CN110013487A - Hydrogen is preparing the purposes in medical prevention and treatment product - Google Patents
Hydrogen is preparing the purposes in medical prevention and treatment product Download PDFInfo
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Abstract
The invention belongs to pharmaceutical technology fields, it is related to hydrogen and is preparing the purposes in medical prevention and treatment product, purposes especially in the preparation and equipment of preparation treatment pressure sore, animal experiment result confirm that, hydrogen intervention may advantageously facilitate the healing that ischemic pays close attention to damaging skin ulcer again, have significant anti-oxidant, anti-inflammatory and rush repairing activity；The oxidative damage and Level of Apoptosis of hydrogen intervention reduction pressure sore tissue, the antioxidant levels of pressure sore mouse can be promoted by activation Nrf2 access, and the proinflammatory microenvironment of ischemical reperfusion injury skin is reversed, and promote the expression of wound repair albumen in ischemical reperfusion injury skin and the formation of promotion wound granulation tissue；The hydrogen intervention effect is positively correlated with density of hydrogen.The present invention is that the application of hydrogen opens new purposes, provides a kind of new intervention stratege for the clinical treatment and prevention and health care of pressure sore, has good potential applicability in clinical practice.
Description
Technical field
The present invention relates to pharmaceutical technology fields, are related to the medical application of hydrogen, and in particular to hydrogen is preparing medical prevention and treatment
Purposes in product, the especially purposes in the preparation and equipment of preparation treatment pressure sore
Background technique
Information, pressure sore are also known as pressure ulcer, be it is a kind of be pressurized for a long time due to body local organization caused by tissue
Gangrenosum acne skin disease.Investigation display, in recent years, with China's urbanization, the quickening of aging process, illness rate is in gradually
Growth trend.Pressure sore its pathogenic mechanism mainly with the factors such as local tissue hypoxia, repeated ischemical reperfusion injury, bacteria planting
It is closely related, it is easy to delay, recurrent exerbation, treatment is difficult, and it is prolonged refractory, it is clinically very common " persistent ailment ", to patient
Quality of Life is very big.Studies have shown that causes skin lesion area to be in chronic ischemia shape since body local organization is pressurized for a long time
State, skin lesions circumferece area, which shows as fibrosis and regional tissue perfusion, to reduce, and it is downright bad to lead to tissue ischemia, and be difficult to heal；Compared with
Histanoxia, tissue ischemia Reperfu- sion are more crucial during pressure sore occurrence and development.Tissue early stage ischemic can reduce cell generation
The level of thanking is to save function, and then to anoxic zone, then easy induced oxidation free radical molecule excessively generates blood reperfusion, opens repeatedly
Dynamic tissue damage events.Again just like acute extensive radical damage can start the apoptosis of vascular endothelial cell, cause endothelium thin
Born of the same parents' skeleton is destroyed and is adhered between damaging cells, and blood vessel endothelium barrier permeability is caused to increase；These damages change, and further promote
Stagnation, adherency, chemotactic and the activation of inflammatory cell, and leukocyte activation can discharge ROS with oxidative burst formula, to enhance accumulative
Oxidative damage aggravates the damage to blood vessel and surrounding tissue, leads to tissue edema and hemodynamic responses；Therefore, ischemic is again
The inflammation damnification that perfusion induces becomes the important pathogenic basis for mediating pressure ulcer occurrence and development.Current clinically used pressure sore
Treatment method is limited, such as intensive care, surgical debridement, physiotherapy, growth factor and dermatoplasty, and the treatment of pressure sore is
As problem generally acknowledged in the industry.
The simplest molecule hydrogen of nature the most, is ignored by living nature and medical field for a long time.In July, 2007,
Japanese Ohta Shigeo professor reports that 2% hydrogen of animal breath can effectively remove free radical, significantly change in Natural Medicine
It is apt to cerebral ischemia re-pouring injured, confirms hydrogen to the selective antiopxidant effect of hydroxyl radical free radical and nitrous acid anion for the first time；
Then clinic shows hydrogen to related oxidative damage, inflammatory reaction, Apoptosis and abnormal angiogenesis etc. with basic research
Illness has therapeutic effect.
Studies have shown that hydrogen obtain in clinical studies another reason for application be its with biological safety, according to
It dives under water and studies according to the hydrogen for one, international submarine medicine more than 70 years of mechanism, it was demonstrated that high pressure hydrogen is without any side effects to human body；
Two, study on intestinal flora is found, big intestinal bacterium escherichia coli can produce hydrogen, have scholar to research and propose acarbose to the heart
It is closely related that dirty protective effect may promote big enteral hydrogen to generate with it；Three, the popularization with hydrogen in clinical studies,
So far, there is no any evidence to show it, there are harmfulness to human body.Currently, hydrogen is classified as food addition by multiple countries
Agent, therefore its bio-safety is unquestionable.
So far, there is not yet in relation to hydrogen in the purposes being used to prepare in the preparation and equipment for the treatment of pressure sore.
Status based on the prior art, present inventor is quasi- to provide hydrogen new medical application, and in particular to hydrogen
Preparing the purposes in medical prevention and treatment product, the especially purposes in the preparation and equipment of preparation treatment pressure sore.
Summary of the invention
The purpose of the present invention is the statuses based on the prior art, provide hydrogen new medical application, are related to hydrogen and are preparing
Purposes in medical prevention and treatment product, the especially purposes in the preparation and equipment of preparation treatment pressure sore.
Hydrogen of the present invention generates for the related hydrogen storage equipment that produces purchased from Shanghai Hui U.S. medical science and technology Co., Ltd.
The present invention has carried out zoopery, the results show that hydrogen intervention may advantageously facilitate ischemic pays close attention to damaging skin again
The healing of ulcer has significant anti-oxidant, anti-inflammatory and rush repairing activity；To mouse pressure caused by ischemical reperfusion injury
Sore, one week ulcer area that can be reduced significantly at early stage skin lesion of hydrogen intervention in advance, 75% hydrogen can be shortened wound healing time；
After ulcer forms mid-term (5-7d after modeling), the DNA damage (8-OXOdG) of hydrogen intervention group mouse skin lesion tissue withers with cell
Rate significant decrease is died, intervention effect is closely related with density of hydrogen, best with 75% hydrogen effect；The oxidation of skin lesion tissue is certainly
It being shown by base and oxidoreducing enzyme detection of expression, hydrogen intervention significantly reduces oxygen free radicals caused by ischemia-reperfusion,
And the significant raising of activity of the antioxidases such as SOD, GPx and CAT；According to the analysis, the antioxidant activity of hydrogen, it may be right derived from its
The activation of the anti-oxidant access of Nrf2 in pressure sore tissue, Nrf2 and downstream key protein HO-1 in hydrogen intervention group skin lesion tissue,
The transcription of AKR1C1 and NQO1 and protein expression level are significantly higher than control group, and it is in positive that regulating effect and hydrogen, which intervene concentration,
It closes；Hydrogen intervention can significantly inhibit the expression of proinflammatory cytokine IL-1 β, TNF-α, IL-6 and IL-8 in pressure sore skin lesion tissue,
Promote the expression of suppression inflammatory cytokine IL-22, the significant proinflammatory microenvironment for reversing ischemia-reperfusion skin histology intervenes effect
Fruit is positively correlated with density of hydrogen；Experimental result of the invention also shows that hydrogen intervention can significantly increase in pressure sore skin lesion tissue
The expression of the wound repairs factor such as VEGF, IGF-1, TGF-β, and significantly inhibit the expression of metalloproteinases MMP9, wound repair
The expression of GAP-associated protein GAP changes the formation for promoting wound granulation tissue, promotees repairing activity with hydrogen and intervenes the close phase of concentration
It closes.
In the present invention, used hydrogen is to human body biological safety with higher, and safety is mainly according to following
Aspect: one, the hydrogen diving research in international submarine medicine more than 70 years of mechanism has shown that high pressure hydrogen is without any side effects to human body；
Two, study on intestinal flora shows that big intestinal bacterium escherichia coli can produce hydrogen, and has scholar to research and propose acarbose
Big enteral hydrogen may be promoted to generate with it protective effect of heart closely related；Three, in clinical studies with hydrogen
It promotes, so far, there is no any evidence to show it, there are harmfulness to human body；And currently, hydrogen by China, Japan,
Multiple countries such as European Union are classified as food additives, and therefore, bio-safety is unquestionable.
In the present invention, the hydrogen can be through the prevention and treatment equipment such as breathing hydrogen, hydrogen-rich bubble bath and intravenous injection hydrogen-rich
The clinical preparations mode such as physiological saline plays Prevention effect, and further, the hydrogen can be used for preparing medical prevention and treatment system
Product, in particular for preparing in the preparation and equipment for the treatment of pressure sore.
The present invention is that the application of hydrogen opens new purposes, provides one kind for the clinical treatment and prevention and health care of pressure sore
New intervention stratege has good potential applicability in clinical practice.
Detailed description of the invention
Fig. 1, various concentration hydrogen breathe the healing that can accelerate pressure sore mouse wound, wherein
A. skin ischemical reperfusion injury mouse skin lesion picture compares；B. mouse pressure sore skin lesion areal analysis；* P <
0.05, * * * P < 0.001；Ctrl is control group.
Fig. 2, hydrogen can significantly reduce the oxidative damage and Level of Apoptosis of pressure sore tissue, wherein
A. after the modeling of each group mouse different time nodes skin lesion tissue 8-oxodG immunohistochemical analysis；B. each group mouse
The Apoptosis of different time nodes skin lesion tissue detects (TUNEL) after modeling；C. mouse tissue apoptosis rate is analyzed；*P <
0.05,**P < 0.01, is compared with control group.
Fig. 3, it is horizontal (A) that hydrogen intervention can significantly reduce oxyradical caused by ischemia-reperfusion, and significantly increases oxygen
Change the expression (B-D) of reductase；*P < 0.05,**P < 0.01.
Fig. 4, hydrogen intervention significantly increase the expression of Nrf2 access key protein in pressure sore mouse skin lesion tissue,
In,
A. real-time quantitative PCR；B. western blot；*P < 0.05,**P < 0.01, is compared with control group.
Fig. 5, hydrogen intervene the significant proinflammatory microenvironment reversed in ischemical reperfusion injury skin histology, wherein
A.ELISA detection；B. western blot；C. immunohistochemistry；*P < 0.05,**P < 0.01.
Fig. 6, hydrogen intervention can significantly promote the expression of wound repair albumen in ischemical reperfusion injury skin, wherein
A. real-time quantitative PCR；B. western blot；*P < 0.05,**P < 0.01, is compared with control group.
Specific embodiment
It elaborates below with reference to embodiment to implementation of the invention, following embodiment is with technical solution of the present invention
Premised under implemented, give detailed embodiment, but protection scope of the present invention is not limited to following embodiments.
1. hydrogen intervention of embodiment is conducive to the healing that ischemic pays close attention to damaging skin ulcer again
24 6-8 week old C57 mouse (this experimental animal Co., Ltd of Changzhou Cavan)；Round ferromagnetic sheet: 12mm diameter,
5mm is thick, area 113mm2, 1000G magnetic force can be formed, external pressure is about 50mmHg, can reduce SkBF 80%, it is sufficient to
Inducing ischemia-reperfusion type cutaneous necrosis；
After C57 mouse anesthesia, conventional treatment is carried out, back depilation is sterilized with 70% alcohol and ANER DIAN, with above-mentioned two
Block magnetic sheet clamps experiment mice skin of back, and magnetic sheet is made to clamp epidermis, corium, subcutaneous fat and part loose connective tissue, but
Not comprising muscle, mark label skin wound edges around magnetic sheet unclamp after 12h；Second day, third day so repeat three
Wheel, i.e., at same position, folder closes 12h release 12h, pressure sore model mice is randomly divided into 3 groups, every group 8 according to grouping: control
Group does not inhale hydrogen, low-concentration hydrogen intervention group 2%H2* 6h/d, high concentration hydrogen intervention group 75%H2* 6h/d, before control group modeling
Hydrogen pretreatment one week in advance；Respectively at 1d, 3d, 5d, 7d, 10d, wound progress of the 12d to every mouse after modeling success
It takes pictures；
Experimental result is as shown in Figure 1,3-5 days after skin Ischemia-Reperfusion Injury Model modeling, hydrogen intervention group mouse pressure
Sore wound area is substantially less than control group, wherein best with 75% hydrogen intervention effect, wound healing is most fast；In addition, 75% hydrogen
It heals within all mouse of gas intervention group 10 days after modeling success, and control group mice at least needs can heal for 12 days；2% hydrogen is dry
It not can be shortened the overall healing time of wounds in mice in advance, but each time point surface of a wound area is below control group, the results showed that, hydrogen
Intervene the healing that especially high concentration hydrogen may advantageously facilitate ischemical reperfusion injury skin ulcer.
2. hydrogen intervention of embodiment can reduce the oxidative damage and Level of Apoptosis of pressure sore tissue
Experimental material: skin paraffin section, dimethylbenzene, 100% ethyl alcohol, 95% ethyl alcohol, 90 ethyl alcohol, 80% ethyl alcohol, 70 second
Alcohol, 30% hydrogen peroxide, 10 × PBS phosphate buffer, citrate buffer, BSA, I-CAM1 antibody (Abcam), DAB
Developing solution (Zhong Shan Golden Bridge, ZLI-9017), haematoxylin, neutral gum.TUNEL staining kit (Kai Ji, KGA7072)；
Inverted microscope (OLYMPUS, IX71), inversion are taken pictures microscope (Leica company IX71), pressure cooker (Haier),
Electric drying oven with forced convection (the rich news GZX-9240MBE in Shanghai), cold storage refrigerator (Haier), pipettor (Eppendorf)；
Experimental method: immunohistochemistry detection: the skin lesion tissue of each group mouse after modeling success is taken, wax stone is prepared；Dimethylbenzene I
Dewax 30min, the dewaxing of dimethylbenzene II 10min；Graded ethanol rehydration, 100% alcohol 3min, 100% alcohol 3min, 95% alcohol
3min, 85% alcohol 3min, 75% alcohol 3min, 50% alcohol 3min, distilled water 3min, distilled water 3min, PBS 5min；It is interior
Source peroxide enzyme-deactivating, 3%H2O2/ PBS is incubated at room temperature 10min, and PBS rinses 5min, in triplicate；Slice is immersed into antigen
It repairs in liquid in (trisodium citrate buffer), 96 DEG C of 5min；Add 2mL PBS to wash in slice surface with 1mL pipettor, repeats
3 times, each 5min；10%FBS closing, room temperature 15min；Primary antibody, 4 DEG C of overnight incubations are added；It develops a film, is being cut with 1mL liquid-transfering gun
Piece surface adds 2mL PBS to wash, and is repeated 3 times, each 5min；100 μ are added in the 100 μ l pipettors of tissue part of slice
LDAB developing solution, develop the color 30min；Flowing water rinses 5min, and the tissue of 1 drop haematoxylin to slice is added dropwise, and acts on 2min, flowing water punching
Wash 5min；Microscopically observation is taken pictures:
TUNEL detection: by the dewaxing 30min of skin lesion tissue dimethylbenzene I, the dewaxing of dimethylbenzene II 10min；Graded ethanol rehydration
Afterwards, 1%Triton X-100 is added and carries out penetrating 5min, PBS is washed 3 times, each 5min；It prepares and 100 μ l is added dropwise containing difference
The DNase I reaction solution of active unit U, 37 degree of processing 30min；PBS is washed 3 times, each 5min；Prepare TdT enzyme reaction solution；Sample
It is blotted around this with blotting paper, 50 μ l TdT enzyme reaction solutions is added dropwise on each sample, are put into wet box, 37 degree are protected from light 1h；
Sample after reaction is washed 3 times with PBS, and each 5min pays attention to being protected from light；Streptavidin-Fluorescein reagent presses every sample
5 μ l dosages and 45 μ l Labeling Buffer are mixed, instant to match after calculating required total amount, pay attention to being protected from light；Cell culture
50 μ l Streptavidin-Fluorescein marking fluids are added dropwise in hole, are put into wet box, 37 degree are protected from light 30min；After reaction
It is washed 3 times with PBS, each 5min pays attention to being protected from light；Hoechst redyes nucleus, and room temperature, which is protected from light, is incubated for 10min；It washes away
Hoechst dye liquor, fluorescence microscopy are taken pictures under the microscope；
8-OXOdG Showed by immune group result (as shown in Figure 2 A): the expression of 1d each group is similar after modeling, shows each at this time
The DNA damage degree of group is similar；8-OXOdG expression of results prompts when 3d, control group > 2%H2Group > 75%H2Group；5d and 3d class
Seemingly；The 8-OXOdG expression of control group decreases when 7d, but still is significantly higher than hydrogen intervention group, 2%H2The expression of intervention group is bright
Aobvious decline, and 75%H2Group expression is minimum.TUNEL testing result is shown (as shown in Fig. 2 B&2C): three groups of Apoptosis when 1d
Rate is similar；The Apoptosis of 75%H2 intervention group slightly reduces when 3d；The apoptosis rate of hydrogen intervention group, which is substantially less than, when 5d compares
Group；Each group apoptosis rate further decreases when 7d, and hydrogen intervention group is substantially less than control group；
The result shows that hydrogen intervention significantly reduces the DNA damage and apoptosis rate in mouse skin lesion tissue, intervene effect
Fruit is closely related with density of hydrogen, best with 75% hydrogen effect.
3. hydrogen of embodiment promotes the antioxidant levels of pressure sore mouse by activation Nrf2 access
Experimental material: GPx (BioVision#K762-100), CAT (BioVision#K773-100), SOD (build up by Nanjing
A001-3), ROS.Trizol (invitrogen), DEPC handle water (Sigma), chloroform/isopropanol/dehydrated alcohol (Shanghai state
Medicine), SYBRGreen PCR kit (Thermo F-415XL), Reverse Transcriptase kit (Thermo#K1622).BCA albumen is fixed
It measures kit (Biosharp BL521A), pvdf film (millipore HATF00010), HRP marks secondary antibody (Zhong Shan Golden Bridge ZB-
5305)；
Cell incubator (Thermo Scientific 8000), optical microscopy (XDS-1A), microplate reader (DNM-
9602)；Real-time PCR (BioRad), warm refrigerated centrifuge (Sigma 3K15), Real-time detector (ABI-
7500).Electrophoresis apparatus (BIO-RAD, mini protean 3cell), microplate reader (Thermo, MK3), integrated chemical shines into
As instrument (ChemiScope 5300Pro)；
Experimental method: ELISA operates same specification；
Real-Time PCR: total serum IgE is stripped using Trizon extracts kit in organizing, and weighs each group 100mg skin
Skin tissue is added 1ml Trizol and is sufficiently homogenized, and is stored at room temperature 5min；0.2ml chloroform is added, acutely vibrates 15s, stands 3min；
4 DEG C of 12000rpm are centrifuged 10min, take supernatant；0.5ml isopropanol is added, mixes, stands 20-30min on ice；4℃12000rpm
It is centrifuged 10min, abandons supernatant；75% ethyl alcohol of 1ml, washing precipitating is added.4 DEG C of 7500g are centrifuged 5min, abandon supernatant；It is placed at room temperature for and dries in the air
5min or so is dried up in dry or super-clean bench, and suitable Rnase-free H2O dissolution is added；Using the first chain cDNA synthetic agent
Box synthesizes cDNA (the same specification of specific steps)；Synthetic primer, primer used in reverse transcription PCR is by Shanghai Sangon Biotech Company
5.0 software design of Primer Premier, synthesis, using house-keeping gene β-actin as internal reference；It is reacted according to Real time-PCR
System prepares reaction solution, carries out PCR amplification, 94 DEG C of 10min, (94 DEG C 20 seconds, 55 DEG C 20 seconds, 72 DEG C 20 seconds) 40 circulations；
Western blot: taking each group pressure sore mouse skin lesion tissue, RIPA lysate is added after homogenate, and be added suitable
PMSF is placed in and cracks 2h on ice；12000rpm, is centrifuged 10min by 4 DEG C；Supernatant is moved in new EP pipe, carries out quantification of protein
After be stored in -20 DEG C of refrigerators, with BCA protein quantification kit carry out protein quantification, prepare PAGE glue, by each histone sample
(20 μ g) carries out loading and electrophoresis (concentration glue 80V 20min, separation gel 120V 60min)；Dyestuff reaches glue bottom and stops electricity
Swimming carries out transferring film；With 5% skimmed milk power, room temperature closes 1h, sequentially adds primary antibody, secondary antibody is incubated for；It is washed 5 times with TBST,
Each 10min, integrated chemical light-emitting appearance shoot photo；
Experimental result is shown, tests and analyzes living radical molecule (ROS) water of 5d each group mouse skin lesion tissue after modeling
As the result is shown (as shown in Figure 3), the ROS level of control group is higher for flat and oxidoreducing enzyme expression, when giving hydrogen intervention
ROS level is decreased obviously afterwards, and the ROS decline of especially 75% hydrogen group is the most obvious；Superoxide dismutase (SOD), paddy Guang
Sweet fabk polypeptide (GPx) and catalase (CAT) are lower in the activity of control group, and the activity that hydrogen intervenes latter three is able to
It improves, three kinds of enzyme activity of same 75%H2 group increase most obvious；It is produced the result shows that hydrogen intervention significantly reduces ischemia-reperfusion
Raw oxygen free radicals, the activity of the antioxidases such as SOD, GPx and CAT is significant to be increased；
In the present embodiment, it is anti-oxidant regulatory pathway important in eukaryocyte based on Nrf2 access, passes through real-time quantitative
PCR and protein blot experiment further assess the Nrf2 access key protein expression in each group mouse skin lesion tissue (as schemed
Shown in 4), as the result is shown: the mRNA level in-site of NRF2, HO-1, AKR1C1 and NQO1 are minimum in control group, table after intervening with hydrogen
Up to rising, and there is concentration dependent, i.e., 75%H2 group increases most；NRF2 is the activity factor of Antioxidant responsive element,
HO-1 and NQO1 is the enzyme of antioxidation downstream, and oxidative damage level reduces after hydrogen is intervened, and oxidation resistant albumen is corresponding
Expression increases；
The experimental results showed that hydrogen intervention significantly increases the anti-oxidant of pressure sore mouse by the activation anti-oxidant access of Nrf2
Ability, intervention effect are positively correlated with density of hydrogen.
4. hydrogen intervention of embodiment can effectively reverse the proinflammatory microenvironment of ischemical reperfusion injury skin
Experimental material: IL-1 β, IL-6, TNF-α, the cell factors ELISA detection kit such as IL-8, IL-22 (Thermo
Fisher Scientific)；IL-1 β, IL-6, TNF-α, the cytokine antibodies such as IL-8, IL-22 (Invitrogen).Skin
Paraffin section, dimethylbenzene, 100% ethyl alcohol, 95% ethyl alcohol, 90 ethyl alcohol, 80% ethyl alcohol, 70 ethyl alcohol, 30% hydrogen peroxide, 10 ×
PBS phosphate buffer, citrate buffer, BSA, I-CAM1 antibody (Abcam), DAB developing solution (Zhong Shan Golden Bridge, ZLI-
9017), haematoxylin, neutral gum.TUNEL staining kit (Kai Ji, KGA7072)；
Inverted microscope (OLYMPUS, IX71), inversion are taken pictures microscope (Leica company IX71), pressure cooker (Haier),
Electric drying oven with forced convection (the rich news GZX-9240MBE in Shanghai), cold storage refrigerator (Haier), pipettor (Eppendorf).Cell culture
Case (Thermo Scientific 8000), optical microscopy (XDS-1A), microplate reader (DNM-9602)；
Experimental method: the methods of ELISA, western blot and immunohistochemistry are the same；
It is that the skin inflammation damnification that ischemical reperfusion injury causes repeatedly reacts based on pressure sore, in the present embodiment, uses
ELISA, western blot and ImmunohistochemistryMethods Methods comment the inflammatory cytokine after modeling in 5 days each group mouse skin lesion tissues
Estimate (as shown in Figure 5), as the result is shown: control group suppression inflammatory cytokines IL-22 concentration is relatively low, and proinflammatory cytokine IL-1
β, IL-6, TNF-α and IL-8 concentration are relatively high；IL-22 concentration rises after hydrogen is intervened, IL-1 β, IL-6, TNF-α and IL-8
Concentration decline, wherein the inflammatory factor expression variation of 75% hydrogen intervention group is the most obvious；
The result shows that hydrogen intervention can significantly inhibit the expression of proinflammatory cytokine IL-1 β, TNF-α, IL-6 and IL-8,
Promote the expression of suppression inflammatory cytokine IL-22, the significant proinflammatory microenvironment for reversing ischemia-reperfusion skin histology intervenes effect
It should be closely related with density of hydrogen.
5. hydrogen intervention of embodiment promotes the expression of wound repair albumen in ischemical reperfusion injury skin
Experimental material: BCA protein quantification kit (Biosharp BL521A), pvdf film (millipore
HATF00010), HRP marks secondary antibody (Zhong Shan Golden Bridge ZB-5305)；
Cell incubator (Thermo Scientific 8000), optical microscopy (XDS-1A), microplate reader (DNM-
9602)；Cryogenic freezing centrifuge (Sigma 3K15), Real-time detector (ABI-7500).Electrophoresis apparatus (BIO-RAD,
Mini protean 3cell), microplate reader (Thermo, MK3), integrated chemical luminescence imaging instrument (ChemiScope
5300Pro), real-time PCR (BioRad)；
Experimental method: real-time quantitative PCR and western blot method are the same；
The close phase of expression based on skin wound healing usually with the wound repairs factor such as VEGF, IGF-1, TGF-β
It closes, VEGF, IGF-1 and TGF-β play an important role to collagenous fibres synthesis, are wound inflammation advanced stages and repair the main of proliferation period
Acting factor, the present embodiment real-time quantitative PCR and western blot analysis detect the wound repair in each group mouse skin lesion tissue
Factor expression level (as shown in Figure 6), as the result is shown: the mRNA level in-site of the wound repairs factor such as VEGF, IGF-1, TGF-β is right
It is minimum according to organizing, it expresses and rises after intervening with hydrogen, and there is concentration dependent, is i.e. 75% hydrogen intervention group increases most aobvious
It writes；Metalloproteinases MMP9 is primarily involved in degradation basilar memebrane or fiber, and mRNA level in-site is in control group highest, after hydrogen is intervened
Expression is remarkably decreased, and the decline of high concentration hydrogen intervention group is the most obvious, and western blot result is completely the same with RT-PCR result；Knot
Fruit shows that in pressure sore mouse, hydrogen is intervened mainly by adjusting the wound repairs phases such as VEGF, IGF-1, TGF-β and MMP9
The expression of the factor is closed, the formation of wound granulation tissue is promoted, intervention effect is positively correlated with density of hydrogen.
Claims (8)
1. hydrogen is preparing the purposes in medical prevention and treatment product.
2. purposes according to claim 1, which is characterized in that the medical prevention and treatment product is to treat the preparation of pressure sore or set
It is standby.
3. purposes according to claim 1, which is characterized in that the preparation of the treatment pressure sore is with high concentration hydrogen 75%H2
For active constituent.
4. purposes according to claim 3, which is characterized in that the hydrogen intervention is conducive to ischemic and pays close attention to damaging skin again
The healing of ulcer.
5. purposes according to claim 3, which is characterized in that the hydrogen intervention reduce the oxidative damage of pressure sore tissue with
Level of Apoptosis.
6. purposes according to claim 3, which is characterized in that the hydrogen promotes pressure sore mouse by activation Nrf2 access
Antioxidant levels.
7. purposes according to claim 3, which is characterized in that the hydrogen intervention reverses ischemical reperfusion injury skin
Proinflammatory microenvironment.
8. purposes according to claim 3, which is characterized in that the hydrogen intervention promotes in ischemical reperfusion injury skin
The expression of wound repair albumen and the formation for promoting wound granulation tissue.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024078544A1 (en) * | 2022-10-11 | 2024-04-18 | 上海氢医医疗科技有限公司 | Hydrogen inhalation intervention for promotion of development of premature infants |
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| WO2024078544A1 (en) * | 2022-10-11 | 2024-04-18 | 上海氢医医疗科技有限公司 | Hydrogen inhalation intervention for promotion of development of premature infants |
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