A siRNA molecule for inhibiting Rab11fip4 gene and its application

Abstract

The present invention belongs to the fields of reproductive biology and molecular biology, and specifically relates to an siRNA molecule that inhibits the Rab11fip4 gene and its application. The present invention first discovered that the Rab11fip4 gene is a molecule that regulates oocyte internalization, that the expression level of the Rab11fip4 gene is closely related to the uptake of nanoparticles, and that siRNA molecules inhibiting Rab11fip4 gene expression can reduce the uptake of harmful nanoparticles by oocytes. This discovery is the first to reveal that siRNA molecules inhibiting Rab11fip4 gene expression can reduce the uptake of harmful nanoparticles by oocytes. This discovery holds promise for developing a novel delivery system targeting the Rab11fip4 gene, providing a new strategy for improving oocyte quality.

Description

Technical Field

[0001] The present invention belongs to the technical field of reproductive biology and molecular biology, and particularly relates to an siRNA molecule for inhibiting the Rab11fip4 gene and an application thereof. Background Art

[0002] High-quality oocytes are the prerequisite and foundation for obtaining high-quality embryos and are crucial for improving the efficiency of assisted reproductive technology and in vitro embryo production. However, reports indicate that freezing can reduce oocyte maturation. Studies have shown that osmotic damage, mechanical damage, and toxicity from high-concentration cryoprotectants during oocyte freezing can all contribute to decreased oocyte quality. Although studies have shown that antioxidants can protect oocytes from cryoprotective damage, their short half-life and high-dose toxicity limit their effectiveness in cryopreservation.

[0003] In recent years, nanoparticles have been increasingly used in the biomedical field due to their unique physicochemical properties. Researchers have synthesized a novel drug delivery system for inhibiting corneal angiogenesis using minocycline-loaded nanohydroxyapatite / poly(lactic-co-glycolic acid) nanoparticles. Furthermore, studies have shown that nanomaterials such as tungsten diselenide poly(vinylpyrrolidone) and soft liquid metal nanoparticles can modulate the thermal properties of solutions at low temperatures and electrostatically attach to water molecules to reduce ice crystal formation. With the widespread use of nanoparticles, concerns about their biosafety are increasing. Polystyrene nanoparticles, also known as PS-NPs, are widely used in cosmetics, toothpaste, abrasives, and detergents. They are thermoplastic and non-biodegradable. Studies have shown that PS-NPs can enter the human body through the skin, respiratory tract, and digestive system. PS-NPs have been observed in various organs, including the colon and lungs, suggesting that they pose a potential threat to human health. Notably, multiple studies have confirmed the harmful effects of PS-NPs on the reproductive system. Studies have found that PS-NPs can penetrate the placental barrier, adversely affecting fetal health. Reports indicate that PS-NPs can accumulate in multiple organs, including the mouse uterus and ovaries, and that exposure to PS-NPs can reduce the number of antral follicles and oocyte quality. Studies have also shown that PS-NPs can enter granulosa cells, causing apoptosis and autophagy, which in turn leads to decreased oocyte quality. PS-NPs can also induce mitochondrial dysfunction, elevated reactive oxygen species (ROS), and lipid peroxidation in oocytes, leading to maturation failure in porcine oocytes. Therefore, developing methods to prevent the entry of harmful nanoparticles is crucial for the safe and effective application of nanoparticles in oocytes.

[0004] Endocytosis is a key process for the internalization of nanoparticles into cells. After endocytosis, nanoparticles are selectively transported to late endosomes, where they are then degraded in lysosomes or transferred to recycling endosomes via exocytosis. Reports indicate that approximately 70%–80% of endocytosed material is recycled from recycling endosomes to the plasma membrane, a process known as the endocytic cycle. The endocytic cycle is central to cellular transport and is influenced by the binding of Ras-related GTPases to RAB proteins. Most RAB protein family members, such as Rab7 and Rab14, are reported to be involved in important biological events such as oocyte meiosis and spindle migration. Studies have shown that Rab11 and its family interactor, Rab11fip4, regulate membrane trafficking during cytokinesis. Therefore, further investigation of Rab11fip4 is necessary to prevent PS-NPs from entering oocytes. Summary of the Invention

[0005] To solve the above problems, the present invention provides an siRNA molecule and its application for inhibiting the Rab11fip4 gene. The siRNA molecule can inhibit the expression of the Rab11fip4 gene, thereby effectively preventing harmful nanoparticles from entering the oocyte and improving the quality of the oocyte.

[0006] To achieve the above objectives, the technical solutions of the present invention are specifically as follows.

[0007] A siRNA molecule for inhibiting the Rab11fip4 gene, wherein the siRNA molecule is siRNA#2, and the siRNA#2 consists of a sense strand having a nucleotide sequence as shown in SEQ ID NO.1 and an antisense strand having a nucleotide sequence as shown in SEQ ID NO.2.

[0008] The present invention uses mouse oocytes as a model and discovers that the Rab11fip4 gene is a key molecule that regulates oocyte internalization. Further research using the Rab11fip4 gene as a target found that inhibiting the expression of the Rab11fip4 gene through siRNA molecules can control the endocytosis cycle process of the oocyte, prevent harmful nanoparticles of polystyrene from being internalized into the cell, and thus prevent harmful nanoparticles of polystyrene from entering the oocyte, thereby solving the problem of oocyte quality decline caused by the entry of polystyrene into the oocyte.

[0009] The second aspect of the present invention provides the use of the siRNA molecule in the preparation of a drug for improving oocyte quality.

[0010] In another preferred embodiment, improving the quality of oocytes refers to reducing the absorption of harmful nanoparticles by oocytes during in vitro culturing of oocytes.

[0011] In another preferred embodiment, the harmful nanoparticles are polystyrene nanoparticles.

[0012] In another preferred embodiment, the drug is an injection.

[0013] In another preferred embodiment, the concentration of the siRNA molecules in the injection is 20 μM to 22 μM.

[0014] In another preferred embodiment, in the application, the drug is injected into the cytoplasm of oocytes cultured in vitro.

[0015] In another preferred embodiment, the oocyte is an oocyte in the germinal vesicle stage.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention uses mouse oocytes as a model and discovers that Rab11fip4 is a key molecule regulating oocyte internalization. Further, using Rab11fip4 as a target, it is found that siRNA molecules can inhibit the expression of Rab11fip4, thereby reducing the oocyte's uptake of harmful polystyrene nanoparticles, thereby effectively solving the problem of oocyte quality decline caused by polystyrene nanoparticles entering the oocyte. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a graph showing the effect of siRNA on Rab11fip4 expression.

[0019] Figure 2 Figure 2 shows the effect of Rab11fip4 on nanoparticle uptake; A shows the internalization of PS-NPs after knocking down Rab11fip4, with a scale of 50 μm; B shows the quantitative analysis of the relative fluorescence intensity of PS-NPs; n represents the number of oocytes used in this test, and * indicates P <0.05, ** indicates P <0.01, *** indicates P <0.001.

[0020] Figure 3 Transmission electron microscopy and immunofluorescence staining images of PLGA-RES entering oocytes; A is a TEM image of the distribution of PLGA-RES in oocytes, the arrow represents PLGA-RES, the scale bar is 1 μm, membrane represents the cell membrane, and nucleus represents the cell nucleus; B is an image of PLGA-RES internalization in oocytes at different times, NP is an image of the oocyte after treatment with PLGA-RES, Hoechst represents the fluorescent staining image of the oocyte nucleus, and Merge represents the image after the synthesis of NP and Hoechst.

[0021] Figure 4 Figure 1 is a quantitative analysis result graph of the relative fluorescence intensity of PLGA-RES at different times and concentrations; among them, A is a quantitative analysis result graph of the relative fluorescence intensity of PLGA-RES at different times; B is a quantitative analysis result graph of the relative fluorescence intensity of PLGA-RES at different PLGA-RES concentrations; C is an image of PLGA-RES internalization in oocytes at different PLGA-RES concentrations; in the figure, NP is an image of oocytes after treatment with PLGA-RES, Hoechst represents the fluorescent staining image of the oocyte nucleus, and Merge represents the image after the synthesis of NP and Hoechst.

[0022] Figure 5 Figure 3 is the effect of PLGA-RES and Rab11fip4 expression on lysosomes; A is the TEM image of lysosomes in oocytes treated with the control group and PLGA-RES, arrows represent lysosomes, the scale bar is 500 nm, 1 is the TEM image of lysosomes in oocytes treated with the control group, 2 is the enlarged image framed by the black dotted line in 1, 3 is the TEM image of lysosomes in oocytes treated with PLGA-RES, and 4 is the enlarged image framed by the black dotted line in 3; B is the quantitative analysis of lysosomes in oocytes treated with the control group and PLGA-RES; C is the quantitative analysis result of the relative mRNA level of LAMP1; n is the number of images under different electron microscope fields used in this experiment, * indicates P <0.05, *** indicates P <0.001. In the figure, control indicates the control group, NP indicates the PLGA-RES-treated group, Rab11fip4-KD indicates the Rab11fip4 knockdown group, and NC indicates the negative control group.

[0023] Figure 6 Figure 1 shows the fluorescence intensity distribution of Rab5 under different antibody treatments, 2 shows the fluorescence intensity distribution of Rab7 under different treatments, 3 shows the fluorescence intensity distribution of LAMP1 under different treatments, and 4 shows the fluorescence intensity distribution of Rab11fip4 under different treatments. In the figure, NP represents the image of oocytes treated with PLGA-RES, DAPI represents the fluorescent staining image of oocyte nuclei, and Merge represents the image of NP combined with DAPI. The white oblique line indicates the pixel intensity measured along the line on the oocyte. The scale bar in the figure is 50 μm.

[0024] Figure 7 for Figure 6 Pixel intensity map at the white oblique line in the middle, where a is the pixel intensity map corresponding to 1, b is the pixel intensity map corresponding to 2, c is the pixel intensity map corresponding to 3, and d is the pixel intensity map corresponding to 4.

[0025] Figure 8 This is an analysis diagram of the model-predicted binding of PLGA-RES to Rab11fip4. In the figure, 2 is an enlarged view of point 1.

[0026] Figure 9 Figure 2 is a quantitative analysis of Rab11fip4 staining in different groups and the fluorescence intensity and relative mRNA level of Rab11fip4; A is the Rab11fip4 staining image of oocytes in different groups; B is the quantitative analysis image of the relative fluorescence intensity of Rab11fip4 protein; C is the quantitative analysis image of the relative mRNA level of Rab11fip4 gene. F in the figure represents fresh oocytes, V represents frozen oocytes, and NP represents oocytes treated with PLGA-RES.

[0027] Figure 10 Figure 3 is the effect of PLGA-RES on the quality of frozen oocytes; A is the image of GVBD and PBE of oocytes treated with different methods, the scale bar is 100 μm, F in the figure represents fresh oocytes, V represents frozen oocytes, and NP represents oocytes treated with PLGA-RES; GVBD represents germinal vesicle rupture, and PBE represents polar body extrusion; B is the incidence graph of GVBD; C is the incidence graph of PBE.

[0028] Figure 11 Figure 2 is a graph showing chromosome staining and aneuploidy rates in different groups. A is a representative image of euploid and aneuploid chromosomes in different groups. The numbers in the figure indicate the number of labeled chromosomes. The 21 circled in the box indicates an abnormal number of chromosomes, indicating aneuploidy. The scale bar is 10 μm. B is a graph showing aneuploidy rates. n indicates the number of cells used in this experiment. * indicates P <0.05, ** indicates P <0.01, *** indicates P <0.001, ns indicates no significant difference. DETAILED DESCRIPTION

[0029] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0030] The Chinese meanings of the following English abbreviations are as follows: GV stage indicates germinal vesicle stage, COCs indicates cumulus cell complex, GVBD indicates germinal vesicle rupture, MⅡ stage indicates metaphase of the second meiotic division, DPBS indicates Dulbecco’s phosphate-buffered saline, OPS indicates open capillary tubules, DAPI indicates 4′,6-diamidino-2-phenylindole, TEM indicates transmission electron microscopy, PLGA-RES indicates poly(lactic-co-glycolic acid), Hoechst indicates Hoechst fluorescent dye, Merge indicates merge, PBE indicates polar body extrusion, PS-NPs indicates polystyrene nanoparticles, and HX-M199 solution indicates culture medium 199.

[0031] The compositions of the reagents in the following examples are as follows: permeabilization solution contains 0.5 wt% Triton-PBS and 0.1 wt% polyvinyl alcohol resin per liter, with the remainder being double-distilled water; cleaning solution contains 0.1 wt% Triton-PBS and 0.1 wt% polyvinyl alcohol resin per liter, with the remainder being double-distilled water; blocking solution contains 3 wt% bovine serum albumin, 0.1 wt% Triton-PBS, and 0.1 wt% polyvinyl alcohol resin per liter, with the remainder being double-distilled water; M2 solution refers to the in vitro manipulation solution for mouse gametes and embryos.

[0032] The polylactic acid-glycolic acid copolymer of resveratrol in the following examples is the polylactic acid-glycolic acid copolymer of resveratrol disclosed in Publication No. CN117546834A, hereinafter referred to as PLGA-RES.

[0033] Example 1: siRNA molecules inhibited the expression of Rab11fip4 and reduced the uptake of PS-NPs by oocytes.

[0034] 1. Experimental method.

[0035] 1.1. The specific protocol for the Rab11fip4 knockdown experiment is as follows.

[0036] Rab11fip4-siRNA#1 and Rab11fip4-siRNA#2, as well as Control-siRNA, which target and knock down Rab11fip4, were designed and synthesized. The sense and antisense strand sequences of Rab11fip4-siRNA#1, Rab11fip4-siRNA#2, and Control-siRNA are shown in Table 1 below.

[0037] Table 1: siRNA sequences

[0038]

[0039] Note: Each sequence in the table also has base TT connected to its 3' end.

[0040] 1.2. siRNA microinjection.

[0041] siRNA was diluted to 20 μM in M2 solution supplemented with 2.5 μM milrinone. 10 μL of the solution was injected into the cytoplasm of GV-stage oocytes using a FemtoJet 4i microinjector. The injected oocytes were incubated in M2 solution supplemented with 2.5 μM milrinone for 24 hours at 37°C in a 5% CO2 incubator. M2 solution refers to the in vitro manipulation medium for mouse gametes and embryos. The NC group represents oocytes injected with control siRNA, the siRNA#1 group represents oocytes injected with the Rab11fip4-siRNA#1 sequence, and the siRNA#2 group represents oocytes injected with the Rab11fip4-siRNA#2 sequence.

[0042] 1.3. Live cell staining.

[0043] The oocytes after the above treatment were divided into two groups and incubated in M2 solution containing 2.5 μM milrinone with or without PS-NPs, which had been balanced in the incubator in advance. The fluorescence intensity was counted after incubation for 1 hour in an incubator at 37°C and 5% CO2 volume percentage.

[0044] 2. Experimental results.

[0045] 2.1. Effect of siRNA injection on Rab11fip4 expression.

[0046] like Figure 1 As shown, siRNAs in the siRNA#1 and siRNA#2 groups were used to knock down Rab11fip4, and it was found that both groups could significantly reduce the mRNA level of Rab11fip4. Compared with the siRNA#1 group, the knockdown efficiency of the siRNA#2 group was higher, with siRNA#1 being 0.73±0.04 and siRNA#2 being 0.40±0.03. P <0.001, so siRNA#2 was selected for subsequent experiments.

[0047] 2.2. Effect of inhibiting Rab11fip4 expression on oocyte uptake of PS-NPs.

[0048] like Figure 2 As shown, compared with the NC group, the fluorescence intensity of PS-NPs in the siRNA#2 group, designated as Rab11fip4-KD, was significantly reduced, 1.00±0.03 for NC and 0.84±0.02 for Rab11fip4-KD. P <0.05, indicating that inhibition of Rab11fip4 expression can reduce the internalization of harmful nanoparticles.

[0049] Example 2: Analysis of the role of Rab11fip4 in oocyte internalization.

[0050] The present invention further studies the Rab11fip4 protein and finds that poly(lactic acid-co-glycolic acid) copolymer of resveratrol can promote the expression of Rab11fip4 protein and promote the in vitro maturation of frozen oocytes. The specific process is as follows.

[0051] 1. Experimental method.

[0052] 1.1. Oocyte retrieval.

[0053] Female 6-week-old ICR mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0054] Mice were maintained on a 12-hour dark / 12-hour light cycle and allowed ad libitum access to food and water. After a one-week acclimation period, they were used for experiments. Mice were intraperitoneally injected with 10 IU of pregnant mare's serum gonadotropin. 48 hours later, the mice were sacrificed by cervical dislocation. The ovaries were removed and placed in HX-M199 solution that had been pre-equilibrated for 4 hours. The ovaries were then shaken and rinsed to remove excess impurities. The ovaries were then transferred to a clean Petri dish and thoroughly minced with a sterile blade to release the follicular contents. Pre-equilibrated HX-M199 solution was then added, and the cumulus ovary-oocyte complexes were collected using a mouth pipette into fresh pre-equilibrated HX-M199 solution. The COCs were aspirated using a mouth pipette larger than the diameter of the oocyte to completely detach the granulosa cells.

[0055] 1.2. Oocyte maturation in vitro.

[0056] The GV-stage oocytes without granulosa cells were incubated in a culture dish with M16 medium that had been equilibrated in the incubator in advance. After culturing in an incubator at 37°C and 5% CO2 volume percentage for 2 hours, the germinal vesicle rupture rate, i.e., the GVBD incidence rate, was calculated. After culturing for 12 hours, the first polar body extrusion rate, i.e., the PBE incidence rate, was calculated.

[0057] 1.3 Transmission electron microscopy analysis.

[0058] Fresh GV-stage oocytes were collected and divided into a control group without PLGA-RES and a PLGA-RES-treated group with 50 μg / mL PLGA-RES. 80 oocytes per group were washed three times with phosphate buffered saline (PBS) and fixed with 4 wt% paraformaldehyde at 23°C for 1 hour. Oocytes were then fixed in 2.5 wt% glutaraldehyde for 14 hours. Oocytes were washed three times with PBS, with washes every 15 minutes. Oocytes were then fixed on ice with osmium sulfate (1 wt% osmium sulfate and 1.5 wt% potassium ferrocyanide) for 60 minutes. Oocytes were then washed three times with ddH2O, with washes every 15 minutes. Oocytes were then stained with 1 wt% uranyl acetate at 23°C for 1 hour and washed three times with ddH2O, with washes every 15 minutes. Washes may be increased to avoid uranium contamination. Next, the samples were dehydrated using four concentration gradients of ethanol: 50wt%, 70wt%, 80wt%, and 90wt%, followed by washing every 5 minutes, and finally dehydrated three times with 100wt% ethanol. The samples were then immersed in propylene oxide twice, each for 5 minutes. Subsequently, infiltration was performed at room temperature as follows: first, infiltration with 812 resin and propylene oxide at a volume ratio of 1:1 for 8 hours; then, infiltration with a volume ratio of 2:1 for 8 hours; then, infiltration with a volume ratio of 3:1 for 8 hours; and finally, infiltration with pure 812 resin at 23°C for 8 hours. After embedding, the samples were polymerized at 60°C and serially sectioned using an automated microtome with a 100nm section thickness. The sections were placed on glass slides and observed under a microscope to confirm that the oocyte was cut. If so, the section thickness was adjusted to 70nm. Finally, the oocyte sections were mounted on copper grids. The sections were incubated and stained with uranyl acetate before examination.

[0059] 1.4. Live cell staining.

[0060] During in vitro culture, oocytes were incubated with 10 μg / mL, 50 μg / mL, and 250 μg / mL of PLGA-RES in M2 solution supplemented with milrinone for varying periods of time. Following incubation, oocytes were washed three times with M2 solution, then placed in M2 solution and photographed under a laser confocal microscope. To control for variation, imaging parameters and exposure times were kept consistent across groups. Using identical cell staining conditions and imaging parameters, specific regions were selected using NIS-Elements AR software, and statistical analysis was performed on the mean fluorescence intensity per unit area within the selected regions. Finally, the mean of all measurements was calculated, and the final mean fluorescence intensity was compared between groups.

[0061] 1.5. Immunofluorescence staining.

[0062] Place the oocytes in fixative solution and fix at room temperature for at least 1 hour. If the oocytes have lost their zona pellucida, dilute the fixative solution with DPBS at a 1:1 volume ratio and fix the oocytes for 30 minutes. Wash the fixed oocytes three times with wash solution and then permeabilize them in permeabilization solution at 23°C for 1 hour. If the oocytes have lost their zona pellucida, permeabilize them for 20 minutes. Wash the permeabilized oocytes three times with wash solution and then block them in blocking solution at 23°C for 1 hour.

[0063] After blocking, oocytes were directly placed in the prepared primary antibody and incubated overnight at 4°C. Following primary antibody incubation, oocytes were washed three times with wash buffer and then placed in the prepared secondary antibody and incubated at room temperature for 1 hour. Following secondary antibody incubation, oocytes were washed three times with wash buffer and then stained with DAPI for 5 minutes. After nuclear staining, oocytes were transferred to an adhesive slide using a mouth pipette and covered with a coverslip for observation. If oocytes were not to be covered, they were washed three times with M2 solution, placed in a droplet of paraffin oil in a glass-bottomed culture dish, and covered for observation.

[0064] Images were taken using an A1 Confocal laser scanning confocal microscope. If statistical analysis of fluorescence intensity was required, imaging parameters and exposure time were consistent across groups. Using the same immunofluorescence staining conditions and imaging parameters, specific regions were selected using NIS-Elements AR software, and statistical analysis was performed on the mean fluorescence intensity per unit area within those regions. The mean of all measurements was calculated, and the final mean fluorescence intensity was compared between groups.

[0065] 1.6. RT-qPCR.

[0066] (1) Sample collection: Collect oocytes from different treatment groups, wash them three times with phosphate buffer, and place them in 1.5 mL centrifuge tubes, 50 oocytes per group. Use a mouth pipette to aspirate excess liquid and freeze the samples in a -80 °C refrigerator for later use.

[0067] (2) Extract oocyte RNA using Trizol.

[0068] A. Take the sample out of the -80℃ freezer and place it in an ice box.

[0069] B. Add 500 μL of Trizol to each sample, vortex to mix, and place on ice for 5 minutes.

[0070] C. Add 100 μL of chloroform to each sample tube for extraction, vortex, and place on ice for 5 min.

[0071] D. Place the sample in a centrifuge and centrifuge at 14,000 g at 4°C for 10 minutes. Stratification of the liquid in the sample tube is visible. Pipette the supernatant and transfer it to a new 1.5 mL centrifuge tube.

[0072] E. Add an equal volume of isopropanol to a new sample tube, then add 2 μL of glycogen, vortex, and let stand on ice for 5 minutes.

[0073] F. Centrifuge at 14,000 g at 4°C for 10 min, discard the supernatant, and add 1 mL of pre-chilled 75 wt% ice ethanol to the sample tube and vortex.

[0074] G. Centrifuge at 14000g and 4℃ for 5min, discard the supernatant, place the sample on a clean bench, and air-dry the white RNA precipitate at room temperature. Stop air-drying when the precipitate turns transparent.

[0075] H. Add 18 μL of double-distilled water to the sample tube to dissolve the RNA. Mix thoroughly with a pipette and measure the RNA concentration using a Nanodrop spectrophotometer.

[0076] (3) Reverse transcription: The reverse transcription system is shown in Table 2.

[0077] Table 2: Reverse transcription system

[0078]

[0079] The reverse transcription procedure is shown in Table 3.

[0080] Table 3: Reverse transcription procedures

[0081]

[0082] After reverse transcription is completed, the reverse transcribed cDNA sample is stored in a -20°C refrigerator for later use.

[0083] (4) Real-time fluorescence quantitative PCR: The 10 μL qPCR reaction system is shown in Table 4.

[0084] Table 4: qPCR reaction system

[0085]

[0086] Based on the qPCR reaction system, the cDNA template was mixed with the reaction kit premix and loaded into a 96-well qPCR plate. Real-time fluorescence quantitative PCR was performed using a Bio-Rad CFX96 Touch Real-Time PCR instrument. The primer sequences that bind to the cDNA are shown in Table 5 below.

[0087] Table 5: Primer sequences

[0088]

[0089] The qPCR reaction program settings are shown in Table 6.

[0090] Table 6: qPCR reaction program

[0091]

[0092] After the qPCR reaction was completed, the amplification cycle number Ct value of the internal reference gene and the target gene was obtained. PPIA and RPL7 were used as double internal reference genes, and 2- △△Ct The relative expression levels of target genes were calculated using the method.

[0093] GraphPad Prism 8 was used for independent sample t-test and chi-square test analysis. All experiments were performed with at least three biological replicates, and the data are expressed as mean ± standard error. P <0.05 was considered statistically significant. P <0.01 was considered a significant difference. P A value <0.001 was considered to be extremely significant, and ns was considered to be no statistical difference.

[0094] 2. Experimental results.

[0095] 2.1. PLGA-RES can be internalized into oocytes.

[0096] Transmission electron microscopy analysis showed that PLGA-RES was distributed around the oocyte membrane and perinuclear region, confirming that oocytes can take up PLGA-RES, e.g. Figure 3 In addition, immunofluorescence and time-lapse imaging results showed that PLGA-RES was internalized into oocytes in a time- and concentration-dependent manner, as shown in Figure 5A. Figure 3 Figure B and Figure 4 shown.

[0097] 2.2. The Rab11fip4-mediated recycling endosomal pathway plays a key role in oocyte internalization.

[0098] TEM results showed that the number of lysosomes was significantly reduced after PLGA-RES treatment, such as Figure 5 As shown in A and B. To further study the intracellular trafficking of PLGA-RES, the mRNA levels of LAMP1 in different groups were detected, and Rab5, Rab7 and LAMP1 were used as immunofluorescence markers to track early endosomes, late endosomes and lysosomes, respectively. The results are shown in Figure 5 C in Figure 6 and Figure 7As shown, LAMP1 levels were significantly elevated in the Rab11fip4-KD group, and the marker was observed to be distributed in clusters rather than uniformly in the cytoplasm. On the other hand, PLGA-RES and Rab11fip4 showed a colocalization pattern. These results suggest that the Rab11fip4-mediated recycling endosomal pathway regulates the intracellular trafficking of PLGA-RES.

[0099] Example 3: Increasing the expression of Rab11fip4 can promote the in vitro maturation of frozen oocytes.

[0100] 1. Experimental method.

[0101] 1.1 Molecular docking experiment

[0102] The crystal structure of Rab11fip4 (ID: Q8BQP8) was downloaded from the UniProt database. Molecular docking studies were performed using AutoDock Vina. Cluster analysis of the docking results was performed, and the model with the lowest binding energy was selected as the predicted binding site for PLGA-RES. The final docking results were visualized using the PyMOL molecular graphics system.

[0103] 1.2. Preparation of oocyte freezing and thawing solutions.

[0104] (1) Pretreatment solution: Add 1 mL of ethylene glycol to 8 mL of DPBS, shake well, add 1 mL of dimethyl sulfoxide, shake well, add 30 mg of bovine serum albumin, let it stand until fully dissolved, and then store in a refrigerator at 4°C for use.

[0105] (2) Freezing solution: Add 3 g of polysucrose, 1.712 g of sucrose, and 0.021 g of bovine serum albumin to 7.02 mL of DPBS to obtain a mixed solution, which is recorded as FS solution. Then, take 7 mL of FS solution, add 1.5 mL of ethylene glycol and 1.5 mL of dimethyl sulfoxide, let it stand until fully dissolved, and then store it in a refrigerator at 4°C for future use.

[0106] (3) Thawing solution: Add 1.9193 g of sucrose and 30 mg of bovine serum albumin to 10 mL of DPBS, let it stand to dissolve, and then store in a refrigerator at 4°C.

[0107] 1.3. Oocyte acquisition in each group.

[0108] The fresh oocyte group was designated as Group F, and the oocytes were obtained according to step 1.1 of Example 2.

[0109] The frozen oocyte group was designated as Group V. The oocytes were vitrified using open capillaries (OPS) at the GV stage. The oocytes were treated in pretreatment solution for 30 seconds and freezing solution for 25 seconds. The OPS tube containing the oocytes was then placed directly into liquid nitrogen for freezing and stored for at least three days. Upon thawing, the OPS tube was removed from the liquid nitrogen and incubated in thawing solution at 37°C for 5 minutes. Finally, the tube was rinsed three times in M2 medium containing milrinone and then transferred to M2 medium containing milrinone for 1 hour to recover. Group V oocytes were obtained for subsequent experiments.

[0110] The oocyte group treated with PLGA-RES was designated as the NP group. 50 μg / mL PLGA-RES was added to the pretreatment solution, freezing solution, thawing solution, and M2 recovery solution after thawing. The rest of the steps were the same as those in the V group.

[0111] 1.4. RT-qPCR.

[0112] Sample collection, RNA extraction, reverse transcription, real-time fluorescence quantitative PCR, qPCR reaction procedures and gene relative expression calculation method were the same as in Example 2. The Rab11fip4 primer sequences are shown in Table 7.

[0113] Table 7: Rab11fip4 primer sequences

[0114]

[0115] 2. Experimental results.

[0116] 2.1. PLGA-RES can promote the expression of Rab11fip4.

[0117] Molecular docking analysis showed that PLGA-RES can interact with Rab11fip4 by forming hydrogen bonds at the ASP-450 residue. The docking score was -6.895 kcal / mol, indicating a strong binding affinity between PLGA-RES and Rab11fip4. Figure 8 In addition, fluorescence staining and RT-PCR results further confirmed that PLGA-RES could significantly increase the expression of Rab11fip4 in frozen oocytes, as shown in Figure 2. Figure 9 shown.

[0118] 2.2. PLGA-RES can improve the in vitro maturation of frozen oocytes.

[0119] like Figure 10As shown in the results, the NP group supplemented with 50 μg / mL PLGA-RES significantly increased the GVBD ratio compared with the V group. The GVBD ratio of the V group was 59.40%±2.67%, while the GVBD ratio of the NP group was 80.2%±0.97%. Given that the increased aneuploidy rate is the main factor leading to the decline in oocyte quality, the effects of PLGA-RES on the aneuploidy rate and spindle positioning were further studied. Figure 11 As shown, compared with the aneuploidy rate of 2.56%±2.56% in group F, the aneuploidy rate of group V was 27.9%±2.86%, which was significantly increased. The aneuploidy rate of group NP was 7.33%±4.13%, which was significantly reduced compared with the aneuploidy rate of group V (27.9%±2.86%). P <0.05.

[0120] In summary, these results demonstrate that Rab11fip4 is a key molecule regulating oocyte internalization, and that PLGA-RES can enhance Rab11fip4 expression in cryopreserved oocytes, thereby promoting in vitro maturation of frozen oocytes. Furthermore, siRNA molecules were prepared, and the siRNA approach effectively inhibited Rab11fip4 expression, thereby reducing oocyte uptake of harmful nanoparticles. This invention is of great significance in revealing the oocyte internalization pathway, improving the quality of cryopreserved oocytes, and promoting the safe and effective application of nanoparticles.

[0121] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of an siRNA molecule in the preparation of a drug for reducing oocyte absorption of polystyrene nanoparticles, characterized in that: The siRNA molecule is siRNA#2, which consists of a sense strand having a nucleotide sequence as shown in SEQ ID NO.1 and an antisense strand having a nucleotide sequence as shown in SEQ ID NO.

2.

2. Use of the siRNA molecule according to claim 1 in the preparation of a drug for reducing oocyte absorption of polystyrene nanoparticles, characterized in that: The medicine is an injection.

3. Use of the siRNA molecule according to claim 2 in the preparation of a drug for reducing oocyte absorption of polystyrene nanoparticles, characterized in that: In the injection, the concentration of siRNA molecules is 20 μM to 22 μM.

4. Use of the siRNA molecule according to claim 1 in the preparation of a drug for reducing oocyte absorption of polystyrene nanoparticles, characterized in that: In the application, the drug is injected into the cytoplasm of oocytes cultured in vitro.

5. Use of the siRNA molecule according to claim 4 in the preparation of a drug for reducing oocyte absorption of polystyrene nanoparticles, characterized in that: The oocyte is an oocyte in the germinal vesicle stage.

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