CN109880794A - The method of tissue block method's culture Yangtze River Delta White goat hair follicle stem cells - Google Patents
The method of tissue block method's culture Yangtze River Delta White goat hair follicle stem cells Download PDFInfo
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Abstract
The present invention provides the methods of tissue block method's culture Yangtze River Delta White goat hair follicle stem cells, it is good using this method separation cell line growth splitting status, typical paving stone shape is presented, primary cell climbs out of the fast speed of tissue block, third day can observe that cell climbs out of tissue block growth, can observe in field of microscope within 4th day has more cell to climb out of tissue block around every piece of tissue block, it can observe within 5th day that large stretch of cell mass is centered around around tissue block under field of microscope, carry out first time secondary culture at this time.Constructed Yangtze River Delta White goat hair follicle stem cells system flushes, hair follicle stem cells marker positive rate is high, it can regulate and control for research Skin Cell gene expression and access, interaction mechanism research between the growth and differentiation mechanism of Skin appendages, especially skin main cell provides good utility model.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of simple, quick separating tissue block method culture the Changjiang river triangle
Continent Bai Shanyang (Haimen goat) hair follicle stem cells method.
Background technique
The hair of mammal is from intrafollicular adult stem cells, and experience falls off and regenerate two mistakes
Journey, hair follicle are the ideal models for studying adult stem cell.Hair follicle stem cells widely exist in Follicle traits, are hair follicles
In initial cell, have multi-lineage potential, can by itself division growth generation body needed for cell.Hair follicle is dry thin
Born of the same parents are located at Hair Follicle Bulge portion, closely related to the maintenance, tissue maintenance and update, skin injury reparation of hair cycle etc..Hair follicle
It (HF) is the idealized system for studying adult stem cell, hair follicle stem cells and hair follicle cycle state are closely related, hair follicle stem cells exist
By environmental stimuli such as skin injury or cropping etc., hair follicle stem cells are raised, and skin repair and hair is promoted to generate.
Current most of separation methods are all using mouse antenna hair follicle and human hair as experimental material, both experimental materials
Hair follicle tissue it is larger, by naked eyes easily can observe and be separately cultured.Main separation method has mechanical phonograph recorder separation and enzyme to disappear
Change method, although two methods can obtain hair follicle stem cells cell, there are still tissue block attach loosely, cell because of enzymic digestion and
It is mechanically decoupled and the deficiencies of damage, rarely have method to describe goat hair follicle stem cells separation process both at home and abroad at present, have focused largely on
Fine-wool sheep and down producing goat, and the hair follicle structure of sheep and goat has difference in microscope size, form, and it is dry thin to allow for hair follicle
The separation method of born of the same parents cannot be applicable in completely between species.Yangtze River Delta White goat is the mountain that can uniquely produce three classes pen material hair
Sheep has weight for China's Yangtze River Delta White goat industry development for the research of Yangtze River Delta White goat hair follicle stem cells
The meaning wanted can not only be established solid for researchs such as Yangtze River Delta White goat Wool growth regulation mechanism, hair follicle growth rules
Theoretical basis, and can be supported for the important theory and technology of providing of wool quality, yield and quality.Therefore, it looks for
It is raw for goats hair growth regulating mechanism, hair follicle to a kind of preparation method of suitable Yangtze River Delta White goat hair follicle stem cells
The researchs such as long rule have important role.
Summary of the invention
The purpose of the present invention is being directed to the deficiency of existing goat hair follicle stem cell isolation techniques, a kind of tissue block method's training is provided
The method for supporting Yangtze River Delta White goat hair follicle stem cells is the produced three classes pen material hair growth of research Yangtze River Delta White goat
Adjustment mechanism, hair follicle growth rule provide experimental material, and this method in a short time can body by shirtsleeve operation step
The Yangtze River Delta White goat hair follicle stem cells being separately cultured outside.
Technical scheme is as follows:
The method of tissue block method's culture Yangtze River Delta White goat hair follicle stem cells, characterized in that the following steps are included:
1) sample chooses the Yangtze River Delta White goat of 120 days gestational ages, takes back experiment after acquiring its nape of the neck skin irrigation and disinfection
Room；
2) sample in step 1) is cut into the tissue block of 1mm × 1mm size, is put into 0.25% trypsase, it is full at 37 DEG C
90min is digested in the incubator of humidity；
3) it takes out immediately and with the culture solution for containing 10% fetal calf serum, rinses tissue block, and tissue block is attached to I type rat-tail
On the coated culture dish of collagen；
4) culture dish is put into incubator after 37 DEG C of digestion incubation 30min, culture solution of the addition containing 10% fetal calf serum, 37 DEG C
Liquid observation cell growth status was changed in saturated humidity culture every 3 days；
5) when observe has large stretch of primary cell to migrate out tissue block under the microscope around every piece of tissue block, passage training is carried out
It supports.
Preferably, in step 1), sample collection first scrapes clean surface with scalpel in the state-run Haimen City's sheep stud in Jiangsu Province
Wool, disinfect in alcohol, then the intact skin histology sample of 2cm × 2cm is cut off with scalpel, the skin histology sample include epidermis
Layer, skin corium, subcutaneous connective tissue are cleaned with physiological saline, again with 5% dual anti-physiology of addition after 75% alcohol disinfecting 30s
Salt water rinses 3 times and is put into serum-free medium, and laboratory is taken back in ice chest, is separately cultured in 12h.
Preferably, in step 2), the skin histology sample acquired in step 1) is impregnated into 30s through 75% alcohol, 1 × PBS is rinsed
Completely, it is put into culture dish, skin histology sample is cut into 1mm along the direction of hair growth with knife blade in super-clean bench
× 1mm, is put into culture dish, and 0.25% trypsase is added and is allowed to do not have skin histology sample, and 37 DEG C of saturated humidities digest 90min.
Preferably, in step 3), the coated culture dish coating process of the I type Collagen type-I being previously mentioned is as follows: sterile
0.5ml I type Collagen type-I drop is drawn in the culture dish of diameter 6cm with pipettor in super-clean bench, is allowed to even spread and culture
Ware bottom.
Preferably, in step 5), culture dish is put into incubator after 37 DEG C of digestion incubation 30min, careful addition contains
The culture solution of 10% fetal calf serum not allow tissue block to swim in culture solution.
Preferably, in step 5), when the tissue block cultivated in step 4) is observed under the microscope around every piece of tissue block
When having large stretch of primary cell to migrate out tissue block, 1 × PBS is rinsed cell 1 time, and 0.25% trypsase is added to not having training just
Ware surface is supported, 37 DEG C of digestion 2min see the cell mass that bulk is dissociated under microscope, blown and beaten with pipettor, use aseptic nipper
Folder removes tissue block, and supernatant is removed in 1200r/min centrifugation, according to 1.0 × 106The concentration of a/ml is cultivated in culture bottle, and every 3 days more
Change culture solution.
Preferably, after step 5), logarithmic growth phase cell is selected, after 37 DEG C of digestion 2min, 1200r/min centrifugations,
1 × PBS of 10ml is added, cell suspension is made, supernatant is abandoned in 1200r/min centrifugation, and cells frozen storing liquid is then added and adjusts to 1.0
×106A/ml is distributed into sterile cryopreservation tube, is sealed, label；Cryopreservation tube is placed in after freezing storing box is put into ultra low temperature freezer for 24 hours
It moves into liquid nitrogen and saves cell for a long time；When recovery cell, trained with 37 DEG C of warm water quick-thawing cells, and with the cell of 9 times of volumes
Cell is collected by centrifugation after being incubated for 10min in nutrient solution mixing, by 5 × 105The cell density of a/mL is inoculated in culture bottle.
Preferably, the frozen stock solution is containing 10%DMSO and 90%FBS.
Beneficial effects of the present invention are as follows:
(1) operation of the present invention is simple, quick, has successfully started up Yangtze River Delta White goat hair follicle stem cells using enzyme digestion
Originally culture has been successfully established Yangtze River Delta White goat hair follicle stem cells system.
(2) present invention utilizes 0.25%(volumetric concentration) trypsase digestion process skin histology under the conditions of 37 DEG C, it can
Make hair follicle stem cells be easier to migrate out tissue block, compare and direct cultured tissue block, shortens hair follicle stem cells and migrate out group
It knits the time of block, while hair follicle stem cells migration can be accelerated by the coated culture dish of I type Collagen type-I with fixing organization blocks
The speed of tissue block out.
(3) good using this method separation cell line growth splitting status, the primary cell speed of growth is very fast, and third day exists
It can observe that a small amount of cell migration goes out tissue block (as shown in figure 1 shown in a) under microscope, it the 4th day can be under the microscope
Seeing around every piece of tissue block has more cell to climb out of tissue block (as shown in figure 1 shown in b), the 5th day can be in microscope
Observing under the visual field has large stretch of cell mass to be centered around around tissue block (as shown in figure 1 shown in c) around every piece of tissue block.
(4) through results tables such as the analysis of above-mentioned cell chromosome, hair follicle stem cells label analyte detection and cell viability detections
Bright, constructed Yangtze River Delta White goat hair follicle stem cells system flushes, hair follicle stem cells marker positive rate is high, can be
It studies Skin Cell gene expression and access regulates and controls, the growth and differentiation mechanism of Skin appendages, especially skin is mainly thin
The interaction mechanism research of intercellular provides good utility model.
Detailed description of the invention
Fig. 1 is the procedure chart that hair follicle stem cells climb out of tissue block in the present invention；
Fig. 2 is hair follicle stem cells H.E colored graph in the present invention；
Fig. 3 is immunofluorescence dyeing figure in the present invention；
Fig. 4 is hair follicle stem cells marker positive rate schematic diagram in the present invention；
Fig. 5 is that rupture of membranes detects hair follicle stem cells marker positive rate schematic diagram in the present invention；
Fig. 6 is CD49F positive hair follicle stem cells screening figure in the present invention；
Fig. 7 is cell viability detection figure in the present invention；
Fig. 8 is cloning efficiency detection figure in the present invention；
Fig. 9 is recovery survival rate detection figure in the present invention.
Specific embodiment
The method of tissue block method's culture Yangtze River Delta White goat hair follicle stem cells:
Firstly, the selection of goat skin tissue sample, sample source used is in Yangtze River Delta White goat (Haimen goat), choosing
The tire sheep for taking 120 days gestational ages or so takes back laboratory after acquiring the nape of the neck skin irrigation and disinfection with scissors；
It is cut into the tissue block of 1mm size with scalpel, is put into trypsase, is digested in the incubator of 37 DEG C of saturated humidities
90min；It takes out and is rinsed with the culture solution changed containing 10% fetal calf serum immediately, and removed subcutaneously under stereomicroscope with tweezers
Connective tissue and epidermis leave the skin corium of skin histology；
Tissue block is attached in the coated culture dish of Collagen type-I, incubator is put into and is incubated for 30min, then careful addition contains
Liquid sight was changed in the culture (not allow tissue block to swim in culture solution) of 10% fetal calf serum, 37 DEG C of saturated humidity cultures every 3 days
Examine cell growth status；
When there are large stretch of primary hair follicle stem cells to climb out of tissue block in field of microscope around every piece of tissue block, passage training is carried out
It supports；It carries out first using 0.25% trypsin digestion 2min when secondary culture, presss from both sides out tissue block with sterile tweezers, regather cell
Carry out first time passage.
It is good and easy to operate, quick using this method separation cell line growth splitting status, reduce primary cell point
From possibility contaminated in incubation.
The process for establishing Yangtze River Delta White goat hair follicle stem cells is as follows:
(1) sample collection is in the state-run Haimen City's sheep stud in Jiangsu Province, and choosing 120 gestational ages, (the sheep normal time gestational period is 146 to arrive
157 days, averagely getting off was 150 days, was here exactly the goat of production not yet.) tire sheep the nape of the neck skin, first use scalpel
The wool for scraping clean surface, disinfects in alcohol, then cuts off the intact skin histology sample of 2cm × 2cm size (comprising table with scalpel
Cortex, skin corium, subcutaneous connective tissue), cleaned with physiological saline, 75%(volumetric concentration) after alcohol disinfecting 30s again with being added
The normal saline flushing of 5% dual anti-(volume ratio for referring to that mycillin accounts for mycillin mixed liquor is 5%) 3 times is simultaneously put into serum-free
Culture solution takes back laboratory in ice chest, is separately cultured in 12h；
(2) by the skin histology sample acquired in step (1) through 75%(volumetric concentration) alcohol impregnates 30s, and 1 × PBS(is
0.01mol/L PBS) rinse well, be put into culture dish, in super-clean bench with knife blade by tissue block be cut into 1mm ×
1mm size, is put into culture dish, and 0.25% trypsase is added and is allowed to do not have tissue block, and 37 DEG C of saturated humidities digest 90min；
(3) when with the culture solution containing 10% fetal calf serum, (culture solution in specification is each meant step (2) postdigestive tissue block
Containing 10% fetal calf serum, bFGF 10ng/ml, EGF 10ng/ml, IGF-1 10ng/ml, 1% dual anti-, I type Collagen type-I 1%
DMEM/F12 culture solution, 10% fetal calf serum refer to that every 100ml culture solution contains 10ml fetal calf serum) rinsing tissue block 2 times；
(4) tissue block in step (3) by rinsing removes epidermis and subcutaneous connective with aseptic nipper under stereomicroscope
Tissue, is attached to tissue block on the culture dish that I type Collagen type-I was coated with, and each tissue block gap is not more than 1cm, is placed in
It is incubated for 30min in incubator, is carefully added into culture solution and is allowed to not cross tissue block, be placed in 37 DEG C, 5%(volumetric concentration) CO2, saturation
Humidified incubator is cultivated, and is changed liquid (culture solution) every three days and is observed upgrowth situation；
(5) the coated culture dish coating process of the I type Collagen type-I being previously mentioned in step (4) is as follows, in sterile super-clean bench
0.5ml I type Collagen type-I drop is drawn with the culture dish of diameter 6cm with pipettor, is allowed to even spread and culture dish bottom；
(6) when observe has large stretch of primary cell to migrate out tissue block under the microscope in step (4) around every piece of tissue block,
1 × PBS is rinsed cell 1 time, 0.25% trypsase is added to not having culture dish surface just, 37 DEG C of digestion 2min, under microscope
See the free cell mass of bulk, is blown and beaten with pipettor, press from both sides out tissue block with aseptic nipper, supernatant is removed in 1200r/min centrifugation,
According to 1.0 × 106The concentration of a/ml is cultivated in culture bottle, every 3 days replacement culture solutions；
(7) select logarithmic growth phase cell that 1 × PBS of 10ml is added and is made carefully after 37 DEG C of digestion 2min, 1200r/min centrifugations
Supernatant is abandoned in born of the same parents' suspension, 1200r/min centrifugation, and cells frozen storing liquid is then added and adjusts to 1.0 × 106A/ml is distributed into sterile
It in cryopreservation tube, seals, label.By cryopreservation tube be placed in freezing storing box be put into ultra low temperature freezer moved into afterwards in liquid nitrogen for 24 hours save for a long time it is thin
Born of the same parents.When recovery cell, with 37 DEG C of warm water quick-thawing cells, and mix with the cell culture fluid of 9 times of volumes be incubated for 10min after from
The heart collects cell, by 5 × 105The cell density of a/mL is inoculated in culture bottle；The culture solution that freezes is that volume containing 10%(is dense
Degree) DMSO and 90%(volumetric concentration) FBS.
In Fig. 1: a is the Yangtze River Delta White goat hair follicle stem cells 72h(tri- using vitro tissue block culture of the present invention
It) after under the microscope it can be seen that have a small amount of cell migration go out tissue block growth；B is to use the method for the present invention 96h(tetra- days)
Can see under the microscope has more cell to climb out of tissue block around tissue block；C is using the method for the present invention 120h(five
It) it can observe there is large stretch of cell mass to be centered around around tissue block around every piece of tissue block under field of microscope,
Secondary culture is carried out at this time.
Fig. 2 is the Yangtze River Delta White goat hair follicle stem cells using the method for the present invention secondary culture to the 3rd generation, cell warp
Typical paving stone shape is presented after crossing H.E dyeing, a.50 × 10；b.20×10；(microscope magnification, before multiplication sign is object
Mirror is followed by eyepiece).
Fig. 3 is to be passaged to the Yangtze River Delta White goat hair follicle stem cells fluorescence detection of the third generation using the method for the present invention
Hair follicle stem cells marker CD49F, CD34；A1, a2, a3 are CD49F, and a4, a5, a6 are control groups；B1, b2, b3 are CD34,
B4, b5, b6 are control groups；
Wherein the green fluorescence of a1, a2, a3 kind indicates the expression of CD49F；The green fluorescence of b1, b2, b3 kind indicates CD34
Expression, blue-fluorescence is DAPI nuclear targeting situation；CD34 and CD49F high expression in the cell, CD34 ratio
CD49F expression quantity is slightly higher.
Fig. 4 is to be passaged to the Yangtze River Delta White goat hair follicle stem cells flow cytometer detection of the third generation using the method for the present invention
Dry hair follicle stem cells marker CD49F, CD34；Flow cytometer detection is the result shows that the positive rate of CD34 albumen is zero, CD49F positive rate
It is 52.9%.
Fig. 5 is to be passaged to the Yangtze River Delta White goat hair follicle stem cells rupture of membranes streaming of the third generation using the method for the present invention
Dry hair follicle stem cells marker CD34 is detected, flow cytometer detection is the result shows that the positive rate in CD34 albumen is 52.7%.
Fig. 6 is to be passaged to the Yangtze River Delta White goat hair follicle stem cells airflow classification of the third generation using the method for the present invention
Dry hair follicle stem cells marker CD49F, a, blank control group, b, experimental group, CD49F positive rate are up to 89.9%.
Fig. 7 is to choose P3, P6, P10 respectively using the method for the present invention, and P13 cell detects 0,1,2,3,4,5,6,7,8d
Cell viability, with increasing for cell passage number, the proliferative capacity of cell dies down.
Fig. 8 is to be chosen P3, P6, P10, P13 cell using the method for the present invention, the measurement of cloning efficiency is done, with cell
Passage number increases, and cloning efficiency gradually decreases, and the decline of the tenth generation is obvious.
Fig. 9 is to choose P3, P6, P10, P13 cell using the method for the present invention, the measurement of recovery survival rate is done, as cell passes
Generation number increases, and recovery survival rate gradually decreases, and the 13rd generation cell still can achieve 40% or more.The present invention provides
A kind of quick and easy method-Isolation and culture Yangtze River Delta White goat hair follicle stem cells, this method is simple to operate,
The hair follicle stem cells system that only can be obtained Isolation and culture by shirtsleeve operation, reduces the step that primary cell is separately cultured
Suddenly, contaminated possibility during Isolation and culture is reduced, it is good using this method separation cell line growth splitting status
It is good, typical paving stone shape is presented, primary cell climbs out of the fast speed of tissue block, and third day can observe that cell is climbed
Tissue block is grown out, and can observe in field of microscope within the 4th day has more cell to climb out of around every piece of tissue block
Tissue block can observe on the 5th day that large stretch of cell mass is centered around around tissue block under field of microscope, at this time into
Row first time secondary culture.Through results such as cell chromosome analysis, hair follicle stem cells label analyte detection and cell viability detections
Show constructed Yangtze River Delta White goat hair follicle stem cells system flush, hair follicle stem cells marker positive rate it is high, can
Regulate and control for research Skin Cell gene expression and access, the growth and differentiation mechanism of Skin appendages, especially skin is main
Intercellular interaction mechanism research provides good utility model.
Claims (8)
1. the method for tissue block method's culture Yangtze River Delta White goat hair follicle stem cells, characterized in that the following steps are included:
1) sample chooses the Yangtze River Delta White goat of 120 days gestational ages, takes back experiment after acquiring its nape of the neck skin irrigation and disinfection
Room；
2) sample in step 1) is cut into the tissue block of 1mm × 1mm size, is put into 0.25% trypsase, it is full at 37 DEG C
90min is digested in the incubator of humidity；
3) it takes out immediately and with the culture solution for containing 10% fetal calf serum, rinses tissue block, and tissue block is attached to I type rat-tail
On the coated culture dish of collagen；
4) culture dish is put into incubator after 37 DEG C of digestion incubation 30min, culture solution of the addition containing 10% fetal calf serum, 37 DEG C
Liquid observation cell growth status was changed in saturated humidity culture every 3 days；
5) when observe has large stretch of primary cell to migrate out tissue block under the microscope around every piece of tissue block, passage training is carried out
It supports.
2. the method for tissue block method's culture Yangtze River Delta White goat hair follicle stem cells according to claim 1, feature
It is that in step 1), sample collection first scrapes the wool of clean surface with scalpel, use wine in the state-run Haimen City's sheep stud in Jiangsu Province
Essence disinfection, then cut off with scalpel the intact skin histology sample of 2cm × 2cm, the skin histology sample include epidermis, skin corium,
Subcutaneous connective tissue is cleaned with physiological saline, again with dual anti-normal saline flushing 3 times of addition 5% after 75% alcohol disinfecting 30s
And it is put into serum-free medium, laboratory is taken back in ice chest, is separately cultured in 12h.
3. the method for tissue block method's culture Yangtze River Delta White goat hair follicle stem cells according to claim 2, feature
It is in step 2), the skin histology sample acquired in step 1) to be impregnated into 30s through 75% alcohol, 1 × PBS is rinsed well, is put into training
It supports in ware, skin histology sample is cut into 1mm × 1mm along the direction of hair growth with knife blade in super-clean bench, is put into
In culture dish, 0.25% trypsase is added and is allowed to do not have skin histology sample, 37 DEG C of saturated humidities digest 90min.
4. the method for tissue block method's culture Yangtze River Delta White goat hair follicle stem cells according to claim 3, feature
It is that in step 3), the coated culture dish coating process of the I type Collagen type-I being previously mentioned is as follows: with shifting in sterile super-clean bench
Liquid device draws 0.5ml I type Collagen type-I drop in the culture dish of diameter 6cm, is allowed to even spread and culture dish bottom.
5. the method for tissue block method's culture Yangtze River Delta White goat hair follicle stem cells according to claim 1, feature
It is in step 5), culture dish to be put into incubator after 37 DEG C of digestion incubation 30min, careful addition is containing 10% fetal calf serum
Culture solution not allow tissue block to swim in culture solution.
6. the method for tissue block method's culture Yangtze River Delta White goat hair follicle stem cells according to claim 1, feature
It is in step 5), to observe around every piece of tissue block have sheet primary thin under the microscope when the tissue block cultivated in step 4)
When born of the same parents migrate out tissue block, 1 × PBS is rinsed cell 1 time, 0.25% trypsase is added to not having culture dish surface just, and 37 DEG C
2min is digested, the free cell mass of bulk is seen under microscope, is blown and beaten with pipettor, removes tissue block with aseptic nipper folder,
Supernatant is removed in 1200r/min centrifugation, according to 1.0 × 106The concentration of a/ml is cultivated in culture bottle, every 3 days replacement culture solutions.
7. the method for tissue block method's culture Yangtze River Delta White goat hair follicle stem cells according to claim 6, feature
It is after step 5), to select logarithmic growth phase cell, after 37 DEG C of digestion 2min, 1200r/min centrifugations, addition 10ml 1 ×
Cell suspension is made in PBS, and supernatant is abandoned in 1200r/min centrifugation, and cells frozen storing liquid is then added and adjusts to 1.0 × 106A/ml,
It is distributed into sterile cryopreservation tube, seals, label；By cryopreservation tube be placed in freezing storing box be put into ultra low temperature freezer for 24 hours afterwards move into liquid nitrogen in
Long-term cell preservation；When recovery cell, with 37 DEG C of warm water quick-thawing cells, and mixes and incubate with the cell culture fluid of 9 times of volumes
Cell is collected by centrifugation after educating 10min, by 5 × 105The cell density of a/mL is inoculated in culture bottle.
8. the method for tissue block method's culture Yangtze River Delta White goat hair follicle stem cells according to claim 7, feature
It is that the frozen stock solution is containing 10%DMSO and 90%FBS.
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| CN110157736A (en) * | 2019-06-03 | 2019-08-23 | 扬州大学 | A method of promoting the stem cells hyperplasia of goat hair follicle |
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