CN108103013B - Enzyme digestion method primary culture and identification method for smooth muscle cells of esophageal-gastric junction - Google Patents
Enzyme digestion method primary culture and identification method for smooth muscle cells of esophageal-gastric junction Download PDFInfo
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Abstract
The invention relates to an enzyme digestion method primary culture and identification method of smooth muscle cells at the junction of esophagus and stomach, which comprises the following steps: obtaining a smooth muscle tissue muscle strip at the esophagus-stomach junction; digesting the smooth muscle tissue muscle strips by using digestive juice to obtain a smooth muscle tissue cell suspension; and placing the smooth muscle tissue cell suspension in a culture container pre-paved with levorotatory polylysine, and culturing until adherent cells account for more than 90% of the bottom of the culture container or local cells grow crowded. The enzyme digestion method of the invention is simple, complete and effective in primary culture of smooth muscle cells at the esophagus-stomach junction, and the identification method can identify the unique phenotype of the smooth muscle cells.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to an enzyme digestion method primary culture and identification method of smooth muscle cells at the junction of esophagus and stomach.
Background
Primary esophageal motor dysfunction diseases (PEMD) including achalasia cardia (EAC), Diffuse Esophageal Spasm (DES), walnut-clamp esophagus (NE), esophageal sphincter hypertension (H L ES), and the like, all occur in the middle-lower segment of the esophagus to the esophageal-gastric junction (EGJ), all have pathological esophageal hypertension type motility abnormality, and weakening or disappearance of esophageal driving force to the esophagus, and all have clinical manifestations of chronic intermittent chest pain and dysphagia.
Disclosure of Invention
Aiming at the technical problems of complicated experimental steps and lack of detailed identification of related connective tissue cells in the prior art, the invention provides an enzyme digestion primary culture method of smooth muscle cells at the junction of esophagus and stomach.
And a method for identifying smooth muscle cells obtained by the enzyme digestion primary culture method of the smooth muscle cells at the esophageal-gastric junction
In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:
an enzyme digestion primary culture method of smooth muscle cells at an esophageal-gastric junction part comprises the following steps:
step a, obtaining a smooth muscle tissue muscle strip at an esophagogastric junction;
b, digesting the smooth muscle tissue muscle strips by using digestive juice to obtain a smooth muscle tissue cell suspension;
and c, placing the smooth muscle tissue cell suspension in a culture container pre-paved with levorotatory polylysine, and culturing until adherent cells account for more than 90% of the bottom of the culture container or local cells grow crowded.
Compared with the prior art, the enzyme digestion method for primary culture of the smooth muscle cells at the esophageal-gastric junction is simple, complete and effective, and the identification method can identify the unique phenotype of the smooth muscle cells. The enzyme digestion primary culture method of the smooth muscle cells at the esophageal-gastric junction lays a foundation for the research of primary esophageal motor dysfunction diseases and esophageal tissue engineering, and the obtained smooth muscle cells can be applied to the research of tissue engineering and regenerative medicine so as to prepare human body substitute tissues in the future.
The embodiment of the invention also provides an identification method of the smooth muscle cells obtained by the primary culture method of the smooth muscle cells at the esophagus-stomach junction by the enzyme digestion method, wherein α -SMA, Vimentin, Desmin, CD90, SM22 α and PCNA protein are used as markers.
The method for identifying the smooth muscle cells provided by the invention identifies the growth rule and the phenotype conversion characteristics of the human-derived smooth muscle cells of the digestive tract in the in-vitro culture process in detail, lays a foundation for the research on the corresponding physiological functions and physiological mechanisms of the smooth muscle cells of the digestive tract, and further lays a foundation for the research on the motor dysfunction and the reconstruction of the digestive tract.
Drawings
FIG. 1 is a diagram of enzyme digestion method for obtaining primary smooth muscle cells of the esophageal-gastric junction;
FIG. 2 is a comparison of the number of adherent smooth muscle cells obtained by 6 different enzymatic digestions;
FIG. 3 is a comparison of the time taken for smooth muscle cells to grow to the first vial by 6 different enzymatic digestions;
FIG. 4 shows the morphology of each group of cells after subculture of smooth muscle cells;
FIG. 5 is a smooth muscle cell population growth morphology;
FIG. 6 is a nodular elevation of smooth muscle cells;
FIG. 7 shows the proliferation of 3 rd generation smooth muscle cells in both SMCS and 10% -F12 media;
FIG. 8 shows an example of immunohistochemical staining of 6 smooth muscle tissue markers at the esophagogastric junction;
FIG. 9 shows the amplification curve and the lysis curve of mRNA expression of 6 smooth muscle tissue marker proteins at the esophageal-gastric junction;
FIG. 10 shows the mRNA expression levels of 6 smooth muscle tissue marker proteins at the esophageal-gastric junction;
FIG. 11 is a fluorescent identification of smooth muscle cells;
FIG. 12 is marker protein expression from smooth muscle cells;
FIG. 13 shows fluorescence data for marker protein expression in smooth muscle cells.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The embodiment of the invention provides an enzyme digestion primary culture method of smooth muscle cells at the junction of esophagus and stomach, which comprises the following steps:
step a, obtaining a smooth muscle tissue muscle strip at an esophagogastric junction;
b, digesting the smooth muscle tissue muscle strips by using digestive juice to obtain a smooth muscle tissue cell suspension;
and c, placing the smooth muscle tissue cell suspension in a culture container pre-paved with levorotatory polylysine, and culturing until adherent cells account for more than 90% of the bottom of the culture container or local cells grow crowded.
Compared with the prior art, the enzyme digestion method for primary culture of the smooth muscle cells at the esophagus-stomach junction is simple, complete and effective, and the obtained smooth muscle cells can be applied to tissue engineering and regenerative medicine research so as to make human body substitute tissues in the future.
Specifically, the step a specifically includes the following operations: taking tissues of an esophagus and stomach junction part cut in an operation, turning over to a mucous membrane surface, scrubbing the mucous membrane surface by iodophor, separating the mucous membrane layer sharply, shearing a thin transparent connective tissue layer on the surface, shearing a smooth muscle tissue, preparing a muscle strip of the smooth muscle tissue, and quickly placing the muscle strip in a container containing a culture medium and antibiotics to obtain the oral liquid.
Preferably, the tissue of the esophagus and stomach junction excised in the operation is taken, the blood supply vessel at the near end is separated for no more than 40min, the tissue is separated for no more than 15min in vitro, so that the tissue preservation and survival activity is ensured, the cell activity is kept in the subsequent operation time, the plane size of the muscle strip of the smooth muscle tissue is 5-15 mm × 5-10 mm, the size is favorable for shearing and preservation of the smooth muscle strip and ensures the basic tissue demand of the subsequent experiment, the culture medium is DMEM/F-12 without bovine serum, has the comprehensive advantages of rich F-12 nutrient components and higher DMEM nutrient component concentration, is more suitable for culturing the cells of the smooth muscle tissue, the antibiotic is streptomycin, belongs to broad-spectrum antibiotic, can be used for preventing bacterial pollution of cell culture, particularly pollution of gram positive and negative bacteria, and the whole operation process is completed within 10min so as to avoid the influence of the activity of the muscle strip in vitro due to overlong time.
The digestive enzymes in the step b comprise pancreatin/EDTA, collagenase I, collagenase II, collagenase V, collagenase VIII, collagenase XI, elastase, papain, deoxyribonuclease I, neutral protease/dispase and soybean trypsin inhibitor, and all the digestive enzymes can be used for digesting the smooth muscle cells, so that more choices can be provided for researchers when obtaining the smooth muscle cells.
Further preferably, the digestive enzyme is collagenase II, the concentration of the collagenase II is 125-200 CDU/mg, the concentration of the collagenase II in the digestive fluid is 0.5-1.0 mg/ml, the collagenase II has a digestion effect on intercellular substances, collagen components in connective tissues can be hydrolyzed, the first bottling time of cells is obviously shortened, the digestive enzyme also comprises 0.25% of pancreatin/EDTA, the pancreatin/EDTA is a reagent suitable for the smooth muscle cell culture, the cells can be separated by degrading intercellular proteins, and the smooth muscle cell membrane can be damaged by too high concentration of the digestive enzyme, so that the activity of the smooth muscle cell is influenced, and the adherence and proliferation of the cells are not facilitated.
Further preferably, the step b specifically comprises the following operations of cutting the smooth muscle tissue muscle strips obtained in the step a into tissue blocks with the plane size of 5-8 mm × 5mm to increase the contact area of the tissue blocks and digestive juice to facilitate digestion, rinsing with PBS + antibiotic mixed liquor, rinsing with PBS to remove residual culture medium, tissue exudates and the like, dissolving digestive enzyme with DMEM/F-12 stock solution to prepare digestive juice, placing the tissue blocks into a container filled with the digestive juice, sucking part of the digestive juice into the tissue blocks to fill the tissue blocks, placing the tissue blocks into an environment at 4 ℃ for digestion for 14-24 hours, wherein digestion effect is poor if digestion time is too short, smooth muscle cell activity is affected due to damage of cell membranes caused by the digestive enzyme if digestion time is too long, adhesion and proliferation are not facilitated, adding newborn calf serum when digestion is stopped, polishing with a tip and a pipette with a large opening to separate the tissue blocks from single cells, filtering the suspension to obtain the smooth muscle tissue cells which basically only contain single cells and can be directly cultured.
Further preferably, the antibiotic is streptomycin, belonging to the broad spectrum of antibiotics, useful for preventing bacterial contamination of cell cultures, in particular of gram-positive and negative bacteria; the volume ratio of PBS to antibiotics in the mixed solution is 5: 1; the rinsing times by the PBS + antibiotic mixed solution are 2 times, each rinsing time is 3min, and after rinsing, residual culture medium and exudates in tissue blocks can be removed; the number of times of rinsing with PBS is 1, the rinsing time is 3min, and residual culture medium and antibiotics can be removed after rinsing; the consumption of the digestive juice is 5-6 times of the volume of the tissue block, so that the tissue block can completely enter the digestive juice; the digestion process is interrupted and soft vibration is performed, so that the digestion is fully performed; the volume of the newborn calf serum is 20-25% of the volume of the digestive juice; the beating time is 5min, so that single smooth muscle cells are separated from tissues; the filter mesh is a nylon mesh with a pore size of 200 μm, and can be filtered to remove residual tissue, leaving single cells separated.
The step c specifically comprises the following operations: c, centrifuging the smooth muscle tissue cell suspension obtained in the step b, removing supernatant, and carrying out heavy suspension on the precipitate by using a smooth muscle cell culture medium to obtain single cell suspension; placing the single cell suspension in each hole of a multi-hole plate pre-paved with L-polylysine, shaking to uniformly disperse the cells in the single cell suspension, and placing in a ventilation atmosphere with 5% CO2Standing for 48 hours in a constant-temperature cell culture box at 37 ℃ to ensure that cells adhere to the wall; then gently flushed with PBSWashing each hole and adding a smooth muscle culture medium, so that cells which are not attached to the wall can be washed away, and the attached cells begin to grow and propagate in the culture medium; and carrying out primary flask distribution and passage when the cell gaps become small and the bottom of a multi-hole plate is fully paved to 90% or local cells grow crowded.
Further preferably, in the primary culture method of smooth muscle cells of esophagogastric junction by the enzyme digestion method described in the present application, the smooth muscle of esophagogastric junction includes esophageal circular muscle, esophageal longitudinal muscle, interlocking fiber, hook fiber, perigastric circular muscle near lasso fiber and perigastric circular muscle near hook fiber.
The embodiment of the invention also provides an identification method of the smooth muscle cells obtained by the enzyme digestion primary culture method of the smooth muscle cells at the esophagus-stomach junction, which takes α -SMA, Vimentin, Desmin, CD90, SM22 α and PCNA protein as markers, α -SMA, Vimentin, Desmin, CD90 and SM22 α can be used for identifying smooth muscle tissues and cells, wherein α -SMA and SM22 α have the strongest specificity, PCNA is mainly synthesized and stored in cell nucleus and participates in DNA synthesis, the expression is obviously increased in the G1-S phase of cell proliferation, and the PCNA is an important index for evaluating the proliferation state of the cells.
The identification method of the present invention allows the identification of a phenotype characteristic of smooth muscle cells, relative to the prior art. The enzyme digestion method for primary culture and identification of smooth muscle cells at the esophageal-gastric junction provided by the invention lays a foundation for the research of primary esophageal motor dysfunction diseases and esophageal tissue engineering.
In order to better illustrate the embodiments of the present invention, the following examples are further illustrative.
Example 1
The embodiment of the invention provides an enzyme digestion method primary culture and identification method of smooth muscle cells at the junction of esophagus and stomach, which comprises the following steps:
1. obtaining smooth muscle tissue muscle strips of esophagus and stomach junction
The smooth muscle tissue is obtained from tissues of an esophagogastric junction excised from a patient during surgery under the conditions approved by the ethical committee and informed consent of the patient himself or a legal authorizer, blood vessels at the proximal end are isolated for no more than 40min, the tissues are isolated for no more than 15min, the specimen is turned over to a mucosal surface, and after all mucosal tissues are scrubbed by iodophors, the mucosal layer is separated by sterile tissue shearing acutely, and the esophageal circular muscle (EC), esophageal longitudinal muscle (esophageal longitudinal muscle, E L), locked fiber (S), hook fiber (clasping long fiber, C), perigastric circular muscle (namely, fundus circular muscle, gastric circular muscle, etc.), gastric circular muscle-lateral gastric circular muscle (namely, primary gastric circular muscle-lateral muscle, GC-S) and lateral gastric circular muscle (namely, lateral gastric circular muscle-lateral gastric circular muscle, etc.) are prepared by placing smooth muscle tissue of smooth muscle-loop tissue, such as large curved muscle, smooth muscle-lateral muscle, GC-5, GC-32, GC-5, and GC-10. 5, 10. 5. 3. two kinds of each of these tissues are prepared by using transparent and one of tissue.
2. Digesting with digestive juice to obtain smooth muscle tissue cell suspension
Shearing a muscle strip of a smooth muscle tissue into a size of 5-8 mm × 5mm, rinsing with 1ml of PBS and 200 μ l of a streptomycin admixture for 2 times, 3min each time, then rinsing with 1ml of PBS for 1 time for 3min, dissolving collagenase II (collagenase II) with a DMEM/F12 stock solution to prepare a collagenase II digestive juice, wherein the concentration of the collagenase II digestive juice is 0.5mg/ml and 1mg/ml (collagenase II is not less than 125CDU/mg), transferring the smooth muscle tissue to a 2ml round bottom centrifuge tube, containing 5-6 times of the collagenase II digestive juice of the volume of a tissue block, sucking a proper amount of digestive juice by a 1ml syringe, injecting the tissue block into the tissue block to fill the tissue block, incubating the tissue block to 4 ℃ for 14-24 h at constant temperature, shaking gently during incubation, adding 400-600 μ l of newborn calf serum when the digestion is stopped, polishing the newborn calf serum mixture with a tip, filtering out a large-opening pipette, and gently blowing the aperture of the newborn calf serum mixture for about 5min, separating a single smooth muscle cell, and sieving a nylon tissue suspension with 200 μm of the smooth muscle cell to obtain a smooth muscle cell suspension.
3. Transferring the smooth muscle tissue cell suspension to a culture container for primary culture
And centrifuging the obtained smooth muscle tissue cell suspension for 5min at the centrifugal radius of 10cm at 1000r/min, removing the supernatant, and re-suspending the precipitate in 1ml of smooth muscle culture medium to obtain the single cell suspension. The single cell suspensions were placed in six well plates pre-plated with l-polylysine and gently shaken to re-disperse the cells evenly. Placing the six-hole plate in aeration with 5% CO2The cell is kept still in a constant temperature cell culture box at 37 ℃ so as to adhere to the wall, and the cell cannot shake within 48 hours. After 48h each well was rinsed gently with PBS to clear nonadherent cells and a small amount of fibrous tissue and 1ml of smooth muscle medium was added. When the cell gap becomes smaller and the bottom of the culture dish is fully paved by nearly 90 percent, or when local cells grow crowded, the first flask division and passage are carried out.
The samples with collagenase II digestion solution concentration of 0.5mg/ml are set as 0.5-EI-4 group, the samples with collagenase II digestion solution concentration of 1mg/ml are set as 1-EI-4 group, enzyme digestion experiments are carried out under other conditions, the samples are compared, smooth muscle tissue blocks are cut into 1-3 mm × 1-3 mm fragments, then the samples are digested for 1h at 37 ℃ by 1mg/ml collagenase II digestion solution, the groups are set as 1-C-37 group, the smooth muscle tissue blocks are cut into 1-3 mm × 1-3 mm fragments, then the samples are digested for 14-24 h at 4 ℃ by 0.5mg/ml collagenase II digestion solution, the groups are set as 0.5-C-4 group, the smooth muscle tissue blocks are cut into 1-3 mm × 1-3 mm fragments, then the samples are digested for 1h at 37 ℃ by 0.25% pancreatin EDTA digestion solution, the groups are set as 0.25-T-37 group, the smooth muscle tissue blocks are cut into 1-3 mm × 1 mm fragments, then the samples are cut into 0.125-4% pancreatin EDTA digestion solution, and the groups are set as 0.4-4% pancreatin EDTA digestion solution, the groups.
4. Biological characterization of smooth muscle cells
(1) Cell viability assay
5 drops of the cell suspension are taken, 1 drop of 0.2 percent trypan blue solution is added into each drop of the cell suspension, the mixture is mixed evenly and counted within 1min, when the cells die, the cells are dyed into red, and the living cells are not dyed, and the cell survival rate is calculated by the following formula, namely the cell survival rate (%) -total number of active cells/(number of active cells + number of dead cells) × 100 percent.
As shown in figure 1, all smooth muscle cells can grow in an adherent manner, after the cells stretch out and adhere to the wall, the cells are uniformly distributed in the background and can be seen scattered in tiny tissues and cell fragments, the cells are mostly in a fusiform or long fusiform shape, two ends of the cells are slightly blunt, the cells are partially rod-shaped, meanwhile, fibroblasts mixed with partial two ends and pseudo toe length are seen, and the survival rate of the cells is over 90% as determined by trypan blue solution.
As shown in fig. 2 and 3, the number of adherent cells visible per 200-fold field of the endoscope was analyzed by comparing the numbers of the enzyme digestions of the respective groups, and no statistical difference was observed (P ═ 0.864). The cells obtained by the enzyme digestion method of each group are compared until the first bottle splitting time, the 0.5-EI-4 group has statistical difference (P values are 0.00 and 0.01 respectively) with the 0.25-T-37 group and the 0.5-C-4 group, and the rest groups have no statistical difference.
(2) Smooth muscle cell proliferation assay
At 25cm2(50ml) Disposable flasks cultured the cells. As can be seen in fig. 4, the cells were significantly larger than the primary adiposity after the passage of digestion. As can be seen in fig. 5, the cells grew themselves in a certain direction, appearing as a peak-valley like pattern. With the increase of the number of passages, the volume of the smooth muscle cells is gradually increased, and synapses are obvious and gradually lose fusiform structures. As shown in fig. 6, when the cells grow densely, the crowded cells surround layer by layer, and the center of the crowded cells can be raised to form a knot.
And (3) passaging the cells obtained by the enzyme digestion method to the 3 rd generation, culturing the cells in a professional smooth muscle cell culture medium (SMCS) or a DMEM/F12 culture medium (10% -F12) containing 10% newborn calf serum respectively, and recording the growth condition of the cells in time.
And inoculating the 3 rd generation smooth muscle cells into a 96-well plate by taking 2000 cells per well as a standard, wherein 3-4 auxiliary measurement wells are required for each sample. The 96-well plate was placed statically in a cell culture box, and cell adherence and expansion were observed after 6 hours. The suspension was prepared by adding 10. mu.l of CCK-8 reagent per 100. mu.l of medium. After removing the medium from each well to be tested, 100. mu.l of the suspension was added to each well. Standing the 96-well plate in an incubator for 2h, taking out, placing the 96-well plate on a microplate oscillator, oscillating for 1min, transferring the 96-well plate to an enzyme-labeling instrument, reading data of each well at a wavelength of 450nm, and recording. The wells after measurement of absorbance were not reused. The absorbance of the experimental wells was measured every 24h for 9 consecutive days (216 h).
As shown in FIG. 7, the smooth muscle cells obtained in this example showed a typical S-shaped cell proliferation curve in SMCS, but slowly or even almost arrested proliferation in 10% -F12.
5. Identification of smooth muscle cells
(1) Immunohistochemical (IHC) identification
Expression of α -SMA, SM22 α, Vimentin, Desmin, CD90 and PCNA proteins was observed in paraffin sections of 6 esophagogastric junction smooth muscle tissues, as shown in FIG. 8, wherein α -SMA, Vimentin and Desmin were strongly or weakly positive in cytoplasm, SM22 α and CD90 were positive or weakly positive in cytoplasm, and PCNA was strongly or positively positive in part of nuclei.
(2) Realtime RT-PCR experiment
Whether the primers of the related genes have amplification curves or not and a melting curve are drawn according to ABI 7500Real-Time PCR (polymerase chain reaction), and mRNA (messenger ribonucleic acid) expressing α -SMA, Vimentin, Desmin, CD90, SM22 α and PCNA in smooth muscle cells of EGJ 6 smooth muscle tissues can be definitely judged, and is shown in figure 9.
The mRNA expression levels of 6 smooth muscle tissues α -SMA, SM22 α, Vimentin, Desmin, CD90 and PCNA at the esophageal-gastric junction are shown in FIG. 10.
(3) Immunofluorescence identification of smooth muscle cells
The primary cells obtained by the enzyme digestion method can be observed that most cells begin to stretch 48 hours after the cells are attached to the wall, immunofluorescence experiments are carried out after the primary cells are passaged (generation 2), and the expression of six proteins of α -SMA, Vimentin, Desmin, CD90, SM22 α and PCNA can be seen by using the corresponding receptor expression of a confocal microscope (L SM510, ZEISS, Germany), as shown in figure 11.
(4) Incellwestern method identification
Expression of α -SMA, Vimentin, Desmin, CD90, SM22 α and PCNA six proteins in the smooth muscle cells (3 rd generation) obtained by the enzyme digestion method can be detected by the Incellwestern method, as shown in FIG. 12 and FIG. 13.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (7)
1. An enzyme digestion primary culture method of smooth muscle cells at the junction of esophagus and stomach is characterized in that: the method comprises the following steps:
step a, obtaining a smooth muscle tissue muscle strip at an esophagogastric junction;
b, digesting the smooth muscle tissue muscle strips by using digestive juice to obtain a smooth muscle tissue cell suspension; the method specifically comprises the following operations:
b, cutting the smooth muscle tissue muscle strips obtained in the step a into tissue blocks with the plane size of 5-8 mm × 5 mm;
rinsing with PBS + antibiotic mixed solution, and rinsing with PBS;
dissolving digestive enzyme by DMEM/F-12 stock solution to prepare digestive fluid, placing the tissue block in a container filled with the digestive fluid, sucking part of the digestive fluid and injecting the digestive fluid into the tissue block to enable the tissue block to be full;
placing the mixture in an environment at 4 ℃ for digestion for 14-24 hours, adding newborn bovine serum, and blowing the mixture by a suction tube with a polished tip;
filtering to obtain smooth muscle tissue cell suspension;
the digestive enzyme is collagenase II, the concentration of the collagenase II is 125-200 CDU/mg, and the concentration of the collagenase II in the digestive juice is 0.5-1.0 mg/ml;
and c, placing the smooth muscle tissue cell suspension in a culture container pre-paved with levorotatory polylysine, and culturing until adherent cells account for more than 90% of the bottom of the culture container or local cells grow crowded.
2. The primary culture method of esophagogastric junction smooth muscle cells by enzyme digestion according to claim 1, wherein: the antibiotic is streptomycin;
the volume ratio of PBS to antibiotics in the mixed solution is 5: 1;
the rinsing times by PBS + antibiotic mixed liquid are 2 times, and the rinsing time is 3min each time;
the number of times of rinsing with PBS is 1, and the rinsing time is 3 min;
the volume of the digestive juice in the container filled with the digestive juice is 5-6 times of the volume of the tissue block;
the digestion process is interrupted and the vibration is soft;
the volume of the newborn calf serum is 20% -25% of the volume of the digestive juice;
the blow-beating time is 5 min;
the filter screen for filtering is a nylon screen, and the aperture is 200 mu m.
3. The primary culture method of esophagogastric junction smooth muscle cells by enzyme digestion according to claim 1, wherein: the c specifically comprises the following operations:
c, centrifuging the smooth muscle tissue cell suspension obtained in the step b, removing supernatant, and carrying out heavy suspension on the precipitate by using a smooth muscle cell culture medium to obtain single cell suspension;
placing the single cell suspension in each hole of a multi-hole plate pre-paved with L-polylysine, shaking to uniformly disperse the cells in the single cell suspension, and placing in a ventilation atmosphere with 5% CO2After standing for 48 hours in a constant-temperature cell culture box at 37 ℃, gently washing each hole by PBS and adding a smooth muscle cell culture medium;
and carrying out primary flask distribution and passage until the cell gaps become small, the bottom of a multi-hole plate is fully paved by 90 percent or local cells grow crowded.
4. The primary culture method of esophagogastric junction smooth muscle cells by enzyme digestion according to claim 1, wherein: the step a specifically comprises the following operations:
taking tissues of an esophagus and stomach junction part cut in an operation, turning over to a mucous membrane surface, scrubbing the mucous membrane surface by iodophor, separating the mucous membrane layer sharply, shearing a thin transparent connective tissue layer on the surface, shearing a smooth muscle tissue, preparing a muscle strip of the smooth muscle tissue, and quickly placing the muscle strip in a container containing a culture medium and antibiotics to obtain the oral liquid.
5. The primary culture method of esophagogastric junction smooth muscle cells by enzyme digestion according to claim 4, wherein: the tissue of the esophagus and stomach junction cut in the operation is taken, the blood supply vessel at the near end is not disconnected for more than 40min, and the tissue is not separated for more than 15 min;
the plane size of the smooth muscle tissue muscle strips is 5-15 mm × 5-10 mm;
the culture medium is DMEM/F-12 without bovine serum;
the antibiotic is streptomycin;
the whole operation process is completed within 10 min.
6. The method for enzyme digestion primary culture of smooth muscle cells of the esophagogastric junction according to any one of claims 1 to 5, wherein the smooth muscle of the esophagogastric junction comprises esophageal circular muscle, esophageal longitudinal muscle, interlocking fiber, hook fiber, perigastric circular muscle near lasso fiber and perigastric circular muscle near hook fiber.
7. A method for identifying smooth muscle cells obtained by the primary culture method of smooth muscle cells at the esophageal-gastric junction according to any one of claims 1 to 6, which comprises using α -SMA, Vimentin, Desmin, CD90, SM22 α and PCNA protein as markers.
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| CN109762796A (en) * | 2018-12-25 | 2019-05-17 | 中国医学科学院北京协和医院 | Composition and its preparation method and application containing trypsin inhibitor |
| CN109666625A (en) * | 2018-12-25 | 2019-04-23 | 中国医学科学院北京协和医院 | Single celled method is separated from tissue |
| CN109694845A (en) * | 2018-12-25 | 2019-04-30 | 中国医学科学院北京协和医院 | For separating single celled kit and its application |
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