CN100360185C - Preparation method of medicine for treating pelada - Google Patents
Preparation method of medicine for treating pelada Download PDFInfo
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- CN100360185C CN100360185C CNB2005100488707A CN200510048870A CN100360185C CN 100360185 C CN100360185 C CN 100360185C CN B2005100488707 A CNB2005100488707 A CN B2005100488707A CN 200510048870 A CN200510048870 A CN 200510048870A CN 100360185 C CN100360185 C CN 100360185C
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Abstract
The present invention relates to a method for preparing a medicine, particularly to a method for preparing a medicine for treating alopecia areata. The precursor of the medicine is basic culture media which contain 15% of human AB type serum. The basal culture media are formed from components with the proportion: 15 ml of human AB type serum, 85 ml of low-sugar culture media (DMEM), 0.24g of hepes, 0.2g of sodium bicarbonate and 0.03gL of glutamine. The basal culture media are used for cultivating low passage papillary cells of human hair, culture supernatant of the low passage papillary cells of human hair is collected as conditioned media for the papillary cells of human hair, and the collected conditioned media for the papillary cells of human hair are prepared into dry powder. The medicine prepared by the present invention has significant therapeutic effect during the treatment of alopecia areata, and the therapeutic effect is obviously better than the therapeutic effect of other medicines, such as hormone on the alopecia areata. In addition, the cells and the culture media, which are used by the medicine do not have antigenicity to human bodies. When the medicine is used for the human bodies, the human bodies do not have any allergic reaction. The present invention overcomes the side effect which is brought to the human bodies by the hormone therapy of the alopecia areata.
Description
Technical field
The present invention relates to a kind of preparation method of medicine, particularly relate to a kind of preparation method for the treatment of the alopecia areata medicine.
Technical background
Alopecia areata is one of clinical common disease of department of dermatologry, account for 10～15% of department of dermatologry outpatient service amount, because the quickening of rhythm of life, this numeral also has further trend of rising, alopecia areata further develops and can develop into alopecia universalis and alopecia totalis, and this brings serious burden for patient's body and mind.Alopecia areata treatment in, how with whole body or topical application hormone, but the life-time service hormone can produce a series of significant side effects as: Ku Xinshi levies, thinning of skin etc.How to reduce or be present problem demanding prompt solution without the hormone therapy alopecia areata.
It is generally acknowledged that (dermal papilla cells is DPC) in the growth of hair follicle and periodically play an important role in the adjusting and controlling growth for human hair papilla cell.Illustrate human hair papilla cell to regulating the mechanism of hair follicle growth and growth, must set about from the excretory biological activity of human hair papilla cell self, this also is one of important content of hair follicle research from now on.We are setting up on the human hair papilla cell in-vitro culture model basis, application is passed at the low and is made conditioned medium for the culture supernatant of human hair papilla cell, observe the influence of its human hair papilla cell growth conditions that difference is gone down to posterity, discovery human dermal papilla cells conditioned medium (dermal papilla cellconditional medium DPCCM) can effectively be induced the human hair papilla cell of non-coagulation growth and is the compendency growth conditions, and promotes cell proliferation.Be biologic activity in the further proteic body of analyst's hair papilla cell growth activity, we make lyophilized powder with human dermal papilla cells conditioned medium, and Preliminary Applications finds that in the alopecia areata patient it has definite therapeutical effect.
Summary of the invention
The object of the present invention is to provide a kind of preparation method for the treatment of the alopecia areata medicine.
The scheme of medication preparation of the present invention: realize by following steps:
1. reagent and material
Low sugar culture-medium (DMEM): available from Sigma company.Hepes: available from BoehringerMannheim company.Healthy people AB type serum: southwest hospital of Third Military Medical University Blood Transfusion Department provides.
2. the method for medication preparation
The preparation of basal medium:
1). the preparation of people AB type serum
A) gather healthy blood donor (getting rid of HIV, HBV, HCV, infection by Treponema pallidum) whole blood 200ml and go into not have the blood bag of anticoagulant through looking into;
B) will place 1 hour in 37 ℃ of water tanks of whole blood blood bag;
C) 2000rpm is centrifugal 30 minutes;
D) with serum separator separation of human AB type serum, it is standby that isolating people AB type serum can be put into-20 ℃ of household freezers, routine step h under also can directly going);
E) people AB type serum bag is taken out from-20 ℃ of household freezers, put into 4 ℃ of cold closetes 12 hours;
F) people AB type serum room temperature was placed 2 hours;
G) people AB type serum was placed 1 hour in 37 ℃ of water tanks;
H) people AB type serum is placed l hour removal complement in 56 ℃ of water tanks, noticed that its upper and lower temperature difference should be less than 0.5 ℃;
I) on super-clean bench with 2% iodine tincture sterilization serum bag drain pipe 5 minutes, empty doing;
J) use the shears that has disinfected to cut off and seal medicated cap, serum is poured in the 100ml bottle that has disinfected;
K) be packed as the 15ml/ bottle with suction pipe ,-20 ℃ frozen standby.
2). the preparation of basal medium: with 15ml people AB type serum and the low sugar culture-medium (DMEM) of 85ml, 0.24gHepes the 0.2g sodium bicarbonate is behind the 0.03g L-glutaminate mixing, with the filtering with microporous membrane degerming of 0.22 micron pore size, directly use or-20 ℃ frozen standby.
3). the collection of human dermal papilla cells conditioned medium
(1) get a palm concentric reducer skin behind normal health youth's (getting rid of HIV, HBV, HCV, infection by Treponema pallidum) pillow of unexpected death through looking into, with two step enzyme method separation of human hair papilla cells (referring to the clock white jade, Yang Xichuan, Yang soldier, etc.Two step enzyme digestion separation of human scalp hair papilla cells [J].Chinese journal of dermatology, 2003; 36 (7): 406.)；
In the 50ml culture bottle, add the 5ml basal medium when (2) cultivating, add the 7ml basal medium in the 100ml culture bottle, changed one time basal medium in 3 days;
(3) open super-clean bench and also use disinfection by ultraviolet light 30 minutes, contagion gown during operation, wear disposable breathing mask and medicated cap, with on super-clean bench, passing at the low behind the 75% alcohol disinfecting both hands for the hair papilla cell culture supernatant from culture bottle in the aseptic centrifuge tube of the aseptic 50ml of pouring into, cover centrifuge tube and tighten with revolving a flap, 3000rpm is centrifugal 10 minutes in IK16 type centrifuge (U.S. SIGMA company);
(4) on super-clean bench, open centrifuge tube,, move in the aseptic glass container with the supernatant in the aseptic straw sucking-off centrifuge tube (carefully can not stir bottom precipitation), add aseptic rubber closure after, perform labelling ,-20 ℃ frozen standby.
4). the preparation of human dermal papilla cells conditioned medium lyophilized powder
(1) cryodesiccation chamber's disinfection by ultraviolet light is 4 hours;
Contagion gown when (2) operating, wear disposable breathing mask and medicated cap,, cover bottle stopper and reserve the lyophilizing hole with on super-clean bench, human dermal papilla cells conditioned medium being divided into 3ml/ lyophilizing bottle behind the 75% alcohol disinfecting both hands with aseptic straw;
(3) the lyophilizing bottle is put into freeze dryer;
(4) freeze-drying process:
I-40 ℃ 4 hours
II-20 ℃ 3 hours
III-5 ℃ 10 hours
5 ℃ of IV 4 hours
15 ℃ of V 3 hours
25 ℃ of VI 5 hours
(5) under vacuum environment, with hand downforce lid, vacuum package lyophilizing bottle is finished;
(6) open freeze dryer cryodesiccation chamber door, take out the lyophilizing bottle, add bottle cap and pressurize the package bottle stopper;
(7) labelled, indicate sign and date, 4 ℃ of preservations are standby.
The physicochemical property of treatment alopecia areata medicine is:
Color: white;
Form: Powdered;
PH value: 7.35;
Dissolubility: every bottle can dissolve in 3 seconds fully with the 3ml normal saline, and dissolving back liquid is orange red.
The beneficial effect of advantage of the present invention and generation is:
Human hair papilla cell is the secreted cell, when In vitro culture, can secrete numerous protein, these protein can promote the g and D of hair follicle, we have proved that it has definite biologic activity at experiment in vitro, but it is applied to the clinical report that yet there are no, we are on the basis of original cultivation human hair papilla cell, improve its culture medium, personnel selection AB type blood serum substituting calf serum is successfully turned out human hair papilla cell, and we collect and cultivated the basal medium of passing at the low for human hair papilla cell, be used for the clinical treatment alopecia areata, observe its therapeutic effect, finding has definite therapeutical effect, has obtained benign economic benefit and social benefit.
The medicine of using the present invention's preparation has tangible curative effect in the treatment alopecia areata, obviously be better than the therapeutic effect of other medicines (as hormone) to alopecia areata, and used cell of this medicine and cultivation serum are all to the equal no antigen of human body, be used for human body and do not have any anaphylaxis, the present invention's base culture base human hair papilla cell that contains people AB type serum, cultivate human hair papilla cell with calf serum than before and obtained breakthrough technically, this just lays a good foundation in clinical for this medicinal application, and this also is the core place of key technology of the present invention.
The specific embodiment
The preparation of medicine: every bottle of lyophilized powder 3ml physiological saline solution (recovering the preceding original concentration of lyophilizing).The biological safety embodiment of medicine
1). acute toxicity test in mice: we select 7～9 ages in week, body weight for use is the Kunming mouse of 18～22g.
Set up 5 dosage groups such as 50.0ml/kg, 40.0ml/kg, 32.0ml/kg, 25.6ml/kg, 20.5ml/kg by trial test common ratio 0.8, every group of 10 mices, male and female half and half.Toxic reaction, death condition and the time of animal are observed, write down to single intraperitoneal injection immediately behind the medicine, observed continuously 14 days.
2). the rabbit pyrogen testing: selecting 3 body weight is the Japanese white big ear rabbit of 1.8～2.4kg.Press 10ml/kg ear vein drug administration by injection.Measure rabbit anus body temperature, the degree of depth is 6 centimetres, later on every 1 hour take temperature 1 time, and totally 3 times.
3). antibacterial culturing: randomly draw 5 lyophilizing bottles, the physiological saline solution dissolving, 4 road methods of scoring are inoculated on the common blood plate, cultivate observed result after 48 hours.
4). fungal culture: randomly draw 5 lyophilizing bottles, the physiological saline solution dissolving is inoculated on the husky Bao Shi culture medium, cultivates observed result after 10 days.
Result of the test: after acute toxicity test in mice was presented at lumbar injection various dose human dermal papilla cells conditioned medium sample solution, each treated animal was not found clear and definite poisoning symptom, whole test no animal dead or other unusual sign in the time limit.After the rabbit pyrogen testing was presented at ear vein injection human dermal papilla cells conditioned medium sample solution, the body temperature of each animal changed the requirement that all meets pyrogen testing.Antibacterial and fungal culture there is no antibacterial and conk.
The clinical implementation example of medicine
Selecting for 50 example ages is human dermal papilla cells conditioned medium treatment group 12～64 years old alopecia areata patient, wherein 26 examples are multiple alopecia areata patient in the human dermal papilla cells conditioned medium treatment group, select with human dermal papilla cells conditioned medium treatment affected part distance at the affected part more than 2 centimetres as omcilon treatment group; Select with the human dermal papilla cells conditioned medium distance at the affected part more than 5 centimetres as blank own control affected part, more than subcutaneous multi-point injection treatment behind 3 groups of equal routine disinfection pars affecta skins, observe the curative effect after February.
Table 1 is respectively organized situation (%) after alopecia areata treatment February
| n | Cure rate (example) | Total effective rate (example) | |
| Blank group omcilon treatment group human dermal papilla cells conditioned medium treatment group | 10 26 50 | 10(1) 54(14) 74(37 *) | 30(3) 73(19) 96(48 *) |
*Compare with the blank group, P＜0.01,
*Compare P＜0.01 with the omcilon group
Human dermal papilla cells conditioned medium is after identifying by bio-safety, and we are applied to affected part under the situation of patient's informed consent.Among the alopecia areata patient of 50 routine human dermal papilla cells conditioned medium treatments, all the other all have-the constant current modulation effect except that 2 examples are invalid, and this and omcilon treatment group have relatively obtained good curative effect.There are 29 examples to be recurrent alopecia areata patients and repeatedly clinical treatment is invalid in the human dermal papilla cells conditioned medium treatment group, all the other all have certain curative effect except that 1 example is invalid in this 29 routine patient, illustrate that human dermal papilla cells conditioned medium obviously is better than the patient and treated used method in the past on treatment recurrent alopecia areata.In 50 routine alopecia areata patients of human dermal papilla cells conditioned medium treatment, we select 26 routine multiple alopecia areata patients, use human dermal papilla cells conditioned medium and omcilon respectively at the alopecia areata affected part that same patient's head is different, find that human dermal papilla cells conditioned medium is when the treatment alopecia areata, its speed that promotes natural on-off cycles of hair growth is obviously faster than omcilon treatment group, and new piliation also obviously is better than omcilon treatment group on glossiness.The growth of the stylish piliation of human dermal papilla cells conditioned medium treatment alopecia areata patient was island growth before this, omcilon then is to present Plain shape growth, the growth that hair is described is that certain mutual promoting action is arranged, and also may rely on relevant with certain hair papilla growth activity protein concentration.We make blank with the lignocaine normal saline solution, discovery has 3 routine New Development alopecia areata patients that certain curative effect is arranged, and the recurrent alopecia areata none is effective, explanation is for the alopecia areata of New Development, with certain stimulation is that certain curative effect is arranged, then effect is relatively poor with common stimulation for the recurrent alopecia areata, and this also used external curing alopecia areata results such as Rhizoma Zingiberis Recens, hot pepper paste, carbolic acid consistent clinically with the past.
Claims (2)
1, a kind of preparation method for the treatment of the alopecia areata medicine is characterized in that: pass at the low for human hair papilla cell with the base culture base that contains people AB type serum, collect and pass at the low for the human hair papilla cell culture supernatant, collected supernatant is prepared into lyophilized powder;
(1) step of above-mentioned people AB type serum preparation is:
A) 200ml that gathers not being had anticoagulant whole blood blood bag placed in 37 ℃ of water tanks 1 hour;
B) 2000rpm is centrifugal 30 minutes;
C) with serum separator separation of human AB type serum;
D) people AB type serum is placed 1 hour removal complement in 56 ℃ of water tanks, its upper and lower temperature difference is less than 0.5 ℃;
E) on super-clean bench with 2% iodine tincture sterilization serum bag drain pipe 5 minutes, empty doing;
F) use the shears that has disinfected to cut off and seal medicated cap, serum is poured in the 100ml bottle that has disinfected;
G) be packed as the 15ml/ bottle with suction pipe ,-20 ℃ frozen standby;
(2) preparation method of above-mentioned basal medium:
Basal medium: with the low sugar culture-medium (DMEM) of 15ml people AB type serum and 85ml, 0.24gHepes, the 0.2g sodium bicarbonate, behind the 0.03g L-glutaminate mixing, with the filtering with microporous membrane degerming of 0.22 micron pore size, directly use or-20 ℃ are frozen standby;
(3) collection process of human dermal papilla cells conditioned medium is:
A) the normal health youth who gets unexpected death rests the head on back one palm concentric reducer skin, with two step enzyme method separation of human hair papillas;
In inoculating the 50ml culture bottle of human hair papilla cell, add 5ml human hair papilla cell basal medium when b) cultivating, inoculated and added 7ml human hair papilla cell basal medium in the 100ml culture bottle of human hair papilla cell, changed one time the human hair papilla cell basal medium in 3 days;
C) open super-clean bench and also use disinfection by ultraviolet light 30 minutes, contagion gown during operation, wear disposable breathing mask and medicated cap, with on super-clean bench, passing at the low behind the 75% alcohol disinfecting both hands for the human hair papilla cell culture supernatant from culture bottle in the aseptic centrifuge tube of the aseptic 50ml of pouring into, cover centrifuge tube and tighten with revolving a flap, centrifugal in centrifuge;
D) open centrifuge tube on super-clean bench, the supernatant with in the aseptic straw sucking-off centrifuge tube moves in the aseptic glass container, add aseptic rubber closure after, perform labelling ,-20 ℃ frozen standby;
(4) freeze-drying process of human dermal papilla cells conditioned medium is:
A) cryodesiccation chamber's disinfection by ultraviolet light is 4 hours;
Contagion gown when b) operating, wear disposable breathing mask and medicated cap,, cover bottle stopper and reserve the lyophilizing hole with on super-clean bench, medicine being divided into 3ml/ lyophilizing bottle behind the 75% alcohol disinfecting both hands with aseptic straw;
C) the lyophilizing bottle is put into freeze dryer;
D) continuous freeze-drying process:
I-40 ℃ 4 hours
II-20 ℃ 3 hours
III-5 ℃ 10 hours
5 ℃ of IV 4 hours
15 ℃ of V 3 hours
25 ℃ of VI 5 hours
E) under vacuum environment, with hand downforce lid, vacuum package lyophilizing bottle is finished;
F) open freeze dryer cryodesiccation chamber door, take out the lyophilizing bottle, add bottle cap and pressurize the package bottle stopper;
G) labelled, indicate sign and date, 4 ℃ of preservations are standby.
2, a kind of preparation method for the treatment of the alopecia areata medicine according to claim 1 is characterized in that: the step of above-mentioned people AB type serum preparation also can be:
A) 200ml that gathers not being had anticoagulant whole blood blood bag placed in 37 ℃ of water tanks 1 hour;
B) 2000rpm is centrifugal 30 minutes;
C) with serum separator separation of human AB type serum, it is standby that isolating AB type serum is put into-20 ℃ of household freezers;
D) people AB type serum bag is taken out from-20 ℃ of household freezers, put into 4 ℃ of cold closetes 12 hours;
E) people AB type serum room temperature was placed 2 hours;
F) people AB type serum was placed 1 hour in 37 ℃ of water tanks;
G) people AB type serum is placed 1 hour removal complement in 56 ℃ of water tanks, its upper and lower temperature difference should be less than 0.5 ℃;
H) on super-clean bench with 2% iodine tincture sterilization serum bag drain pipe 5 minutes, empty doing;
I) use the shears that has disinfected to cut off and seal medicated cap, serum is poured in the 100ml bottle that has disinfected;
J) be packed as the 15ml/ bottle with suction pipe ,-20 ℃ frozen standby.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100488707A CN100360185C (en) | 2005-12-31 | 2005-12-31 | Preparation method of medicine for treating pelada |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100488707A CN100360185C (en) | 2005-12-31 | 2005-12-31 | Preparation method of medicine for treating pelada |
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| CN1824310A CN1824310A (en) | 2006-08-30 |
| CN100360185C true CN100360185C (en) | 2008-01-09 |
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| CNB2005100488707A Expired - Fee Related CN100360185C (en) | 2005-12-31 | 2005-12-31 | Preparation method of medicine for treating pelada |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109554454A (en) * | 2017-09-26 | 2019-04-02 | 东莞自然衡健康科技有限公司 | A method of freeze-dried powder hair regrowth is evaluated by measurement cytokine content |
| CN108992466B (en) * | 2018-09-30 | 2021-07-16 | 浙江卫未生物医药科技有限公司 | Preparation method of injection for treating androgenetic alopecia by using hair papilla cell exosomes |
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2005
- 2005-12-31 CN CNB2005100488707A patent/CN100360185C/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| 人毛乳头细胞条件培养基对毛乳头细胞的作用. 罗洋，郝飞，钟白玉，宋志强，杨希川.临床皮肤科杂志，第33卷第12期. 2000 * |
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