CN111450119A - Perinatal tissue-derived extracellular matrix hydrogel preparation for promoting organ injury repair - Google Patents

Abstract

The invention is a perinatal tissue-derived extracellular matrix hydrogel preparation for promoting organ damage repair. Organ damage includes skin damage, lower extremity ischemia, kidney damage, myocardial infarction and other organ damage models. The preparation is prepared from perinatal tissue-derived extracellular matrix hydrogel, and is administered by in situ injection. The preparation can not only be used as a preparation for promoting organ repair, but also as a carrier for cells, drugs, etc. Promote the recovery of damaged organ tissue structure and function. This preparation has low immunogenicity, is rich in bioactive components, and can provide cells with a microenvironment closer to the natural tissue, thereby promoting organ damage repair.

Description

A perinatal tissue-derived extracellular matrix hydrogel that promotes repair of organ damage glue preparation

technical field

The invention relates to utilizing an extracellular matrix hydrogel preparation derived from perinatal tissues to enhance its therapeutic effect in organ damage, and belongs to the technical field of biological material treatment and regenerative medicine.

Background technique

The extracellular matrix (extracellular matrix) is an intricate network composed of a variety of biologically active macromolecules, and its main material components include collagen, non-collagen, elastin, proteoglycans and glycosaminoglycans. The extracellular matrix can not only support the survival of cells and provide a suitable place for the life activities of cells, but also further regulate the proliferation, metabolism, function and migration of cells by affecting the signal transmission and information exchange between cells. There are two main forms of extracellular matrix. It exists in the form of basement membrane in the basal part of epithelial cells, and in the form of intercellular adhesion structure in the form of interstitial connective tissue. The biological functions of the extracellular matrix are mainly reflected in three aspects: first, it can play physical functions such as supporting cell survival and proliferation, protecting the basic structure of cells, and maintaining intercellular connections; second, as a product of cell secretion and metabolism, cells The extracellular matrix can dynamically regulate the biological behavior of cells; thirdly, through the interaction with cells, it maintains normal cell metabolism, proliferation, differentiation and information exchange between cells.

Extracellular matrix plays an important role in tissue injury repair and regenerative medicine. Extracellular matrix refers to the extracellular matrix components that remain after the cellular components of a tissue or organ are completely removed by physical, chemical or biological methods. Compared with other biological materials, extracellular matrix has extremely low immunogenicity, is rich in bioactive components, and can provide cells with a microenvironment closer to natural tissues, so it is more suitable for promoting tissue cell proliferation and seeding cells. growth.

The perinatal tissue is an important organ for material exchange between the fetus and the mother. Among them, the placenta is an inter-mother tissue-binding organ formed by the joint growth of the embryonic membrane and the maternal endometrium during human pregnancy. The umbilical cord is a tubular connecting fetus and placenta. structure. Perinatal tissues such as placenta and umbilical cord are rich in extracellular matrix and basement membrane, and the extracellular matrix is rich in growth factors, such as epidermal growth factor (EGF, epidermal growth factor), fibroblast growth factor (bFGF) , basic fibroblast growth factor), transforming growth factor (TGF, transforming growth factor), vascular endothelial growth factor (VEGF, vascular endothelial growth factor), hepatocyte growth factor (HGF, hepatocyte growth factor), so it can be used to promote tissue and organ damage repair .

Organ damage includes skin damage, lower extremity ischemia, kidney damage, myocardial infarction and other organ damage models. Tissue damage repair is a complex and interactive biological process in which a large number of cells and cytokines are involved. As a material that can promote tissue damage repair, hydrogel has shown to be a biomaterial with great potential in the field of biology and medicine. Collagen and glycosaminoglycan, the main components of extracellular matrix, are also widely distributed in tissues and organs. Among them, glycosaminoglycans have very important biological functions. Chondroitin sulfate not only acts as a component of the extracellular matrix in connective tissue, but also participates in the growth and development of nerves, wound healing, and anticoagulation through the fibrinogen system. Physiological or pathological processes such as growth factor signal transduction; heparan sulfate can not only regulate FGF signal and other growth factors to promote wound healing, but also be used in biomedical materials and has obvious effect; hyaluronic acid can induce angiogenesis, It is the preferred substance for promoting tissue damage repair hydrogel. There are many kinds of hydrogels currently used to promote tissue damage repair, although each has its own characteristics, but there are always some defects, and perinatal tissue-derived extracellular matrix hydrogel preparations have significant tissue damage repair and medical regeneration. Function, collagen, glycosaminoglycan and bioactive factors contained in it are the key to the healing of organ damage, and the treatment of organ damage in the form of hydrogel preparations can be more convenient and widely used, which is the key to tissue damage repair and its treatment. Treatment provides new ideas and methods.

SUMMARY OF THE INVENTION

The invention is a perinatal tissue-derived extracellular matrix hydrogel preparation for promoting organ damage repair.

The invention is a perinatal tissue-derived extracellular matrix hydrogel preparation, which is delivered to target cells and target organs by the method of in situ injection, thereby achieving the effect of promoting organ damage and repair.

The extracellular matrix is an extracellular matrix derived from perinatal tissues, and the tissues include placenta, umbilical cord and the like.

The invention can effectively enhance the therapeutic effect after organ damage, and the effective molecules in the extracellular matrix can interact with the damaged organ, release molecules with biological activity and therapeutic effect, and promote the recovery of the structure and function of the damaged tissue.

The organ damage includes skin damage, lower limb ischemia, kidney damage, myocardial infarction and other organ damage models.

Description of drawings

Figure 1A is the state of the extracellular matrix hydrogel; Figure 1B is the rheological identification of the extracellular matrix hydrogel; Figure 1C is the scanning electron microscope identification of the extracellular matrix hydrogel; Figure 1D is the extracellular matrix hydrogel Figure 1E is the identification of the total sugar content of the extracellular matrix hydrogel, Figure 1F is the identification of the protein content of the extracellular matrix hydrogel, which is a human placenta-derived extracellular matrix hydrogel glue.

Figure 2 shows that the placenta-derived extracellular matrix hydrogel preparation promotes the repair of tissue structure and function of skin damage. Figure 2A is H&E staining, showing the recovery of tissue structure in the extracellular matrix hydrogel treatment group; Figure 2B is Masson staining, showing the degree of fibers after skin wound healing.

Fig. 3A shows that the placenta-derived extracellular matrix hydrogel preparation promotes angiogenesis in skin lesions; 3B is the quantitative analysis of luciferase in the skin injury wound area after the placenta-derived extracellular matrix hydrogel preparation was treated with Living Image software The expression status shows the improvement of skin injury after extracellular matrix hydrogel treatment; 3C is RT-PCR detection of the expression of angiogenesis-related genes at the injury site.

Detailed ways

In the following examples, unless otherwise specified, the methods used are conventional methods, and the reagents used can be obtained from commercial sources.

Embodiment 1, the present invention provides a preparation method of tissue-derived extracellular matrix hydrogel.

Reagents such as sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, trihydroxymethylaminomethane, ethylenediaminetetraacetic acid were purchased from Solarbio; double antibody, nystatin, pepsin and other reagents were all purchased from Solarbio It was purchased from Gibco Company; the serum used was Hyclone Company.

(1) Take the tissue for homogenization and centrifugation to remove the blood in the tissue;

(2) Add sterile PBS solution to (1), fully shake, wash, and centrifuge, and retain the precipitated part;

(3) At room temperature, add Tris-EDTA buffer (pH=7.4) to the precipitate, shake and wash for 24 hours;

(4) Centrifuge, add SDS solution to the pellet to resuspend, shake and wash at room temperature for 24 hours;

(5) Take sterile PBS solution to wash the precipitated part three times, transfer the precipitated fraction after centrifugation to a preheated 37°C FBS solution (containing 1% double antibody and 1% nystatin), shake and wash for 3 hours . Centrifuge, wash the precipitate repeatedly with PBS solution until FBS is completely removed, centrifuge to obtain white flocculent precipitate;

(6) collecting the precipitated part, lyophilizing it, and grinding to obtain tissue extracellular matrix powder;

(7) Dissolving the obtained powder with a hydrochloric acid-pepsin solution, shaking to a dissolved state, adjusting the pH value to 7.4, and then placing the solution at 37° C. to form a hydrogel colloid.

Embodiment 2, the present invention provides a method for identifying properties of tissue-derived extracellular matrix hydrogel preparations.

Determination of DNA content in extracellular matrix hydrogels: DNA from placental tissue before and after decellularization was extracted using a tissue genomic DNA extraction kit. After the DNA was extracted, it was detected by a microplate reader at 260 nm, and normal placental tissue was used as a control to compare the changes of DNA content in placental tissue before and after decellularization.

Determination of protein content in extracellular matrix hydrogels: protein concentration was detected by BCA method. Mix solution B and solution A at a ratio of 50:1 and prepare an appropriate volume of working solution. Add 10 μL of protein sample to each 200 μL of BCA solution, react at 37°C for 30 minutes, and immediately use a spectrophotometer for colorimetric measurement, record the spectrophotometric value, and substitute it into the standard curve to obtain the protein concentration.

Determination of total sugar content in extracellular matrix hydrogel: Take the galactose standard and accurately configure it into standard solutions of 0.02 mg/mL, 0.04 mg/mL, 0.06 mg/mL, 0.08 mg/mL, and 0.10 mg/mL, respectively. 100 μL, add 200 μL of 6% phenol solution, 1.5 mL of concentrated sulfuric acid, shake and mix, react at 100 °C for 10 minutes, detect the absorbance at 490 nm after cooling, and draw a standard curve. The lyophilized sample to be tested was prepared into a 0.2 mg/mL solution, 100 μL was taken, the absorbance was detected according to the above steps, and the total sugar concentration was calculated by the standard curve regression equation.

Rheological measurement of extracellular matrix hydrogels: The prepared samples were placed between rheometer plates with a diameter of 20 mm, a spacing of 1 mm, and a frequency of 1 Hz. The storage modulus (G') and loss modulus (G″) of the samples were recorded from 0°C to 40°C to explore the stability of the hydrogel.

SEM identification of extracellular matrix hydrogel: The extracellular matrix was sprayed with gold in a vacuum state to make it coated with gold-palladium, and then observed and photographed by SEM under the condition of an accelerating voltage of 10 kV. Finally, ImagJ software (National Institute of Health, USA) was used to calculate and measure the pore size.

Example 3, the present invention provides methods for constructing mouse models of excisional skin injury, lower limb ischemia, kidney injury and myocardial infarction.

Construction of excisional skin lesions in mice: The mice were weighed, and the mice were anesthetized with an intraperitoneal injection of 4% chloral hydrate (330 mg/kg); the anesthetized mice were fixed in the prone position, and the back hair was removed using a shaver. The skin of the surgical area was sterilized; sterile ophthalmic scissors were used to form a full-thickness skin injury wound with a diameter of about 1 cm on the back, deep to the fascia; 3 mm thick annular silicone was sutured on the wound with nylon sutures to prevent The wound was non-pathologically contracted; after the operation, the mice were placed supine on a heating pad for rewarming, and then returned to the rearing cage after recovery.

Construction of mouse lower extremity ischemia model: weigh the mice and anesthetize the mice with an intraperitoneal injection of 4% chloral hydrate (330 mg/kg); make an incision along the medial knee of the right leg to the midpoint of the groin, and bluntly dissect the subcutaneous Connective tissue; use ophthalmic surgical forceps to separate and expose the femoral artery and the great saphenous artery, the external circumflex iliac artery and vein and the muscular branch of the femoral artery and vein, gently separate the accompanying nerves, ligate both ends of the femoral artery with 8/0 wire The middle section was removed; the skin incision was sutured and the animal was placed on a heating pad until awake.

The establishment of a mouse model of acute kidney injury: the weight of the mice was weighed, and the mice were anesthetized by intraperitoneal injection of 4% chloral hydrate (330 mg/kg). 1 cm), the subcutaneous connective tissue was separated, the back muscle was severed into the abdominal cavity, the left kidney was explored and exposed, and the normal kidney was bright red; the perirenal fat and fascia were freed with sharp forceps, and the kidney was gently pulled to the left to expose the kidney. The renal pedicle was clamped slightly close to the kidney with a miniature vascular clip, and the kidney appeared purple-black with congestion in about 1 minute; during renal ischemia, the incision was covered with gauze dipped in warm saline and kept the gauze moist, and a warm light was placed above the mouse. Irradiated and kept the heating pad temperature (37°C) constant; the vessel clip was removed after 40 minutes of ischemia, and the color of the kidney immediately changed to red; after injection of the extracellular matrix hydrogel preparation under the renal capsule, 4- 0 Muscle and skin are sutured layer by layer with silk sutures. The dorsal incision is closed and the animal is placed on a heating pad until awake.

Construction of the mouse myocardial infarction model: pre-anesthetize the mice with 5% isoflurane gas and fix them, cut the neck trachea, connect the anesthesia ventilator to positive pressure ventilation after tracheal intubation, and adjust the isoflurane content to about 1%-1.5%, the respiratory rate is 120 breaths/min; a thoracotomy is performed, and the anterior wall of the left ventricle of the heart is exposed at the fourth intercostal space; the lower part of the origin of the left coronary artery is about 1 mm away from the left atrial appendage; The left anterior descending coronary artery was ligated with 7-0 suture; the desired drugs were injected at 1 mm at the lower left and lower right of the ligation site; the chest was closed, the muscles and skin were sutured, and the animals were placed on a heating pad until they woke up.

Embodiment 4, the present invention provides a method for detecting the effect of extracellular matrix hydrogel hydrogel on the treatment function of organ damage.

Hematoxylin & eosin staining to evaluate the recovery of damaged tissue structure: extracellular matrix hydrogel was used to treat excised skin injury model mice. On the 7th and 14th days, the mice were sacrificed, and the damaged tissue was collected, and paraffin sections were made. Hematoxylin & eosin staining was performed to evaluate the necrosis of muscle fibers and inflammatory infiltration in the injured tissue, indicating that the extracellular matrix hydrogel can reduce the necrosis of the injured tissue, inhibit the inflammatory response, and promote the recovery of the injured tissue structure (Figure 2A).

Masson staining to evaluate the fibrosis of injured tissue: extracellular matrix hydrogel excised skin injury model mice were used. On the 7th and 14th days, the mice were sacrificed, and the injured tissue was collected, paraffin sections were made, and Masson staining was performed on them. The level of fibrosis in the injured tissue was assessed, indicating that the extracellular matrix hydrogel can reduce the fibrosis of the injured tissue and promote the recovery of the structure and function of the injured tissue (Fig. 2B).

Real-time fluorescence quantitative PCR: evaluate the changes in the expression of skin inflammatory factor genes by extracellular matrix hydrogel; use TRIzol method to extract total intracellular RNA, obtain cDNA by reverse transcription, and analyze the RNA level of inflammation-related genes by Real-time PCR detection. The results showed that the extracellular matrix hydrogel could better inhibit the expression of skin inflammatory genes (Fig. 3C).

Claims (6)

1. The active ingredient of the preparation is tissue-derived extracellular matrix, which is extracted from perinatal tissues and carries bioactive molecules such as collagen, glycoproteins, proteoglycans, and glycosaminoglycans in the extracted tissues. , exert a therapeutic effect by transferring the molecule to the target organ. 2 . The extracellular matrix according to claim 1 , wherein the extracellular matrix is derived from mammalian tissues, including perinatal tissues from different sources (placenta, umbilical cord, etc.). 3 . 3. The treatment injury of the preparation is organ injury, which is characterized in that: the organ injury includes skin injury, lower extremity ischemia, kidney injury, myocardial infarction and other organ injury models. 4. A perinatal tissue-derived extracellular matrix hydrogel preparation for promoting organ damage repair is to inject the extracellular matrix derived from human perinatal tissue according to claim 2 to claim 3 by in situ injection In the organ damage model of claim 1, the therapeutic effect is exerted. 5. According to claim 4, this preparation can not only be used as a preparation for promoting organ repair, but also can be used as a carrier for cells, drugs, etc., to provide cells with an environment similar to that in the body, and to promote damage to organ tissue structure and function. recover. 6. The technical means according to claim 5, characterized in that: the technology can enhance the retention rate and stability of cells and drugs at the damaged site, and can realize the biocompatibility of extracellular matrix, and further enhance the cell Therapeutic effects of extracellular matrix.

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