CN109908179A - A kind of injection medicament of hair follicle progenitor cells - Google Patents
A kind of injection medicament of hair follicle progenitor cells Download PDFInfo
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- CN109908179A CN109908179A CN201910174273.0A CN201910174273A CN109908179A CN 109908179 A CN109908179 A CN 109908179A CN 201910174273 A CN201910174273 A CN 201910174273A CN 109908179 A CN109908179 A CN 109908179A
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- 239000003814 drug Substances 0.000 title claims abstract description 25
- 238000002347 injection Methods 0.000 title claims abstract description 22
- 239000007924 injection Substances 0.000 title claims abstract description 22
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- 102000004190 Enzymes Human genes 0.000 claims abstract description 12
- 238000000338 in vitro Methods 0.000 claims abstract description 9
- 210000000642 hair follicle dermal papilla cell Anatomy 0.000 claims abstract description 8
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 7
- 210000002950 fibroblast Anatomy 0.000 claims abstract description 6
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- 230000029087 digestion Effects 0.000 claims description 33
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention belongs to hair regeneration fields, disclose a kind of injection medicament of hair follicle progenitor cells.The injection medicament of the hair follicle progenitor cells includes the acquisition of hair follicle progenitor cells and the formation of hair follicle progenitor cells cluster.The present invention carries out various processing in vitro hair follicle progenitor cells, hair follicle progenitor cells abundance, it is digested by separating enzyme first, then Hair Follicle Bulge portion is cut, knuckle portion is digested by trypsase again, and it is separated, cultivated, separated again, is further cultured for, special fibroblast in the cutin progenitor cells and hair follicle corium in hair follicle epidermis after collecting culture again, that is hair follicle dermal papilla cell, hair follicle progenitor cells are obtained by culture, further culture forms hair follicle progenitor cells cluster.Hair follicle progenitor cells cluster injection medicament prepared by the present invention is suitble to plant in the scalp of people, obvious to baldness therapeutic effect, and less generation immunological rejection.
Description
Technical field
The invention belongs to hair regeneration field more particularly to a kind of injection medicaments of hair follicle progenitor cells.
Background technique
Currently, the prior art commonly used in the trade is such that the phenomenon that alopecia refers to epilation.The hair normally to fall off
It is at the hair of catagen and stand-down, is constantly in dynamically flat due to entering catagen and the new hair into growth period
Weighing apparatus, therefore the hair of normal quantity can be maintained.Pathologic alopecia refers to that hair is abnormal or excessive and falls off that there are many reason: (1) male
Hormonal alopecia, is autosomal dominant inheritance, and hereditary feature need to just show under androgenic effect；(2) nerve
Usually there is alopecia and increases in alopecia when stress is excessive, under the action of stress, human body arrectores pilorum is shunk, hair is straight
Vertical, vegetative nerve or nervous centralis function get muddled, and hair follicle papilla changes and malnutrition, raw so as to cause hair
Long function inhibitio, hair enter stand-down and alopecia occur；(3) endocrine alopecia, hair growth is by a variety of endocrine hormones
It influences, so causing hair loss disorders when cryptorrhea occurs, such as postpartum, climacteric alopecia more；(4) trophism alopecia, hair
Hair is the external manifestation of physical condition, and body malnutrition and pathobolism can cause the change of hair quality and color development, seriously
Malnutrition even results in alopecia generalisata；(5) physical alopecia, the common physical factor for causing alopecia includes mechanicalness
Stimulation and contact radioactive substance；(6) alopecia of chemical origin, chemical factor can cause hair color to change even alopecia；(7) feel
Metachromia alopecia, the infection of various pathogen are a kind of key factors in hair follicle disease, mainly include bacterium, virus, fungi, spiral
The infection such as body, helminth；(8) symptomatic alopecia, certain systematicness or local diseases can all occur together alopecia；(9) congenital alopecia,
Hair caused by developmental defect lacks or sparse completely, and the common hair sparse of patient is tiny, or hair is normal when birth, soon
It just falls off and does not regenerate, isolated defects and other deformities can be divided into；(10) seasonal alopecia, general summer is easy alopecia, because of the summer
The high pore expansion of its temperature leads to alopecia, and alopecia is not easy when the autumn and winter, because of this period temperature decline pore closure.Wherein, male
Property region baldness is a kind of common disease, is treated often through the transfer operation of hair.In the process, from non-bald
The intracutaneous hair follicle in hair region is removed, and realizes the dream sent out entirely all over the face by transplanting again.In fact,
During this, since the limited amount of the hair follicle for redistribution can be obtained, new hair is not formed.
In conclusion problem of the existing technology is: male's regionality baldness takes place frequently, and it is general to treat this baldness at present
Hair transplant is taken to treat.In the process, since the limited amount of the hair follicle for redistribution can be obtained, do not have
To form new hair.
Solve the difficulty and meaning of above-mentioned technical problem: the present invention carries out various processing, hair in vitro hair follicle progenitor cells
Capsule progenitor cells abundance first digests it by separating enzyme, then cuts Hair Follicle Bulge portion, then pass through trypsase
Knuckle portion is digested, and it is separated, cultivate, separate again, is further cultured for, the upper layer after collecting culture again does not attach
Keratinocyte and hair follicle dermal fibroblast, obtain hair follicle progenitor cells by culture, and further culture forms hair follicle ancestral
Cell cluster.Hair follicle progenitor cells cluster injection medicament prepared by the present invention is suitble to plant in the scalp of people, obvious to baldness therapeutic effect,
And less generation immunological rejection.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of injection medicament of hair follicle progenitor cells,
The invention is realized in this way a kind of injection medicament of hair follicle progenitor cells, preparation step include: hair follicle progenitor cells
Acquisition and hair follicle progenitor cells cluster formation.
Further, hair follicle progenitor cells acquisition the following steps are included:
Step 1: the first digestion is carried out in vitro hair follicle using separation enzyme and is cultivated；
Step 2: the Hair Follicle Bulge portion for cutting hair follicle after digesting for the first time carries out the second digestion culture；
Step 3: the Hair Follicle Bulge portion obtained after being digested using pancreatin to second is carried out third time digestion and cultivated；
Step 4: terminating digestion, is collected by centrifugation to obtain hair to by the Hair Follicle Bulge portion that digestion process obtains three times
Bladder cell；
Step 5: hair follicle cell is cultivated in the culture solution containing various amino acid and glucose of serum-free, is carried out just
Step separation, obtains initial gross separation product；
Step 6: initial gross separation product being placed in culture dish and is cultivated, and is collected after centrifugation to obtain the cutin in hair follicle epidermis
Special fibroblast, i.e. hair follicle dermal papilla cell in progenitor cells and hair follicle corium；
Step 7: it takes upper layer hair follicle dermal papilla cell to be cultivated, obtains hair follicle progenitor cells.
Further, in step 1, the first digestion is carried out in vitro hair follicle using separation enzyme and is cultivated, specifically:
(1) it takes hair several, utilizes 75% alcohol rinse 2~secondary；
(2) hair follicle tissue for intercepting growth period, shreds, and using 0.35% separation enzyme, 34 DEG C~36 DEG C, digests 35-
40min；
(3) 25 DEG C, 1000r/min is centrifuged 2~4min, removes supernatant, the cell kind of hair follicle tissue and digestion is entered 36 holes
In plate, 6%CO2 incubator culture.
Further, in step 2, Hair Follicle Bulge portion is obtained using step 1 is cut under a dissecting microscope.
Further, in step 3, the Hair Follicle Bulge portion obtained after being digested using pancreatin to second carries out third time digestion
Culture, specifically:
(1) the Hair Follicle Bulge portion obtained after second of digestion is taken, is shredded, 0.20% pancreatin+0.06% of 0.65ml is utilized
EDTA, 37 DEG C of digestion 5min are repeatedly blown and beaten；
(2) 25 DEG C, 800r/min is centrifuged 6~8min, and supernatant is taken to set in EP pipe, and 0.65mlDMEM/F12 culture is added
Liquid；
(3) using 0.65ml0.20% pancreatin+0.06%EDTA digest 3min, 25 DEG C, 800-1200r/min centrifugation 2~
4min removes supernatant, the cell kind of hair follicle tissue and digestion is entered in 36 orifice plates, 6%CO2Incubator culture.
Further, in step 5, initial gross separation preferably uses density gradient centrifugation.
Further, in step 6, the condition of culture is preferably that 30 square centimeters of collagens are equipped in culture dish, culture
Temperature is 37 DEG C, 5%CO2。
Further, in step 7, culture solution is preferably the culture medium containing various amino acid and glucose of 20% serum
(DMEM) conditioned medium；In incubation, a subculture is changed within preferably every 2 days.
Further, hair follicle progenitor cells cluster formation the following steps are included:
Step 1: taking hair follicle progenitor cells, is cultivated using RPMl1640 culture medium, amplifying hair follicle progenitor cell population；
Step 2: the hair follicle progenitor cells through cultivating are made to form cell cluster；
Step 3: using syringe collecting hair follicle progenitor cells cluster, and medicament is made, and refrigerates spare.
In conclusion advantages of the present invention and good effect are as follows: the present invention carries out various places in vitro hair follicle progenitor cells
Reason, hair follicle progenitor cells abundance first digest it by separating enzyme, then cut Hair Follicle Bulge portion, then pass through pancreas
Protease digests knuckle portion, and is separated, cultivated, separated again, be further cultured for it, collects the upper layer after culture again
Special fibroblast, i.e. hair follicle dermal papilla cell in cutin progenitor cells and hair follicle corium in hair follicle epidermis, by training
It supports and obtains hair follicle progenitor cells, further culture forms hair follicle progenitor cells cluster.Hair follicle progenitor cells cluster injection medicament prepared by the present invention
It is suitble to plant in the scalp of people, it is obvious to baldness therapeutic effect, and less generation immunological rejection.
Detailed description of the invention
Fig. 1 is the acquisition operating procedure flow chart of hair follicle progenitor cells provided in an embodiment of the present invention
Fig. 2 is the forming step flow chart of hair follicle progenitor cells cluster provided in an embodiment of the present invention
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
The injection medicament of hair follicle progenitor cells provided in an embodiment of the present invention, preparation step include: obtaining for hair follicle progenitor cells
Take the formation with hair follicle progenitor cells cluster.
The principle of the present invention is described further with reference to the accompanying drawing:
As shown in Figure 1, the acquisition of hair follicle progenitor cells provided in an embodiment of the present invention the following steps are included:
S101: the first digestion is carried out in vitro hair follicle using separation enzyme and is cultivated；
S102: the Hair Follicle Bulge portion for cutting hair follicle after digesting for the first time carries out the second digestion culture；
S103: the Hair Follicle Bulge portion obtained after being digested using pancreatin to second is carried out third time digestion and cultivated；
S104: terminating digestion, is collected by centrifugation to obtain hair follicle to by the Hair Follicle Bulge portion that digestion process obtains three times
Cell；
S105: hair follicle cell is cultivated in the culture solution containing various amino acid and glucose of serum-free, is carried out preliminary
Separation obtains initial gross separation product；
S106: initial gross separation product being placed in culture dish and is cultivated, and is collected after centrifugation to obtain the cutin ancestral in hair follicle epidermis
Special fibroblast, i.e. hair follicle dermal papilla cell in cell and hair follicle corium；
S107: it takes upper layer hair follicle dermal papilla cell to be cultivated, obtains hair follicle progenitor cells.
It is provided in an embodiment of the present invention that the first digestion culture is carried out in vitro hair follicle using separation enzyme in step S101,
Specifically:
(1) it takes hair several, utilizes 75% alcohol rinse 2~secondary；
(2) hair follicle tissue for intercepting growth period, shreds, and using 0.35% separation enzyme, 34 DEG C~36 DEG C, digests 35-
40min；
(3) 25 DEG C, 1000r/min is centrifuged 2~4min, removes supernatant, the cell kind of hair follicle tissue and digestion is entered 36 holes
In plate, 6%CO2Incubator culture.
In step S102, use provided in an embodiment of the present invention cuts step 1 under a dissecting microscope and obtains Hair Follicle Bulge
Portion.
In step S103, it is provided in an embodiment of the present invention digested using pancreatin to second after the Hair Follicle Bulge portion that obtains into
Row third time digestion culture, specifically:
(1) the Hair Follicle Bulge portion obtained after second of digestion is taken, is shredded, 0.20% pancreatin+0.06% of 0.65ml is utilized
EDTA, 37 DEG C of digestion 5min are repeatedly blown and beaten；
(2) 25 DEG C, 800r/min is centrifuged 6~8min, and supernatant is taken to set in EP pipe, and 0.65mlDMEM/F12 culture is added
Liquid；
(3) using 0.65ml0.20% pancreatin+0.06%EDTA digest 3min, 25 DEG C, 800-1200r/min centrifugation 2~
4min removes supernatant, the cell kind of hair follicle tissue and digestion is entered in 36 orifice plates, 6%CO2Incubator culture.
In step S105, initial gross separation provided in an embodiment of the present invention preferably uses density gradient centrifugation.
In step S106, the condition of culture provided in an embodiment of the present invention is preferably that 30 square centimeters are equipped in culture dish
Collagen, cultivation temperature are 37 DEG C, 5%CO2。
In step S107, culture solution provided in an embodiment of the present invention is preferably 20% serum containing various amino acid and grape
Culture medium (DMEM) conditioned medium of sugar；In incubation, a subculture is changed within preferably every 2 days.
As shown in Fig. 2, the formation of hair follicle progenitor cells cluster provided in an embodiment of the present invention the following steps are included:
S201: taking hair follicle progenitor cells, is cultivated using RPMl1640 culture medium, amplifying hair follicle progenitor cell population；
S202: the hair follicle progenitor cells through cultivating are made to form cell cluster；
S203: using syringe collecting hair follicle progenitor cells cluster, and medicament is made, and refrigerates spare.
Application principle of the invention is further described combined with specific embodiments below；
Embodiment 1；
After skin is warmed with stupe, hair follicle progenitor cells medicament is injected into the place for wishing natural on-off cycles of hair growth.
As a result:
About 8 weeks after injection, the regeneration of hair follicle was completed, and patient can start to experience new, organizational project head
The growth of hair, the hair of the growth are completely normal and as the hair of donor site.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (9)
1. a kind of injection medicament of hair follicle progenitor cells, which is characterized in that the injection medicament of the hair follicle progenitor cells includes hair follicle
The acquisition of progenitor cells and the formation of hair follicle progenitor cells cluster.
2. the injection medicament of hair follicle progenitor cells as described in claim 1, which is characterized in that the acquisition packet of the hair follicle progenitor cells
Include following steps:
Step 1: the first digestion is carried out in vitro hair follicle using separation enzyme and is cultivated；
Step 2: the Hair Follicle Bulge portion for cutting hair follicle after digesting for the first time carries out the second digestion culture；
Step 3: the Hair Follicle Bulge portion obtained after being digested using pancreatin to second is carried out third time digestion and cultivated；
Step 4: terminating digestion, thin to being collected by centrifugation to obtain hair follicle by the Hair Follicle Bulge portion that three times digestion process obtains
Born of the same parents；
Step 5: hair follicle cell is cultivated in the culture solution containing various amino acid and glucose of serum-free, is tentatively divided
From acquisition initial gross separation product；
Step 6: initial gross separation product being placed in culture dish and is cultivated, and the cutin ancestral for being collected after centrifugation to obtain in hair follicle epidermis is thin
Special fibroblast, i.e. hair follicle dermal papilla cell in born of the same parents and hair follicle corium；
Step 7: it takes upper layer hair follicle dermal papilla cell to be cultivated, obtains hair follicle progenitor cells.
3. the injection medicament of hair follicle progenitor cells as described in claim 1, which is characterized in that in the step 1, utilize separation
Enzyme carries out the first digestion in vitro hair follicle and cultivates, specifically:
(1) it takes hair several, utilizes 75% alcohol rinse 2~secondary；
(2) hair follicle tissue for intercepting growth period, shreds, and using 0.35% separation enzyme, 34 DEG C~36 DEG C, digests 35-40min；
(3) 25 DEG C, 1000r/min is centrifuged 2~4min, removes supernatant, the cell kind of hair follicle tissue and digestion is entered in 36 orifice plates,
6%CO2 incubator culture.
4. the injection medicament of hair follicle progenitor cells as described in claim 1, which is characterized in that in the step 2, using solving
It cuts open and cuts step 1 under microscope and obtain Hair Follicle Bulge portion.
5. the injection medicament of hair follicle progenitor cells as described in claim 1, which is characterized in that in the step 3, utilize pancreatin
The Hair Follicle Bulge portion obtained after digesting to second carries out third time digestion culture, specifically:
(1) the Hair Follicle Bulge portion obtained after second of digestion is taken, is shredded, using 0.20% pancreatin+0.06%EDTA of 0.65ml,
37 DEG C of digestion 5min are repeatedly blown and beaten；
(2) 25 DEG C, 800r/min is centrifuged 6~8min, and supernatant is taken to set in EP pipe, and 0.65mlDMEM/F12 culture solution is added；
(3) 3min is digested using 0.65ml0.20% pancreatin+0.06%EDTA, 25 DEG C, 800-1200r/min is centrifuged 2~4min,
Supernatant is removed, the cell kind of hair follicle tissue and digestion is entered in 36 orifice plates, 6%CO2Incubator culture.
6. the injection medicament of hair follicle progenitor cells as described in claim 1, which is characterized in that in the step 5, initial gross separation
It is preferred that using density gradient centrifugation.
7. the injection medicament of hair follicle progenitor cells as described in claim 1, which is characterized in that in the step 6, the item of culture
Part is preferably that 30 square centimeters of collagens are equipped in culture dish, and cultivation temperature is 37 DEG C, 5%CO2。
8. the injection medicament of hair follicle progenitor cells as described in claim 1, which is characterized in that in the step 7, culture solution is excellent
It is selected as the culture medium condition culture solution containing various amino acid and glucose of 20% serum；In incubation, change within preferably every 2 days
One subculture.
9. the injection medicament of hair follicle progenitor cells as described in claim 1, which is characterized in that the formation of the hair follicle progenitor cells cluster
The following steps are included:
Step 1: taking hair follicle progenitor cells, is cultivated using RPMl1640 culture medium, amplifying hair follicle progenitor cell population；
Step 2: the hair follicle progenitor cells through cultivating are made to form cell cluster；
Step 3: using syringe collecting hair follicle progenitor cells cluster, and medicament is made, and refrigerates spare.
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| CN201910174273.0A CN109908179A (en) | 2019-03-08 | 2019-03-08 | A kind of injection medicament of hair follicle progenitor cells |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910174273.0A CN109908179A (en) | 2019-03-08 | 2019-03-08 | A kind of injection medicament of hair follicle progenitor cells |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1553793A (en) * | 2001-01-29 | 2004-12-08 | 阿德兰斯研究院有限公司 | Hair follicle neogenesis by injection of follicle progenitor cells |
| CN102586177A (en) * | 2012-02-15 | 2012-07-18 | 北京雍禾植发技术研究院 | Culture method of hair follicle stem cell for treating baldness, as well as syringe |
| CN107164310A (en) * | 2017-06-29 | 2017-09-15 | 山东省口腔医院（山东大学口腔医院） | Method for reconstructing hair follicle in vivo |
-
2019
- 2019-03-08 CN CN201910174273.0A patent/CN109908179A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1553793A (en) * | 2001-01-29 | 2004-12-08 | 阿德兰斯研究院有限公司 | Hair follicle neogenesis by injection of follicle progenitor cells |
| CN102586177A (en) * | 2012-02-15 | 2012-07-18 | 北京雍禾植发技术研究院 | Culture method of hair follicle stem cell for treating baldness, as well as syringe |
| CN107164310A (en) * | 2017-06-29 | 2017-09-15 | 山东省口腔医院（山东大学口腔医院） | Method for reconstructing hair follicle in vivo |
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