CN108546675A - Stem cell is promoted to be divided into the preparation method of the extracellular matrix of liver cell - Google Patents
Stem cell is promoted to be divided into the preparation method of the extracellular matrix of liver cell Download PDFInfo
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Abstract
The embodiment of the present invention provides the preparation method for the extracellular matrix for promoting stem cell to be divided into liver cell, it is related to biomedical sector, make the extracellular matrix of preparation that there is the advantages of adjusting mesenchymal stem cell microenvironment, inducing bone mesenchymal stem cell breaks up to mature hepatocytes.It can be used for studying hepatopathy mechanism screening drug in scientific research and prepare liver cell before clinical hepatocyte transplantation treats hepatopathy.It is described to promote the preparation method of the stem cell extracellular matrix that is divided into liver cell to include：Step 1：Prepare porcine hepatocyte epimatrix；Step 2：The separation and culture of autologous patient mesenchymal stem cell；Step 3：The mesenchymal stem cells differentiation of external 3D cultures is liver cell；Step 4：The functional evaluation of liver cell.
Description
Technical field
The present invention relates to biomedical sectors, more particularly to the extracellular matrix for promoting stem cell to be divided into liver cell
Preparation method.
Background technology
There are about 80 ten thousand to one hundred two ten ten thousand people to die of hepatopathy in the world every year, accounts for about total dead person-time of 1.3%-2%.In
Severely afflicated area of the state as hepatopathy high infectious rate occurred frequently, it is an extremely urgent times to provide cost-effective means treatment hepatopathy
Business.Hepatopathy according to the variation of pathogenesis and liver function can be divided into the liver fibrosis of mid-term early period and the hepatic sclerosis in latter stage and
Liver decompensation caused by whole latter stage, by taking hepatitis B as an example, early period, mid-term even latter stage antiviral drugs can pass through suppressing virus replication
Or hepatopathy is effectively relieved.However since liver autosynthesis ability is strong, prehepatic mid-term phenotype unobvious, once it finds as latter stage
It is later in the majority.
Healthy liver is entered hepatic failure in patient body by liver transplant by surgical graft, is that clinic is controlled all the time
Treat the main or even unique effective means of End-stage liver disease.However limited healthy liver supply, expensive surgery cost, limit
The application of liver transplant is made.And liver transplant can not thoroughly cure patient, lifelong serious immunological rejection and patient
Various complication after immunosupress etc. factor makes the service life of the average 5 years longests to 10 to 35 years of postoperative patient extension.
It is at present hepatocyte transplantation grinding the therapy for being expected to substitute liver transplant.Utilize autologous stem cells or immunogenicity
If weak Derived from Mesenchymal Stem Cells becomes liver cell, immunological rejection after transplanting can be solved the problems, such as.Hepatocyte transplantation
Key limiting link is to obtain the ripe cell with liver function in vitro.Mescenchymal stem cell is each in adult human body
All existing stem cell, such as fat mesenchymal stem cell, mesenchymal stem cell etc. in histoorgan.Due to there is no ethics to strive
View, derives from a wealth of sources, and is easy to obtain, and immunogenicity is extremely low, is the optimal selection of vitro differentiation liver cell.Nowadays promote mesenchyma
The method of stem cell differentiation and maturation liver cell is mostly to add the cell factor of a kind of or various high concentrations, and such as most common liver is thin
The intracellular growth factor（HGF）.Since cell factor is expensive, and this method differentiated hepatocellular is inefficient, differentiates
Cellular metabolic activity is weak compared with normal liver cell, directly limits the development and application of hepatocyte transplantation therapy.
As that studies cell differentiation gos deep into, it has been found that the extracellular matrix in hepatic tissue source can be largely
Upper promotion liver differentiation.Collagen on extracellular matrix etc. can be responded as signal and activate be attached to it is thin on extracellular matrix
Receptor on after birth divides to which the signal path in active cell maintains Cell Homeostasis that cell is promoted to grow when needed
Change.Extracellular matrix that is after the de- cell of tissue and carrying out frozen dried eliminates immunogenicity, and it is attached mainly to leave collagenous fibres
Cell factor complex, has not needed to additionally add cell factor.Therefore using basic non-immunogenicity after de- cell
Extracellular matrix, can replace directly addition cell factor method come promote Derived from Mesenchymal Stem Cells be liver cell.
Chinese patent CN201510937399.0 discloses a kind of training that liver cell vitro differentiation phenotype and function can be improved
The method of supporting.Liver cell, liver source property interstitial cell and extracellular matrix has been used to be inoculated on porous silk albumen holder and simulate
Hepatocytes culture in vetro microenvironment is cultivated.The invention significantly improves the hepatocyte function that differentiation is got, and can be used for commenting
Influence and hepatotoxicity wind agitation detection of the valence drug to hepatic metabolism enzyme.However liver cell has sound surface antigen expression, liver source property
The separation of mesenchymal cell will obtain people source in addition compared with the mescenchymal stem cell in other sources and more cumbersome and negligible amounts
Hepatic tissue cell need to carry out major trauma operation, therefore, this method for improving liver cell vitro differentiation phenotype and function is not
The hepatopathy of people is treated suitable for hepatocyte transplantation, and preferably makees in vitro study purposes.Chinese patent CN201310203436.6
It has invented a kind of mesenchymal stem cell autocrine extracellular matrix and it is induced to become the method for liver cell.Using by marrow
Matrix that is secreted by mescenchymal stem cell and eliminating mesenchymal stem cell, induced efficiency is high, and differentiation effect is good, lures
The Hepatocyte Glycogen synthesis capability and urea synthesizing ability led are strong.However mesenchymal stem cell autocrine obtain it is thin
The amount that the amount of extracellular matrix more directly takes off extracellular matrix prepared by cell method with model animal tissue will lack several orders of magnitude, and
Process is relatively complicated, and time-consuming takes cell, needs first through mesenchymal stem cell culture extracellular matrix secretion, then detach cell
Epimatrix, then inoculation mesenchymal stem cell induces its differentiation to become liver cell again.Therefore, expense relatively using cell because
Sub- inducing hepatocyte differentiation is more expensive, is unfavorable for large-scale production and application.
Liver is maximum internal organs in human body, is located on the right side of human body epigastrium under diaphragm, on the right of gall-bladder front end
In front of kidney.Facies superior hepatis is divided into two leaves of left and right by ligament, and it is vena portae hepatica, arteria hepatica, common hepatic duct, nerve and lymph to have traverse furrow below
The place that pipe comes in and goes out.Hepatic tissue forms basic unit by countless lobuli hepatis, while receiving to come from hepatic arterial blood and portal vein
In come from gastral blood.Liver carries decomposition of glycogen synthesis storage, secretes blood plasma egg as extremely active body of gland is metabolized
In vain, urea is secreted, bile absorbs and the waste in degradation blood circulation and the vital effect such as toxin.Cell packet in liver
Most of liver parenchymal cells are included, liver non-parenchymal cell such as responds Kupffer cell, that is, sternzellen of inflammatory signals, keeps liver
The hepatic sinusoidal endothelial cells of blood sinus mass exchange permeability, necessity are to promote regenerated liver mesenchyma stem cell of liver various types of cells etc.
Deng.
Human body contains the manganese of about 12mg, most of in bone, liver and kidney.Manganese ion be human development metabolism and
Ingredient necessary to antioxidant system needs confactor of the manganese ion as enzyme reaction（Coenzyme）Enzyme have oxidoreducing enzyme man
Race shifts enzyme family, hydrolase family, topological enzyme family, connection enzyme family etc..Manganese element is also used as compound form（I.e.
Manganese sulfate）It is present in eucaryote mitochondria, detoxifies when generating peroxide on hydrogen ion transfer chain as aerobe
Neccessary composition.Our research, which demonstrates suitable Mn ions, can help the medulla mesenchyma being attached on extracellular matrix
The efficiency of enzyme needed for expansion of stem cells and differentiation simultaneously reduces injury of the peroxide for cell in metabolic process, to promote liver
The differentiation of cell simultaneously maintains good cell micro-environment.
In conclusion cost dearly since existing hepatocyte differentiation method and step is cumbersome, and cannot be in clinical application
Angle goes the problem of figuring out how to obtain the ripe available liver cell of no immunological rejection, therefore, develops a step and succinctly uses
Material is economical, and the method conducive to the liver cell of the treatment hepatopathy of Clinical practice is very necessary.
Invention content
An embodiment of the present invention provides the preparation methods that promotion stem cell is divided into the extracellular matrix of liver cell, make
The extracellular matrix of preparation, which has, adjusts mesenchymal stem cell microenvironment, and inducing bone mesenchymal stem cell is thin to ripe liver
The advantages of born of the same parents break up.It can be used for studying hepatopathy mechanism screening drug in scientific research and before clinical hepatocyte transplantation treats hepatopathy
Prepare liver cell.
In order to achieve the above objectives, the embodiment of the present invention adopts the following technical scheme that：
The embodiment of the present invention provides a kind of preparation method for the extracellular matrix for promoting stem cell to be divided into liver cell, described
The preparation method of the stem cell extracellular matrix that is divided into liver cell is promoted to include：
Step 1：Prepare porcine hepatocyte epimatrix；
Step 2：The separation and culture of autologous patient mesenchymal stem cell；
Step 3：The mesenchymal stem cells differentiation of external 3D cultures is liver cell；
Step 4：The functional evaluation of liver cell.
Further, the step 1, specifically includes：
Complete fresh pig liver is obtained to leave behind vena cave and open as outlet, arteria hepatica and hepatic duct ligation by conduit
After being inserted into portal vein, peristaltic pump (model such as Millipore, Billerica, MA, USA are used immediately）With 100mL/min's
The sterile PBS solution perfusion pork liver that 20L contains 5000IU/L heparin is removed blood in liver by rate；
0.01% lauryl sodium sulfate being gradually pre-chilled in sequence with 150L at 4 DEG C（SDS）Solution, 150L precoolings
0.1% SDS solution, the Triton X-100 perfusion pork livers that the 1% SDS solution and 50L 1% of 50L precoolings are pre-chilled are thin to make it take off
Born of the same parents have finally taken off the liver of cell using the perfusion cleaning of 100L distilled water, and remaining surfactant and cell are removed with this
Residual；
Graininess will be worn into using rotating blade grinder after pork liver progress lyophilized processing after de- cell, the powder that will be obtained
End is resuspended with the concentration of 50mg/L in sterile PBS solution, is used in combination Syrup-homogenizing instrument mixing to form porcine hepatocyte epimatrix emulsion, is made
With 60 Co gamma radiation（15kGy）To porcine hepatocyte epimatrix emulsion carry out sterilization treatment, be stored in after sterilizing 4 DEG C with
Standby rear use.
Further, the step 2, specifically includes：
According to the jawbone bone chip clast that the method for orthognatic surgery or implant operation takes patient disease-free, it is placed in α-MEM cultures
4 DEG C of preservations, are cleaned three times with sterile PBS solution in liquid, and 800rpm centrifuges 5min and obtains cell precipitation and bone chip under room temperature；With
Cell precipitation and bone chip mixture are gently blown and beaten mixing by the α-MEM culture solutions containing 10% fetal calf serum, are inoculated into the training of six holes
It supports in plate.At 37 DEG C, 5% CO2It is cultivated 3-7 days under the conditions of saturated humidity, until there is cell to be climbed out of from organization edge.Cell is close
When degree is up to 80%, 2.5x10 is arrived in 0.25% pancreatin passage digestion, gradually amplification5。
Further, the step 3, specifically includes：
By 2.5x105The digestion centrifugation of Bone Marrow of Patients mescenchymal stem cell is resuspended in the outer base of ready porcine hepatocyte before 200 μ L
It in matter emulsion and a Collagen Type VI isodensity mixed liquor, is seeded in 24 hole plates, 50 μM of Mn is added2+, at 37 DEG C, 5% CO2
So that colloid is solidified with 30min is cultivated under the conditions of saturated humidity, adds the α-MEM that 500 μ L contain 10% fetal calf serum to arrive each hole, often
A culture solution is changed within two days, sampling progress paraffin fixed test glycogen after culture 2 weeks（PAS is dyed）, seralbumin（It is immune glimmering
Light）Equal liver function indexs.
Further, the step 4, specifically includes：
The human marrow mesenchymal stem cell and pig source liver cell epimatrix mixture for taking step 3 culture are thoroughly ground by liquid nitrogen,
The rna expression that RNA carries out reverse transcription and quantitative fluorescent PCR verifies liver cell marker gene is extracted according to TRIZOL specifications, separately
The outer human marrow mesenchymal stem cell and pig source liver cell epimatrix mixture that step 3 culture is taken according to protein cleavage specification,
After being cleaned three times with sterile PBS solution, protein lysate is added and is used as Western blot detections, verifies the liver cell mark of differentiation
Will protein expression.
It is verified in zoopery：It is generally acknowledged using the hepatic fibrosis animal model of tetrachloro-methane induction
Screening treatment hepatopathy caused by fibrosis medicine experiment made on the living model.Article is published in Journal of Tissue
Engineering Regeneration Medicine(Doi:10.1002/term.2161.) its concrete operation step is：Often
All SD rats by intraperitoneal injection 1mL/kg weight for 4 weeks twice to weighing about 250 ± 20g, rapeseed oil is dissolved in 60% concentration
In carbon tetrachloride solution, carry out induced rat liver fibrosis.After the injection of last time carbon tetrachloride finishes for 24 hours, by fibrosis
Rat is divided into control group, mesenchymal stem cell group, manganese ion activated mesenchymal stem cell group.Control group is only
The rat of the sterile PBS solutions of 0.5mL is injected, it is solution that mesenchymal stem cell group, which is with the sterile PBS solutions of 0.5mL, is passed through
Introportal infusion 3 × 106The rat of mesenchymal stem cell, manganese ion activated mesenchymal stem cell group are 50 μ
M manganese ions processing 3 × 106It is dissolved in after mesenchymal stem cell 16h in the sterile PBS solutions of 0.5mL and passes through introportal infusion
Rat.Every group contains at least 10 rats.Serology, histopathology, the inspection of RNA and albumen are carried out after treatment four weeks
It surveys.
It is verified in cell experiment：It objectively has detected a different Collagen Type VIs and extracellular matrix emulsion is mixed
Composition and division in a proportion breaks up inducing bone mesenchymal stem cell the influence of hepatoblast, and the selection for the cell treatment process of optimization provides
Theoretical foundation.Its concrete operation step is：After control group, mesenchymal stem cell group cell are collected, with sterile PBS solution
Cleaning three times, 1ml Trizol lysates is added per hole, and sample is received in no enzyme 1.5ml EP pipes after standing 5min.According to Trizol
Kits A cell sample RNA extraction steps, carry out the extracting of RNA.After the completion of extracting, RNA concentration and the survey of A260/A280 are carried out
It is fixed.It is inverse that RNA is carried out according to the specification of PrimeScript 1st Strand cDNA Synthesis kit Reverse Transcriptase kits
Turn, and using cDNA as template RT-PCR testing goal gene expressions.
The preparation method of extracellular matrix provided by the present invention, it is advantageous that：
1, the method can obtain mesenchymal stem cell with from patient itself jawbone, then the pig being lyophilized after being handled with de- cell
Derived cell epimatrix is added appropriate manganese ion mixed culture and induces differentiation into acquaintance liver cell, thoroughly solves cell transplantation and exempt from
The problem of epidemic disease is repelled.
2, the method extracellular matrix of drawing materials from miniature pig facilitates economy, later stage Fiber differentiation need not add cell because
Son further reduces costs, easy to utilize, both can be used for breaking up as scientific research and has screened the cell of liver disease drug,
The hepatocyte origin of hepatopathy can be treated as clinical hepatocyte transplantation.
Description of the drawings
Fig. 1 is the external pig source liver cell matrix hepatoblast testing result of the present invention；
Fig. 2 is that mesenchymal stem cell manganese ion activated in the present invention is treated from introportal infusion by tetrachloro-methane induction
Rat acute hepatic failure design sketch.
Specific implementation mode
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention is retouched in detail
It states.
Embodiment 1
Step 1：Prepare porcine hepatocyte epimatrix
The adult miniature swine that weight is 60kg to 80kg is anaesthetized, complete fresh liver is obtained.By arteria hepatica and hepatic duct knot
It pricks, leaves behind vena cave and open as outlet.After inserting the catheter into portal vein, using peristaltic pump immediately, (model is such as
Millipore, Billerica, MA,USA）20L is contained to the sterile PBS of 5000IU/L heparin with the rate of 100mL/min
Infusion pork liver removes blood in liver.
0.01% lauryl sodium sulfate being gradually pre-chilled successively with 150L at 4 DEG C in sequence（SDS）Solution, 150L
Pork liver is perfused to make in 0.1% SDS solution of precooling, the Triton X-100 that the 1%SDS solution and 50L 1% of 50L precoolings are pre-chilled
Its de- cell.Finally, the liver of cell has been taken off using the perfusion cleaning of 100L distilled water, remaining surfactant is removed with this
And cell residue.It such as needs to carry out quality testing, after the completion of de- cell step, fritter can be taken off to the liver after cell at 4 DEG C
Leaf sample is fixed overnight with 4% paraformaldehyde and carries out group change detection de- a cell effect and cytokine analysis etc..
In order to produce gluey porcine hepatocyte epimatrix, rotation is used after the pork liver after de- cell is carried out lyophilized processing
Blade grinder wears into graininess.Obtained powder is resuspended with the concentration of 50mg/L in sterile PBS solution, Syrup-homogenizing instrument is used in combination
Mixing forms porcine hepatocyte epimatrix emulsion.It is radiated using 60 Co gamma（15kGy）To porcine hepatocyte epimatrix emulsion
Carry out sterilization treatment.4 DEG C are stored in after sterilizing for future use.In following steps unless specifically indicated, the otherwise outer base of porcine hepatocyte
Matter emulsion is all to be mixed with a Collagen Type VI equal portions to form the gluey holder cultivated for 3D.
Step 2：The separation and culture of autologous patient mesenchymal stem cell
According to the jawbone bone chip clast that the method for orthognatic surgery or implant operation takes patient disease-free, it is placed in α-MEM cultures
4 DEG C of preservations, are cleaned three times with sterile PBS solution in liquid, and 800rpm centrifuges 5min and obtains cell precipitation and bone chip under room temperature.With
Cell precipitation and bone chip mixture are gently blown and beaten mixing by the α-MEM culture solutions containing 10% fetal calf serum, are inoculated into the training of six holes
It supports in plate.At 37 DEG C, 5% CO2It is cultivated 3-7 days under the conditions of saturated humidity, until there is cell to be climbed out of from organization edge.Cell is close
When degree is up to 80%, 2.5x10 is arrived in 0.25% pancreatin passage digestion, gradually amplification5(needing about 3-5 times passage).
Step 3：The mesenchymal stem cells differentiation of external 3D cultures is liver cell
By 2.5x105The digestion centrifugation of Bone Marrow of Patients mescenchymal stem cell is resuspended in the outer base of ready porcine hepatocyte before 200 μ L
It in matter emulsion and a Collagen Type VI isodensity mixed liquor, is seeded in 24 hole plates, 50 μM of Mn is added2+, at 37 DEG C, 5% CO2
Colloid is set to solidify with 30min is cultivated under the conditions of saturated humidity.α-the MEM that 500 μ L contain 10% fetal calf serum are added to arrive each hole, often
Change within two days a culture solution.It can sample after cultivating 2 weeks and carry out paraffin fixed test glycogen（PAS is dyed）, seralbumin（Exempt from
Epidemic disease fluorescence）Equal liver function indexs.
Step 4：The functional evaluation of liver cell
In order to detect the liver cell of differentiation, take the human marrow mesenchymal stem cell of step 3 culture and pig source liver cell epimatrix mixed
It closes object thoroughly to grind by liquid nitrogen, extracting RNA according to TRIZOL specifications carries out reverse transcription and quantitative fluorescent PCR verification liver cell
The rna expression of marker gene.Human marrow mesenchymal stem cell and the pig of step 3 culture are taken according further to protein cleavage specification
Source liver cell epimatrix mixture after being cleaned three times with sterile PBS solution, is added protein lysate and is used as Western blot inspections
It surveys, verifies the liver cell marker protein expression of differentiation.
It is verified in zoopery：It is generally acknowledged using the hepatic fibrosis animal model of tetrachloro-methane induction
Screening treatment hepatopathy caused by fibrosis medicine experiment made on the living model.Article is published in Journal of Tissue
Engineering Regeneration Medicine(Doi:10.1002/term.2161.) its concrete operation step is：Often
All SD rats by intraperitoneal injection 1mL/kg weight for 4 weeks twice to weighing about 250 ± 20g, rapeseed oil is dissolved in 60% concentration
In carbon tetrachloride solution, carry out induced rat liver fibrosis.After the injection of last time carbon tetrachloride finishes for 24 hours, by fibrosis
Rat is divided into control group, mesenchymal stem cell group, manganese ion activated mesenchymal stem cell group.Control group is only
The rat of the sterile PBS solutions of 0.5mL is injected, it is solution that mesenchymal stem cell group, which is with the sterile PBS solutions of 0.5mL, is passed through
Introportal infusion 3 × 106The rat of mesenchymal stem cell, manganese ion activated mesenchymal stem cell group are 50 μ
M manganese ions processing 3 × 106It is dissolved in after mesenchymal stem cell 16h in the sterile PBS solutions of 0.5mL and passes through introportal infusion
Rat.Every group contains at least 10 rats.Serology, histopathology, the inspection of RNA and albumen are carried out after treatment four weeks
It surveys.
It is verified in cell experiment：It objectively has detected a different Collagen Type VIs and extracellular matrix emulsion is mixed
Composition and division in a proportion breaks up inducing bone mesenchymal stem cell the influence of hepatoblast, and the selection for the cell treatment process of optimization provides
Theoretical foundation.Its concrete operation step is：After control group, mesenchymal stem cell group cell are collected, with sterile PBS solution
Cleaning three times, 1ml Trizol lysates is added per hole, and sample is received in no enzyme 1.5ml EP pipes after standing 5min.According to Trizol
Kits A cell sample RNA extraction steps, carry out the extracting of RNA.After the completion of extracting, RNA concentration and the survey of A260/A280 are carried out
It is fixed.It is inverse that RNA is carried out according to the specification of PrimeScript 1st Strand cDNA Synthesis kit Reverse Transcriptase kits
Turn, and using cDNA as template RT-PCR testing goal gene expressions.
As shown in Figure 1, for the external pig source liver cell matrix hepatoblast testing result of the present invention；
Figure 1A：It is with or without the three dimensional gel shape supporting structure overall diagram of pig source liver cell matrix.Figure 1B：Increased by MTT cells
Value detection illustrates that Apoptosis L929 is cultivated from the leaching liquor obtained in L-ECM, competence for added value enhancing.Figure
1C,D：To having（Fig. 1 C）Or do not have（Fig. 1 D）The mesenchymal stem cell of the dimensional culture of pig source cell epimatrix carries out PAS
Glycogen synthesis situation is checked in dyeing.Black arrow is directed toward glycogen storage point.Visible dimensional culture is thin for having after 14 days in figure
The cell of extracellular matrix addition, glycogen can be detected.And for the cell of acellular epimatrix addition, glycogen
It can not be detected.Fig. 1 E, F：Immuno-fluorescence assay is having（E）Extracellular matrix is added, or only collagen does not have cell
Epimatrix（F）Carry out the seralbumin of the hepatocytes secrete broken up in the cell of dimensional culture.Seralbumin quilt in figure
White arrow is marked.The study show that addition extracellular matrix is for promoting mesenchymal stem cell to liver cell
Differentiation plays conclusive effect, and liver cell obtained by differentiation has glycogen storage and secretes sero-abluminous effect.
As shown in Fig. 2, being treated from introportal infusion by four for mesenchymal stem cell manganese ion activated in the present invention
The rat acute hepatic failure design sketch of chlorination carbon induction；It is injected intraperitoneally after 60% carbon tetrachloride 1mL/kg rat body weights 24 hours, by
Introportal infusion passes through or without manganese ion activated mesenchymal stem cell.By rat after 7 days（n=10）Materials detection.Figure
2A, 2B, 2C, 2D are sterile PBS solution group respectively from top to bottom, normal bone marrow mescenchymal stem cell group, and are swashed by manganese ion
The mesenchymal stem cell group lived.Fig. 2A：Use 7 days after the above-mentioned three kinds of solution of rat acute hepatic failure mold injection livers
Overall diagram.Scale indicates the length of 5mm in figure.Fig. 2 B, 2C：Hepatolith wax fix after H＆E dyeing and（2B）：Masson’s
Trichrome is dyed（2C）.Show to compare with the control group for only having injected sterile PBS solution, it is dry thin to have injected medulla mesenchyma
Born of the same parents are reduced and collagen deposition degree by location of necrosis in the hepatic tissue of manganese ion activated mesenchymal stem cell
It reduces.Scale represents 200 μm.Fig. 2 D：Sterile PBS solution is injected in the rat of acute hepatic failure respectively, medulla mesenchyma is dry thin
Born of the same parents carry out TUNEL by the hepatic tissue of manganese ion activated mesenchymal stem cell and detect Apoptosis situation.Our research
Show that a large amount of apoptosis can be observed in the control group for having injected sterile PBS solution, followed by mesenchymal stem cell
Group, and in by manganese ion activated mesenchymal stem cell, it is nearly no detectable the cell of apoptosis.Scale represents in figure
200μm.The study show that in the Hepatic Fibrosis of Animal experiment of tetrachloro-methane induction, manganese ion can be withered by reducing cell
It dies, reduces necrosis and collagen deposition effectively improves the effect of mesenchymal stem cell is for liver fibrosis and acute hepatic failure.
The above, the only specific implementation mode of the application, but the protection domain of the application is not limited thereto, it is any
Change or replacement in the technical scope that the application discloses, should all cover within the protection domain of the application.Therefore, this Shen
Protection domain please should be based on the protection scope of the described claims.
Claims (5)
1. a kind of preparation method for the extracellular matrix for promoting stem cell to be divided into liver cell, which is characterized in that the side
Method includes：
Step 1：Prepare porcine hepatocyte epimatrix；
Step 2：The separation and culture of autologous patient mesenchymal stem cell；
Step 3：The mesenchymal stem cells differentiation of external 3D cultures is liver cell；
Step 4：The functional evaluation of liver cell.
2. the preparation method of the extracellular matrix according to claim 1 for promoting stem cell to be divided into liver cell,
It is characterized in that, the step 1 specifically includes：
Complete fresh pig liver is obtained to leave behind vena cave and open as outlet, arteria hepatica and hepatic duct ligation by conduit
After being inserted into portal vein, 20L is contained to the Sterile phosphate of 5000IU/L heparin using peristaltic pump with the rate of 100mL/min immediately
Pork liver is perfused to remove blood in liver in buffer salt PBS solution；
0.01% lauryl sodium sulfate being gradually pre-chilled in sequence with 150L at 4 DEG C（SDS）Solution, 150L precoolings
0.1% SDS solution, the Triton X-100 perfusion pork livers that the 1% SDS solution and 50L 1% of 50L precoolings are pre-chilled are thin to make it take off
Born of the same parents have finally taken off the liver of cell using the perfusion cleaning of 100L distilled water, and remaining surfactant and cell are removed with this
Residual；
Graininess will be worn into using rotating blade grinder after pork liver progress lyophilized processing after de- cell, the powder that will be obtained
End is resuspended with the concentration of 50mg/L in sterile PBS solution, is used in combination Syrup-homogenizing instrument mixing to form porcine hepatocyte epimatrix emulsion, is made
With 60 Co gamma radiation（15kGy）To porcine hepatocyte epimatrix emulsion carry out sterilization treatment, be stored in after sterilizing 4 DEG C with
Standby rear use.
3. the preparation method of the extracellular matrix according to claim 1 for promoting stem cell to be divided into liver cell,
It is characterized in that, the step 2 specifically includes：
According to the jawbone bone chip clast that the method for orthognatic surgery or implant operation takes patient disease-free, it is placed in α-MEM cultures
4 DEG C of preservations, are cleaned three times with sterile PBS solution in liquid, and 800rpm centrifuges 5min and obtains cell precipitation and bone chip under room temperature；With
Cell precipitation and bone chip mixture are gently blown and beaten mixing by the α-MEM culture solutions containing 10% fetal calf serum, are inoculated into the training of six holes
It supports in plate；At 37 DEG C, 5% CO2It is cultivated 3-7 days under the conditions of saturated humidity, until there is cell to be climbed out of from organization edge；Cell is close
When degree is up to 80%, 2.5x10 is arrived in 0.25% pancreatin passage digestion, gradually amplification5。
4. the preparation method of the extracellular matrix according to claim 1 for promoting stem cell to be divided into liver cell,
It is characterized in that, the step 3 specifically includes：
By 2.5x105The digestion centrifugation of Bone Marrow of Patients mescenchymal stem cell is resuspended in the outer base of ready porcine hepatocyte before 200 μ L
It in matter emulsion and a Collagen Type VI isodensity mixed liquor, is seeded in 24 hole plates, 50 μM of Mn is added2+, at 37 DEG C, 5% CO2
So that colloid is solidified with 30min is cultivated under the conditions of saturated humidity, adds the α-MEM that 500 μ L contain 10% fetal calf serum to arrive each hole, often
A culture solution is changed within two days, sampling progress paraffin fixed test glycogen after culture 2 weeks（PAS is dyed）, seralbumin（It is immune glimmering
Light）Equal liver function indexs.
5. the preparation method of the extracellular matrix according to claim 1 for promoting stem cell to be divided into liver cell,
It is characterized in that, the step 4 specifically includes：
The human marrow mesenchymal stem cell and pig source liver cell epimatrix mixture for taking step 3 culture are thoroughly ground by liquid nitrogen,
The rna expression that RNA carries out reverse transcription and quantitative fluorescent PCR verifies liver cell marker gene is extracted according to TRIZOL specifications, separately
The outer human marrow mesenchymal stem cell and pig source liver cell epimatrix mixture that step 3 culture is taken according to protein cleavage specification,
After being cleaned three times with sterile PBS solution, protein lysate is added and is used as Western blot detections, verifies the liver cell mark of differentiation
Will protein expression.
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