Establishment method of umbilical cord mesenchymal stem cells highly expressing IGF-1 and application of umbilical cord mesenchymal stem cells in premature ovarian failure

Abstract

The invention provides an establishment method of umbilical cord mesenchymal stem cells with high expression of IGF-1 (Insulin-like Growth Factor 1) and application of the umbilical cord mesenchymal stem cells in premature ovarian failure. The construction method of the umbilical cord mesenchymal stem cells with high expression of IGF-1 comprises the step of transfecting the umbilical cord mesenchymal stem cells with the IGF-1 gene, so that the umbilical cord mesenchymal stem cells highly express IGF-1. The invention also provides application of the umbilical cord mesenchymal stem cell with high expression of IGF-1 in preparation of drugs for treating premature ovarian failure. According to the invention, the IGF-1 gene is transfected to the hUC-MSC, so that the hUC-MSC highly expresses the IGF-1, and the hUC-MSC secretes a large amount of IGF-1. The ovarian function is improved through the immunoregulation effect of hUC-MS, and meanwhile, the effect of resisting apoptosis of granular cells is achieved through IGF-1 secreted by hUC-MS. And the treatment effect on the premature ovarian failure is achieved through synergism of the two effects.

Description

Technical Field

[0001] The invention belongs to the field of cell therapy, and specifically relates to a method for establishing umbilical cord mesenchymal stem cells that highly express IGF-1 and an application thereof in premature ovarian failure. Background Art

[0002] Premature ovarian insufficiency (POI), also known as premature ovarian failure, refers to the ovarian dysfunction in women before the age of 40, mainly manifested by menstrual abnormalities (amenorrhea, rare or frequent menstruation), elevated follicle-stimulating hormone levels (FSH>25U / L), and fluctuating decreases in estrogen levels. The pathophysiological characteristics of POI are: premature exhaustion of ovarian follicles. POI often leads to some serious consequences, such as the risk of cardiovascular disease, osteoporosis, cognitive decline and mood disorders, and reduced quality of life for patients.

[0003] There is still no effective way to protect and restore ovarian function for POI patients. Hormone replacement therapy is the main strategy for treating POI patients, but it cannot effectively improve ovarian function; and hormone therapy increases the risk of tumor occurrence and development. Therefore, a safe and effective new method to improve ovarian function is urgently needed in the clinic to replace traditional treatment.

[0004] A large number of studies have shown that umbilical cord mesenchymal stem cells (hUC-MSC) have immunomodulatory effects, can reduce inflammatory responses in ovarian tissue, improve the microenvironment of ovarian tissue, and improve ovarian function. For example, hUC-MSC can regulate cell subsets, inhibit the differentiation of Th1 and Th17 subsets, and promote the proliferation of Treg subsets; hUC-MSC can inhibit the secretion of inflammatory factor TNF-α and promote the secretion of anti-inflammatory factor IL-10. However, a large number of studies have shown that in addition to inflammatory responses, patients with premature ovarian insufficiency also have apoptosis of granulosa cells in the ovaries, which leads to premature ovarian failure.

[0005] Studies have shown that IGF-1 is an anti-apoptotic protein that can promote the expression of insulin-like growth factor 1 receptor (IGF1R), thereby activating the PI3K / Akt / FOXO3a signaling pathway and promoting the proliferation of ovarian granulosa cells and the development of primordial follicles. However, research data show that hUC-MSCs basically have no ability to secrete IGF-1 (see Table 1 for details). Therefore, when hUC-MSCs are injected into ovaries with ovarian dysfunction, they can only play an immunomodulatory role and have limited effect on the apoptosis of granulosa cells in the ovaries.

[0006] There is still no effective way to protect and restore ovarian function for POI patients. Hormone replacement therapy is the main strategy for treating POI patients, but it cannot effectively improve ovarian function; and hormone therapy increases the risk of tumor occurrence and development. Therefore, a safe and effective new method to improve ovarian function is urgently needed in the clinic to replace traditional treatment. Summary of the invention

[0007] In order to solve the above technical problems existing in the prior art, the present invention provides a method for establishing umbilical cord mesenchymal stem cells that highly express IGF-1 and application thereof in premature ovarian failure.

[0008] The specific technical solutions of the present invention are as follows:

[0009] The first object of the present invention is to provide a method for constructing umbilical cord mesenchymal stem cells that highly express IGF-1, wherein the method comprises transfecting the IGF-1 gene into the umbilical cord mesenchymal stem cells so that the umbilical cord mesenchymal stem cells highly express IGF-1.

[0010] Furthermore, the method comprises the following steps:

[0011] S1, obtain IGF-1 gene;

[0012] S2, transfer the IGF-1 gene into the lentiviral packaging plasmid;

[0013] S3, transfecting the umbilical cord mesenchymal stem cells with the IGF-1 gene lentivirus obtained in S2 to obtain the umbilical cord mesenchymal stem cells that highly express IGF-1.

[0014] Furthermore, the IGF-1 gene is amplified from human blood sample DNA.

[0015] Further, the amplification primers are:

[0016] Upstream primer: 5′-GCAGTCTTCCAACCCAA-3′ (SEQ ID No. 1);

[0017] Downstream primer: 5'-ACATCTCCAGCCTCCTTA-3' (SEQ ID No. 2).

[0018] Furthermore, the lentiviral packaging plasmid is selected from pUC57, pGP, pVSVG, and PLVX-EF1a.

[0019] The second object of the present invention is to provide umbilical cord mesenchymal stem cells that highly express IGF-1 obtained by the aforementioned construction method, wherein the constructed umbilical cord mesenchymal stem cells highly express IGF-1.

[0020] The third object of the present invention is to provide the use of the aforementioned umbilical cord mesenchymal stem cells that highly express IGF-1 in the preparation of a drug for treating premature ovarian failure.

[0021] The beneficial effects of the present invention are:

[0022] In order to obtain hUC-MSCs that can secrete a large amount of IGF-1, hUC-MSCs have both paracrine function and immunomodulatory effect, and improve premature ovarian failure through two pathways. The present invention constructs an IGF-1 gene lentiviral vector, and transfects it into hUC-MSCs, and detects the expression of IGF-1 protein in the cells by Western Blot to verify whether the IGF-1 gene can be highly expressed in hUC-MSCs.

[0023] After successfully constructing hUC-MSCs that highly express IGF-1, in order to confirm its effect on the animal model of premature ovarian failure, an animal model of premature ovarian failure was constructed. IGF was injected intraperitoneally, and hUC-MSCs and IGF-1hUC-MSCs were injected in situ into the ovaries. The pharmacodynamic indicators of the animal model of premature ovarian failure in each experimental group were compared to evaluate the effect of IGF-1hUC-MSCs on the animal model of premature ovarian failure.

[0024] The present invention transfects hUC-MSC with IGF-1 gene, so that hUC-MSC highly expresses IGF-1 and secretes a large amount of IGF-1. The ovarian function is improved through the immunomodulatory effect of hUC-MSC, and the IGF-1 secreted by hUC-MSC simultaneously exerts an anti-apoptosis effect on granulosa cells. The above two effects synergistically exert a therapeutic effect on premature ovarian failure.

[0025] Therefore, the IGF-1 gene was integrated into the hUC-MSC genome through lentiviral transfection, and hUC-MSC was endowed with the ability to highly express IGF-1. An animal model of premature ovarian insufficiency was constructed, and hUC-MSC highly expressing IGF-1 was injected into the ovaries. The immunomodulatory effect of hUC-MSC and the anti-apoptotic effect of IGF-1 protein jointly improved ovarian function, thereby playing a therapeutic role in premature ovarian insufficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 .Ovarian index of each experimental group;

[0027] Among them: Mean±SD, *Compared with the blank group, ***P<0.001; #Compared with the model group, ##P<0.01, ###P<0.001; &Compared with the IGF-1 group, &P<0.05;

[0028] Figure 2 .HE staining pathological photos of ovaries of rats in each group;

[0029] Figure 3 .Serum estradiol content in the serum of rats in each group;

[0030] Figure 4 .Follitropin levels in serum of rats in each group. DETAILED DESCRIPTION

[0031] The present invention is further explained below with reference to the examples, but the examples do not limit the present invention in any form.

[0032] Example 1

[0033] 1. Operation steps

[0034] (1) Extract DNA from human blood samples;

[0035] (2) IGF-1 gene sequencing: After designing IGF-1 primers, PCR amplification was performed using human blood sample DNA as a template, and the amplified product was sequenced.

[0036] Upstream primer: 5′-GCAGTCTTCCAACCCAA-3′;

[0037] Downstream primer: 5′-ACATCTCCAGCCTCCTTA-3′.

[0038] (3) Lentivirus preparation:

[0039] 1) Resuscitate hUC-MSCs into T25 cell culture flasks and place in a 37°C, 5% CO2 incubator for 48 hours overnight.

[0040] 2) After adding 5 mL of PBS to rinse hUC-MSCs, add 2 mL of trypsin to digest the cells for 5 minutes, and add FBS to terminate the digestion.

[0041] 3) RNA extraction, reverse transcription and target gene amplification: RNA of hUC-MSCs was extracted, reverse transcribed into cDNA, and used as a template for polymerase chain reaction to amplify the IGF-1 target gene.

[0042] 4) The above PCR product was subcloned into PLVX-EF1a vector (product name: Lenti-X Expression System (EF1α Version), purchased from clontech) to construct the PLVX-IGF1 vector.

[0043] 5) Prepare a 15 cm dish, inoculate 5×106 293T cells (purchased from ATCC), add complete culture medium (DMEM high glucose, 10% FBS, double antibody), place in a 37°C, 5% CO2 incubator, and culture overnight.

[0044] 6) Take out 100μM linearized polyethyleneimine (Polyethylenimine Linear, PEI) and lentiviral packaging plasmids (PLVX-IGF1, pGP, pVSVG) from the refrigerator, thaw at room temperature, and mix thoroughly by pipetting up and down. Take out the PBS buffer and warm it to room temperature. Take 2mL PBS to one well of a 6-well plate, add 10μg PLVX-IGF1, 4μg pGP, and 2μg pVSVG respectively, pipette up and down to mix thoroughly, add 18μL 100μM PEI, immediately pipette up and down to mix, and let stand at room temperature for 10 minutes.

[0045] 7) Add the above DNA / PEI complex dropwise into a 15 cm culture dish, gently shake the dish to mix thoroughly. Place the dish in a 37°C, 5% CO2 incubator, and after 6-8 hours of culture, remove the culture medium containing the transfection reagent and replace it with fresh complete culture medium.

[0046] 8) After continuous culture for 48 hours, collect the supernatant of the culture medium containing the virus in the culture dish, filter it with a 0.45μm filter membrane, add 20% volume of 50% polyethylene glycol (PEG) 6000 solution, incubate at 4 degrees for 2 hours, transfer to a centrifuge tube, balance it, and centrifuge it at 3000xg at 4℃ for 0.5 hours. After the centrifugation, carefully remove the liquid in the centrifuge tube in the biosafety cabinet, add 500μL PBS buffer to resuspend the precipitate, and obtain the Leti-IGF-1 lentivirus, which is stored at -80℃.

[0047] (4) IGF-1 gene lentivirus transfection of hUC-MSCs: hUC-MSCs were seeded in 6-well plates, and when the cell density reached 60%, the cells were transfected with PLVX-IGF1 lentivirus.

[0048] (5) Identification of IGF-1 gene expression in hUC-MSC: 48 h after transfection, the transfected hUC-MSC cells were collected and the expression of IGF-1 protein was detected by Western blot.

[0049] (6) Construction of premature ovarian failure model: 50 SD rats were intraperitoneally injected with cyclophosphamide (CTX) every day for 15 consecutive days. The first injection dose was 50 mg / kg, and then the dose was continuously injected at 50 mg / kg for 14 consecutive days to construct an animal model of premature ovarian failure; 10 SD rats were intraperitoneally injected with phosphate buffer as a blank control group.

[0050] (7) In situ injection of hUC-MSC cells transfected with IGF-1 into the ovaries: The experiment was divided into 6 groups: blank control group, model control group, positive control group, IGF-1 group, hUC-MSC group, and IGF-1-hUC-MSC group. The blank control group received no treatment; the model control group received in situ injection of hUC-MSC culture medium into the ovaries; the positive control group received oral administration of 0.1 mg / kg estradiol valerate and 0.8 mg / kg medroxyprogesterone acetate; the IGF-1 group received intraperitoneal injection of IGF-1 solution at a dose of 10 μg / kg.d for 7 consecutive days; the hUC-MSC group received hUC-MSC injection at 3.0×105 / ovary on both sides of the ovaries; the IGF-1-hUC-MSC group received IGF-1-hUC-MSC injection at 3.0×105 / ovary on both sides of the ovaries.

[0051] (8) Pharmacodynamic evaluation: Fifteen days after administration, blood was collected from the orbital venous plexus and serum was collected for the detection of estradiol (E2) and follicle stimulating hormone (FSH) levels. The ovaries on both sides were weighed and the ovarian index was calculated. The ovaries were then immersed in formalin fixative to analyze the number of follicles at different levels in the ovaries.

[0052] Experimental Example 1

[0053] The IGF-1 secretion ability of IGF-1-hUC-MSC and hUC-MSC was compared. Compared with the blank culture medium, the IGF-1 secretion content of hUC-MSC culture supernatant was only slightly increased, which showed that hUC-MSC basically did not secrete IGF-1; the IGF-1 content in the IGF-1-hUC-MSC culture supernatant was 49.85ng / ml, which was about 10 times the IGF-1 content in the hUC-MSC culture supernatant. Therefore, it was concluded that IGF-1-hUC-MSC had the ability to highly express IGF-1 and IGF-1-hUC-MSC was an umbilical cord mesenchymal stem cell that highly expressed IGF-1. See Table 1 for details.

[0054] Table 1. Comparison of IGF-1 secretion between IGF-1-hUC-MSC and hUC-MSC

[0055]

[0056] Experimental Example 2

[0057] The ovarian weight of the ovarian group injected with IGF, hUC-MSC and IGF-1-hUC-MSC was significantly improved compared with the model group, and the ovarian weight of the IGF-1-hUC-MSC group was higher than that of the IGF-1 and hUC-MSC groups, and there was a significant difference compared with the IGF group. Figure 1shown.

[0058] Experimental Example 3

[0059] In situ injection of IGF-1-hUC-MSC into the ovaries can significantly improve the number of dominant follicles in the ovarian tissue and reduce the number of atretic ovaries. It can be seen that in situ injection of IGF-1-hUC-MSC into the ovaries can significantly improve ovarian function compared with injection of IGF and hUC-MSC, and its therapeutic effect is the most obvious. See Table 2 for details.

[0060] Table 2. Statistical data of ovarian follicle counts in rats of each group

[0061]

[0062]

[0063] Experimental Example 4

[0064] In situ injection of IGF, hUC-MSC and IGF-1-hUC-MSC into the ovaries can significantly increase the number of mature follicles in the ovaries and reduce the number of atretic follicles. The number of mature follicles in the ovaries of the IGF-1-hUC-MSC injection group was significantly higher than that of the IGF-1 and hUC-MSC groups, and the number of atretic follicles was significantly lower than that of the IGF-1 and hUC-MSC groups. This shows that in situ injection of hUC-MSC transfected with the IGF-1 gene into the ovaries can maximize the therapeutic effect of premature ovarian failure ( Figure 2 ).

[0065] Experimental Example 5

[0066] The serum E2 levels of the ovarian orthotopic injection of IGF, hUC-MSC and IGF-1-hUC-MSC groups were higher than those of the model group, and compared with the IGF-1 group, the serum E2 levels of the hUC-MSC and IGF-1-hUC-MSC groups were significantly increased (P<0.01), indicating that hUC-MSC and hUC-MSC transfected with IGF-1 can increase the serum E2 content of POI rats. Figure 3 shown.

[0067] Experimental Example 6

[0068] The serum follicle-stimulating hormone levels in the ovarian orthotopic injection IGF and IGF-1-hUC-MSC groups were lower than those in the model group, and the serum follicle-stimulating hormone level in the IGF-1-hUC-MSC group was significantly lower than that in the IGF group and hUC-MSC group. hUC-MSC transfected with IGF-1 can reduce the serum follicle-stimulating hormone level in rats with premature ovarian failure. Figure 4 shown.

[0069] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing umbilical cord mesenchymal stem cells that highly express IGF-1, characterized in that: The method comprises the steps of transfecting the IGF-1 gene into umbilical cord mesenchymal stem cells, so that the umbilical cord mesenchymal stem cells highly express IGF-1.

2. The construction method according to claim 1, characterized in that: The method comprises the following steps: S1, obtain IGF-1 gene; S2, transfer the IGF-1 gene into the lentiviral packaging plasmid; S3, transfecting the umbilical cord mesenchymal stem cells with the IGF-1 gene lentivirus obtained in S2 to obtain the umbilical cord mesenchymal stem cells that highly express IGF-1.

3. The construction method according to claim 2, characterized in that: The IGF-1 gene is amplified from human blood sample DNA.

4. The construction method according to claim 3, characterized in that: The amplification primers are: Upstream primer: 5′-GCAGTCTTCCAACCCAA-3′; Downstream primer: 5′-ACATCTCCAGCCTCCTTA-3′.

5. The construction method according to claim 2, characterized in that: The lentiviral packaging plasmid is selected from pGP, pVSVG, and PLVX-EF1a.

6. Umbilical cord mesenchymal stem cells highly expressing IGF-1 obtained by the construction method of claim 1, characterized in that: The constructed umbilical cord mesenchymal stem cells highly express IGF-1.

7. Use of the umbilical cord mesenchymal stem cells highly expressing IGF-1 according to claim 6 in the preparation of a drug for treating premature ovarian failure.

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