Glial engineering cell and construction method and application thereofTechnical Field
The invention belongs to the field of construction and culture of engineering cells, and particularly relates to glial engineering cells for transplanting schwann cells into mitochondria of myocardial cells, and a construction method and application thereof.
Background
Mitochondria (mitochondrion) are a type of organelle that is present in most cells and is coated with two membranes, and are structures that produce energy in cells, which are the primary sites for cells to breathe aerobically, called "energy factories" of cells. The diameter of which is about 0.5 to 1.0 micron. Mitochondria are commonly existing in various cells of mammals, have wide sources and play an irreplaceable role in the cells. In addition to powering cells, mitochondria are involved in processes such as cell differentiation, cell information transfer, and apoptosis, and possess the ability to regulate cell growth and cell cycle.
Cardiomyocytes, also known as myocardial fibers, are one of the most active cells in the organism. The cardiomyocytes are classified into two main types according to their functions, one type is working cells, which are mainly present in atria and ventricles, and are rich in myofibrils to perform the contractile function of heart, and the other type is self-discriminant cells, which are specially differentiated cardiomyocytes and have the characteristics of excitability, conductivity, spontaneous rhythmicity and the like. Cardiomyocytes are adapted to the sustained rhythmic contractile activity of the heart muscle and contain a large number of glycogen particles and mitochondria within the cell to meet the energy supply.
With the rapid development of the current social traffic, construction industry and manufacturing industry, the occurrence rate of spinal cord injury and other types of nerve injury rises year by year, and the sequelae caused by the nerve injury afflicts the remaining life of patients, and brings heavy burden to the life and families of individuals, so that the related problems of nerve repair are more remarkable. There are few effective nerve injury repair methods, and in recent years, initial progress has been made in the treatment of spinal cord injury. In the rat experiment, the damaged spinal cord can be effectively restored by transplanting the schwann cells to the damaged part, but the effect of the normal schwann cell transplantation is not satisfactory, and the effect of the normal schwann cell transplantation does not reach an ideal state although the effect of the normal schwann cell transplantation has the effect of promoting the restoration of the damage. How the transplanted schwann cells can be passaged in a large amount in a short time, it becomes important to secrete more neurotrophic factors for repairing damaged nerves. Therefore, it is important to be able to obtain the number of schwann cells required for transplantation in a shorter time, and every second the process is shortened, meaning that the patient suffering from nerve damage can suffer from one second less affliction.
Disclosure of Invention
Aiming at the problems and the defects in the prior art, the invention provides a glial engineering cell which is constructed by transplanting schwann cells through myocardial cell mitochondria, has a faster growth speed, can grow to the cell number required by experiments or treatments in a shorter time, and can stably secrete a large amount of neurotrophic factors for a long time.
The invention also provides a construction method of the glial engineering cell.
The invention also provides application of the engineering cell in preparing a medicament for repairing nerve injury.
In order to achieve the above purpose, the specific technical scheme related to the invention is as follows:
The invention provides a glial engineering cell, which is constructed by grafting a cardiac muscle cell mitochondria into a schwann cell.
The invention also provides a construction method of the glial engineering cell, which comprises the following steps:
(1) 200mg of myocardial tissue is put into a PBS solution precooled at 4 ℃, sheared, fragments are washed, filter paper is sucked dry, and high-purity mitochondria are prepared after grinding and homogenizing;
(2) The mitochondria were resuspended in 100-200 μl DMEM cell culture medium and immediately used;
(3) Adding the extracted purified myocardial cell mitochondria into the cultured adherent schwann cells, and incubating and culturing to allow the myocardial cell mitochondria to enter the schwann cells.
Further, in the step (1), the specific preparation process of the high-purity mitochondria comprises the following steps:
(1) Placing the washed fragments into a small glass homogenizer;
(2) Adding 1mL of precooled Lysis Buffer into a glass homogenizer, and grinding and homogenizing on ice at 0 ℃ to obtain a homogenized substance;
(3) Transferring the ground homogenate into a 1.5mL centrifuge tube, centrifuging at 4 ℃ for 1000g multiplied by 5min, and collecting supernatant;
(4) Transferring the supernatant to a new centrifuge tube, centrifuging again at 4 ℃ for 1000g multiplied by 5min, and collecting the supernatant;
(5) Transferring the supernatant obtained by centrifugation into a new 1.5ml EP tube, centrifuging at 4 ℃ for 12000g multiplied by 10min, discarding the supernatant, and reserving the precipitate to obtain a crude extract of myocardial cell mitochondria;
(6) Adding 0.5-1.0mL Wash Buffer into the mitochondrial crude extract, re-suspending, centrifuging at 4deg.C for 1000g×5min, and collecting supernatant;
(7) Transferring the supernatant into a new centrifuge tube, centrifuging at 4deg.C for 12000g×10min, and discarding the supernatant to obtain high purity mitochondria.
Further, in the step (3), the adherent schwann cells are cultured in a 6-well plate with the inoculum size of 1.5 multiplied by 105/mL, 3mL of 5% CO2 is added to each well of the 6-well plate, and the culture is carried out in a 37 ℃ incubator for 12-24 hours until the cells are adherent.
Further, in step (3), the cardiomyocyte mitochondria were added to Schwann cells in an amount of 20. Mu.l per well in a 6-well plate by resuspending 200mg of purified mitochondria extracted from myocardial tissue with 120. Mu.l of DMEM cell culture medium.
Further, in the step (3), the incubation and culture conditions are 5% CO2 and 37 ℃ incubator, and the incubation is carried out for 12 hours.
The invention also provides application of the glia engineering cells in preparing medicines for repairing nerve injury.
The myocardial tissue used in the invention is obtained by the following steps:
1) 4-week-old Wistar rats were anesthetized after 3-5min by intraperitoneal injection of 10% chloral hydrate at 3 mL/kg;
2) The anesthetized rats were surgically sheared along the ventral midline to expose the heart, and approximately 200mg of myocardial tissue was cut with ophthalmic scissors and minced into 3-5mm pieces in 4 ℃ pre-chilled PBS solution.
The beneficial effects of the invention are as follows:
(1) The schwann cell engineering cells cultured by the invention have a faster growth speed and can reach the cell number required by experiments or treatments in a shorter time.
(2) The engineering cell construction method is simple and has low cost of raw materials.
(3) The engineering cell constructed by the invention has the same cell property as the original cell line, and can better meet the experiment and other requirements.
(4) Compared with the original cell line, the engineering cell constructed by the invention can stably secrete more neurotrophic factors for a longer period.
(5) The engineering cell constructed by the invention can well inhibit the expression of inflammatory factors at the damaged part and promote the expression of the inflammatory factors when being used for repairing spinal cord injury, has the advantages of simple repairing method, lower raw material cost, no toxic or side effect and less secondary injury to organisms.
Drawings
FIG. 1 is a two-photon laser confocal microscope image of an engineered cell prepared according to the present invention.
FIG. 2 is a graph of the cell viability of engineered cells prepared according to the present invention.
FIG. 3 is a graph showing the content of various neurotrophic factors secreted by the engineering cells prepared by the invention.
FIG. 4 shows the secretion of various neurotrophic factors from engineering cells prepared according to the present invention.
FIG. 5 is a graph showing the extent of expression of the pro-inflammatory factors IL-10 and IL-1β after application to engineered cells.
FIG. 6 is a graph comparing the quantitative analysis of the anti-inflammatory factor IL-10 and the pro-inflammatory factor IL-1β.
FIG. 7 is a diagram showing the structure of the spinal cord at the damaged part after the repair of the engineering cells prepared by the invention.
Detailed Description
The present invention will be described in further detail by the following detailed description, but it should not be construed that the scope of the invention is limited to the following examples. Various substitutions and alterations are also within the scope of this disclosure, as will be apparent to those of ordinary skill in the art and by routine experimentation, without departing from the spirit and scope of the invention as defined by the foregoing description.
The schwann cells used in the present invention can be directly extracted by the methods described in the prior art.
Example 1
(1) The 4-week-old Wistar rats were anesthetized with chloral hydrate, hearts were removed, myocardial tissue was cut with an ophthalmic scissors, placed in 4 ℃ precooled PBS solution, minced into pieces 3-5mm in diameter, washed with blood, blotted with filter paper, and placed in a small glass homogenizer.
(2) 1ML of precooled Lysis Buffer was added to the glass homogenizer and ground 20-30 times on 0℃ice.
(3) The ground homogenate was transferred to a 1.5mL centrifuge tube, centrifuged at 4℃for 1000 g.times.5 min and the supernatant was collected.
(4) The supernatant was transferred to a new centrifuge tube, centrifuged again at 4℃and 1000 g.times.5 min, and the supernatant was collected.
(5) The supernatant obtained by centrifugation was transferred to a new 1.5ml EP tube, centrifuged at 4 ℃,12000g×10min, the supernatant discarded, and the pellet was retained, which was precipitated as a crude extract of cardiomyocyte mitochondria.
(6) To the crude mitochondrial extract, 0.5-1.0mL Wash Buffer was added, resuspended, and centrifuged at 4℃for 1000 g.times.5 min to obtain the supernatant.
(7) Transferring the supernatant into a new centrifuge tube, centrifuging at 4deg.C for 12000g×10min, and discarding the supernatant to obtain high purity mitochondria.
(8) Mitochondria were resuspended in 100-200. Mu.l DMEM cell culture medium and used immediately.
(9) Culturing the Schwann cells, namely 1.5X105/mL Schwann cells, adding 3mL of 5% CO2 into each hole of a 6-hole plate, and culturing in a 37 ℃ incubator for 12-24 hours until the cells adhere to the wall;
(10) Purified mitochondria extracted from 200mg myocardial tissue were resuspended in 120. Mu.l DMEM cell culture medium, added 20. Mu.l per well in 6-well plates and incubated for 12 hours in a 5% CO2, 37℃incubator. And the mitochondria of the myocardial cells enter the schwann cells to obtain engineering cells.
Effect example 1 myocardial cell mitochondria enter Schwann cells by co-incubation
The mitochondria of the extracted and purified cardiomyocyte cells are immediately marked by a mitochondrial red fluorescent probe, in order to prevent the excessive fluorescent probe from influencing the experimental result, the marked mitochondria are resuspended by a DMEM culture medium and centrifuged again, washed for three times, superfluous fluorescent dye is washed off, the cell is incubated with the Schwann cells for 12 hours, and then the nuclei are stained by a DAPI reagent for cell positioning and observed under a two-photon laser confocal microscope. As shown in fig. 1. From the two-photon confocal microscope image, it can be seen that near the nucleus (blue), the aggregated stained cardiomyocyte mitochondria (red, white arrow) have entered schwann cells.
Effect example 2 MTT assay
For the extracted and purified myocardial cell mitochondria, rat schwann cells are introduced through co-incubation, and the MTT experiment is used for determining the cell viability. The experimental results are shown in FIG. 2.
Experimental results show that the schwann cells transplanted with myocardial cell mitochondria have stronger cell activity compared with common schwann cells and activated schwann cells, which is about 1.2 times of common schwann cells, and have extremely obvious difference through statistical analysis.
Effect example 3 measurement of neurotrophic factor secretion amount
After the constructed schwann cell engineering cells are cultured for 3 days, taking a cell culture solution, adopting an ELISA neurotrophic factor kit to measure the contents of various neurotrophic factors in the cell culture solution, and taking the cell culture solution at 6, 9, 12, 15 and 18 days to measure the contents of various neurotrophic factors. The results are shown in FIGS. 3 and 4.
Experimental results show that the schwann cells transplanted with myocardial cell mitochondria have obvious or extremely obvious differences compared with common schwann cells in secretion of three kinds of neurotrophic factors, namely NT-3 (neurotrophic factor 3), BDNF (brain-derived neurotrophic factor) and NGF (nerve growth factor), have no obvious differences compared with activated schwann cells, have higher secretion of neurotrophic factors, and save the activation stage of the damaged schwann cells. And the secretion of the neurotrophic factors in the cell culture medium does not obviously fluctuate within 18 days, so that the cell culture medium is stable.
Effect example 3 repair of spinal cord injury
(1) Digesting the Schwann cells transplanted with myocardial cell mitochondria by pancreatin, adding DMEM to resuspend the cells, and adjusting the cell concentration to 1X 106/mL;
(2) The cell suspensions were injected into the center of spinal cord injury, 5mm above and below the center, using 10. Mu.l microinjectors, with a depth of about 1-2mm for each injection of 2. Mu.l of the cell suspension.
Experiment one, a Wistar rat T10 section spinal cord injury model is constructed, and a self-made Kirschner wire striker is adopted, wherein the striking strength is 30g multiplied by 3cm. The experiments were divided into four groups, sham, trauma (Injury), schwann cell transplantation (SCs), mitochondrial transplantation in combination with schwann cell transplantation (MT-SCs). The Sham group only performs vertebral plate elimination and the other does not perform any treatment, the Injury group does not perform any treatment after striking the spinal cord of the T10 section with a striking force of 30g multiplied by 3cm, the SCs group performs injection of 2 mu l of common Schwann cell suspension into three sites at the center of the rat spinal cord injury and 5mm above and below the injury center respectively at 24 hours after injury, and the needle penetration depth is about 1-2mm by using a 10 mu l microinjector. MT-SCs groups were injected with 2. Mu.l of mitochondrial-transplanted Schwann cell suspension at the very center of spinal cord injury in rats, and at 5mm above and below the center of injury, at three sites, using 10. Mu.l microinjectors, and needle penetration depths of about 1-2mm, 24 hours after injury. After two weeks of treatment, about 1-1.5cm of spinal cord at the injured part is taken, a sample is prepared, and the expression degrees of the anti-inflammatory factors IL-10 and the pro-inflammatory factors IL-1 beta are measured by a Western Blot method. The results are shown in FIG. 5 and FIG. 6.
Experimental results show that compared with the SCs group and Injury groups, the MT-SCs group has extremely obvious difference and obvious difference in the expression quantity of the anti-inflammatory factor IL-10 at the spinal cord injury position, and can well inhibit the expression of the pro-inflammatory factor by transplanting the Schwann cells containing myocardial cell mitochondria in the aspect of the expression quantity of the pro-inflammatory factor IL-1 beta. Therefore, by transplanting the Schwann cells containing myocardial cell mitochondria to the spinal cord injury part, the inflammatory reaction of the injury part can be well inhibited, and the injury caused by inflammation after spinal cord injury can be reduced.
And secondly, constructing a Wistar rat T10 section spinal cord injury model, and adopting a self-made Kirschner wire striker with the striking strength of 30g multiplied by 3cm. The experiments were divided into four groups, sham, trauma (Injury), schwann cell transplantation (SCs), mitochondrial transplantation in combination with schwann cell transplantation (MT-SCs). The Sham group only performs vertebral plate elimination and the other does not perform any treatment, the Injury group does not perform any treatment after striking the spinal cord of the T10 section with a striking force of 30g multiplied by 3cm, the SCs group performs injection of 2 mu l of common Schwann cell suspension into three sites at the center of the rat spinal cord injury and 5mm above and below the injury center respectively at 24 hours after injury, and the needle penetration depth is about 1-2mm by using a 10 mu l microinjector. MT-SCs groups were injected with 2. Mu.l of mitochondrial-transplanted Schwann cell suspension at the very center of spinal cord injury in rats, and at 5mm above and below the center of injury, at three sites, using 10. Mu.l microinjectors, and needle penetration depths of about 1-2mm, 24 hours after injury. One month after injury, sections were taken for spinal cord injury centers, HE stained, and spinal cord structures at the injury site were observed. The results are shown in FIG. 7.
Experimental results show that the Sram group (FIG. 7A) has complete spinal cord tissue structure, full spinal cord artery shape, no obvious spinal cavities and cracks, complete myelin sheath (blue arrow) structure, close fitting with spinal cord, injury group (FIG. 7B) has disordered spinal cord injury part structure tissue, large-area spinal cavities (red arrow), myelin sheath shedding (blue arrow) accompanied by massive inflammatory cell infiltration, and the SCs group (FIG. 7C) and MT-SCs group (FIG. 7D) have obviously reduced cavity area at the injured part after the treatment of the spinal cord injury by adopting the Schwann cell transplantation, especially the MT-SCs group, the spinal cord tissue is orderly arranged, the tissue shape is restored, myelin sheath is thinner, but the structure is complete, the spinal cord fitting is close, and inflammatory cells are obviously reduced. The results show that the schwann cells transplanted with myocardial cell mitochondria can better promote the preservation of tissue morphology at the damaged part, reduce infiltration of inflammatory cells and promote the recovery of the tissue morphology of the spinal cord compared with the common schwann cells aiming at the treatment of spinal cord injury.