CN105907703B - A kind of method that inducing bone mesenchymal stem cell breaks up to hepatic lineage - Google Patents
A kind of method that inducing bone mesenchymal stem cell breaks up to hepatic lineage Download PDFInfo
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
- C12N2506/1353—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from bone marrow mesenchymal stem cells (BM-MSC)
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Abstract
The present invention provides a kind of method that inducing bone mesenchymal stem cell breaks up to hepatic lineage, includes：Bone marrow mesenchymal stem cells are separately cultured, collect the serum of liver regeneration 12h, liver tissue homogenate, bone marrow mesenchymal stem cells Fiber differentiation in the serum and the liver tissue homogenate is made with the liver of regeneration 12h.The method that inducing bone mesenchymal stem cell provided by the invention breaks up to hepatic lineage, the hepatic lineage after induction have liver cell feature completely.
Description
Technical field
The present invention relates to Cell. Mol field more particularly to a kind of inducing bone mesenchymal stem cell are thin to liver sample
The method of born of the same parents' differentiation.
Background technology
Liver transplant is to treat the effective ways of various End-stage liver diseases at present, but since donor source is limited, expense is held high
The factors such as expensive, immunological rejection and be difficult to large-scale popularization, thus limit the application of Clinical Liver Transplantation.With the rise of organizational project
And development, cell replacement therapy will be as capturing the new way of some difficult and complicated illness.Mesenchymal stem cell (bone
Mesenchymal stem cells, BMSCs) there is powerful proliferative capacity, stronger multi-lineage potential, immunological rejection
It reacts low, derives from a wealth of sources, materials are easy, and damage is small, there is no ethics problem, and with good histocompatbility, are expected to
As a kind of Novel seed cell for the treatment of End-stage liver disease.Therefore, various inducing bone mesenchymal stem cells are explored to liver
The method of cell differentiation has great significance for the popularization of clinical treatment.
In the prior art, the method that inducing bone mesenchymal stem cell breaks up to liver cell, what it is due to induction is not most
Good physiological environment, therefore the hepatic lineage after induction mostly not exclusively has the feature of liver cell.
Invention content
The object of the present invention is to provide a kind of method that inducing bone mesenchymal stem cell breaks up to hepatic lineage, after induction
Hepatic lineage completely have liver cell feature.
In order to solve the above technical problem, the present invention provides inducing bone mesenchymal stem cell break up to hepatic lineage
What method was realized in：
A kind of method that inducing bone mesenchymal stem cell breaks up to hepatic lineage, including：Bone marrow mesenchymal is separately cultured to do
Cell collects the serum of liver regeneration 12h, liver tissue homogenate is made with the liver of regeneration 12h, the bone marrow mesenchymal stem cells are in institute
State Fiber differentiation in serum and the liver tissue homogenate.
Optionally, bone marrow mesenchymal stem cells bone described in Fiber differentiation in the serum and the liver tissue homogenate
Marrow mesenchymal stem cells are 3 generation cells.
Optionally, a concentration of the 10% of the serum.
Optionally, a concentration of the 10% of the liver tissue homogenate.
Optionally, the bone marrow mesenchymal stem cells use adherent method culture.
Optionally, the liver with regeneration 12h is made liver tissue homogenate and includes：After liver regeneration 12h hours, liver is cut off
It is dirty, culture medium is added in the ratio of 200mg hepatic tissues/ml L-DMEM, is homogenized on ice, 4 DEG C, 12 000 r/min centrifugations
25min takes supernatant that damage liver tissue homogenate is made.
The method that inducing bone mesenchymal stem cell provided by the invention breaks up to hepatic lineage, serum are by plasma removing
Fibrinogen and a kind of very complicated mixture formed, contain various plasma proteins, polypeptide, fat, carbohydrate, life
The long factor, hormone, inorganic matter etc.；In rats'liver, DNA about 12h after partially hepatectomized start to replicate, and peak for 24 hours；
12h Cell differentiation inducing activities, the factor being proliferated will be more than for 24 hours after partially hepatectomized；Use the serum of liver regeneration 12h and regeneration 12h
Liver liver tissue homogenate is made as induced environment, closer to the physiological status of rat, result of the test shows induced environment
AFP and ALB is not expressed in the BMSCs cells of liver tissue homogenate and serum are not added in.AFP is adding in serum and liver tissue homogenate
14 d expression quantity of Fiber differentiation is most, is reduced with the extension expression quantity of time；ALB is adding in serum and liver tissue homogenate's induction
It is most to cultivate 21 d expression quantity；7 days detectable urea is induced, with the extension of induction time, urea expression quantity rises；Therefore originally
The method that invention inducing bone mesenchymal stem cell breaks up to hepatic lineage, induced environment is closer to physiological status, Fiber differentiation
Hepatic lineage it is more ripe closer to physiological status, hepatic lineage.
Adhere-wall culture mesenchymal stem cell of the present invention has easy to operate, quick, small on cell activity influence.
Description of the drawings
Fig. 1 is mesenchymal stem cell provided by the invention Western blot immunoblottings first tire egg after induction
White and albumin expression figure；
Fig. 2 is that mesenchymal stem cell provided by the invention liver function energy metabolism after induction detects urea content curve pair
Than figure.
Specific embodiment
Present invention aims at the suitable hepatocyte origins for finding hepatocyte transplantation needs, study different methods for inducing pair
Mesenchymal stem cell (bone marrow mesenchymal stem cells, BMSCs) is divided into liver cell in vitro
The influence of like cell.
Material used herein, L-DMEM culture mediums are purchased from Gibco companies of the U.S., and fetal calf serum is purchased from the U.S.
Hyclone companies, trypsase are purchased from Sigma Co., USA, albumin (Albumin, ALB) and alpha-fetoprotein (α-
Fetoprotein, FP or AFP) monoclonal antibody is purchased from Santa Cruz companies of the U.S., and SD rats are purchased from Xinxiang College of Medical Science's reality
Test animal center, cleaning grade meets animal ethics standard in experimentation to animal disposition.
The present invention sets four groups of experiments respectively, and A groups is only use fetal calf serum（FBS, fetal bovine serum) make
For culture medium, second group of B is using the liver tissue homogenate of FBS and regeneration 12h as the medium of induction BMSCs, and C third groups are with liver regeneration
The serum of 12h is the medium for inducing BMSCs, and liver tissue homogenate is made with the liver of the serum of liver regeneration 12h and regeneration 12h in D groups
To induce the medium of BMSCs, reference compares the method that best inducing bone marrow mesenchymal stem cells support cell differentiation to liver.Specifically such as
Under：
First, separation, the culture of rat BMSCs
Cervical dislocation puts to death 4-6 week old male and healthy SD rats（Weight 100-150 g）, detached under aseptic condition double
Side lower limb femur, shin bone go out marrow in centrifuge tube with culture medium, and single cell suspension is made in piping and druming.1 000 r/min, from
5 min of the heart, abandons supernatant, is resuspended with culture medium.By cell inoculation in culture bottle, be placed in 37 DEG C, volume fraction be 0.05
It is cultivated in CO2 incubators.48 h half, which are measured, changes liquid, and every 3 days full doses change liquid later.It can be carried out when cell confluent cultures bottle about 80%
Passage.General 1:3 inoculation passages.
2nd, prepared by serum and liver tissue homogenate
Etherization rat opens abdominal cavity under aseptic condition at about 1-2cm below xiphoid-process along ventrimeson, cuts off left lobe of liver
And the middle period（Account for about the 68% of full liver weight）, sewing up a wound and sprinkling sulfanilamide (SN) prevents from infecting, postoperative routine feeding.Heart extracts after 12h
Whole blood prepares serum, and -20 DEG C save backup；Regenerating Liver of Rat tissue is taken out, in the ratio of 200mg hepatic tissues/ml L-DMEM
Culture medium is added in, is homogenized on ice, 4 DEG C, 12 000 r/min centrifugation 25min take supernatant to be used as and damage liver tissue homogenate, and 0.22
- 20 DEG C of preservations after μm membrane filtration.
3rd, induction BMSCs differentiation
The 3rd generation cell is taken, by 2 × 104A cell inoculation is in the culture dish of 35mm, 37 DEG C, 5% CO2Culture, the experiment of A groups
Culture medium be the L-DMEM containing 100 U/mL of penicillin, 100 U/mL+10%FBS of streptomysin；B groups experiment culture medium be
L-DMEM containing 100 U/mL of penicillin, 100 U/mL+10% Liver Regeneration of Rat 12h liver tissue homogenates of streptomysin；C groups are tested
Culture medium be the L-DMEM containing 100 U/mL of penicillin, 100 U/mL+10% Liver Regeneration of Rat 12h serum of streptomysin；D groups
The culture medium of experiment is that the serum+10% containing 100 U/mL of penicillin, 100 U/mL+10% Liver Regeneration of Rat 12h of streptomysin is big
The L-DMEM of mouse liver regeneration 12h liver tissue homogenates.Induction 0,7,14,21d observe cellular morphology under inverted microscope, are changed per 3d
Liquid 1 time.
Specific detection is as follows：
Detection statistics credit is analysed：It is inspection level with α=0.05, is analyzed with SPSS13.0 statistical softwares, using duplicate measurements
Two analysis of variance of data.
First, Immunofluorescence test
7,14,21 d respectively at induction collect cell, make cell smear.10min is fixed with 4% paraformaldehyde solution,
PBS is rinsed 3 times, and 0.1% Triton X-100 are incubated 10min, the closing of 10% sheep blood serum room temperature 30 min, 0.3% H202Processing 15
Min, is separately added into 4 DEG C of ALB or AFP primary antibodies overnight incubation, PBS added in after rinsing the secondary antibody that mark through Biotin with
Streptavidin-FITC is incubated 60min, observes and takes pictures under fluorescence microscope (Japanese Nicon companies).Statistical result is shown
Show, for AFP and ALB positive cell rates in four groups, D groups are most, and B groups are better than C groups, and A groups do not express AFP and ALB, as shown in table 1.
Positive cell rates of table 1 AFP and ALB in different methods for inducing
-:It is negative; ± :It is a small amount of positive; +:20%-30% is positive; ++:30%-40% is positive; +++: 40%-50%
It is positive；++++:50%-60% is positive
2nd, Western blot immunoblotting assays
It is taken at the expression of cell detection ALB, AFP of 0,7,14,21 d of induction.12%SDS-PAGE gel electrophoresises, 80mV
2h goes to protein electricity on pvdf membrane, after ALB, AFP are incubated 1h respectively, adds in the secondary antibody of horseradish peroxidase-labeled,
ECL methods develop the color.
The cell of second group of experiment and third group experiment different time points is collected, Western blot detections ALB, AFP's
Expression.The results show that AFP and ALB positive cell rates are in four groups, D groups are most, and B groups are better than C groups, A groups do not express AFP with
ALB, as shown in table 1.
In four groups, D group expression quantity is more by ALB, AFP, and B groups are more than C groups, and A groups are not expressed.As shown in Figure 1.
3rd, hepatocyte function detects
0,7,14,21 d in induction collect culture cell supernatant, are detected in supernatant using glutamate dehydrogenase enzyme process
Urea content observes its dynamic change trend, draws urea content change curve.As shown in Figure 2.
Urea concentration in each time point supernatant of each group is depicted as line chart.A groups can't detect in Each point in time
Urea.Its excess-three group is with the extension of induction time, and urea expression quantity rises, but the urea content of D groups induction is more than B groups and C groups
（P <0.05）.
The cell that rat liver only has about 0.0012%~0.01% under normal physiological status carries out mitosis.
It damages or performs the operation caused by poisonous substance after excision, liver cell can start hyperplasia rapidly and restore liver quickly.And hepatomegaly part is cut
Except postoperative regenerating hepatic tissue homogenate may then include whole stimulating factors and nutritional ingredient.And in rats'liver, DNA is in part
About 12h starts to replicate after hepatectomy, peaks for 24 hours；12h Cell differentiation inducing activities, the factor being proliferated will after partially hepatectomized
More than for 24 hours.Serum is by plasma removing fibrinogen and a kind of very complicated mixture for being formed, and serum composition and content are normal
It is different and different with the gender of blood supply animal, age, physiological condition and nutritional condition.In serum containing various plasma proteins, polypeptide,
Fat, carbohydrate, growth factor, hormone, inorganic matter etc., composition may have as many as hundreds of kinds.
Therefore, the present invention uses liver tissues of rats homogenate and partially hepatectomized 12h rat liver blood after partially hepatectomized 12h
Induction differentiation is carried out clearly, the factor for promoting hepatic cell growth, hormone and other regulatory factors is allowed to play a role jointly, the liver of induction
Like cell is closer to physiological status.Concrete analysis is as follows：
AFP is a kind of plasmosin, is secreted by liver precursor, is disappeared as cell is gradually ripe, ripe liver is thin
Born of the same parents do not express AFP.ALB is the protein that content is most in human normal plasma, and after ALB is synthesized in liver cell, secretion enters blood
Liquid recycles and is distributed to body everywhere.ALB is the most frequently used and most reliable hepatocyte function Testing index because ALB mainly by
Liver cell synthesis secretion, the secretion of other histocytes are few.The ammonia synthesis urea that liver can generate amino acid metabolism, through kidney
It is dirty to excrete.Urea is mainly synthesized in liver, other organs such as kidney and brain effect are little.Ours the experimental results showed that without
The BMSCs of partially hepatectomized liver tissues of rats homogenate induction does not express AFP, ALB and urea.Through fetal calf serum and regeneration for 24 hours
Liver of the expression of AFP, ALB and urea of liver tissue homogenate's induction less than the serum through liver regeneration 12h and regeneration 12h is made
The expression of AFP, ALB and urea of liver tissue homogenate's induction.Liver group is made in the liver of serum and regeneration 12h through liver regeneration 12h
AFP peaks when knitting 14 d of BMSCs of homogenate induction, is reduced with the extension expression quantity of time；ALB and urea exist
Liver tissue homogenate, which is made, in the liver of serum and regeneration 12h for adding in liver regeneration 12h, which cultivates 7 d, a small amount of expression, with the time
Extend expression quantity raising, 21 d expression quantity are most.The joint detection results of multiple indexs support BMSCs to mature hepatocytes side
To differentiation, and with the extension of induction time, there is hepatocytes secrete albumin, the cell of urea synthesis gradually increases,
It prompts such cell that there are mature hepatocytes, successfully completes differentiation of the external evoked rat BMSCs to liver cell.
Multiple indexs of two aspects of hepatocytic phenotype that this experiment is detected and hepatocyte function, during with Fiber differentiation
Between extension, mark gradually appears and tends to ripe.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement made within refreshing and principle etc., should all be included in the protection scope of the present invention.
Claims (4)
1. a kind of method that inducing bone mesenchymal stem cell breaks up to hepatic lineage, which is characterized in that including：It is separately cultured bone
Marrow mesenchymal stem cells collect the serum of liver regeneration 12h, liver tissue homogenate are made with the liver of regeneration 12h, the bone marrow mesenchymal is done
Cell Fiber differentiation in the serum and the liver tissue homogenate；A concentration of the 10% of the serum, the liver tissue homogenate
A concentration of 10%.
2. according to the method that the inducing bone mesenchymal stem cell described in claim 1 breaks up to hepatic lineage, feature exists
In bone marrow mesenchymal stem cells bone marrow mesenchymal stem cells described in Fiber differentiation in the serum and the liver tissue homogenate
For 3 generation cells.
3. the method that inducing bone mesenchymal stem cell according to claim 1 or 2 breaks up to hepatic lineage, feature exist
In the liver with regeneration 12h is made liver tissue homogenate and includes：It after liver regeneration 12h hours, hepatectomizes, by 200mg liver groups
Knit/ratio of ml L-DMEM adds in culture medium, it is homogenized on ice, 4 DEG C, 12 000 r/min centrifugation 25min take supernatant to be made
Damage liver tissue homogenate.
4. the method that inducing bone mesenchymal stem cell according to claim 3 breaks up to hepatic lineage, which is characterized in that
The bone marrow mesenchymal stem cells use adherent method culture.
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| 损伤肝组织匀浆与骨髓间充质干细胞的分化;侯军霞 等;《中国组织工程研究》;20120701;第16卷(第27期);摘要 * |
| 损伤肝组织匀浆诱导间充质干细胞甲胎蛋白和白蛋白表达;王莹 等;《中国老年学杂志》;20081231;第28卷;摘要，第2431页右栏第1.3-1.5节 * |
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