LU508779B1 - The application of SCTCs-ABs in promoting the survival of ultra-long random skin flaps - Google Patents

Abstract

The present invention relates to the field of biomedical technology, particularly to the application of SCTCs-ABs in promoting the survival of ultra-long random skin flaps. At a concentration of 1 mg/ml, dorsal skin flaps were lifted and injected in situ in C57BL6 mice: 8-week-old males weighing approximately 20 g. The dorsal skin flaps were then raised and injected in situ with only one treatment. The beneficial effects of the SCTCs-ABs proposed in the present invention for promoting the survival of extra-long skin flaps are: SCTCs-ABs have a clear anti-ischemic necrosis effect of skin flaps, have a clear effect on improving skin flap blood flow, have a better effect on promoting collagen repair during skin flap survival, and have a good effect on promoting skin flap microangiogenesis. Figure 1

Description

The application of SCTCs-ABs in promoting the survival of ultra-long LU508779 random skin flaps

Technical area

The present invention relates to the field of biomedical technology, particularly to the application of SCTCs-ABs in promoting the survival of ultra-long random skin flaps.

Technology in the background

In recent years, the random flap has become a common reconstruction flap used to repair skin defects caused by various reasons (e.g., trauma, congenital diseases, cancer resection, diabetes mellitus).

However, if the length to width ratio of the flap exceeds 1.5:1, the distal end of the

The flap is poorly perfused, leading to varying degrees of necrosis, which severely limits its clinical application. Prevention and treatment of ischemic necrosis at the distal end of the randomization flap is key to successful surgery.

However, current treatment methods are not ideal. Recent studies have shown that apoptotic bodies (ABs) produced in neighboring tissues can be taken up by the surrounding cells and

promote cell proliferation. This suggests that ABs derived from skin connective tissue cells (SCTCs) may have great therapeutic potential. Administration of staurosporine (Staurosporine, STS) triggered apoptosis to

to produce SCTCs-ABs, which were isolated and purified after differential centrifugation. It was also found that SCTCs-ABs improved blood flow,

Promoted collagen repair and microangiogenesis. Furthermore, there are no reports that SCTCs-ABs can affect the survival of random skin flaps.

Therefore, the role of SCTCs-ABs in promoting survival over longer

lobe. For this reason, it is necessary to conduct a study on the role of

To propose SCTCs-ABs in promoting the survival of ultra-long random skin flaps.

Content of the invention

The aim of the present invention is to provide the application of SCTCs-ABs to promote the survival of ultra-long skin flaps in order to solve the above-mentioned problems of the prior art, with the aim of promoting the survival of ultra-long skin flaps.

To achieve the above-mentioned goal, the present invention provides the following technical solution: the application of SCTCs-ABs in promoting the survival of ultra-long random skin flaps is used as a single treatment at a concentration of 1 mg/ml in C57BL6 mice: males, 8 weeks old, weighing about 20 g, dorsal flaps raised and injected in situ only once.

Preferably, the SCTCs-ABs are epithelial connective tissue cells of

Mice cultured in a high-glucose medium. After 12 hours

Stimulation by administration of Astrocystin STS 0.5 uM, the cell trimmers are removed by differential centrifugation at 300 g*5 minutes and the supernatant at 2000 g*30 minutes, and the precipitate is resuspended in PBS and applied as soon as possible at 4 °C or kept at -80 °C for long-term storage.

Preferably, the drug is configured as a concentration of SCTCs-ABs-U508779 which is measured with BCA hair and diluted with PBS to a concentration of 1 mg/ml.

Preferably, the active ingredient is applied in situ at an average of 8 sites with a

microinjection needle after the dorsal skin flap was raised in mice.

Preferably, the role of SCTCs-ABs in promoting the viability of ultra-long, arbitrary flaps to improve blood flow will be investigated.

Preferably, the role of SCTCs-ABs in promoting collagen repair is included in promoting the viability of ultra-long, irregular skin flaps.

Preferably, the role of SCTCs-ABs in promoting collagen repair is

Promoting the viability of ultra-long random lobes included.

The advantageous effect of the present invention compared to the prior art is:

The present invention proposes the application of SCTCs-ABs in the promotion of

survival of ultra-long random skin flaps, through which SCTCs-ABs have obvious anti-ischemic necrosis effect of random skin flaps, obvious effect on improving blood flow of random skin flaps, better effect on promoting collagen repair during survival of random skin flaps, and good effect on promoting microangiogenesis of random skin flaps.

Description of the attached drawings

Figure 1 shows the scanning electron microscopy and marker identification of SCTCs-ABs of the present invention;

Figure 2 shows the comparison of the flap survival area in the control group, the

PBS group and SCTCs-ABs treatment group 7 days after the operation of the present invention;

Figure 3 shows a comparison of the blood perfusion volume in the control group, the

PBS group and SCTCs-ABs treatment group 7 days after the operation of the present invention;

Figure 4 shows a comparison of the promotion of collagen repair in the control group, the PBS group and the SCTCs-ABs treatment group 7 days after the operation of the present invention;

Figure 5 shows a comparative graph of the promotion of microvascular improvement in the control group, the PBS group and the SCTCs-ABs treatment group 7 days after

Operation of the present invention.

Detailed description

In order to more clearly understand the purpose of the present invention, the technical solutions for a clear and complete description, and the advantages, the following

Embodiments of the present invention in detail in conjunction with the attached

drawings. It should be understood that the specific

Embodiments described herein are part of the embodiments of the present

Invention, not all of the embodiments, only for the explanation of the embodiments of the present invention, and is not used to limit the embodiments of the present invention, all other embodiments by the normal

skilled in the art without any creative work under the premise of the protection4U508779 of the scope of the present invention.

Embodiment 1

The present invention offers a technical solution: the application of

SCTCs-ABs in promoting the survival of ultra-long random skin flaps under

Using a concentration of 1 mg/ml in C57BL6 mice: male, 8 weeks old, weighing approximately 20 g, dorsal lobes were raised and injected in situ for a single treatment. SCTCs-ABs were mouse epithelial connective tissue cells cultured in high-glucose medium and stimulated by the administration of 0.5 μM Astrocystin STS for 12 hours. Cellular residues were determined by differential

Centrifugation at 300g*5min, and the supernatant was separated at 2000g*30min, the precipitate was resuspended in PBS and applied as soon as possible at 4°C or stored for longer periods at -80°C.

The drug was configured as a concentration of SCTCs-ABs that

BCA hair was measured and diluted with PBS to a concentration of 1 mg/ml.

The method of administration consisted of treating an average of 8 sites in situ with a

microinjection needle after the dorsal lobe of the mice was raised.

Embodiment 2

Starting from embodiment 1, the following is proposed:

Stimulation of SCTCs induced the production of ABs, which were mediated by differential

centrifugation were isolated and purified.

The subcutaneous connective tissue cell line L-wnt3A was selected, cultured in DMEM medium with high glucose content (10% fetal bovine serum) and incubated for 12 hours with 0.5 uM

Stellatospondin (STS). The remaining cell trimmers were stimulated by differential

Centrifugation at 300 g * 5 minutes, and the supernatant was centrifuged at 2000 g * 30

minutes; the precipitate was resuspended in PBS and either applied as quickly as possible at 4 °C or stored at -80 °C for a longer period.

Scanning electron microscopy: Centrifugation to collect the SCTCs-ABs after

Discard the medium and rinse gently with PBS, discard PBS plus

Electron microscope fixative and inflating the cells suspended in the fixative, which were fixed at room temperature for 2 hours. The fixed samples were rinsed three times with 0.1 M phosphate buffer PBS (pH 7.4) for 15 minutes each.

PBS (pH 7.4) containing 1% osmic acid was fixed at room temperature and protected from light for 1 to 2 hours. 0.1 M phosphate buffer PBS (pH 7.4) was rinsed three times for 15 minutes each. The tissues were dehydrated sequentially for 15 minutes each in 30%-50%-70%-80%-90%-95%-100%-100% alcohol and for 15 minutes in isoamyl acetate. The samples were then placed in a critical point dryer for drying. The samples were then attached to double-sided conductive tape.

A carbon film was glued and placed in the sample stage of the ion sputtering device for approximately 30 seconds for gold spraying. Finally, the sample was examined under a scanning electron microscope. (See Figure 1)

Western blotting for the detection of surface markers: extracted SCTCs-ABs and

SCTCs were treated with 50ul RIPA cell lysate + 1ul PMSF per 100ug, incubated for 30 minutes on

Lysed on ice and then lysed five times 5s*3 times with Ss interval using ultrasound.

Centrifuge at 12000 rpm for 30 minutes to remove the precipitate, and the

Supernatant was prepared in a standard amount of 20ug protein per 20ul volume according to BCA for did 508779

Protein concentration was distributed. The proteins were

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and

Electrotransfer to a polyvinylidene difluoride (PVDF) membrane. The membranes were sealed with 5% skim milk and incubated overnight at 4°C with the following

Antibodies detected: H3 (1:1000), H2B (1:1000), CIQC (1:1000), C3B (1:1000),

Capase-3 (CASP-3, 1:1000), B-actin (1:1000). After 2 hours of incubation with secondary

The bands were visualized using the ECL Plus kit. The intensity of the individual bands was measured using Image Lab 3.0 software (Bio-Rad, Hercules, CA, USA). (See Figure 1)

Preparation of an ultra-long skin flap model randomly: 90 healthy male C57BL/6 mice weighing 20-30 g were obtained from the Experimental Animal

Centre of Wenzhou Medical University, SCXK[ZJ]2005-0019. The

Mice were divided into three groups according to the random number table: control group, 30

Mice in the PBS control group and 30 mice in the SCTCs-ABs treatment group.

Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital, in

Fixed in the prone position, depilated, disinfected with iodophor and dried, and in the middle of the

A rectangular caudolateral random-type flap with a width of 1.5 cm and a length of 4.5 cm was created on the back, with the connecting line between the two

The iliac crests of the tail of the mice were used as tibias. The skin and subcutaneous tissue were incised along the design line to create the superficial

layer of the deep fascia, the subcutaneous capillary network was preserved, and the subcutaneous tissue was separated from the superficial surface of the deep fascia, encountering known blood vessels, which were ligated with 4-0 silk sutures. After the

Once the skin flap was fully raised, the bleeding was completely stopped and the

The skin flap was immediately closed with interrupted 4-0 medical mousse sutures.

Each flap was divided into three equal zones: Zone I (the caudal part closest to the loose flap), Zone II and Zone III (the most distal

part), and the peri-incision area was sterilized with iodine-povidone (see Figure 1).

SCTCs-ABs treatment group: 1 mg/ml SCTCs-ABs was injected in situ using a microinjection needle and evenly distributed over the flap; PBS group: the same dose was injected in situ; Control group: only modeling was performed without any

Treatment was performed. The injection was given only once intraoperatively. To reduce surgical errors, all procedures were performed by a

person. (see Figure 1).

Embodiment 3

Based on Embodiment 2, the following is presented: Determination of the

lobe survival area ratio.

On the 7th postoperative day, the flatness of the flap was assessed using high-quality

Photographs were evaluated. Surviving and ischemic areas were evaluated using the Imag-Pro Plus

imaging software. The percentage of surviving area was calculated as [(surviving area)/(total surviving ischemic area)] x 100%.

Survival rate of the SCTCS-ABs treatment group was significantly better than that of the

PBS group. The median survival of the SCTCs-ABs treatment group, the

PBS group and the control group was (83.98+4.91)%, (56.08+5.84)%, respectively.

(59.53+6.25)%. If one compares the ratio of the flap survival area between the with/508779

SCTCs-ABs treated group and the PBS group, the difference was statistically significant (p < 0.05). Comparing the ratio of flap survival area between the

control and PBS groups, the difference was not statistically significant (p > 0.05). 5 (See Figure 2).

Embodiment 4

Based on Embodiment 3, the following is proposed: The

Laser Doppler flowmetry is used to measure neovascularization.

To measure blood flow, six mice per group were anesthetized seven days postoperatively and scanned with a laser Doppler device. Blood flow was measured with a

perfusion device and calculated using the Moor LDI Review software. Blood flow improved in the SCTCs-ABs treatment group, and the quantitative

Blood flow measurements were significantly improved compared to the PBS group.

Blood flow in the SCTCs-ABs treatment group, the PBS group and the

Control group was (377.40+40.92) PU, (361.40+14.46) PU and (257.60+21.80) PU respectively.

When comparing the SCTCs-ABs treatment group with the PBS group, the

Difference statistically significant (p < 0.05). When comparing the control and the

In the PBS group, the difference was not statistically significant (p > 0.05). (See Figure 3).

Embodiment 5

Based on embodiment 4, it is proposed that: the flap collagen was observed by Masson staining (Masson).

Seven days after surgery, after the animals were euthanized, six 1 cm x 1 cm tissue samples were taken from area II (middle position) of the skin flap in each group.

The tissues were immersed in 4% paraformaldehyde for fixation, dehydrated, and embedded in paraffin. The samples were then sectioned transversely to create 4-μm sections, and the sections, mounted on poly-L-lysine-coated sections, were subjected to

Matson staining was performed. Under a 200x light microscope, six areas of each set of sections were selected, and Image J calculated the percentage of collagen in the total dermal layer to quantify the amount of collagen contained in the dermis. The collagen density in the SCTCs-ABs treatment group was significantly higher than that in the PBS group: the collagen percentage in the SCTCs-ABs treatment group, the PBS group, and the control group was (35.10 + 4.36) percent, (16.27 + 3.80) percent, and (16.20 + 3.39) percent, respectively. The difference was statistically significant (p < 0.05) when the

SCTCs-ABs treated group was compared with the PBS group, and not statistically significant (p > 0.05) when the control group was compared with the PBS group. (See

Figure 4).

Embodiment 6

Based on Embodiment 5, the following is proposed:

Immunofluorescence observation.

Neovascularization is a key determinant of flap survival, and by demonstrating the distribution of CD31/EMCN within the flap, we investigated whether the pro-survival effect of SCTCs-ABs on the flap is related to endogenous neovascularization. The above-mentioned paraffin sections were deparaffinized with xylene and subjected to a graded ethanol bath. The sections were placed in sodium citrate buffer for antigen repair at 95°C for 20 minutes and 100508779

minutes with 10% (w/v) bovine serum albumin phosphate buffer. The sections were then incubated with antibodies at 4°C. The primary antibodies were

hours at room temperature for primary staining and then with the secondary

Anti-rabbit antibody DAPI was incubated for 1 hour at 37 °C. The cells in the

Preparations were examined with a fluorescence microscope and a

DP2-TWAN imaging system (Olympus Corporation, Tokyo, Japan). Six homogeneously stained areas from three groups of sections were randomly selected, and the flap tissues were imaged with the DP2TWAN imaging system at 100x magnification, and CD31/EMCN-positive vascular expression was assessed with

ImageProPlus. The number of CD31/EMCN-positive microvessels was (85.89+4.83)/mm2, (51.19+4.31)/mm2, and (52.21+5.44)/mm2 in the

SCTCs-ABs treatment group, the PBS group, and the control group, respectively. The difference was statistically significant (p < 0.05) when the SCTCs-ABs treatment group was compared with the

PBS group and not statistically significant (p > 0.05) when the

Control group was compared with the PBS group. (See Figure 4) (See Figure 5).

Although embodiments of the present invention have been shown and described, those skilled in the art will recognize that numerous changes, modifications,

Substitutions and variations may be made to these embodiments without departing from the principle and spirit of the present invention, the scope of which is limited by the appended claims and their equivalents.

Claims (7)

Claims LU508779 1. The application of SCTCs-ABs in promoting the survival of ultra-long random skin flaps, characterized in that the method of application is a concentration of 1 mg/ml in C57BL6 mice: male, 8 weeks old, weighing about 20 g, dorsal skin flap lifted and injected in situ, only one treatment. 2. The application of SCTCs-ABs in promoting the survival of ultra-long random skin flaps according to claim 1, characterized in that: SCTCs-ABs are mouse epithelial connective tissue cells cultured in high-glucose medium and stimulated with astrocystin STS 0.5 μM for 12 hours, differential centrifugation was performed at 300 g * 5 min to remove cell debris and 2000 g * 30 min to remove the supernatant, and the precipitate was resuspended with PBS and applied as soon as possible at 4 °C or stored for a long time at -80 °C. 3. The application of SCTCs-ABs in promoting the survival of ultra-long random skin flaps according to claim 2, characterized in that: the medicament is configured as the concentration of SCTCs-ABs measured by BCA hair and diluted with PBS to the concentration of 1 mg/ml. 4. The application of SCTCs-ABs in promoting the survival of ultra-long random skin flaps according to claim 3, characterized in that: the method of administering the drug consists in using a microinjection needle to inject an average of 8 sites in situ after the dorsal skin flaps of mice have been raised. 5. The use of SCTCs-ABs in promoting the survival of ultra-long random skin flaps according to claim 4, characterized in that: the effect of SCTCs-ABs on improving blood flow in promoting the survival of ultra-long skin flaps comprises. 6. The use of SCTCs-ABs in promoting the survival of ultra-long random skin flaps according to claim 4, characterized in that it comprises the role of SCTCs-ABs in promoting the survival of an extra-long random flap in promoting collagen repair. 7. The use of SCTCs-ABs in promoting the survival of ultra-long random skin flaps according to claim 4, characterized in that it comprises the role of SCTCs-ABs in promoting the survival of ultra-long random flaps in promoting collagen repair.