CN102732478A - Inducer for inducing directional differentiation of umbilical cord mesenchymal stem cells into bladder smooth muscle cells and preparation and application thereof - Google Patents
Inducer for inducing directional differentiation of umbilical cord mesenchymal stem cells into bladder smooth muscle cells and preparation and application thereof Download PDFInfo
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Abstract
本发明公开了基质细胞衍生因子-1在制备诱导脐带间充质干细胞定向分化为膀胱平滑肌细胞的诱导剂中的用途。本发明还公开了一种诱导脐带间充质干细胞定向分化为膀胱平滑肌细胞的诱导剂，以及该诱导剂的用途和促进脐带间充质干细胞定向分化为膀胱平滑肌细胞的方法。本发明提供的诱导剂可以体外促进脐带间充质干细胞定向分化为膀胱平滑肌细胞，可用于膀胱损伤的修复，具有良好的应用前景和经济效益。
The invention discloses the use of stromal cell-derived factor-1 in preparing an inducer for inducing directional differentiation of umbilical cord mesenchymal stem cells into bladder smooth muscle cells. The invention also discloses an inducer for inducing umbilical cord mesenchymal stem cells to differentiate into bladder smooth muscle cells, use of the inducer and a method for promoting umbilical cord mesenchymal stem cells to differentiate into bladder smooth muscle cells. The inducer provided by the invention can promote the directional differentiation of umbilical cord mesenchymal stem cells into bladder smooth muscle cells in vitro, can be used for repairing bladder damage, and has good application prospects and economic benefits.
Description
Technical field
The present invention relates to the agent of a kind of promotion umbilical cord mesenchymal stem cells cell directional induced differentiation, particularly a kind of external evoked human umbilical cord mesenchymal stem cells is divided into the inductor of smooth muscle of bladder cell.
Background technology
The various diseases of bladder, for example: the damage of congenital abnormality, acquired character, serious inflammation infection etc., all can cause the forfeiture of its structure or function, need repair and rebuild.Traditional method mainly is to utilize patient self stomach intestinal tissue to carry out cystectasy; These methods exist such as complication such as new bladder capacity minimizing, metabolism disorder, intestinal obstruction, formation calculus, perforation even malignant changes, and cause the bladder reconstruction is the difficult point of Urology Surgery clinical position always.The rise of tissue engineering technique and brought new hope to the bladder reparation with rebuilding in the successful Application of medical science different field; Its core comprises that the interaction and the inside and outside of development, cell and timbering material of cultivation, the timbering material of seed cell makes up engineering tissue etc.; Wherein, seed cell is to make up to organize the most basic key element of bladder body.At present, comparatively common to make up the organizational project bladder as seed cell from body smooth muscle of bladder cell.But; As the cell of differentiation and maturation; Smooth muscle of bladder cells in vitro multiplication capacity is limited; And when bladder is in whole latter stage (like tumour, severe trauma, gangrenous cystitis, tuberculosis of bladder, bladder fibrosis, contracture of bladder etc.), can't obtain the cultivation amplification that normal bladder body is used for cell.Thereby the seed cell source is not enough to have become the bottleneck that limits the further research and development of organizational project bladder, and seeking ideal bladder body engineering seed cell is the emphasis of studying at present.
Stem cell is one type and has self, the highly propagation and the special cells of multidirectional differentiation potential.After stem cell induced,, then be expected to solve the organizational project bladder seed cell insufficient problem of originating if can form newborn bladder body.At present; The common initiating cell that the preparation cellular system engineering is cultivated smooth muscle of bladder cell seed cell is mesenchymal stem cells MSCs, bone marrow stroma stem cell, fat mesenchymal stem cell, people's amnion mesenchymal cell, characterizes urine derived stem cells etc., and common inducible factor is TGF-β 3,1% DMSO 99.8MIN. (DMSO) and rat bladder tissue homogenate supernatant etc.Wherein, the stem cell that utilizes derived from bone marrow is the focus of studying at present as seed cell; Its acquisition methods is comparatively ripe; And more animal experiment and Study of Clinical Application report are arranged, but mesenchymal stem cells MSCs has its inevitable defective, as; The method that quantity of stem cell and proliferation and differentiation ability can descend, obtain marrow gradually along with the growth at donor age is traumatic, causes its limited use.
Umbilical cord blood mesenchymal stem cells; It is the mescenchymal stem cell that exists in the neonatal umbilical cord; Compare its special advantages than the mescenchymal stem cell in marrow and other sources: annual all have ten hundreds of newborn infants to be born; The source is abundant, and obtains Shi Buhui the donor is caused new misery and wound, does not have the ethics restriction; Umbilical cord mesenchymal stem cells is a primary stem cell comparatively, has extremely strong propagation and multidirectional differentiation capability; Lower immunogenicity.But at present in the domestic and foreign literature, it is actually rare that the research human umbilical cord mesenchymal stem cells is divided into the experiment of smooth muscle of bladder cell; Rarely seen Deng Li, " people's umbilical cord blood mesenchymal stem cells becomes the flesh differentiation to reach the experimental study to the sphincter urethrae function effect ", Third Military Medical University; Obstetrics and gynecology; 2007, Ph D dissertation reported, utilize genetic engineering technique with myogenic regulatory factor Myocardin gene transfection people's umbilical cord blood mesenchymal stem cells (UB-MSCs) after; UB-MSCs has the ability to the smooth muscle cell differentiation under the Myocardin regulation and control.This method need be gene constructed to eukaryon expression plasmid pEGFP-Myocl with myogenic regulatory factor Myocardin; Transfection is in people's umbilical cord blood mesenchymal stem cells (UB-MSCs); Cause people's umbilical cord blood mesenchymal stem cells to contain foreign gene,, control the expression of exogenous gene regulation and control and remain a big difficult point though present engineered working method is comparatively ripe; As organizational project bladder seed cell, there is potential safety hazard with the smooth muscle of bladder cell that contains foreign gene.Directly inducing human umbilical cord mesenchymal stem cells to be divided into the smooth muscle of bladder cell with inducible factor, is safer a kind of mode, therefore, is badly in need of seeking a kind of inducible factor and is used to induce human umbilical cord mesenchymal stem cells to be divided into the smooth muscle of bladder cell.
Stroma cell derivative factor-1 (SDF-1, or CXCL12) is the amino acid whose member who attracts 68 in the chemokine family of tranquillization T lymphocyte, monocyte and CD34+ stem cell.SDF-1 during fetal development and after the stem cell transplantation hemopoietic stem cell go back to the nest and to marrow, play a crucial role; SDF-1 is also very important in heart generation and vasculogenesis; In addition; SDF-1 can improve patient especially diabetic subject's wound or ulcer healing, but does not see that the external evoked mescenchymal stem cell of SDF-1 is divided into the report of smooth muscle of bladder cell, does not see that more the external evoked human umbilical cord mesenchymal stem cells of SDF-1 is divided into the report of smooth muscle of bladder cell.
Summary of the invention
In order to address the above problem, the invention provides the inductor that a kind of external evoked human umbilical cord mesenchymal stem cells is divided into the smooth muscle of bladder cell.
The invention provides stroma cell derivative factor-1, to induce the umbilical cord mesenchymal stem cells directed differentiation in preparation be the purposes in the inductor of smooth muscle of bladder cell.
Stroma cell derivative factor-1 (SDF-1) finds that usually it has two kinds of different form SDF-1 α and SDF-1 β, and the difference of existence is that SDF-1 β has 4 amino acid at C-terminal (extension Arg-Phe-Lys-Met), this difference are that the mRNA montage causes.The SDF-1 of two kinds of forms is initial with the signal peptide of 21 amino acid lengths, and signal peptide is cut to form bioactive peptide, and SDF-1 of the present invention is the activity form (promptly after cleavable signal peptide) of said peptide, comprises SDF-1 α and SDF-1 β.
Further, comprising concentration in the said inductor is 25 ~ 75 μ g/ml stroma cell derivative factors-1.
Further, the concentration of said stroma cell derivative factor-1 is 50 μ g/ml.
It is the inductor of smooth muscle of bladder cell that the present invention also provides a kind of umbilical cord mesenchymal stem cells directed differentiation of inducing, and it comprises concentration is 25 ~ 75 μ g/ml stroma cell derivative factors-1.
Further, the concentration of said stroma cell derivative factor-1 is 50 μ g/ml.
It is the application in the smooth muscle of bladder cell inducing the umbilical cord mesenchymal stem cells directed differentiation that the present invention also provides aforementioned inductor.
The present invention provides also that a kind of to induce interband mesenchymal stem cells directed differentiation be the method for smooth muscle of bladder cell, and it comprises the steps:
(1) prepares aforementioned inductor;
(2) get umbilical cord mesenchymal stem cells, subculture in vitro separately is cultivated, and in nutrient solution, adds the inductor of step (2) preparation, and inducing culture is after 7 days, and umbilical cord mesenchymal stem cells is divided into the smooth muscle of bladder cell.
Inductor provided by the invention can be divided into the smooth muscle of bladder cell at external evoked human umbilical cord mesenchymal stem cells, for the organizational project bladder makes up seed cell is provided, and has important clinical application value.
Obviously, according to foregoing of the present invention,,, can also make modification, replacement or the change of other various ways not breaking away under the above-mentioned basic fundamental thought of the present invention prerequisite according to the ordinary skill knowledge and the customary means of this area.
Below, foregoing of the present invention is remake further detailed description through the embodiment of embodiment form.But should this be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following instance.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
Description of drawings
Under Fig. 1 inverted phase contrast microscope, umbilical cord mesenchymal stem cells form, 200 times
Fig. 2 flow cytometer detects the CD44 expression
Fig. 3 flow cytometer detects the CD166 expression
Fig. 4 flow cytometer detects the CD34 expression
Fig. 5 flow cytometer detects the CD40L expression
Under Fig. 6 inverted phase contrast microscope, smooth muscle of bladder cellular form, 200 times
When Fig. 7 cultivates 7 days, the immunocytochemistry result of cell expressing α-SMA situation
When Fig. 8 cultivates 7 days, the immunocytochemistry result of cell expressing SM-MHC situation
When Fig. 9 cultivates 14 days, the immunocytochemistry result of cell expressing α-SMA situation
When Figure 10 cultivates 14 days, the immunocytochemistry result of cell expressing SM-MHC situation
Embodiment
Embodiment 1 human umbilical cord mesenchymal stem cells external evoked
(1) gets stroma cell derivative factor-1;
(2) get umbilical cord mesenchymal stem cells, subculture in vitro separately is cultivated, and nutrient solution is to contain in the L-DMEM nutrient solution of 10% foetal calf serum, in nutrient solution, adds stroma cell derivative factor-1, and the concentration of stroma cell derivative factor-1 is 25 μ g/ml, inducing culture 7 days.
Embodiment 2 human umbilical cord mesenchymal stem cells external evoked
(1) gets stroma cell derivative factor-1;
(2) get umbilical cord mesenchymal stem cells, subculture in vitro separately is cultivated, and nutrient solution is to contain in the L-DMEM nutrient solution of 10% foetal calf serum, in nutrient solution, adds stroma cell derivative factor-1, and the concentration of stroma cell derivative factor-1 is 50 μ g/ml, inducing culture 7 days.
Embodiment 3 human umbilical cord mesenchymal stem cells external evoked
(1) gets stroma cell derivative factor-1;
(2) get umbilical cord mesenchymal stem cells, subculture in vitro separately is cultivated, and nutrient solution is to contain in the L-DMEM nutrient solution of 10% foetal calf serum, in nutrient solution, adds stroma cell derivative factor-1, and the concentration of stroma cell derivative factor-1 is 75 μ g/ml, inducing culture 7 days.
Experimental example 1 human umbilical cord mesenchymal stem cells external evoked
(1) experimental technique
1, the cultivation of umbilical cord mesenchymal stem cells
(1) former be commissioned to train foster (tissue mass cell culture) of umbilical cord mesenchymal stem cells
1) get the about 10cm of FTNB umbilical cord, after the aseptic collection, 4 ℃ of preservations in preserving liquid;
2) in the ultra-clean work, under the sterile state, earlier umbilical cord is cut into the segment of 1.5cm in petridish, washes umbilical cord repeatedly with 4 ℃ of PBS liquid, particularly the blood in umbilical vein is used irrigation with syringe, wash to PBS liquid more limpid till.Cut umbilical cord open with the sterile scissors stringer, remove umbilical vein and artery, separate stripping the magnificent general formula glue tissue in the umbilical cord, glue tissue is cut into the fritter of 1mm * 1mm, attention is moistening with PBS liquid maintenance tissue block.
3) tissue block is shredded after, evenly be tiled under the aseptic condition at the bottom of the culturing bottle of T25, at interval approximately about 1cm, will cultivate bottleneck and make progress, be inverted into 37 ° of incubators about 2 hours, make tissue block adherent;
4) take out culturing bottle, slowly add the about 5ml of L-DMEM nutrient solution that contains 10% foetal calf serum under the inversion state, slowly set level culturing bottle then, make sure to keep in mind not that communications centre plays tissue block, put into 5%CO
2, cultivate in 37 ℃ of saturated humidity incubators, be positioned over after 3 days to observe inverted microscope under to have and acellularly climb out of, and change nutrient solution first from the tissue block edge, changed liquid once in per afterwards three days, treat that cell grows to the cultivation of going down to posterity after the 80%-90% fusion.
(2) cultivation of going down to posterity of umbilical cord mesenchymal stem cells
The umbilical cord mesenchymal stem cells primary cultured cell reaches the cultivation of going down to posterity after 80%-90% merges:
1) in super clean bench, clean the nutrient solution in former generation culturing bottle with suction pipe, use the rinsing of PBS liquid more once, draw PBS liquid;
2) in culturing bottle, add the about 1.5ml of 0.25% trypsinase;
3) place the situation of observation of cell digestion constantly under the inverted microscope immediately, treat that the intercellular substance increases, tailfiber disappears, and cellular contraction is a similar round, adds the about 5ml of the substratum that contains foetal calf serum immediately and stops trysinization;
4) use suction pipe suction substratum, make attached cell become single cell suspension at the bottom of blowing and beating culturing bottle repeatedly gently, cell suspension is transferred in the centrifuge tube of 15ml; With 1200rpm/min, centrifugal 3min, abandoning supernatant; Add fresh perfect medium; Aspirate substratum repeatedly with suction pipe, blow and beat the centrifuge tube bottom gently, make it become single cell suspension;
5) go down to posterity with the 1:2 ratio, plant culturing bottle, be positioned over CO in T25
2Cultivate in the incubator, changed liquid once in per afterwards three days, the back of at every turn going down to posterity later on is with the cultivation of going down to posterity of 1:3 ratio.
(3) flow cytometry is identified the immunophenotype of culturing cell
Get the cell of the 3rd generation separation and Culture in the umbilical cord, with the expression of CD44, CD166, CD34 and CD40L in the conventional flow cytometry identification of cell.
2, umbilical cord mesenchymal stem cells is to the differentiation of inducing of organizational project smooth muscle of bladder cell
Get the growth conditions umbilical cord mesenchymal stem cells in good P3 generation; In the L-DMEM nutrient solution that contains 10% foetal calf serum, cultivate; When treating that its growth reaches the 80%-90% fusion; The agent of adding cytokine induction--the external evoked differentiation of stroma cell derivative factor (SDF) 50 μ g/ml does not add inductor as control group.Changed a nutrient solution in per two days, observe stem cell growth state and metamorphosis situation every day under inverted microscope, treats that cell grows to the cultivation of going down to posterity when 80-90% merges, and experiments such as row immunocytochemistry, RT-PCR detect in the time of the 8th day, 14 days.
(2) experimental result
1, umbilical cord mesenchymal stem cells form and evaluation
As shown in Figure 1, under inverted microscope, observe, the cell of separation and Culture from umbilical cord tissue, form is irregular, is the fibroblast-like cells of corynebacterium, the visible a plurality of projections that have, kernel is obvious, and refractivity is good; Shown in Fig. 2 ~ 5; Identify through flow cytometry, by cell high expression level CD44, the CD166 of separation and Culture in the umbilical cord, faint expression CD34, CD40L; According to the expression of morphological specificity and CD44, CD166, CD34 and CD40L, explain that the present invention has obtained umbilical cord mesenchymal stem cells.
2, cultivating the external evoked back of umbilical cord mesenchymal stem cells detects
As shown in Figure 6, when inducing the 8th day, under inverted microscope, to observe, cell is spindle shape, and karyomorphism is irregular.Shown in Fig. 7 ~ 8, when cultivating the 8th day, cell expressing α-SMA positive cells number increases, and it is yellow that the positive cell endochylema is, and induce preceding color burn, and SM-MHC begins to appear faint expression, and control group does not have considerable change; Shown in Fig. 9 ~ 10; Showed increased when the a-SMA positive cells was than 8 days when cultivating 14 days, and the positive cell endochylema is deep yellow, and SM-MHC when cultivating 8 days positive cell number also showed increased and expressing strengthen; The positive cell endochylema is light yellow, and control group does not have considerable change.Experimental result explanation, induce 7 days after, the umbilical cord mesenchymal stem cells directed differentiation is the smooth muscle of bladder cell.
It is damaged that resulting smooth muscle of bladder cell can be used to repair bladder.
To sum up; Experiment proof inductor provided by the invention can external evoked umbilical cord mesenchymal stem cells directed differentiation be the smooth muscle of bladder cell; For clinical tissue engineering bladder makes up a kind of safe and reliable seed cell source is provided, has had a good application prospect and economic benefit.
Claims (7)
1. to induce the umbilical cord mesenchymal stem cells directed differentiation in preparation be the purposes in the inductor of smooth muscle of bladder cell to stroma cell derivative factor-1.
2. purposes according to claim 1 is characterized in that: comprising concentration in the said inductor is 25 ~ 75 μ g/ml stroma cell derivative factors-1.
3. purposes according to claim 2 is characterized in that: the concentration of said stroma cell derivative factor-1 is 50 μ g/ml.
One kind to induce the umbilical cord mesenchymal stem cells directed differentiation be the inductor of smooth muscle of bladder cell, it is characterized in that: it comprises concentration is 25 ~ 75 μ g/ml stroma cell derivative factors-1.
5. inductor according to claim 4 is characterized in that: the concentration of said stroma cell derivative factor-1 is 50 μ g/ml.
6. the described inductor of claim 4 ~ 5 is the application in the smooth muscle of bladder cell inducing the umbilical cord mesenchymal stem cells directed differentiation.
One kind to induce the umbilical cord mesenchymal stem cells directed differentiation be the method for smooth muscle of bladder cell, it is characterized in that: it comprises the steps:
(1) the described inductor of preparation claim 4 ~ 5;
(2) get umbilical cord mesenchymal stem cells, subculture in vitro separately is cultivated, and in nutrient solution, adds the inductor of step (2) preparation, and inducing culture is after 7 days, and umbilical cord mesenchymal stem cells is divided into the smooth muscle of bladder cell.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111269881A (en) * | 2020-02-12 | 2020-06-12 | 中国人民解放军第四军医大学 | Cell culture method for inducing differentiation of dental pulp stem cells to bladder smooth muscle cells in vitro |
| CN112391341A (en) * | 2020-11-30 | 2021-02-23 | 张川 | Application of SDF-1 protein activator in promoting human umbilical cord mesenchymal stem cell proliferation and differentiation in vitro |
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- 2012-06-15 CN CN2012101989661A patent/CN102732478A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111269881A (en) * | 2020-02-12 | 2020-06-12 | 中国人民解放军第四军医大学 | Cell culture method for inducing differentiation of dental pulp stem cells to bladder smooth muscle cells in vitro |
| CN112391341A (en) * | 2020-11-30 | 2021-02-23 | 张川 | Application of SDF-1 protein activator in promoting human umbilical cord mesenchymal stem cell proliferation and differentiation in vitro |
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Application publication date: 20121017 |