CN104946580A - Isolated culturing and identifying method for lung microvascular endothelial cells of rat - Google Patents
Isolated culturing and identifying method for lung microvascular endothelial cells of rat Download PDFInfo
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Abstract
The invention discloses an isolated culturing and identifying method for lung microvascular endothelial cells of a rat. The lung microvascular endothelial cells of the rat are cultured through an ECM culture through medium special for the endothelial cells, other types of mixed cells are few, the purity of PMVECs cultured throught the ECM culture medium special for the endothelial cells is up to 99.28%, one generation can be circulated averagely two to three days for the growth speed of the endothelial cells, the subsequent experimental study can be better met, and according to the isolated culturing and identifying method for the lung microvascular endothelial cells of the rat, materials are convenient to draw, operation is simple, the reproduction speed is high, and purity is high.
Description
Technical field
The invention belongs to biological field, be specifically related to a kind of Isolation and identification method of Pulmonary Microvascular Endothelial Cells in Rats.
Background technology
PMEC (Pulmonary Microvascular Endothelial Cells) (pulmonary microvascular endothelial cells, PMVECs) plays an important role in the research of the disease such as acute lung injury, pulmonary hypertension, is the effector cell activated the earliest in lung.Capillary endothelium lining invests blood vessel, be positioned the capillary blood vessel carrying out blood-tissue exchange, it is not only the important component forming barrier between vascular tissue, and conditioner internal milieu stable, maintain normal physiological and immunologic function, and disease mediated generation, develop and the aspect such as to lapse to and all play main effect.
Vascular endothelial cell not only has barrier action, and can make different responsing reactions to various stimulation, all plays an important role under physiology and pathologic condition.Research display, the structure and function of lung great vessels and PMEC (Pulmonary Microvascular Endothelial Cells) also exists difference, and PMEC (Pulmonary Microvascular Endothelial Cells) has stronger adaptability compared with great vessels to surrounding environment.Organs except lungs is relevant with the generation of the disease such as adult respiratory distress syndrome, pulmonary hypertension, thus cultivate and study of lung capillary endothelium then very necessary.The article of current studies in China this respect is less, and may be not easy original cuiture with this cell has certain relation.
Because PMEC (Pulmonary Microvascular Endothelial Cells) relates generally to the pathogenesis of various diseases, the capillary endothelium of application vitro culture, kinds of experiments model can be built and come the gene of capillary endothelium under studying physiological and pathologic condition, phenotype and function, inquire into various histocyte and interact and regulatory mechanism.The capillary endothelium of Different Organs and tissue origin is in form, gene, phenotype and function aspects difference to some extent.Therefore, research betides the microangiopathies of a certain organ or tissue, the capillary endothelium of this organ or tissue should be adopted as the cell model of research, and other vascular endothelial cells of originating should not be used to replace.
But, because capillary endothelium is cultivated, difficulty is comparatively large, research cost is higher, thus the universal of it and application are subject to certain restrictions, vitro culture PMVECs is to build experimental model, significant to the gene of PMVECs, phenotype, changes of function and regulatory mechanism thereof under further investigation physiology and pathologic condition.Although at present both at home and abroad existing many reports about PMVECs Isolation and culture and authentication method, the primary cell culture difficulty of PMVECs is still comparatively large, and cell cultures success ratio is lower, poor repeatability, and cost is higher.
Summary of the invention
For above-mentioned prior art Problems existing, the present invention adopts ECM endotheliocyte special culture media to cultivate Pulmonary Microvascular Endothelial Cells in Rats, establish a kind ofly to draw materials conveniently, the Isolation and identification method of a kind of Pulmonary Microvascular Endothelial Cells in Rats that simple to operate, reproduction speed is fast, purity is high.
To achieve these goals, the technical solution used in the present invention is: a kind of Pulmonary Microvascular Endothelial Cells in Rats Isolation and identification method, comprises the following steps:
(1) histiocytic separation: the SD rat of getting surrounding age, male and female regardless of, according to 50mg/kg ratio abdominal injection 2% vetanarcol, after anesthesia, in super clean bench, carry out the cleaning disinfection of thorax abdomen after thorax abdomen shaves hair with 75% alcohol, open abdominal cavity, the bloodletting of abdomen cardinal vein is also changed and is opened thoracic cavity, cut off left auricle of heart, release the blood in pulmonary circulation; Take out cardiopulmonary, put in DMEM nutrient solution (containing 100U/mL penicillin, 100U/mL Streptomycin sulphate) and wash three times, each 1min; Removing heart, lung tissue is moved into new DMEM nutrient solution (containing 100U/mL penicillin, 100U/mL Streptomycin sulphate) in carry out cleaning 2 times, after cleaning, lung tissue is moved in desiccation culture ware, add DMEM nutrient solution (containing 100U/mL penicillin, 100U/mL Streptomycin sulphate), just not have lungs to be advisable, tear the pleura on lung surface off with thin curved tweezer, cut the lung outward flange tissue block that 2-3mm is wide; The edge tissues block cut is moved in dry culture dish, 2 times are cleaned with PBS liquid, after cleaning, tissue block is moved in aseptic antibiotic bottle, and add 2ml DMEM nutrient solution (containing 10%FCS) and be advisable just not have tissue block, be cut into the tissue block of about 1mm × 1mm × 1mm size with eye scissors, be then inoculated in 100mm culture dish, in 5%CO with connector bend dropping tube aspirates tissue block
2cultivate under 37 DEG C of conditions in incubator.
(2) cultivation of cell: after 2h, adds 5-6ml ECM nutrient solution (containing 5%FBS, 1%ECGS), and nutrient solution continues to cultivate to change 1 subculture every 48h, removes tissue block, replaced medium, continue to cultivate after 72h; General cultivation is after 3-5 days, and cell can merge into individual layer, can be used for cultivating P3 for cell; After primary cell culture 3-5 days, cell confluency becomes individual layer, carries out Secondary Culture; Discard the nutrient solution in culturing bottle, wash 1 time by PBS solution, add the trysinization of 1.5mL 0.125%, under microscope, see that cell starts shrinkage and becomes bowlder, at the bottom of piping and druming culturing bottle bottle, make the completely de-wall of cell, add the appropriate blood serum medium that contains and stop digestion; The cell of digestion is transferred in sterile centrifugation tube, 1000 revs/min, centrifugal 5min; Abandon supernatant, with the endotheliocyte special culture media re-suspended cell containing 5% foetal calf serum, move into 75cm
2secondary Culture in culturing bottle, after 24h first time change liquid, afterwards interval 2-3d change liquid 1 time and under inverted microscope observation of cell growing state.
(3) cell purity qualification: the PMVECs two collecting P3 generation by sterile test tube manages, often pipe about 2 X 106 cells, and a pipe adds APC and marks anti-human CD31 antibody, and another pipe adds homotype negative control, and 4 DEG C of lucifuges hatch 30min; PBS washs, 1000 turns/min, centrifugal 10min; Add 0.5mL PBS re-suspended cell after abandoning supernatant liquor again, the sieved elimination of 200 order cell is except cell mass; Flow cytomery cell purity.
The invention has the beneficial effects as follows: the present invention adopts ECM endotheliocyte special culture media to cultivate Pulmonary Microvascular Endothelial Cells in Rats, establish a kind ofly to draw materials conveniently, the Isolation and identification method of a kind of Pulmonary Microvascular Endothelial Cells in Rats that simple to operate, reproduction speed is fast, purity is high.
Accompanying drawing explanation
Fig. 1 is FB(flow block) of the present invention.
Fig. 2 is pulmonary vascular endothelial cell original cuiture of the present invention; Wherein, figure a is that after edge lung tissue sterilization, cut into the tissue block of 1mm × 1mm × 1mm size, PMEC (Pulmonary Microvascular Endothelial Cells) 30h cultivates in tissue block method; Figure b is uterus tissue pieces 48h; Figure c is for cultivating 60h; Figure d is that primary cell is cultivated through the ECM of 5%FBS+1%ECGS, 5 days cytogamy.
Fig. 3 is flow cytometer purity detecting result; The cell-surface antigens CD31 of result display 99.28% expresses positive, the PMVECs high purity 99.28% namely cultivated.
Embodiment
Below in conjunction with accompanying drawing, the invention will be further described.
Embodiment 1
Get the SD rat in surrounding age, male and female all can, according to 50mg/kg ratio abdominal injection 2% vetanarcol, after anesthesia, in super clean bench, carry out the cleaning disinfection of thorax abdomen after thorax abdomen shaves hair with 75% alcohol, open abdominal cavity, the bloodletting of abdomen cardinal vein is also changed and is opened thoracic cavity, cut off left auricle of heart, release the blood in pulmonary circulation; Take out cardiopulmonary, put in DMEM nutrient solution (containing 100U/mL penicillin, 100U/mL Streptomycin sulphate) and wash three times, each 1min; Removing heart, lung tissue is moved into new DMEM nutrient solution (containing 100U/mL penicillin, 100U/mL Streptomycin sulphate) in carry out cleaning 2 times, after cleaning, lung tissue is moved in desiccation culture ware, add DMEM nutrient solution (containing 100U/mL penicillin, 100U/mL Streptomycin sulphate), just not have lungs to be advisable, tear the pleura on lung surface off with thin curved tweezer, cut the lung outward flange tissue block that 2-3mm is wide; The edge tissues block cut is moved in dry culture dish, 2 times are cleaned with PBS liquid, after cleaning, tissue block is moved in aseptic antibiotic bottle, and add 2ml DMEM nutrient solution (containing 10%FCS) and be advisable just not have tissue block, be cut into the tissue block of about 1mm × 1mm × 1mm size with eye scissors, be then inoculated in 100mm culture dish, in 5%CO with connector bend dropping tube aspirates tissue block
2cultivate under 37 DEG C of conditions in incubator.After 2h, add 5-6ml ECM nutrient solution (containing 5%FBS, 1%ECGS), nutrient solution continues to cultivate to change 1 subculture every 48h, removes tissue block, replaced medium, continue to cultivate after 72h; General cultivation is after 3-5 days, and cell can merge into individual layer, can be used for cultivating P3 for cell; Primary cell culture is after 3 days, and cell confluency becomes individual layer, carries out Secondary Culture; Discard the nutrient solution in culturing bottle, wash 1 time by PBS solution, add the trysinization of 1.5mL 0.125%, under microscope, see that cell starts shrinkage and becomes bowlder, at the bottom of piping and druming culturing bottle bottle, make the completely de-wall of cell, add the appropriate blood serum medium that contains and stop digestion; The cell of digestion is transferred in sterile centrifugation tube, 1000 revs/min, centrifugal 5min; Abandon supernatant, with the endotheliocyte special culture media re-suspended cell containing 5% foetal calf serum, move into 75cm
2secondary Culture in culturing bottle, after 24h first time change liquid, afterwards interval 2 change liquid 1 time and under inverted microscope observation of cell growing state.The PMVECs two collecting P3 generation by sterile test tube manages, often pipe about 2 X 106 cells, and a pipe adds APC and marks anti-human CD31 antibody, and another pipe adds homotype negative control, and 4 DEG C of lucifuges hatch 30min; PBS washs, 1000 turns/min, centrifugal 10min; Add 0.5mL PBS re-suspended cell after abandoning supernatant liquor again, the sieved elimination of 200 order cell is except cell mass; Flow cytomery cell purity, as shown in Figure 3, the PMVECs high purity 99.28% of cultivation.
Embodiment 2
Get the SD rat in surrounding age, male and female all can, according to 50mg/kg ratio abdominal injection 2% vetanarcol, after anesthesia, in super clean bench, carry out the cleaning disinfection of thorax abdomen after thorax abdomen shaves hair with 75% alcohol, open abdominal cavity, the bloodletting of abdomen cardinal vein is also changed and is opened thoracic cavity, cut off left auricle of heart, release the blood in pulmonary circulation; Take out cardiopulmonary, put in DMEM nutrient solution (containing 100U/mL penicillin, 100U/mL Streptomycin sulphate) and wash three times, each 1min; Removing heart, lung tissue is moved into new DMEM nutrient solution (containing 100U/mL penicillin, 100U/mL Streptomycin sulphate) in carry out cleaning 2 times, after cleaning, lung tissue is moved in desiccation culture ware, add DMEM nutrient solution (containing 100U/mL penicillin, 100U/mL Streptomycin sulphate), just not have lungs to be advisable, tear the pleura on lung surface off with thin curved tweezer, cut the lung outward flange tissue block that 2-3mm is wide; The edge tissues block cut is moved in dry culture dish, 2 times are cleaned with PBS liquid, after cleaning, tissue block is moved in aseptic antibiotic bottle, and add 2ml DMEM nutrient solution (containing 10%FCS) and be advisable just not have tissue block, be cut into the tissue block of about 1mm × 1mm × 1mm size with eye scissors, be then inoculated in 100mm culture dish, in 5%CO with connector bend dropping tube aspirates tissue block
2cultivate under 37 DEG C of conditions in incubator.After 2h, add 5-6ml ECM nutrient solution (containing 5%FBS, 1%ECGS), nutrient solution continues to cultivate to change 1 subculture every 48h, removes tissue block, replaced medium, continue to cultivate after 72h; General cultivation is after 3-5 days, and cell can merge into individual layer, can be used for cultivating P3 for cell; Primary cell culture is after 4 days, and cell confluency becomes individual layer, carries out Secondary Culture; Discard the nutrient solution in culturing bottle, wash 1 time by PBS solution, add the trysinization of 1.5mL 0.125%, under microscope, see that cell starts shrinkage and becomes bowlder, at the bottom of piping and druming culturing bottle bottle, make the completely de-wall of cell, add the appropriate blood serum medium that contains and stop digestion; The cell of digestion is transferred in sterile centrifugation tube, 1000 revs/min, centrifugal 5min; Abandon supernatant, with the endotheliocyte special culture media re-suspended cell containing 5% foetal calf serum, move into 75cm
2secondary Culture in culturing bottle, after 24h first time change liquid, afterwards interval 2 change liquid 1 time and under inverted microscope observation of cell growing state.The PMVECs two collecting P3 generation by sterile test tube manages, often pipe about 2 X 106 cells, and a pipe adds APC and marks anti-human CD31 antibody, and another pipe adds homotype negative control, and 4 DEG C of lucifuges hatch 30min; PBS washs, 1000 turns/min, centrifugal 10min; Add 0.5mL PBS re-suspended cell after abandoning supernatant liquor again, the sieved elimination of 200 order cell is except cell mass; Flow cytomery cell purity, the PMVECs high purity 99.22% of cultivation.
Embodiment 3
Get the SD rat in surrounding age, male and female all can, according to 50mg/kg ratio abdominal injection 2% vetanarcol, after anesthesia, in super clean bench, carry out the cleaning disinfection of thorax abdomen after thorax abdomen shaves hair with 75% alcohol, open abdominal cavity, the bloodletting of abdomen cardinal vein is also changed and is opened thoracic cavity, cut off left auricle of heart, release the blood in pulmonary circulation; Take out cardiopulmonary, put in DMEM nutrient solution (containing 100U/mL penicillin, 100U/mL Streptomycin sulphate) and wash three times, each 1min; Removing heart, lung tissue is moved into new DMEM nutrient solution (containing 100U/mL penicillin, 100U/mL Streptomycin sulphate) in carry out cleaning 2 times, after cleaning, lung tissue is moved in desiccation culture ware, add DMEM nutrient solution (containing 100U/mL penicillin, 100U/mL Streptomycin sulphate), just not have lungs to be advisable, tear the pleura on lung surface off with thin curved tweezer, cut the lung outward flange tissue block that 2-3mm is wide; The edge tissues block cut is moved in dry culture dish, 2 times are cleaned with PBS liquid, after cleaning, tissue block is moved in aseptic antibiotic bottle, and add 2ml DMEM nutrient solution (containing 10%FCS) and be advisable just not have tissue block, be cut into the tissue block of about 1mm × 1mm × 1mm size with eye scissors, be then inoculated in 100mm culture dish, in 5%CO with connector bend dropping tube aspirates tissue block
2cultivate under 37 DEG C of conditions in incubator.After 2h, add 5-6ml ECM nutrient solution (containing 5%FBS, 1%ECGS), nutrient solution continues to cultivate to change 1 subculture every 48h, removes tissue block, replaced medium, continue to cultivate after 72h; General cultivation is after 3-5 days, and cell can merge into individual layer, can be used for cultivating P3 for cell; Primary cell culture is after 5 days, and cell confluency becomes individual layer, carries out Secondary Culture; Discard the nutrient solution in culturing bottle, wash 1 time by PBS solution, add the trysinization of 1.5mL 0.125%, under microscope, see that cell starts shrinkage and becomes bowlder, at the bottom of piping and druming culturing bottle bottle, make the completely de-wall of cell, add the appropriate blood serum medium that contains and stop digestion; The cell of digestion is transferred in sterile centrifugation tube, 1000 revs/min, centrifugal 5min; Abandon supernatant, with the endotheliocyte special culture media re-suspended cell containing 5% foetal calf serum, move into 75cm
2secondary Culture in culturing bottle, after 24h first time change liquid, afterwards interval 2 change liquid 1 time and under inverted microscope observation of cell growing state.The PMVECs two collecting P3 generation by sterile test tube manages, often pipe about 2 X 106 cells, and a pipe adds APC and marks anti-human CD31 antibody, and another pipe adds homotype negative control, and 4 DEG C of lucifuges hatch 30min; PBS washs, 1000 turns/min, centrifugal 10min; Add 0.5mL PBS re-suspended cell after abandoning supernatant liquor again, the sieved elimination of 200 order cell is except cell mass; Flow cytomery cell purity, the PMVECs high purity 99.11% of cultivation.
The present invention adopts ECM endotheliocyte special culture media to cultivate Pulmonary Microvascular Endothelial Cells in Rats, other cell type mixes few, during as adopted 10% traditional or 20%FCS/DMEM culture medium culturing, the Growth of Cells such as same suitable synthon, therefore endotheliocyte easily mixes cell, purity is lower, and the PMVECs purity that endotheliocyte special culture media ECM cultivates as shown in Figure 3, the cell-surface antigens CD31 of 99.28% expresses positive, the PMVECs high purity 99.28% namely cultivated.
Endothelial cell growth speed on average can pass a generation in 2-3 days soon, subsequent experimental research can be better met, the present invention establish a kind ofly to draw materials conveniently, the separation of a kind of Pulmonary Microvascular Endothelial Cells in Rats that simple to operate, reproduction speed is fast, purity is high, cultivation and authentication method.
The above; be only the present invention's preferably embodiment, but protection scope of the present invention is not limited thereto, is anyly familiar with those skilled in the art in the technical scope that the present invention discloses; the change that can expect easily or replacement, all should be encompassed within protection scope of the present invention.
Claims (5)
1. a Pulmonary Microvascular Endothelial Cells in Rats isolation cultivation method, is characterized in that: comprise the following steps:
(1) the SD rat in surrounding age is got, male and female all can, according to 50mg/kg ratio abdominal injection 2% vetanarcol, after anesthesia, in super clean bench, carry out the cleaning disinfection of thorax abdomen after thorax abdomen shaves hair with 75% alcohol, open abdominal cavity, the bloodletting of abdomen cardinal vein is also changed and is opened thoracic cavity, cut off left auricle of heart, release the blood in pulmonary circulation;
(2) take out cardiopulmonary, put in DMEM nutrient solution (containing 100U/mL penicillin, 100U/mL Streptomycin sulphate) and wash three times, each 1min;
(3) heart is removed, lung tissue is moved into new DMEM nutrient solution (containing 100U/mL penicillin, 100U/mL Streptomycin sulphate) in carry out cleaning 2 times, after cleaning, lung tissue is moved in desiccation culture ware, add DMEM nutrient solution (containing 100U/mL penicillin, 100U/mL Streptomycin sulphate), just not have lungs to be advisable, tear the pleura on lung surface off with thin curved tweezer, cut the lung outward flange tissue block that 2-3mm is wide;
(4) the edge tissues block cut is moved in dry culture dish, 2 times are cleaned with PBS liquid, after cleaning, tissue block is moved in aseptic antibiotic bottle, and add 2ml DMEM nutrient solution (containing 10%FCS) and be advisable just not have tissue block, be cut into the tissue block of about 1mm × 1mm × 1mm size with eye scissors, be then inoculated in 100mm culture dish, in 5%CO with connector bend dropping tube aspirates tissue block
2cultivate under 37 DEG C of conditions in incubator.
(5) after 2h, add 5-6ml ECM nutrient solution (containing 5%FBS, 1%ECGS), nutrient solution continues to cultivate to change 1 subculture every 48h, removes tissue block, replaced medium, continue to cultivate after 72h;
(6) cultivate after 3-5 days, cell can merge into individual layer, can be used for cultivating P3 for cell;
(7) after primary cell culture 3-5 days, cell confluency becomes individual layer, carries out Secondary Culture;
(8) nutrient solution in culturing bottle is discarded, wash 1 time by PBS solution, add the trysinization of 1.5mL 0.125%, under microscope, see that cell starts shrinkage and becomes bowlder, make the completely de-wall of cell at the bottom of piping and druming culturing bottle bottle, add appropriate containing blood serum medium termination digestion;
(9) cell of digestion is transferred in sterile centrifugation tube, 1000 revs/min, centrifugal 5min;
(10) abandon supernatant, with containing the endotheliocyte special culture media re-suspended cell of 5% foetal calf serum, move into Secondary Culture in 75cm2 culturing bottle, after 24h, first time changes liquid, afterwards interval 2-3d change liquid 1 time and under inverted microscope observation of cell growing state.
2. a kind of Pulmonary Microvascular Endothelial Cells in Rats isolation cultivation method according to claim 1, is characterized in that: rat used is the SD rat in surrounding age.
3. a kind of Pulmonary Microvascular Endothelial Cells in Rats isolation cultivation method according to claim 1, it is characterized in that: the edge tissues block cut is moved in dry culture dish, 2 times are cleaned with PBS liquid, after cleaning, tissue block is moved in aseptic antibiotic bottle, and add 2ml DMEM nutrient solution (containing 10%FCS) and be advisable just not have tissue block, the tissue block of about 1mm × 1mm × 1mm size is cut into eye scissors, then be inoculated in 100mm culture dish, in 5%CO with connector bend dropping tube aspirates tissue block
2cultivate under 37 DEG C of conditions in incubator.
4. a kind of Pulmonary Microvascular Endothelial Cells in Rats isolation cultivation method according to claim 1, it is characterized in that: after 2h, add 5-6ml ECM nutrient solution (containing 5%FBS, 1%ECGS), ECM nutrient solution is vascular endothelial cell special culture solution, continue to cultivate to change 1 subculture every 48h, remove tissue block after 72-90h, replaced medium, continue to cultivate.
5. adopt the Pulmonary Microvascular Endothelial Cells in Rats Purity method as claim 1 obtains, it is characterized in that: comprise the following steps:
(1) collect the PMVECs two in P3 generation by sterile test tube to manage, often pipe is about 2X106 cell, and a pipe adds APC and marks anti-human CD31 antibody, and another pipe adds homotype negative control, and 4 DEG C of lucifuges hatch 30min;
(2) PBS washing, 1000 turns/min, centrifugal 10min;
(3) add 0.5mL PBS re-suspended cell after abandoning supernatant liquor again, the sieved elimination of 200 order cell is except cell mass;
(4) flow cytomery cell purity.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104371968A (en) * | 2014-06-25 | 2015-02-25 | 北京农学院 | Method for separating and purifying mouse lung microvascular endothelial cell |
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| CN104371968A (en) * | 2014-06-25 | 2015-02-25 | 北京农学院 | Method for separating and purifying mouse lung microvascular endothelial cell |
Non-Patent Citations (3)
| Title |
|---|
| 胡国栋: "一种改良的肺微血管内皮细胞培养方法", 《南方医科大学》 * |
| 蔡维霞等: "活性氧对烧伤大鼠血清诱导肺微血管内皮细胞凋亡的影响", 《中华烧伤杂志》 * |
| 贾建桃等: "肺微血管内皮细胞原代培养方法的建立", 《细胞与分子免疫学杂志》 * |
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