CN110179807A - Application of the trehalose in the drug that preparation promotes ultra long random flap survival - Google Patents
Application of the trehalose in the drug that preparation promotes ultra long random flap survival Download PDFInfo
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- CN110179807A CN110179807A CN201910413999.5A CN201910413999A CN110179807A CN 110179807 A CN110179807 A CN 110179807A CN 201910413999 A CN201910413999 A CN 201910413999A CN 110179807 A CN110179807 A CN 110179807A
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Abstract
The invention discloses application of the trehalose in the drug that preparation promotes ultra long random flap survival.The concrete application of trehalose is dissolved in 1 milliliter of physiological saline by every 1 gram of trehalose by the way of intraperitoneal injection, is configured to 1g/ml solution, and application method is daily intraperitoneal injection dosage 2g/kg, and preoperative 12 to postoperative 7 days as one therapeutic course.It is drawn a conclusion by a large amount of zoopery, the survival area ratio for using trehalose that can make flap significantly improves, and can promote the survival of overlength ischemic Survival of Random Flap well.
Description
Technical field
The present invention relates to the new applications of trehalose, and in particular to trehalose promotes the medicine of ultra long random flap survival in preparation
Application in object.
Background technique
In recent years, with the fast development of the industries such as industry, traffic and building, the damage of large area and tissue defect
Patient is more common, and patient is after debridement for this type, often will appear skin tissue defects, can not directly be sutured, and needs
Flap is wanted to cover.And Survival of Random Flap is taken conveniently because its is flexible in application, is restored the advantages that good and is widely used in clinic.So
And when the length-width ratio of Survival of Random Flap is more than 1.5:1, distal end blood supply is bad to make it different degrees of necrosis occur.
This undoubtedly brings very big limitation to the application of Survival of Random Flap clinically, therefore how to increase the survival of Survival of Random Flap, reduces
Flap necrosis, mono- important project of Cheng Liao.After after study, promotes the formation of Survival of Random Flap capilary, increase the blood of flap
Perfusion improves tissue edema, and alleviating oxidative stress etc. is the effective therapy target of survival for promoting Survival of Random Flap.
Trehalose is a kind of non-reducing disaccharide, is made of two glucose molecules, by some bacteriums, fungi, plant, nothing
Vertebrate synthesis.Recent studies have found that trehalose has multiple biological activities: trehalose can induce the activation of cell autophagy,
To promote heart reconstruction after myocardial ischemia infarct；In addition, under the conditions of trehalose can be by inhibiting oxidative stress to inhibit Anoxia
Nerve cell death, treat neurodegenerative disease；However, there is presently no can influence Survival of Random Flap survival about trehalose
Report.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides the drugs that trehalose promotes ultra long random flap to survive in preparation
In application.The concrete application of trehalose is dissolved in 1 milliliter of physiological saline by every 1 gram of trehalose by the way of intraperitoneal injection,
It is configured to 1g/ml solution, application method is daily intraperitoneal injection dosage 2g/kg, preoperative 12 to postoperative 7 days as one therapeutic course.
It is drawn a conclusion by a large amount of zoopery, the survival area ratio for using trehalose that can make flap obviously mentions
Height can promote the survival of overlength ischemic Survival of Random Flap well.
Detailed description of the invention
Fig. 1 is the Flap survival comparison diagram of postoperative 3 days trehalose groups, physiological saline group, sucrose group；
Fig. 2 is the Flap survival comparison diagram of postoperative 7 days trehalose groups, physiological saline group, sucrose group；
Fig. 3 is the Flap survival column comparison diagram of postoperative 7 days trehalose groups, physiological saline group, sucrose group；
Fig. 4 is the flap oedema degree comparison diagram of postoperative 7 days trehalose groups, physiological saline group, sucrose group；
Fig. 5 is the flap oedema degree column comparison diagram of postoperative 7 days trehalose groups, physiological saline group, sucrose group；
Fig. 6 is the flap blood perfusion amount comparison diagram of postoperative 7 days trehalose groups, physiological saline group, sucrose group；
Fig. 7 is the flap blood perfusion amount column comparison diagram of postoperative 7 days trehalose groups, physiological saline group, sucrose group；
Fig. 8 is the flap microvessel quantity comparison diagram of trehalose group, physiological saline group, sucrose group；
Fig. 9 is the flap microvessel quantity column comparison diagram of trehalose group, physiological saline group, sucrose group；
Figure 10 is the vascular endothelial growth factor expression amount comparison diagram of trehalose group, physiological saline group, sucrose group；
Figure 11 is the vascular endothelial growth factor expression amount column comparison diagram of trehalose group, physiological saline group, sucrose group；
Figure 12 is the SOD1 expression quantity comparison diagram of trehalose group, physiological saline group, sucrose group；
Figure 13 is the SOD1 expression quantity column comparison diagram of trehalose group, physiological saline group, sucrose group；
Figure 14 is the HO1 expression quantity comparison diagram of trehalose group, physiological saline group, sucrose group；
Figure 15 is the HO1 expression quantity column comparison diagram of trehalose group, physiological saline group, sucrose group；
Figure 16 is the eNOS expression quantity comparison diagram of trehalose group, physiological saline group, sucrose group；
Figure 17 is the eNOS expression quantity column comparison diagram of trehalose group, physiological saline group, sucrose group.
Specific embodiment
Trehalose can significantly improve the viability of overlength ischemic Survival of Random Flap.In the mistake for promoting the survival of ischemic Survival of Random Flap
In journey by the way of intraperitoneal injection, it is dissolved in 1 milliliter of physiological saline by every 1 gram of trehalose, is configured to 1g/ml solution, uses
Method is that every kilogram of mouse is injected intraperitoneally 2g trehalose daily, preoperative 12 to postoperative 7 days as one therapeutic course.The following are trehaloses to promote
Into ischemic Survival of Random Flap survive in evaluation of effect:
1, ischemic Survival of Random Flap model foundation
Male C57BL/6 mouse 90 for selecting health, are provided, cleaning grade, SCXK by Wenzhou Medical University's Experimental Animal Center
[ZJ] 2005-0019, weight 20-30g.Mouse is divided into 3 groups according to table of random number hair, saline control group 30, sugarcane
Sugared control group 30, trehalose experimental group 30.Using the flap production method of improvement McFlane, 1% penta bars of ratios of intraperitoneal injection
Appropriate sodium anesthetized mice, animal prone position is fixed, depilation, iodophor disinfection, drape, using two interiliac line of rat tails as the base of a fruit,
One width 1.5cm of back center design, long 4.5cm rectangle caudal random pattern skin flap, design lines cut skin, subcutaneous tissue, up to deeply
Fascia shallow-layer retains Subdermal vascular plexus net, separates subcutaneous tissue in deep fascia shallow-layer surface, encounters well-known blood vessel i.e. with 4-
The ligation of 0 silk thread.After flap starts completely, thoroughly stops blooding, admire silk suture interrupted suture with 4-0 is medical immediately.On each flap
It is divided into three equal regions: the area I (closest to the end section of Survival of Random Flap), the area II and the area III (distalmost end).Notch
Surrounding iodophor disinfection.
Sucrose group: intraperitoneal injection 2g/kg sucrose；Trehalose group: intraperitoneal injection trehalose 2g/kg；Physiological saline group: identical
Dosage intraperitoneal injection.One time a day, by preoperative 12 days to postoperative 7 days, continuous injection 19 days.To reduce surgical procedure bring
Error, all operations are completed by 1 people.
2, observation index
The detection of 2.1 Flap survival ratios
Postoperative 3rd day and the 7th day, its flatness was evaluated with high quality camera work.Known using Imag-Pro Plus imaging software
It Cun Huo not area and ischemic region.Area percentage of surviving calculates are as follows: [(survival area)/(the survival ischemic gross area)] x 100%.Art
3 days afterwards, without obvious necrosis, but there is oedema, pale in flap distal end (IIIth area) in each group flap.Indefiniteness is poor between test group
It is different.Postoperative 7 days, although each group flap proximal end (Ith area) obviously survives, IIIth area started the necrosis sign such as dark-coloured, hardening, incrustation occur
As some similar phenomenons also occur in the flap in intermediate region (area II).
The survival rate of trehalose group is substantially better than physiological saline group and sucrose group, and without aobvious between physiological saline group and sucrose group
Write difference.Trehalose group, physiological saline group, sucrose group the average survival time area be respectively (69.47 ± 3.35) % and (56.34 ±
2.93) % and (54.27 ± 2.67) %.Compare the Skin Flaps face of trehalose group and physiological saline group, trehalose group and sucrose group
Product ratio, as shown in Figs. 1-3.
The detection of 2.2 oedema degree comparison
Postoperative 7 days, the flap of every group of 6 animals was picked-off and is weighed with acquisition " weight in wet base ".Flap is placed on 50 degrees Celsius
It is dehydrated in high-pressure bottle, stablizes 2 days, weigh again, obtained " dry weight ".Oedema journey is calculated with postoperative 7 days percentage of water content
Degree, calculation formula are as follows: ([weight in wet base-dry weight]/weight in wet base) × 100%.
Physiological saline group and sucrose group have apparent oedema and venae subcutaneae extravasated blood, and trehalose group is then relatively light.Seaweed
The water content of sugar group is lower than physiological saline group and sucrose group, and then without significant difference between physiological saline group and sucrose group.3 groups random
Flap water content is respectively (39.90 ± 2.70) % and (54.57 ± 2.94) %, respectively (50.69 ± 2.47) %, compares seaweed
Sugar group and physiological saline group, the water content of trehalose group and sucrose group, as illustrated in figures 4-5.
2.3 laser Doppler flowmetries measure new vessels
Postoperative 7 days, to measure blood flow, by every group of 6 mouse anesthesias, Laser doppler seanning was scanned.Use device for casting
Blood flow is quantified, Moor LDI censorware is calculated.Every animal is scanned and measures 3 times, and average value is used for into one
The statistical analysis of step.
Trehalose group improving blood flow, quantitative blood flow measurements have compared with physiological saline group and sucrose group obviously to be changed
It is kind, without significant difference between physiological saline group and sucrose group.Trehalose group, physiological saline group, sucrose group blood perfusion amount are respectively
(402.95 ± 20.62) PU and (282.20 ± 23.91) PU and (266.48 ± 20.26) PU.Compare trehalose group and
The blood perfusion amount of physiological saline group, trehalose group and sucrose group, as shown in fig. 6-7.
2.4 h and Es dye (H&E) and observe flap microvessel quantity
Postoperative 7 days, for animal after euthanasia, every group took 6 1cm × 1cm tissue specimens from IIth area of flap (middle position).By group
It knits to be immersed in and be fixed in 4% paraformaldehyde, it is after paraffin embedding, sample is crosscutting, 4 μm of slices are made, rely poly- L- is mounted on
The slice of propylhomoserin coating carries out H&E dyeing.Under 200 times of optical microscopies, 6 regions of every group of slice, unit of account are chosen
Area number of microvessels (/mm2), to quantify vessel density.
In terms of angiogenesis, trehalose group microvessel density is apparently higher than physiological saline group and sucrose group, physiological saline
Group is compared with sucrose group without significant difference.Algae sugar group, physiological saline group, sucrose group microvessel quantity be respectively (263.63 ±
35.89)/mm2 and (168.65 ± 12.67)/mm2 and (178.49 ± 19.94)/mm2, as Figure 8-9.
2.5 immunohistochemical observation
Angiogenesis is the key factor for determining survival length of flap, through detection vascular endothelial growth factor (VEGF) in flap
Distribution, whether we explore trehalose related with endogenous angiogenesis to the rush survival effect of flap.By above-mentioned paraffin section
It is separated with dimethylbenzene, and immunohistochemical analysis is carried out by classification ethanol bath.With 3% (v/v) H2O2 after slice is clean
Closing.Slice is placed in sodium citrate buffer solution 20 min under 95 DEG C of environment, slow with 10% (w/v) bovine serum albumin(BSA) phosphate
10 min of fliud flushing.Finally, 4 DEG C at a temperature of be sliced with antibody incubation.Slice is incubated for HRP combination secondary antibodies, hematoxylin
Dyeing, resin mounting.6 even dyeing regions for randomly choosing 3 groups of slices, using DP2-TWAN image capturing system to flap
Tissue has carried out 200 times of amplification imagings, evaluates vegf expression index using Image-Pro Plus.
In trehalose group, expression of the VEGF in flap II area's vascular endothelial cell and stroma cell is significantly higher than physiology
Salt water group and sucrose group.Compare 3 groups of vegf expression amounts, as shown in figs. 10-11.
2.6 immunoblottings (Western blotting)
It plays a major role in survival length of flap in view of oxidative stress, we have also investigated trehaloses whether to adjust oxidative stress.Art
7 days afterwards, 6 0.5cm × 0.5cm skin samples are chosen from IIth area middle part, sarcolemma layer is removed, claims quality, dilute, made in ice-water bath
At 10% tissue homogenate of volume fraction, supernatant, BCA kits each group sample total protein concentration are taken after centrifugation.By egg
White sample is uniformly mixed with 5 times of SDS-PAGE albumen sample-loading buffers, and hot bath heating makes albuminous degeneration.It is solidifying through SDS-PAGE row
Gel electrophoresis turns pvdf membrane, and confining liquid is added, and closes 2h.Right SOD1 to be given, 4 DEG C of eNOS and HO1 primary antibody slowly shake incubation
Overnight, after washing film, secondary antibody is incubated at room temperature 2h, washes film, is placed in darkroom exposure, development, analyzes development item using Image J software
Band, with the relative expression quantity of destination protein and GAPDH band gray value ratio albumen as a purpose.
Trehalose group SOD 1 is horizontal to be higher than physiological saline group and sucrose group, and physiological saline group and sucrose group SOD 1 express nothing
Difference (p> 0.05).Compared with physiological saline group and sucrose group, trehalose group HO-1 and eNOS protein level is also significantly raised, and
Without significant difference between physiological saline group and sucrose group, as shown in Figure 12-17.
By above-mentioned control experiment it can be concluded that
1, trehalose can promote the survival of ultra long random flap；
2, the mechanism of trehalose promotion survival length of flap may be by promoting the expression of VEGF, to promote the increasing of new vessels
It is raw, it is related to increase blood flow；
3, trehalose promotes the mechanism of survival length of flap may be related with oxidative stress is inhibited.
Claims (1)
1. application of the trehalose in the drug that preparation promotes ultra long random flap survival.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1973828A (en) * | 2006-11-10 | 2007-06-06 | 中国人民解放军第二军医大学 | VEGF slowly releasing injection microsphere support and its prepn and use |
| CN108685948A (en) * | 2018-05-30 | 2018-10-23 | 北京壹典壹生生物技术有限公司 | A kind of preparation method of new medical cell repair agent |
-
2019
- 2019-05-17 CN CN201910413999.5A patent/CN110179807A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1973828A (en) * | 2006-11-10 | 2007-06-06 | 中国人民解放军第二军医大学 | VEGF slowly releasing injection microsphere support and its prepn and use |
| CN108685948A (en) * | 2018-05-30 | 2018-10-23 | 北京壹典壹生生物技术有限公司 | A kind of preparation method of new medical cell repair agent |
Non-Patent Citations (9)
| Title |
|---|
| A. K. KITAHARA ET AL: "Evaluation of new improved solution containing trehalose in free skin flap storage", 《BRITISH JOURNAL OF PLASTIC SURGERY》 * |
| HONGQIANG WU ET AL: "Trehalose promotes the survival of random-pattern skin flaps by TFEB mediated autophagy enhancement", 《CELL DEATH AND DISEASE》 * |
| MICHELA PALMIERI ET AL: "mTORC1-independent TFEB activation via Akt inhibition promotes cellular clearance in neurodegenerative storage diseases", 《NATURE COMMUNICATIONS》 * |
| 侯春林主编: "《皮瓣外科学》", 31 January 2013, 上海科学技术出版社 * |
| 曾炳芳主审: "《创伤骨科软组织治疗手册》", 31 October 2013, 山东科学技术出版社 * |
| 金鑫等: "海藻糖抑制短暂缺氧性神经损伤的体外研究", 《SHENZHEN JOURNAL OF INTEGRATED TRADITIONAL CHINESE AND WESTERN MEDICINE》 * |
| 钟剑平主编: "《中国外科年鉴（2008）》", 31 March 2008, 第二军医大学出版社 * |
| 闫迎军等: "皮瓣缺血再灌注损伤的药物治疗进展", 《中国误诊学杂志》 * |
| 齐战等: "海藻糖在器官组织保存中的应用", 《国外医学呼吸系统分册》 * |
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