SiRNA molecule for inhibiting Rab11fip4 gene and application

Abstract

The invention belongs to the technical field of reproductive biology and molecular biology, and particularly relates to a siRNA molecule for inhibiting a Rab11fip4 gene and application of the siRNA molecule. According to the application, it is found for the first time that the Rab11fip4 gene is a molecule for regulating and controlling the internalization of the oocytes, the expression quantity of the Rab11fip4 gene is closely related to uptake of nanoparticles, it is found for the first time that the siRNA molecule can reduce uptake of harmful nanoparticles by the oocytes by inhibiting expression of the Rab11fip4 gene, and a novel delivery system with the Rab11fip4 gene as a target is expected to be developed; a new strategy is provided for improving the oocyte quality.

Description

Technical Field

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

[0002] High-quality oocytes are the premise and basis for obtaining high-quality embryos, and are of great significance to the improvement of assisted reproductive technology and the efficiency of embryo production in vitro. However, reports indicate that freezing can reduce the ability of oocytes to mature. Studies have found that osmotic damage, mechanical damage, and high-concentration cryoprotectant toxicity during oocyte freezing can all cause oocyte quality to decline. Although studies have shown that antioxidants can protect oocytes from freezing damage, antioxidants have a short half-life and high-dose toxicity, which limits their application efficiency in frozen oocytes.

[0003] In recent years, nanoparticles have been increasingly used in the biomedical field due to their unique physical and chemical properties. Researchers used minocycline-loaded nanohydroxyapatite / polylactic-co-glycolic acid nanoparticles to synthesize a new drug delivery system that can be used to inhibit corneal angiogenesis. In addition, studies have found that nanomaterials such as tungsten diselenide polyvinyl pyrrolidone and soft liquid metal nanoparticles can regulate the thermal properties of solutions at low temperatures and electrostatically attach to water molecules to reduce ice crystal formation. With the widespread application of nanoparticles, people's attention to their biosafety has also been increasing. Polystyrene nanoparticles, also known as PS-NPs, are widely used in cosmetics, toothpaste, abrasives, detergents and other commodities. They are thermoplastic and non-biodegradable. Studies have found that PS-NPs can enter the human body through the skin, respiratory tract and digestive system. PS-NPs have been observed in multiple organs such as the colon and lungs. It can be seen that PS-NPs pose a potential threat to human health. It is worth noting that many studies have confirmed the harm of PS-NPs to the reproductive system. Studies have found that PS-NPs can penetrate the placental barrier and have adverse effects on fetal health. Reports have also pointed out that PS-NPs can accumulate in multiple organs such as the mouse uterus and ovaries, and PS-NPs exposure can reduce the number of antral follicles and the quality of oocytes. Studies have shown that PS-NPs can enter granulosa cells, causing apoptosis and autophagy in granulosa cells, which in turn leads to a decrease in oocyte quality. PS-NPs can also induce abnormal mitochondrial function in oocytes, increased levels of reactive oxygen species, and lipid peroxidation, leading to the failure of pig oocyte maturation. Therefore, the development of methods to prevent the entry of harmful nanoparticles is of great significance for the safe and effective application of nanoparticles in oocytes.

[0004] Endocytosis is the key process for nanoparticles to be internalized into cells. After endocytosis, nanoparticles are selectively transported to late endosomes, and then degraded by lysosomes or transferred to recycling endosomes by exocytosis. Reports indicate that about 70% to 80% of endocytosed materials are recycled from recycling endosomes to the plasma membrane, a process called endocytic recycling. The endocytic recycling is the core of cellular material transport and is affected by the binding of GTPases associated with the Ras gene to RABs proteins. It has been reported that most members of the RAB protein family, such as Rab7 and Rab14, are involved in important biological events such as oocyte meiosis and spindle migration. Studies have found that Rab11 and its family interacting molecule Rab11fip4 regulate membrane transport in cytokinesis. Therefore, in order to prevent PS-NPs from entering oocytes, further research on Rab11fip4 is needed. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a siRNA molecule for inhibiting the Rab11fip4 gene and its application. 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 Rab11fip4 gene, wherein the siRNA molecule is siRNA#2, and the siRNA#2 is composed of a sense strand having a nucleotide sequence such as SEQ ID NO.1 and an antisense strand having a nucleotide sequence such as SEQ ID NO.2.

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

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

[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 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: The present invention uses mouse oocytes as a model and finds that Rab11fip4 is a key molecule that regulates oocyte internalization. Further, using Rab11fip4 as a target, it is found that siRNA molecules can inhibit the expression of Rab11fip4, thereby reducing the uptake of harmful polystyrene nanoparticles by oocytes, thereby effectively solving the problem of decreased oocyte quality due to the entry of polystyrene nanoparticles into oocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a graph showing the effect of siRNA on Rab11fip4 expression.

[0018] Figure 2 Figure 2 is the effect of Rab11fip4 on nanoparticle uptake; A is the PS-NPs internalization diagram after knocking down Rab11fip4, with a scale of 50 μm; B is the quantitative analysis of the relative fluorescence intensity of PS-NPs; n represents the number of oocytes used in this test, and * represents P <0.05, ** indicates P <0.01, *** indicates P <0.001.

[0019] Figure 3 The transmission electron microscopy and immunofluorescence staining images of PLGA-RES entering the oocyte; A is the TEM image of the distribution of PLGA-RES in the oocyte, the arrow represents PLGA-RES, the scale bar is 1μm, membrane represents the cell membrane, and nucleus represents the nucleus; B is the image of PLGA-RES internalization in the oocyte at different times, NP is the 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.

[0020] Figure 4The figures are the quantitative analysis results of the relative fluorescence intensity of PLGA-RES at different times and concentrations; wherein, A is the quantitative analysis results of the relative fluorescence intensity of PLGA-RES at different times; B is the quantitative analysis results of the relative fluorescence intensity of PLGA-RES at different PLGA-RES concentrations; C is the image of PLGA-RES internalization in oocytes at different PLGA-RES concentrations; in the figure, NP is the 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 5 The effects 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 the PLGA-RES group, 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 represents the number of images under different electron microscope fields used in this experiment, and * represents P <0.05, *** indicates P <0.001. In the figure, control represents the control group, NP represents the PLGA-RES treatment group, Rab11fip4-KD represents the Rab11fip4 knockdown group, and NC represents the negative control group.

[0022] Figure 6 The fluorescence intensity distribution diagram under different antibody treatments, 1 represents the fluorescence intensity distribution diagram of Rab5 under different treatment conditions, 2 represents the fluorescence intensity distribution diagram of Rab7 under different treatment conditions, 3 represents the fluorescence intensity distribution diagram of LAMP1 under different treatment conditions, 4 represents the fluorescence intensity distribution diagram of Rab11fip4 under different treatment conditions; in the figure, NP represents the image of the oocyte after PLGA-RES treatment, DAPI represents the fluorescent staining image of the oocyte nucleus, and Merge represents the image after NP and DAPI are synthesized. The white oblique line is the pixel intensity measured along the line on the oocyte, and the scale bar in the figure is 50μm.

[0023] 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.

[0024] Figure 8 This is an analysis diagram of the model predicting the binding of PLGA-RES to Rab11fip4, in which 2 is an enlarged view of point 1.

[0025] Fig. 9 The figures are the quantitative analysis graphs of Rab11fip4 staining in different groups and the fluorescence intensity and relative mRNA level of Rab11fip4; A is the Rab11fip4 staining graph of oocytes in different groups; B is the quantitative analysis graph of the relative fluorescence intensity of Rab11fip4 protein; C is the quantitative analysis graph of the relative level of Rab11fip4 gene mRNA, in which F represents fresh oocytes, V represents frozen oocytes, and NP represents oocytes treated with PLGA-RES.

[0026] Fig.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 100μm, F in the figure represents fresh oocytes, V represents frozen oocytes, 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.

[0027] Fig.11 The chromosome staining images and aneuploidy rate results in different groups; A is a representative image of euploid and aneuploid chromosomes in different groups, the numbers in the figure represent the number of labeled chromosomes, the 21 circled in the box is the abnormal number of chromosomes, indicating aneuploidy, the scale bar is 10μm; B is the aneuploidy rate result, n represents the number of cells used in this experiment, * represents P <0.05, ** indicates P <0.01, *** indicates P <0.001, ns means no significant difference. DETAILED DESCRIPTION

[0028] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but 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.

[0029] 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 tubule, 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.

[0030] The compositions of the reagents in the following embodiments are as follows: each liter of the permeation solution contains 0.5wt% Triton-PBS and 0.1wt% polyvinyl alcohol resin, and the balance is double distilled water; each liter of the cleaning solution contains 0.1wt% Triton-PBS and 0.1wt% polyvinyl alcohol resin, and the balance is double distilled water; the blocking solution contains 3wt% bovine serum albumin, 0.1wt% Triton-PBS and 0.1wt% polyvinyl alcohol resin, and the balance is double distilled water; M2 solution refers to the in vitro operation solution for mouse gametes and embryos.

[0031] 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.

[0032] Example 1: siRNA molecules inhibited the expression of Rab11fip4 and reduced the uptake of PS-NPs by oocytes.

[0033] 1. Experimental method.

[0034] 1.1. The specific protocol for the Rab11fip4 knockdown experiment is as follows.

[0035] 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.

[0036] Table 1: siRNA sequences

[0037] Note: Each sequence in the table has a TT base connected to its 3' end.

[0038] 1.2. siRNA microinjection.

[0039] The siRNA was diluted to 20 μM in M2 solution containing 2.5 μM milrinone, and 10 pL was injected into the cytoplasm of GV stage oocytes using a FemtoJet 4i microinjector. The injected oocytes were incubated in M2 solution containing 2.5 μM milrinone at 37°C and CO. 2 The cells were cultured in an incubator with a volume fraction of 5% M2 for 24 hours. M2 solution refers to the in vitro operation solution 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.

[0040] 1.3. Live cell staining.

[0041] The oocytes treated as above were divided into two groups and incubated in M2 solution containing 2.5 μM milrinone with or without PS-NPs, which was balanced in advance in the incubator, at 37°C and CO. 2 The fluorescence intensity was counted after incubation in an incubator with a volume percentage of 5% for 1 hour.

[0042] 2. Experimental results.

[0043] 2.1. Effect of siRNA injection on Rab11fip4 expression.

[0044] like Figure 1 As shown, siRNAs in 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 siRNA#1 group, the knockdown efficiency of siRNA#2 group was higher, 0.73±0.04 for siRNA#1 and 0.40±0.03 for siRNA#2. P <0.001, so siRNA#2 was selected for subsequent experiments.

[0045] 2.2. The effect of inhibiting Rab11fip4 expression on the uptake of PS-NPs by oocytes.

[0046] like Figure 2 As shown, compared with the NC group, the fluorescence intensity of PS-NPs in the siRNA#2 group, denoted 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.

[0047] Example 2: Analysis of the role of Rab11fip4 in oocyte internalization.

[0048] The present invention further studies the Rab11fip4 protein and finds that the polylactic acid-glycolic acid copolymer of resveratrol can promote the expression of the Rab11fip4 protein and promote the in vitro maturation of frozen oocytes. The specific process is as follows.

[0049] 1. Experimental method.

[0050] 1.1. Oocyte acquisition.

[0051] Female 6-week-old ICR mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0052] The mice were maintained on a 12-h dark / 12-h light cycle and allowed to eat and drink freely. They were used for experiments after a one-week adaptation period. The mice were intraperitoneally injected with 10 IU of pregnant mare serum gonadotropin. After 48 hours, the mice were killed by cervical dislocation. The ovaries were removed and placed in HX-M199 solution pre-equilibrated for 4 hours, and shaken to rinse to clean excess impurities. The ovaries were then transferred to a clean culture dish, and the ovaries were fully minced using a sterile blade to release the contents of the follicles. The pre-equilibrated HX-M199 solution was then added, and the cumulus-oocyte complex was collected into a new pre-equilibrated HX-M199 solution using a mouth pipette. COCs were blown and aspirated with a mouth pipette larger than the diameter of the oocyte to completely detach the granulosa cells.

[0053] 1.2. Oocyte maturation in vitro.

[0054] The GV-stage oocytes without granulosa cells were incubated in a culture dish with M16 medium that had been balanced in advance in the incubator at 37°C and CO. 2 After culturing in a 5% volume incubator for 2 hours, the germinal vesicle rupture rate was calculated, which was the GVBD incidence rate, and after culturing for 12 hours, the first polar body extrusion rate was calculated, which was the PBE incidence rate.

[0055] 1.3. Transmission electron microscopy analysis.

[0056] Fresh GV stage oocytes were collected and divided into a control group without PLGA-RES and a PLGA-RES treatment group with 50 μg / mL PLGA-RES, 80 oocytes in each group, washed three times with phosphate buffer, fixed with 4 wt% paraformaldehyde at 23 °C for 1 h, then placed in a 2.5 wt% glutaraldehyde fixative for 14 h, washed three times with PBS, washed every 15 min, and then fixed with osmium acid obtained by mixing 1 wt% osmium acid and 1.5 wt% potassium ferrocyanide on ice for 60 min, followed by ddH2O. 2Wash three times with 15-min intervals. Stain with 1 wt% uranyl acetate at 23°C for 1 h, then rinse with ddH 2 O three times, washing once every 15 minutes. To avoid uranium contamination, the number and time of washing can be increased according to the actual situation. Next, dehydrate with four concentration gradient ethanol solutions of 50wt%, 70wt%, 80wt%, and 90wt%, wash once every 5 minutes, and finally dehydrate with 100wt% ethanol for 3 times. The sample was soaked in propylene oxide twice, 5 minutes each time. Subsequently, room temperature infiltration was performed according to the following steps: first, 812 resin and propylene oxide were infiltrated at a volume ratio of 1:1 for 8 hours; then, the volume ratio was 2:1 for infiltration for 8 hours; then, the volume ratio was 3:1 for infiltration for 8 hours; finally, pure 812 resin was used for infiltration at 23℃ for 8 hours. After the sample was embedded, it was polymerized at 60℃, and continuous sectioning was performed using an automatic slicer. The section thickness was set to 100nm, and the section was placed on a slide. Observe under a microscope whether the oocyte was cut. If it was cut, the section thickness was adjusted to 70nm, and finally the oocyte section was fixed on a copper grid. The sections were incubated and stained with uranyl acetate and then observed on a microscope.

[0057] 1.4. Live cell staining.

[0058] During in vitro culture, 10 μg / mL, 50 μg / mL, and 250 μg / mL of PLGA-RES were co-incubated with oocytes in M2 solution supplemented with milrinone for different periods of time. After incubation, the oocytes were washed three times with M2 solution, then placed in M2 solution, and photographed under a laser confocal microscope. In order to control variables, the photographing parameters and exposure time were kept consistent between the groups when photographing. Under the same cell staining conditions and photographing parameters, specific areas were selected using NIS-Elements AR software, and the mean fluorescence intensity per unit area in the specific area was statistically analyzed. Finally, the average of all measurements was calculated, and the final mean fluorescence intensity was compared between different groups.

[0059] 1.5. Immunofluorescence staining.

[0060] Place the oocyte in the fixative and fix it at room temperature for more than 1 hour. If the oocyte has lost its zona pellucida, dilute the fixative with DPBS at a volume ratio of 1:1 and fix the oocyte for 30 minutes. Wash the fixed oocytes with washing solution 3 times, then place them in the permeabilization solution and permeabilize them at 23℃ for 1 hour. If the oocyte has lost its zona pellucida, permeabilize them for 20 minutes. Wash the permeabilized oocytes with washing solution 3 times, then place them in the blocking solution and block them at 23℃ for 1 hour.

[0061] The blocked oocytes were directly placed in the prepared primary antibody and incubated overnight at 4°C. After the primary antibody incubation, the oocytes were washed 3 times with the cleaning solution, and then placed in the prepared secondary antibody and incubated at room temperature for 1 hour. After the secondary antibody incubation, the oocytes were washed 3 times with the cleaning solution, and then the nuclei were stained with DAPI for 5 minutes. The oocytes after the nuclear staining were transferred to the adhesion slide with a mouth pipette and observed with a cover glass. If the oocytes are not pressed, the oocytes after the nuclear staining are washed 3 times with M2 solution, placed in a droplet of a glass bottom culture dish, and covered with paraffin oil for observation.

[0062] An A1 Confocal laser confocal microscope was used for photographic observation. If statistical analysis of fluorescence intensity was required, the imaging parameters and exposure time between groups should be kept consistent. Under the same immunofluorescence staining conditions and photographic parameters, specific areas were selected using NIS-Elements AR software, and statistical analysis was performed on the mean fluorescence intensity per unit area in the specific areas. The mean values ​​of all measured values ​​were calculated, and the final mean fluorescence intensity between different groups was compared.

[0063] 1.6. RT-qPCR.

[0064] (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.

[0065] (2) Extract oocyte RNA using Trizol.

[0066] A. Take the sample out of the -80℃ freezer and place it in an ice box.

[0067] B. Add 500 μL Trizol to each sample, vortex to mix, and place on ice for 5 min.

[0068] C. Add 100 μL of chloroform to each set of sample tubes for extraction, vortex and place on ice for 5 min.

[0069] D. Place the sample in a centrifuge and centrifuge at 14,000 g at 4°C for 10 min. The liquid in the sample tube will be separated into layers. Then aspirate the supernatant and transfer it to a new 1.5 mL centrifuge tube.

[0070] E. Add an equal volume of isopropanol to a new sample tube, then add 2 μL of glycogen, vortex and place on ice for 5 min.

[0071] F. Centrifuge at 14000g at 4℃ for 10 min, discard the supernatant, add 1mL of pre-cooled 75wt% ice ethanol to the sample tube, and vortex.

[0072] G. Centrifuge at 14000g, 4℃ for 5min, discard the supernatant, place the sample on a clean bench, air-dry the white RNA precipitate at room temperature, and stop air-drying when the precipitate turns transparent.

[0073] H. Add 18 μL of double distilled water to the sample tube to dissolve the RNA, mix thoroughly with a pipette, and use a Nanodrop spectrophotometer to detect the RNA concentration.

[0074] (3) Reverse transcription: The reverse transcription system is shown in Table 2.

[0075] Table 2: Reverse transcription system

[0076] The reverse transcription procedure is shown in Table 3.

[0077] Table 3: Reverse transcription procedures

[0078] After the reverse transcription is completed, the reverse transcribed cDNA sample is stored in a -20°C refrigerator for later use.

[0079] (4) Real-time fluorescence quantitative PCR: The 10 μL qPCR reaction system is shown in Table 4.

[0080] Table 4: qPCR reaction system

[0081] According to the qPCR reaction system, the cDNA template was mixed with the reaction kit premix, and then loaded into a 96-well plate for qPCR, and a real-time fluorescence quantitative PCR reaction 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.

[0082] Table 5: Primer sequences

[0083] The qPCR reaction program settings are shown in Table 6.

[0084] Table 6: qPCR reaction procedure

[0085] 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 level of the target gene was calculated by this method.

[0086] 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 results are expressed as mean ± standard error. P <0.05 was considered statistically significant. P <0.01 was considered a significant difference. P <0.001 was considered to be a very significant difference, and ns was considered to be no statistical difference.

[0087] 2. Experimental results.

[0088] 2.1. PLGA-RES can be internalized into oocytes.

[0089] 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 As shown in A. In addition, immunofluorescence and time-lapse imaging results showed that PLGA-RES was internalized into oocytes in a time- and concentration-dependent manner. Figure 3 Figure B and Figure 4 shown.

[0090] 2.2. The Rab11fip4-mediated recycling endosomal pathway plays a key role in oocyte internalization.

[0091] 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 in Figure 3. To further study the intracellular transport 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 7 As shown, the level of LAMP1 was significantly increased in the Rab11fip4-KD group, and the above markers were observed to be distributed in clusters rather than evenly distributed in the cytoplasm. On the other hand, PLGA-RES and Rab11fip4 showed a co-localization distribution pattern. The above results indicate that the Rab11fip4-mediated recycling endosomal pathway regulates the intracellular trafficking of PLGA-RES.

[0092] Example 3: Increasing the expression of Rab11fip4 can promote the in vitro maturation of frozen oocytes.

[0093] 1. Experimental method.

[0094] 1.1 Molecular docking experiment.

[0095] The crystal structure ID of Rab11fip4: Q8BQP8 was downloaded from the UniProt database. Molecular docking studies were performed using AutoDockVina. The docking results were clustered and the model with the lowest binding energy was selected as the predicted binding position of PLGA-RES. The final docking results were visualized using the PyMOL molecular graphics system.

[0096] 1.2. Preparation of oocyte freezing and thawing solutions.

[0097] (1) Pretreatment solution: Add 1 mL of ethylene glycol to 8 mL of DPBS. Shake well and add 1 mL of dimethyl sulfoxide. Shake well and add 30 mg of bovine serum albumin. After standing still and dissolving, store in a refrigerator at 4°C for future use.

[0098] (2) Freezing solution: Add 3 g polysucrose, 1.712 g sucrose, and 0.021 g bovine serum albumin to 7.02 mL of DPBS to obtain a mixed solution, referred to 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 stand until fully dissolved, and store in a refrigerator at 4°C for later use.

[0099] (3) Thawing solution: Add 1.9193 g of sucrose and 30 mg of bovine serum albumin to 10 mL of DPBS, let stand to dissolve, and store in a refrigerator at 4°C.

[0100] 1.3. Acquisition of oocytes from each group.

[0101] The fresh oocyte group was designated as Group F, and the oocytes were obtained by referring to step 1.1 of Example 2.

[0102] The frozen oocyte group was marked as group V, and its oocytes were treated with open capillaries as OPS. The GV stage oocytes were vitrified and frozen. The specific method was as follows: the oocytes were treated in the pretreatment solution for 30 s and in the freezing solution for 25 s, and then the OPS tube containing the oocytes was directly put into liquid nitrogen for freezing, and stored for at least 3 days. When thawing, the OPS tube was taken out of the liquid nitrogen, treated in the thawing solution at 37°C for 5 minutes, and finally rinsed in the M2 solution containing milrinone for 3 times, and transferred to the M2 culture solution containing milrinone for recovery for 1 hour, and the oocytes of group V were obtained for subsequent experiments.

[0103] 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 of the V group.

[0104] 1.4. RT-qPCR.

[0105] 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 sequence is shown in Table 7.

[0106] Table 7: Rab11fip4 primer sequences

[0107] 2. Experimental results.

[0108] 2.1. PLGA-RES can promote the expression of Rab11fip4.

[0109] Molecular docking analysis showed that PLGA-RES could interact with Rab11fip4 by forming hydrogen bonds at the ASP-450 residue. The docking score was -6.895 kcal / mol, indicating that there was a strong binding affinity between PLGA-RES and Rab11fip4, such as Figure 8 In addition, the results of fluorescence staining and RT-PCR further confirmed that PLGA-RES could significantly increase the expression of Rab11fip4 in frozen oocytes, as shown in Figure 2. Fig. 9 shown.

[0110] 2.2. PLGA-RES can improve the in vitro maturation of frozen oocytes.

[0111] like Fig.10 As shown in the figure, compared with group V, the NP group with 50 μg / mL PLGA-RES significantly increased the GVBD ratio, which was 59.40%±2.67% in group V and 80.2%±0.97% in group NP. Given that the increase in aneuploidy rate is the main factor leading to the decline in oocyte quality, the effect of PLGA-RES on aneuploidy rate and spindle positioning was further studied. Fig.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 lower than that of group V (27.9%±2.86%). P <0.05.

[0112] In summary, the above results show that Rab11fip4 is a key molecule regulating oocyte internalization, and PLGA-RES can increase the expression of Rab11fip4 in frozen oocytes, thereby promoting the in vitro maturation of frozen oocytes. In addition, siRNA molecules were prepared, and the siRNA method can effectively inhibit the expression of Rab11fip4, thereby reducing the absorption of harmful nanoparticles by oocytes. The present invention is of great significance for revealing the internalization pathway of oocytes, improving the quality of frozen oocytes, and promoting the safe and effective application of nanoparticles.

[0113] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A siRNA molecule for inhibiting the Rab11fip4 gene, characterized in that: The siRNA molecule is siRNA#2, and the siRNA#2 consists of a sense strand having a nucleotide sequence such as SEQ ID NO.1 and an antisense strand having a nucleotide sequence such as SEQ ID NO.

2.

2. Use of the siRNA molecule according to claim 1 in the preparation of a drug for improving oocyte quality.

3. The use of the siRNA molecule according to claim 2 in the preparation of a drug for improving oocyte quality, characterized in that: Improving the quality of oocytes refers to reducing the absorption of harmful nanoparticles by oocytes during in vitro culture of oocytes.

4. Use of the siRNA molecule according to claim 3 in the preparation of a drug for improving oocyte quality, characterized in that: The harmful nanoparticles are polystyrene nanoparticles.

5. Use of the siRNA molecule according to claim 2 in the preparation of a drug for improving oocyte quality, characterized in that: The medicine is an injection.

6. Use of the siRNA molecule according to claim 5 in preparing a drug for improving oocyte quality, characterized in that: In the injection, the concentration of siRNA molecules is 20 μM to 22 μM.

7. Use of the siRNA molecule according to claim 5 in preparing a drug for improving oocyte quality, characterized in that: In the application, the drug is injected into the cytoplasm of oocytes cultured in vitro.

8. Use of the siRNA molecule according to claim 7 in the preparation of a drug for improving oocyte quality, characterized in that: The oocyte is an oocyte in the germinal vesicle stage.

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