CN110946877A - Stem cell biological product for treating liver cirrhosis and preparation method and application thereof - Google Patents
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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Abstract
In order to overcome the problem of insufficient effect of the conventional treatment method for cirrhosis, the invention provides a stem cell biological product, which comprises mesenchymal stem cells, wherein the mesenchymal stem cells are introduced with a vector through transfection, and the vector comprises a CCR9 gene sequence. Meanwhile, the invention also discloses a preparation method and application of the stem cell biological product. According to the stem cell biological product provided by the invention, the CCL25 is combined with the receptor CCR9 to achieve a cell chemotaxis effect, so that the specific homing capacity of the mesenchymal stem cells at a liver cirrhosis focus position is improved, the number of local stem cells is increased, and the treatment effect of the mesenchymal stem cells on liver cirrhosis is effectively improved.
Description
Technical Field
The invention belongs to the technical field of stem cells, and particularly relates to a stem cell biological product for treating cirrhosis, and a preparation method and application thereof.
Background
Cirrhosis is a clinically common chronic progressive liver disease characterized by diffuse liver lesions formed by long-term or repeated action of one or more etiologies, diffuse fibrosis of the liver tissue, pseudolobules, and regenerative nodules. In China, the majority of the patients are posthepatitic cirrhosis, and the minority of the patients are alcoholic cirrhosis and schistosomiasis cirrhosis. The peak age of the disease is 35-48 years old, and the proportion of male and female is about (3.6-8): 1.
research shows that the chemotactic factor CCL25 is expressed in normal liver tissues, liver fibrosis and liver tissues of liver cirrhosis patients, when the liver is subjected to fibroproliferative damage, the content of CCL25 in the liver is obviously increased, and the CCL25 content in the liver tissues of the liver cirrhosis patients is the highest along with the deepening of the liver fibrosis to the liver cirrhosis course. Bone marrow-derived liver stem cells can migrate and colonize the damaged part of the liver under the chemotaxis of CCL25, supplement the number of liver cells reduced due to disease damage, and repair the damaged liver tissue structure due to disease damage.
Disclosure of Invention
Aiming at the problem of insufficient effect of the conventional treatment method for liver cirrhosis, the invention provides a stem cell biological product for treating liver cirrhosis and a preparation method and application thereof.
The technical scheme adopted by the invention for solving the technical problems is as follows:
in one aspect, the invention provides a stem cell biologic comprising a mesenchymal stem cell transfected with a vector comprising a CCR9 gene sequence.
Optionally, the mesenchymal stem cell is a mesenchymal stem cell isolated in vitro of a mammal, and the mesenchymal stem cell includes one or more of an umbilical cord mesenchymal stem cell, a placenta mesenchymal stem cell, an adipose mesenchymal stem cell, a bone marrow mesenchymal stem cell and a dental pulp mesenchymal stem cell.
Optionally, the vector is selected from the group consisting of plasmid vectors comprising one or more of pcDNA3.1+, pcDNA3.1-HisA, B, C, pcDNA3.1-3xflag and pcDNA6/MYC-HIS A, B, C.
Optionally, the CCR9 gene sequence comprises a sequence shown as SEQ ID NO.9, and the vector transferred into the CCR9 gene sequence comprises a sequence shown as SEQ ID NO. 10.
In another aspect, the present invention provides a method for preparing a stem cell biological product as described above, comprising the following operations:
obtaining a CCR9 target gene;
constructing a vector comprising a CCR9 gene sequence;
obtaining mesenchymal stem cells;
the vector transfects mesenchymal stem cells.
Optionally, the step of obtaining the CCR9 target gene comprises the following operations:
collecting a liver tissue sample, extracting total RNA, designing an amplification primer, amplifying the full length of a CCR9 gene by a reverse transcription-polymerase chain reaction PCR method, carrying out electrophoresis, and purifying a CCR9 target gene fragment.
Optionally, after the "construction of the vector comprising the CCR9 gene sequence", the vector comprising the CCR9 gene sequence is transfected into prokaryotic cells for expansion culture, and the vector comprising the CCR9 gene sequence is extracted.
Optionally, the route of "obtaining mesenchymal stem cells" includes obtaining umbilical cord, placenta, fat, bone marrow or dental pulp, culturing and separating to obtain mesenchymal stem cells.
Alternatively, the means for "transfection of mesenchymal stem cells with vector" includes electric shock method, calcium phosphate method, liposome-mediated method and virus-mediated method.
In another aspect, the invention provides the use of a stem cell biologic as described above in the preparation of a medicament for the treatment of cirrhosis.
According to the stem cell biological product provided by the invention, a vector with a CCR9 gene sequence is transferred into the mesenchymal stem cells in a gene transfection manner, so that the high expression of a chemokine receptor CCR9 in the mesenchymal stem cells is promoted, and as the inflammatory part highly expresses CCL25 and the CCL 35 25 is a chemokine, the combination of the CCL25 and the receptor CCR9 has a cell chemotactic effect, so that the specific homing capacity of the mesenchymal stem cells at a liver cirrhosis focus part is improved, the number of local stem cells is increased, and the treatment effect of the mesenchymal stem cells on liver cirrhosis is effectively improved.
Drawings
FIG. 1 shows the results of western blot assay for CCR9 protein expression.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention provides a stem cell biological product which comprises mesenchymal stem cells, wherein the mesenchymal stem cells are transfected and introduced with a vector, and the vector comprises a CCR9 gene sequence.
Chemokine receptor 9(CCR9) is a member of the chemokine receptor family, CCR9 is a class of small molecule G protein-coupled receptors, with only one known ligand, chemokine 25(CCL 25). CCR9 is mainly expressed on cell membranes of lymphocytes, dendritic cells, mononuclear macrophages and the like, and research proves that CCR9 is involved in the occurrence and development of various inflammatory diseases, including inflammatory bowel diseases, acute hepatitis, rheumatoid arthritis and other inflammatory diseases. The mesenchymal stem cells provided by the embodiment can promote high expression of a receptor CCR9 by introducing a vector comprising a CCR9 gene sequence through transfection, thereby promoting the treatment effect and promoting the homing of the mesenchymal stem cells at the focus position.
The vector in the mesenchymal stem cell is not a naturally occurring substance but a result of manual manipulation by a genetic engineering method.
In some embodiments, the mesenchymal stem cells comprise one or more of umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells and dental pulp mesenchymal stem cells.
In a preferred embodiment, the mesenchymal stem cells are selected from umbilical cord mesenchymal stem cells.
In some embodiments, the mesenchymal stem cell is a mesenchymal stem cell isolated in vitro from a mammal.
The mammals include humans and other mammals.
In some embodiments, the vector is selected from the group consisting of plasmid vectors comprising one or more of pcDNA3.1+, pcDNA3.1-HisA, B, C, pcDNA3.1-3xflag, and pcDNA6/MYC-HIS A, B, C.
In other embodiments, the vector may also be a viral vector.
In some embodiments, the CCR9 gene sequence includes the sequence shown as SEQ ID No. 9.
In some embodiments, the vector into which the sequence of the CCR9 gene is transferred comprises the sequence shown in SEQ ID No. 10.
It should be noted that the CCR9 gene sequence in the present invention is not limited to the sequence shown in SEQ ID No.9 used in the examples, and the vector is not limited to the sequence shown in SEQ ID No.10, and other CCR9 sequences and CCR9 sequences and their variants that may be newly found or other vectors can be used through routine verification, and CCR9 gene sequences can be prepared by using sequence information disclosed in the present specification or the attached sequence listing, and sequence information registered in public databases (e.g., DDBJ/EMBL/GenBank) as references, using standard genetic engineering methods, molecular biological methods, biochemical methods, and the like. Other CCR9 sequences, as well as possible newly discovered CCR9 sequences and variants thereof, or other vectors, corresponding primers and molecular biological experimental manipulations can also be performed by means of techniques conventional in the molecular biology field (e.g., SammBruk molecular cloning, A laboratory Manual), and are intended to be included within the scope of the present invention.
In view of the degeneracy of codons, the DNA sequence encoding the same protein (i.e., CCR9 receptor) is functionally equivalent to the DNA sequence shown in SEQ ID NO.9, and can be used as the CCR9 gene in the present invention. In general, when a part of the amino acid sequence of a certain protein is modified, the modified protein may have the same function as the protein before modification. That is, the function of the protein may not be substantially affected by the change in the amino acid sequence, and the function of the protein may be maintained before and after the change. Therefore, a DNA sequence of a protein having a homologous amino acid sequence to the protein encoded by the DNA sequence shown in SEQ ID NO.9 and acting as a CCR9 receptor (hereinafter also referred to as "homologous protein") can also be used as the CCR9 gene of the present invention. The term "homologous amino acid sequence" as used herein means an amino acid sequence which is partially different from the amino acid sequence shown in SEQ ID NO.9, but which has no substantial effect on the function of the protein (in this case, CCR9 receptor activity).
"partial difference in amino acid sequence" typically means that an amino acid sequence is mutated (changed) by deletion, substitution, or addition, insertion, or combination of one to several amino acids constituting the amino acid sequence. The difference in amino acid sequence here is permissible as long as the CCR9 receptor activity is not greatly reduced. The position where the amino acid sequence differs is not particularly limited as long as the condition is satisfied, and the amino acid sequence may differ at a plurality of positions. The plurality herein is, for example, a number corresponding to less than about 30%, preferably a number corresponding to less than about 20%, more preferably a number corresponding to less than about 10%, still more preferably a number corresponding to less than about 5%, and most preferably a number corresponding to less than about 1% of all amino acids. That is, the homologous protein has, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more, still more preferably about 95% or more, and most preferably about 99% or more homology with the amino acid sequence of SEQ ID NO. 15.
Another embodiment of the present invention provides a method for preparing a stem cell biological product as described above, comprising the following operations:
obtaining a CCR9 target gene;
constructing a vector comprising a CCR9 gene sequence;
obtaining mesenchymal stem cells;
the vector transfects mesenchymal stem cells.
In some embodiments, the "obtaining a CCR9 target gene" comprises the following operations:
collecting a liver tissue sample of a patient, extracting total RNA, designing an amplification primer, amplifying the full length of a CCR9 gene by a reverse transcription-polymerase chain reaction PCR method, carrying out electrophoresis, and purifying a CCR9 target gene fragment.
In some embodiments, the CCR9 gene sequence is cleaved at Kpn1 and EcoR1, and amplification primers for CCR9 gene are designed using primer6.0 software, and the primer sequences are as follows:
(1) when the vector is selected from pcDNA3.1+, the primer sequences are designed as follows:
CCR9-Kpn1-F1:ggggtaccat gacacccaca gacttcaca(SEQ ID NO.1)
CCR9-EcoR1-R1:cggaattctc agagggagag tgctcctg(SEQ ID NO.2)
(2) when the vector is selected from pcDNA3.1-HisA, B, C, the primer sequences are designed as follows:
CCR9-Kpn1-F2:ggggtaccaa tgacacccac agacttcaca(SEQ ID NO.3)
CCR9-EcoR1-R1:cggaattctc agagggagag tgctcctg(SEQ ID NO.4)
(3) when the vector is selected from pcDNA3.1-3xflag, the primer sequences are designed as follows:
CCR9-EcoR1-F3:cggaattcat gacacccaca gacttcaca(SEQ ID NO.5)
CCR9-Kpn1-R3:ggggtacccg gagggagagt gctcctgagg(SEQ ID NO.6)
(4) when the vector is selected from pcDNA6/MYC-HIS A, B, C, the primer sequences are designed as follows:
CCR9-Kpn1-F1:ggggtaccat gacacccaca gacttcaca(SEQ ID NO.7)
CCR9-EcoR1-R4:cggaattccg gagggagagt gctcctgagg(SEQ ID NO.8)。
in some embodiments, the "constructing a vector comprising the CCR9 gene sequence" comprises the following operations:
carrying out double enzyme digestion on the vector by using restriction enzyme, adding a CCR9 target gene and ligase, and transferring the CCR9 target gene into the enzyme digestion site of the vector.
When the CCR9 target gene is transferred, a functional sequence (e.g., a selection marker or an enhancer) may be added to the vector for further modification.
For the procedures, reagents, conditions, and the like required for constructing the expression vector of the present invention, reference may be made to, for example, Molecular Cloning (third edition, Cold spring harbor laboratory Press, New York) or Molecular biology laboratory method compilation (Frederick M. Ausubel et al, 1987).
In some embodiments, following the "construction of a vector comprising the CCR9 gene sequence", the vector comprising the CCR9 gene sequence is transfected into prokaryotic cells for expansion culture and the vector comprising the CCR9 gene sequence is extracted.
The prokaryotic cell is used for expanding and culturing the vector comprising the CCR9 gene sequence, so that the subsequent purification of the vector comprising the CCR9 gene sequence and the detection of whether the expression of the vector is successful or not can be facilitated.
The prokaryotic cell is selected from prokaryotic bacteria of Escherichia coli or other suitable culture carriers.
In some embodiments, the "obtaining mesenchymal stem cells" comprises obtaining umbilical cord, placenta, fat, bone marrow or dental pulp, culturing and isolating to obtain mesenchymal stem cells.
In some embodiments, the means by which the "vector transfects mesenchymal stem cells" includes electroporation, calcium phosphate, liposome-mediated methods, and virus-mediated methods.
In a preferred embodiment, the mesenchymal stem cells are treated using liposome-mediated methods to successfully transfect the vector into the mesenchymal stem cells.
Another embodiment of the present invention provides the use of a stem cell biologic as described above in the preparation of a medicament for the treatment of cirrhosis of the liver.
The stem cell biological product can be injected by intravenous injection or local injection at a lesion site.
The present invention will be further illustrated by the following examples.
Example 1
This example illustrates a stem cell biological product and a method for preparing the same, comprising the following steps:
step one, construction of recombinant plasmid vector carrying target gene CCR9
CCR9-Kpn1-F1:ggggtaccat gacacccaca gacttcaca(SEQ ID NO.1)
CCR9-EcoR1-R1:cggaattctc agagggagag tgctcctg(SEQ ID NO.2)
mu.g of total RNA was used to reverse transcribe cDNA using OligodT (20) and the full length of CCR9 gene was amplified by RT-PCR.
PCR conditions were as follows: denaturation at 98 deg.C for 15s, annealing at 68 deg.C and extension for 3min, circulating for 35 times, and extending for 15min at 72 deg.C after the last circulation. And (5) observing by agarose gel electrophoresis, cutting gel, recovering and purifying the target gene fragment.
And 2, carrying out double enzyme digestion on the vector. Sterilized EP tube, 5ug of plasmid vector pcDNA3.1+, double digestion with Kpn1 and EcoR1, 1.5ul Kpn1, 1.5ul EcoR 1; by dd H2O supplement system is 50ul, enzyme digestion is carried out for 20min at 37 ℃, enzyme digestion product 6ul is taken for 1.5% agarose gel electrophoresis for 30 min; placing the gel under an ultraviolet lamp, judging the position of the target fragment to carry out gel cutting recovery, placing the recovered gel into a sterile EP tube, and recovering the DNA target fragment by using a trace agarose gel DNA recovery kit.
And 3, connecting the target fragment with the vector. The target fragment CCR9 is linked with a commercial plasmid vector pcDNA3.1+, and the reaction system comprises the following steps: pcDNA3.1+100ng, CCR 9100 ng, 5 CE II Buffer 4ul, Clonexpress II One StepCringing Kit ligase 2 ul; dd H2O is added to a total volume of 20 ul; ligation was performed at 37 ℃ for 30min in an ice bath for 5 min.
And 4, converting the ligation product into a competent cell. Thawing competent cells 150ul in ice, adding 2ul ligation product after thawing, shaking up and ice-bathing for 30min, heat shocking in 42 ℃ water bath for 45s, and rapidly ice-bathing for 2 min. Then placing the mixture into 500ul LB culture medium, culturing the mixture for 1h in a shaking incubator at 37 ℃ and 225r/min, and uniformly coating 50ul, 100ul and 150ul of bacteria solution with gradient increasing quantity on 3 agar plates containing Amp antibiotics (100 mu g/ml). The cells were placed in an incubator at 37 ℃ for overnight incubation in an inverted state. A single colony is picked and cultured in 3 tubes (about 5ml per tube) of Amp + LB (100 mu g/ml) culture medium for 12-16 h, and plasmids are extracted when the OD value is determined to be about 2-3.
And 5, plasmid extraction. Taking 5ml of the bacteria culture solution, placing in a clean sterile centrifuge tube, centrifuging at 8000r/min for 2min, and blotting to dry the culture medium; adding 250 μ l Buffer P1/RNaseA mixed solution, and thoroughly suspending the bacterial precipitate by using a vortex oscillator; adding 250 mu l of Buffer P2, immediately mixing the centrifuge tube for 5-10 times in a gentle and reversed manner, and standing for 2-4 min at room temperature; adding 350 mu l of buffer P3 to generate white precipitates, immediately and flatly inverting the centrifuge tube for 5-10 times, uniformly mixing, centrifuging at 12000r/min for 12min, completely transferring the supernatant into an adsorption column, centrifuging at 8000r/min for 30s, and pouring out the liquid in the collection tube; adding 500 mu l buffer DW1 into the adsorption column, centrifuging at 8000r/min for 30s, and pouring off the liquid in the collection tube; adding 500 μ l Washsolution into adsorption column, centrifuging at 10000r/min for 30s, and pouring off liquid in collection tube; washing once with 500 μ l Wash Solution, centrifuging at 10000r/min for 30s, and pouring out the liquid in the collecting tube. Centrifuging the air adsorption column at 8000r/min for 1 min; the adsorption column is placed into a new 1.5ml centrifuge tube, 65 mul of Elution buffer preheated to 65 ℃ is added to the center of the adsorption membrane, in order to improve the Elution rate, the mixture is kept stand for 2min at room temperature, centrifuged for 1min at 8000r/min, and the obtained plasmid DNA solution is stored at-20 ℃ for subsequent in vitro transcription experiments.
And 6, carrying out enzyme digestion identification on the plasmid. Carrying out double enzyme digestion by using restriction enzyme Knp1 and EcoR1, wherein the enzyme digestion reaction system is 10 multiplied by Buffer 5 ul; pcDNA3.1-CCR 92 μ g; restriction enzyme Knp1 and EcoR1 each 1 ul; adding enzyme-free water to make up to 20ul of the total volume; the digestion was carried out at 37 ℃ for 20 min. After the enzyme digestion, 6ul 15000Marker and 6ul enzyme digestion product are taken for 1.5 percent agarose gel electrophoresis detection. After the agarose gel electrophoresis identification is finished, the DNA sequence of the plasmid is determined by Shanghai worker, and the sequencing result is compared with the sequence SEQ ID NO.9, thereby proving that the pcDNA3.1-CCR9 eukaryotic expression vector is successfully constructed.
Step two, isolated culture of umbilical cord mesenchymal stem cells
Step 1: taking down the umbilical cord from the operating table, immersing the umbilical cord in 0.9% physiological saline containing antibiotics, and storing at 4 ℃;
step 2: taking out umbilical cord from super clean bench, washing residual blood in umbilical artery and umbilical vein with physiological saline, removing the above blood vessel with hemostatic forceps and scissors, and cutting umbilical cord into tissue blocks with size of 1mm ^ 3;
and step 3: adding collagenase type II with the mass/volume ratio of 0.1% to completely cover the tissue block, placing the tissue block in an incubator for continuous digestion for 6 hours, and filtering and collecting cells by a 100-mesh screen;
and 4, step 4: washing cells with physiological saline for 8min at 2000rpm for 3 times;
and 5: resuspending cells in DMEM/F12 culture medium containing 10% FBS, adjusting cell density to 1 × 106cells/ml, inoculating into T.25 culture flask, placing at 37 deg.C, saturated humidity, and 5% C02Culturing in an incubator;
step 6: changing the culture solution to MesenPRORSrMMedium after 3 days, discarding the non-adherent cells and impurities, and changing the culture solution every 3 days according to the growth condition of the cells.
Step three, transiently transfecting umbilical cord mesenchymal stem cells by using liposome
Umbilical cord mesenchymal stem cells in MesenPRORSrMMedium culture medium with 5% CO at 37 DEG C2After culturing until the cell density reaches 80%, transfecting pcDNA3.1-CCR9DNA to the umbilical cord mesenchymal stem cells by using a Lipofectamine TM 2000 kit, wherein the specific steps refer to the kit specification. After 24h, the cells were transfected with 5. mu.g of pcDNA3.1-CCR9 DNA.
Example 2
This example illustrates a stem cell biological product and a method for preparing the same according to the present disclosure, including most of the steps of example 1, except that:
in the first step, the amplification primers designed in the step 1 are:
CCR9-Kpn1-F2:ggggtaccaa tgacacccac agacttcaca(SEQ ID NO.3)
CCR9-EcoR1-R1:cggaattctc agagggagag tgctcctg(SEQ ID NO.4)；
Constructing pcDNA3.1-HisA, B, C-CCR9 expression vector.
In the third step, the umbilical cord mesenchymal stem cells are transfected by pcDNA3.1-HisA, B, C-CCR 9.
Example 3
This example illustrates a stem cell biological product and a method for preparing the same according to the present disclosure, including most of the steps of example 1, except that:
in the first step, the amplification primers designed in the step 1 are:
CCR9-EcoR1-F3:cggaattcat gacacccaca gacttcaca(SEQ ID NO.5)
CCR9-Kpn1-R3:ggggtacccg gagggagagt gctcctgagg(SEQ ID NO.6)；
and step 3, linking the target fragment CCR9 with a commercial plasmid vector pcDNA3.1-3 xflag.
Constructing pcDNA3.1-3xflag-CCR9 expression vector.
In the third step, the umbilical cord mesenchymal stem cells are transfected by pcDNA3.1-3xflag-CCR 9.
Example 4
This example illustrates a stem cell biological product and a method for preparing the same according to the present disclosure, including most of the steps of example 1, except that:
in the first step, the amplification primers designed in the step 1 are:
CCR9-Kpn1-F1:ggggtaccat gacacccaca gacttcaca(SEQ ID NO.7)
CCR9-EcoR1-R4:cggaattccg gagggagagt gctcctgagg(SEQ ID NO.8)；
Constructing pcDNA6/MYC-HIS A, B, C-CCR9 expression vector.
In the third step, the umbilical cord mesenchymal stem cells are transfected by pcDNA6/MYC-HIS A, B, C-CCR 9.
Comparative example 1
This comparative example, which is used for comparative illustration of the stem cell bioproduct and the preparation method thereof disclosed in the present invention, includes most of the operation steps in example 1, except that:
constructing a recombinant plasmid vector carrying the target gene CCR9 without the step I;
and in the third step, the umbilical cord mesenchymal stem cells are treated by adopting a serum-free culture medium to replace a Lipofectamine TM 2000 kit.
Comparative example 2
This comparative example, which is used for comparative illustration of the stem cell bioproduct and the preparation method thereof disclosed in the present invention, includes most of the operation steps in example 1, except that:
constructing a recombinant plasmid vector carrying the target gene CCR9 without the step I;
in the third step, the umbilical cord mesenchymal stem cells are cultured in MesenPRORSrMMedium culture solution at 37 ℃ with 5% CO2After culturing until the cell density reaches 80%, transfecting pcDNA3.1 to the umbilical cord mesenchymal stem cells by using a Lipofectamine TM 2000 kit, wherein the specific steps refer to the kit specification. After 24h, the cells were transfected with 5. mu.g of pcDNA3.1DNA.
Performance testing
Detection of CCR9 expression condition of transfected umbilical cord mesenchymal stem cells
Adding a sample buffer solution into a 50-80 mu g protein sample according to a proportion, carrying out boiling water bath for 10min, cooling and centrifuging, carrying out sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12% separation gel, carrying out wet transfer to a PVDF membrane by a wet transfer method at 110V 90min, sealing for 1h at room temperature by using phosphate buffer solution and 5% skimmed milk powder dissolved in TBST, incubating a primary antibody [ rabbit-derived anti-CCR 9 monoclonal antibody (1: 500), rabbit-derived anti- β -actin monoclonal antibody (1: 1000) ] for 4 ℃ overnight, washing for 4 times (8 min each time) the next day by using TBST, then incubating a horseradish peroxidase-labeled secondary antibody (mouse anti-rabbit monoclonal antibody IgG 1: 5000) for 2h at room temperature, and finally detecting the protein expression condition on the PVDF membrane by using ECL.
The test results are shown in fig. 1, in which reference numeral 1 is the electrophoresis result of comparative example 1, reference numeral 2 is the electrophoresis result of comparative example 2, reference numeral 3 is the electrophoresis result of example 1, reference numeral 4 is the electrophoresis result of example 2, reference numeral 5 is the electrophoresis result of example 3, and reference numeral 6 is the electrophoresis result of example 4.
From the test results of fig. 1, it can be seen that examples 1 to 4 can promote high expression of CCR9 protein in umbilical cord mesenchymal stem cells by transfecting the CCR9 gene into umbilical cord mesenchymal stem cells by means of a plasmid vector, compared to the control group of comparative example 1 and comparative example 2.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
<110> Shenzhen Hospital of parakeen regenerative medicine science and technology Limited company Beijing university
<120> stem cell biological product for treating liver cirrhosis and preparation method and application thereof
<160>10
<170>SIPOSequenceListing 1.0
<210>1
<211>29
<212>DNA
<213> Artificial sequence
<400>1
ggggtaccat gacacccaca gacttcaca 29
<210>2
<211>28
<212>DNA
<213> Artificial sequence
<400>2
cggaattctc agagggagag tgctcctg 28
<210>3
<211>30
<212>DNA
<213> Artificial sequence
<400>3
ggggtaccaa tgacacccac agacttcaca 30
<210>4
<211>28
<212>DNA
<213> Artificial sequence
<400>4
cggaattctc agagggagag tgctcctg 28
<210>5
<211>29
<212>DNA
<213> Artificial sequence
<400>5
cggaattcat gacacccaca gacttcaca 29
<210>6
<211>30
<212>DNA
<213> Artificial sequence
<400>6
ggggtacccg gagggagagt gctcctgagg 30
<210>7
<211>29
<212>DNA
<213> Artificial sequence
<400>7
ggggtaccat gacacccaca gacttcaca 29
<210>8
<211>30
<212>DNA
<213> Artificial sequence
<400>8
cggaattccg gagggagagt gctcctgagg 30
<210>9
<211>1110
<212>DNA
<213> CCR9 gene
<400>9
atgacaccca cagacttcac aagccctatt cctaacatgg ctgatgacta tggctctgaa 60
tccacatctt ccatggaaga ctacgttaac ttcaacttca ctgacttcta ctgtgagaaa 120
aacaatgtca ggcagtttgc gagccatttc ctcccaccct tgtactggct cgtgttcatc 180
gtgggtgcct tgggcaacag tcttgttatc cttgtctact ggtactgcac aagagtgaag 240
accatgaccg acatgttcct tttgaatttg gcaattgctg acctcctctt tcttgtcact 300
cttcccttct gggccattgc tgctgctgac cagtggaagt tccagacctt catgtgcaag 360
gtggtcaaca gcatgtacaa gatgaacttc tacagctgtg tgttgctgat catgtgcatc 420
agcgtggaca ggtacattgc cattgcccag gccatgagag cacatacttg gagggagaaa 480
aggcttttgt acagcaaaat ggtttgcttt accatctggg tattggcagc tgctctctgc 540
atcccagaaa tcttatacag ccaaatcaag gaggaatccg gcattgctat ctgcaccatg 600
gtttacccta gcgatgagag caccaaactg aagtcagctg tcttgaccct gaaggtcatt 660
ctggggttct tccttccctt cgtggtcatg gcttgctgct ataccatcat cattcacacc 720
ctgatacaag ccaagaagtc ttccaagcac aaagccctaa aagtgaccat cactgtcctg 780
accgtctttg tcttgtctca gtttccctac aactgcattt tgttggtgca gaccattgac 840
gcctatgcca tgttcatctc caactgtgcc gtttccacca acattgacat ctgcttccag 900
gtcacccaga ccatcgcctt cttccacagt tgcctgaacc ctgttctcta tgtttttgtg 960
ggtgagagat tccgccggga tctcgtgaaa accctgaaga acttgggttg catcagccag 1020
gcccagtggg tttcatttac aaggagagag ggaagcttga agctgtcgtc tatgttgctg 1080
gagacaacct caggagcact ctccctctga 1110
<210>10
<211>1531
<212>DNA
<213> Artificial plasmid
<400>10
cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg 60
taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat 120
aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac 180
gactcactat agggagaccc aagctggcta gcgtttaaac ttaagcttgg taccatgaca 240
cccacagact tcacaagccc tattcctaac atggctgatg actatggctc tgaatccaca 300
tcttccatgg aagactacgt taacttcaac ttcactgact tctactgtga gaaaaacaat 360
gtcaggcagt ttgcgagcca tttcctccca cccttgtact ggctcgtgtt catcgtgggt 420
gccttgggca acagtcttgt tatccttgtc tactggtact gcacaagagt gaagaccatg 480
accgacatgt tccttttgaa tttggcaatt gctgacctcc tctttcttgt cactcttccc 540
ttctgggcca ttgctgctgc tgaccagtgg aagttccaga ccttcatgtg caaggtggtc 600
aacagcatgt acaagatgaa cttctacagc tgtgtgttgc tgatcatgtg catcagcgtg 660
gacaggtaca ttgccattgc ccaggccatg agagcacata cttggaggga gaaaaggctt 720
ttgtacagca aaatggtttg ctttaccatc tgggtattgg cagctgctct ctgcatccca 780
gaaatcttat acagccaaat caaggaggaa tccggcattg ctatctgcac catggtttac 840
cctagcgatg agagcaccaa actgaagtca gctgtcttga ccctgaaggt cattctgggg 900
ttcttccttc ccttcgtggt catggcttgc tgctatacca tcatcattca caccctgata 960
caagccaaga agtcttccaa gcacaaagcc ctaaaagtga ccatcactgt cctgaccgtc 1020
tttgtcttgt ctcagtttcc ctacaactgc attttgttgg tgcagaccat tgacgcctat 1080
gccatgttca tctccaactg tgccgtttcc accaacattg acatctgctt ccaggtcacc 1140
cagaccatcg ccttcttcca cagttgcctg aaccctgttc tctatgtttt tgtgggtgag 1200
agattccgcc gggatctcgt gaaaaccctg aagaacttgg gttgcatcag ccaggcccag 1260
tgggtttcat ttacaaggag agagggaagc ttgaagctgt cgtctatgtt gctggagaca 1320
acctcaggag cactctccct ctgagaattc tgcagatatc cagcacagtg gcggccgctc 1380
gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag 1440
ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact 1500
gtcctttcct aataaaatga ggaaattgca t 1531
Claims (10)
1. A stem cell biologic comprising a mesenchymal stem cell transfected with a vector comprising a CCR9 gene sequence.
2. The stem cell biologic of claim 1, wherein said mesenchymal stem cell is a mesenchymal stem cell isolated outside the body of a mammal, said mesenchymal stem cell comprising one or more of an umbilical cord mesenchymal stem cell, a placental mesenchymal stem cell, an adipose mesenchymal stem cell, a bone marrow mesenchymal stem cell, and a dental pulp mesenchymal stem cell.
3. The stem cell biologic of claim 1, wherein said vector is selected from the group consisting of plasmid vectors comprising one or more of pcDNA3.1+, pcDNA3.1-HisA, B, C, pcDNA3.1-3xflag, and pcDNA6/MYC-HIS A, B, C.
4. The stem cell biologic according to claim 1, wherein said CCR9 gene sequence comprises the sequence shown as SEQ ID No.9, and the vector into which CCR9 gene sequence is transferred comprises the sequence shown as SEQ ID No. 10.
5. The method for producing a stem cell biological product according to any one of claims 1 to 4, comprising the following operations:
obtaining a CCR9 target gene;
constructing a vector comprising a CCR9 gene sequence;
obtaining mesenchymal stem cells;
the vector transfects mesenchymal stem cells.
6. The stem cell biological product according to claim 5, wherein the step of obtaining CCR9 target gene comprises the following operations:
collecting a liver tissue sample, extracting total RNA, designing an amplification primer, amplifying the full length of a CCR9 gene by a reverse transcription-polymerase chain reaction PCR method, carrying out electrophoresis, and purifying a CCR9 target gene fragment.
7. The stem cell biological product according to claim 5, wherein after the "construction of the vector comprising the CCR9 gene sequence", the vector comprising the CCR9 gene sequence is transfected into prokaryotic cells for expansion culture, and the vector comprising the CCR9 gene sequence is extracted.
8. The stem cell biological product of claim 5, wherein the route of obtaining the mesenchymal stem cells comprises obtaining umbilical cord, placenta, fat, bone marrow or dental pulp, culturing and separating to obtain the mesenchymal stem cells.
9. The stem cell biologic according to claim 5, wherein said means for "transfecting mesenchymal stem cells with a vector" comprises electroporation, calcium phosphate method, liposome-mediated method, and virus-mediated method.
10. Use of a stem cell bioproduct according to any one of claims 1 to 4 for the preparation of a medicament for the treatment of cirrhosis.
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