CN102168062B - Human pulmonary artery smooth muscle cell separation and culturing method and application of same - Google Patents
Human pulmonary artery smooth muscle cell separation and culturing method and application of same Download PDFInfo
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Abstract
The invention relates to a method of separating and culturing human pulmonary artery smooth muscle cells from strips of PTE and the cells' application in studies of diseases related to lung circulation. The method comprises treating cell membranes by single enzymic digestion method to decrease damage degree of the cells, thereby benefiting survival of the cells. The culturing method has stable technology and good repeatability, the cultured cells have uniform forms, and the growth of the cells is good. At the same time, the cells obtained provide rich material source with strong singularity for further study on lung circulation diseases, particularly for experiments on Chronic Thromboembolic Pulmonary Hypertension patients' pathogenesis, diseases generation and development processes and PTE prognosis.
Description
Technical field
The present invention relates to a kind of from people's treatment of pulmonary thromb-endarterectomy (PTE) overburden, the separation and the method for cultivator arteria pulmonalis smooth muscle cells and the smooth muscle cell that is obtained by the method, and the application of this kind cell in the research of pulmonary circulation relative disease, belong to biological technical field.
Background technology
Human pulmonary artery smooth muscle cells is the important cells material of the disease incidence mechanism such as study of lung circular correlation disease, especially pulmonary hypertension.The arteria pulmonalis smooth muscle cells source that is used at present experimental study both at home and abroad is in the majority with animal, wherein mainly is to derive from rat.But because kind is different, intercellular differing greatly is so the arteria pulmonalis smooth muscle cells in mouse source is used for the cell that the meaning of people's pulmonary hypertension and study of incident mechanism thereof is significantly less than employment.There is some scholars to adopt the arteria pulmonalis smooth muscle cells of induced labor foetus (in 18 weeks) to carry out experimental study for this reason, because the fetal cell multiplication capacity is strong and be easy to cultivate, but because Fetal Lung is not yet reached maturity, its arteria pulmonalis smooth muscle cells and adult's cell is difference to some extent also, and the cultural method of therefore seeking simple adult's pulmonary arterial smooth muscle cell just seems very important.In addition, because difference, the pathogenetic difference of morbid state, cell can have different functions and characteristic, therefore successfully separate and cultivate pulmonary hypertension patient pulmonary arterial smooth muscle cell and since the conversion medical research for the pathogenesis of inquiring into targetedly this kind disease, to the possible means of seeking to improve pulmonary hemodynamics and stoping the lung vascular remodeling, seek new treatment target spot, prevention and treatment CTEPH tool are of great significance tool are of great significance.
That chronic thromboembolic states pulmonary hypertension (Chronic Thromboembolic Pulmonary Hypertension, CTEPH) refers to is relevant with the PTE that once or repeatedly occurs, take resistance of pulmonary blood flow increase and the pulmonary artery pressure rising as the clinical disease of feature.Because its morbidity concealment, rapid, the mortality ratio very high (America NI H data shows that its mean survival time only is 2.8 years) of progress have become quite concerned public health problem at present.
The research of nearly more than 10 years relevant pulmonary hypertensions has obtained marked improvement, but very deficient about the research contents of the pathophysiological mechanism of CTEPH genesis.Especially part patient
The mechanism of lung vascular remodeling is also studied very few, and wherein one of important reason is exactly the pulmonary arterial smooth muscle cell that lacks patient CTEPH that can supply the research application.
Treatment of pulmonary thromb-endarterectomy (PTE) is that the modes such as chest, extracorporeal circulation, profound hypothermia are opened in a kind of employing, complete removing thromboembolic states patients with pulmonary hypertension pulmonary thrombosis, strip off the inner membrance that thickens, unobstructed to reach the recovery pulmonary arterial vascular, reduce pulmonary artery pressure, thereby improve heart function, prolong patient's life, recover the operation of patients ' life quality.Surgical procedure is that rete divests blood vessel occlusion is complete from pulmonary artery, we have found that the sample of PTE operation contains pulmonary artery endothelial cell, endothelial progenitor cells, inoblast and smooth muscle cell.Because operating difficulty is very big, thereby sample is very precious.Only have at present the only a few scholar from PTE postoperative patient sample, to obtain human pulmonary artery smooth muscle cells, the method of the cultivator arteria pulmonalis smooth muscle cells of taking comprises composite collagen enzyme process or Explant culture, but simple Explant culture culture cycle is long, the used enzyme class of composite collagen enzyme process is too much, not only the comparatively small amt of the initiating cell of the uneconomical but also work that obtains.Therefore it is easy to operate to set up a cover, culture cycle is short, purity is high, the separation from the overburden for the treatment of of pulmonary thromb-endarterectomy (PTE) that survival rate is high and the method for cultivator arteria pulmonalis smooth muscle cells thereof are very important, for the pathogenesis of research thromboembolic states patients with pulmonary hypertension, understand migration, propagation and the secreting function of smooth muscle cell in the disease development process variation with and the signal transduction process, understand patient's prognosis and have very important significance.
Summary of the invention
The purpose of this invention is to provide a kind of from the overburden for the treatment of of pulmonary thromb-endarterectomy (PTE), the separation and the method for cultivator arteria pulmonalis smooth muscle cells.The method is consistent, good reproducibility, and cultured cells form homogeneous, well-grown, and have form and the characteristics of typical smooth muscle cell.
The objective of the invention is to be achieved through the following technical solutions:
A kind ofly from treatment of pulmonary thromb-endarterectomy (PTE) overburden, separate, the method for cultivator arteria pulmonalis smooth muscle cells, step is as follows:
1. obtain the arteria pulmonalis smooth muscle cells of former culture: from the overburden for the treatment of of pulmonary thromb-endarterectomy (PTE), separate human smooth muscle cell, and carry out former culture, obtain the human pulmonary artery smooth muscle cells of former culture;
2. obtain the arteria pulmonalis smooth muscle cells that goes down to posterity and cultivate: the primary cell in step 1. grows to the logarithmic growth after date, after the taking-up primary cell cleans with PBS solution, add 33-37 ℃ of digestion of 0.2-0.5ml 0.1-0.2% (w/v) trypsin solution (containing 0.01-0.05%EDTA) 1～2 minute, when retraction appears in cell, when the intercellular substance increases, the sucking-off trypsin solution, add 5～7ml complete culture solution, gentle concussion makes it to form cell suspension, be seeded in the new culturing bottle, changed liquid once in per two days, namely finish passage cell and cultivate, obtain the chronic thromboembolic states hypertensive pulmonary arterial disease human pulmonary artery smooth muscle cells that goes down to posterity and cultivate.
The described concrete steps of obtaining the arteria pulmonalis smooth muscle cells of former culture in the described step 1 are:
1) the lung vascular tissue sample that strips down in the Endarterectomy art places 4 ℃ of preservations of HBSS liquid;
2) super clean bench uviolizing 30-40min, 75% alcohol wipe, all apparatuses, vessel autoclaving;
3) preheating Digestive system, HBSS and complete culture solution to 37 ℃;
4) sample is placed 37 ℃ of vessel that contain HBSS clean 8-15min;
5) it is stand-by the 10ml Digestive system to be filtered to small beaker with 2.2 μ m;
6) white sample that will the wide strip of the long 1mm of about 1.5cm is cut into the fragment of about 1mm * 1mm * 1mm with aseptic scissors in Digestive system, continue digestion 25-35min;
7) the sample fragment of tissue is transferred in the aseptic centrifuge tube room temperature 280-350g gradient centrifugation 12-20min;
8) the sucking-off supernatant liquor discards, and the sucking-off cell suspension is collected in another aseptic centrifuge tube, adds complete culture solution and adjusts cell density 3-4 * 10
5/ ml, and divide kind to 6 orifice plates with this density, 37 ℃, 5%CO put
2Leave standstill cultivation in the incubator;
9) postdigestive fragment of tissue was planted to 6 orifice plates with perfect medium in resuspended rear minute again.
10) observe next day, 3-4 days afterwards all or half amount change nutrient solution.
11) when Growth of Cells to logarithmic phase, namely finish primary cell culture, obtain the human pulmonary artery smooth muscle cells of former culture;
In the above-mentioned steps HBSS solution be: calcium chloride 1.0-1.5mM, sodium-chlor 120-150mM, Repone K 5.0-6.0mM, magnesium chloride 0.3-1.0mM, dipotassium hydrogen phosphate 0.2-1.0mM, sal epsom 0.2-1.0mM, sodium bicarbonate 3.5-4.5mM, Sodium phosphate dibasic 0.1-0.5mM, sugar-free glucose 4.0-7.0mM and HEPES 5.0-10.0mM, the pH value of this solution is 7.2-7.4, tests preparation in front 24 hours and high pressure degerming.
Digestive system is: adding II Collagenase Type is formulated in the HBSS balanced salt solution, and ratio is the 8-12mlHBSS balanced salt solution: 8-30mg II Collagenase Type.
Complete culture solution is: by M231 basic medium (SMBS), medium supplement (SMGS), the microbiotic of penicillin and Streptomycin sulphate is formulated, wherein the volume ratio of M231 basic medium and medium supplement SMGS is 90-100: 1-5, the final concentration of penicillin is 80-100U/ml, and the final concentration of Streptomycin sulphate is 80-100mg/ml.
The invention still further relates to a kind of human pulmonary artery smooth muscle cells that is obtained by above-mentioned cultural method.
The invention still further relates to a kind of described human pulmonary artery smooth muscle cells in the application of study of lung circular correlation disease.
In the technical scheme of the present invention, the pulmonary circulation relative disease is pulmonary hypertension.
In the technical scheme of the present invention, described pulmonary hypertension is the thromboembolic states pulmonary hypertension.
The inventive method compared with prior art has following beneficial effect:
1. the degree of damage by common instruments, utilization single enzyme digestion method cell membrane is light, is beneficial to cell survival, has solved the technical barrier of separation and Culture human pulmonary artery smooth muscle cells from treatment of pulmonary thromb-endarterectomy (PTE) overburden.
2. cultural method is consistent, good reproducibility, cultured cells form homogeneous, well-grown.
3. the cell that obtains identifies to have typical arteria pulmonalis smooth muscle cells form and characteristics through microscopic examination and immunocytochemistry, and derive from the thromboembolic states patients with pulmonary hypertension because of it, therefore be further to further investigate disease of pulmonary circulation, especially the pathogenesis of thromboembolic states patients with pulmonary hypertension, the disease development process with and the experiment such as PTE prognosis the material source of abundant high specificity is provided.
Description of drawings
Fig. 1 is the overburden (PTA) (1 *) for the treatment of of pulmonary thromb-endarterectomy (PTE)
Fig. 2 is the former culture arteria pulmonalis smooth muscle cells of thromboembolic states patients with pulmonary hypertension under the common inverted phase contrast microscope (40 *);
Fig. 3 is the thromboembolic states patients with pulmonary hypertension cultivation arteria pulmonalis smooth muscle cells that goes down to posterity under the fluorescent microscope (20 *), occurs blue positive fluorescent reaction in the Visible Core endochylema, the smooth muscle cell slurry SM-α-positive fluorescent reaction of the rubescent look of Actin antigen;
Fig. 4 is that the thromboembolic states patients with pulmonary hypertension goes down to posterity and cultivates arteria pulmonalis smooth muscle cells slurry SM-α-positive fluorescent reaction of the rubescent look of Actin antigen under the fluorescent microscope (20 *);
Fig. 5 is that the thromboembolic states patients with pulmonary hypertension goes down to posterity to cultivate in the arteria pulmonalis smooth muscle cells nuclear and blue positive fluorescent reaction occurs under the fluorescent microscope (20 *);
Embodiment
The present invention is described in further detail below in conjunction with example and accompanying drawing, but embodiments of the present invention are not limited to this, according to foregoing of the present invention, ordinary skill knowledge and customary means according to this area, not breaking away under the above-mentioned basic fundamental thought of the present invention prerequisite, can also make modification, replacement or the change of other various ways.
Embodiment 1
1, main experiment material
(1) Specimen origin: the overburden that obtains in the thromboembolic states patients with pulmonary hypertension treatment of pulmonary thromb-endarterectomy (PTE).
(2) HBSS balanced salt solution: add 8.0g sodium-chlor, 0.4g Repone K, 0.1g magnesium chloride, 1.0g glucose 0.14g calcium chloride 0.06g potassium primary phosphate in the 1000ml tri-distilled water, 0.1g sal epsom, 0.35g sodium bicarbonate, 0.09g Sodium phosphate dibasic and 2.08gHEPES, testing preparation in front 24 hours and filtration sterilization or autoclaving pH value is 7.4.
(3) Digestive system: adding 25mg II Collagenase Type is formulated in the 10mlHBSS balanced salt solution.
(4) complete culture solution is: by the M231 basic medium (SMBS) of 46.5ml, and the medium supplement of 2.5ml (SMGS), the microbiotic of the penicillin of 100U/ml, the Streptomycin sulphate of 100mg/ml is formulated.
2, operation steps
(1) obtain overburden in the thromboembolic states patients with pulmonary hypertension treatment of pulmonary thromb-endarterectomy (PTE):
1) removing thromboembolic states patients with pulmonary hypertension pulmonary thrombosis at operating table is overburden in the treatment of pulmonary thromb-endarterectomy (PTE), inserts in the ice chest cryopreservation and sends into as early as possible the laboratory super clean bench and test in 4 hours.
2) tissue is sent in the super clean bench operated, contain HBSS with 37 ℃ and clean 10min, remove blood stains.
(2) obtain the arteria pulmonalis smooth muscle cells of former culture thromboembolic states patients with pulmonary hypertension.
1) white sample that will the wide strip of the long 1mm of about 1.5cm shreds sample with aseptic eye scissors as far as possible, to the about 1mm * 1mm of fritter * 1mm, digests 30min with Digestive system, and the visible tissue piece is soft, and the edge is scared.
2) sample is transferred in the aseptic centrifuge tube the centrifugal 15min of room temperature 300g;
3) sucking-off supernatant Digestive system discards gently, and the sucking-off cell suspending liquid is collected in another aseptic centrifuge tube, adds the complete culture solution re-suspended cell and adjusts cell density 3～4 * 10
5/ ml also divides kind to 6 orifice plates with this density, puts 37 ℃, 5%CO
2Leave standstill cultivation in the incubator;
4) postdigestive fragment of tissue was planted to 6 orifice plates with perfect medium in resuspended rear minute again.
5) observe next day, all changed afterwards complete culture solution or half and measure the replacing complete culture solution in 3-4 days.When Growth of Cells to logarithmic phase, namely finish primary cell culture, obtain the human pulmonary artery smooth muscle cells of former culture.
(3) obtain the arteria pulmonalis smooth muscle cells of the thromboembolic states patients with pulmonary hypertension of cultivating of going down to posterity:
After the primary cell stand density reaches 90%, can go down to posterity.When the time comes, clean cell 3 times with 2ml PBS solution, add 35 ℃ of digestion of 0.3ml 0.1% (w/v) trypsin solution (containing 0.02%EDTA) 1～2 minute, when retraction, intercellular substance increase appear in cell, absorb trypsin solution, add the 5ml complete culture solution, gently the cell on the piping and druming bottle wall, make it to form cell suspending liquid, with 2 * 10
5The cell density of individual/ml is inoculated in the new culturing bottle, place 37 ℃, 5% carbonic acid gas incubator to cultivate, changed liquid once in per 2 days, to Growth of Cells to logarithmic phase, the arteria pulmonalis smooth muscle cells of the thromboembolic states patients with pulmonary hypertension of cultivating of obtaining to go down to posterity.
3, cultivation results
(1) observes under the inverted phase contrast microscope, just rounded, bright, the single even distribution of cell of plantation.2-3 days left and right sides visible part cell attachments of the arteria pulmonalis smooth muscle cells of the thromboembolic states patients with pulmonary hypertension of former culture growth, to launch cellular form various, is prismatic, polygon, star or irregular shape, and size is slightly variant; Endochylema is abundant; It is monokaryon that karyon mostly is the most cells of oval, and a few cell has double-core or three nuclears, and one or more kernels are arranged.The cell quantity showed increased is the characteristic of concentration growth after 4-6 days, and iuntercellular often has projection to link to each other.Growth of Cells reached the logarithmic phase growth in 7-10 days, and subregion cell overlap accumulated growth forms " peak ", and the cell number of plies is gradually few between " peak " and " peak ", forms " paddy ".When Growth of Cells after merge, the iuntercellular bunchy that is arranged parallel to each other, height rises and falls, and forms typical smooth muscle cell growth feature " peak " " paddy " shape growth (referring to Fig. 2).
(2) immunofluorescence of SM-α-Actin antigen detects: adopt unstriated muscle SM-α-Actin monoclonal antibody that cultured cells is identified, and adopt international redyeing process commonly used to dye, with hochest all cells nuclear is dyeed, guarantee the reliability that cell purity calculates.At the fluorescence microscopy Microscopic observation, the cytoplasm unstriated muscle SM-α of the visible rhodamine mark-rubescent look fluorescence of Actin antigen, the nucleus of the hochest mark look fluorescence (Fig. 3) that turns blue.Negative control is without adding primary antibodie, and the nucleus that only detects the hochest mark does not detect the SM-α-Actin of rhodamine mark, so rarely seen look fluorescence (Fig. 5) that turns blue.Low power lens calculates cell purity, and every sheet is got 5 visuals field at random, and 200 cells are detected in each visual field at least.Calculate SM-α in each visual field-Actin and express the ratio that positive cell accounts for this total cell count in visual field, be smooth muscle cell purity average out to more than 95%.
Embodiment 2
1, main experiment material
(1) Specimen origin: the overburden that obtains in the thromboembolic states patients with pulmonary hypertension treatment of pulmonary thromb-endarterectomy (PTE).
(2) HBSS balanced salt solution: add 7.01g sodium-chlor, 0.37g Repone K, 0.06g magnesium chloride, 0.79g glucose 0.11g calcium chloride, 0.03g potassium primary phosphate, 0.05g sal epsom in the 1000ml tri-distilled water.0.29g sodium bicarbonate, 0.04g Sodium phosphate dibasic and 1.19gHEPES, testing preparation in front 24 hours and filtration sterilization or autoclaving pH value is 7.2.
(3) Digestive system: adding 30mg II Collagenase Type is formulated in the 8mlHBSS balanced salt solution.
(4) complete culture solution is: by the M231 basic medium (SMBS) of 90ml, and the medium supplement of 1ml (SMGS), the microbiotic of the penicillin of 80U/ml, the Streptomycin sulphate of 80mg/ml is formulated.
2, operation steps
(1) obtain overburden in the thromboembolic states patients with pulmonary hypertension treatment of pulmonary thromb-endarterectomy (PTE):
1) removing thromboembolic states patients with pulmonary hypertension pulmonary thrombosis at operating table is overburden in the treatment of pulmonary thromb-endarterectomy (PTE), inserts in the ice chest cryopreservation and sends into as early as possible the laboratory super clean bench and test in 4 hours.
2) tissue is sent in the super clean bench operated, contain HBSS with 37 ℃ and clean 15min, remove blood stains.
(2) obtain the arteria pulmonalis smooth muscle cells of former culture thromboembolic states patients with pulmonary hypertension.
1) white sample that will the wide strip of the long 1mm of about 1.5cm shreds sample with aseptic eye scissors as far as possible, and the about 1mm * 1mm of fritter * 1mm digests 35min with Digestive system, and the visible tissue piece is soft, and the edge is scared.
2) sample is transferred in the aseptic centrifuge tube the centrifugal 15min of room temperature 350g;
3) sucking-off supernatant Digestive system discards gently, and the sucking-off cell suspending liquid is collected in another aseptic centrifuge tube, adds the complete culture solution re-suspended cell and adjusts cell density 3～4 * 10
5/ ml also divides kind to 6 orifice plates with this density, puts 37 ℃, 5%CO
2Leave standstill cultivation in the incubator;
4) postdigestive fragment of tissue was planted to 6 orifice plates with complete culture solution in resuspended rear minute again.
5) observe next day, all changed afterwards complete culture solution or half and measure the replacing complete culture solution in 3-4 days.When Growth of Cells to logarithmic phase, namely finish primary cell culture, obtain the human pulmonary artery smooth muscle cells of former culture;
(3) obtain the arteria pulmonalis smooth muscle cells of the thromboembolic states patients with pulmonary hypertension of cultivating of going down to posterity:
After the primary cell stand density reaches about 90%, can go down to posterity.When the time comes, clean cell 3 times with 2mlPBS solution, add 37 ℃ of digestion of 0.5ml 0.1% (w/v) trypsin solution (containing 0.05%EDTA) 1～2 minute, when retraction, intercellular substance increase appear in cell, absorb trypsin solution, every hole adds the 2ml complete culture solution, gently the cell on the piping and druming bottle wall, make it to form cell suspending liquid, with 2.5 * 10
5The cell density of individual/ml is inoculated in the new culturing bottle, place 37 ℃, 5% carbonic acid gas incubator to cultivate, changed liquid once in per 2 days, to Growth of Cells to logarithmic phase, the arteria pulmonalis smooth muscle cells of the thromboembolic states patients with pulmonary hypertension of cultivating of obtaining to go down to posterity.
3, cultivation results
(1) observes under the inverted phase contrast microscope, just rounded, bright, the single even distribution of cell of plantation.4 days left and right sides visible part cell attachments of the arteria pulmonalis smooth muscle cells of the thromboembolic states patients with pulmonary hypertension of former culture growth, to launch cellular form various, is prismatic, polygon, star or irregular shape, and size is slightly variant; Endochylema is abundant; It is monokaryon that karyon mostly is the most cells of oval, and a few cell has double-core or three nuclears, and one or more kernels are arranged.The cell quantity showed increased is the characteristic of concentration growth after 4-6 days, and iuntercellular often has projection to link to each other.Growth of Cells reached the logarithmic phase growth in 8-10 days, and subregion cell overlap accumulated growth forms " peak ", and the cell number of plies is gradually few between " peak " and " peak ", forms " paddy ".When Growth of Cells after merge, the iuntercellular bunchy that is arranged parallel to each other, height rises and falls, and forms the growth of typical smooth muscle cell growth feature " peak " " paddy " shape.
(2) immunofluorescence of unstriated muscle α-Actin antigen detects: adopt SM-α-Actin monoclonal antibody that cultured cells is identified, and adopt international redyeing process commonly used to dye, with hochest all cells nuclear is dyeed, guarantee the reliability that cell purity calculates.At the fluorescence microscopy Microscopic observation, the cytoplasm SM-α of the visible rhodamine mark-rubescent look fluorescence of Actin antigen, the nucleus of the hochest mark look fluorescence that turns blue.Negative control is without adding primary antibodie, and the nucleus that only detects the hochest mark does not detect the SM-α-Actin of rhodamine mark, so rarely seen look fluorescence that turns blue.Low power lens calculates cell purity, and every sheet is got 5 visuals field at random, and 200 cells are detected in each visual field at least.Calculate SM-α in each visual field-Actin and express the ratio that positive cell accounts for this total cell count in visual field, be smooth muscle cell purity average out to more than 90%.
Embodiment 3
1, main experiment material
(1) Specimen origin: the overburden that obtains in the thromboembolic states patients with pulmonary hypertension treatment of pulmonary thromb-endarterectomy (PTE).
(2) HBSS balanced salt solution: add 8.77g sodium-chlor, 0.45g Repone K, 0.2g magnesium chloride, 1.39g glucose 0.17g calcium chloride, 0.136g potassium primary phosphate, 0.25g sal epsom in the 1000ml tri-distilled water.0.38g sodium bicarbonate, 0.18g Sodium phosphate dibasic and 2.38gHEPES, testing preparation in front 24 hours and filtration sterilization or autoclaving pH value is 7.4.
(3) Digestive system: adding 20mg II Collagenase Type is formulated in the 12mlHBSS balanced salt solution.
(4) complete culture solution is: by the M231 basic medium of 100ml, and the medium supplement SMGS of 5ml, the microbiotic of the penicillin of 90U/ml, the Streptomycin sulphate of 90mg/ml is formulated.
2, operation steps
(1) obtain overburden in the thromboembolic states patients with pulmonary hypertension treatment of pulmonary thromb-endarterectomy (PTE):
1) removing thromboembolic states patients with pulmonary hypertension pulmonary thrombosis at operating table is overburden in the treatment of pulmonary thromb-endarterectomy (PTE), inserts in the ice chest cryopreservation and sends into as early as possible the laboratory super clean bench and test in 4 hours.
2) tissue is sent in the super clean bench operated, contain HBSS with 37 ℃ and clean 8min, remove blood stains.
(2) obtain the arteria pulmonalis smooth muscle cells of former culture thromboembolic states patients with pulmonary hypertension.
1) white sample that will the wide strip of the long 1mm of about 1.5cm shreds sample with aseptic eye scissors as far as possible, and the about 1mm * 1mm of fritter * 1mm digests 25min with Digestive system, and the visible tissue piece is soft, and the edge is scared.
2) sample is transferred in the aseptic centrifuge tube the centrifugal 20min of room temperature 280g;
3) sucking-off supernatant Digestive system discards gently, and the sucking-off cell suspending liquid is collected in another aseptic centrifuge tube, adds the complete culture solution re-suspended cell and adjusts cell density 3～4 * 10
5/ ml also divides kind to 6 orifice plates with this density, puts 37 ℃, 5%CO
2Leave standstill cultivation in the incubator;
4) postdigestive fragment of tissue was planted to 6 orifice plates with perfect medium in resuspended rear minute again.
5) observe next day, all changed afterwards complete culture solution or half and measure the replacing complete culture solution in 3-4 days.When Growth of Cells to logarithmic phase, namely finish primary cell culture, obtain the human pulmonary artery smooth muscle cells of former culture;
(3) obtain the arteria pulmonalis smooth muscle cells of the thromboembolic states patients with pulmonary hypertension of cultivating of going down to posterity:
After the primary cell stand density reaches about 90%, can go down to posterity.When the time comes, clean cell 3 times with 2mlPBS solution, add 33 ℃ of digestion of 0.2ml 0.2% (w/v) trypsin solution (containing 0.01%EDTA) 1～2 minute, when cell retraction occurs, when the intercellular substance increases, absorbs trypsin solution, clean cell 3 times with 2mlPBS solution, add the 5ml complete culture solution, cell on the piping and druming bottle wall makes it to form cell suspending liquid, with 3.0 * 10 gently
5The cell density of individual/ml is inoculated in the new culturing bottle, place 37 ℃, 5% carbonic acid gas incubator to cultivate, changed liquid once in per 2 days, to Growth of Cells to logarithmic phase, the arteria pulmonalis smooth muscle cells of the thromboembolic states patients with pulmonary hypertension of cultivating of obtaining to go down to posterity.
3, cultivation results
(1) observes under the inverted phase contrast microscope, just rounded, bright, the single even distribution of cell of plantation.3 days left and right sides visible part cell attachments of the arteria pulmonalis smooth muscle cells of the thromboembolic states patients with pulmonary hypertension of former culture growth, to launch cellular form various, is prismatic, polygon, star or irregular shape, and size is slightly variant; Endochylema is abundant; It is monokaryon that karyon mostly is the most cells of oval, and a few cell has double-core or three nuclears, and one or more kernels are arranged.The cell quantity showed increased is the characteristic of concentration growth after 4-5 days, and iuntercellular often has projection to link to each other.Growth of Cells reached the logarithmic phase growth in 7-9 days, and subregion cell overlap accumulated growth forms " peak ", and the cell number of plies is gradually few between " peak " and " peak ", forms " paddy ".When Growth of Cells after merge, the iuntercellular bunchy that is arranged parallel to each other, height rises and falls, and forms the growth of typical smooth muscle cell growth feature " peak " " paddy " shape.
(2) immunofluorescence of SM-α-Actin antigen detects: adopt unstriated muscle α-Actin monoclonal antibody that cultured cells is identified, and adopt international redyeing process commonly used to dye, with hochest all cells nuclear is dyeed, guarantee the reliability that cell purity calculates.At the fluorescence microscopy Microscopic observation, the cytoplasm SM-α of the visible rhodamine mark-rubescent look fluorescence of Actin antigen, the nucleus of the hochest mark look fluorescence that turns blue.Negative control is without adding primary antibodie, and the nucleus that only detects the hochest mark does not detect the SM-α-Actin of rhodamine mark, so rarely seen look fluorescence that turns blue.Low power lens calculates cell purity, and every sheet is got 5 visuals field at random, and 200 cells are detected in each visual field at least.Calculate SM-α in each visual field-Actin and express the ratio that positive cell accounts for this total cell count in visual field, be smooth muscle cell purity average out to more than 92%.
Embodiment 4
The separation of employing example 1 and cultural method and application number are that the plurality of enzymes digestion method of 200710033016.2 patents proposition and the plurality of enzymes digestion method of foreign literature employing compare test, the result shows that smooth muscle cell that patient's arteria pulmonalis smooth muscle cells that experimental technique that this patent adopts obtains obtains than plurality of enzymes digestion method reaches the time average 24 hours ahead of time of logarithmic phase from the overburden for the treatment of of pulmonary thromb-endarterectomy (PTE), the degree of damage that shows the single enzyme digestion method cell membrane that this patent adopts is lighter, is beneficial to cell survival and growth.
Embodiment 5
The human pulmonary artery smooth muscle cells of patient's arteria pulmonalis smooth muscle cells and non-pulmonary hypertension in the overburden of the method separation treatment of pulmonary thromb-endarterectomy (PTE) of employing example 1, cultivation results shows that the non-pulmonary hypertension patient's of patient's arteria pulmonalis smooth muscle cells volume ratio unstriated muscle is large, slightly variant on the form, and smooth muscle cell " peak " " paddy " shape growth characteristics also owes obvious than the smooth muscle cell in other source.Show that the experimental specimen that this patent adopts has more specificity to diseases such as study of lung arterial hypertensions.
Embodiment 6
According to the cultural method of example 1-3, this research team separates patient's arteria pulmonalis smooth muscle cells more than 20 examples from the overburden for the treatment of of pulmonary thromb-endarterectomy (PTE), and test repeatability is good, cultured cells form homogeneous, well-grown.
Claims (5)
1. a method of stripping off separation and Culture human pulmonary artery smooth muscle cells the thing from people's treatment of pulmonary thromb-endarterectomy is characterized in that, described method comprises the steps:
One) obtain the arteria pulmonalis smooth muscle cells of former culture:
1) the lung vascular tissue sample that strips down in the Endarterectomy art places 4 ℃ of preservations of HBSS solution;
2) super clean bench uviolizing 30-40min, 75% alcohol wipe, all apparatuses, vessel autoclaving;
3) preheating Digestive system, HBSS and complete culture solution to 37 ℃;
4) sample is placed 37 ℃ of vessel that contain HBSS clean 8-15min;
5) it is stand-by the 10ml Digestive system to be filtered to small beaker with 2.2 μ m;
6) the white sample of the wide strip of the long 1mm of 1.5cm is cut into the fragment of 1mm * 1mm * 1mm with aseptic scissors in Digestive system, continues digestion 25-35min;
7) the sample fragment of tissue is transferred in the aseptic centrifuge tube room temperature 280-350g gradient centrifugation 12-20min;
8) abandon supernatant, cell suspension is collected in another aseptic centrifuge tube, add complete culture solution and adjust cell density 3-4 * 10
5/ ml, and divide kind to 6 orifice plates with this density, 37 ℃, 5%CO put
2Leave standstill cultivation in the incubator;
9) postdigestive fragment of tissue was planted to 6 orifice plates with complete culture solution in resuspended rear minute again;
10) observe 3-4 days later half amounts or all change complete culture solution next day;
11) when Growth of Cells to logarithmic phase, namely finish primary cell culture, obtain the human pulmonary artery smooth muscle cells of former culture; Two) obtain the arteria pulmonalis smooth muscle cells that goes down to posterity and cultivate: work as step 1) in primary cell grow to the logarithmic growth after date, after the cleaning of PBS solution, add 33-37 ℃ of digestion of 0.2-0.5ml0.1-0.2% (w/v) trypsin solution 1～2 minute, wherein above-mentioned trypsin solution contains 0.01-0.05%EDTA, when retraction appears in cell, when the intercellular substance increases, the sucking-off trypsin solution, add 5～7ml complete culture solution, soft concussion makes it to form cell suspension, be seeded in the new culturing bottle, changed liquid once in per two days, namely finish passage cell and cultivate, obtain the chronic thromboembolic states hypertensive pulmonary arterial disease human pulmonary artery smooth muscle cells that goes down to posterity and cultivate; Described HBSS solution composition is: calcium chloride 1.0-1.5mM, sodium-chlor 120-150mM, Repone K 5.0-6.0mM, magnesium chloride 0.3-1.0mM, dipotassium hydrogen phosphate 0.2-1.0mM, sal epsom 0.2-1.0mM, sodium bicarbonate 3.5-5.0mM, sodium hydrogen phosphate 0.1-0.5mM, glucose 4.0-7.0mM and HEPES7.0-10.0mM, the pH value of this solution is 7.2-7.4, tests preparation in front 24 hours, and the high pressure degerming;
Described Digestive system is: adding II Collagenase Type is formulated in the described HBSS solution, and ratio is 8-12mlHBSS solution: the 20-30mgII Collagenase Type;
Described complete culture solution is: by the M231 basic medium, medium supplement SMGS, the microbiotic of penicillin and Streptomycin sulphate is formulated, wherein the volume ratio of M231 basic medium and medium supplement SMGS is 90-100: 1-5, the final concentration of penicillin is 80-100U/ml, and the final concentration of Streptomycin sulphate is 80-100mg/ml.
2. cultural method as claimed in claim 1, wherein said HBSS solution is: calcium chloride 1.3mM, sodium-chlor 136.9mM, Repone K 5.4mM, magnesium chloride 0.5mM, dipotassium hydrogen phosphate 0.4mM, sal epsom 0.4mM, sodium bicarbonate 4.2mM, sodium hydrogen phosphate 0.3mM, glucose 5.5mM and HEPES8.0mM, the pH value of this solution are 7.4, test preparation in front 24 hours, and the high pressure degerming.
3. cultural method as claimed in claim 1, the ratio of HBSS solution and II Collagenase Type is 10ml: 25mg in the wherein said Digestive system.
4. cultural method as claimed in claim 1, the M231 basic medium in the wherein said complete culture solution and the volume ratio of medium supplement SMGS are 93: 5.
5. such as any described cultural method of claim 1-4, wherein, the concrete steps of obtaining the arteria pulmonalis smooth muscle cells of former culture are:
1) the lung vascular tissue sample that strips down in the Endarterectomy art places 4 ℃ of preservations of HBSS liquid;
2) super clean bench uviolizing 30min, 75% alcohol wipe, all apparatuses, vessel autoclaving;
3) preheating Digestive system, HBSS and complete culture solution to 37 ℃;
4) sample is placed 37 ℃ of vessel that contain HBSS clean 10min;
5) it is stand-by the 10ml Digestive system to be filtered to small beaker with 2.2 μ m;
6) the white sample of the wide strip of the long 1mm of 1.5cm is cut into the fragment of 1mm * 1mm * 1mm with aseptic scissors in Digestive system, continues digestion 30min;
7) the sample fragment of tissue is transferred in the aseptic centrifuge tube room temperature 300g gradient centrifugation 15min;
8) the sucking-off supernatant liquor discards, and the sucking-off cell suspension is collected in another aseptic centrifuge tube, adds perfect medium and adjusts cell density 3 * 10
5/ ml, and divide kind to 6 orifice plates with this density puts in 37 ℃, 5%CO2 incubator and leaves standstill cultivation;
9) postdigestive fragment of tissue was planted to 6 orifice plates with perfect medium in resuspended rear minute again;
10) observe next day, change nutrient solution after 3-4 days or partly measure replaced medium;
11) when Growth of Cells to logarithmic phase, namely finish primary cell culture, obtain the human pulmonary artery smooth muscle cells of former culture.
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