CN114699429A - Application of BMSCs in inhibition of bile duct scar formation through inhibition of bile duct epithelial cell EMT - Google Patents
Application of BMSCs in inhibition of bile duct scar formation through inhibition of bile duct epithelial cell EMT Download PDFInfo
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Abstract
The invention discloses an application of BMSCs in inhibiting bile duct scarring by inhibiting bile duct epithelial cells EMT. The BMSCs are applied to preparing the medicine for inhibiting the formation of bile duct scars under the effect of bile duct epithelial cells EMT. BMSCs inhibit the signaling pathway of biliary epithelial EMT.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of BMSCs in inhibiting bile duct scarring by inhibiting bile duct epithelial cells EMT.
Background
Repair and healing after bile duct injury is one of the major problems facing the medical community today, because wound healing, while restoring tissue integrity, is often accompanied by varying degrees of scarring, with excessive scarring leading to luminal narrowing and tissue fibrosis. Under the conditions of iatrogenic biliary tract injury, abdominal biliary tract trauma, bile duct stones and infection, bile duct tumor, bile duct repair of liver transplantation, bile duct and bile duct anastomosis or bile duct and intestine anastomosis, recurrent cholangitis and the like, scar hyperplasia contracture in the bile duct repair and healing process is a main cause of bile duct stenosis, and even serious complications such as obstructive jaundice, multi-organ function damage and even death caused by the scar hyperplasia contracture are main factors influencing the curative effect and prognosis of a patient, so the method has great significance for researching bile duct scar contracture.
The formation mechanism and cause of scar contracture of biliary tract and bile duct stenosis are not clear, and bile duct fibroblasts are one of the important causes in the formation of scar stenosis of bile duct. Epithelial-mesenchymal phenotypic transformation (EMT) may be another important mechanism of fibrotic scarring of organs, involved in physiological and pathological processes such as cell invasion and metastasis, tissue healing and organ fibrosis [1 ]. EMT is mainly characterized by the loss of polarity of epithelial cells, which are transformed into mesenchymal-like cells with a specific phenotype or function. The epithelial cells lose the specific cell markers, obtain the mesenchymal cell markers, and mainly comprise the down regulation of the protein levels of E-cadherin, keratin and Z0-1 of the epithelial cells and the up regulation of the expression of the markers FSP-1/S100A4, alpha-SMA and Vimentin (Vimentin) of the mesenchymal cells. In addition, cells undergoing EMT can also produce large amounts of extracellular matrix proteins, such as fibronectin, type I/iii collagen, metallo-matrix proteases. The integrity of the morphology and function of the epithelial cells is an important factor for preventing and treating bile duct scar stenosis. Relevant studies have shown that epithelial-mesenchymal phenotypic cell transformation is ubiquitous in organs and tissues and may also play an important role in biliary tract disorders. In vitro and in vivo experimental results of our earlier animals have confirmed that in the healing process of scar of damaged bile duct, the epithelial cells of bile duct generate EMT, and express a specific marker alpha-SMA of myofibroblasts, which indicates that the epithelial cells can be transformed to myofibroblast and participate in the formation of bile duct scar [5,6 ]. The mechanism of EMT generation may be mainly through two signal pathways of TGF-beta/Smad and Wnt/beta-catenin, and in addition to the two pathways, the mechanism may also be involved through other signal pathways of Notch, ERKl/2, RhoA and the like. However, which gene or genes are the major genes or other genes that are first involved in signaling in the pathway remain to be investigated.
At present, the research on the inhibition method of bile duct scar contracture mainly focuses on chemotherapy drugs, change bile components, stem cells and the like. Wherein the stem cells show potential advantages in inhibiting bile duct scarring. Bone marrow mesenchymal stem cells (BMSCs) have the advantages of easy separation and culture, self-renewal, strong differentiation potential to various cells, autograft without immunological rejection and ethical problems [8-11], and the like, are considered as donor cells with the most treatment potential, and provide rational donor cells for our experiments. The bone marrow mesenchymal stem cells have the characteristics of multidirectional differentiation potential, hematopoietic support, stem cell implantation promotion, immunoregulation, self-replication and the like, can be differentiated into various tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium and the like under in vivo or in vitro specific induction conditions, still have multidirectional differentiation potential after continuous subculture and cryopreservation, and can be used as ideal seed cells for repairing tissue and organ injuries caused by aging and pathological changes. BMSCs can secrete a variety of cytokines, one is growth factors such as Hepatocyte Growth Factor (HGF), basic fibroblast growth factor (bFGF), hematopoietic growth factors (such as IL-6, IL-11, LIF, M-CSF, SCF and the like), the other is anti-fibrosis factors such as HGF, granulocyte colony-stimulating factor (G-CSF), matrix metalloproteinase-9 (MMP-9), IL-10, TNF-alpha and the like, and BMSCs have been used for the clinical treatment of hepatic fibrosis and cirrhosis. The plasticity of the adult stem cells is the basis of the current clinical therapeutic application, and has good treatment effect on the treatment of ischemic diseases of cardiovascular, central nervous system, lower limbs and the like. In the field of skin burn and trauma, the purposes of reducing scar formation and realizing functional repair of skin are shown in the aspects of inducing skin epidermal stem cells, reconstructing sweat glands, sebaceous glands and hair follicles by applying the epidermal stem cells, inhibiting keloid formation caused by excessive scar hyperplasia and the like. Research shows that the mesenchymal stem cell conditioned medium contains various bioactive substances related to the effects of tissue repair, angiogenesis, cell survival, anti-inflammation and the like. The nitric oxide secreted by the compound neutralizes active oxygen, and the liver cell growth factor, interleukin 10, basic fibroblast growth factor, vascular endothelial growth factor A, adrenomedullin and the like have the functions of inhibiting fibroblast proliferation and differentiation and collagen synthesis, promoting extracellular matrix remodeling, improving tissue re-epithelization, resisting inflammation, promoting vascular regeneration and the like, and the mesenchymal stem cells can be differentiated into various skin cells to play an important role in the wound healing process. The mesenchymal stem cell conditioned medium can inhibit proliferation of hypertrophic scar fibroblast and collagen production by secreting anti-fibrosis bioactive factors. The research shows that in a New Zealand rabbit body, marrow mesenchymal stem cells can effectively inhibit the formation of bile duct scars, meanwhile, in the inhibition process, the expression of alpha-SMA and vimentin (vimentin) is down-regulated, and the E-cadherin level is up-regulated, and the three proteins are markers of EMT, so the previous research results preliminarily show that the marrow mesenchymal stem cell culture solution has the effect of inhibiting the expression of bile duct epithelial cells alpha-SMA, and further has the effect of inhibiting the bile duct epithelial EMT. (the research results are shown in the early foundation of fund). How BMSCs regulate EMT action mechanism is not clear, whether BMSCs are directly differentiated into terminal cells to participate in, or indirectly secrete cytokines to participate in, or participate in two ways, and whether the BMSCs intervene through signal paths, which are problems to be further researched.
Disclosure of Invention
To address the above-described deficiencies, the present invention provides for the use of BMSCs to inhibit scarring of the bile duct by inhibiting EMT of the bile duct epithelial cells.
In order to achieve the above purpose, the invention provides the following technical scheme: the BMSCs can inhibit bile duct scar formation by inhibiting bile duct epithelial cells EMT.
Further, the BMSCs are applied to preparing the medicine for inhibiting the formation of bile duct scars through the effect of bile duct epithelial cells EMT.
Further, BMSCs inhibit the signaling pathway of biliary epithelial EMT.
Furthermore, after the bile duct is scarred, the expression of E-cadherin genes and proteins is reduced, and the expression of alpha-SMA, N-cadherin, snai1 and vimentin is obviously enhanced, which indicates that the bile duct epithelial cells generate EMT change; after the bone marrow mesenchymal stem cells and the bile duct epithelial cells are co-cultured, the occurrence of EMT of the bile duct epithelial cells is inhibited.
Further, TGF- β/Smad is one of important pathways of EMT, which is inhibited by mesenchymal stem cells.
The beneficial effects are that: the invention completely defines the formation of bile duct scars inhibited by the BMSC through the effect of inhibiting the bile duct epithelial cells EMT for the first time. The BMSC is firstly studied to inhibit possible signal pathways of bile duct epithelial cell EMT and important conduction factors in the pathways.
Compared with the prior art, the invention has the following advantages: (1) the chitosan membrane of the composite bone marrow mesenchymal stem cells is prepared by using the experience of preparing the chitosan membrane in the early stage, and whether the bone marrow mesenchymal stem cells can inhibit the formation of scars by inhibiting the bile duct epithelium from generating EMT or not is further researched on the basis; the main signaling pathway of bone marrow mesenchymal stem cells for inhibiting the occurrence of EMT in bile duct epithelium and the main regulatory proteins in the pathway are researched.
(2) The invention is closely related to clinic and has strong practicability. Meanwhile, the method can also be applied to the repair and treatment of the injured scars of other tissues and organs, and has wide application prospect. Therefore, the invention is applied to the treatment of biliary duct scar stenosis by combining with the subsidy of special fund so as to further understand the complex mechanism of the biliary duct epithelial cells for EMT and whether the BMSC can inhibit the biliary duct epithelial cells EMT to better prevent and treat the biliary duct scar hyperproliferation contracture and provide a more effective treatment method for the biliary duct scar stenosis.
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FIG. 1 is a graph of growth of various groups of HIBEPIC detected by the MTT method of the present invention, data are presented as mean + Standard Deviation (SD), and data difference is statistically analyzed by One-way ANOVA (One-way ANOVA) with p < 0.05; p < 0.01; p <0.001 fig. 1 shows that HBMSCS induces proliferation of hibipic.
FIG. 2 is a graph of the present invention showing the apoptosis of each group by flow-testing, the data is presented as mean value + Standard Deviation (SD), and the data difference is statistically analyzed by One-way ANOVA (One-way ANOVA) with p < 0.05; p < 0.01; p <0.001. FIG. 2 shows that HBMSCS does not induce HIBEPIC apoptosis.
FIG. 3 is a QPCR detection scheme of the present invention for the expression of the α -SMA gene in each group of cells. Data are presented as mean + Standard Deviation (SD), data differences are statistically analyzed using One-way anova,. p < 0.05; p < 0.01; p <0.001. FIG. 3 shows the demonstration from the genetic level that HBMSCS inhibits the phenotypic shift of biliary epithelial cells to myofibroblasts.
FIG. 4 is a QPCR detection graph of the expression of the E-cadherin gene in each group of cells. Data are presented as mean + Standard Deviation (SD), data differences are statistically analyzed using One-way ANOVA,. p < 0.05; p < 0.01; p <0.001. FIG. 4 shows that it was confirmed from the gene level that TGF-. beta.1 causes decreased expression of bile duct epithelial cells E-cadherin, decreased cell adhesion junctions, causing cell detachment, and HBMSCS inhibits bile duct epithelial cell detachment.
FIG. 5 is a QPCR detection of the expression of the N-cadherin gene in each group of cells according to the invention. Data are presented as mean + Standard Deviation (SD), data differences are statistically analyzed using One-way ANOVA, p < 0.05; p < 0.01; p <0.001. FIG. 5 shows that it is confirmed from the gene level that TGF-beta 1 causes high expression of bile duct epithelial cell N-cadherin, promotes cell separation, and HBMSCS inhibits bile duct epithelial cell separation.
FIG. 6 is a QPCR detection map of the present invention for detecting the expression of snail gene in each group of cells. Data are presented as mean + Standard Deviation (SD), data differences are statistically analyzed using One-way anova with p < 0.05; p < 0.01; p <0.001. FIG. 6 shows that from the gene level, TGF-beta 1 causes high expression of bile duct epithelial cell snail, activates transcription channel, promotes EMT generation, and HBMSCS inhibits bile duct epithelial cell EMT.
FIG. 7 is a QPCR assay of the present invention showing the expression of vimentin genes in each cell group. Data are presented as mean + Standard Deviation (SD), data differences are statistically analyzed using One-way anova,. p < 0.05; p < 0.01; p <0.001. FIG. 7 shows that from the gene level, it is confirmed that TGF-beta 1 causes bile duct epithelial cell vimentin to be highly expressed, cytoskeleton is changed, cell separation is promoted, and HBMSCS inhibits bile duct epithelial cell separation.
FIG. 8 is an expression diagram of the Westernblot for detecting alpha-SMA protein in each group of cells. Data are presented as mean + Standard Deviation (SD), data differences are statistically analyzed using One-way ANOVA, p < 0.05; p < 0.01; p <0.001. FIG. 8 shows demonstration of HBMSCS inhibiting the phenotypic shift of biliary epithelial cells to myofibroblasts from the protein level.
FIG. 9 is an expression diagram of the Westernblot for detecting E-cadherin protein in each group of cells of the invention. Data are presented as mean + Standard Deviation (SD), data differences are statistically analyzed using One-way ANOVA,. p < 0.05; p < 0.01; p <0.001. FIG. 9 shows that TGF-. beta.1 causes decreased bile duct epithelial cell E-cadherin expression, decreased cell adhesion junctions, resulting in cell detachment, and HBMSCS inhibits bile duct epithelial cell detachment, as demonstrated from the protein level.
FIG. 10 is a Westernblot expression profile for detecting N-cadherin protein in each cell group of the invention. Data are presented as mean + Standard Deviation (SD), data differences are statistically analyzed using One-way ANOVA,. p < 0.05; p < 0.01; p <0.001. FIG. 10 shows that TGF-. beta.1 causes bile duct epithelial cell N-cadherin to be highly expressed from protein level, promoting cell separation, and HBMSCS inhibits bile duct epithelial cell separation.
FIG. 11 is a Westernblot expression profile for detecting Snail proteins in various groups of cells in accordance with the present invention. Data are presented as mean + Standard Deviation (SD), data differences are statistically analyzed using One-way ANOVA,. p < 0.05; p < 0.01; p <0.001. FIG. 6 shows that from the protein level, TGF-beta 1 causes bile duct epithelial cell snail high expression, activates transcription channel, promotes EMT generation, and HBMSCS inhibits bile duct epithelial cell EMT.
FIG. 12 is a Westernblot expression chart for detecting Vimentin proteins in various groups of cells. Data are presented as mean + Standard Deviation (SD), data differences are statistically analyzed using One-way ANOVA,. p < 0.05; p < 0.01; p <0.001. FIG. 12 shows that TGF-. beta.1 causes bile duct epithelial cell vimentin to be highly expressed from the protein level, cytoskeleton is changed, cell separation is promoted, and HBMSCS inhibits bile duct epithelial cell separation, as shown in FIG. 12.
FIG. 13 is a bile duct HE staining pattern of the present invention. FIG. 13 shows that BMSCS can reduce the degree of disorder of cell arrangement in the bile duct epithelium, decrease the number of cell layers, and inhibit scarring of the bile duct.
FIG. 14 is a bile duct Masson staining pattern of the present invention. Fig. 14 shows that BMSCS was able to alleviate bile duct collagen, inhibiting bile duct scarring.
Detailed Description
For better understanding of the present invention, the following examples are given to further illustrate the present invention, but the present invention is not limited to the following embodiments. The experimental methods used therein are conventional methods unless otherwise specified.
The BMSCs can inhibit bile duct scar formation by inhibiting bile duct epithelial cells EMT.
The invention relates to application of BMSCs in preparing a medicament for inhibiting the formation of bile duct scars through the action of bile duct epithelial cells EMT.
BMSCs inhibit the signaling pathway of biliary epithelial cells EMT and important regulatory proteins in the pathway.
After the bile duct is scarred, the expression of E-cadherin genes and proteins is reduced, and the expression of alpha-SMA, N-cadherin, snai1 and vimentin is obviously enhanced, which indicates that the epithelial cells of the bile duct have EMT change; after the mesenchymal stem cells of the bone marrow and the epithelial cells of the bile duct are co-cultured, the generation of EMT of the epithelial cells is inhibited.
TGF-beta/Smad is one of important pathways of EMT, and bone marrow mesenchymal stem cells can inhibit pathway proteins of EMT.
Example 1
Research method and research index
(1) In vitro cell experiments demonstrated whether BMSC could inhibit bile duct scarring by inhibiting biliary epithelial EMT and through which possible signaling pathways BMSC inhibit biliary epithelial EMT:
the generation of bile duct epithelial cell EMT is induced by TGF-beta, and the experiment is divided into 4 groups: bile duct epithelial cell group, bile duct epithelial cell + TGF-beta group, bile duct epithelial cell + BMSC co-culture + TGF-beta group. Four groups of cells were tested using the following method:
1) the mRNA expression levels of EMT related factors alpha-SMA, E-Cadherin, N-Cadherin, snail and Vimentin are detected by a Real-time fluorescence quantitative polymerase chain amplification method (Real-time PCR).
2) And detecting the protein expression levels of EMT related factors alpha-SMA, E-Cadherin, N-Cadherin, snail and Vimentin by using a western blot method.
3) And detecting the apoptosis condition of the epithelial cells by adopting an Annexin V-FITC/PI double staining method.
4) The growth of epithelial cells was examined by the MTT method.
(2) Animal experiments verified whether BMSC can inhibit bile duct scarring by inhibiting bile duct epithelial cell EMT:
the experiment was divided into 4 groups (3 per group): the chitosan membrane group, the chitosan membrane and EMT inducer group, the chitosan membrane-BMSC group and the chitosan membrane-BMSC and EMT inducer group. The front wall of the common bile duct of each group of rats is incised by an operation to prepare a bile duct injury model, a 4-0 strip needle is used as a mucous membrane to perform eversion and full anastomosis of the mucous membrane, and the anastomosis opening is checked to have no leakage after the operation. Then, each prepared group of chitosan films are wrapped at the anastomotic opening of the common bile duct, and the wrapping sleeve 1-2 needles can be added for fixation at intervals when necessary. And closing the abdomen. After operation, water and food can be freely fed. Intramuscular pioneer V (20mg/kg) prevents infection for 3 days. Animal diet, activity, hair color, scleral condition were observed. After 1 week, 2 weeks and 1 month, 5 animals were respectively taken and placed into the abdomen from the original incision. Separating the adhesion, exposing the common bile duct, completely taking down the bile duct tissue from the bifurcation of the common hepatic duct to the oddi sphincter, removing the wrapping sleeve and exposing the repair area. The 4 sets of models were tested at the previous time points:
1) and observing the conventional conditions of cholestasis, calculus formation, stenosis and the like in each group under a normal visual field.
2) The gross pathology of bile ducts and the arrangement of submucosal collagen fibers were observed by an optical microscope.
(II) results of the experiment
1. In vitro cell experiments verify whether the BMSC can inhibit the formation of bile duct scar by inhibiting the bile duct epithelial cell EMT and which signal path the BMSC can inhibit the bile duct epithelial cell EMT
(1) MTT method for detecting growth of HIBEpiC in each group
As can be seen from Table 1, the OD values of the HIBEPIC + HBMSCs group, the HIBEPIC + HBMSCs + TGF-. beta.1 group were increased (P < 0.05) compared to the HIBEPIC group, wherein there was no statistical difference between the HIBEPIC + HBMSCs group and the HIBEPIC + HBMSCs + TGF-. beta.1 group (P > 0.05).
TABLE 1
Note: *: compared to the hibipic group,: is less than 0.05; **: less than 0.01; ***: is less than 0.001.
(2) Flow detection of apoptosis of each group of cells
As can be seen from Table 2, the apoptosis rates of the HIBEPIC + TGF-. beta.1, HIBEPIC + HBMSCs group, HIBEPIC + HBMSCs + TGF-. beta.1 group were reduced (P < 0.05) compared to the HIBEPIC group, wherein there was no statistical difference (P >0.05) between the HIBEPIC + TGF-. beta.1, HIBEPIC + HBMSCs, and HIBEPIC + HBMSCs + TGF-. beta.1 groups.
TABLE 2
Note: *: compared to the hibipic group,: less than 0.05; **: less than 0.01; ***: is less than 0.001.
(3) QPCR detection of expression of alpha-SMA gene in each group of cells
As can be seen from Table 3, the amount of α -SMA gene in the HIBEPIC + TGF-. beta.1 group was significantly increased (P < 0.05) compared to the HIBEPIC group, in which there was no statistical difference between the HIBEPIC + HBMSCs group and the HIBEPIC + HBMSCs + TGF-. beta.1 group (P > 0.05).
TABLE 3
Note: *: compared to hibipic group,: less than 0.05; **: is less than 0.01; ***: is less than 0.001.
(4) QPCR detection of E-cadherin gene expression in various groups of cells
As can be seen from Table 4, the amounts of E-cadherin genes in the HIBEPIC + TGF-. beta.1 group, the HIBEPIC + HBMSCs + TGF-. beta.1 group were decreased (P < 0.05) as compared with the HIBEPIC group, wherein the decreases in the HIBEPIC + TGF-. beta.1 group, and the HIBEPIC + HBMSCs + TGF-. beta.1 group were more significant (P < 0.05) as compared with the HIBEPIC group.
TABLE 4
Note: *: compared to the hibipic group,: is less than 0.05; **: less than 0.01; ***: is less than 0.001.
(5) QPCR detection of expression of N-cadherin genes in various groups of cells
As can be seen from Table 5, the amounts of N-cadherin genes in the HIBEPIC + TGF-. beta.1 group and the HIBEPIC + HBMSCs + TGF-. beta.1 group were increased (P < 0.05) as compared with the HIBEPIC group, and the amounts of N-cadherin genes in the HIBEPIC + TGF-. beta.1 group were more significantly increased (P < 0.05) as compared with the HIBEPIC group.
TABLE 5
Note: *: compared to the hibipic group,: is less than 0.05; **: less than 0.01; ***: is less than 0.001.
(6) QPCR (quantitative polymerase chain reaction) detection of snai1 gene expression in each group of cells
As is clear from Table 6, the amount of snai1 gene was increased in the HIBEPIC + TGF-. beta.1 group and in the HIBEPIC + HBMSCs + TGF-. beta.1 group as compared with the HIBEPIC group (P < 0.05).
TABLE 6
Note: *: compared to the hibipic group,: is less than 0.05; **: less than 0.01; ***: is less than 0.001.
(7) QPCR detection of vimentin Gene expression in Each group of cells
As can be seen from Table 7, the amount of viral genes in the HIBEPIC + TGF-. beta.1 group was increased (P < 0.05) as compared with the HIBEPIC group, and the amounts of viral genes in the HIBEPIC + HBMSCs group, the HIBEPIC + HBMSCs + TGF-. beta.1 group were not statistically different (P >0.05) as compared with the HIBEPIC group.
TABLE 7
Note: *: compared to the hibipic group,: less than 0.05; **: is less than 0.01; ***: is less than 0.001.
(8) Western blot detection of expression of alpha-SMA protein in each cell group
As can be seen from Table 8, the amounts of α -SMA proteins in the HIBEPIC + TGF-. beta.1 group and the HIBEPIC + HBMSCs + TGF-. beta.1 group were increased as compared with the HIBEPIC group (P < 0.05).
TABLE 8
Note: *: compared to the hibipic group,: less than 0.05; **: less than 0.01; ***: is less than 0.001.
(9) Western blot detection of expression of E-cadherin proteins in cells of each group
As can be seen from Table 9, the amounts of E-cadherin proteins in the HIBEPIC + TGF-. beta.1 group were decreased (P < 0.05) compared with the HIBEPIC group, and the amounts of E-cadherin proteins in the HIBEPIC + HBMSCs, HIBEPIC + HBMSCs + TGF-. beta.1 group were not statistically different (P >0.05) compared with the HIBEPIC group. .
TABLE 9
Note: *: compared to hibipic group,: is less than 0.05; **: less than 0.01; ***: is less than 0.001.
(10) Western blot detection of expression of N-cadherin proteins in cells of each group
As is clear from Table 10, HIBEPIC + TGF-. beta.1 group, HIBEPIC + HBMSCs + TGF-. beta.1 group
The amount of the N-cadherin protein in the group is increased compared with that in the HIBEPIC group (P is less than 0.05), and the amount of the E-cadherin protein in the HIBEPIC + HBMSCs group is not statistically different (P is more than 0.05) compared with that in the HIBEPIC group.
Note: *: compared to the hibipic group,: less than 0.05; **: is less than 0.01; ***: is less than 0.001.
(11) Western blot detection of expression of Snai1 protein in each group of cells
As can be seen from Table 11, the amount of Snai1 protein in the HIBEPIC + TGF-. beta.1 group was increased (P < 0.05) compared with the HIBEPIC group, and the amount of Snai1 protein in the HIBEPIC + HBMSCs, HIBEPIC + HBMSCs + TGF-. beta.1 group was not statistically different (P >0.05) compared with the HIBEPIC group.
TABLE 11
Note: *: compared to the hibipic group,: less than 0.05; **: less than 0.01; ***: is less than 0.001.
(12) Western blot detection of expression of Vimentin proteins in cells of each group
As can be seen from Table 12, the amounts of Vimentin proteins in the HIBEPIC + TGF-. beta.1 group and the HIBEPIC + HBMSCs + TGF-. beta.1 group were increased (P < 0.05) as compared with the HIBEPIC group, and the amounts of Vimentin proteins in the HIBEPIC + HBMSCs group were not statistically different (P >0.05) as compared with the HIBEPIC group.
TABLE 12
Note: *: compared to the hibipic group,: less than 0.05; **: less than 0.01; ***: is less than 0.001.
(13) Bile duct HE staining
In the control group (scar group), the arrangement of cells on the epithelial layer of the bile duct is disordered, the number of cell layers is increased, the epithelial layer is thickened, and obvious inflammatory cell infiltration is not seen; in the scar + BMSCS group, the degree of disorder of the cell arrangement of the bile duct epithelial layer is light, the number of the cell layers is small and is close to the normal epithelial structure, and the epithelial layer is not obviously thickened and is not obviously infiltrated by inflammatory cells.
(14) Bile duct Masson staining
Bile duct collagen was significantly increased in the control group (scar group), while bile duct collagen was decreased in the scar + BMSCS group.
(III) conclusion
1. After the bile duct is scarred, the expression of E-cadherin genes and proteins is reduced, and the expression of alpha-SMA, N-cadherin, snai1 and vimentin is obviously enhanced, which indicates that the epithelial cells of the bile duct have EMT change; after the bone marrow mesenchymal stem cells and the bile duct epithelial cells are co-cultured, the occurrence of EMT of the bile duct epithelial cells is inhibited.
TGF-beta/Smad is one of important pathways for EMT, and mesenchymal stem cells can inhibit pathway proteins of EMT.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (5)
1. Use of BMSCs to inhibit scarring of the bile ducts by inhibiting EMT of the bile duct epithelial cells.
2. Use according to claim 1, characterized in that: the BMSCs are applied to preparing the medicine for inhibiting the formation of bile duct scars under the effect of bile duct epithelial cells EMT.
3. Use according to claim 1, characterized in that: BMSCs inhibit the signaling pathway of biliary epithelial EMT.
4. Use according to claim 1, characterized in that: after the bile duct is scarred, the expression of E-cadherin genes and proteins is reduced, and the expression of alpha-SMA, N-cadherin, snai1 and vimentin is obviously enhanced, which indicates that the epithelial cells of the bile duct have EMT change; after the mesenchymal stem cells of the bone marrow and the epithelial cells of the bile duct are co-cultured, the epithelial cells of the bile duct are inhibited from generating EMT.
5. Use according to claim 1, characterized in that: TGF-beta/Smad is one of important pathways for EMT, and bone marrow mesenchymal stem cells can inhibit the pathway of EMT.
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| CN (1) | CN114699429A (en) |
Citations (4)
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| WO2002083864A2 (en) * | 2001-04-13 | 2002-10-24 | Anterogen Co., Ltd. | Methods and reagents for cell transplantation |
| WO2010081152A2 (en) * | 2009-01-12 | 2010-07-15 | University Of Southern California | Keloid derived precursor cells and its applications of use thereof |
| US20170095511A1 (en) * | 2015-08-03 | 2017-04-06 | The Johns Hopkins University | In vitro pre-conditioned bone marrow-derived mesenchymal stem cells and uses thereof |
| CN110892061A (en) * | 2017-06-19 | 2020-03-17 | 剑桥企业有限公司 | Method for amplifying bile duct cells |
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2022
- 2022-04-28 CN CN202210460810.XA patent/CN114699429A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002083864A2 (en) * | 2001-04-13 | 2002-10-24 | Anterogen Co., Ltd. | Methods and reagents for cell transplantation |
| WO2010081152A2 (en) * | 2009-01-12 | 2010-07-15 | University Of Southern California | Keloid derived precursor cells and its applications of use thereof |
| US20170095511A1 (en) * | 2015-08-03 | 2017-04-06 | The Johns Hopkins University | In vitro pre-conditioned bone marrow-derived mesenchymal stem cells and uses thereof |
| CN110892061A (en) * | 2017-06-19 | 2020-03-17 | 剑桥企业有限公司 | Method for amplifying bile duct cells |
Non-Patent Citations (5)
| Title |
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| YING LI等: "Implantation of Bone Marrow Mesenchymal Stem Cells into Small Intestinal Submucosa Improves Bile Duct Injury in Rabbits", TISSUE ENG REGEN MED, vol. 18, no. 5, XP037563764, DOI: 10.1007/s13770-021-00351-2 * |
| ZHENGZHOU SHI等: "Extracellular vesicles produced by bone marrow mesenchymal stem cells attenuate renal fibrosis, in part by inhibiting the RhoA/ROCK pathway, in a UUO rat model", 《STEM CELL RES THER.》, vol. 11, no. 1, pages 11 - 12 * |
| 宋飞;向盈盈;黄明;张小文;: "紫杉醇壳聚糖缓释膜抑制兔胆管上皮细胞EMT的实验", 昆明医科大学学报, no. 12 * |
| 宋飞等: "紫杉醇抗良性胆管瘢痕纤维化的最佳抵抗因素研究", 《中国生化药物杂志》, vol. 34, no. 3, pages 4 * |
| 王曙亮;王少江;李永刚;: "紫杉醇-壳聚糖缓释膜通过抑制TGFβ/Smad信号通路抵抗良性胆管瘢痕纤维化", 中国现代普通外科进展, no. 01 * |
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