CN114796279A - Application of Hypo-ABs in promotion of survival of overlength random skin flap - Google Patents
Application of Hypo-ABs in promotion of survival of overlength random skin flap Download PDFInfo
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Abstract
The invention discloses application of Hypo-ABs in promoting the survival of an overlength random flap. Wherein Hypo-Abs are cultured by mouse epithelial connective tissue cells in 10% fetal bovine serum DMEM low-sugar medium, 1% O2, 94% N2 and 5% CO2 oxygen-controlled incubator for 24h, followed by 0.5. mu.M stimulation with staurosporine (STS) for 12h, then cell debris residues are removed by differential centrifugation at 300g x 5mins, supernatant is removed at 2000g x 30mins, and pellet is resuspended in PBS. The method of use was an average injection of six sites in situ after flap lift at a concentration of 1 mg/ml. The data demonstrate that Hypo-ABs has significant effects in promoting the survival of the overlength involuntary flap, and in the course of treatment, has significant effects in eliminating edema, improving blood flow, and promoting microangiogenesis.
Description
Technical Field
The invention relates to a biological medicine, in particular to application of Hypo-ABs in promoting survival of an ultralong random skin flap.
Background
In recent years, casual skin flaps are common prosthetic skin flaps that are often used to repair skin defects caused by a variety of causes (e.g., trauma, congenital disease, cancer resection, diabetes). However, when the aspect ratio of the voluntary skin flap exceeds 1.5: 1, the poor blood supply at the far end of the voluntary skin flap causes necrosis of the voluntary skin flap to occur to different degrees, which brings great limitation to the clinical application of the voluntary skin flap.
Hypoxic apoptotic bodies (Hypo-ABs) are hypoxia-energized extracellular vesicles that have the ability to resist ischemic and Hypoxic necrosis. Based on the original ABs, hypoxia energization is given before apoptosis induction, so that the blast cells massively express anti-hypoxia genes, Staurosporine (STS) is subsequently given to induce apoptosis to generate ABs, and the Hypo-ABs is separated and purified after differential centrifugation. The hypoxia-energized ABs contains more anti-hypoxia capability on the original basis, and has better effect of promoting the survival of tissue cells in the environment of ischemia and hypoxia. However, no research has been conducted on whether Hypo-Abs can be used to promote survival of long random flaps.
Disclosure of Invention
In response to the deficiencies of the prior art, the present invention is directed to promoting survival of an overlength voluntary flap.
In order to achieve the purpose, the invention provides the following technical scheme: application of Hypo-ABs in promoting the survival of overlength skin flap at will.
Wherein, the Hypo-Abs with the concentration of 1mg/ml is injected in situ after the flap is lifted in the application process.
Wherein, the administration method is that six sites are averagely injected in situ by using a micro-injection needle after the flap is lifted.
Wherein the drug was formulated at a Hypo-ABs concentration measured by BCA method, diluted to a concentration of 1mg/ml with PBS.
Among them, Hypo-ABs is included for the reduction of edema in the promotion of the ultralong voluntary flap survival.
Among them, Hypo-ABs has an effect of promoting the blood flow improvement in the survival of an ultralong voluntary skin flap.
Among them, Hypo-ABs has an effect on angiogenesis in promoting the survival of an ultralong random flap.
Wherein the acquisition method of the Hypo-Abs comprises the following steps: after 24 hours of hypoxic culture in an incubator filled with nitrogen and without sugar, epithelial connective tissue cells were subjected to 0.5. mu.M stimulation with staurosporine (STS) for 12 hours, cell debris residues were removed by differential centrifugation at 300 g.times.5 mins, supernatant was removed at 2000 g.times.30 mins, and the pellet was resuspended in PBS.
Wherein the Hypo-Abs storage environment obtained is-80 ℃.
The invention has the beneficial effects that:
1. ABs with low oxygen endowing effect has obvious anti-anoxia effect.
2. Has obvious effect on the survival of the random skin flap.
3. Has better effect on eliminating edema in the survival process of the random skin flap.
4. Has obvious effect on improving blood flow in the survival process of the random skin flap.
5. Has obvious effect on the micro-angiogenesis in the survival process of the random skin flap.
Drawings
FIG. 1 is a scanning electron micrograph of Hypo-ABs;
FIG. 2 is Hypo-ABs marker identification;
FIG. 3 is a graph showing the comparison of the survival area of the skin flap in PBS group and in Hypo-ABs treatment group at 3 days and 7 days after operation;
FIG. 4 is a graph comparing the degree of edema of the skin flap in the PBS group and the Hypo-ABs treatment group at 7 days after surgery;
FIG. 5 is a graph comparing the perfusion rate of skin flaps in PBS group and Hypo-ABs treatment group after 7 days of operation;
FIG. 6 is a comparison of the number of cutaneous valve microvasculature in PBS group and Hypo-ABs treated group.
Detailed Description
The invention will be further described in detail with reference to the following examples, which are given in the accompanying drawings.
As shown with reference to figures 1-6,
Hypo-ABs is subjected to low-oxygen energization and stimulation induction, and is subjected to differential centrifugation and purification
Selecting a subcutaneous connective tissue cell line L-wnt3A, culturing in a 10% fetal bovine serum DMEM low-sugar culture medium, an oxygen-controlled culture box with 1% O2, 94% N2 and 5% CO2 for 24h, then giving 0.5 mu M of staurosporine (STS) for stimulation for 12h, removing cell debris residues by using a differential centrifugation method of 300g x 5mins, removing supernatant by using 2000g x 30mins, re-suspending the precipitate by using PBS, applying the precipitate as soon as possible at 4 ℃, or storing the precipitate for a long time at-80 ℃.
Scanning electron microscope
The culture medium was collected by centrifugation, Hypo-ABs was discarded, the medium was gently rinsed with PBS, the PBS was discarded and the cell suspension was blown open and fixed in a fixative at room temperature for 2 h. The fixed samples were rinsed 3 times for 15min each with 0.1M phosphate buffer PB (pH 7.4). 0.1M phosphate buffer PB (pH7.4) was incubated with 1% osmic acid in the dark at room temperature for 1-2 h. 0.1M PBS PB (pH7.4) was rinsed 3 times for 15min each. The tissue is dehydrated by sequentially adding 30-50-70-80-90-95-100% alcohol and isoamyl acetate for 15min each time. And then putting the sample into a critical point dryer for drying. And then, putting the sample tightly attached to the double-sided adhesive of the conductive carbon film on a sample table of an ion sputtering instrument for spraying gold for about 30 s. And finally, observing and acquiring the picture under a scanning electron microscope. (see FIG. 1)
Western blotting detection of surface markers
After extraction, 50ul RIPA cell lysate +1ul PMSF is added to each 100ug of Hypo-ABs and L-wnt3A cells, and the cells are lysed on ice for 30min, then the intensity of ultrasonic lysis is five, 5s x 3 times, and the interval is 5 s. The pellet was removed by centrifugation at 12000 for 30min and the supernatant was pooled to standard amounts of 20ug protein per 20ul volume after BCA assay protein concentration. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE), electrotransferred to polyvinylidene fluoride (PVDF) membrane, blocked with 5% skim milk, and detected overnight at 4 ℃ with the following antibodies: h3 (1: 1000), H2B (1: 1000), C1QC (1: 1000), C3B (1: 1000), GAPDH (1: 1000). After 2h incubation with secondary antibody, bands were visualized with ECL Plus kit. The intensity of each band was measured using Image Lab 3.0 software (Bio-Rad, Hercules, Calif., USA). (see FIG. 1)
Establishment of ischemia random skin flap model
60 healthy male C57BL/6 mice were selected and provided by the university of Wenzhou medical laboratory animal center, clean grade, SCXK [ ZJ ] 2005-. Mice were divided into 2 groups according to the random number table, 30 in the PBS control group, and 30 in the Hypo-ABs treatment group. A skin flap manufacturing method of improved McFlane is adopted, a 1% sodium pentobarbital is injected into an abdominal cavity to anaesthetize a mouse, an animal prone position is fixed, unhairing, iodophor disinfection and towel paving are carried out, a rectangular tail side random skin flap with the width of 1.5cm and the length of 4.5cm is designed in the middle of the back by taking the connecting line of two iliac crests at the tail of the mouse as a pedicle, the skin and subcutaneous tissues are cut along the designed line to reach the deep fascia shallow layer, a subdermal capillary network is reserved, the subdermal tissues are separated from the surface of the deep fascia shallow layer, and the skin flap is ligated by a 4-0 silk thread when a known blood vessel is met. After the flap is completely lifted, hemostasis is completely realized, and intermittent suture is immediately carried out by using 4-0 medical mousse suture. Three equal areas are divided on each flap: zone I (the end portion closest to the flap at random), zone II and zone III (the most distal). And sterilizing the periphery of the incision by iodophor. (see FIG. 1)
Hypo-ABs treatment group: 1mg/ml of Hypo-ABs was injected in situ with a microneedle evenly distributed over the skin flap.
PBS group: the same dose was injected in situ. Only once in the operation. In order to reduce the error brought by the operation, all the operations are completed by 1 person. (see FIG. 1)
Example 1 detection of the ratio of area of flap survived
On days 3 and 7 after the operation, the flatness was evaluated by high-quality photography. The surviving and ischemic regions were identified using Imag-Pro Plus imaging software. Percent surviving area was calculated as: [ (area surviving)/(total area surviving ischemic area) ] x 100%. After 3 days of operation, there was no obvious necrosis of each group of skin flap at the distal end (zone III) of the flap, but edema and pallor appeared. There was no qualitative difference between the experimental groups. After 7 days of operation, although the proximal end of each flap group (zone I) survived significantly, necrosis signs such as darkening, hardening, scabbing began to appear in zone III, and some similar phenomena appeared in the flap in the middle zone (zone II).
The survival rate of the Hypo-ABs treated group was significantly better than that of the PBS group. The average survival areas of the Hypo-ABs treatment group and the PBS group were (67.00. + -. 3.28)% and (40.37. + -. 2.70)%, respectively. Comparison of the ratio of the area of flap survival between the Hypo-ABs treated group and the PBS group revealed that the difference was statistically significant (p < 0.01) (see FIG. 2)
Example 2 detection of edema level contrast
7 days post-surgery, skin flaps of 6 animals per group were removed and weighed to obtain a "wet weight". The skin flap was dehydrated in a high pressure vessel at 50 degrees celsius, stabilized for 2 days, and weighed again to obtain "dry weight". The edema level was calculated as percent water content for 7 days post-surgery as follows: ([ wet weight-dry weight ]/wet weight) × 100%.
Both the PBS and Hypo-ABs treatment groups had significant edema and subcutaneous venous stasis, while the Hypo-ABs treatment group was milder. The water content of the free flap of the Hypo-ABs treated group and the PBS group was (40.33. + -. 2.60)% and (54.67. + -. 2.91)%, respectively, and the difference was statistically significant (p < 0.05). (see FIG. 3)
Example 3 measurement of neovasculature by laser Doppler rheometer
After 7 days of operation, 6 mice per group were anesthetized for blood flow measurement and scanned with a laser doppler. Blood flow was quantified using a perfusion apparatus and calculated using the Moor LDI audit software. Each animal was scanned and measured 3 times and the mean was used for further statistical analysis.
The Hypo-ABs treated group showed improved blood flow, and the quantitative blood flow measurements were significantly improved compared to the PBS group. The blood flow perfusion rates of the Hypo-ABs treatment group and the PBS group were (379.36. + -. 12.36) PU and (231.76. + -. 18.29) PU, respectively. The differences were statistically significant (p < 0.01) when comparing the Hypo-ABs treatment group to the PBS group. (see FIG. 4)
Example 4 hematoxylin and eosin staining (H & E) Observation of the number of skin flap microvasculature
After 7 days of surgery, animals were euthanized and 6 tissue specimens of 1cm × 1cm were taken from flap II area (middle position) per group.
Tissues were soaked in 4% paraformaldehyde for fixation, dehydrated and paraffin embedded, specimens were cross-sectioned to make 4 μm sections, and sections mounted on poly-L-lysine coated sections were H & E stained. Under a 200-fold optical microscope, 6 regions of each group of sections were selected and the number of microangioses per unit area (/ mm2) was calculated to quantify the vascular density.
In terms of angiogenesis, the Hypo-ABs treated group had significantly higher microvascular density than the PBS group. The number of microvessels in the Hypo-ABs treatment group and PBS group were (283.33. + -. 13.02)/mm2 and (158.33. + -. 14.53)/mm2, respectively. The difference was statistically significant (p < 0.01) in comparison between the Hypo-ABs treatment group and the PBS group. (see FIG. 5)
Example 5 immunofluorescence Observation
Angiogenesis is a key factor determining the survival of the skin flap, and by detecting the distribution of CD34/ACTA2 in the skin flap, we explored whether the pro-survival effect of Hypo-ABs on the skin flap is related to endogenous angiogenesis. The paraffin sections were dewaxed with xylene and subjected to a graded ethanol bath. The sections were placed in sodium citrate buffer for antigen retrieval at 95 ℃ for 20min and blocked with 10% (w/v) bovine serum albumin phosphate buffer for 10 min. Finally, the sections were incubated with the antibody at a temperature of 4 ℃. The primary antibody was incubated at room temperature for 2h for primary staining, followed by anti-rabbit secondary antibody DAPI staining for 1h, and incubated at 37 ℃. Cells in the specimen were observed with a fluorescence microscope and a DP2-TWAN image acquisition system (Olympus, Tokyo, Japan). 6 areas of uniform staining were randomly selected from 3 sections, and skin flap tissue was imaged at 100-fold magnification using DP2-TWAN Image acquisition system, and positive vascular expression of CD34/ACTA2 was assessed using Image-ProPlus. (see FIG. 6)
The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.
Claims (9)
- Use of Hypo-ABs for promoting the survival of an ultralong voluntary flap.
- 2. The use according to claim 1, wherein the Hypo-Abs is administered in situ after lifting of the flap by injection at a concentration of 1mg/ml during use.
- 3. The use according to claim 2, wherein the administration is by average injection of six sites in situ using a microneedle after flap up.
- 4. The use according to claim 1, 2 or 3, wherein the medicament is formulated for a Hypo-ABs concentration as measured by BCA method, diluted to a concentration of 1mg/ml with PBS.
- 5. The use as claimed in claim 1, comprising Hypo-ABs for the reduction of oedema in promoting the survival of an ultralong voluntary flap.
- 6. The use as claimed in claim 1, comprising the effect of Hypo-ABs on blood flow improvement in promoting the survival of an ultralong voluntary flap.
- 7. The use according to claim 1, characterized by comprising the effect of Hypo-ABs on angiogenesis in promoting the survival of an ultra-long voluntary flap.
- 8. The use according to claim 1, wherein the Hypo-Abs is obtained by a method comprising: after 24 hours of hypoxic culture in a sugar-free medium filled with nitrogen, epithelial connective tissue cells were subjected to 0.5. mu.M stimulation with Asterosporine (STS) for 12 hours, cell debris residues were removed by differential centrifugation at 300 g.times.5 mins, supernatants were removed at 2000 g.times.30 mins, and the pellet was resuspended in PBS.
- 9. The use according to claim 8, wherein the Hypo-Abs storage environment is obtained at-80 degrees celsius.
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| US20210244700A1 (en) * | 2018-05-15 | 2021-08-12 | Duke University | Compositions and methods for preventing and/or reducing ischemia after surgical incisions |
| CN113876693A (en) * | 2021-10-25 | 2022-01-04 | 鸳鸯故里(屏南)生态农业科技有限公司 | Pilose antler polypeptide monomer gel preparation for promoting wound healing and preparation method thereof |
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| US20210244700A1 (en) * | 2018-05-15 | 2021-08-12 | Duke University | Compositions and methods for preventing and/or reducing ischemia after surgical incisions |
| CN113876693A (en) * | 2021-10-25 | 2022-01-04 | 鸳鸯故里(屏南)生态农业科技有限公司 | Pilose antler polypeptide monomer gel preparation for promoting wound healing and preparation method thereof |
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