CN105274050A - Culture method of human airway epithelial cells for treating bronchial asthma - Google Patents
Culture method of human airway epithelial cells for treating bronchial asthma Download PDFInfo
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- CN105274050A CN105274050A CN201510771532.XA CN201510771532A CN105274050A CN 105274050 A CN105274050 A CN 105274050A CN 201510771532 A CN201510771532 A CN 201510771532A CN 105274050 A CN105274050 A CN 105274050A
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Abstract
The invention discloses a culture method of human airway epithelial cells for treating bronchial asthma and belongs to the technical field of biology. Human trachea is digested for collecting airway epithelial cells first, the cells are washed with isopropyl alcohol solution and placed in a culture dish for cleaning, digesting is performed then by a digestion method before digestive juice is collected, the digestive juice is then centrifugally treated, supernate is collected and added into fetal calf serum and ACD medium, centrifugal treatment is performed after mixing to obtain a skin cell medium, the human airway epithelial cell medium is inoculated to a culture board, and human airway epithelial cells for treating bronchial asthma are obtained by culturing. This embodiment proves that the culture method is simple to operate and easy to implement, no pollution occurs during operation, no damage is caused to the cells, and the survival rate of the cells is up to higher than 90%.
Description
Technical field
The invention discloses a kind of epithelial cultural method of human airway for the treatment of bronchial asthma, belong to biological technical field.
Background technology
Asthma is a kind of chronic airway inflammation disease, shows as the symptoms such as the panting of repeated relapsing, shortness of breath, uncomfortable in chest, cough, be everlasting night and outbreak aggravation in early morning.Patient's speech utterance is difficult to continuously, and severe patient even can not be talked.Most of patients can be alleviated through treatment, but runs into risk factor, again can recurrent exerbation.Asthma is many falls ill infant period, because can not effect a radical cure, is slowly transformed into chronic disease, frequently-occurring disease, and this disease also can be lifelong with patient.It is very complicated that it brings out the cause of disease: have environmental pollution, Climate Anomalies, respiratory tract disease, contactant is irritated, food medicine is irritated, the weak immunizing power of physique is low, hereditary etc.
Bronchial asthma is often reinvented with the structure of air flue, one of its characteristic feature be under airway epithelia inoblast activity strengthen produce basal membrane thickening, air flue conformability reduce, cause persistence Raw air way resistance to increase, be difficult to reverse ventilatory function damage.The mechanism of Airway Remodeling is still unclear at present, is therefore difficult to clinically obtain effectively preventing method, so inquiring into the epithelial cultural method of a kind of human airway is significantly.At present, the cultural method of human airway epithelial cells mainly contains: pancreatin and/or protease digestion are in conjunction with mechanically peel mucous membrane method and/or mechanical wiper method, all there is following shortcoming in these methods: (1) mechanically peel mucous membrane method/mechanical wiper method adopts the apparatuses such as tweezers on tracheae or segmental bronchus, carry out blunt separation, its operation easier is large, and in stripping process easy damaged epithelium, the survivaling cell obtained is few, and cell viability is poor.(2) trysinization/protease digestion method adopts pancreatin/protein enzyme solution to carry out digestion process to tracheae or segmental bronchus, to obtain epithelial cell, but the eupepsy of pancreatin/proteolytic enzyme is very strong, and digestion time is not easily grasped, easily epithelial cell is caused damage, cell pick-up rate is few, and cell survival rate is low and vigor is poor.In addition, remain and cause being that the cell cultures of originating is restricted with human airway epithelial cells in following reason: 1. lungs are open internal organs, therefore the air flue obtained easily pollutes in the process of carrying out cell cultures, simultaneously contains the dregs such as a large amount of Saliva Orthanas due to patient airway and adds the difficulty of cellular segregation; 2. can be used for the sample of separation gas tract epithelial cell, it is all the position of the tool focus of getting off from excision, sample limited amount, and normal tracheae in sample or segmental bronchus little, human airway epithelial cells cultural method as adopted,, pick-up rate large to cell injury is low adds cell attachment ability, and the cell quantity finally survived is few; 3., in human airway epithelial cells original cuiture, the most easily introduce inoblast and pollute, fibroblastic growth is fast, vigor good, has growth vigor, finally causes primary human airway epithelial cells to cultivate unsuccessfully.
Summary of the invention
The technical problem that the present invention mainly solves: at present in the epithelial culturing process of human airway, large, the easy damaging cells of traditional mechanically peel mucous membrane method operation easier, and trysinization is difficult to control, cell survival rate is low and vigor is poor, and easily occur in the process of cell cultures to pollute to cause chrotoplast to cultivate failed shortcoming, provide a kind of epithelial cultural method of human airway for the treatment of bronchial asthma.First the method is got person's windpipe digestion and is collected human airway epithelial cells, and put into culture dish decontamination with aqueous isopropanol is clean, Digestive system is collected after adopting digestion method to digest afterwards, subsequently Digestive system is carried out centrifugal treating, get supernatant liquor and add foetal calf serum and the rear recentrifuge process of ACDE substratum mixing, obtain human body and play skin cells substratum, finally skin cell culture medium on human airway is seeded on culture plate, after cultivation, obtains a kind of human airway epithelial cell for the treatment of bronchial asthma.Not only operation is simple for the method, do not have any pollution in operating process, and can not damaging cells, improves cell survival rate.
In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is:
(1) in vitro people's tracheae or segmental bronchus is got, human airway epithelial cells is collected after digesting 30 ~ 32h by digest medium at 3 ~ 5 DEG C, use the aqueous isopropanol separation cleaning tissue sample of 90 ~ 95% afterwards, then tissue sample is put into sterile petri dish, under dissecting microscope, with dissecting fat residual in tweezers and scissors removal tissue sample;
(2) by the tissue sample after above-mentioned process, adopt airtight tube chamber digestion method, with Digestive system, active removal blood and mucus are carried out to human airway epithelial cells, 5 ~ 15h is digested at 37 ~ 38 DEG C, and frequently shake, make tissue digestion evenly, completely, the Digestive system afterwards after collection and treatment, described Digestive system is the IV substratum containing collagenase;
(3) Digestive system after above-mentioned obtaining is put into whizzer, with the rotating speed of 500 ~ 800r/min, centrifugal 15 ~ 20min, get supernatant liquor afterwards, and add foetal calf serum and the ACDE substratum of 10 ~ 15%, stir, put into whizzer again, raising rotating speed is 550 ~ 850r/mn, after centrifugal 10 ~ 15min, gets supernatant liquor and obtains skin cell culture medium on human airway;
(5) by skin cell culture medium on human airway obtained above, with 2 ~ 5 × 10
6on after the density of cell/mL is inoculated in sterilization, to be covered with collagen protein and agar culture plate, set temperature is 35 ~ 37 DEG C, at 5 ~ 10%CO
2quiescent culture 10 ~ 15 days under normal oxygen environment, can obtain a kind of human airway epithelial cell for the treatment of bronchial asthma.
Described IV, ACDE substratum making step is:
(1) first in beaker, 1L distilled water is poured into;
(2) get 10 ~ 12g inositol respectively, 0.05 ~ 0.08g nicotinic acid, 0.01 ~ 0.03g vitamin, 30 ~ 50g sucrose is poured in distilled water, by the abundant stirring and dissolving of stirring rod;
(3) draw 0.2mg indolylacetic acid with adjustable hydrodynamic device and put into above-mentioned solution;
(4) by precision test paper or acidometer adjustment pH to 5.7 ~ 5.8;
(5) take 6g agar powder to be poured in the above-mentioned solution prepared, be placed on electric furnace and be heated to boiling, until agar powder dissolves;
(6) cooling is rear a little loads in culture vessel, and puts into sterilization pot sterilizing 20 ~ 30min;
(7) from Autoclave, take out substratum after sterilizing, wait its cooled and solidified.
Application method of the present invention is: when there is lung bronchogenic carcinoma, chronic bronchitis, bronchiectasis, bronchostenosis, can be studied by the human airway epithelial cell got obtained by the present invention, by reconstruction of tissue and organ or can reproduce, not only make cell reproduce rear survival rate to more than 85%, and basis and clinic study and application are of great importance.
The invention has the beneficial effects as follows:
(1) preparation method of the present invention is easy, consistent, does not pollute;
(2) safe and reliable, can carry out under prevailing experimental conditions, be with a wide range of applications;
(3) any damage can not be had to cell in treating processes, make the survival rate of cell up to more than 90%.
Specific embodiments
First in vitro people's tracheae or segmental bronchus is got, human airway epithelial cells is collected after digesting 30 ~ 32h by digest medium at 3 ~ 5 DEG C, use the aqueous isopropanol separation cleaning tissue sample of 90 ~ 95% afterwards, then tissue sample is put into sterile petri dish, under dissecting microscope, with dissecting fat residual in tweezers and scissors removal tissue sample; Subsequently by the tissue sample after above-mentioned process, adopt airtight tube chamber digestion method, with Digestive system, active removal blood and mucus are carried out to human airway epithelial cells, 5 ~ 15h is digested at 37 ~ 38 DEG C, and frequently shake, make tissue digestion evenly, completely, the Digestive system afterwards after collection and treatment, described Digestive system is the IV substratum containing collagenase; Next the Digestive system after above-mentioned obtaining is put into whizzer, with the rotating speed of 500 ~ 800r/min, centrifugal 15 ~ 20min, get supernatant liquor afterwards, and add foetal calf serum and the ACDE substratum of 10 ~ 15%, stir, put into whizzer again, raising rotating speed is 550 ~ 850r/mn, after centrifugal 10 ~ 15min, gets supernatant liquor and obtains human airway subepithelium's skin cell culture medium; Next by skin cell culture medium on human airway obtained above, with 2 ~ 5 × 10
6on after the density of cell/mL is inoculated in sterilization, to be covered with collagen protein and agar culture plate, set temperature is 35 ~ 37 DEG C, at 5 ~ 10%CO
2quiescent culture 10 ~ 15 days under normal oxygen environment, can obtain a kind of human airway epithelial cell for the treatment of bronchial asthma.First wherein said IV, ACDE substratum making step for pour 1L distilled water in beaker; Then get 10 ~ 12g inositol respectively, 0.05 ~ 0.08g nicotinic acid, 0.01 ~ 0.03g vitamin, 30 ~ 50g sucrose is poured in distilled water, by the abundant stirring and dissolving of stirring rod; Draw 0.2mg indolylacetic acid with adjustable hydrodynamic device subsequently and put into above-mentioned solution; Afterwards by precision test paper or acidometer adjustment pH to 5.7 ~ 5.8; Next take 6g agar powder to be poured in the above-mentioned solution prepared, be placed on electric furnace and be heated to boiling, until agar powder dissolves; Load in culture vessel after cooling a little, and put into sterilization pot sterilizing 20 ~ 30min; From Autoclave, take out substratum after last sterilizing, wait its cooled and solidified.
Example 1
First in vitro people's tracheae or segmental bronchus is got, human airway epithelial cells is collected after digesting 30h by digest medium at 3 DEG C, use the aqueous isopropanol separation cleaning tissue sample of 90% afterwards, then tissue sample is put into sterile petri dish, under dissecting microscope, with dissecting fat residual in tweezers and scissors removal tissue sample; Subsequently by the tissue sample after above-mentioned process, adopt airtight tube chamber digestion method, with Digestive system, active removal blood and mucus are carried out to human airway epithelial cells, 5h is digested at 37 DEG C, and frequently shake, make tissue digestion evenly, completely, the Digestive system afterwards after collection and treatment, described Digestive system is the IV substratum containing collagenase; Next the Digestive system after above-mentioned obtaining is put into whizzer, with the rotating speed of 500r/min, centrifugal 15min, get supernatant liquor afterwards, and add foetal calf serum and the ACDE substratum of 10%, stir, put into whizzer again, raising rotating speed is 550r/mn, after centrifugal 10min, gets supernatant liquor and obtains skin cell culture medium on human airway; Next by skin cell culture medium on human airway obtained above, with 2 × 10
6on after the density of cell/mL is inoculated in sterilization, to be covered with collagen protein and agar culture plate, set temperature is 35 DEG C, at 5%CO
2quiescent culture 10 days under normal oxygen environment, can obtain a kind of human airway epithelial cell for the treatment of bronchial asthma.First wherein said IV, ACDE substratum making step for pour 1L distilled water in beaker; Then get 10g inositol respectively, 0.05g nicotinic acid, 0.01g vitamin, 30g sucrose is poured in distilled water, by the abundant stirring and dissolving of stirring rod; Draw 0.2mg indolylacetic acid with adjustable hydrodynamic device subsequently and put into above-mentioned solution; Afterwards by precision test paper or acidometer adjustment pH to 5.7; Next take 6g agar powder to be poured in the above-mentioned solution prepared, be placed on electric furnace and be heated to boiling, until agar powder dissolves; Load in culture vessel after cooling a little, and put into sterilization pot sterilizing 20min; From Autoclave, take out substratum after last sterilizing, wait its cooled and solidified.
This example is simple, make cell survival rate up to 90%, when there is lung bronchogenic carcinoma, chronic bronchitis, bronchiectasis, bronchostenosis, can be studied by the human airway epithelial cell got obtained by the present invention, by reconstruction of tissue and organ or can reproduce, not only make cell reproduce rear survival rate to 85%, and basis and clinic study and application are of great importance.
Example 2
First in vitro people's tracheae or segmental bronchus is got, human airway epithelial cells is collected after digesting 31h by digest medium at 4 DEG C, use the aqueous isopropanol separation cleaning tissue sample of 93% afterwards, then tissue sample is put into sterile petri dish, under dissecting microscope, with dissecting fat residual in tweezers and scissors removal tissue sample; Subsequently by the tissue sample after above-mentioned process, adopt airtight tube chamber digestion method, with Digestive system, active removal blood and mucus are carried out to human airway epithelial cells, 10h is digested at 37.5 DEG C, and frequently shake, make tissue digestion evenly, completely, the Digestive system afterwards after collection and treatment, described Digestive system is the IV substratum containing collagenase; Next the Digestive system after above-mentioned obtaining is put into whizzer, with the rotating speed of 750r/min, centrifugal 18min, get supernatant liquor afterwards, and add foetal calf serum and the ACDE substratum of 13%, stir, put into whizzer again, raising rotating speed is 750r/mn, after centrifugal 13min, gets supernatant liquor and obtains skin cell culture medium on human airway; Next by skin cell culture medium on human airway obtained above, with 4 × 10
6on after the density of cell/mL is inoculated in sterilization, to be covered with collagen protein and agar culture plate, set temperature is 36 DEG C, at 8%CO
2quiescent culture 13 days under normal oxygen environment, can obtain a kind of human airway epithelial cell for the treatment of bronchial asthma.First wherein said IV, ACDE substratum making step for pour 1L distilled water in beaker; Then get 11g inositol respectively, 0.07g nicotinic acid, 0.02g vitamin, 40g sucrose is poured in distilled water, by the abundant stirring and dissolving of stirring rod; Draw 0.2mg indolylacetic acid with adjustable hydrodynamic device subsequently and put into above-mentioned solution; Afterwards by precision test paper or acidometer adjustment pH to 5.75; Next take 6g agar powder to be poured in the above-mentioned solution prepared, be placed on electric furnace and be heated to boiling, until agar powder dissolves; Load in culture vessel after cooling a little, and put into sterilization pot sterilizing 25min; From Autoclave, take out substratum after last sterilizing, wait its cooled and solidified.
This example is simple, make cell survival rate up to 91%, when there is lung bronchogenic carcinoma, chronic bronchitis, bronchiectasis, bronchostenosis, can be studied by the human airway epithelial cell got obtained by the present invention, by reconstruction of tissue and organ or can reproduce, not only make cell reproduce rear survival rate to 86%, and basis and clinic study and application are of great importance.
Example 3
First in vitro people's tracheae or segmental bronchus is got, human airway epithelial cells is collected after digesting 32h by digest medium at 5 DEG C, use the aqueous isopropanol separation cleaning tissue sample of 95% afterwards, then tissue sample is put into sterile petri dish, under dissecting microscope, with dissecting fat residual in tweezers and scissors removal tissue sample; Subsequently by the tissue sample after above-mentioned process, adopt airtight tube chamber digestion method, with Digestive system, active removal blood and mucus are carried out to human airway epithelial cells, 15h is digested at 38 DEG C, and frequently shake, make tissue digestion evenly, completely, the Digestive system afterwards after collection and treatment, described Digestive system is the IV substratum containing collagenase; Next the Digestive system after above-mentioned obtaining is put into whizzer, with the rotating speed of 800r/min, centrifugal 20min, get supernatant liquor afterwards, and add foetal calf serum and the ACDE substratum of 15%, stir, put into whizzer again, raising rotating speed is 850r/mn, after centrifugal 15min, gets supernatant liquor and obtains skin cell culture medium on human airway; Next by skin cell culture medium on human airway obtained above, with 5 × 10
6on after the density of cell/mL is inoculated in sterilization, to be covered with collagen protein and agar culture plate, set temperature is 37 DEG C, at 10%CO
2quiescent culture 15 days under normal oxygen environment, can obtain a kind of human airway epithelial cell for the treatment of bronchial asthma.First wherein said IV, ACDE substratum making step for pour 1L distilled water in beaker; Then get 12g inositol respectively, 0.08g nicotinic acid, 0.03g vitamin, 50g sucrose is poured in distilled water, by the abundant stirring and dissolving of stirring rod; Draw 0.2mg indolylacetic acid with adjustable hydrodynamic device subsequently and put into above-mentioned solution; Afterwards by precision test paper or acidometer adjustment pH to 5.8; Next take 6g agar powder to be poured in the above-mentioned solution prepared, be placed on electric furnace and be heated to boiling, until agar powder dissolves; Load in culture vessel after cooling a little, and put into sterilization pot sterilizing 30min; From Autoclave, take out substratum after last sterilizing, wait its cooled and solidified.
This example is simple, make cell survival rate up to 92%, when there is lung bronchogenic carcinoma, chronic bronchitis, bronchiectasis, bronchostenosis, can be studied by the human airway epithelial cell got obtained by the present invention, by reconstruction of tissue and organ or can reproduce, not only make cell reproduce rear survival rate to 87%, and basis and clinic study and application are of great importance.
Claims (2)
1. treat the epithelial cultural method of human airway of bronchial asthma, it is characterized in that concrete operation step is:
(1) in vitro people's tracheae or segmental bronchus is got, human airway epithelial cells is collected after digesting 30 ~ 32h by digest medium at 3 ~ 5 DEG C, use the aqueous isopropanol separation cleaning tissue sample of 90 ~ 95% afterwards, then tissue sample is put into sterile petri dish, under dissecting microscope, with dissecting fat residual in tweezers and scissors removal tissue sample;
(2) by the tissue sample after above-mentioned process, adopt airtight tube chamber digestion method, with Digestive system, active removal blood and mucus are carried out to human airway epithelial cells, 5 ~ 15h is digested at 37 ~ 38 DEG C, and frequently shake, make tissue digestion evenly, completely, the Digestive system afterwards after collection and treatment, described Digestive system is the IV substratum containing collagenase;
(3) Digestive system after above-mentioned obtaining is put into whizzer, with the rotating speed of 500 ~ 800r/min, centrifugal 15 ~ 20min, get supernatant liquor afterwards, and add foetal calf serum and the ACDE substratum of 10 ~ 15%, stir, put into whizzer again, raising rotating speed is 550 ~ 850r/mn, after centrifugal 10 ~ 15min, gets supernatant liquor and obtains skin cell culture medium on human airway;
(5) by human airway subepithelium's skin cell culture medium obtained above, with 2 ~ 5 × 10
6on after the density of cell/mL is inoculated in sterilization, to be covered with collagen protein and agar culture plate, set temperature is 35 ~ 37 DEG C, at 5 ~ 10%CO
2quiescent culture 10 ~ 15 days under normal oxygen environment, can obtain a kind of human airway epithelial cell for the treatment of bronchial asthma.
2. a kind of epithelial cultural method of human airway for the treatment of bronchial asthma according to claim 1, is characterized in that: described IV, ACDE substratum making step is:
(1) first in beaker, 1L distilled water is poured into;
(2) get 10 ~ 12g inositol respectively, 0.05 ~ 0.08g nicotinic acid, 0.01 ~ 0.03g vitamin, 30 ~ 50g sucrose is poured in distilled water, by the abundant stirring and dissolving of stirring rod;
(3) draw 0.2mg indolylacetic acid with adjustable hydrodynamic device and put into above-mentioned solution;
(4) by precision test paper or acidometer adjustment pH to 5.7 ~ 5.8;
(5) take 6g agar powder to be poured in the above-mentioned solution prepared, be placed on electric furnace and be heated to boiling, until agar powder dissolves;
(6) cooling is rear a little loads in culture vessel, and puts into sterilization pot sterilizing 20 ~ 30min;
(7) from Autoclave, take out substratum after sterilizing, wait its cooled and solidified.
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Citations (5)
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|---|---|---|---|---|
| WO2006030241A2 (en) * | 2004-09-13 | 2006-03-23 | University Of Southampton | Growth factor treatment for asthma |
| CN102174573A (en) * | 2011-03-03 | 2011-09-07 | 张彦明 | Porcine trachea epithelial cell line and building method thereof |
| CN102433296A (en) * | 2011-12-13 | 2012-05-02 | 广州医学院第一附属医院 | Method for culturing human airway epithelial cells |
| CN102787095A (en) * | 2012-07-06 | 2012-11-21 | 广州医学院第一附属医院 | Method for cultivating rat primary airway epithelial cells |
| JP2013000121A (en) * | 2011-06-16 | 2013-01-07 | Tokyo Women's Medical College | Epithelial cell culture medium, method for culturing epithelial cell using the same, and epithelial cell obtained therefrom |
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2015
- 2015-11-12 CN CN201510771532.XA patent/CN105274050A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006030241A2 (en) * | 2004-09-13 | 2006-03-23 | University Of Southampton | Growth factor treatment for asthma |
| CN102174573A (en) * | 2011-03-03 | 2011-09-07 | 张彦明 | Porcine trachea epithelial cell line and building method thereof |
| JP2013000121A (en) * | 2011-06-16 | 2013-01-07 | Tokyo Women's Medical College | Epithelial cell culture medium, method for culturing epithelial cell using the same, and epithelial cell obtained therefrom |
| CN102433296A (en) * | 2011-12-13 | 2012-05-02 | 广州医学院第一附属医院 | Method for culturing human airway epithelial cells |
| CN102787095A (en) * | 2012-07-06 | 2012-11-21 | 广州医学院第一附属医院 | Method for cultivating rat primary airway epithelial cells |
Non-Patent Citations (1)
| Title |
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| 翟春媛 等: "猪呼吸道上皮细胞的体外原代培养研究", 《黑龙江畜牧兽医》 * |
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