Application of mesenchymal stem cells in drugs for treating premature ovarian failure

Abstract

The present invention relates to the application of mesenchymal stem cells in drugs for treating premature ovarian failure. Through previous research, the inventors of the present invention found that hsa_circ_0032434 has a significant difference in expression in patients with premature ovarian failure and has the advantage of being able to serve as a target for treating premature ovarian failure. In response to this, in the present invention, the inventors continued to analyze the true difference of hsa_circ_0032434 in patients with premature ovarian failure by quantitative PCR, and targeted the design of siRNA for down-regulating hsa_circ_0032434, and constructed umbilical cord mesenchymal stem cells transfected with siRNA, confirming that the umbilical cord mesenchymal stem cells prepared in the present invention have good potential for treating premature ovarian failure.

Description

Technical Field

[0001] The present invention relates to the technical field of the treatment of premature ovarian failure, and particularly relates to the application of mesenchymal stem cells in drugs for treating premature ovarian failure. Background Art

[0002] Premature ovarian failure (POF) refers to the phenomenon of amenorrhea before the age of 40 caused by ovarian function failure. Its characteristics are primary or secondary amenorrhea accompanied by elevated levels of serum gonadotropins and decreased estrogen levels, and a series of hypoestrogen symptoms to varying degrees, such as hot flashes, flushing, decreased libido, etc. The etiology and pathogenesis of premature ovarian failure are relatively complex and not yet fully understood. The treatment of premature ovarian failure mainly uses hormone therapy. However, clinical studies have found that treating premature ovarian failure with hormones will increase the incidence of breast cancer, endometrial cancer, cardiovascular diseases, and stroke.

[0003] In recent years, with the gradual in-depth research on stem cells by global medical researchers, rich achievements have also been made in clinical scientific research on intervening and repairing premature ovarian failure in this way. A large number of research results show that stem cells enable women with premature ovarian failure to restore their fertility and delay the menopause of patients. However, the mesenchymal stem cells reported currently mainly come from bone marrow, and the defect of stem cells is that the treatment effect is poor and not ideal, and it is still necessary to improve the stem cells to overcome their technical defects. Summary of the Invention

[0004] Aiming at the technical problems existing in the prior art, the present invention provides the application of mesenchymal stem cells in drugs for treating premature ovarian failure. Through preliminary research, the present invention found that hsa_circ_0032434 has a significant expression difference in patients with premature ovarian failure and has the advantage of being able to be used as a target for treating premature ovarian failure. In view of this, in the present invention, the inventor continued to analyze the real difference of hsa_circ_0032434 in patients with premature ovarian failure by quantitative PCR, and designed siRNA for down-regulating hsa_circ_0032434 by targeting, and constructed umbilical cord mesenchymal stem cells transfected with siRNA, and confirmed that the umbilical cord mesenchymal stem cells prepared by the present invention have good potential for treating premature ovarian failure.

[0005] One of the purposes of the present invention is the use of hsa_circ_0032434 in the preparation of drugs for preventing or treating premature ovarian failure, and the sequence of hsa_circ_0032434 is shown in SEQ ID NO.1.

[0006] Preferably, the drug is a substance for down-regulating the expression of hsa_circ_0032434.

[0007] Furthermore, the present invention provides a kind of umbilical cord mesenchymal stem cells, and the mesenchymal stem cells are transfected with a substance that down-regulates the expression of hsa_circ_0032434.

[0008] Preferably, the substance includes but is not limited to siRNA, shRNA, ASO, CRISPR gene editing system, etc.

[0009] Preferably, the substance is siRNA.

[0010] More preferably, the sequence of the siRNA is shown as any one of SEQ ID NO.6-8.

[0011] Furthermore, the present invention also provides the use of the above-mentioned mesenchymal stem cells in the preparation of drugs for preventing or treating premature ovarian failure.

[0012] Preferably, the mesenchymal stem cells are administered by intravenous injection.

[0013] More preferably, the dosage of the intravenous injection is 1×10 5 -1×10 9 cells per time.

[0014] Furthermore, the present invention also provides a method for treating premature ovarian failure with mesenchymal stem cells, and the method includes administering the mesenchymal stem cells of the present invention as described above.

[0015] The advantages of the present invention are as follows: The present invention discovers for the first time that hsa_circ_0032434 has a significant difference in expression in patients with premature ovarian failure, and has the advantage of being able to be used as a target for treating premature ovarian failure. And siRNA designed to target and down-regulate hsa_circ_0032434 is constructed, and umbilical cord mesenchymal stem cells transfected with siRNA are prepared, which proves that the umbilical cord mesenchymal stem cells prepared by the present invention have good potential for treating premature ovarian failure. Brief Description of the Drawings

[0016] Figure 1 . Expression of hsa_circ_0032434 in patients with premature ovarian failure;

[0017] Figure 2 . Analysis of the inhibitory effect of the siRNA of the present invention. Detailed Embodiments

[0018] The following further detailed description of the present invention is made in conjunction with specific embodiments, so that those skilled in the art can understand the present invention more clearly.

[0019] Example 1 Detection of the expression of hsa_circ_0032434 in patients with premature ovarian failure by RT-PCR.

[0020] I. Preparation of experimental materials:

[0021] Collect blood samples from 50 patients diagnosed with early ovarian failure by clinicians from January 2022 to December 2022 for backup; simultaneously collect blood samples from 50 healthy individuals as controls.

[0022] II. Test methods

[0023] 1) Extraction of total RNA from venous blood: Use an RNA extraction kit (purchased from Yeasen Biotechnology (Shanghai) Co., Ltd., product number 19241ES50) to extract total RNA from serum samples, including circRNA, and store it at -20°C for backup;

[0024] 2) Synthesis of reverse transcribed cDNA: Operate according to the instructions of the ReverTraAce qPCR RT MasterMix with gDNA Remover reverse transcription kit from TOYOBO Company to synthesize cDNA, and store it at -20°C for backup.

[0025] 3) RT-PCR: Design specific primers according to the nucleic acid sequence of hsa_circ_0032434 (SEQ ID NO.1). Among them, the upstream primer and downstream primer of hsa_circ_0032434 are SEQ ID NO.2 and SEQ ID NO.3 respectively. The internal reference gene is GAPDH, and its upstream and downstream primer sequences are SEQ ID NO.4 and SEQ ID NO.5 respectively.

[0026] The 25 μL PCR reaction system includes: 9 μL 10×PCR Buffer, 2 μL SYBR Green I, 4 μL dNTP, 1 μL each of the upstream and downstream primers of hsa_circ_0032434, 1 μL each of the upstream and downstream primers of the internal reference GAPDH, 2 μL Taq DNA polymerase, and 4 μL cDNA; The PCR reaction program is pre-denaturation at 95°C for 10 min; 95°C for 45 s, 55°C for 45 s, 72°C extension for 45 s, a total of 40 cycles; 72°C extension for 5 min;

[0027] According to formula 2 -ΔΔCt Calculate the relative expression level of hsa_circ_0032434, and the results are shown in Figure 1 . It can be seen from Figure 1 that compared with healthy individuals, the expression level of hsa_circ_0032434 detected in the blood samples of 50 patients with premature ovarian failure is significantly up-regulated and the difference is significant.

[0028] Example 2 Targeted inhibition of hsa_circ_0032434 for improving symptoms of premature ovarian failure

[0029] I. Preparation of premature ovarian failure mouse model

[0030] Take 50 BALB / C mice and intraperitoneally inject cisplatin at a dose of 1.5 mg / kg once a day for 7 consecutive days.

[0031] II. Test method

[0032] 2.1 Preparation of umbilical cord mesenchymal stem cells

[0033] 1) Take fresh umbilical cord, wash it with sterile PBS solution, and cut it into small pieces for standby;

[0034] 2) Add a digestive solution containing 1.5% (v / v) hyaluronidase and 0.5% (v / v) trypsin to the small pieces of umbilical cord obtained in step 1) for enzymatic digestion, and incubate at 37 °C for 2 h for standby;

[0035] 3) Add an equal volume of DMEM / F12 complete medium to the digestive solution to terminate digestion, centrifuge to discard the supernatant, and wash the cell pellet 3 times with DMEM / F12 complete medium for standby;

[0036] 4) After continuously passaging the cells isolated from the umbilical cord in step 3) 5 times, perform morphological and immunophenotypic identification. After passing the identification, centrifuge and collect for standby.

[0037] 2.2 siRNA design of hsa_circ_0032434

[0038] According to the full-length sequence of hsa_circ_0032434, select a suitable region as the target of siRNA, and obtain 3 siRNAs with the top scores through optimization scoring with the design software in this field. Their sequences are shown in SEQ ID NO.6 - 8 in sequence. Taking CHO cells as an example, analyze the inhibitory effect of siRNA on hsa_circ_0032434.

[0039] 2.3 Modification of umbilical cord mesenchymal stem cells

[0040] Taking the siRNA (SEQ ID NO.6) of hsa_circ_0032434 as an example, use Lipofectamine TM 3000 transfection reagent (purchased from Invitrogen TM , product number L3000015) to complete the transfection of siRNA into umbilical cord mesenchymal stem cells. The specific transfection process can be adjusted according to the transfection reagent instruction manual. After the transfection, change to fresh low-glucose DMEM medium, and inoculate the cell suspension into a culture flask for continuous culture for standby.

[0041] 2.4 Efficacy evaluation of umbilical cord mesenchymal stem cells

[0042] The mouse models were randomly divided into three groups. Among them, the mice in the treatment group were intravenously injected with 1×10 5 umbilical cord mesenchymal stem cells transfected with siRNA, the mice in the control group were intravenously injected with 1×10 5 umbilical cord mesenchymal stem cells, and the mice in the blank group were intravenously injected with an equal amount of normal saline during the period. The ovarian indexes of the mice were observed and recorded 7 days after the injection.

[0043] The results showed that the external morphology of the umbilical cord mesenchymal stem cells isolated by the method of the present invention presented a typical spindle-like fibroblast morphology, and CD29, CD44, and CD59 were specifically expressed on the cell surface, while CD14, CD34, and CD45 were lowly expressed. It was confirmed that the cells isolated by the method of the present invention were umbilical cord mesenchymal stem cells, which could meet the subsequent experimental analysis. Analysis of the inhibitory effect of the siRNA of the present invention found that all three siRNAs could effectively inhibit hsa_circ_0032434 in cells, and the results are shown in Figure 2 the content. And as can be seen from Table 1, compared with the mice with healthy ovaries, the E2 level of the mice in the model group decreased significantly, and the levels of FSH and LH hormones increased significantly, confirming that the mouse model of premature ovarian failure of the present invention was successfully constructed. Taking the model mice as an example, after intravenous injection of umbilical cord mesenchymal stem cells, the results found that: unmodified umbilical cord mesenchymal stem cells had a certain anti-premature ovarian failure effect, but the ability was limited and could not very effectively restore the hormone levels of the mice. After intravenous injection of umbilical cord mesenchymal stem cells transfected with the siRNA of the present invention, the results found that the hormone levels of the mouse model of premature ovarian failure had basically returned to the state of healthy mice, and compared with the control group, the difference in this effect was significant, with an obvious anti-premature ovarian failure advantage.

[0044] Table 1 Analysis of mouse hormone levels

[0045] E2 (pmol / L) FSH (IU / L) LH (mIU / mL) Healthy group 28.79±6.15 19.66±2.36 20.57±2.59 Blank group 15.25±3.26 30.46±7.15 29.86±6.57 Control group 18.78±4.38 28.45±6.25 27.25±4.59 Treatment group 29.93±5.54 20.48±1.25 20.56±4.85

[0046] The role of the above embodiments is to illustrate the substantial content of the present invention, but does not limit the protection scope of the present invention. Those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and protection scope of the technical solutions of the present invention.

Claims

1. An umbilical cord mesenchymal stem cell, characterized in that: The mesenchymal stem cells are transfected with a substance that downregulates the expression of hsa_circ_0032434, wherein the substance includes siRNA, and the sequence of the siRNA is any one of SEQ ID NO.6-8 or a combination thereof.

2. Use of the umbilical cord mesenchymal stem cells according to claim 1 in preparing a composition for preventing or treating premature ovarian failure, characterized in that: The mesenchymal stem cells are administered by intravenous injection.

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