CN1673357A - Method for digesting blood smooth muscle - Google Patents
Method for digesting blood smooth muscle Download PDFInfo
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- CN1673357A CN1673357A CN 200510042438 CN200510042438A CN1673357A CN 1673357 A CN1673357 A CN 1673357A CN 200510042438 CN200510042438 CN 200510042438 CN 200510042438 A CN200510042438 A CN 200510042438A CN 1673357 A CN1673357 A CN 1673357A
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Abstract
The digestion process of smooth muscle of blood vessels belongs to the field of biomedical technology. The digestion process includes the following steps: adding 0.15 % concentration collagenase II solution compounded with DMEM into blood vessel smooth muscle tissue block and acting in water bath for 1.5 hr until forming floccular tissue; cleaning with 0.02 % EDTA solution, adding 0.125 % trypsase solution and water bath vibrating to digest at 37 deg.c to obtain dispersed smooth muscle cells; terminating enzyme action with 10 % new born ox serum culture liquid; regulating cell concentration with 20 % new born ox serum culture liquid to 50,000-500,000/L, inoculation, and culturing in culture tank with 5 % CO2 and first 37 deg.c to cell fusion of 70-80 %; and subculturing conventionally. The process uses less enzyme varieties, is low in cost and simple, and may be used in medicine, biology and other fields.
Description
(1) technical field
The present invention relates to a kind of digestion method of vascular smooth muscle, belong to field of biomedicine technology.
(2) background technology:
Vascular smooth muscle cell is cultivated method commonly used has Explant culture (explant) and enzymolysis from method (enzymedisperse).Explant culture has the smooth muscle cell of acquisition purity height, and amount is many, advantages of simple operation.But easily make myofilament forfeiture in the smooth muscle cell kytoplasm with Explant culture, subcellular organelle increases.On the contrary, enzymolysis is from method, because the effect of enzyme makes iuntercellular lose connection, myofilament content is abundant in the cell, and so the type that remains retracted state and form well differentiated are the factor that can be used for studying the apparent variation of cell and influence cellular contraction.
Carry out enzymolysis and cultivate vascular smooth muscle cell, at first must solve the digestive problems of vascular smooth muscle from method.Existing digestion method mainly contains:
1, is coated with immortality etc. " II Collagen Type VI enzyme/elastin enzyme digestion is cultivated rat smooth muscle cells ", " Chinese arteriosclerosis magazine " 2001,9 (5): propose among the 438-440., vascular smooth muscle digests at twice, at first artery is used Hanks liquid rinsing 2～3 times, and cut off blood vessel external fat and reticular tissue, put into 2g/L collagenase II (1ml/aorta) Digestive system and digest 20～30min.Then, clamp adventitia with No. 5 tweezers, soft round about pullling removed adventitia.Artery (aorta) placed contain 10% new-born calf serum (Newborn Calf Serum is hatched 24h among DMEM NCS) (Dulbcco ' sModifed Eagle Medium, Eagle ' the s substratum of Dulbecco ' s improvement).Take out artery then, put into the compound Digestive system that contains 2g/L collagenase II and 0.25g/L elastoser again, the amount of the Digestive system that every artery is required is 1.5ml, at 5%CO
2, hatch about 2h in 37 ℃ of incubators.
2, Gao Pingjin etc. " protein kinase C a and vascular smooth muscle cell phenotype transform ", " Chinese cardiovascular diseases magazine " 1999,27 (6): propose among the 453-455., separate aorta, use collagenase 3mg/ml, elastoser 1mg/ml, Trypsin inhibitor SBTI 1mg/ml, bovine serum albumin 1mg/ml separating blood vessel smooth muscle cell.
3, Li Zhi etc. " cultivation of pulmonary artery smooth muscle ", " Chinese Medical Sciences University's journal " 1996,25 (3): propose among the 221-224., feeding 95%O
2And 5%CO
2The ice bath Hanks liquid of mixed gas in, with Sprague-Dawley (SD) induced lung artery scissors into about 1～2mm
3Fragment, this fragment is through elastoser (1: the 1) 3ml of 0.2% collagenase and 0.0125% digestion, the supernatant liquor that will contain cell and other tissue hatch 20min in 37 ℃ isothermal vibration water-bath after sops up, precipitation adds 0.1% collagenase and 0.0125% elastoser (1: 1) 3ml is hatched 40min again, and hatching for twice needs to feed mixed gas in the process.Supernatant 10%FBS (foetal calf serum) PNM (0.1M NaH2PO4,0.1M Na2HPO4 (pH8.0), 0.1%Nonidet P-40; 5%nonfat dry milk; 0.02%sodium azide) liquid stopped reaction; Precipitation adds 0.125% trypsinase 1ml mixing, and the carbonic acid gas incubator is hatched 5min for 37 ℃ again.
4, " 2 kinds of enzyme digestions are cultivated the comparison " Third Military Medical University's journal " 1999 of rat smooth muscle cells growth characteristics; 21 (10): propose among the 765-767.; use collagenase digesting separately: with the clock and watch tweezer artery is torn into about 1mm * 1mm little earlier; put into the collagenase liquid that fills 2～3ml0.2% in advance; cover tight bottle stopper, put and wave 37 ℃ of 25 times/min runout, 10～12h on the instrument for Wang Guansong etc.Prozyme digestion: with the clock and watch tweezer artery is torn into about 1mm * 1mm little earlier, puts into the compound digestive ferment liquid (comprising collagenase, elastoser and trypsinase) of 2ml 0.2%, in 37 ℃ of CO
2Incubator was cultivated 30～40 minutes.
More than several digestion methods, used enzyme class and the quantity that have are many, cost is too high, complex steps is cultivated vascular smooth muscle as: Gao Pingjin etc. with collagenase, elastoser, Trypsin inhibitor SBTI and bovine serum albumin digestion; The digestion time that has is long, makes cell exposure duration in the enzyme environment long, is unfavorable for the survival of cell, as: Wang Guansong etc. put vascular smooth muscle and wave 37 ℃ of 25 times/min runout, 10～12h on the instrument; What have is exposed to vascular smooth muscle overlong time in the trypsinase environment, cause a large amount of cell membrane damages, as: be coated with immortality etc. vascular smooth muscle is put into the compound Digestive system (1.5ml/aorta) that contains 2g/L collagenase II and 0.25g/L elastoser, at 5%CO
2, hatch about 2h in 37 ℃ of incubators.
(3) summary of the invention:
The invention provides a kind of digestion method of vascular smooth muscle, the kind of the enzyme of use is few, and cost is low; Step is easy.
Method steps of the present invention is as follows:
A. the vascular smooth muscle tissue block of handling well is placed centrifuge tube, add the volume 8-10 of smooth muscle tissue doubly, with 0.15% collagenase II solution of DMEM preparation, place 37 ℃ of water bath effect 1.2h-1.5h to be cotton-shaped, abandoning supernatant to organizing.
Above-mentioned DMEM (Dulbcco ' s Modifed Eagle Medium) be Eagle ' the s substratum of Dulbecco ' s improvement.
B. use the volume 3-5 of smooth muscle tissue 0.02% edathamil (ethylene diaminetetra-acetic acid doubly again, EDTA) cleaning is digested to cotton-shaped smooth muscle tissue, to eliminate from divalent ions all in the substratum, so that keep tryptic activity.
C. add the volume 3-5 of smooth muscle tissue 0.125% trypsin solution doubly, in 37 ℃ of water-bath vibration digestion 1-2 time, each 10min-15min gets dispersive smooth muscle cell suspension.
D. in dispersive smooth muscle cell suspension, add new-born calf serum again, through the centrifugal 7min-8min of 3000r, abandoning supernatant.
Above-mentioned new-born calf serum is dew living bovine serum or the DMEM nutrient solution that contains the 10-20% new-born calf serum.
The 1/15-1/10 that dew living bovine serum consumption is the trypsinase amount.
Add the DMEM nutrient solution that contains the 10-20% new-born calf serum amount be the 1/3-1/2 of trypsinase amount.
E. add the DMEM nutrient solution that contains 20% new-born calf serum, and blow and beat cell dispersion with suction pipe, use the tally counting cells, regulating cell density is 5 * 10
4～5 * 10
5Individual/ml.
F. will adjust the cell suspension 2～3ml of density, be inoculated in the 25ml culturing bottle.Put into 5%CO
2, cultivate in 37 ℃ of incubators.A small amount of adherent smooth muscle cell is arranged behind the 24h, changed fresh medium in 3-4 days, merge, get final product when cell reaches 70%～80%.
Above-mentioned fresh medium is the DMEM nutrient solution that contains 10% new-born calf serum.
More than each component concentrations all be weight percentage.
The vascular smooth muscle that digestion is good is used for going down to posterity according to a conventional method.
Shortened the digestion time-histories through this digestion method of experiment confirm, the degree of damage of cell membrane is light, is beneficial to the survival of cell; And two kinds of enzyme action compensatings, digestion are fully, and the cell quantity of collection is many, purity is high; The proterties and the metabolism of isolated cells have been protected simultaneously better.
Because the kind of the enzyme that this method is used is few, cost is low; Step is easy, and effect is better than existing other digestion method, can improve working feasibility, accuracy and repeatability greatly, has unique advantage carrying out enzymolysis when the method vascular smooth muscle is cultivated.
The present invention is applied widely, can be used for fields such as medical science, biology.
Compare with other method, present method mainly contains following excellent results:
1. vascular smooth muscle exposure duration in trypsinase short, shortened the digestion time-histories, the degree of damage of cell membrane is light, is beneficial to the survival of cell.
2. two kinds of enzyme action compensatings digest fully, and the cell quantity of collection is many.
3. has good selectivity, the cell purity height.
4. protect the proterties and the metabolism of isolated cells better.
(4) embodiment
Embodiment:
1 materials and methods
1.1 reagent
Collagenase II type (Sigma): 0.15%; Trypsin Sigma): 0.125%; Each composition of D-Hanks liquid is commercially available analytical pure; New-born calf serum (NCF) and DMEM are the blue Tokushima biotech firm (Grand IslandBiological Company (GIBCO)) of brother product.All be weight percentage.
1.2 draw materials
The SD rat is available from medical college of Shandong University Experimental Animal Center.In aseptic super clean bench, open the thoracic cavity, cut the heart bloodletting, take out thoracic aorta (arise under the left clavicle, terminate in the position that artery enters diaphragm) rapidly, be soaked in the culture dish that aseptic DMEM is housed, rinsing 2～3 times, and cut off blood vessel external fat and reticular tissue.
1.3 digestion
The vascular smooth muscle tissue block 0.2ml that handles well is placed the 10ml centrifuge tube, add 0.15% collagenase II solution 1.8ml, in 37 ℃ of water baths, act on 1.5h, be cotton-shaped, abandoning supernatant to organizing with the DMEM preparation; Be digested to cotton-shaped smooth muscle tissue with the 1ml0.02%EDTA cleaning, then, add 0.125% trypsin solution 1ml,, can obtain the dispersive smooth muscle cell in 37 ℃ of water-baths vibration digestion 10min.All be weight percentage.
1.4 cultivate
In the graduated centrifuge tube that fills cell suspension, add 10% new-born calf serum 0.5ml with the DMEM preparation, stop the effect of enzyme.Cross 200 order nets, the centrifugal 7min of 3000r, abandoning supernatant.Adding is with the nutrient solution of 20% new-born calf serum of DMEM preparation, piping and druming gently, and use the tally counting cells, regulate (the big lattice total cellular score in cell density=cell count/milliliter stoste=4/4 * 10,000 * extension rate is regulated cell density by changing extension rate) cell density is 5 * 10
4～5 * 10
5Individual/L, with adjusting the cell suspension 2～3ml of density, be inoculated in the 25ml culturing bottle, put into 5%CO
2, cultivate in 37 ℃ of incubators.Just can see a small amount of adherent smooth muscle cell behind 24 h, change fresh medium (with 10% new-born calf serum of DMEM preparation) on the 4th day.All be weight percentage.Can go down to posterity according to a conventional method when cell reached 75% fusion in the 6th day.Get 3-4 and carry out the biological characteristics evaluation for cell.
1.5 identify
The vascular smooth muscle cell of cultivating is dripped kind on the cover glass that coats poly-lysine in advance in the culture dish, after 2～3 days adherent growth, take turns doing following processing: 10% Paraformaldehyde 96 (the tri-distilled water preparation that adds 1/3 nutrient solution volume in the culture dish, pH7.2), pre-fix 15min (incubation in 37 ℃ of incubators), PBS (phosphate buffered saline buffer) rinsing of incline nutrient solution and usefulness 0.01M 3 times, each 5 minutes, with the fixing 20min of 10% Paraformaldehyde 96, wash with PBS then again.Adopt mouse anti rat α-Actin immunohistochemical staining, identify the vascular smooth muscle cell Actin muscle, with the positive result of the pale brown look of endochylema.
1.6 passage cell surviving rate
9 of the vascular smooth muscle cell suspensions that cancellationization goes down to posterity add 1 0.4% trypan blue liquid, draw 1 behind the mixing on cell counting count board, put microscopically and observe, and all cytochromes are undesired cell or dead cell.Amount to several 1000 cells, calculate cell survival rate.
2 results
2.1 cell growing state
Inoculate the 2nd day, can see cell attachment, adherent primary cell differs in size, and shape is various, and fusiformis, trilateral or star etc. are arranged.Nuclear is positioned at central authorities, and 1 or several not of uniform size, dark kernels are arranged in the nuclear, and endochylema is abundant.Passage cell is inoculated in culturing bottle, and the adherent stretching, extension of most cells during 24h along with the cumulative height of prolongation transparency of time, and is in contact with one another, and is typical " Gu Yufeng " growth.
2.2 the immunohistochemical staining of Actin muscle
Use streptomycete avidin-peroxidase (SP) method to vascular smooth muscle Actin muscle immunohistochemical staining, 96% the cell dyeing positive.More bar filament is arranged in the visible kytoplasm under the inverted microscope, be pale brown look, be Actin muscle.Karyon is light blue.
2.3 trypan blue coloration result
Amount to several 1000 cells, wherein 30 positive, have the cell dyeing more than 97% to be negative, show that cultured cells had digestive transfer culture surviving rate is higher.
Claims (5)
1. the digestion method of a vascular smooth muscle, step is as follows:
A. the vascular smooth muscle tissue block of handling well is placed centrifuge tube, add the volume 8-10 of smooth muscle tissue doubly, with 0.15% collagenase II solution of DMEM preparation, place 37 ℃ of water bath effect 1.2h-1.5h to be cotton-shaped, abandoning supernatant to organizing;
B. clean with the volume 3-5 of smooth muscle tissue 0.02% edathamil doubly again and be digested to cotton-shaped smooth muscle tissue; Then
C. add the volume 3-5 of smooth muscle tissue 0.125% trypsin solution doubly, in 37 ℃ of water-bath vibration digestion 1-2 time, each 10min-15min gets dispersive smooth muscle cell suspension;
D. in dispersive smooth muscle cell suspension, add new-born calf serum again, through the centrifugal 7min-8min of 3000r, abandoning supernatant;
E. add the nutrient solution that contains 20% new-born calf serum with the DMEM preparation, use the tally counting cells, regulating cell density is 5 * 10
4～5 * 10
5Individual/ml;
F. will adjust the cell suspension 2～3ml of density, be inoculated in the 25ml culturing bottle, put into 5%CO
2, cultivate in 37 ℃ of incubators, changed fresh medium in 3-4 days, cell reaches 70%～80% and merges, and gets final product;
More than each component concentrations all be weight percentage.
2. the digestion method of vascular smooth muscle as claimed in claim 1 is characterized in that, the new-born calf serum described in the steps d is dew living bovine serum or the DMEM nutrient solution that contains weight percent 10-20% new-born calf serum.
3. the digestion method of vascular smooth muscle as claimed in claim 1 is characterized in that, when the new-born calf serum described in the steps d was dew living bovine serum, consumption was the 1/15-1/10 of trypsinase amount.
4. the digestion method of vascular smooth muscle as claimed in claim 1 is characterized in that, the new-born calf serum described in the steps d is that consumption is the 1/3-1/2 of trypsinase amount when containing the DMEM nutrient solution of weight percent 10-20% new-born calf serum.
5. the digestion method of vascular smooth muscle as claimed in claim 1 is characterized in that, the fresh medium described in the step f is the DMEM nutrient solution that contains weight percent 10% new-born calf serum.
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| CN 200510042438 CN1250713C (en) | 2005-02-03 | 2005-02-03 | Method for digesting blood smooth muscle |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104988112A (en) * | 2015-07-22 | 2015-10-21 | 南阳师范学院 | SD rat thoracic artery smooth muscle cell separation and culture method |
| CN112255221A (en) * | 2020-10-16 | 2021-01-22 | 航科中投生物技术（北京）有限公司 | Colorimetric identification method for concentration and spraying effect of biological protection filter medium |
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2005
- 2005-02-03 CN CN 200510042438 patent/CN1250713C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104988112A (en) * | 2015-07-22 | 2015-10-21 | 南阳师范学院 | SD rat thoracic artery smooth muscle cell separation and culture method |
| CN112255221A (en) * | 2020-10-16 | 2021-01-22 | 航科中投生物技术（北京）有限公司 | Colorimetric identification method for concentration and spraying effect of biological protection filter medium |
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