CN103933547A - Application of recombinant lucid ganoderma immune regulation protein in preparing medicine for treating skin tissue senility and wound - Google Patents
Application of recombinant lucid ganoderma immune regulation protein in preparing medicine for treating skin tissue senility and wound Download PDFInfo
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Abstract
The invention relates to an application of recombinant lucid ganoderma immune regulation protein (rLZ-8) in preparing a medicine for treating skin tissue senility caused by endogenous and exogenous simulation factors and skin wound caused by external force, sports, postoperation and the like. According to the application disclosed by the invention, a skin senility model and a skin wound model of mice are respectively established. Through rLZ-8 administration treatment, an experimental result shows that the rLZ-8 obviously improves the physical indexes of the skin senility model in the aspect of skin senility and can promote growth factor secretion of the skin tissue and obviously accelerate healing of the skin tissue wound of mice in the skin wound repair. The conclusion that the rLZ-8 can effectively repair skin tissue senility and wound of mice can be obtained.
Description
Technical field
The present invention relates to field of biological pharmacy, emphasis relates to the application of recombinant Ganoderma lucidum immunoregulation protein (rLZ-8) in preparing skin histology aging and wound medicine, is specifically related to the foundation of skin aging rat model and skin trauma rat model; The design for the treatment of procedure and the statistical analysis of therapeutic outcome, emphasis relates to the application of recombinant Ganoderma lucidum immunoregulation protein in treatment skin aging rat model and skin trauma rat model.
Background technology
Skin aging, mainly by internal cause and external cause, caused, wherein, intrinsic factor: 1) naturally go down for example sweat gland, the glandular integumentaria function of skin of skin accessory organ's function reduce, secretions reduces, make skin membrane, the horny layer of skin lack moist and be dried, cause dry stricture of vagina, decortication phenomenon.2) because skin metabolism slows down, intradermal Moisture factor reduces, and makes intradermal elastic fibers and collagen fiber hypofunction, causes skin tension, and elastic force weakens, and makes skin easily occur wrinkle.3) skin of face is thin compared with other position skins of health.Due to the malnutrition of skin, subcutaneous fat is stored gradually and reduce, cell and fibrous tissue malnutrition, hydraulic performance decline.4) organism enzyme reduces gradually, and health each side technical ability goes down, and makes a large amount of free radicals destroy human body cell, makes cell death.Extrinsic factor: 1) maintain improperly, lack the nursing to skin, or incorrect skin nursing program.2) cold, dry weather, makes every hypofunction of skin, and skin lacks moisture.3) over-exposure in the sun, causes skin aging.4) causing the main cause of skin aging is due to pore, to be usually subject to the obstruction of dead cell, affects due to metabolism.
Skin trauma, first degree wound only limits to skin epidermis, can heal by epithelium regeneration.Slightly severe one has skin and subcutaneous tissue fracture, and occurs wound; Serious wound can have fracture and the fracture of muscle, flesh key, nerve.Skin appendages (hair follicle, sweat gland and sebaceous gland) destroys completely as met with, can not holomorphosis, and there is scar repairing.After tendon rupture, the initial stage is also scar repairing, but the constantly reconstruction along with functional exercise, collagen fiber can be arranged by original tendon machine direction, reach holomorphosis.Skin wound healing process:
1. the early changes wound part of wound has tissue necrosis and rupture of blood vessel in various degree hemorrhage, just occurs inflammatory reaction in a few hours, shows as hyperemia, serosity oozes out and leukocytoplania, therefore local red and swollen.Blood and the Fibrinogen in transudate in wound solidify formation grumeleuse very soon, and some grumeleuse dry tack frees form crust, and grumeleuse and crust play a part protection wound.
2. the antemarginal whole layer skin of wound contraction 2～3 days and subcutaneous tissue move to center, then wound dwindle rapidly, until stop about 14 days.The meaning of wound contraction is to dwindle wound surface.
Granulation tissue hyperplasia and cicatrization approximately since the 3rd day from wound bottom and edge grow granulation tissue and fill and lead up wound.Within 5th～6 days, play fibroblast and produce collagen fiber, collagen fiber formation in one week is thereafter very active, slows down gradually later.Along with collagen fiber are more and more, there is Process of Forming Scar, about latter one month cicatrix of wound, be completed into greatly.
4. epidermis and other tissue regeneration wound occurred in 24 hours, and the basal cell of edge of wound starts hypertrophy, and towards wound center, moved under grumeleuse, formed simple epithelium, were covered in the surface of granulation tissue.When these cells meet each other, stop migration, and hypertrophy, differentiation become squamous epithelial cancer.Healthy granulation tissue is very important to promoting epidermization, because it can provide the required nutrition of epithelium regeneration and somatomedin.If granulation tissue can not be filled and led up wound form cicatrix for a long time, epithelium regeneration will delay; In another case, due to stimulations such as foreign body and infection, the granulation tissue (Exuberant granulation) of undue growth, above skin surface, also can stop promoting epidermization, therefore clinically often need be excised.If wound excessive (it is generally acknowledged when diameter surpasses 20cm), the epidermis of regenerating is difficult to wound to cover completely, often needs skin-grafting.
Ganoderma lucidum immunoregulation protein (Fungal Immunomodulatory Protein of Ganoderma lucidium, LZ-8) is that Kino in 1989 etc. are separated to first fungal immunomodulatory protein from Ganoderma mycelium extract.Former studies shows, LZ-8 has the reaction of the mice of inhibition general anaphylaxis, the immunologic competence that the cytokines such as stimulating human peripheral lymphocyte proliferation and IL-2, TNF-α and IFN-γ produce.Utilize the recombinant expressed rLZ-8 of Pichia pastoris GS115 bacterial strain and the corresponding pilot scale purifying process that have built to obtain recombinant Ganoderma lucidum immunoregulation protein (rLZ-8).RLZ-8, as recombiant protein immunomodulator, has panimmunity function, a series of toxicities that it can avoid chemotherapy to bring in anticancer, the cancer suppressing action of performance.
Summary of the invention
The object of the invention is to provide emphasis and relates to the application of recombinant Ganoderma lucidum immunoregulation protein (rLZ-8) in preparation skin repair medicine, high spot reviews the impact of skin aging rat model and skin trauma rat model therapeutic effect, skin aging rat model and skin trauma rat model have been set up, adopt mathematical statistics software to data processings of testing, show that rLZ-8 has significant curative effect in skin aging rat model and skin trauma rat model are treated.Concrete summary of the invention is as follows:
Skin repair model is set up: 1) skin aging rat model: select healthy female Wistar rats, the modeling of the subcutaneous injection D-of nape portion galactose, injects several weeks continuously.Microscopic examination model is set up situation.2) skin trauma rat model: select healthy female sd inbred rats, between body weight 240-290 gram, 5% Na for rat
2the depilation of S back, causes 1 full pachydermia Wound Defect in back, diameter is 1.5cm, deeply reaches flesh layer.
Experiment grouping and administering mode: 1) skin aging rat: 78 female Wistar rats, are divided into 6 groups (n=13) at random: dosage group, rLZ-8 high dose group in Normal group, aging model group, positive controls, rLZ-8 low dose group, rLZ-8; Normal group and aging model group injecting normal saline (0.1ml), positive controls injection vitamin E (80mg/kg/d), rLZ-8 low dose group (8ug/kg/d), dosage group (16ug/kg/d) in rLZ-8, rLZ-8 high dose group (32ug/kg/d).Injection is 14 days continuously.2) skin trauma rat model: wound matched group adopts the normal saline of equivalent, treatment group rat wound carries out direct drug treatment and (medicine is directly applied in to rat traumatic wounds place, rLZ-8 administration concentration is 125ug/kg, 250ug/kg, 500ug/kg), 4 drug treatments of periphery (are got 4 of rat wound peripheries and are carried out drug treatment, rLZ-8 administration concentration is 125ug/kg, 250ug/kg, 500ug/kg) and gastric infusion, rLZ-8 administration concentration is 125ug/kg, 250ug/kg, 500ug/kg, each group all adopts independent raising pattern, administration every day 1 time, continue 2 weeks.
Test item and method: 1) skin aging model: main test item is MDA content, body lipofuscin (LF) content and the pathological change of HE dyeing skin histology Microscopic observation etc. in SOD content, body in hyaluronic acid contents, body in hydroxyproline content, skin in skin moisture content, skin.Wherein adopt superoxide dismutase (SOD), ELISA detection kit (R & D company) is measured the SOD content in rat plasma and skin histology, according to SOD test kit description method, operates; Utilize ELISA test kit (R & D company) to measure the HA content in rat plasma and skin histology, according to test kit description method, operate; Use Coomassie brilliant blue protein determination kit to measure skin protein content, according to test kit description method, operate; Utilize hydroxyproline (Hyp) ELISA test kit (R & D company) to measure the Hyp content in rat plasma and skin histology; With malonaldehyde (MDA) ELISA test kit (R & D company), measure the MDA content in rat plasma and skin histology.2) skin trauma model: the general histidine content of hydroxyl that main test item is rat EGF ELISA (EGF), rat VEGF (VEGF), rat fibroblast somatomedin (FGF) somatomedin and granulation tissue etc.Wherein the project such as FGF, EGF, VEGF adopts corresponding euzymelinked immunosorbent assay (ELISA) to measure, and the hydroxyproline content of granulation tissue utilizes hydroxyproline (Hyp) ELISA test kit (R & D company) to measure.
Experimental result statistics: the present invention is mainly by skin moisture content, hydroxyproline content in skin, hyaluronic acid contents in skin, SOD content in body, MDA content in body, the indexs such as body lipofuscin (LF) content and the pathological change of HE dyeing skin histology Microscopic observation are investigated the impact of the therapeutic effect of rLZ-8 rat skin aging model, by rat EGF ELISA (EGF), rat VEGF (VEGF), the therapeutic effect of rLZ-8 to rat skin trauma model investigated in the detection of the general histidine content of hydroxyl of rat fibroblast somatomedin (FGF) somatomedin and granulation tissue.The experimental data of These parameters of take is carried out data analysis as basis adopts SPSS statistical software, according to carrying out analytical control between each treatment group, show that statistical result evaluates the application of rLZ-8 in skin repair medicine.
Skin aging aspect: rLZ-8 can improve hyaluronic content in skin histology, suppresses the reduction of the skin histology moisture content that causes due to aging; RLZ-8 can improve the important extracellular matrix components of skin histology---the content of-collagen, thereby improves the elasticity of skin, reduces lipofuscin level in skin histology, delaying senility; RLZ-8 can strengthen the level of superoxide dismutase (SOD) in rat body, reduces the level of lipid peroxide metabolite malonaldehyde (MDA), and the rLZ-8 aging of Skin Cell due to antagonism D-galactose is effectively described.
Skin trauma aspect: three kinds of route of administration can be put the therapeutic effect that reaches consistent at one time, experimental result shows, after rat wound, rLZ-8 can significantly regulate the expression of rat EGF ELISA (EGF), rat VEGF (VEGF), rat fibroblast somatomedin (FGF) somatomedin in 7 days, promote repair in trauma, steadily improve the hydroxyproline content of granulation tissue simultaneously, by measuring the content of the content indirect reaction collagen of hydroxyproline, understand collagenic supersession situation in wound.Along with the synthetic continuous increase of collagen, defective tissue is filled up gradually, and wound surface dwindles, and then healing.
Accompanying drawing explanation:
Under 40 times of amplifications of Fig. 1, respectively organize the contrast of rat model skin histology HE dyeing photo
Under 200 times of amplifications of Fig. 2, respectively organize the contrast of rat model skin histology HE dyeing photo
The specific embodiment
The therapeutical effect of embodiment 1 rLZ-8 to rat skin aging
1.1 experimental technique
1.1.1 experiment reagent and material:
RLZ-8, vitamin E, normal saline, D-galactose, distilled water, 1mL disposable syringe, superoxide dismutase (SOD) ELISA detection kit, malonaldehyde (MDA) ELISA test kit, Coomassie brilliant blue protein determination kit, hydroxyproline (Hyp) ELISA test kit, HA ELISA test kit (R & D company)
1.1.2 instrument and equipment:
Analytical balance, vertical pressure steam sterilizer, centrifuge, double superclean bench, inverted microscope, 4 ℃ of refrigerators, cryogenic refrigerator (80 ℃), common microplate reader.
1.1.3 laboratory animal:
Healthy adult Wistar rat, female, body weight 240-260 gram, SPF level.Under the SPF of Jilin University level condition, raise.Experimental session is controlled room temperature in (20 ± 2) ℃, alternately illumination in 12 hours, and mice freely drinks water, takes food.
1.1.4 experiment is divided into groups and treatment sequence:
78 female Wistar rats, are divided into 6 groups (n=13) at random: dosage group, rLZ-8 high dose group in Normal group, aging model group, vitamin E positive controls, rLZ-8 low dose group, rLZ-8; The subcutaneous injection normal saline 0.lml/10g BW of Normal group nape every day portion, all the other respectively organize the subcutaneous injection D-of nape portion galactose 1000mg/kg every day (being equivalent to injection capacity is 0.1ml/l0g BW) modeling, totally 7 weeks.Set up rat subacute aging model.Administration group simultaneously gavage gives vitamin E or lumbar injection rLZ-8, and Normal group and aging model group give equal-volume normal saline.Drug dose is as shown in the table:
Table 1 skin aging rat model treatment grouping and treatment sequence
1.2 experimental results:
The present invention mainly investigates the impact of the therapeutic effect of rLZ-8 rat skin aging model by indexs such as MDA content, body lipofuscin (LF) content and the pathological changes of HE dyeing skin histology Microscopic observation in SOD content, body in hyaluronic acid contents, body in hydroxyproline content, skin in skin moisture content, skin, concrete detection method and result are as follows:
1.2.1 the impact of rLZ-8 on rat skin aging model moisture content
Get mouse back 2cm * 2cm size skin, accurately claim weight in wet base.Put 80 ℃ of drying baker and dry 12 hours, claim dry weight record.Calculate moisture content.
The moisture content of skin=(skin weight in wet base one skin dry weight)/skin weight in wet base * l00%.
Skin moisture content result shows, utilize after the modeling of D-galactose, the moisture content of rat skin significantly lower than matched group (
p<0.05), other each groups are by giving V
eor the rLZ-8 of high, medium and low various dose, can significantly improve the moisture content in skin, and return to normal level (table 2).
Table 2. is respectively organized the skin moisture content of rat model
| Group | Moisture content |
| Normal group | 61.69±7.99 ※ |
| Aging model group | 43.76±14.13 ＃ |
| Positive drug group (VE) | 60.93±5.82 ※ |
| RLZ-8 low dosage | 61.54±2.59 ※ |
| Dosage in rLZ-8 | 57.22±12.60 ※ |
| RLZ-8 high dose | 59.29±9.47 ※ |
#: with Normal group ratio,
p<0.05; ※: with model group ratio,
p<0.05.
1.2.2 the impact of rLZ-8 on hydroxyproline content in rat skin aging model skin
The present invention adopts hydroxyproline (Hyp) ELISA test kit (R & D company) to measure the Hyp content in rat plasma and skin histology, according to test kit description method, operates.Result shows, except VE positive controls, the content of hydroxyproline in the rat skin of other each experimental group (Hyp) all significantly lower than Normal group (
p<0.05), but by giving the rLZ-8 of high dose and low dosage, can significantly improve the content of hydroxyproline in rat skin, in these two groups of rat skins, the content of hydroxyproline is significantly higher than model group (table 3).Prompting rLZ-8 can promote the synthetic of skin collagen, makes skin be tending towards rejuvenation and changes.
Table 3. is respectively organized hydroxyproline content in rat skin (ng/g)
| Group | Hyp |
| Normal group | 7.70±0.41 b |
| Aging model group | 4.64±1.35 a |
| Positive drug group (VE) | 7.17±0.74 b |
| RLZ-8 low dosage | 6.60±1.16 a ， b |
| Dosage in rLZ-8 | 5.40±0.75 a |
| RLZ-8 high dose | 6.17±0.63 a ， b |
A: with Normal group ratio,
p<0.05; B: with model group ratio,
p<0.05.
1.2.3 the impact of rLZ-8 on hyaluronic acid contents in rat skin aging model skin
The present invention adopts ELISA test kit (R & D company) to measure the HA content in rat plasma and skin histology, according to test kit description method, operates.Result shows, in aging model group skin, hyaluronic content is compared with Normal group, there is no significant difference.But in high concentration and low concentration group skin hyaluronic content be significantly higher than model group and Normal group (
p<0.05) concrete outcome is in Table 4.
Table 4 is respectively organized hyaluronic acid contents in rat skin (ng/g)
| Group | HA |
| Normal group | 5.68±0.48 |
| Aging model group | 6.03±0.95 |
| Positive drug group (VE) | 5.83±0.96 |
| RLZ-8 low dosage | 8.58±0.16 a ， b |
| Dosage in rLZ-8 | 6.02±0.46 |
| RLZ-8 high dose | 7.42±0.34 a ， b |
A: with Normal group ratio, p<0.05; B: with model group ratio, p<0.05.
1.2.4 rLZ-8 is in rat skin aging model blood plasma and the impact of SOD content in skin histology
The present invention adopts superoxide dismutase (SOD) ELISA detection kit (R & D company) to measure the SOD content in rat model blood plasma and in skin histology, according to SOD test kit description method, operates.Experimental result shows, compares with Normal group, and the content of SOD decreases in aging model group blood plasma and in skin histology, but difference significance not; But the content of SOD is all significantly higher than model group and Normal group (p<0.05) in each dosage group blood plasma of rLZ-8 and in tissue, and VE organizes higher than model group (table 5).Show that rLZ-8 can strengthen the safeguard function of skin to free radical.
Table 5. is respectively organized SOD content in rat body
| Group | SOD content (ng/ml) in blood plasma | SOD content (ng/g) in skin |
| Normal group | 276.62±75.91 | 5.61±0.93 |
| Aging model group | 224.47±48.72 | 4.98±0.50 |
| Positive drug group (VE) | 302.22±64.14 b | 6.29±0.81 b |
| RLZ-8 low dosage | 472.00±142.63 a ， b | 6.48±0.77 a ， b |
| Dosage in rLZ-8 | 543.77±117.83 a ， b | 7.47±0.87 a ， b |
| RLZ-8 high dose | 412.84±92.46 a ， b | 7.63±2.48 a ， b |
A: with Normal group ratio,
p<0.05; B: with model group ratio,
p<0.05.
1.2.5 the impact of rLZ-8 on malonaldehyde (MDA) content in rat skin aging model body
The present invention adopts with malonaldehyde (MDA) ELISA test kit (R & D company) and measures the MDA content in rat plasma and skin histology, according to test kit description method, operates.Experimental result shows, the content of MDA in aging model group body is significantly higher than Normal group (p<0.05), after giving rLZ-8, MDA content in blood plasma and skin histology all significantly reduces, wherein in concentration and low concentration group even lower than Normal group (p<0.05) (table 6).Prompting rLZ-8 can reduce lipid peroxidation product and pile up.
Table 6. is respectively organized MDA content in rat body
| Group | MDA content (ng/ml) in blood plasma | MDA content (ng/g) in skin |
| Normal group | 376.12±69.67 b | 4.93±1.13 b |
| Aging model group | 491.31±90.85 a | 6.78±0.86 a |
| Positive drug group (VE) | 359.75±79.80 b | 5.62±0.74 b |
| RLZ-8 low dosage | 179.94±89.69 a ， b | 3.61±0.53 a ， b |
| Dosage in rLZ-8 | 284.74±53.82 a ， b | 3.67±0.47 a ， b |
| RLZ-8 high dose | 315.20±143.42 b | 5.30±0.53 b |
A: with Normal group ratio,
p<0.05; B: with model group ratio,
p<0.05.
1.2.6 the impact of rLZ-8 on rat skin aging model body lipofuscin (LF) content
The present invention adopts with lipofuscin (LF) ELISA test kit (R & D company) and measures the LF content in rat plasma and skin histology, according to test kit description method, operates.Experimental result shows, the lipofuscin level of model group is significantly higher than Normal group, and after giving the rLZ-8 of Three doses, the lipofuscin level in skin histology significantly reduces, the content of the LF in high and low dose group skin wherein, with Normal group quite (table 7).
Table 7 is respectively organized LF content in rat body
| Group | LF content (ng/ml) in blood plasma | FL content (ng/g) in skin |
| Normal group | 370.10±45.45 b | 6.03±0.43 b |
| Aging model group | 624.90±144.60 a | 8.35±1.24 a |
| Positive drug group (VE) | 468.41±95.20 | 5.52±0.95 b |
| RLZ-8 low dosage | 318.07±113.54 b | 5.97±0.89 b |
| Dosage in rLZ-8 | 457.10±61.72 a ， b | 7.26±0.50 a ， b |
| RLZ-8 high dose | 441.06±184.47 a | 6.30±1.58 b |
A: with Normal group ratio,
p<0.05; B: with model group ratio,
p<0.05.
1.2.7 respectively organizing rat skin histopathology changes
The present invention adopts HE staining that rat model is carried out histiocyte dyeing and observed, concrete grammar: get mouse back center 0.5cm * 0.5cm size skin, put into fixedly 12-24h of 4% paraformaldehyde, conventional dehydration, transparent, waxdip, embedding, paraffin section, thickness 5um, paster is to microscope slide.Dimethylbenzene dewaxing, ethanol rehydration is to washing step by step, and haematoxylin dyeing, washes from the beginning, acidic alcohol differentiation, tap water soaks, HE dyeing, conventional dehydration, transparent neutral natural gum mounting, optical microphotograph Microscopic observation, photomicrography.Result shows: rats in normal control group skin thickness is normal, epidermal tissue's structural integrity, and cell layering is clear, and skin corium is thicker, and visible wavy collagen fiber are arranged fine and close; Compare with Normal group, the remarkable attenuation of aging model group rat skin skin corium, collagen fiber reduce, and arrange and loosen, and vitamin E group is compared with aging model group, and rat skin corium is thicker, and collagen fiber are more, arrange finer and close; RLZ-8 group is compared with aging model group, and rat skin corium obviously thickens, and collagen fiber showed increased is arranged densification, approaches with Normal group.Visible, rLZ-8 can significantly improve the degree of aging (Fig. 1,2) of skin histology.
The application of embodiment 2 rLZ-8 in the treatment of rat skin repair in trauma
2.1 experimental technique
2.1.1 experiment reagent and material:
RLZ-8, normal saline, distilled water, 1mL disposable syringe, sodium bicarbonate, sodium chloride, potassium chloride, sodium hydrogen phosphate, potassium dihydrogen phosphate, formaldehyde, dehydrated alcohol, EGF somatomedin enzyme linked immunological kit, hydroxyproline determination test kit, VEGF somatomedin enzyme linked immunological kit, FGF somatomedin enzyme linked immunological kit, determining the protein quantity test kit
2.1.2 instrument and equipment:
Constant temperature blender with magnetic force, high-pressure steam sterilizing pan, low speed centrifuge, Roberval's balance, digital display thermostat water bath, tissue homogenate instrument, electronic balance, plastic centrifuge tube, EP pipe, rifle head, tissue embedding machine, histotome, electric heating constant temperature water temperature case, Constant Temp. Oven, electric drying oven with forced convection, microplate reader, ultraviolet spectrophotometer, low-temperature and high-speed centrifuge, analytical balance, vertical pressure steam sterilizer, double superclean bench, general optics inverted microscope, 4 ℃ of refrigerators, cryogenic refrigerator (80 ℃), common microplate reader.
2.1.3 laboratory animal:
Healthy adult SD rat, female, body weight 240-260 gram, SPF level.Under the SPF of Jilin University level condition, raise.Experimental session is controlled room temperature in (20 ± 2) ℃, and alternately illumination in 12 hours, freely drinks water, takes food.
2.1.4 experiment is divided into groups and treatment sequence:
100 of male SD rats, amount to 200.Be divided at random 10 groups, 20 of model group (M group), administration is divided into dosage group (B group) in rLZ-8 low dose group (A group), rLZ-8, rLZ-8 high dose group (C group); Every group 20,3 kinds of administering modes amount to 9 groups.Weigh, labelling.Concrete treatment sequence is in Table 8.
Table 8 rat skin trauma model experiment grouping and treatment sequence (n=20)
2.1.5 test test item and method:
After rat modeling the 3rd day, the 7th day, the 14th day with etherization, put to death 6 rats for every group and carry out that eyeground vein clump is got the granulation tissue of blood, rat skin wound surface, wound surface skin around carries out the detection of every somatomedin and some functional parameters.After serum, skin, granulation tissue sample treatment, all place in-80 degree refrigerators and preserve, standby.Detection method is provided by commercially available every somatomedin detection kit.
2.2 experimental results:
The present invention mainly investigates the impact of rLZ-8 on the repairing and treating effect of rat skin trauma model by indexs such as hydroxyproline content, rat EGF ELISA (EGF) content, rat fibroblast somatomedin (FGF) content, rat VEGF (VEGF) content in Wound healing rate, wound surface granulation tissue, and concrete detection method and result are as follows:
2.2.1 the impact of rLZ-8 on rat skin trauma model healing rate
The present invention adopts wound healing standard: the complete flap coverage of the granulation of take is criterion of cure, and reaching the wound healing standard time is wound healing time; Record is respectively organized after modeling to wound healing required time.
Wound healing rate: Wound healing rate=(original wound surface area-residual wound area)/original wound surface area * 100%.In postoperative 3d, 7d and 14d, with template, accurately describe respectively to organize the form of granulation flap, the other scale of placing of wound surface, takes a picture, by wound surface image document input computer, by image analysis software, Photoshop is analyzed, obtain data, obtain data, calculate Wound healing rate, Wound healing rate 100% is wound healing, specific experiment result following (table 9):
Table 9 rLZ-8 is on the impact of rat skin trauma model healing rate (% n=6)
▲experimental group and model group relatively have significant difference
p<0.05.
Experimental result shows, directly administration and other medicine-feeding ways relatively can be found out, after modeling the 3rd of administration the day, rLZ-8 low dose group healing rate was the fastest, remarkable with model group comparing difference, have statistical significance (
p<0.05).And after modeling the 7th of administration the day, in 4 route of administration, rLZ-8 low dose group is had outstanding performance, healing rate reaches 59.2 ± 10.5%, remarkable with model group comparing difference, have statistical significance (
p<0.05), after modeling the 14th of administration the day, we found that the healing rate of this group, still for the highest, reaches average more than 95.0%; Simultaneously we find that gastric infusion approach contrasts and there is no outstanding behaviours with above-mentioned two kinds of route of administration, with model group more also for there is significant difference, do not have statistical significance (
p> 0.05), illustrate that in the treatment route of administration of rat skin trauma model directly administration and 4 dose regimens are more applicable.Meanwhile, we can also see the healing raising Wound healing rate that can promote fast wound surface in rLZ-8 treatment rat skin trauma model process.
2.2.2 the impact of rLZ-8 on hydroxyproline content in rat skin wound surface granulation tissue
The present invention adopts commercially available hydroxyproline (Hyp) ELISA test kit (R & D company) to measure hydroxyproline content in rat skin wound surface granulation tissue, according to hydroxyproline (Hyp) ELISA test kit description method, operate, concrete testing result is in Table 10.
The impact (μ g/mg) (x ± s, n=6) of table 10rLZ-8 on hydroxyproline content in rat skin wound surface granulation tissue
▲experimental group and model group relatively have significant difference
p<0.05.
Experimental result shows: after modeling, respectively organize hydroxyprolin levels and increase gradually, to the 14th day, reach the highest.After modeling the 3rd of administration the day, directly the rLZ-8 low dose group hydroxyproline expression in route of administration increases obviously, relatively have significant difference with model group, after modeling, administration is the 7th day, in each dosage group relative model group granulation tissue of rLZ-8 hydroxyproline content be all significantly increased (
p<0.05); After modeling, administration is the 14th day, can find out, only has hydroxyproline content in rLZ-8 low dose group in direct route of administration and model group comparison granulation tissue to significantly improve, have statistical significance (
p<0.05).Illustrated that under direct administration effect, rLZ-8 has the effect that in obvious promotion granulation tissue, hydroxyproline is expressed, when the reparation of skin trauma is had to positive facilitation, also illustrated that the administering mode of direct administration under comparing is applicable to skin trauma repairing and treating.
2.2.3 the impact of rLZ-8 on rat EGF ELISA (EGF) content
The present invention adopts commercially available rat EGF ELISA (EGF) enzyme linked immunological kit, measure the changes of contents of somatomedin in Rat Epidermis, according to epidermal growth factor (EGF) enzyme linked immunological kit description method, operate, concrete testing result is in Table 11.
Table 11 rLZ-8 is on the impact of rat EGF ELISA (EGF) content (unit: ug/L) (x ± s, n=6)
▲experimental group and model group relatively have significant difference
p<0.05.
Experimental result shows: after modeling drug treatment, there is reducing early stage the phenomenon that the later stage increases in rat EGF ELISA (EGF), time relationship, Trauma Rats skin EGF expression is a slower change procedure, higher in the time of the 3rd day, minimum in the time of the 7th day, then rise gradually, in the time of the 14th day, raise to some extent during compared with 7 days, but expression while also not reaching 3 days; From drug effect dosage, after wound, the content rLZ-8 low dose group of EGF is better than middle and high dosage, and from route of administration relatively, 4 administrations and gastric infusion administration after modeling were had outstanding performance in the time of 14 days, relatively there is significant difference with model group, have statistical significance (
p<0.05).
2.2.4 the content influence of rLZ-8 to rat fibroblast somatomedin (FGF)
The present invention adopts commercially available rat fibroblast somatomedin (FGF) enzyme linked immunological kit, measure the changes of contents of rat fibroblast somatomedin (FGF), according to fibroblast growth factor (FGF) enzyme linked immunological kit description method, operate, concrete testing result is in Table 12.
Table 12 rLZ-8 is to the content influence of rat fibroblast somatomedin (FGF) (unit: ug/L) (x ± s, n=6)
Experimental result shows: after rat wound, from time relationship, Trauma Rats skin FGF expression and other factors are different, be the process raising gradually, within the 3rd day, FGF level is minimum, starts to raise in the time of the 7th day, to the 14th day time, FGF level no longer raises, basicly stable; From drug effect dosage, in the different time periods, low dose group FGF level is the highest, secondly in dosage group, high dose group FGF level is minimum, but various dose group FGF level is all higher than model group, difference is not remarkable; From route of administration, the impact of Trauma Rats skin FGF expression and route of administration is little, between each group, does not occur significant difference with model group contrast.
2.2.5 the impact of rLZ-8 on rat VEGF (VEGF) content
The present invention adopts commercially available rat VEGF (VEGF) enzyme linked immunological kit, measure the changes of contents of rat VEGF (VEGF), according to rat VEGF (VEGF) enzyme linked immunological kit description method, operate, concrete testing result is in Table 13.
Table 13 rLZ-8 is on the impact of rat VEGF (VEGF) content (unit: ug/L) (x ± s, n=6)
Experimental result shows: time relationship, Trauma Rats skin vegf expression level is a slower change procedure, higher in the time of the 3rd day, minimum in the time of the 7th day, then rise gradually, and expression while substantially reaching 3 days in the time of the 14th day; From drug effect dosage, after wound, VEGF concentration rLZ-8 low dose group will be higher than middle and high dosage, but each dosage group VEGF level is more not remarkable lower than model group difference than all with model, not statistically significant, has illustrated that rLZ-8 is less on the impact of rat VEGF (VEGF) content.From route of administration relatively, between each route of administration group, there is not significant difference, not statistically significant in the VEGF concentration of each time point of administration after modeling.
2.2.6 rLZ-8 organizes the impact of albumin content on rat skin
The present invention adopts commercially available albumin detection kit, measures albuminous changes of contents in rat skin tissue, according to albumin content detection kit description method, operates, and concrete testing result is in Table 14.
Table 14 rLZ-8 organizes the impact (unit: ug/L) (x ± s, n=6) of albumin content on rat skin
Experimental result shows: from the wound healing time, experiment respectively organize skin histology albumin content all along with healing time increases and reduce.After bibliographical information wound, Human Serum Albumin content is early stage slight rising, remarkable downward trend of later stage.The ferrum that wound causes and associated protein thereof etc. are lost compensatory and are changed.Experimental result is consistent in related documents report.From drug effect dosage, between each dosage group, protein content is lower than model, positive drug group, but no difference of science of statistics (
p> 0.05).From different way of administration, between each experimental group of different way of administration, protein content and model, positive drug group significantly do not change, no difference of science of statistics (
p> 0.05).
Claims (5)
1. the application of recombinant Ganoderma lucidum immunoregulation protein in preparation treatment skin histology aging and wound medicine.
2. skin aging as claimed in claim 1, is characterized in that this symptom comprises the skin aging that endogenous and exogenous stimulation factor cause.
3. skin trauma as claimed in claim 1, is characterized in that this symptom comprises external force, motion, the skin trauma causing such as postoperative.
4. medicine as claimed in claim 1, is characterized in that this pharmaceutical preparation nucleus is comprised of fusion rotein claimed in claim 1 and the optional acceptable adjuvant of pharmacy.
5. pharmaceutical preparation route of administration as claimed in claim 1 is oral and parenterai administration, wherein, and the oral oral liquid that comprises, tablet, pill and capsule; Parenterai administration comprises medicine for external use and injection.
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| PCT/CN2015/071882 WO2015158177A1 (en) | 2014-04-16 | 2015-01-30 | Uses of recombinant ganoderma lucidum immunoregulatory protein in preparing medicines for treating skin tissue aging and wounds |
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| WO2015158177A1 (en) * | 2014-04-16 | 2015-10-22 | 张喜田 | Uses of recombinant ganoderma lucidum immunoregulatory protein in preparing medicines for treating skin tissue aging and wounds |
| CN112316108A (en) * | 2019-07-17 | 2021-02-05 | 蘑法生物科技股份有限公司 | Compositions and methods for promoting and treating chronic wound healing |
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| JP2024529557A (en) * | 2021-08-09 | 2024-08-06 | チャンチュン インテリクラウン ファーマシューティカル カンパニー，リミティド | Novel mutants of recombinant Ganoderma lucidum immunomodulatory protein and their applications |
| CN113462632B (en) * | 2021-08-13 | 2023-08-29 | 徐州医科大学 | Balsam pear exosome, extraction method and application thereof in preparation of medicines for treating burns and scalds |
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| 丁生晨: "重组灵芝免疫调节蛋白对帕金森病模型小鼠的作用及其机制研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》, no. 09, 15 September 2013 (2013-09-15) * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015158177A1 (en) * | 2014-04-16 | 2015-10-22 | 张喜田 | Uses of recombinant ganoderma lucidum immunoregulatory protein in preparing medicines for treating skin tissue aging and wounds |
| CN112316108A (en) * | 2019-07-17 | 2021-02-05 | 蘑法生物科技股份有限公司 | Compositions and methods for promoting and treating chronic wound healing |
| TWI773927B (en) * | 2019-07-17 | 2022-08-11 | 蘑法生物科技股份有限公司 | Composition and methods for promoting and treating chronic wound healing |
| CN112316108B (en) * | 2019-07-17 | 2024-04-16 | 蘑法生物科技股份有限公司 | Compositions and methods for promoting and treating chronic wound healing |
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