CN112048479A - Animal model for protecting high oxygen exposure lung and brain injury by umbilical cord mesenchymal stem cells - Google Patents
Animal model for protecting high oxygen exposure lung and brain injury by umbilical cord mesenchymal stem cells Download PDFInfo
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Abstract
The invention belongs to the technical field of models for science, medicine or mathematics, and discloses an animal model for protecting high-oxygen exposed lung and brain injury by umbilical cord mesenchymal stem cells. The invention uses slow virus carrier with GFP to transfect UC-MSc, and adopts 3 transplantation methods of trachea, nasal cavity and tail vein injection UC-MSc for comparison. Transplanting UC-MSc through trachea, nasal cavity and tail vein injection, tracking the colonization condition of the UC-MSc in lung and brain, and exploring the simultaneous treatment effect of the UC-MSc on SCI rat hyperoxic lung and brain injury.
Description
Technical Field
The invention belongs to the technical field of models for science, medicine or mathematics, and particularly relates to an animal model for protecting high-oxygen exposure lung and brain injury by umbilical cord mesenchymal stem cells.
Background
Currently, the current state of the art commonly used in the industry is such that: higher concentrations and longer duration of oxygen therapy can lead to acute lung injury or bronchopulmonary dysplasia (BPD), with a worldwide incidence of increased BPD year by year, with BPD occurring in premature infants at 6% -57% and mortality rates of 20.8%. Premature infants are mostly resuscitated and respiratory managed, and their antioxidant defense mechanisms are immature, and are particularly susceptible to toxicity of oxygen therapy, and in addition to the effects on the lungs, hyperoxia also leads to cell death of the developing brain, leading to motor and cognitive deficits, and causing severe economic and psychological burdens on society and families. Clinical studies have shown that BPD is an independent risk factor for delayed neural development and cerebral palsy in premature infants, and although steroids can reduce BPD, it may be associated with long-term adverse neurological consequences, or with increased neonatal mortality, and to date there is no specific or effective treatment. Therefore, how to prevent and treat respiratory tract and nerve dysfunction caused by hyperoxic lung and brain injury is a difficult point for research in the intensive care field of neonates. Stem cell therapy is expected to be a new breakthrough for the treatment of complex and multifactorial refractory and devastating neonatal diseases. The close link between hyperoxic lung and brain injury in premature infants suggests that lung protective therapy may also have a neuroprotective synergistic effect in premature infants. The etiology of BPD is closely related to hyperoxic lung injury, and during the critical period of lung development, neonatal exposure to high oxygen for a long time can block alveolar septal formation, reduce alveolar number, alveolitis and pulmonary vessel injury, manifested as pulmonary development obstruction, and exhibit the characteristics of normal lung growth "reprogramming", including airway hyperreactivity, abnormal pulmonary function examination, and emphysema change, secondary pulmonary hypertension and the like which in some cases persist into adulthood, increase mortality, even develop adult chronic obstructive pulmonary disease, with lifelong consequences. And hyperoxia-induced brain injury mainly occurs in the most fragile development stage, namely 23-32 weeks equivalent to the gestational age of human, and the antioxidase of P14 in mature rats can protect the newborn brain from oxidation and subsequent inflammation injury. Periventricular leukomalacia (PVL) is the result of encephalitis and oxidative damage based on BPD in clinically premature infants. The death or the subsequent neurological dysfunction and cerebral palsy of the serious patients occur, the specific mechanism is not known, and the specific mechanism is supposed to be mediated by a plurality of factors, including aging hyperoxia injury per se, inflammation, oxidative stress, apoptosis, secretory effect and growth factors such as VEGF and the like, and hyperoxia can destroy the blood brain barrier for a long time, is more easily influenced by paracrine, and even shows the change of development and behavior. It is very urgent to seek better treatment strategies to prevent and treat BPD, PVL and related complications to ensure the normal development of children. Hyperoxia can lead to growth arrest of developing normal organs such as the lung and brain by producing excessive amounts of oxygen radicals. Recent evidence suggests that loss of endogenous stem/progenitor cells required for normal cell differentiation and tissue repair may be the pathologic biological basis of this damage. Stem cells are primitive cells that can undergo self-renewal and have the potential to differentiate into multiple cell types, critical for normal development and to maintain normal physiology. Both basic and clinical studies support that loss of circulating and tissue progenitor cells may be the mechanism for abnormal developmental growth patterns of diseases such as BPD and PVL. Mesenchymal Stem Cells (MSCs) have been extensively studied in preclinical models of disease and in human transplantation studies. They have the ability to self-renew and their progeny have the ability to differentiate into various cell lineages, the sources of which include bone marrow, adipose tissue, umbilical cord blood, placenta, etc. The test result in a hyperoxia-induced neonatal rat BDP model shows that bone marrow-derived MSCs can inhibit inflammation, improve alveolar and pulmonary vascular injury and related pulmonary arterial hypertension, and umbilical mesenchymal stem cells (UC-MSc) are transplanted into the trachea, so that the protection effect is only in the early stage of inflammation, and the long-term protection effect can be continued for 100 days artificially. In phase I clinical trials, however, it has also been shown that trachea cannula allogeneic human ucmscs are safe and feasible in premature infants without long-term adverse reactions. Meanwhile, animal experiments also prove that the bone marrow-derived MSC can be used for permanent planting of high-oxygen damaged lung tissues in an environment similar to indoor air control environment, and the structure and the function of the lung tissues are recovered. However, the protective effect of MSC transplantation on hyperoxic brain injury is only rarely reported, and although the treatment effects of MSC spatial learning improvement and sensory motor function recovery have been confirmed in a rat model of traumatic brain injury, no clinical test using MSC in neonatal brain injury is shown. Whether intratracheal MSC transplantation has potential neuroprotective effects has not been verified. Successful transplantation methods of MSCs have been reported to date to include Intravenous (IV), Intratracheal (IT) or peritoneal (IP), local intracerebroventricular and Intranasal (IN) transplants, and the like, all of which have shown therapeutic efficacy. Although the optimal route of transplantation remains to be determined, a recent study has shown that IT transplantation using the neonatal rat hyperoxic model appears to be more effective, while IVMSC injection has no effect on lung histology or dysfunction. At present, the research of MSC in a body mostly passes through a venous route, but the MSC transplanted by intravenous injection cannot pass through a blood brain barrier and enters blood circulation through nasal cavity or respiratory mucosa absorption, the acting speed is high, the first-pass elimination can be avoided, the bioavailability is complete, and the toxic and side effects are generally less than that of systemic medication. The most effective method of delivering MSCs to injured brain appears to be the intranasal route, with intranasal delivery of hWJ-MSCs in rats preventing demyelination and microglial cell proliferation in the WMI model in the brain of preterm rats; intranasal administration of MSCs in mouse models has shown that treated animals show significant improvement in sensorimotor and cognitive functions, and that the protective effects caused by MSC treatment persist for life, with good safety, and no systemic pathological damage or neoplasia of the nose, brain or other organs is seen. Therefore, intratracheal or nasal MSC transplantation may be more effective than systemic venous route of MSC, but the aggregation, colonization and differentiation condition of MSC entering the body through the tracheal or nasal cavity route, the mechanism of homing to lung or brain after entering the body and how to further exert the injury repair function, and the critical treatment time window of transplantation needs to be studied deeply.
In summary, the problems of the prior art are as follows:
(1) how to administer the medicine by nasal route realizes the colonization of stem cells in the brain.
(2) Hyperoxia stimulates a hyperoxia mouse model of how to achieve simultaneous brain and lung injury.
The difficulty and significance for solving the technical problems are as follows: how the stem cells enter the brain for colonization along the olfactory nerve pathway, and how to select the injection position of the stem cells can ensure that the stem cells enter the olfactory nerve channel.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an animal model for protecting the high oxygen exposure lung and brain injury by umbilical cord mesenchymal stem cells.
The invention is realized in such a way that the establishment method of the UCMSc cell line marked by the lentivirus-mediated GFP comprises the following steps:
step one, constructing a GFP over-expression virus vector by using a lentivirus vector to obtain a titer of 1 × 108TU/mL of virus particles;
step two, separating and culturing UCMSc from umbilical cord, and standing at 37 deg.C and 5% CO2Culturing in an incubator, and identifying by flow cytometry.
Another object of the present invention is to provide a lentivirus-mediated GFP-tagged ucmscs cell line established by the establishment method of the lentivirus-mediated GFP-tagged ucmscs cell line.
Another object of the present invention is to provide a hyperoxic rat model constructed from the lentivirus-mediated GFP-tagged ucmscs cell line.
Another object of the present invention is to provide a method for constructing the rat model with hyperoxia, which comprises: 96 newborn full-term SD rats are randomly divided into 48 high-oxygen groups and 48 air groups within 6h after birth; placing the high-oxygen group newborn rats in an automatic oxygen generation box 30min after the birth, continuously inputting oxygen, maintaining the oxygen concentration of the oxygen box at more than 95%, and killing and taking materials after 7 days to the end; detecting oxygen concentration with oxygen concentration detector, absorbing CO in oxygen box with soda lime2Maintenance of CO2The concentration is less than 5%, the water vapor is absorbed by allochroic silica gel, and the humidity is kept between 50 and 60 percent; opening the box at regular time every day for 30min, adding water, feed and replacement padding, and exchanging the surrogate mother rats in the high oxygen group and the air group; the air group is placed in the indoor air.
Another objective of the invention is to provide a GFP-UC-MSc transplantation therapeutic hyperoxic rat model constructed by the lentivirus-mediated GFP-marked UCMSc cell line.
Another objective of the present invention is to provide a method for constructing a rat model for transplant therapy of GFP-UC-mscs, where the method for constructing the rat model for transplant therapy of GFP-UC-mscs comprises:
GFP-UC-MSc transplantation is performed through trachea local injection, after a rat is anesthetized, 0.1mL of GFP-UC-MSc suspension is instilled in a trachea, and PBS buffer solution with a corresponding volume is injected into a control group; performing nasal cavity injection stem cell transplantation, injecting 0.1mL of GFP-UC-MSc suspension into the left lower abdomen, and injecting PBS buffer solution with the corresponding volume into a control group; injecting stem cells into tail vein, injecting corresponding cells into rats 2 × 10 after anesthesia6In each control group, the tail vein of the control group is injected with PBS buffer solution with corresponding volume; finally, 96 rats are divided into 8 groups according to intervention, the hyperoxic group and the air group are respectively divided into four groups of IT, IN, IV and PBS, and each group comprises 12 rats; and (5) dead materials are obtained at 2d and 8w after intervention respectively, and corresponding detection is carried out.
In summary, the advantages and positive effects of the invention are: continuous high oxygen (90%, 14d) is used for inducing lung and brain injury in a rat model, meanwhile, a lentivirus vector mediated Green Fluorescent Protein (GFP) is constructed to mark human umbilical cord blood mesenchymal stem cells (UC-MSc), trachea, nasal cavity and vein transplantation is adopted, UC-MSc colonization, growth tracking and apoptosis detection are carried out, and histological evaluation, oxidative stress and inflammatory factor detection and function evaluation are carried out in parallel. The proper route, dosage and time window of stem cell administration are determined, the colonization of the human umbilical cord mesenchymal stem cell transplantation in the lung and the brain is confirmed, and the treatment effect of the human umbilical cord blood mesenchymal stem cell transplantation on hyperoxic lung and brain injury rats is finally clarified.
The invention uses slow virus carrier with GFP to transfect UC-MSc, and adopts 3 transplantation methods of trachea, nasal cavity and tail vein injection UC-MSc for comparison. The nasal cavity transplantation is initiated at home, and the self-check of three Chinese databases is not reported. Transplanting UC-MSc through trachea, nasal cavity and tail vein injection, tracking the colonization condition of lungs and brain, and exploring the simultaneous treatment effect of UC-MSc on SCI rat hyperoxic lung and brain injury.
Drawings
Fig. 1 is a flow chart of a construction method of an animal model for protecting umbilical cord mesenchymal stem cells from high oxygen exposure lung and brain injury provided by the embodiment of the invention.
FIG. 2 is a flow chart of an implementation of a method for constructing an animal model for protecting high oxygen exposure lung and brain injury by umbilical cord mesenchymal stem cells provided by an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention constructs a lentivirus-mediated GFP-labeled UC-MSc cell line; the transplantation of vein, trachea and nasal cavity is adopted to introduce the rat with high oxygen exposure, the field planting of human umbilical cord mesenchymal stem cell transplantation in lung and brain is confirmed, the treatment effect of the human umbilical cord blood mesenchymal stem cell transplantation on the rat with high oxygen lung and brain injury is clarified, and the action mechanism and clinical application of the rat are further disclosed to lay a foundation. Aiming at the nervous system and related diseases of the newborn, the basic and clinical research of cell therapy is carried out by using umbilical cord mesenchymal stem cells, the mechanism of the stem cell transplantation participating in the functional recovery of organs is researched, and the safety and the effectiveness of the cell therapy are evaluated.
The following detailed description of the principles of the invention is provided in connection with the accompanying drawings.
The animal model for protecting the high oxygen exposure lung and brain injury by the umbilical cord mesenchymal stem cells provided by the embodiment of the invention is 96 newborn term SD rats, and is randomly divided into 48 high oxygen groups and 48 air groups within 6h after the birth. Placing the high-oxygen group newborn rats in an automatic oxygen generation box 30min after the birth, continuously inputting oxygen, maintaining the oxygen concentration of the oxygen box at more than 95%, and killing and taking materials after 7 days to the end; detecting oxygen concentration with oxygen concentration detector, absorbing CO in oxygen box with soda lime2Maintenance of CO2The concentration is less than 5%, and the water vapor is absorbed by allochroic silica gel, and the humidity is kept between 50 and 60 percent. Opening the box at regular time every day for 30min, adding water, feed and replacement padding, and exchanging the surrogate mother mice in the high oxygen group and the air group to avoid the reasonHigh oxygen causes a decrease in feeding ability.
As shown in fig. 1, the method for constructing an animal model for protecting the high oxygen exposure lung and brain injury by umbilical cord mesenchymal stem cells provided by the embodiment of the present invention comprises the following steps:
s101: the rats are transplanted by the local Injection (IT) of GFP-UC-MSc in the trachea at 5 days after the birth, and 0.1mL (1 multiplied by 10 cells containing) of GFP-UC-MSc suspension is instilled in the trachea after the rats are anesthetized6) The control group was injected with the corresponding volume of PBS buffer;
s102: nasal injection for stem cell transplantation (IN), and left lower abdomen injection for GFP-UC-MSc suspension 0.1mL (containing cells 1X 10)6) The control group was injected with the corresponding volume of PBS buffer;
s103: the stem cell transplantation (IV) is carried out by tail vein injection, and after anesthesia, rats are respectively injected with corresponding cells by 2 multiplied by 10 through tail vein injection6In each control group, the tail vein of the control group is injected with PBS buffer solution with corresponding volume;
s104: 96 rats were divided into 8 groups by intervention, i.e. hyperoxic and air groups were each divided into four groups of IT, IN, IV, PBS, 12 per group; and (5) dead materials are obtained at 2d and 8w after intervention respectively, and corresponding detection is carried out.
The application of the principles of the present invention will now be described in further detail with reference to specific embodiments.
(1) Establishment of lentivirus-mediated GFP-labeled UCMSc (LV-GFP-UCMSc) cell line
Firstly, a GFP over-expression viral vector is constructed by using a lentiviral vector, and the company # Biotech is entrusted to construct and synthesize the GFP over-expression viral vector, and finally the titer is 1 multiplied by 108TU/mL of virus particles.
② separating and culturing UCMSc from umbilical cord, and placing at 37 deg.C and 5% CO2Culturing in an incubator, and identifying by flow cytometry.
(2) Hyperoxia rat model making and grouping
Neonatal full-term Sprague-Dawley (SD) rats 96, 48 each within 6h after birth were randomized into hyperoxic and air groups. Placing the high-oxygen group newborn rats in an automatic oxygen generation box 30min after the birth, continuously inputting oxygen, maintaining the oxygen concentration of the oxygen box at more than 95%, and killing and taking materials after 7 days to the end; detection with oxygen concentration measuring instrumentOxygen concentration, CO absorption by soda lime in oxygen tank2Maintenance of CO2The concentration is less than 5%, and the water vapor is absorbed by allochroic silica gel, and the humidity is kept between 50 and 60 percent. Opening the box at regular time for 30min every day, adding water, feed and replacement padding, and exchanging the surrogate mother rats in the hyperoxic group and the air group so as to avoid the reduction of feeding capacity caused by hyperoxic. The air group is arranged in indoor air, and the specific method and experimental control factors are the same as those of the hyperoxia group.
(3) GFP-UC-MSc transplantation treatment hyperoxia rat model
The 2 groups of rats are transplanted with UC-MSc at 5 days after birth by the following method: firstly, by means of trachea local Injection (IT) GFP-UC-MSc transplantation, after rat anesthesia, 0.1mL GFP-UC-MSc suspension (containing 1 multiplied by 10 cells) is instilled in trachea6) The control group was injected with the corresponding volume of PBS buffer; ② nasal injection stem cell transplantation (IN), and injecting GFP-UC-MSc suspension 0.1mL (containing cells 1X 10)6) The control group was injected with the corresponding volume of PBS buffer; ③ carrying out stem cell transplantation (IV) by tail vein injection, and respectively injecting corresponding cells 2 multiplied by 10 into rats after anesthesia by tail vein injection6In each control group, the tail vein was injected with a corresponding volume of PBS buffer.
Finally 96 rats were divided into 8 groups by intervention, i.e. hyperoxic and air groups were each divided into four IT, IN, IV, PBS groups of 12 rats each. And (5) dead materials are obtained at 2d and 8w after intervention respectively, and corresponding detection is carried out.
(4) Tissue morphology and cytokine detection
The lung and brain tissues were taken 2 days and 8 weeks after cell transplantation for pathology to assess injury, and tissue sections were examined by light microscopy using HE staining for Radioactive Alveolar Counts (RAC).
Tracing lung and brain tissues by a GFP positive cell fluorescence microscope, taking corresponding tissues 2 days and 8 weeks after cell transplantation, and detecting myelin sheath change by adopting MBP to mark positive nerve fibers; the immunofluorescence double-label detects astrocytes (GFAP), microglia (Iba1) and activated microglia/macrophages (ED1), and an ELISA system is adopted to detect the expression levels of IL-1 beta, IL-6, IL-10 and TNF-alpha in lung and brain tissues.
(5) Apoptosis detection
Rats in each group were sacrificed and brain and lung injury areas were cryosectioned, TUNEL for apoptosis level WB for caspase-3 protein expression.
(6) Behavioral assessment
Behavioral assessment on mNSS scoring before and after rat transplantation to evaluate the recovery condition of the nerve function; after transplantation, rats were tested for learning and memory using the Morris water maze.
The application principle of the present invention will be further described with reference to experiments.
The invention utilizes GFP-carrying lentivirus vector to transfect UC-MSc to construct GFP-labeled cell line. UC-MSc enters the rat body through tracheal injection, peritoneal cavity injection and tail vein injection, and the cells are traced through GFP to study cell homing.
(1) The UC-MSc is transfected by a lentivirus vector carrying GFP to construct a GPF-marked cell line, and the lentivirus carries green fluorescent protein, so that the differentiation and proliferation of the UC-MSc can be conveniently observed.
(2) Transplanting UC-MSc by tracheal injection, nasal injection and tail vein injection, and tracking and comparing UC-MSc introduction method for planting and growing UC-MSc.
(3) The evaluation and detection method comprises the steps of behavioral evaluation of brain function change and morphological and ELISA detection of dynamic change of related cells, apoptosis and cytokine levels.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (6)
1. A method for establishing a lentivirus-mediated GFP-labeled UCMSc cell line is characterized by comprising the following steps of:
step one, constructing a GFP over-expression virus vector by using a lentivirus vector to obtain a titer of 1 × 108TU/mL of virus particles;
step two, from the umbilicusSeparating and culturing UCMSc at 37 deg.C and 5% CO2Culturing in an incubator, and identifying by flow cytometry.
2. A lentivirus-mediated GFP-tagged ucmscs cell line established by the method of establishing a lentivirus-mediated GFP-tagged ucmscs cell line of claim 1.
3. A hyperoxic rat model constructed from the lentivirus-mediated GFP-tagged ucmscs cell line of claim 2.
4. The method for constructing the rat model with high oxygen content according to claim 3, wherein the method for constructing the rat model with high oxygen content comprises the following steps: 96 newborn full-term SD rats are randomly divided into 48 high-oxygen groups and 48 air groups within 6h after birth; placing the high-oxygen group newborn rats in an automatic oxygen generation box 30min after the birth, continuously inputting oxygen, maintaining the oxygen concentration of the oxygen box at more than 95%, and killing and taking materials after 7 days to the end; detecting oxygen concentration with oxygen concentration detector, absorbing CO in oxygen box with soda lime2Maintenance of CO2The concentration is less than 5%, the water vapor is absorbed by allochroic silica gel, and the humidity is kept between 50 and 60 percent; opening the box at regular time every day for 30min, adding water, feed and replacement padding, and exchanging the surrogate mother rats in the high oxygen group and the air group; the air group is placed in the indoor air.
5. A GFP-UC-MSc transplantation therapeutic hyperoxic rat model constructed from the lentivirus-mediated GFP-tagged ucmscs cell line of claim 2.
6. The method for constructing a GFP-UC-MSc transplant hyperoxygenated rat model of claim 5, wherein the GFP-UC-MSc transplant hyperoxygenated rat model is constructed by:
GFP-UC-MSc transplantation is performed through trachea local injection, after a rat is anesthetized, 0.1mL of GFP-UC-MSc suspension is instilled in a trachea, and PBS buffer solution with a corresponding volume is injected into a control group; the stem cell transplantation is performed by nasal cavity injection, GFP-UC-MSc suspension is injected into the left lower abdomen for 0.1mL, and PBS with the corresponding volume is injected into the control groupA buffer solution; injecting stem cells into tail vein, injecting corresponding cells into rats 2 × 10 after anesthesia6In each control group, the tail vein of the control group is injected with PBS buffer solution with corresponding volume; finally, 96 rats are divided into 8 groups according to intervention, the hyperoxic group and the air group are respectively divided into four groups of IT, IN, IV and PBS, and each group comprises 12 rats; and (5) dead materials are obtained at 2d and 8w after intervention respectively, and corresponding detection is carried out.
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