CN113577394B - Method for preparing biological gel-cell polymer system by umbilical cord mesenchymal stem cells and application of umbilical cord mesenchymal stem cells in low oxygen condition - Google Patents
Method for preparing biological gel-cell polymer system by umbilical cord mesenchymal stem cells and application of umbilical cord mesenchymal stem cells in low oxygen condition Download PDFInfo
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Abstract
The invention relates to the technical field of mesenchymal stem cells, in particular to a method for preparing a biogel-cell polymer system by umbilical cord mesenchymal stem cells and application of the biogel-cell polymer system under a low oxygen condition, which comprises the following steps: s1: separating, culturing and identifying human umbilical cord mesenchymal stem cells; s2: culturing and identifying umbilical cord mesenchymal stem cell polymers; s201 culturing umbilical cord mesenchymal stem cell polymers; s202, HE staining is carried out on umbilical cord mesenchymal stem cell aggregates; s203, observing the umbilical cord mesenchymal stem cell polymer by using a scanning electron microscope; s204, performing immunohistochemical staining on the umbilical cord mesenchymal stem cell polymer; s3: constructing a biological gel-umbilical cord mesenchymal stem cell polymer system; s301, separating and culturing human umbilical vein endothelial cells; identifying S302 hUVECs; s303, establishing a biogel-hUCMSCs polymer system; s304 biogel-hUCMSCs polymer system HE staining. By the method provided by the invention, complete repair of the function and the appearance of the defective muscle tissue can be efficiently and nondestructively obtained under the low oxygen condition.
Description
Technical Field
The invention relates to the technical field of mesenchymal stem cells, in particular to a method for preparing a biogel-cell polymer system by using umbilical cord mesenchymal stem cells and application of the biogel-cell polymer system under a low oxygen condition.
Background
The integrity of the mouth, jaw and limb function and the aesthetic appearance of patients have been affected by massive skeletal muscle defects caused by genetic diseases, dysplasias, surgical removal of tumors or trauma. The current clinical commonly used repair method comprises prosthesis made of various bioactive-free materials and transplantation of free or associated muscle skin flap, but the treatment means have some limitations, such as failure to recover muscle stretching function, requirement of transplantation operation on defect part or damage of donor part tissue, etc., while the altitude of 3500-5000 m high oxygen content is only 12.9-15.3%, and a large amount of clinical facts and scientific researches show that the hypoxia condition can seriously hinder the repair and regeneration of the tissue such as muscle, etc. Therefore, under the condition of hypoxia, the efficient and lossless complete repair of the function and the appearance of the defective muscle tissue becomes a new problem.
Disclosure of Invention
In view of the above, the present invention provides a method for preparing a biogel-cell polymer system from umbilical cord mesenchymal stem cells and an application of the system under a hypoxic condition, wherein the method can efficiently and nondestructively obtain complete repair of functions and shapes of defective muscle tissues under the hypoxic condition.
The invention solves the technical problems by the following technical means:
a method for preparing a bio-gel-cell polymer system by umbilical cord mesenchymal stem cells comprises the following steps:
s1: separating, culturing and identifying human umbilical cord mesenchymal stem cells;
s101, separating and culturing human umbilical cord mesenchymal stem cells;
s102, osteogenesis and adipogenesis induced differentiation;
s103, testing the clone formation rate;
s104 MTT growth Curve test
S105, identifying the flow cell surface marker;
s2: culturing and identifying umbilical cord mesenchymal stem cell polymers;
s201, culturing an umbilical cord mesenchymal stem cell polymer;
s202, staining an umbilical cord mesenchymal stem cell polymer HE;
s203, observing the umbilical cord mesenchymal stem cell polymer by using a scanning electron microscope;
s204, performing immunohistochemical staining on the umbilical cord mesenchymal stem cell polymer;
s3: constructing a biological gel-umbilical cord mesenchymal stem cell polymer system;
s301, isolated culture of human umbilical vein endothelial cells;
identifying S302 hUVECs;
establishing an S303 biological gel-hUCMSCs polymer system;
s304 biogel-hUCMSCs polymer system HE staining.
Further, in step S201, the specific steps of culturing the umbilical cord mesenchymal stem cell polymer are as follows: counting the number of the third generation hUCMSCs, then respectively inoculating the hUCMSCs into a six-hole culture plate, adding an alpha-MEM culture medium containing 10% FBS for culture, after the cells grow in an adherent manner and are converged to 90%, changing a polymer inducing solution for induction to form a polymer mature edge tilting, and harvesting the polymer by using a cell scraper.
Further, in step S202, the specific steps of staining the umbilical cord mesenchymal stem cell aggregate HE are as follows: washing a mature umbilical cord mesenchymal stem cell polymer by PBS, fixing paraformaldehyde, dehydrating by a dehydrator, embedding paraffin, continuously slicing by a slicer, performing gradient dewaxing to water by xylene and ethanol, staining by eosin hematoxylin, and sealing.
Further, in step S203, the specific steps of the scanning electron microscope observation of the umbilical cord mesenchymal stem cell polymer are as follows: washing a mature umbilical cord mesenchymal stem cell polymer by PBS, fixing glutaraldehyde, dehydrating by a vacuum dehydrator, melting and spraying pure gold, and observing and taking a picture by a scanning electron microscope.
Further, in step S204, the specific steps of the immunohistochemical staining of the umbilical cord mesenchymal stem cell aggregate are as follows: washing a mature umbilical cord mesenchymal stem cell polymer by PBS, fixing paraformaldehyde, dehydrating by a dehydrator, embedding by paraffin, continuously slicing by a slicer, performing gradient dewaxing to water by xylene and ethanol, repairing by citric acid solution under high pressure, treating by hydrogen peroxide, washing by PBS, soaking by Tween, sealing by goat serum, adding anti-integrin-beta 1, col-I, fibnectin primary antibody, passing through the night at 4 ℃, washing by PBS, adding rabbit/rat anti-human universal secondary antibody, incubating at 37 ℃, dyeing by DAB color developing solution, immediately stopping color development after dyeing under an observation mirror, and sealing by resin.
Further, in step S301, the specific steps of isolating and culturing the human umbilical vein endothelial cells are: taking an umbilical cord, breaking and flushing the umbilical cord vein tube, and emptying liquid after flushing; clamping the lower end of the umbilical cord, adding type II collagenase, sealing the upper end, and digesting in a CO2 incubator at 37 ℃; after digestion, loosening an umbilical cord at one end in a super clean bench, extruding digestive juice into a sterile culture dish, and washing the umbilical cord; collecting the digestive juice and the flushing liquid together into a centrifuge tube, centrifuging and settling cells, and removing supernatant; adding a special UVECs culture solution, uniformly blowing, inoculating the mixture into a culture dish for culture, culturing until the cell grows until the fusion rate reaches about 80%, carrying out passage according to the method in the step S101, and changing the special culture solution every three days.
Further, in step S302, the specific method for identifying the hvuecs includes: detecting specific expression proteins CD31 and factor VIII of rat umbilical vein endothelial cells by using cellular immunofluorescence, inoculating cells on a small sterilized creeper in a culture plate, sucking a culture solution when the cells grow to 80%, washing by using PBS, and sucking the cells; fixing the slide by using paraformaldehyde, and soaking and washing the slide by using PBS; treating the cells with Triton X-100 in PBS at room temperature; washing the slide with PBS, sucking to dry, and sealing cells on the slide with goat serum at room temperature; removing the sealing liquid without cleaning, dripping the diluted primary antibody on a climbing sheet, covering cells, putting the climbing sheet into a wet box, and incubating overnight in a refrigerator at 4 ℃; washing the slide by PBS, sucking out residual liquid, dropwise adding diluted fluorescent secondary antibody, incubating in a light-resistant wet box at 37 ℃, and washing by PBS; staining cell nucleuses on the creepers with DAPI, incubating in a dark place, washing with PBS, and washing the redundant fluorescent dye; sucking residual liquid on the climbing sheet, sealing the sheet by using a special sealing liquid containing the anti-fluorescence quenching agent, and observing and photographing under a fluorescence microscope.
Further, in step S303, the specific method for establishing the biogel-hutmscs polymer system is as follows: placing the Matrixgel in a refrigerator at 4 ℃ for freezing and thawing overnight, centrifugally sucking a supernatant, uniformly mixing the Matrixgel by using a precooled gun head, placing a pore plate on an ice surface, sucking the liquid Matrixgel, slowly adding the liquid Matrixgel into one of the pores along the pore wall, adding a serum-free alpha-MEM culture medium, uniformly mixing the culture medium with the gel in equal volume, adding the third generation of hUVECs into the liquid Matrixgel mixed solution, fully and uniformly mixing, placing the mixture in a 37 ℃ CO2 incubator, and solidifying the gel; taking down hUCMSCs cell aggregates from a pore plate, dripping alpha-MEM culture medium containing 10% FBS on the surface of the solid gel for infiltration, flatly paving the cell aggregates on the surface of the gel, dripping culture solution again, and standing in an incubator until the cell aggregates are attached to the solid gel;
and uniformly mixing the liquid Matrixgel with the equal volume of the serum-free alpha-MEM culture medium in the other hole, adding the third generation of hUVECs into the liquid Matrixgel mixed solution, fully and uniformly mixing, adding all the mixed solution into the first hole containing the solid gel and the cell aggregate by using a precooled gun head when the mixed solution is in a liquid state until all the liquid gel is uniformly spread on the solid gel-cell aggregate, placing the mixed solution in an incubator at 37 ℃, and waiting for the upper layer of biological gel to solidify.
Further, in step S304, the specific steps of HE staining the biogel-hutmscs polymer system are as follows: washing a biogel-hUCMSCs polymer system by PBS, fixing paraformaldehyde, dehydrating by a dehydrator, embedding by paraffin, continuously slicing by a slicer, performing gradient dewaxing to water by xylene and ethanol, staining by eosin hematoxylin, and sealing.
The invention also discloses an application of the umbilical cord mesenchymal stem cells in preparing a biogel-cell polymer system, and the biogel-cell polymer system is applied to muscle tissue repair and regeneration.
The invention has the beneficial effects that: the biological gel-cell polymer system prepared by the invention can promote the efficient regeneration of the defected muscle tissue, can also promote the regeneration of the defected muscle tissue when being applied to the hypoxia condition, has no damage to the function and the shape of the regenerated muscle tissue, and can completely repair the function and the shape of the muscle tissue at the defected part.
Drawings
FIG. 1 is an inverted microscope photograph of osteogenic induced differentiation in a method for preparing a bio-gel-cell polymer system from umbilical cord mesenchymal stem cells according to the present invention;
fig. 2 is an inverted microscope view of adipogenic induced differentiation in a method for preparing a biogel-cell aggregate system from umbilical cord mesenchymal stem cells according to the present invention;
FIG. 3 is an inverted microscope photograph of the colony formation rate in a method for preparing a bio-gel-cell aggregate system from umbilical cord mesenchymal stem cells according to the present invention;
FIG. 4 is a MTT growth curve test chart in a method for preparing a bio-gel-cell polymer system from umbilical cord mesenchymal stem cells according to the present invention;
FIG. 5 is a scanning electron microscope image of the umbilical cord mesenchymal stem cell polymer in the method for preparing the biogel-cell polymer system by using umbilical cord mesenchymal stem cells;
FIG. 6 is a fluorescence microscope image of hUVECs identification in a method for preparing a bio-gel-cell polymer system from umbilical cord mesenchymal stem cells according to the present invention;
FIG. 7 is a picture of a normal group in a rat Tibialis Anterior (TA) large volume defect repair experiment in application of hypoxic condition of a bio-gel-cell polymer system prepared from umbilical cord mesenchymal stem cells according to the present invention;
FIG. 8 is a picture of a 12-week postoperative control group in a rat Tibialis Anterior (TA) large-volume defect repair experiment in application of hypoxic conditions for a bio-gel-cell polymer system prepared from umbilical cord mesenchymal stem cells according to the present invention;
FIG. 9 is a drawing of a 12-week postoperative experimental group in rat Tibialis Anterior (TA) large-volume defect repair experiment in application of hypoxic condition for preparing a biogel-cell polymer system from umbilical cord mesenchymal stem cells according to the present invention;
FIG. 10 is a cross-sectional view of a normal group in a rat Tibialis Anterior (TA) large volume defect repair experiment in application of hypoxic condition of a bio-gel-cell polymer system prepared from umbilical cord mesenchymal stem cells according to the present invention;
FIG. 11 is a longitudinal sectional view of a normal group in a rat Tibialis Anterior (TA) large volume defect repair experiment in application of hypoxic condition of a bio-gel-cell polymer system prepared from umbilical cord mesenchymal stem cells according to the present invention;
FIG. 12 is a cross-sectional view of a 12-week postoperative control group in a rat Tibialis Anterior (TA) large volume defect repair experiment in application of hypoxic conditions for a bio-gel-cell polymer system prepared from umbilical cord mesenchymal stem cells according to the present invention;
FIG. 13 is a longitudinal section of a 12-week postoperative control group in rat Tibialis Anterior (TA) large volume defect repair experiment in application of hypoxic condition for a bio-gel-cell polymer system prepared from umbilical cord mesenchymal stem cells of the present invention;
FIG. 14 is a cross-sectional view of a 12-week postoperative experimental group in a rat Tibialis Anterior (TA) large-volume defect repair experiment in application of hypoxic conditions for preparing a biogel-cell polymer system from umbilical cord mesenchymal stem cells according to the present invention;
FIG. 15 is a longitudinal section of a 12-week postoperative experimental group in a rat Tibialis Anterior (TA) large-volume defect repair experiment in application of hypoxic condition of a bio-gel-cell polymer system prepared from umbilical cord mesenchymal stem cells according to the present invention;
Detailed Description
The invention will be described in detail below with reference to the following figures and specific examples:
the invention relates to a method for preparing a biogel-cell polymer system by umbilical cord mesenchymal stem cells, which comprises the following steps:
s1: isolated culture and identification of human umbilical cord mesenchymal stem cells
S101, separating and culturing human umbilical cord mesenchymal stem cells;
taking umbilical cord of a healthy and suitable-age puerpera, cutting the umbilical cord along veins by using an autoclave instrument, peeling and cutting Wharton's jelly into tissue blocks with the diameter of about 1mm3, inoculating the tissue blocks into a six-hole plate, wherein the culture solution is alpha-MEM containing 15% fetal calf serum, incubating the tissue blocks in a CO2 cell incubator for 5-7 days at the temperature of 37 ℃, and changing the culture solution once every three days until a large number of cells climb out from the edges of the tissue blocks and a plurality of colonies are formed; performing monoclonal screening on cultured cells by adopting a limiting dilution method, digesting primary cells by adopting 0.25% trypsin, counting by using a cell counter, adding a certain amount of diluted cell suspension into a 96-well cell culture plate to enable the number of cells in each well to be about 1, and then putting the cell culture plate into a 37 ℃ CO2 incubator for incubation; and after culturing for 8 hours, observing the growth condition of cells in the holes under a mirror, adding culture solution into the culture holes only containing one adherent cell, marking, continuously observing, digesting and passing to a larger culture plate when the cells in the marked holes occupy half of the area of the bottom of the holes, and gradually increasing the area of the bottom of the culture plate step by step to effectively amplify the cell amount.
S102, osteogenesis and adipogenesis induced differentiation;
the third generation of hUCMSCs were inoculated into six-well culture plates (2 x 105/well), after the cells had grown until about 80% confluency, changing into osteogenesis inducing solution (osteogenesis inducing solution comprises 250mL of alpha-MEM culture medium, 10% of fetal bovine serum, 5mM of beta sodium glycerophosphate, 100nM dexamethasone and 50mg/mL of ascorbic acid) or adipogenesis inducing solution (adipogenesis inducing solution comprises 250mL of alpha-MEM culture medium, 10% of fetal bovine serum, 2mM of glutamine, 100U/mL of penicillin, 100mM of ascorbic acid, 0.5mM of hydrocortisone and 60mM of indomethacin), changing the solution every three days, observing under a microscope to find calcified nodules or fat drops, PBS washing, paraformaldehyde fixation, alizarin red or oil red O staining, 4 ℃ overnight, followed by PBS washing 2 times, and photographing by observation under an inverted microscope, as shown in FIG. 1 and FIG. 2.
S103, testing the clone formation rate;
third generation hUCMSCs were inoculated at 1000 cells per dish (100 mm diameter) and cultured in α -MEM medium containing 10% FBS for 7-9 days. After washing twice with PBS, fixation was performed at 4 ℃ with 4% paraformaldehyde, then washing three times with PBS, staining with toluidine blue dye, then washing with PBS, and photographing was observed under an inverted microscope, as shown in fig. 3, and cell colonies of at least more than 100 cells were marked as one clone.
S104 MTT growth curve test;
the third generation of hUCMSCs were digested, counted and plated in ninety-six well plates (5X 103/well) with 6 wells per row for a total of 7 rows, incubated with α -MEM medium containing 10% FBS fetal bovine serum for 7 days per well, one entire row of medium was aspirated each day, PBS washed 2 times, 20 μ L of thiazole blue solution (5 μ g/mL) was added to each well of the first 4 wells of one row, the remaining two wells were set as controls, incubated at 37 ℃ in an incubator for 4 hours, PBS washed 2 times, 150 μ L of DMSO was added to each well, shaken in the dark for 10 minutes, absorbance was measured at 490nm wavelength in an enzyme linked immunosorbent assay, 2 control wells of 4 experimental wells per day was measured for 7 days, as shown in FIG. 4.
S105, identifying a flow cell surface marker;
digesting third-generation hUCMSCs, centrifuging, collecting, counting, washing and suspending by PBS, centrifuging to remove supernatant, resuspending cells by PBS containing 3% fetal bovine serum, subpackaging into EP tubes, wherein 200 mu L of liquid in each EP tube ensures that the number of cells in each tube is more than 2X 105, adding 2 mu L (1: 500) of CD29, CD31, CD34, CD44, CD45, CD105 and CD90 antibodies into each EP tube under an ice bath environment, incubating for 2 hours in a refrigerator in a dark place at 4 ℃, washing the cells by PBS for 3 times, resuspending the cells by 200 mu L of PBS containing 3% FBS, and detecting the expression condition of a cell surface marker by using a flow cytometer, wherein the expression condition of the cell surface marker is as follows: .
S2: culture and identification of umbilical cord mesenchymal stem cell polymer
S201 culturing umbilical cord mesenchymal stem cell polymers;
counting the number of the third generation hUCMSCs, then respectively inoculating cells into a six-well culture plate according to the number of 2 multiplied by 106 per well, adding alpha-MEM culture medium containing 10% FBS for culturing for 7-9 days, after the cells grow adherently and are converged to 90%, changing the polymer inducing liquid (the components of the polymer inducing liquid are 250mL of alpha-MEM culture medium, 10% FBS and 50mg/mL ascorbic acid) for inducing for 14 days, forming polymer mature edge tilting, and harvesting the polymer by cell scraping.
S202, HE staining is carried out on umbilical cord mesenchymal stem cell aggregates;
washing a mature umbilical cord mesenchymal stem cell polymer by PBS, fixing by 4% paraformaldehyde for 4 hours, dehydrating by a dehydrator, embedding by paraffin, continuously slicing by a slicer, performing gradient dewaxing to water by using 98%, 95%, 90%, 85%, 75%, 60% and 50% ethanol after dimethylbenzene, staining by eosin hematoxylin, and sealing.
S203, observing the umbilical cord mesenchymal stem cell polymer by using a scanning electron microscope;
washing the mature umbilical cord mesenchymal stem cell polymer by PBS, fixing by 2.5% glutaraldehyde for 4 hours, dehydrating by a vacuum dehydrator, melting and spraying pure gold, and taking a picture by scanning electron microscope observation, as shown in figure 5.
S204, performing immunohistochemical staining on the umbilical cord mesenchymal stem cell aggregate;
washing a mature umbilical cord mesenchymal stem cell polymer by PBS, fixing 4% paraformaldehyde for 4 hours, dehydrating by a dehydrator, embedding paraffin, continuously slicing by a slicer, performing gradient dewaxing by xylene and ethanol until water is obtained, repairing by citric acid solution under high pressure, treating by 3% hydrogen peroxide for 15 minutes, washing by PBS for three times, 5 minutes each time, soaking tween for a moment, sealing by 2% goat serum for 1 hour, adding anti-integrin beta 1, col-I and fibnectin primary antibody (1: 500) with proper concentration, standing overnight at 4 ℃, washing by PBS for three times, 5 minutes each time, adding rabbit/rat anti-human universal secondary antibody, incubating for 2 hours at 37 ℃, dyeing by DAB developing solution, immediately stopping developing after dyeing under an observation lens, and sealing by resin.
S3: construction of biogel-umbilical cord mesenchymal stem cell polymer system
S301, isolated culture of human umbilical vein endothelial cells;
taking umbilical cord of a parturient with a healthy and suitable age, processing a round and blunt syringe needle, carefully pricking into an umbilical vein tube, washing with sterile PBS for 3-5 times until the flushed liquid is free from blood color, and then pushing air to empty the liquid; clamping the lower end of the umbilical cord by using a hemostatic forceps, adding 2-3ml of type II collagenase (1mg/ml), sealing the upper end by using the hemostatic forceps, digesting for 10-15 minutes in a CO2 incubator at 37 ℃, and shaking once every 5 minutes; loosening one end of the hemostatic forceps in a super clean bench after digestion is finished to loosen one end of the umbilical cord, squeezing digestive juice into a sterile culture dish, and washing the umbilical cord for 3-5 times by PBS; collecting the digestive juice and the flushing liquid into a centrifuge tube, centrifuging (1000rpm, 8min) to settle cells, and removing supernatant; adding special UVECs culture solution (a special UVECs culture solution of Lonza, USA) to the culture medium, uniformly blowing, inoculating the culture medium to a 10mm culture dish for culture, changing the culture solution after 24 hours, washing with PBS, continuously culturing until the cell growth reaches about 80% of fusion rate, and then carrying out passage according to the method in the step S101, and changing the special culture solution every 3 days.
Identifying S302 hUVECs;
detecting specific expression proteins CD31 and factor VIII of rat umbilical vein endothelial cells by using cellular immunofluorescence, inoculating the cells on a small sterilized climbing piece in a 24-hole culture plate, sucking culture solution when the cells grow to 80%, washing by using PBS, and sucking the cells; fixing the slide with 4% paraformaldehyde at 4 ℃ for 30 minutes, and soaking and washing the slide with PBS for 3 times; cells were treated with Triton X-100 (0.5%) in PBS at room temperature for about 15 minutes; washing the slide with PBS for 3 times, sucking to dry, sealing the cells on the slide with goat serum, and standing at room temperature for about 30 min; spin-drying or sucking off the sealing liquid, not washing, dripping diluted proper amount of primary antibody on a climbing sheet, covering cells, putting into a wet box, and incubating overnight in a refrigerator at 4 ℃; washing the slide with PBS for 3 times, sucking out residual liquid, dropwise adding diluted fluorescent secondary antibody, incubating for 2 hours in a light-proof wet box at 37 ℃, washing with PBS for 3 times, and paying attention to the light-proof operation of the fluorescent secondary antibody; staining cell nucleuses on the creepers with DAPI, incubating for 5 minutes in a dark place, staining the nucleuses on the specimens, washing 3 times with PBS, and thoroughly washing the redundant fluorescent dye; residual liquid on the creeper is sucked dry, and the piece is sealed by special sealing liquid containing the anti-fluorescence quenching agent, and the piece is observed and photographed under a fluorescence microscope, as shown in figure 6.
Establishing an S303 biological gel-hUCMSCs polymer system;
placing Matrixgel in a refrigerator at 4 ℃ for freezing and thawing overnight, centrifuging at 14000rpm for 20min, sucking supernatant, uniformly mixing the Matrixgel by using a precooled gun head, placing a 12-hole plate on an ice surface, sucking 250 mu L of liquid Matrixgel, slowly adding 250 mu L of a serum-free alpha-MEM culture medium along the hole wall to avoid bubble generation, then adding 250 mu L of a serum-free alpha-MEM culture medium into the gel in equal volume, uniformly mixing, then adding third-generation hUVECs into the liquid Matrixgel mixed solution according to 1 x 106/ML, fully mixing, placing in a 37 ℃ CO2 incubator for 1 hour, solidifying the gel, then gently taking out hUCMSCs cell aggregates from the hole plate, dripping 2-3 drops of the alpha-MEM culture medium containing 10% of FBS on the surface of the solid gel for infiltration, then spreading the cell aggregates on the surface of the gel, dripping 5-6 drops of the culture solution again, and standing in the incubator for 4 hours to wait for the polymer cells to be solid gel;
and mixing 250 mu L of liquid Matrixgel with the same volume of serum-free alpha-MEM medium in another hole, adding the third generation of hUVECs into the liquid Matrixgel mixture according to 1 × 106/ML, mixing the mixture fully, when the mixture is in a liquid state, lightly adding the whole mixture into the first hole containing the solid gel and the cell aggregate by using a precooled gun head, and taking care that the mixed gel liquid is slowly dripped downwards from the center of the hole when the mixed gel liquid is added, so that the cell aggregate attached to the solid gel is prevented from being washed up by the added liquid until the whole liquid gel is uniformly laid on the solid gel-cell aggregate. Placing the mixture in an incubator at 37 ℃ for 1 hour until the biological gel on the upper layer is solidified.
S304 biogel-hUCMSCs polymer system HE staining;
the bio-gel-hucsmcs polymer system was washed with PBS, fixed with 4% paraformaldehyde for 4 hours, dehydrated with a dehydrator, embedded in paraffin, sliced continuously with a microtome, and gradient-dewaxed to water with 98%, 95%, 90%, 85%, 75%, 60%, and 50% ethanol in sequence after xylene. Staining with eosin and sealing.
Application of biological gel-cell polymer system in rat Tibialis Anterior (TA) large-volume defect repair experiment
The operation is prepared and implemented according to the operation specification of experimental animals as follows:
1) firstly, weighing a rat, and carrying out intraperitoneal injection anesthesia on 1% pentobarbital sodium, wherein the dosage is 50 mg/kg;
2) the sterilization scalpel makes a longitudinal incision of about 10mm on the anterior-lateral side of the lower hind limb of a rat, the distance between the sterilization scalpel and a tibia is about 1-2mm, then the skin and the fascia are separated bluntly, a longitudinal incision of about 10mm is made on the fascia covering TA, and then the fascia is separated bluntly carefully to keep the integrity of the fascia as much as possible;
3) after the TA is exposed, marking the area to be cut by using a precision surgical steel ruler and a medical skin marking pen, wherein the area is 1/3 positioned in the middle of the TA, the length is about 10mm, the width is about 5mm, the TA is separated in a blunt way by using small hemostatic forceps, and a thin stainless steel plate is inserted below the TA;
4) Firstly, making horizontal incisions at two ends of the defect along a marking line by using an operation blade, wherein the incision depth is about 1/3-1/2 of the thickness of muscle, so as to avoid cutting off the tendon, then making two longitudinal incisions along the marking line, wherein the distance from the near-far middle edge of the TA is about 2-3mm, and the four incisions are connected end to end;
5) lifting superficial tissues at the edge of the region to be excised by using a medical forceps, then separating and excising by using a surgical blade while turning up, weighing excised muscles, and then continuing to excise until the weight of excised muscles reaches about 20 percent of the total weight of TA;
6) the prosthesis is properly cut and then placed at the defect, fascia and muscle are sewn on the 6-0 absorbable nylon thread layer by layer, and the bandage is bandaged for 5-10 minutes after the iodophor is disinfected.
The normal group is directly sutured without defects after TA separation, the control group is pure Matrixgel, and the experimental group is a biogel-hUCMSCs polymer system. After the operation, the rats were raised in a hypoxic state with an oxygen content of 5%, and TA on both sides of the rats were taken under anesthesia 12 weeks after the operation.
As shown in fig. 7-15, the experimental results show that: general morphology of tibialis anterior muscle after 12 weeks of repair was observed by surgery, and it was seen that normal group had full muscle morphology and smooth surface without scar tissue. The muscle form of the control group is plump and the surface is uneven when the control group is seen in 12 weeks after the operation under the hypoxia condition; the experimental group had a full muscle form, smooth surface and no scar.
The muscle tissue grown by rat Tibialis Anterior (TA) experiment under the condition of hypoxia breeding is subjected to H & E staining, and the H & E staining result can be seen under an electron microscope: the normal group of the muscle tissues with transverse and longitudinal sections has smooth surface without connective tissues and complete structure. The surface of the residual muscle of the control group is flat after 12 weeks of operation, the residual muscle is rich in obvious fibrous connective tissue, and the residual muscle can be scattered in the dense fibrous tissue; in the experimental group, the diameter of the fiber of the new muscle tissue is slightly smaller than that of the original tissue, but the fiber is orderly arranged, and a small amount of fiber tissue is distributed around the muscle fiber.
The technical scheme can promote the efficient regeneration of the defective muscle tissue under the low oxygen condition, and the function and the appearance of the regenerated muscle tissue are not damaged, so that the function and the appearance of the muscle tissue at the defective part are completely repaired.
Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims. The techniques, shapes, and configurations not described in detail in the present invention are all known techniques.
Claims (8)
1. A method for preparing a biological gel-cell polymer system by umbilical cord mesenchymal stem cells is characterized by comprising the following steps:
s1: separating, culturing and identifying human umbilical cord mesenchymal stem cells;
s101, separating and culturing human umbilical cord mesenchymal stem cells;
s102, osteogenesis and adipogenesis induction differentiation are carried out;
s103, testing the clone formation rate;
s104MTT growth Curve test
S105, identifying a flow cell surface marker;
s2: culturing and identifying umbilical cord mesenchymal stem cell polymers;
s201 culturing umbilical cord mesenchymal stem cell polymers;
s202, HE staining is carried out on umbilical cord mesenchymal stem cell aggregates;
s203, observing the umbilical cord mesenchymal stem cell polymer by using a scanning electron microscope;
s204, performing immunohistochemical staining on the umbilical cord mesenchymal stem cell aggregate;
s3: constructing a biological gel-umbilical cord mesenchymal stem cell polymer system;
s301, isolated culture of human umbilical vein endothelial cells;
identifying S302 hUVECs;
establishing an S303 biological gel-hUCMSCs polymer system;
in the step S303, the specific method for establishing the biogel-hUCMSCs polymer system is as follows: placing the Matrixgel inFreezing and thawing in a refrigerator at 4 ℃ overnight, centrifugally sucking supernatant, uniformly mixing Matrixgel by using a precooled gun head, placing a pore plate on the ice surface, sucking liquid Matrixgel, slowly adding the liquid Matrixgel into one of the pores along the pore wall, adding a serum-free alpha-MEM culture medium, uniformly mixing with the gel in equal volume, adding third-generation hUVECs into the liquid Matrixgel mixed solution, fully and uniformly mixing, placing the mixture in a 37 ℃ CO (carbon monoxide) system, and uniformly mixing 2 In the incubator, the gel is solidified; taking down the hUCMSCs cell polymer from a pore plate, dripping alpha-MEM culture medium containing 10% FBS on the surface of the solid gel for infiltration, flatly paving the cell polymer on the surface of the gel, dripping culture solution again, and standing in an incubator until the cell polymer is attached to the solid gel;
uniformly mixing the liquid Matrixgel with the equal volume of the serum-free alpha-MEM culture medium in the other hole, adding the third generation of hUVECs into the liquid Matrixgel mixed solution, fully and uniformly mixing, adding all the mixed solution into the first hole containing the solid gel and the cell aggregate by using a precooled gun head when the mixed solution is in a liquid state until all the liquid gel is uniformly spread on the solid gel-cell aggregate, placing the mixed solution in an incubator at 37 ℃, and waiting for the upper layer of biological gel to solidify;
s304 biogel-hUCMSCs polymer system HE staining.
2. The method for preparing a biogel-cell polymer system from umbilical cord mesenchymal stem cells according to claim 1, wherein in the step S201, the specific steps of umbilical cord mesenchymal stem cell polymer culture are as follows: counting the number of the third generation hUCMSCs, then respectively inoculating the hUCMSCs into a six-hole culture plate, adding an alpha-MEM culture medium containing 10% FBS for culture, after the cells grow in an adherent manner and are converged to 90%, changing a polymer inducing solution for induction to form a polymer mature edge tilting, and harvesting the polymer by using a cell scraper.
3. The method for preparing a biogel-cell aggregate system by using umbilical cord mesenchymal stem cells according to claim 2, wherein in the step S202, HE staining of umbilical cord mesenchymal stem cell aggregates comprises the following specific steps: washing a mature umbilical cord mesenchymal stem cell polymer by PBS, fixing paraformaldehyde, dehydrating by a dehydrator, embedding by paraffin, continuously slicing by a slicer, performing gradient dewaxing to water by xylene and ethanol, staining by eosin hematoxylin, and sealing.
4. The method for preparing a biogel-cell polymer system from umbilical cord mesenchymal stem cells according to claim 3, wherein in the step S203, the specific steps of scanning electron microscope observation of umbilical cord mesenchymal stem cell polymers are as follows: washing a mature umbilical cord mesenchymal stem cell polymer by PBS, fixing glutaraldehyde, dehydrating by a vacuum dehydrator, melting and spraying pure gold, and observing and photographing by a scanning electron microscope.
5. The method for preparing a biogel-cell aggregate system by using umbilical cord mesenchymal stem cells according to claim 4, wherein in the step S204, the specific steps of immunohistochemical staining of umbilical cord mesenchymal stem cell aggregate are as follows: washing a mature umbilical cord mesenchymal stem cell polymer by PBS, fixing paraformaldehyde, dehydrating by a dehydrator, embedding by paraffin, continuously slicing by a slicer, performing gradient dewaxing to water by xylene and ethanol, repairing by citric acid solution under high pressure, treating by hydrogen peroxide, washing by PBS, soaking by Tween, sealing by goat serum, adding anti-integrin-beta 1, col-I, fibnectin primary antibody, passing through the night at 4 ℃, washing by PBS, adding rabbit/rat anti-human universal secondary antibody, incubating at 37 ℃, dyeing by DAB color developing solution, immediately stopping color development after dyeing under an observation mirror, and sealing by resin.
6. The method for preparing a biogel-cell polymer system from umbilical cord mesenchymal stem cells according to claim 5, wherein in the step S301, the specific steps of the isolated culture of the human umbilical cord vein endothelial cells are as follows: taking an umbilical cord, breaking and flushing the umbilical cord vein tube, and emptying liquid after flushing; clamping the lower end of the umbilical cord, adding collagenase type II, sealing the upper end, and introducing CO at 37 deg.C 2 Digesting in an incubator; after digestion, loosening an umbilical cord at one end in a super clean bench, extruding digestive juice into a sterile culture dish, and washing the umbilical cord; collecting digestive juice and washing liquid together into centrifuge tube, centrifuging and precipitatingReducing cells, and removing supernatant; adding a special UVECs culture solution, uniformly blowing, inoculating the mixture into a culture dish for culture, culturing until the cell grows until the fusion rate reaches about 80%, carrying out passage according to the method in the step S101, and changing the special culture solution every three days.
7. The method for preparing a bio-gel-cell polymer system from umbilical cord mesenchymal stem cells according to claim 6, wherein the specific method for identifying the hUVECs in step S302 is as follows: detecting specific expression proteins CD31 and factor VIII of rat umbilical vein endothelial cells by using cellular immunofluorescence, inoculating cells on a small sterilized creeper in a culture plate, sucking a culture solution when the cells grow to 80%, washing by using PBS, and sucking the cells; fixing the slide by using paraformaldehyde, and soaking and washing the slide by using PBS; treating the cells with TritonX-100 prepared with PBS at room temperature; washing the slide with PBS, sucking to dry, and sealing cells on the slide with goat serum at room temperature; removing the sealing liquid without cleaning, dripping the diluted primary antibody on a climbing sheet, covering cells, putting the climbing sheet into a wet box, and incubating overnight in a refrigerator at 4 ℃; washing the slide by PBS, dripping diluted fluorescent secondary antibody after sucking residual liquid, incubating in a light-resistant wet box at 37 ℃, and washing by PBS; staining cell nucleuses on the creepers with DAPI, incubating in a dark place, washing with PBS, and washing the redundant fluorescent dye; sucking residual liquid on the climbing sheet, sealing the sheet by using a special sealing liquid containing the anti-fluorescence quenching agent, and observing and photographing under a fluorescence microscope.
8. The method for preparing a bio-gel-cell aggregate system from umbilical cord mesenchymal stem cells according to claim 7, wherein the specific steps of HE staining the bio-gel-hUCMSCs aggregate system in step S304 are as follows: washing a biogel-hUCMSCs polymer system by PBS, fixing paraformaldehyde, dehydrating by a dehydrator, embedding by paraffin, continuously slicing by a slicer, performing gradient dewaxing to water by xylene and ethanol, staining by eosin hematoxylin, and sealing.
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