CN107164331B - Liver injury targeted mesenchymal stem cell based on miR-221 and preparation method and application thereof - Google Patents

Abstract

The invention discloses a liver injury targeting mesenchymal stem cell based on miR-221 and a preparation method and application thereof, wherein the liver injury targeting mesenchymal stem cell is prepared by transfecting or infecting nucleic acid by the mesenchymal stem cell, the high expression miR-221 of the liver injury targeting mesenchymal stem cell is specifically prepared by transfecting a miR-221 analogue (mimic or agomir) by a human mesenchymal stem cell or preparing a virus for infecting and expressing miR-221; in order to further improve the efficiency of targeting MSCs to the site of liver injury, and improve the effects of liver function and tissue architecture recovery, mesenchymal stem cells can be treated with basic fibroblast growth factor. The liver injury targeting mesenchymal stem cell has efficient targeting on liver injury, is beneficial to relieving liver fibrosis and improving liver function, has high stability, is convenient to store and transport, is safe to use, and can bring gospel to patients with liver diseases.

Description

Liver injury targeted mesenchymal stem cell based on miR-221 and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a liver injury targeting mesenchymal stem cell based on miR-221, and a preparation method and application thereof.

Background

Acute and chronic liver injury can be caused by various reasons such as virus, parasite infection, ethanol, drug or chemical toxicity, immune dysfunction and the like, and cirrhosis can be caused even to liver failure and end-stage liver disease by long-term or repeated action. Once the liver disease progresses to the terminal stage, the conventional treatment can only relieve the clinical symptoms of the patient and has poor curative effect. Mesenchymal Stem Cells (MSCs) are mesoderm-derived adult stem cells with a multipotentiality, which are mainly present in connective tissues and organ interstitium, most abundant in bone marrow tissues. The mesenchymal stem cells have the unique unlimited proliferation and self-renewal capacity of the stem cells, and can still keep dry after in vitro subculture and cryopreservation recovery; the cell has multi-differentiation potential, and can be differentiated into various types of cells such as osteoblasts, chondrocytes, adipocytes and the like under appropriate in-vivo and in-vitro environments; the surface antigen is not obvious, has low immunogenicity and has an immunoregulation function, so that the surface antigen is widely applied to tissue and organ damage repair caused by aging and pathological changes as an ideal seed cell. The scholars at home and abroad apply the MSCs from different sources to liver disease animal models and treatment of clinical and preclinical liver diseases, and obtain some meaningful findings and favorable treatment effects. Research shows that MSCs can be induced to differentiate into cells with morphological, phenotypic and functional characteristics of hepatocytes in vitro, and the cells can express specific markers of hepatic cells and cholangiocytes such as albumin (albumin), alpha-fetoprotein (AFP), Cytokeratins (CK) CK8 and CK 18.

However, the treatment of liver diseases by MSCs transplantation is only in the theoretical stage, and many problems to be solved still exist in clinical application, especially the curative effect is not stable, and the individual difference is large; further studies have shown that MSCs transplantation has no effect, even negative effects, on the repair of liver function, tissue architecture. On the one hand, on the other hand, the transplanted MSCs can only perform the treatment and repair effects after being planted/homing to the lesion part, but in vivo research finds that only a small amount of transplanted cells are enriched to the injury or lesion part; secondly, transplanted MSCs are transplanted to the liver injury part, and are differentiated into hepatophagy cells (kupffer cells), and the hepatophagy cells have promotion effects on the activation, proliferation, migration, survival and the like of hepatic stellate cells (hepatic stellate cells), and are not beneficial to the repair of inflammation and fibrosis; some myofibroblasts (myofibroblast) are gathered to promote collagen secretion and fibrosis; some can transdifferentiate into functional hepatic stellate cells and myofibroblasts, and secrete more extracellular matrix such as collagen, thereby further enhancing fibrotic damage of the liver (see: ZHao Q, Ren H, Zhu D, Han Z. Stem/promoter cells in liver deficiency and regeneration. biolCell, 2009, 101(10): 557 571; Ji J, He BP, Dheen ST, Tay SS. Interactions of chemins and chemo receptors in the differentiation of metabolic Stem cells to the amplified site in the fibrous extracellular mineral in Stem cells 2004, 22(3): 427).

microRNA (micro RNA, miRNA) is an endogenous non-coding small RNA molecule with the length of about 20-24 nucleotides, and is generated by processing a single-stranded RNA precursor with a hairpin structure and the size of about 70-90 nucleotides by Dicer enzyme. miRNA binds to 3'-untranslated region (3' -UTR) of target mRNA by base pairing, inhibits its translation, negatively regulates expression of target mRNA, and further regulates various biological behaviors such as development process, stem cell differentiation, cell proliferation and apoptosis diseases, and tumorigenesis. In recent years, miR-221 has been studied to show that it is overexpressed in various tumor cells, and is also associated with angiogenesis. miR-9 is primarily expressed in the brain of vertebrates to regulate neural differentiation. The hypoxic environment can induce the expression of miR-26b, which is up-regulated in smooth muscle cell differentiation and neurogenesis and has different expression in different tumors. These results show that different miRNAs have different functions and are related to the environments and cell types, and whether miR-221 can promote MSCs to target liver injury areas in vivo and play a therapeutic role has not been reported.

Disclosure of Invention

The invention aims to provide a high-efficiency liver injury targeting mesenchymal stem cell and a preparation method thereof, wherein the number of MSCs (mesenchymal stem cells) homing to a focus part is increased based on miR-221, and the colonization capacity of the mesenchymal stem cell at the focus part is improved; is beneficial to improving the targeting of the MSCs to liver injury and improving the treatment and repair effects of the MSCs to liver function and liver tissue structure.

The invention adopts the following technical scheme to realize the purpose of the invention:

a liver injury targeting mesenchymal stem cell is prepared by transfecting or infecting nucleic acid of the mesenchymal stem cell; the nucleic acid is miR-221. Preferably, the liver injury targeting mesenchymal stem cell is obtained by transfecting or infecting nucleic acid with mesenchymal stem cells and then treating with alkaline fibroblast growth factor.

In the above technical solution, the mesenchymal stem cells are derived from bone marrow, adipose tissue, dental pulp, umbilical cord blood, amniotic fluid or placenta. The mesenchymal stem cells in the invention have wide sources, can be autologous or allogeneic, have sufficient sources, have excellent capacities in various aspects of cell proliferation, survival, differentiation, migration, synthesis, factor secretion and the like, have no ethical problem, have low immunogenicity and have wide application prospect.

The invention also discloses a preparation method of the hepatic injury targeted mesenchymal stem cell, wherein miR-221 is transferred into the mesenchymal stem cell by a transfection reagent to obtain the hepatic injury targeted mesenchymal stem cell; or transferring the miR-221 into the mesenchymal stem cells through a virus vector to obtain the liver injury targeted mesenchymal stem cells. The method for realizing high expression of miR-221 by MSCs comprises the steps of transfecting miR-221 by using various transfection reagents, including liposome transfection reagents such as Lipofectamine2000 and the like; and viruses which are clinically proved to be safe and effective, such as adeno-associated virus AAV, Adenovirus Adenoviral, retrovirus, lentivirus and the like, are utilized to construct recombinant viruses for expressing miR-221 precursor to infect MSCs. And after 24-48 h of transfection or infection, carrying out qRT-PCR detection on the miR-221 significant high expression to obtain the liver injury targeted mesenchymal stem cells.

In the technical scheme, the step of transferring miR-221 into mesenchymal stem cells through a transfection reagent to obtain the liver injury target mesenchymal stem cells comprises the steps of diluting the miR-221 simulant and the transfection reagent respectively with L-DMEM, mixing, and standing at room temperature to obtain a transfection mixed solution; then washing the mesenchymal stem cells cultured to the 4 th-8 th generation and with the confluence degree of 80% -90% by PBS, and then adding transfection mixed solution and L-DMEM; then incubating in an incubator at 37 ℃ for 5-6 h, replacing the transfection mixed solution with a complete culture medium, and continuously culturing for 36-48 h to obtain the liver injury targeted mesenchymal stem cells; transferring miR-221 into mesenchymal stem cells through a virus carrier to obtain the liver injury targeted mesenchymal stem cells, namely washing the mesenchymal stem cells which are cultured to the 4 th-8 th generation and have the confluence degree of 80% -90% by PBS, then adding virus supernatant for expressing miR-221, infecting for 90min at 37 ℃, and replacing the virus supernatant with a complete culture medium to continuously culture for 24-48 h to obtain the liver injury targeted mesenchymal stem cells. For example, 50 nM miR-221 mice and 5 μ l Lipofectamine2000 are respectively diluted by 250 μ l L-DMEM, and the two are combined after standing for 10 min at room temperature and standing for 30 min at room temperature. Washing 2 times with MSCs PBS cultured to the 4 th-8 th generation and with the confluence degree of 80% -90%, adding the transfection mixed solution and 500 mu l L-DMEM, incubating for 6 h in an incubator at 37 ℃, changing into a complete culture medium, and continuing culturing for 48 h. Or selecting MSCs cultured to the 4 th-8 th generation and with the confluence degree reaching 80% -90%, washing with PBS for 2 times, adding virus supernatant for expressing miR-221, infecting for 90min at 37 ℃, and then replacing with a complete culture medium to continue culturing for 24-48 h. And (3) detecting miR-221 by qRT-PCR (quantitative reverse transcription-polymerase chain reaction) to obtain the mesenchymal stem cell targeted by the hepatic injury, wherein the miR-221 is remarkably high in expression and good in cell state.

In the technical scheme, miR-221 is transferred into mesenchymal stem cells through a transfection reagent, and then the mesenchymal stem cells are treated by basic fibroblast growth factor (bFGF) to obtain liver injury targeted mesenchymal stem cells; or transferring the miR-221 into the mesenchymal stem cells through a virus vector, and then treating the mesenchymal stem cells through alkaline fibroblast growth factors to obtain the hepatic injury targeted mesenchymal stem cells. For example, the mesenchymal stem cells transferred into miR-221 are placed in a culture medium with an alkaline fibroblast growth factor and are treated for 0.5-24 h to obtain liver injury targeted mesenchymal stem cells; in the culture medium with the basic fibroblast growth factor, the concentration of the basic fibroblast growth factor is 5-20 ng/mL; the application of bFGF to treatment of MSCs can further improve the efficiency of targeting MSCs to liver injury parts and improve the effects of liver function and tissue structure recovery.

Micrornas (miRNAs) are small, siRNA-like nucleic acid molecules encoded by a genome, and the expression profiles and expression levels of miRNAs molecules vary greatly among different tissue cell types, and thus the functions of different miRNAs may vary greatly, even though the same miRNA may function in different tissue cell types. The affinity of the mesenchymal stem cells in different modification states and in vivo cytokines is different, so that the chemotaxis and the planting quantity are different, the mesenchymal stem cells planted in the liver are different in quantity, the differentiation, transdifferentiation efficiency, paracrine and other effects are different, the biological effects exerted by the mesenchymal stem cells are different, and the improvement on diseases is different.

The liver injury targeted mesenchymal stem cells disclosed by the invention can be efficiently planted at the liver injury part, avoid blood flow scouring or natural falling, increase the amount of homing cells, and underexpress p27 and PTEN proteins, thereby being beneficial to the repair effect after liver injury. Therefore, the invention also discloses the application of the liver injury targeted mesenchymal stem cells in the preparation of targeted drugs for treating liver injury; the method for causing liver injury mainly comprises viral infection, parasitic infection, ethanol toxicity, drug toxicity, chemical toxicity or immunologic dysfunction, the liver injury in animals mainly shows that cells at a focus part wither and fall, tissues are necrotic and the like, and the repair of the liver injury mainly comprises the steps of stably protecting damaged liver cells by various methods, promoting recovery of diseased cells, stimulating DNA synthesis of normal liver cells, promoting regeneration of the liver cells and relieving hepatic fibrosis, so that a normal liver tissue structure is reconstructed, and the liver function is repaired. The mesenchymal stem cell medicine is generally an injection type, is infused through blood vessels and reaches a focus part along with blood circulation. Compared with the in vitro method, the blood environment in the animal body is complex, various substances and factors exist, the microenvironment difference of different parts is large, and the transfer of the mesenchymal stem cells is obviously influenced; and the damaged part of the liver is an active tissue, has self-metabolism, is influenced by blood flow and an immune system, and is not beneficial to the field planting and survival of foreign cells. The main reasons for the poor colonization ability and the small number of homing cells of the mesenchymal stem cells with good in-vitro effects (strong proliferation and migration capabilities) at the liver injury part in vivo in the prior art are also shown.

The invention discloses a preparation method of a targeted drug for treating liver injury, which is used for preparing the targeted drug for treating liver injury by mixing the liver injury targeted mesenchymal stem cells with pharmaceutical excipients. The invention discloses a targeted medicine for treating liver injury, which comprises the liver injury targeted mesenchymal stem cells; also comprises medicinal auxiliary materials. The pharmaceutical adjuvants are available in the prior art, such as buffer solution, physiological saline, etc.

The mesenchymal stem cells disclosed by the invention can obviously improve the number of transplanted cells homing to the damaged part of the liver, improve the colonization ability of the cells and provide a good foundation for effectively repairing the damaged liver.

In the invention, microRNA-221(miR-221) regulates the directional enrichment of MSCs, miR-221 is expressed in the MSCs, and the capability of the MSCs towards liver injury parts is remarkably promoted; particularly, after being treated by 5-20 ng/mL (preferably 10 ng/mL) of bFGF, the capability of MSCs (messenger ribonucleic acid) expressing miR-221 to tend to liver injury parts is remarkably promoted, and the tissue structure and the function of the injury parts are repaired; and the expression of E-cadherin protein in the MSCs expressing miR-221 is reduced, so that the fibrosis promoting risk of transplanted MSCs is reduced, and the liver injury repair is facilitated. According to the embodiment of the invention, a recombinant adenovirus (Ad-221) expressing miR-221 is constructed, after the MSCs are infected with Ad-22148 h, the recombinant adenovirus is transplanted into a mouse body of an acute and chronic liver injury model, cells are treated by a culture medium containing bFGF for 24 h one day before transplantation, blood of the mouse is collected after 7d and 14 d transplantation for analyzing liver functions, the mouse is killed, liver tissues are taken for frozen section and paraffin section, immunofluorescence staining, HE staining or Masson staining is carried out, the number and distribution of the transplanted cells in the liver tissues are observed, and pathological repair conditions such as liver tissue inflammation, fibrosis and the like are analyzed; the result shows that the MSCs with high miR-221 expression transplantation has good treatment and repair effects on liver injury, and theoretical and experimental basis is provided for clinical application.

Drawings

FIG. 1 is an expression diagram of human umbilical cord-derived mesenchymal stem cells carrying blue fluorescence and high expression of miR-221 and miR-221 in example I;

FIG. 2 shows that human umbilical cord-derived mesenchymal stem cells highly expressing miR-221 are transplanted into CCl by tail vein injection in example I₄Cryo-section of liver after 7d in induced acute liver injury mice;

FIG. 3 shows an example of transplantation of human umbilical cord-derived mesenchymal stem cells highly expressing miR-221 into CCl by tail vein injection₄Statistical graphs of mouse liver indices after 7d in induced acute liver injury mice;

FIG. 4 is an expression diagram of human umbilical cord-derived mesenchymal stem cells carrying green fluorescence and miR-221 with high miR-221 expression prepared in example II;

FIG. 5 is the human umbilical cord-derived mesenchymal stem cells of example two, which highly express miR-221, transplanted into CCl by tail vein injection₄Cryo-section of liver after 7d in induced chronic liver injury mice;

FIG. 6 shows the human umbilical cord-derived mesenchymal stem cells highly expressing miR-221 transplanted into CCl by tail vein injection in example II₄Statistical graphs of mouse liver indices after 7d in induced acute liver injury mice;

FIG. 7 shows the human umbilical cord-derived mesenchymal stem cells of miR-221-expressing in example III transplanted into CCl by tail vein injection₄HE staining pattern of paraffin sections of liver after 7d in induced acute liver injury mice;

FIG. 8 shows the human umbilical cord-derived mesenchymal stem cells of miR-221-expressing in example III transplanted into CCl by tail vein injection₄Cryo-section of liver after 7d in induced chronic liver injury mice;

FIG. 9 shows the human umbilical cord-derived mesenchymal stem cells of miR-221-expressing in example III transplanted into CCl by tail vein injection₄HE staining pattern of paraffin sections of liver after 7d in induced chronic liver injury mice;

FIG. 10 shows the transplantation of human umbilical cord-derived mesenchymal stem cells expressing miR-221 by tail vein injection into CCl in example₄Masson staining pattern of paraffin sections of liver after 7d in induced chronic liver injury mice.

Detailed Description

The present invention will be further described with reference to the following examples.

Example A human umbilical cord-derived mesenchymal stem cell highly expressing miR-221 is transplanted to a chronic liver injury mouse model

1. Preparation of chronic liver injury experimental animal model

Kunming mouse, 40 mice, male. The random number table method was divided into a normal control group (10) and a model group (50). The model groups were administered 3 ml/kg of a mixture of CCl4 and olive oil at a volume of 1:1 subcutaneously 2 times a week (fixed time points at 14:00 pm on tuesdays and thursdays), and normal control groups were administered with an equal dose of olive oil subcutaneously. After 6 weeks of continuous injection, 1 injection was performed once a week. Until week 10. The transplantation experiment was performed 24 hours after the last molding at the 10 th week of the modeling.

2. And (4) separating, culturing and identifying the MSCs.

Human umbilical cord-derived mesenchymal stem cells (HUCMSCs) were used. Collecting umbilical cord of newborn about 10 cm, removing external membrane layer and internal artery and vein of umbilical cord, reserving Wharton jelly, mechanically digesting, cutting to about 1 mm × 1 mm × 1 mm, spreading and inoculating to 75 cm²Flask, 10% FBS + L-DMEM, 37 ℃, saturated humidity, 5% CO₂Culturing in an incubator, observing by inverting a phase difference microscope day by day, changing liquid after 3-5 days, changing liquid by using L-DMEM containing 10% FBS for every 3 days, removing tissue blocks after the cells are fused to 50%, continuously culturing, and carrying out passage after the cells are fused to 80% -90%. Taking 1-5 generations of passage cells to prepare a single cell suspension, taking 0.4% trypan blue as a staining agent, counting live cells and dead cells by using a cell counting plate for 3 times, taking an average value of 3 times, and calculating the cell survival rate: viable cell rate (%) = (number of unstained cells/total number of observed cells) × 100%. The activity rate is more than 90%.

The morphology of HUCMSCs with different growth cycles has different characteristics, and the growth condition and the morphological characteristics of cells are observed by using an inverted phase contrast microscope and a fluorescence microscope.

Adherent cells are conventionally digested with 0.25% trypsin, terminated with L-DMEM containing L0% fetal bovine serum, washed by adding PBS containing 1% FBS, labeled with FITC-labeled CD105 (SH2), CD44 antibody and their corresponding isotype control-labeled cells, and detected by flow cytometry.

Cell surface CD molecule detection: the cells of 1 st to 8 th generation were collected and cultured, and divided into 1X 10 cells per EP tube (1.5 ml)⁵Cells were washed and resuspended in 20. mu.l PBS, and mouse anti-human straight-mark PE or FITC monoclonal antibodies CD90, CD44, CD105 and CD73 were added to establish negative controls. Detecting by a flow cytometer, and analyzing the result by Cell Quest software.

3. Preparation of transplanted cells

Diluting the 50 nM miR-221 simulant and 5 mul of transfection reagent Lipofectamine2000 with 250 mul l L-DMEM respectively, standing at room temperature for 10 min, mixing the two, and standing at room temperature for 30 min to obtain a transfection mixed solution; culturing HUCMSCs cultured to the 8 th generation until the culture confluency reaches 80-90%, discarding the culture medium, washing for 2 times by PBS, and adding transfection mixed liquid and 500 mu l L-DMEM; and then incubating in an incubator at 37 ℃ for 5-6 h, replacing the transfection mixed solution with a complete culture medium, and continuing culturing for 36-48 h to obtain cells with high miR-221 expression, wherein the cells can be used for cell transplantation.

4. Preparation of cell suspension for transplantation

(1) HUCMSCs transfect a high-expression miR-221 analogue (221-imic) and a disordered Negative Control (NC), and after the HUCMSCs are cultured for 36 hours, cells with high-expression miR-221 and Control cells are obtained and can be used for cell transplantation. Before transplantation, cells are marked with blue fluorescence by H33258 for 30 min, the original culture medium is discarded, the cells are washed by PBS for 2 times, 0.25% trypsin digestion solution is added for digestion, after the shortening deformation of the cells is observed under a microscope, complete culture medium is added to stop digestion, the cells are blown to prepare single cell suspension, the single cell suspension is centrifuged for 5 min at 1000 r/min, the supernatant is removed, then normal saline is used for resuspension, washing and centrifugation are carried out for 2 times, and the components of the culture solution are removed. Adding physiological saline to resuspend cells, counting cells, adjusting cell density to 2 × 10⁶And/ml, marked as 221-mic-HUCMSCs and NC-HUCMSCs cell suspension, and stored at 4-10 ℃ for later use, and the storage period is 12 h.

In order to further improve the efficiency of targeting MSCs to a damaged liver site and improve the recovery effect of liver function and tissue structure, 221-mic-HUCMSCs and NC-HUCMSCs cells are cultured for 12h in a culture medium containing 20 ng/ml basic fibroblast growth factor (bFGF) before transplantation, and then labeled, digested, washed and resuspended in physiological saline according to the steps (1) to prepare 2 × 10⁶bFGF-treated 221-mix-HUCMSCs, NC-HUCMSCs cell suspension/ml.

FIG. 1 is a graph of expression quantification of human umbilical cord-derived mesenchymal stem cells highly expressing miR-221 and miR-221, wherein the graph scale bar shows 250 μm; separating cultured human umbilical cord-derived mesenchymal stem cells (HUCMSCs), and after multiple passages, determining the cell viability of each passage to be more than 95% by trypan blue staining; the detection result of the flow cytometry shows that the cells cultured to the 3 rd generation express CD90⁺、CD44⁺、CD105⁺、CD73⁺And NOT-HUCMSCs are negative, and accord with the biological characteristics of HUCMSCs. After 48 hours of miR-221 mimics transfection, the expression quantity of miR-221 is obviously increased compared with that of a transfection negative control group.

5. Cell transplantation

The chronic injury animal model was randomly divided into a cell treatment group (40) and a model control group (10). The cell transplantation treatment group is respectively given 221-imic-HUCMSCs cell suspension, 221-imic-HUCMSCs cell suspension treated by bFGF, NC-HUCMSCs cell suspension and NC-HUCMSCs cell suspension treated by bFGF at one time in tail vein, and the dosage is 1.0X 10⁶In terms of/kg. The tail vein of the model control group was given an equal dose of saline. The tail vein of the normal control group animals is given with normal saline with equal dosage at one time.

Randomly selecting 5 mice respectively at 7d and 14 d after cell transplantation, performing ether anesthesia, collecting eyeball blood, and detecting liver function indexes such as ALT, AST, ALB, TBIL, blood coagulation function, etc.; and (4) killing after blood sampling, weighing the liver, and measuring and calculating the organ index. Then, a part of liver is taken out for freezing section, the distribution condition of blue fluorescence positive cells in the section is observed under a fluorescence microscope and planted in the liver of a mouse, the rest part is fixed by 4 percent paraformaldehyde, the section is embedded by liver tissue paraffin, and the pathological change of the liver is detected by HE staining and albumin immunohistochemistry.

CCl₄After the induced chronic liver injury mice are injected with NC-HUCMSCs and 221-imic-HUCMSCs cell suspension for 7d through tail veins, liver tissues are taken to be frozen sections, the distribution of transplanted blue fluorescence positive cells is observed under a fluorescence microscope, and the blue fluorescence intensity is counted. See fig. 2, in which the graphical scale indicates 250 μm, P < 0.001 compared to the NC-HUCMSCs group. The result shows that 7d after transplantation, the number of HUCMSCs highly expressing miR-221 in the damaged liver tissue is obviously higher than that of control cells, and the miR-221 can promote HUCMSCs to home and colonize in the damaged liver tissue.

FIG. 3 shows that the human umbilical cord-derived mesenchymal stem cells with high miR-221 expression are transplanted to CCl through tail vein injection₄Liver index after 7d in induced chronic liver injury mice. The results show that, compared to the chronic model group (CLI)PIs less than 0.01. The detection result shows that the liver index is not obviously reduced after the mouse with chronic liver injury is injected with NC-HUCMSCs through tail veins, and the liver index is obviously reduced after 221-mim-HUCMSCs are transplanted compared with a chronic model group and tends to a normal control group.

After the cells are transplanted, the liver function index of the mouse can be improved and even recovered to a normal control level, which shows that the stem cells based on the miR-221 can improve the liver function and the effect of recovering the tissue structure, and the ratio of each organ to the body weight is relatively constant in normal conditions.

Example II transplantation of human umbilical cord-derived mesenchymal stem cells highly expressing miR-221 into acute liver injury mouse model

1. Preparation of acute liver injury experimental animal model

Kunming mouse, 40 mice, male. The random number table method was divided into a normal control group (10) and a model group (50). The model groups are all injected with 5 ml/kg mixed solution of CCl4 and olive oil at one time (1:1) subcutaneously, and normal control groups are injected with olive oil at equal dose subcutaneously. After 24 h of modeling, a transplantation experiment was performed.

2. Construction of miR-221 recombinant adenovirus expression vector (Ad-221)

Using rno-miR-221 primer (SEQ ID No.1: 5'-TGGTCGACATTTCCTTATCTGTACTTC-3'; SEQ ID No.2: 5'-TCGCTCGAGGCATGTGAGACTGTTTTAG-3') with Sal I and Xho I enzyme cutting sites, using mesenchymal stem cell genome DNA as template PCR to obtain target gene fragment, recovering target gene fragment, connecting with pMD-19-T vector, transforming DH5 α competent cell, picking up single clone, overnight shaking culture and amplification, extracting plasmid, Sal I and Xho I double enzyme cutting identification, identifying correct positive clone, sending Shanghai biological sequencing identification, using Sal I and Xho I preset to make double enzyme cutting directional cloning, product transforming to Top10 competent cell, overnight shaking culture and amplification, extracting plasmid Sal I and Xho I double enzyme cutting identification, identifying correct recombinant shuttle vector, naming as pAdTrack-221, then connecting pAdTrack-221 and adenovirus skeleton-sensitive vector in BdAmJ-5183, and transferring the recombinant adenovirus skeleton of pAdAm-5183 into active recombinant adenovirus vector, so as to obtain the recombinant adenovirus vector⁺The recombinant strain was cultured and shaken under resistance to prepare competent cells. The recombinant shuttle vector pAdTrack-221 was linearized with Pme I restriction enzyme to expose its homologous recombination site, and the cleavage product was transformed into BJ5183 competent cells containing the pAdEasy-1 vector in Kan⁺And (2) culturing on a resistant LB plate, selecting a monoclonal, shaking, extracting plasmids, detecting the extracted plasmids by agarose gel electrophoresis, identifying the plasmids by Pac I single enzyme digestion, wherein the banding pattern and the size of an electrophoresis strip are similar to and close to those of pAdEasy-1 and can be positive clones with correct recombination, identifying the plasmids by Pac I single enzyme digestion, obtaining the recombinant adenovirus expressing miR-221 with complete correctness, and naming the recombinant adenovirus as pAdEasy-221, and respectively preserving the plasmids and bacterial liquid at the temperature of-80 ℃.

The recombinant adenovirus backbone plasmid pAdEasy-221 constructed above is subjected to single enzyme digestion by Pac I, a small amount of enzyme is taken for electrophoresis detection, and a product subjected to complete enzyme digestion is transfected into 293A cells with 70% confluence by Lipofectamine2000 according to the procedures of a reagent instruction and packaged. First wheel bagHarvesting cells after 7-10 days, repeatedly freezing and thawing for three times to release viruses in the cells, centrifuging for 5 min at 4 ℃ and 5000 rpm, and collecting supernatant. Sucking up the 293A cells which are up to 70% confluent, washing with PBS for 2 times, adding the collected virus supernatant for the first round of infection, harvesting the cells after more than 50% of the cells become round and shed, repeatedly freezing and thawing for three times, collecting the virus supernatant, and performing the next round of infection according to the steps. After 3-5 rounds of infection, high-titer virus liquid can be obtained, and 293A cells subjected to amplification culture can be infected to amplify viruses, so that more recombinant adenoviruses expressing miR-221 with higher titer are generated, and the recombinant adenoviruses are named as Ad-221. The virus titer is detected by a dilution method or a qRT-PCR method and reaches 10⁷The pfu/ml can be used for infecting host cells, so that the host cells highly express miR-221.

3. Green Fluorescent Protein (GFP) marked HUCMSCs cell with high miR-221 expression

Selecting HUCMSCs cultured to the 4 th generation, culturing with confluency of 80-90%, discarding the culture medium, washing with PBS for 2 times, adding L-DMEM containing about 20 μ L Ad-221 virus solution (determined by virus titer), saturating at 37 deg.C with humidity of 5%, and CO₂Infecting the incubator for 1.5-2 h, removing virus liquid, adding a complete culture medium, continuously culturing for 24-48 h, observing under a fluorescence microscope, wherein GFP positive cells reach more than 70% -80%, and the cells are good in state and can be used for cell transplantation. HUCMSCs infected with empty virus Ad were used as control cells.

4. Preparation of cell suspension for transplantation

(1) HUCMSCs infect the virus vector (Ad-221) with high miR-221 expression and a control virus (Ad), and cells (Ad-221-HUCMSCs) with high miR-221 expression and control cells (Ad-HUCMSCs) are obtained after continuous culture for 48h and can be used for cell transplantation. Before transplantation, discarding the original culture medium from the cells, washing the cells for 2 times by PBS, adding 0.25% trypsin digestion solution for digestion, observing the cell shrinkage deformation under a microscope, adding the complete culture medium to stop digestion, blowing the cells to prepare single cell suspension, centrifuging the single cell suspension at the speed of 1000 r/min for 5 min, removing the supernatant, then re-suspending the suspension by using physiological saline, washing and centrifuging the single cell suspension for 2 times, and removing the components of the culture solution. Adding physiological saline to resuspend cells, counting cells, adjusting cell density to 2 × 10⁶Perml, labelled Ad-221-HUCMSCs and Ad-HUCMSCs cell suspension are stored at 4-10 ℃ for standby, and the storage period is 12 h.

In order to further improve the efficiency of targeting MSCs to liver injury sites and improve the recovery effect of liver function and tissue structure, Ad-221-HUCMSCs and Ad-HUCMSCs cells are cultured for 12h in a culture medium containing 10 ng/ml basic fibroblast growth factor (bFGF) before transplantation, and then digested, washed and resuspended in normal saline according to the steps (1) to prepare 2 × 10⁶bFGF-treated Ad-221-HUCMSCs, Ad-HUCMSCs cell suspension/ml.

FIG. 4 is a quantitative expression chart of the human umbilical cord-derived mesenchymal stem cells highly expressing miR-221 and miR-221, wherein the graphical scale bar in the chart shows 250 μm; separating cultured human umbilical cord-derived mesenchymal stem cells (HUCMSCs), and after multiple passages, determining the cell viability of each passage to be more than 95% by trypan blue staining; the detection result of the flow cytometry shows that the cells cultured to the 3 rd generation express CD90⁺、CD44⁺、CD105⁺、CD73⁺And NOT-HUCMSCs are negative, and accord with the biological characteristics of HUCMSCs. After 48h of infection of recombinant adenovirus (Ad-221) expressing miR-221, the expression level of miR-221 is remarkably increased compared with that of a control group infected with empty virusP＜0.001）。

5. Cell transplantation

The tail vein is administered with Ad-221-HUCMSCs cell suspension, bFGF-treated Ad-221-HUCMSCs cell suspension, Ad-HUCMSCs cell suspension, bFGF-treated Ad-HUCMSCs cell suspension at a dose of 1.0 × 10⁶In terms of/kg. The tail vein of the model control group was given an equal dose of saline. The tail vein of the normal control group animals is given with normal saline with equal dosage at one time.

Respectively randomly selecting 5 mice at 7d and 14 d after cell transplantation, taking eyeballs after ether anesthesia, taking blood, detecting liver function indexes such as ALT, AST, ALB, TBIL, blood coagulation function and the like, and if the liver function indexes of the mice can be improved or even restored to a normal control level after the cell transplantation, the miR-221 can further improve the efficiency of HUCMSCs targeting the liver injury part and improve the effects of liver function and tissue structure restoration; and (3) killing the blood after taking the blood, weighing the liver, measuring and calculating the organ index, and comparing the ratio of each organ to the body weight in normal conditions to be constant. After the animal is infected with the virus, the weight of the damaged organs can be changed, so the organ coefficient is changed accordingly. The increased organ coefficient indicates congestion, edema, hyperplasia and hypertrophy of organs; decreased organ coefficient, indicating atrophy and other degenerative changes of the organs; and (3) taking a part of liver frozen sections, observing the implantation distribution condition of green fluorescent positive cells in the sections in the mouse liver under a fluorescent microscope, counting the cell number or representing the homing and implantation condition of miR-221 on HUCMSCs by green fluorescent intensity. The remaining liver tissue was then fixed with 4% paraformaldehyde, paraffin embedded sections, HE stained and albumin immunohistochemistry to detect liver pathological changes.

CCl₄After injecting Ad-HUCMSCs and Ad-221-HUCMSCs cell suspension 7d into induced acute liver injury mice via tail vein, taking liver tissues as frozen sections, observing the distribution of transplanted green fluorescence positive cells under a fluorescence microscope, and counting the number of cells or the green fluorescence intensity. See FIG. 5, compare with Ad-HUCMSCs groupP< 0.01, and in the figure, the graphical scale shows 100 μm. The results show that 7d after transplantation, the number of HUCMSCs highly expressing miR-221 in the damaged liver tissue is obviously higher than that of control cells, and the miR-221 can promote HUCMSCs to home and colonize in the acute damaged liver tissue.

FIG. 6 shows the liver index of mice with acute liver injury after tail vein injection of HUCMSCs cell suspension for 7d, compared with acute model group (ALI)P＜0.01，***PIs less than 0.001. The detection result shows that the liver index of the mouse with the acute liver injury model after the mesenchymal stem cell transplantation with miR-221 is reduced and tends to a normal control group, and the liver index of the mouse with the acute liver injury model after the transplantation of the control HUCMSCs is not obviously different from that of the mouse with the acute liver injury model.

FIG. 7 is a graph of HE staining of paraffin sections of liver, in which the scale bar is 100 μm. The result shows that after the HUCMSCs 7d with high miR-221 expression are transplanted to the mouse model with acute liver injury, the symptoms of mouse hepatocyte swelling and degeneration, hepatic sinus stenosis, liver inflammatory cell infiltration and the like are obviously relieved, and the HUCMSCs with high miR-221 expression have obvious curative effect on the acute liver injury.

Example III transplantation of human umbilical cord-derived mesenchymal Stem cells highly expressing miR-221 into a mouse model of chronic liver injury

1. Preparation of chronic liver injury experimental animal model

The animal model of chronic injury was prepared as described in example one.

2. Preparation of transplanted cells

Ad-221-HUCMSCs cell suspension, bFGF-treated Ad-221-HUCMSCs cell suspension, Ad-HUCMSCs cell suspension, bFGF-treated Ad-HUCMSCs cell suspension were prepared as in example two; wherein the concentration of basic fibroblast growth factor (bFGF) is 10 ng/ml, and the treatment time is 18 h.

3. Cell transplantation

Acute injury animal models were randomly divided into cell-treated groups (40) and model control groups (10). The cell transplantation treatment group was administered to Ad-221-HUCMSCs cell suspension, bFGF-treated Ad-221-HUCMSCs cell suspension, Ad-HUCMSCs cell suspension, bFGF-treated Ad-HUCMSCs cell suspension at a dose of 1.0X 10⁶In terms of/kg. The tail vein of the model control group is given with equal dose of normal saline at one time. The tail vein of the normal control group animals is given with normal saline with equal dosage at one time. Collecting blood at 7d and 14 d after transplanting cells to detect liver function index and organ index; freezing the liver to be sliced, and observing the colonization distribution condition of the transplanted blue fluorescent cells in the liver of the mouse under a fluorescent microscope; the pathological changes of the liver tissues are detected by paraffin-embedded sections of the liver tissues, HE staining and Masson staining.

CCl₄After injecting Ad-HUCMSCs and Ad-9-HUCMSCs cell suspension 7d into the induced acute liver injury mouse through tail vein, taking liver tissues as frozen sections, observing the distribution of transplanted GFP positive cells under a fluorescence microscope, and counting the number of cells. See FIG. 8, compare with Ad-HUCMSCs groupP< 0.01, and in the figure, the graphical scale shows 100 μm. The results show that 7d after transplantation, the number of HUCMSCs highly expressing miR-221 in the damaged liver tissue is obviously higher than that of control cells, and the results show that miR-221 can promote HUCMSCs homing and colonizationIn chronically damaged liver tissue.

FIG. 9 is a graph of HE staining of paraffin sections of liver, in which the scale bar is 100 μm. The result shows that after HUCMSCs 7d with high miR-221 expression is transplanted to a chronic liver injury mouse model, the swelling of liver cells of the mouse is obviously reduced, and although part of liver cells are degenerated and necrotized, the structural damage of liver lobules and inflammatory cell infiltration are obviously reduced along with the regeneration of the liver cells. The HUCMSCs with high miR-221 expression are shown to have obvious curative effect on chronic liver injury.

FIG. 10 is a Masson staining of paraffin sections of liver, compared to the Chronic model group (CLI)P＜0.01，***P< 0.001, and in the figure, the graphical scale indicates 100 μm. The result shows that after HUCMSCs 7d with high miR-221 expression are transplanted to a mouse model with chronic liver injury (hepatic fibrosis), the thickness and the range of fibrous tissues are obviously reduced compared with those of transplanted control cells and a chronic model group, and the HUCMSCs with high miR-221 expression can obviously reduce the fibrosis degree of the chronic injury liver tissues.

Example four human umbilical cord-derived mesenchymal stem cells highly expressing miR-221 were transplanted into an alcoholic liver disease mouse model

1. Preparation of alcoholic liver disease mouse model

Kunming mouse, 40 mice, male. The random number table method was divided into a normal control group (10) and a model group (50). The model group mice were given 8 g/kg body weight of 40% alcohol per day with gavage divided into 3 times. The control group was given an equal volume of physiological saline and gavaged 3 times daily. After 4 weeks of continuous gavage, transplantation experiments were performed.

2. Preparation of cell suspension for transplantation

Ad-221-HUCMSCs cell suspension, bFGF-treated Ad-221-HUCMSCs cell suspension, Ad-HUCMSCs cell suspension, bFGF-treated Ad-HUCMSCs cell suspension were prepared as in example three; wherein the concentration of basic fibroblast growth factor (bFGF) is 15ng/ml, and the treatment time is 8 h.

5. Cell transplantation

The tail vein is given with Ad-221-HUCMSCs cell suspension and bFGF treated Ad-221-HUCMSCs cell suspension at one timeAd-HUCMSCs cell suspension, bFGF-treated Ad-HUCMSCs cell suspension at a dose of 1.0X 10⁶In terms of/kg. The tail vein of the model control group was given an equal dose of saline. The tail vein of the normal control group animals is given with normal saline with equal dosage at one time. Collecting blood at 7d and 14 d after transplanting cells to detect liver function index and organ index; the pathological changes of the liver tissues are detected by paraffin-embedded sections of the liver tissues, HE staining and Masson staining. The miR-221 can promote HUCMSCs to home and colonize in the chronic damaged liver tissue, and HUCMSCs with high miR-221 expression can remarkably reduce the fibrosis degree of the chronic damaged liver tissue.

Claims (6)

1. The application of the liver injury targeting mesenchymal stem cells in preparing the targeting drug for treating liver injury is characterized in that: the liver injury targeting mesenchymal stem cells are prepared by transfecting or infecting nucleic acid by the mesenchymal stem cells; the nucleic acid is miR-221; the liver injury targeting mesenchymal stem cells are obtained by transfecting or infecting nucleic acid by mesenchymal stem cells and then treating by alkaline fibroblast growth factors; the concentration of the basic fibroblast growth factor is 10-20 ng/mL.

2. Use according to claim 1, characterized in that: the preparation method of the liver injury targeted mesenchymal stem cell comprises the steps of transferring miR-221 into the mesenchymal stem cell through a transfection reagent, and then treating the mesenchymal stem cell with an alkaline fibroblast growth factor to obtain the liver injury targeted mesenchymal stem cell; or transferring the miR-221 into the mesenchymal stem cells through a virus vector, and then treating the mesenchymal stem cells through alkaline fibroblast growth factors to obtain the hepatic injury targeted mesenchymal stem cells.

3. Use according to claim 2, characterized in that: the transfection reagent is a liposome transfection reagent; the virus is an adeno-associated virus, an adenovirus or a retrovirus.

4. Use according to claim 2, characterized in that: transferring miR-221 into mesenchymal stem cells through a transfection reagent to obtain liver injury targeted mesenchymal stem cells, namely diluting the miR-221 simulant and the transfection reagent by using L-DMEM respectively, mixing, and standing at room temperature to obtain a transfection mixed solution; then washing the mesenchymal stem cells cultured to the 4 th-8 th generation and with the confluence degree of 80% -90% by PBS, and then adding a transfection mixed solution; then incubating in an incubator at 37 ℃ for 5-6 h, replacing the transfection mixed solution with a complete culture medium, and continuously culturing for 36-48 h to obtain the liver injury targeted mesenchymal stem cells;

transferring miR-221 into mesenchymal stem cells through a virus carrier to obtain the liver injury targeted mesenchymal stem cells, namely washing the mesenchymal stem cells which are cultured to the 4 th-8 th generation and have the confluence degree of 80% -90% by PBS, then adding virus supernatant for expressing miR-221, infecting for 85-95 min at 37 ℃, and replacing the virus supernatant with a complete culture medium to continuously culture for 24-48 h to obtain the liver injury targeted mesenchymal stem cells.

5. Use according to claim 1, characterized in that: placing the mesenchymal stem cells transferred into miR-221 into a culture medium with an alkaline fibroblast growth factor, and treating for 0.5-24 h to obtain liver injury targeted mesenchymal stem cells; in the culture medium with the basic fibroblast growth factor, the concentration of the basic fibroblast growth factor is 10-20 ng/mL.

6. A preparation method of a targeted medicine for treating liver injury is characterized by comprising the following steps: mixing the liver injury targeted mesenchymal stem cells with pharmaceutical excipients to prepare a targeted drug for treating liver injury, wherein the liver injury targeted mesenchymal stem cells are prepared by transfecting or infecting nucleic acid with the mesenchymal stem cells; the nucleic acid is miR-221; the liver injury targeting mesenchymal stem cells are obtained by transfecting or infecting nucleic acid by mesenchymal stem cells and then treating by alkaline fibroblast growth factors; the concentration of the basic fibroblast growth factor is 10-20 ng/mL.

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