Umbilical cord mesenchymal source exosome combined with lipstatin

Abstract

The invention discloses an umbilical cord mesenchymal source exosome combined with lipstatin, and relates to the technical field of medical treatment and public health. The method comprises the following steps: firstly, completing collection of an umbilical cord specimen, carrying out isolated culture and identification of mesenchymal stem cells, then collecting third and fourth generation stem cell culture supernatant cultured for 48-72 hours, and carrying out filtering treatment, so as to obtain the umbilical cord mesenchymal stem cell culture supernatant. The exosome is prepared by using an ultracentrifugation method, the form and the particle size of the obtained exosome are identified, and the expression conditions of molecules such as symbolic proteins CD63, CD81 and TG101 of the exosome are detected by Western Blot; the culture supernatant derived from the mesenchymal stem cells and the exosome released by the mesenchymal stem cells are the most powerful alternative methods for novel cell-free treatment, and the lipstatin is an inhibitor of ferroptosis and can generate positive feedback influence on premature ovarian failure by inhibiting ferroptosis.

Description

Technical Field

[0001] The present invention relates to the field of medical and health technology, and in particular to an umbilical cord mesenchymal-derived exosome combined with lipstatin. Background Art

[0002] Premature ovarian failure (POF) refers to a syndrome of ovarian dysfunction in women before the age of 40, which is mainly manifested by increased gonadotropin and decreased estrogen levels, leading to perimenopausal symptoms in women and affecting the functions of multiple organs in the body. Currently, there is no clinical treatment to fundamentally restore ovarian function. Exosomes are extracellular vesicles with a diameter of about 30 to 150 nm secreted by living cells. They are cell signaling molecules that can selectively carry and transport proteins, lipids, microRNA, mRNA, etc. into cells, thereby affecting cell function. Umbilical cord mesenchymal stem cell-derived exosomes (huMSCs-exo) have attracted widespread attention from researchers because of their lack of ethical controversy, rapid onset of action, low immunogenicity, non-tumorigenicity, easy storage and transportation, and higher safety. This study aims to explore the therapeutic effect of huMSCs-exo on the rat POF model, so as to provide a new means to provide a new level of treatment for POF;

[0003] There is no specific drug treatment for premature ovarian failure. A large number of preclinical studies have shown that the use of mesenchymal stem cells to treat premature ovarian failure shows significant efficacy, and clinical trials are still ongoing. However, due to the risk of stem cell tumorigenesis, its clinical application and promotion are affected. In response to the above problems, the inventors proposed a combination of exosomes derived from umbilical cord mesenchyme and lipstatin to solve the above problems. Summary of the invention

[0004] In order to solve the problem that there is no specific drug treatment for premature ovarian failure, the purpose of the present invention is to provide an umbilical cord mesenchymal-derived exosome combined with lipstatin.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solution: an umbilical cord mesenchymal-derived exosome combined with lipstatin, comprising the following steps:

[0006] S1: First, complete the collection of umbilical cord specimens and perform isolation, culture and identification of mesenchymal stem cells. Then, collect the supernatant of the 3rd and 4th generation stem cells cultured between 48-72 hours, filter them, prepare exosomes by ultracentrifugation, identify the morphology and particle size of the obtained exosomes, and detect the expression of exosome marker proteins such as CD63, CD81, TG101, etc. by Western Blot.

[0007] S2: Cisplatin (4 mg / kg) was then used as an inducer to establish the model through intraperitoneal injection. After the model was established, hUCMSCs-exos treatment was performed once a week, with 1 mL of exosomes injected intraperitoneally at a concentration of 0.75 mg / ml, and Lip-1 treatment was performed by intraperitoneal injection at a concentration of 60 mg / kg.

[0008] S3: Finally, the follicle development and immune cell levels of the model mice before and after treatment were analyzed to verify the molecular mechanism of the immunosuppressive function of exosomes. The expression level of the target gene Gpx4 that inhibits ferroptosis changed after treatment, verifying that the effectiveness of Lip-1 treatment is related to the inhibition of ferroptosis.

[0009] Preferably, the method for identifying exosomes can be detected by transmission electron microscopy and Nanosight.

[0010] Preferably, HE staining is performed to observe the development of follicular cells.

[0011] Preferably, the levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2) in serum are detected by ELISA.

[0012] Preferably, the gene expression profile of the cellular components is quantified using the CIBERSORT deconvolution method to identify the infiltration of immune cells in ovarian tissue.

[0013] Preferably, the mRNA expression level of the ferroptosis target gene Gpx4 is detected.

[0014] Preferably, the protein expression level of the ferroptosis target gene Gpx4 is detected by Western Blot.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] 1. The culture supernatant derived from mesenchymal stem cells and the exosomes released by mesenchymal stem cells in the present invention are the most powerful alternative methods for novel cell-free therapy, and lipstatin is an inhibitor of ferroptosis, which can produce a positive feedback effect on premature ovarian failure by inhibiting ferroptosis;

[0017] 2. The present invention studies the exosome combined with Lip- therapy in the mouse model of premature ovarian failure and explores the effectiveness of this treatment regimen and its molecular mechanism, providing new ideas and experimental basis for improving the level of clinical ovarian treatment. DETAILED DESCRIPTION

[0018] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] Embodiment: The present invention provides a technical solution: an umbilical cord mesenchymal-derived exosome combined with lipstatin, comprising the following steps:

[0020] S1: First, complete the collection of umbilical cord specimens and perform isolation, culture and identification of mesenchymal stem cells. Then, collect the supernatant of the 3rd and 4th generation stem cells cultured between 48-72 hours, filter them, prepare exosomes by ultracentrifugation, identify the morphology and particle size of the obtained exosomes, and detect the expression of exosome marker proteins such as CD63, CD81, TG101, etc. by Western Blot.

[0021] S2: Cisplatin (4 mg / kg) was then used as an inducer to establish the model through intraperitoneal injection. After the model was established, hUCMSCs-exos treatment was performed once a week, with 1 mL of exosomes injected intraperitoneally at a concentration of 0.75 mg / ml, and Lip-1 treatment was performed by intraperitoneal injection at a concentration of 60 mg / kg.

[0022] S3: Finally, the follicle development and immune cell levels of the model mice before and after treatment were analyzed to verify the molecular mechanism of the immunosuppressive function of exosomes. The expression level of the target gene Gpx4 that inhibits ferroptosis changed after treatment, verifying that the effectiveness of Lip-1 treatment is related to the inhibition of ferroptosis.

[0023] Among them, exosomes can be identified through transmission electron microscopy and Nanosight detection.

[0024] HE staining was used to observe the development of follicular cells.

[0025] The levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2) in serum were detected by ELISA.

[0026] The CIBERSORT deconvolution method was used to quantify the gene expression profiles of cellular components and identify the infiltration of immune cells in ovarian tissue.

[0027] Among them, the mRNA expression level of Gpx4, the target gene of ferroptosis, was detected.

[0028] Among them, Western Blot detection of the protein expression level of the target gene Gpx4 of ferroptosis. Working principle: The human umbilical cord tissue specimens described in this invention were provided by our hospital, and this study was approved by the ethics committee of our hospital and the informed consent of the donor was obtained. First, the stem cells derived from umbilical cord tissue were isolated and cultured using the tissue adhesion method. The nutrient solution consisted of 5% UltraGRO and α-MEM basal culture medium containing 0.032% medical heparin sodium. The digestion subculture method was used for expansion culture and cell purity identification, and the cell culture supernatant of the 3rd to 4th generations was collected; the human umbilical cord mesenchymal stem cell culture supernatant was centrifuged at 4℃300× for 10 min to remove suspended cells and dead cells; 4℃2000×g was centrifuged for 10 min to remove impurities such as cell debris; the supernatant was filtered through a 0.22um sterile filter to further remove bacteria and other impurities; 4℃100000×g, 120min ultracentrifugation was performed to obtain exosome precipitation, and 200μl PBS was added to wash once after removing the supernatant; 4℃100000×g, 120min ultracentrifugation was performed again, the supernatant was discarded, and 100μl PBS was added to obtain high-purity mesenchymal stem cell-derived exosomes; after aliquoting, it was stored in a -80℃ refrigerator for future use; negative staining method was used for transmission electron microscopy (TEM) detection of exosomes. For negative staining, approximately 10 μl of exosome resuspension was placed on a copper mesh plate and allowed to stand at 4°C for 3 min. The mesh was washed and dried with filter paper, then negatively stained in 1% phosphotungstic acid for 2 min, then washed with distilled water and dried in the dark. The exosome particles were resuspended in 500 μL sterile PBS and filtered with a 0.22 μm syringe filter (Millipore) using a NanoSight NS 300 system (NanoSight Technology, Malvern, UK). The sample was diluted (1:4 000) until individual nanoparticles could be tracked. The number and size of exosomes were directly tracked by the Brownian motion rate of the exosomes, and rapid video capture and data analysis were performed to detect the size and concentration (particles / ml) of the exosomes. The purified exosomes were lysed using a special lysis buffer for exosomes for western blot identification. The protein molecules detected were: CD63, CD9, and HSP70; the rabbit anti-human primary antibodies used were as follows: CD63 monoclonal antibody (1:500), CD9 monoclonal antibody (1:500), and HSP70 monoclonal antibody (1:500); goat anti-rabbit secondary antibody (1:10000) was used to identify the expression of marker protein molecules of exosomes; when constructing the animal model, 25 SD rats were randomly divided into five groups: A, B, C, D, and E, with 5 rats in each group.Among them, the four groups B, C, D, and E were given cisplatin (4 mg / kg) by intraperitoneal injection into rats once a week for modeling for 2 weeks. Group A was used as the normal control group and injected with an equal volume of saline at the same time. After the modeling was completed, treatment began in the third week. The Lip-1 treatment group was injected with 60 mg / kg of Lip-1 by intraperitoneal injection. The hUCMSCs-exos treatment group was treated once a week, and 1 mL of exosomes was injected intraperitoneally at a concentration of 0.75 mg / ml. The treatment doses of the hUCMSCs-exos combined with Lip-1 treatment groups were as follows: hUCMSCs-exos0 (0.75 mg / ml) was injected intraperitoneally, and the model rats of the treatment group injected intraperitoneally with Lip-1. The normal control group was injected intraperitoneally with an equal amount of saline; and the mental state, activity, coat gloss, reaction to the outside world, and food intake of the rats in each group needed to be observed daily. After the treatment, the rats were killed by CO method, and venous blood was collected by periocular venous plexus blood collection method, and ovarian tissue was obtained. Detection and analysis of immune cell infiltration in ovarian tissue, HE staining and PAS staining of tissue sections, blood was collected from the orbits on days 1, 7, 14, 21, and 28 of modeling, and serum was separated. ELISA kit was used to detect the levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) in serum, and left lung tissue was fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin, and sliced ​​at 4 μm. ① Obtain the ovarian tissue of mice, fix it, make paraffin sections, perform HE staining, and observe the differences in immune cells and follicle development in the ovarian tissue of mice in different groups under a microscope. ②Ovarian tissue was obtained, fixed and sliced ​​into paraffin sections for PAS testing, which was mainly used to detect glycogen, glycosaminoglycans and other polysaccharide-rich substances in the tissue; then the ovarian tissue specimens were tested and analyzed by ①extracting ovarian tissue RNA to detect the expression of the ferroptosis marker gene GPX4 in its mRNA; ②Western Blot was used to detect the protein expression of the ferroptosis marker gene GPX4; and ③the infiltration of immune cells in the ovarian tissue.

[0029] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. An umbilical cord mesenchymal-derived exosome combined with lipstatin, characterized in that: The following steps are involved: S1: First, complete the collection of umbilical cord specimens and perform isolation, culture and identification of mesenchymal stem cells. Then, collect the supernatant of the 3rd and 4th generation stem cells cultured between 48-72 hours, filter them, prepare exosomes by ultracentrifugation, identify the morphology and particle size of the obtained exosomes, and detect the expression of exosome marker proteins such as CD63, CD81, TG101, etc. by Western Blot. S2: Cisplatin (4 mg / kg) was then used as an inducer to establish the model through intraperitoneal injection. After the model was established, hUCMSCs-exos treatment was performed once a week, with 1 mL of exosomes injected intraperitoneally at a concentration of 0.75 mg / ml, and Lip-1 treatment was performed with intraperitoneal injection at a concentration of 60 mg / kg. S3: Finally, the follicle development and immune cell levels of the model mice before and after treatment were analyzed to verify the molecular mechanism of the immunosuppressive function of exosomes. The expression level of the target gene Gpx4 that inhibits ferroptosis changed after treatment, verifying that the effectiveness of Lip-1 treatment is related to the inhibition of ferroptosis.

2. The umbilical cord mesenchymal-derived exosomes combined with lipstatin according to claim 1, characterized in that: Among them, exosomes can be identified through transmission electron microscopy and Nanosight detection.

3. The umbilical cord mesenchymal-derived exosomes combined with lipstatin according to claim 1, characterized in that: HE staining was used to observe the development of follicular cells.

4. The umbilical cord mesenchymal-derived exosomes combined with lipstatin according to claim 1, characterized in that: The levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2) in serum were detected by ELISA.

5. The umbilical cord mesenchymal-derived exosomes combined with lipstatin according to claim 1, characterized in that: The CIBERSORT deconvolution method was used to quantify the gene expression profiles of cellular components and identify the infiltration of immune cells in ovarian tissue.

6. The umbilical cord mesenchymal-derived exosomes combined with lipstatin according to claim 1, characterized in that: Among them, the mRNA expression level of Gpx4, the target gene of ferroptosis, was detected.

7. The umbilical cord mesenchymal-derived exosomes combined with lipstatin according to claim 1, characterized in that: Western Blot detection of the protein expression level of Gpx4, a target gene of ferroptosis.