem cells on acute liver failure is fully proved in an acute liver failure model constructed by applying the concanavalin A, the carbon tetrachloride, the galactosamine and the like. The supporting role of metabolism and functions of various cytokines secreted by the microenvironment dependent disease of the mesenchymal stem cells is the key for exerting the treatment potential, and paracrine is an important mechanism. But the mesenchymal stem cells for intravenous use are mostly confined to the lungs and the number of cells that can be recruited to the site of injury is extremely limited. Therefore, improving the homing ability of mesenchymal stem cells to the damaged site may become an important measure for improving the therapeutic ability thereof.
Before in vivo application, the migration capacity and curative effect of the mesenchymal stem cells can be obviously improved through various pretreatment schemes. Wang et al have demonstrated that the ability to home mesenchymal stem cells to damaged liver and treat acute liver failure can be significantly improved by genetically modifying and expressing c-Met. However, the safety problem of genetic engineering and the uncontrollable concentration of effective components in the serum of liver injury severely limit the clinical application of the pretreatment scheme.
In view of the above problems, there is a need to provide mesenchymal stem cells for treating acute liver failure, which is a technical problem to be solved urgently.
Disclosure of Invention
The invention aims to provide a preparation method of mesenchymal stem cells for treating acute liver failure, and aims to solve the problems in the prior art.
In order to achieve the purpose, the invention provides the following technical scheme:
a preparation method of IL-1 β pretreated mesenchymal stem cells comprises the following steps:
1) separating bone marrow mesenchymal stem cells;
2) subculturing to obtain subculture mesenchymal stem cells;
3) selecting subculture mesenchymal stem cells, and culturing by using a culture medium containing IL-1 β to obtain the mesenchymal stem cells pretreated by IL-1 β.
According to an optimized technical scheme, in the step 3), continuous mesenchymal stem cells are selected and cultured in a culture medium, and when the cell fusion degree reaches 60% -70%, the continuous culture is carried out for 48 hours by replacing the culture medium with an IL-1 β culture medium containing 20ng/mL, so that the mesenchymal stem cells pretreated by IL-1 β are obtained.
According to an optimized technical scheme, during the operation of the step 2), the mesenchymal stem cells obtained by the separation in the step 1) are selected, cultured in a culture medium, subcultured when the cell fusion degree reaches 70%, and cultured for 3-5 generations to obtain the subculture mesenchymal stem cells.
According to the optimized technical scheme, in the step 1), the source of the mesenchymal stem cells is rat bone marrow.
In an optimized technical scheme, in the step 1), the method for separating the mesenchymal stem cells is a full bone marrow adherence method.
In an optimized technical scheme, in the step 1), the specific operation steps are as follows:
a) selecting a rat, carrying out anesthesia and sacrifice, carrying out aseptic separation on the femur of the rat, flushing a marrow cavity by using sterile PBS, collecting and scattering the marrow, and inoculating the marrow into a DMEM/F12 culture medium containing 10% fetal calf serum for culture;
b) and (3) after culturing for 72h, replacing the culture medium, removing nonadherent cells, and replacing the culture medium 1 time every 3 days to obtain the mesenchymal stem cells.
According to an optimized technical scheme, cell identification is carried out after the passage mesenchymal stem cells are obtained in the step 2).
In an optimized technical scheme, in the step 2), the cell identification step comprises the following steps:
a) observing the form and growth characteristics of the passage mesenchymal stem cells through an inverted microscope;
b) selecting passage mesenchymal stem cells, and detecting the expression of positive surface markers CD90 and CD105 and negative markers CD45 and CD34 by adopting a flow cytometer;
c) selecting passage mesenchymal stem cells, inducing the cells into fat and osteogenic differentiation by an in vitro induction method, and identifying the cells by oil red O staining and alizarin red staining respectively after inducing for 2 weeks.
According to an optimized technical scheme, the IL-1 β pretreated mesenchymal stem cells are applied to treatment of acute liver failure.
According to the optimized technical scheme, when the mesenchymal stem cells pretreated by IL-1 β are used for treating acute liver failure, 1 × 10 is injected into the vein7Cells/kg mesenchymal stem cells.
Compared with the prior art, the invention has the beneficial effects that:
acute liver failure is a disease that progresses rapidly and is highly fatal, and there is a lack of effective treatment regimens other than liver transplantation. Mesenchymal stem cells have been widely studied as a possible effective treatment for acute liver failure due to their ready availability, lack of ethical concerns, and metabolic and functional support for inflammatory diseases. Numerous preclinical experiments prove that the application of the mesenchymal stem cells can effectively reduce the death rate of acute liver failure and improve the liver function and the pathophysiological process of the liver.
The invention adopts the whole bone marrow wall pasting method which has the advantages of simplest operation, minimum damage to cells and minimum cell loss, the purity of the separated mesenchymal stem cells is higher, the expression rate of CD90 and CD105 is more than 95 percent, the expression amount of CD45 and CD34 is less than 2 percent, and the invention has better multidirectional differentiation potential and meets the requirements of in vivo experiments.
After the bone marrow mesenchymal stem cells are separated and obtained, the invention also carries out in-vitro subculture, the subculture mesenchymal stem cells are obtained after the culture is carried out to 3 rd to 5 th generations, and then the subculture mesenchymal stem cells are pretreated by IL-1 β, so that the IL-1 β pretreated mesenchymal stem cells disclosed by the invention are obtained, and the cells have higher CXCR4 expression amount than the untreated mesenchymal stem cells, thereby having stronger homing capability towards the damaged liver, and after the homing capability is improved, the survival rate and the liver function of the rats with acute hepatic failure can be more effectively improved, the proliferation of the hepatic cells of the damaged liver can be better promoted, and the apoptosis of the hepatic cells can be inhibited.
The invention discloses a preparation method of mesenchymal stem cells for treating acute liver failure, which has simple step operation and reasonable technical scheme, can effectively improve the curative effect of common mesenchymal stem cells in the treatment of the acute liver failure by IL-1 β pretreatment, provides a new direction and theoretical basis for the treatment of the acute liver failure, and has higher practicability.
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In order that the present invention may be more readily and clearly understood, a more particular description of the invention briefly described above will be rendered by reference to specific embodiments that are illustrated in the appended drawings.
FIG. 1 is a schematic diagram of the microscopic observation of P4 generation cells in the example of the present invention;
FIG. 2 is a graph showing the expression rate of CD90 in P4 generation cells according to the present invention;
FIG. 3 is a graph showing the CD105 expression rate of P4 generation cells in examples of the present invention;
FIG. 4 shows the CD45 expression rate of P4 generation cells in examples of the present invention;
FIG. 5 shows the CD34 expression rate of P4 generation cells in examples of the present invention;
fig. 6 is a schematic diagram illustrating the survival rate results in the survival rate experiments according to the embodiment of the present invention, wherein the model control group in the survival rate experiments is shown as Con group, the blank group is shown as NS, the treatment control group is shown as MSCs, and the treatment experimental group is shown as Pre-MSCs;
FIG. 7 is a schematic diagram of alanine aminotransferase in venous serum in a liver function test experiment according to an embodiment of the present invention;
FIG. 8 is a schematic diagram of aspartate aminotransferase in venous serum in a liver function test experiment according to an embodiment of the present invention;
FIG. 9 is a schematic diagram of total bilirubin in venous blood serum in a liver function test experiment according to an embodiment of the present invention;
FIG. 10 is a schematic diagram of albumin in venous blood in a liver function test experiment according to an embodiment of the present invention;
FIG. 11 is a schematic view of HE staining of a liver specimen of a white blood treatment group in an experiment for liver function detection according to an embodiment of the present invention;
FIG. 12 is a schematic view of HE staining of a liver specimen from a treatment control group in a liver function test experiment according to an embodiment of the present invention;
FIG. 13 is a schematic view of HE staining of liver specimen from a treatment group in a liver function test according to an embodiment of the present invention;
FIG. 14 is a schematic diagram of the statistics of relative necrosis area of a liver specimen in a liver function test according to an embodiment of the present invention; fig. 7-14 are liver function test results, in which the model control group is shown as Con group, the blank group is shown as NS, the treated control group is shown as MSCs, and the treated experimental group is shown as Pre-MSCs;
FIG. 15 is a schematic diagram of the statistics of liver TUNEL positive hepatocytes in an in situ apoptosis assay experiment according to embodiments of the present invention;
FIG. 16 is a diagram showing the statistics of liver Ki67 positive hepatocytes in an immunohistochemical assay of Ki67 according to example of the present invention; FIGS. 15-16 show NS as a blank group, MSCs as a treatment control group, and Pre-MSCs as a treatment experimental group in immunohistochemical and in situ apoptosis detection experiments;
FIG. 17 is a diagram illustrating relative fluorescence intensity detection in a homing experiment according to an embodiment of the present invention; wherein the blank group in the homing experiment is shown as NS, the treatment control group is shown as MSCs, the treatment experimental group is shown as Pre-MSCs, and all cells are marked by CM-Dil;
FIG. 18 is a schematic diagram of CXCR4 and c-Met protein expression in a Western blot detection experiment according to an embodiment of the present invention, wherein a mesenchymal stem cell group pretreated by IL-1 β in the Western blot detection experiment is shown as IL-1 β, and a P4-generation mesenchymal stem cell group is shown as Con group.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example (b):
1. male adult SD rats, 6-8 weeks old, weighing 180-: SCXK (threo) 2016-: SYXK (threo) 2015-.
2. Preparation of IL-1 β pretreated mesenchymal stem cells:
s1: and (3) killing the rats by anesthesia, aseptically separating thighbones of the rats, flushing marrow cavities by sterile PBS, collecting and scattering bone marrow, inoculating the bone marrow into a DMEM/F12 culture medium containing 10% Fetal Bovine Serum (FBS) for culture, replacing the culture medium after 72h to remove non-adherent cells, and replacing the culture medium for 1 time every 3 days to obtain the mesenchymal stem cells.
S2: and selecting bone marrow mesenchymal stem cells, culturing in a culture medium, carrying out subculture when the cell fusion degree reaches 70%, and culturing for 4 generations to obtain fourth-generation mesenchymal stem cells marked as P4.
S3, selecting P4 generation cells, changing the cells into a culture medium additionally containing 20ng/mL IL-1 β when the cell fusion degree reaches 60% -70%, and continuing culturing for 48h to obtain the IL-1 β pretreated mesenchymal stem cells.
3. Detection experiment:
the statistical method of the embodiment comprises the following steps: statistical analysis is carried out by adopting SPSS19.0 software, and data results are all calculated as mean +/-standard deviation
Data comparisons were performed by one-way anova, with two comparisons using LSD-t and 2 data only comparisons between groups using t-test, test level α ═ 0.05 (double-sided)>0.05 represents no significant difference in comparison between the data, P < 0.05 represents statistical difference in comparison between the data, and P < 0.01 and P < 0.001 represent more significant statistical difference.
The following experiments were performed according to this statistical method:
(1) identification of P4 passage cells:
selecting P4 generation cells, and observing morphology and growth characteristics through an inverted microscope;
detecting the expression of positive surface markers CD90 and CD105 and negative markers CD45 and CD34 of the P4 generation cells by using a flow cytometer;
selecting P4 generation cells, inducing adipogenic and osteogenic differentiation by an in vitro induction method, and identifying by oil red O staining and alizarin red staining after inducing for 2 weeks.
In conclusion, ① the cells in the P4 generation showed an increase in volume and a long spindle-shaped, polygonal-like growth when observed under an inverted microscope (see FIG. 1).
② flow cytometry results showed that the expression rate of CD90 and CD105 on the cell surface of P4 generation was greater than 95%, while the expression level of CD45 and CD34 was less than 2% (as shown in FIGS. 2-5).
③ the cells cultured by induced osteogenic differentiation can be seen to be formed by lipid droplets stained by oil red O, and the cells cultured by induced osteogenic differentiation can be seen to be stained positive by alizarin red, which indicates that calcium salt is formed.
Through identification experiments, the P4 generation cells prepared by the embodiment are mesenchymal stem cells with high purity, the surfaces of the stem cells highly express CD90 and CD105, but hardly express CD45 and CD34 representing the immunophenotype of hematopoietic cells, and meanwhile, the mesenchymal stem cells have good multidirectional differentiation potential and meet the requirements of in vivo experiments.
(2) Survival rate experiments:
respectively administering 3ml/Kg vegetable oil solution to 10 rats by single intraperitoneal injection to serve as model control groups;
respectively taking 30 rats, respectively giving 3ml/Kg of carbon tetrachloride vegetable oil solution with volume fraction of 50% to a single intraperitoneal injection to construct a rat acute liver failure model, and randomly averting the rats with the acute liver failure into 3 groups after 6 hours of modeling:
blank group: rats were given 5mL/kg of physiological saline by tail vein injection.
Treatment control group: rats were given a 1X 10 injection into the tail vein7Cells/kg of P4 generation mesenchymal stem cells.
Treatment experimental groups: rats were given a 1X 10 injection into the tail vein7Cells/kg of IL-1 β pretreated mesenchymal stem cells.
The survival of rats was recorded by daily observation after treatment to the end of day 6 and the survival rate of rats in each group was calculated.
And (4) conclusion: after observation and recording, the rats in the model control group have no death phenomenon until the observation is finished, the survival rate of the rats in the treated experimental group is as high as 70 percent, the survival rate of the rats in the treated control group is 40 percent, and the survival rate of the rats in the blank group is only 20 percent (as shown in figure 6).
(3) Liver function test experiment:
respectively administering 2.5ml/Kg vegetable oil solution with volume fraction of 50% to 8 rats by single intraperitoneal injection to serve as model control groups;
respectively taking 24 rats, respectively giving 2.5ml/Kg of carbon tetrachloride vegetable oil solution with volume fraction of 50% to the rats for single intraperitoneal injection, constructing an acute liver failure model of the rats, and randomly averting the acute liver failure rats into 3 groups after 6 hours of modeling:
blank group: rats were given 5mL/kg of physiological saline by tail vein injection.
Treatment control group: rats were given a 1X 10 injection into the tail vein7Cells/kg of P4 generation mesenchymal stem cells.
Treatment experimental groups: rats were given a 1X 10 injection into the tail vein7Cells/kg of IL-1 β pretreated mesenchymal stem cells.
Venous blood was collected by the inner canthus vein blood sampling method 24h and 48h after treatment, and serum alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), Total Bilirubin (TBIL) and Albumin (ALB) were detected using a biochemical autoanalyzer.
Rats were sacrificed under anesthesia 48h post-treatment, liver tissue was retained and fixed in 10% volume fraction formalin for 24h, paraffin embedded and cut into sections of 4 μm thickness for eosin (HE) staining and immunohistochemical analysis. HE staining was observed microscopically, 5 fields were randomly selected for photographing at 100 × magnification, and the ratio of the necrotic area of the liver to the photographed area was analyzed using ImageJ.
① the results of biochemical autoanalyzer test show that each index of the model control group remained normal after 24h treatment, ALT, AST, TBIL and ALB of the treatment experimental group were 141.71 + -34.98U/L, 556.14 + -97.32U/L, 3.14 + -0.54 μmol/L and 26.11 + -0.58 g/L, respectively, ALT, AST, TBIL and ALB of the treatment control group were 233.43 + -2.30U/L, 1049.86 + -150.35U/L, 4.67 + -1.28 μmol/L and 26.48 + -1.03 g/L (P < 0.05).
After 48 hours of treatment, all indexes of the model control group are kept normal, and ALT, AST, TBIL and ALB of the treatment experiment groups are respectively 98.29 +/-12.24U/L, 376.14 +/-60.09U/L, 1.37 +/-0.37 mu mol/L and 26.31 +/-0.76 g/L. ALT, AST, TBIL and ALB in the treatment control group are 176.67 + -17.61U/L, 713.83 + -91.52U/L, 1.9 + -0.32 mu mol/L and 24.88 + -0.69 g/L respectively (P is less than 0.01).
The detection results are shown in the attached figures 7-10 of the specification.
② HE staining results show that large-area hepatic tissue necrosis regions are obviously visible in the blank group, the hepatic tissue necrosis conditions of the treatment experimental group and the treatment control group are obviously improved, and the hepatic tissue necrosis conditions of the treatment experimental group are the lightest.
Statistics show that the relative necrosis area of the liver tissue in the treatment control group is 23.72% + -2.88%; the relative necrotic area of liver tissue in the blank group was 28.95% + -3.00%, while the relative necrotic area of liver tissue in the treatment experiment group was 17.64% + -3.07%, which was significantly lower than that in the other two groups (P < 0.01). (as shown in FIGS. 11-14)
According to the survival rate, liver function and liver pathology detection results, the mesenchymal stem cells treated by the IL-1 β can improve the liver function of a rat more obviously, improve the survival rate of the rat and reduce the necrosis of the liver.
(4) Immunohistochemical and in situ apoptosis detection experiments:
taking three groups of liver tissues (blank group, treatment control group and treatment experiment group) reserved in the liver function detection experiment in step (3), and respectively carrying out the following detection:
ki67 antibodies are respectively selected as primary antibodies, and an antigen retrieval method is applied to immunohistochemical staining. In situ apoptosis detection was accomplished using a commercial TUNEL apoptosis kit with reference to the instructions. After staining was complete, the cells were observed microscopically, photographed at 200 × magnification in 5 fields at random, and the number of positive stained cells was counted using Image J.
① TUNEL staining picture and positive cell number analysis show that the number of apoptotic hepatocytes in the blank group is 765.00 + -86.97, the other two groups (treatment control group and treatment experimental group) have significantly reduced TUNEL positive cell number (P < 0.001), and the treatment experimental group has the best therapeutic effect, wherein the number of apoptotic hepatocytes in the treatment experimental group is 225.4 + -51.03, and the number of apoptotic hepatocytes in the treatment control group is 355.4 + -46.01. (P < 0.01)
② Ki67 staining results in completely opposite trend to TUNEL, while the proliferation cell numbers of blank group, treated experimental group and treated control group are 139.4 + -17.18, 189.2 + -29.99 and 391.8 + -46.58 respectively, wherein the proliferation effect of liver cells of the treated experimental group is strongest.
The detection results show that the mesenchymal stem cells pretreated by the IL-1 β can more obviously inhibit the apoptosis of the liver cells and promote the proliferation of the liver cells, and the curative effect is better than that of the mesenchymal stem cells which are not pretreated.
The above detection results are shown in FIGS. 15 to 16.
(5) Cell labeling and homing experiments:
① cell labeling experiment, selecting P4 generation cells, when the fusion degree of the P4 generation cells reaches about 90%, trypsinizing the cells, applying 3 mu M CM-Dil-containing PBS solution to resuspend the cells, incubating for 5min at 37 ℃, then incubating for 15min at 4 ℃, centrifuging, discarding supernatant, washing with PBS for 3 times, finally resuspending the cells with NS and counting to prepare 2 x 106 cells/mL cell solution, adding a small amount of cell solution into a culture medium containing 10% PBS, continuing culturing in a 37 ℃ constant temperature incubator, observing and photographing by using a fluorescence microscope after the cells are stably attached to the wall, and analyzing the labeling rate of the cells by comparing with white light images.
② homing experiment, respectively taking 24 rats, respectively giving 2.5ml/Kg of carbon tetrachloride vegetable oil solution with volume fraction of 50% to the rats for single intraperitoneal injection, constructing an acute liver failure model of the rats, and randomly averting the acute liver failure rats into 3 groups after 6 hours of modeling:
blank group: rats were given 5mL/kg of physiological saline by tail vein injection.
Treatment control group: rats were given a 1X 10 injection into the tail vein7CM-Dil labeled P4 generation mesenchymal stem cells per kg of cells.
Treatment experimental groups: rats were given a 1X 10 injection into the tail vein7Cells/kg of CM-Dil labeled mesenchymal stem cells after IL-1 β medium culture.
After 24h of treatment, the rats were sacrificed by anesthesia, liver tissues were kept frozen at-80 ℃, then sectioned by a cryomicrotome with a thickness of 4 μm, the distribution of labeled cells in the liver tissues was observed under a fluorescence microscope, 5 fields were randomly selected for photographing under a magnification of 200 × and the fluorescence signal density of each group was analyzed by Image J.
In conclusion, ① the cells are marked by using CM-Dil in the cell marking experiment, almost all the cells can be positively marked, and the marking has no obvious influence on the cell morphology.
② in the homing experiment, fluorescence of the frozen sections of rat liver was observed by fluorescence microscope after 24h and analyzed for relative optical density using Image J, which showed no red fluorescence in the blank group, the most significant fluorescence in the treated experimental group, relative fluorescence density of 1.88% + -0.42%, and relative fluorescence density of 0.57% + -0.23% (P < 0.001) in the treated control group (see FIG. 17).
The detection results show that the pretreatment of IL-1 β can remarkably promote the homing capability of mesenchymal stem cells to damaged liver, and further promote the proliferation of liver cells so as to improve the liver function of rats.
(6) Western blot detection:
and (3) collecting the total cell protein of the mesenchymal stem cells and the total cell protein of the mesenchymal stem cells of generation P4 after being pretreated for 48 hours by using 20ng/mL of IL-1 β according to the BCA kit instruction, carrying out Western blot detection by using CXCR4, c-Met and internal reference antibody Tubulin as primary antibodies and corresponding secondary antibodies, and carrying out data analysis on the grey value of protein expression.
The conclusion is that the detection result shows that the expression level of CXCR4 of the mesenchymal stem cells after the IL-1 β pretreatment for 48h is obviously improved, the gray value of CXCR4 is 2.46 times (P is less than 0.001) of the gray value of CXCR4 of the mesenchymal stem cells of generation P4, and the expression of c-Met in the two cells has no significant difference (P is more than 0.5) (as shown in figure 18).
In conclusion, the mesenchymal stem cells with higher purity are successfully separated and cultured by using a full bone marrow wall pasting method, and are subjected to IL-1 β pretreatment, the IL-1 β pretreatment obviously enhances the expression of CXCR4 of the mesenchymal stem cells during treatment, and further improves the homing capacity of the mesenchymal stem cells to damaged livers, so that the improvement effect of the mesenchymal stem cells on the survival rate, the liver function, the hepatic necrosis and the hepatic apoptosis of rats with acute hepatic failure is obviously improved, the hepatic reconstruction is further promoted, and the mesenchymal stem cells obtained by the preparation method have excellent curative effect during the treatment of the acute hepatic failure.
The research proves that the curative effect of the mesenchymal stem cells in the treatment of the acute liver failure can be obviously enhanced by the IL-1 β pretreatment for the first time through experiments, and the enhancement of the curative effect is at least partially caused by that the expression of CXCR4 of the mesenchymal stem cells is improved by the IL-1 β pretreatment, and the homing capability of the mesenchymal stem cells to the damaged liver is further improved.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Claims (10)
1. A preparation method of mesenchymal stem cells for treating acute liver failure is characterized by comprising the following steps: the method comprises the following steps:
1) separating bone marrow mesenchymal stem cells;
2) subculturing to obtain subculture mesenchymal stem cells;
3) selecting subculture mesenchymal stem cells, and culturing by using a culture medium containing IL-1 β to obtain the mesenchymal stem cells pretreated by IL-1 β.
2. The method for preparing the mesenchymal stem cell for treating the acute liver failure according to claim 1, wherein in the step 3), the continuous mesenchymal stem cell is selected and cultured in a culture medium, and the mesenchymal stem cell is continuously cultured for 48h by replacing the culture medium containing 20ng/mL of IL-1 β when the cell fusion degree reaches 60% -70%, so as to obtain the mesenchymal stem cell pretreated by IL-1 β.
3. The method for preparing mesenchymal stem cells for treating acute liver failure according to claim 1, wherein the mesenchymal stem cells are prepared by the following steps: and 2) during operation, selecting the mesenchymal stem cells separated in the step 1), culturing in a culture medium, carrying out subculture when the cell fusion degree reaches 70%, and culturing for 3-5 generations to obtain the subculture mesenchymal stem cells.
4. The method for preparing mesenchymal stem cells for treating acute liver failure according to claim 1, wherein the mesenchymal stem cells are prepared by the following steps: in the step 1), the source of the mesenchymal stem cells is rat bone marrow.
5. The method for preparing mesenchymal stem cells for treating acute liver failure according to claim 4, wherein the mesenchymal stem cells are prepared by the following steps: in the step 1), the method for separating the mesenchymal stem cells is a full bone marrow adherence method.
6. The method for preparing mesenchymal stem cells for treating acute liver failure according to claim 5, wherein the mesenchymal stem cells are prepared by the following steps: in the step 1), the specific operation steps are as follows:
a) selecting a rat, carrying out anesthesia and sacrifice, carrying out aseptic separation on the femur of the rat, flushing a marrow cavity by using sterile PBS, collecting and scattering the marrow, and inoculating the marrow into a DMEM/F12 culture medium containing 10% fetal calf serum for culture;
b) and (3) after culturing for 72h, replacing the culture medium, removing nonadherent cells, and replacing the culture medium 1 time every 3 days to obtain the mesenchymal stem cells.
7. The method for preparing mesenchymal stem cells for treating acute liver failure according to claim 1, wherein the mesenchymal stem cells are prepared by the following steps: and 2) performing cell identification after the passage mesenchymal stem cells are obtained.
8. The method for preparing mesenchymal stem cells for treating acute liver failure according to claim 7, wherein the mesenchymal stem cells are prepared by the following steps: in step 2), the cell identification step comprises:
a) observing the form and growth characteristics of the passage mesenchymal stem cells through an inverted microscope;
b) selecting passage mesenchymal stem cells, and detecting the expression of positive surface markers CD90 and CD105 and negative markers CD45 and CD34 by adopting a flow cytometer;
c) selecting passage mesenchymal stem cells, inducing the cells into fat and osteogenic differentiation by an in vitro induction method, and identifying the cells by oil red O staining and alizarin red staining respectively after inducing for 2 weeks.
9. The use of IL-1 β pretreated mesenchymal stem cells according to any one of claims 1-8, characterized in that the IL-1 β pretreated mesenchymal stem cells are used in the treatment of acute liver failure.
10. The use of IL-1 β pretreated mesenchymal stem cells according to claim 9, wherein the IL-1 β pretreated mesenchymal stem cells are administered intravenously as a 1X 10 injection for the treatment of acute liver failure7Cells/kg mesenchymal stem cells.
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