CN111450119A

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

Info

Publication number: CN111450119A
Application number: CN201910264077.2A
Authority: CN
Inventors: 李宗金; 王晨; 崔凯歌
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2019-01-21
Filing date: 2019-04-03
Publication date: 2020-07-28

Abstract

The invention relates to an extracellular matrix hydrogel preparation derived from perinatal tissue for promoting organ injury repair. The organ injury comprises organ injury models such as skin injury, lower limb ischemia, kidney injury, myocardial infarction and the like. The preparation is prepared from extracellular matrix hydrogel derived from perinatal tissue, is administered by in-situ injection, and can be independently used as a preparation for promoting organ repair, and can also be used as a carrier of cells, medicaments and the like for promoting the recovery of the structure and function of damaged organ tissue. The preparation has low immunogenicity, contains abundant bioactive components, and can provide microenvironment close to natural tissue for cells, thereby promoting organ injury repair.

Description

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

Technical Field

The invention relates to a method for enhancing the treatment effect of a perinatal tissue-derived extracellular matrix hydrogel preparation in organ injury, belonging to the technical field of biomaterial treatment and regenerative medicine.

Background

Extracellular matrix (extracellular matrix) is a complex network of many biologically active macromolecular substances, the major components of which include collagen, non-collagenous proteins, elastin, proteoglycans and glycosaminoglycans. The extracellular matrix not only can support the survival of cells and provide a suitable place for the life activities of the cells, but also can further regulate and control the proliferation, metabolism, functions and migration of the cells by influencing signal transmission and information exchange among the cells. The extracellular matrix exists in two main forms, in the form of basement membrane at the base of epithelial cells, and the intercellular adhesion structure exists in the form of interstitial connective tissue. The biological function of the extracellular matrix is mainly embodied in three aspects: firstly, the cell culture medium can play physical functions of supporting cell survival and proliferation, protecting cell basic structure, maintaining intercellular communication and the like; second, the extracellular matrix is capable of dynamically modulating cellular biological behavior as a product of cellular secretory metabolism; thirdly, through the interaction with cells, the normal metabolism, proliferation, differentiation, intercellular information exchange and the like of the cells are maintained.

Extracellular matrices play an important role in tissue damage repair and regenerative medicine. The extracellular matrix refers to extracellular matrix components remaining after cellular components of a tissue or organ are completely removed by physical, chemical or biological methods. Compared with other biological materials, the extracellular matrix has extremely low immunogenicity, contains abundant bioactive components, can provide a microenvironment which is closer to natural tissues for cells, and is more suitable for promoting the proliferation of tissue cells and the growth of seed cells.

Perinatal tissue is an important organ for the exchange of substances between the fetus and the mother, wherein the placenta is an intermaternal-fetal tissue-bound organ formed by the joint growth of embryonic germ membranes and the mother's endometrium during human pregnancy, and the umbilical cord is a tubular structure connecting the fetus and the placenta. Peripartum tissues such as placenta and umbilical cord are rich in extracellular matrix and basement membrane, and the extracellular matrix contains abundant growth factors, such as Epidermal Growth Factor (EGF), fibroblast growth factor (bFGF), Transforming Growth Factor (TGF), Vascular Endothelial Growth Factor (VEGF), Hepatocyte Growth Factor (HGF), and thus can be used to promote tissue organ damage repair.

The organ injury comprises organ injury models such as skin injury, lower limb ischemia, kidney injury, myocardial infarction and the like. Tissue damage repair is a complex interactive biological process in which a large number of cells and cytokines are involved. Hydrogel, as a material capable of promoting tissue damage repair, has been shown to be a biomaterial having great potential in the biological and medical fields. Collagen and glycosaminoglycans, which are major components of the extracellular matrix, are also widely distributed in tissues and organs. The glycosaminoglycan has very important biological functions, and the chondroitin sulfate not only serves as a component of extracellular matrix in connective tissues, but also participates in physiological or pathological processes such as nerve growth and development, wound healing, anticoagulation exerted by a fibrinogen system, growth factor signal transduction and the like; the heparan sulfate not only can regulate FGF signals and other growth factors to promote wound healing, but also is applied to biomedical materials and has obvious effect; hyaluronic acid is a preferred substance for promoting tissue damage repair hydrogel, and is capable of inducing angiogenesis. The hydrogel for promoting tissue injury repair at present has various varieties and various characteristics, but has some defects, the extracellular matrix hydrogel preparation from perinatal tissue has remarkable tissue injury repair and medical regeneration functions, collagen, glycosaminoglycan and bioactive factors contained in the hydrogel preparation are key for organ injury healing, and the hydrogel preparation for treating organ injury can be more conveniently and widely applied, so that a new thought and method are provided for tissue injury repair and treatment.

Disclosure of Invention

The invention relates to an extracellular matrix hydrogel preparation derived from perinatal tissue for promoting organ injury repair.

The invention relates to an extracellular matrix hydrogel preparation derived from perinatal tissues, which is delivered to target cells and target organs by an in-situ injection method to play a therapeutic role, thereby achieving the effect of promoting the repair of organ injury.

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

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

The organ injury comprises organ injury models such as skin injury, lower limb ischemia, kidney injury, myocardial infarction and the like.

Drawings

FIG. 1A is the state of an extracellular matrix hydrogel; FIG. 1B is a rheological mechanical characterization of extracellular matrix hydrogels; FIG. 1C is a scanning electron microscope identification of extracellular matrix hydrogels; FIG. 1D shows the DNA content characterization of extracellular matrix hydrogels; FIG. 1E shows the identification of total carbohydrate content of an extracellular matrix hydrogel, and FIG. 1F shows the identification of protein content of an extracellular matrix hydrogel derived from human placenta.

Figure 2 is a placenta-derived extracellular matrix hydrogel formulation promoting repair of damaged tissue structure and function of skin. FIG. 2A is a H & E stain showing tissue structure recovery in the extracellular matrix hydrogel treatment group; figure 2B is Masson staining showing the degree of fiber after skin wound healing.

FIG. 3A shows that the placenta-derived extracellular matrix hydrogel preparation promotes angiogenesis of skin damaged tissues, 3B shows that luciferase expression in the wound surface region of skin damaged tissues is quantitatively analyzed by using L eving Image software after the placenta-derived extracellular matrix hydrogel preparation is treated, and the improvement of the skin damaged tissues after the extracellular matrix hydrogel treatment is shown, and 3C shows that the angiogenesis-promoting gene expression of the damaged parts is detected by RT-PCR.

Detailed Description

In the following examples, unless otherwise specified, all methods used are conventional and all reagents used are commercially available.

Example 1 the present invention provides a method for preparing an extracellular matrix hydrogel derived from a tissue.

Sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, tris (hydroxymethyl) aminomethane, ethylenediaminetetraacetic acid and other reagents are all available from Solarbio corporation; reagents such as double antibody, nystatin, pepsin and the like are purchased from Gibco company; the serum used was Hyclone.

(1) Homogenizing the tissue, centrifuging, and removing blood from the tissue;

(2) adding sterile PBS solution into the solution obtained in the step (1), fully oscillating, washing and centrifuging, and reserving a precipitate part;

(3) adding Tris-EDTA buffer (pH = 7.4) to the pellet at room temperature, shaking, and washing for 24 hours;

(4) centrifuging, adding SDS solution into the precipitate for resuspension, oscillating at room temperature, and washing for 24 hours;

(5) the pellet fraction was washed three times with sterile PBS solution, and the centrifuged pellet fraction was transferred to preheated 37 ℃ FBS solution (containing 1% diabody and 1% nystatin) and washed for 3 hours with shaking. Centrifuging, repeatedly washing the precipitate with PBS solution until FBS is completely removed, and centrifuging to obtain white flocculent precipitate;

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

(7) the obtained powder was dissolved using hydrochloric acid-pepsin solution, shaken to a dissolved state, adjusted to pH 7.4, and then the solution was left at 37 ℃ to form a hydrogel colloid.

Example 2, the present invention provides a method for characterizing a tissue-derived extracellular matrix hydrogel preparation.

Determination of DNA content of extracellular matrix hydrogel: DNA was extracted from the placenta tissue before and after the decellularization treatment using a tissue genome DNA extraction kit. After extracting the DNA, detecting the DNA by an enzyme-labeling instrument under the condition of 260 nm, and comparing the change of the DNA content in the placenta tissue before and after the decellularization treatment by taking the normal placenta tissue as a reference.

And (3) measuring the protein content of the extracellular matrix hydrogel, namely detecting the protein concentration by using a BCA method, mixing the solution B and the solution A at a ratio of 50:1 to prepare a working solution with a proper volume, adding a 10 mu L protein sample into every 200 mu L BCA solution, reacting for 30 minutes at 37 ℃, immediately carrying out colorimetric measurement by using a spectrophotometer, recording the spectrophotometric value of the spectrophotometric value, and substituting the spectrophotometric value into a standard curve to obtain the protein concentration.

And (3) measuring the total sugar content of the extracellular matrix hydrogel, namely accurately preparing a galactose standard substance into standard substance solutions of 0.02 mg/m L, 0.04mg/m L, 0.06 mg/m L, 0.08 mg/m L and 0.10 mg/m L, respectively preparing 100 mu L, adding 200 mu L6% of phenol solution and 1.5 m L concentrated sulfuric acid, shaking and uniformly mixing, reacting at 100 ℃ for 10 minutes, cooling, detecting the absorbance at 490 nm, drawing a standard curve, preparing a freeze-dried sample to be detected into a solution of 0.2 mg/m L, preparing 100 mu L, detecting the absorbance according to the steps, and calculating the total sugar concentration by using a regression equation of the standard curve.

Rheological measurement of extracellular matrix hydrogel: the prepared sample is placed between rheometer plates, the diameter of each plate is 20 mm, the distance between the plates is 1 mm, and the frequency is 1 Hz. The storage modulus (G ') and loss modulus (G') of the samples were recorded as a function of temperature from 0 ℃ to 40 ℃ to investigate the stability of the hydrogels.

And (3) identifying the extracellular matrix hydrogel by a scanning electron microscope: the extracellular matrix is sprayed with gold in a vacuum state, so that the extracellular matrix is wrapped by gold-palladium, and then SEM observation and photographic recording are carried out under the condition that the acceleration voltage is 10 kV. Finally, the pore size was calculated and measured using ImagJ software (National Institute of Health, USA).

Example 3, the present invention provides a method for constructing models of mouse ablative skin injury, lower limb ischemia, kidney injury, myocardial infarction, and the like.

Construction of excisional skin lesions in mice: mice were weighed and anesthetized with 4% chloral hydrate (330 mg/kg) intraperitoneally; fixing the anesthetized mouse in a prone position, removing back hair by using a shaver, and sterilizing the skin of an operation area by iodophor; forming a full-thickness wound surface, deep fascia, of skin with a diameter of about 1cm on the back of the patient with sterile ophthalmic scissors; a 3 mm thick ring of silicone was sutured to the wound with nylon sutures to prevent non-pathological contraction of the wound; after the operation, the mouse is laid on the heating pad for rewarming, and is put back into the rearing cage after reviving.

Constructing a mouse lower limb ischemia model: weighing the weight of the mouse, and carrying out intraperitoneal injection on 4% chloral hydrate (330 mg/kg) to anaesthetize the mouse; making an incision from the inner side of the right leg and knee to the midpoint of the groin, and blunt-separating subcutaneous connective tissue; separating and exposing femoral artery and great saphenous artery with ophthalmologic forceps, rotating external iliac artery and vein and branch of femoral arteriovenous muscle, gently separating accompanying nerve, tying two ends of femoral artery with 8/0 # thread and removing middle section; skin incisions were sutured and animals were placed on a heating pad until awakened.

Construction of mouse acute kidney injury model: weighing the weight of the mouse, and carrying out intraperitoneal injection on 4% chloral hydrate (330 mg/kg) to anaesthetize the mouse; longitudinally cutting skin (0.8-1 cm) at the middle part of the back and 0.2 cm left side of the spine, separating subcutaneous connective tissue, cutting off back muscle, introducing into abdominal cavity, probing and exposing left kidney, and observing that normal kidney is bright red; dissociating perirenal fat and fascia by using sharp forceps, slightly pulling the kidney to expose the renal pedicle to the left, slightly closing the renal pedicle by using a miniature vascular clamp, and observing that the kidney is purple black after extravasated blood appears in about 1 minute; during renal ischemia, the incision was covered with gauze soaked with warm saline and kept moist, and the mouse was illuminated with a warm light and kept at a constant heating pad temperature (37 ℃); after 40 minutes of ischemia, the vascular clamps are removed, and the color of the kidney can be seen by naked eyes and instantly turns into red; after the extracellular matrix hydrogel formulation was injected under the kidney capsule, the muscle layer and skin were sutured layer by layer using 4-0 silk to close the back incision, and the animal was placed on a heat pad until awakening

Construction of mouse myocardial infarction model: pre-anaesthetizing a mouse by using 5% isoflurane gas, fixing, cutting off a neck trachea, connecting an anesthesia respirator for positive pressure ventilation after trachea intubation, adjusting the content of isoflurane to be about 1% -1.5%, and adjusting the respiratory frequency to be 120 times/minute; performing a thoracotomy and exposing the anterior wall of the left ventricle of the heart at the fourth intercostal site; the left anterior descending branch of the coronary artery is ligated by 7-0 suture line at the position which is about 1 mm away from the left auricle below the starting end of the left coronary artery; injecting required medicines at the positions of 1 mm below the left and right of the ligation site respectively; close the chest, sew the muscles and skin and place the animal on a heating pad until awakened.

Example 4, the present invention provides a method for detecting the effect of an extracellular matrix hydrogel on the function of organ injury therapy.

Hematoxylin & eosin staining to evaluate the recovery of the damaged tissue structure: the extracellular matrix hydrogel was used to treat the excised skin injury model mice, and on

days

7 and 14, the mice were sacrificed to take the injured tissue, paraffin sections were prepared, and were stained with hematoxylin & eosin, and the myofiber necrosis and inflammatory infiltration of the injured tissue were evaluated, indicating that the extracellular matrix hydrogel could alleviate the necrosis of the injured tissue, inhibit the inflammatory response, and promote the recovery of the injured tissue structure (fig. 2A).

Masson staining evaluates fibrosis of damaged tissues: using an extracellular matrix hydrogel excision skin injury model mouse, at 7 and 14 days, the mouse was sacrificed to take the injured tissue, paraffin sections were prepared and Masson stained, and the fibrosis level of the injured tissue was evaluated, indicating that the extracellular matrix hydrogel could alleviate fibrosis of the injured tissue and promote the restoration of the structure and function of the injured tissue (fig. 2B).

Real-time fluorescent quantitative PCR: evaluating the change of the extracellular matrix hydrogel on the expression condition of the skin inflammatory factor gene; total RNA in cells is extracted by a TRIzol method, cDNA is obtained through reverse transcription, and then RNA level detection is carried out on inflammation related genes through Real-time PCR. The results show that the extracellular matrix hydrogel can better inhibit the expression of skin inflammation genes (figure 3C).

Claims

1. The preparation contains extracellular matrix extracted from perinatal tissue as effective component, carries bioactive molecules such as collagen, glycoprotein, proteoglycan, glycosaminoglycan, etc. in the extracted tissue, and can be transferred to target organ for therapeutic effect.

2. The extracellular matrix according to claim 1, wherein: the extracellular matrix is derived from mammalian tissue, including perinatal tissue (placenta, umbilical cord, etc.) of various origins.

3. The preparation is used for treating organ injury, and is characterized in that: the organ injury comprises organ injury models such as skin injury, lower limb ischemia, kidney injury, myocardial infarction and the like.

4. A perinatal tissue-derived extracellular matrix hydrogel preparation for promoting organ injury repair, which is prepared by injecting the human perinatal tissue-derived extracellular matrix of claim 2 in situ into the organ injury model of claim 3, and exerting therapeutic effect by the method of claim 1.

5. According to claim 4, the preparation not only can be used alone as an organ repair promoting preparation, but also can be used as a carrier of cells, drugs and the like, provides an environment similar to that in vivo for the cells, and promotes the recovery of the tissue structure and function of the damaged organ.

6. The technical means of claim 5, characterized in that: the technology can enhance the retention rate and stability of cells and drugs at the damaged part, realize the biocompatibility of the extracellular matrix and further enhance the treatment effect of the extracellular matrix.