Use of a biological agent in the treatment of placental dysfunction

Abstract

The application aims to provide an application of a biological agent in treating placental dysfunction, and belongs to the technical field of medicine.The biological agent is composed of siRNA for inhibiting expression of a LOC101929800 gene, a transcript sequence of the LOC101929800 gene is SEQ ID NO.1 in the sequence listing, and a sequence of the siRNA is SEQ ID NO.10 and SEQ ID NO.11 in the sequence listing.The application finds that the siRNA can reverse the decrease of the invasion ability of the trophoblast cells caused by hypoxia-reoxygenation to a certain extent by transfecting the siRNA in the cells under hypoxia-reoxygenation, and therefore the siRNA can be used for preparing the biological agent for treating placental injury.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, and in particular relates to the application of a biological agent in the treatment of placental dysfunction. Background Technology

[0002] The placenta is crucial for the fetus during pregnancy. Placental dysfunction can lead to fetal growth restriction and, in severe cases, even fetal death. Normal placental function depends on the normal proliferation and differentiation of trophoblast cells. Disorders in trophoblast cell differentiation can cause placental dysfunction, resulting in pregnancy complications such as preeclampsia.

[0003] Preeclampsia is a major cause of maternal illness, death, perinatal mortality, premature birth, and intrauterine growth retardation. Decreased trophoblast cell infiltration capacity is a major cause of placental dysfunction leading to preeclampsia. Therefore, effectively improving the trophoblast cell infiltration capacity through biological agents will help treat placental dysfunction. Summary of the Invention

[0004] The purpose of this invention is to propose the application of siRNA that inhibits the expression of the LOC101929800 gene in the treatment of placental dysfunction caused by decreased trophoblast cell infiltration capacity.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The application of the LOC101929800 gene as a target in the preparation of biological agents for the treatment of placental dysfunction, wherein the transcript sequence of the LOC101929800 gene is SEQ ID NO.1 in the sequence listing.

[0006] Preferably, the target inhibition is performed using siRNA from the LOC101929800 gene.

[0007] Preferably, the siRNA sequence is SEQ ID NO.10 and SEQ ID NO.11 in the sequence listing.

[0008] The application of siRNA that inhibits the expression of the LOC101929800 gene in the preparation of biological agents for the treatment of placental dysfunction, wherein the transcript sequence of the LOC101929800 gene is SEQ ID NO.1 in the sequence listing.

[0009] Preferably, the siRNA sequence is SEQ ID NO.10 and SEQ ID NO.11 in the sequence listing.

[0010] The application of siRNA that inhibits the expression of the LOC101929800 gene in the preparation of a biological agent that promotes the invasion of trophoblast cells damaged by hypoxia-reoxygenation, wherein the transcript sequence of the LOC101929800 gene is SEQ ID NO.1 in the sequence listing.

[0011] Preferably, the siRNA sequence is SEQ ID NO.10 and SEQ ID NO.11 in the sequence listing.

[0012] The application of siRNA that inhibits the expression of the LOC101929800 gene in the preparation of a biological agent that promotes the expression of MMP2, a protein related to trophoblast cell invasion induced by hypoxia-reoxygenation, is characterized in that the transcript sequence of the LOC101929800 gene is SEQ ID NO.1 in the sequence listing.

[0013] Preferably, the siRNA sequence is SEQ ID NO.10 and SEQ ID NO.11 in the sequence listing.

[0014] The application of a biological agent in the treatment of placental dysfunction, wherein the biological agent is an siRNA that inhibits the expression of the LOC101929800 gene, the transcript sequence of the LOC101929800 gene is SEQ ID NO.1 in the sequence listing; and the sequences of the siRNA are SEQ ID NO.10 and SEQ ID NO.11 in the sequence listing.

[0015] The beneficial effects of this invention are: Hypoxia-reoxygenation is one of the main causes of trophoblast cell damage. This invention found that hypoxia-reoxygenation treatment can increase the expression level of the LOC101929800 gene in trophoblast cells. By transfecting hypoxia-reoxygenated cells with siRNA to inhibit the LOC101929800 gene, it was found that the decrease in trophoblast cell invasion ability caused by hypoxia-reoxygenation could be reversed to a certain extent. Therefore, it can be used to prepare biological agents for the treatment of placental injury. Attached Figure Description

[0016] Figure 1 Expression of the LOC101929800 gene under hypoxia-reoxygenation conditions; Figure 2 The interference effect of siRNAs of the three LOC101929800 genes designed in this invention was detected; Figure 3 The regulatory effect of LOC101929800 gene siRNA on trophoblast cell invasion under hypoxia-reoxygenation conditions; Figure 4Results of siRNA analysis of the LOC101929800 gene on the regulation of MMP2 protein expression in trophoblast cells under hypoxic-reoxygenation conditions. Detailed Implementation

[0017] To clearly illustrate the technical features of this solution, the following detailed implementation method will be used to describe the solution.

[0018] Example 1 (1) HTR-8 / SVneo cells were seeded in culture plates and cultured overnight. Then, cells that did not adhere were washed with PBS to remove them. (2) The cells of the control group were placed in an incubator and cultured for another 48 hours; (3) The cells in the experimental group were first cultured in an incubator with 5% CO2, 2% O2, and 93% N2 for 8 hours, and then cultured in an incubator with 5% CO2, 20% O2, and 75% N2 for 16 hours. This was repeated twice, with 5 replicates in each group. (4) After the treatment, remove the culture medium, wash the cells with PBS, add 1 ml of TRIZOL, blow the cells off with a pipette and collect them in an enzyme-free EP tube; (5) Place at room temperature for 5 min, centrifuge at 12000 rpm / min for 10 min, and aspirate the supernatant into a new enzyme-free EP tube; (6) Add 200 μL of chloroform, shake to mix, place at room temperature for 5 min, centrifuge at 12000 rpm / min for 15 min, and aspirate 400 μL of the uppermost aqueous phase into a new enzyme-free EP tube. (7) Add 400 μL of isopropanol, mix well, let stand at room temperature for 10 min, and then centrifuge at 12000 rpm for 10 min. (8) After centrifugation, discard the supernatant to obtain a white precipitate; (9) After dissolving the precipitate in 500 μL of 70% ethanol prepared with DEPC water, centrifuge at 8000 rpm for 5 min. Repeat this step to remove the supernatant, dry the precipitate in a fume hood, add DEPC water, and obtain the RNA sample. (10) Prepare the following system according to the reverse transcription kit instructions: The reaction is carried out under the following conditions: 37℃ for 15 min, 85℃ for 5 s, and 4℃ for 5 min.

[0019] (11) Prepare the following system according to the instructions of the real-time PCR kit: The reaction is carried out under the following conditions: 95℃ for 5 min; 95℃ for 15 s, 60℃ for 40 s, 40 cycles; The primer sequences are as follows: (12) Use 2 -△△Ct The method processes the data, and the results are displayed. Figure 1 middle.

[0020] The experimental results show that, compared with the control group, the expression level of LOC101929800 in cells treated with hypoxia and reoxygenation was significantly increased, indicating that when trophoblast cells are damaged, the expression level of LOC101929800 in cells is upregulated.

[0021] Example 2 (1) The sequences of the si-RNA (si-LOC101929800-1,2,3) and si-NC of LOC101929800 designed in this invention are shown below: (2) HTR-8 / SVneo was seeded in 6-well plates and cultured overnight. Then, si-NC and si-LOC101929800 were transfected into the cells according to the Lipofectamine 2000 instructions. (3) 48 h after transfection, RNA was extracted according to the method in Example 1, and the interference effects of si-LOC101929800-1, si-LOC101929800-2, si-LOC101929800-3 and si-NC were detected. The blank control group was not treated. Three replicates were set up for each group. The results are as follows: Figure 2 As shown.

[0022] The experimental results show that si-NC has no inhibitory effect, while si-LOC101929800-1 has an inhibition rate of 61.2%, si-LOC101929800-2 has an inhibition rate of 63.9%, and si-LOC101929800-3 has an inhibition rate of 81.7%. In subsequent experiments, si-LOC101929800-3 will be selected for further experiments.

[0023] Example 3 (1) Dissolve the Matrigel in a refrigerator at 4°C overnight; (2) Dilute the matrix gel with RPMI 1640 medium, place the Transwell chamber into a 24-well plate, add 50 μL of diluted matrix gel to each well, and place it in a cell culture incubator to solidify; (3) Control group: vaccinated with 1×10 5 HTR-8 / SVneo cells transfected with si-NC were cultured for 48 h under normal conditions; si-NC group: seeded with 1×10 5 HTR-8 / SVneo cells transfected with si-NC were subjected to two cycles of normoxic (8 h) and reoxygenation (16 h); si-NC LOC101929800-3 group: seeded with 1×10 5 The virus was transfected with si- LOC101929800-3 and subjected to two cycles of normoxic (8h) and reoxygenation (16h). (4) After the treatment is completed, remove the chamber and gently wipe away any excess matrix gel and cells inside the chamber with a cotton swab; (5) After washing the chambers three times with PBS, fix the cells with methanol for 15 min; (6) After fixation, remove the fixative, wash the chamber three times with PBS, and stain with 0.5% crystal violet staining solution for 15 min; (7) After staining, remove the staining solution, take pictures under a microscope, and count the samples from 5 different fields of view. The experimental results are as follows: Figure 3 As shown.

[0024] The experimental results show that hypoxia-reoxygenation treatment reduces the invasive ability of trophoblast cells, while the invasive ability of trophoblast cells transfected with si-LOC101929800-3 is reversed to some extent.

[0025] Example 4 (1) Control group: HTR-8 / SVneo cells transfected with si-NC seeded in 6-well plates were cultured under normal conditions for 48 h; si-NC group: HTR-8 / SVneo cells transfected with si-NC seeded in 6-well plates were subjected to normoxic (8 h) and reoxygenation (16 h) treatment for 2 cycles; si-LOC101929800-3 group: HTR-8 / SVneo cells transfected with si-LOC101929800-3 seeded in 6-well plates were subjected to normoxic (8 h) and reoxygenation (16 h) treatment for 2 cycles; (2) After the treatment, wash the cells with PBS and add 100 μL of protein lysis buffer to each well; (3) After the cells are fully lysed, the lysate is transferred to a centrifuge tube and centrifuged at 12,000 rpm for 15 minutes. After centrifugation, the supernatant is transferred to a new centrifuge tube. (4) Determine the protein concentration of the sample according to the BCA instructions, add loading buffer to adjust the protein concentration to 2ug / ul, boil at 100℃ for 5min to obtain the protein sample; (5) Assemble the electrophoresis tank, add 10 μL of protein sample and protein marker to each well, and perform electrophoresis at a constant voltage of 80 V. After the protein markers separate, adjust the voltage to 120 V until the electrophoresis is finished. (6) Assemble the electric rotary clamp and electric rotary slot according to the "sandwich model", and run the electric rotary at 250mA for 1.5h; (7) After electroporation, remove the membrane and transfer it to 5% skim milk powder. Incubate at room temperature for 1 hour. (8) Wash the membrane three times with TBST for 10 min each time, incubate with the primary antibody dilution of MMP2 and β-actin overnight at 4°C on a shaker; (9) After incubation, recover the antibody, wash the membrane three times with TBST for 10 min each time, and incubate the secondary antibody for 1 h; (10) In a dark room, quickly add the luminescent liquid droplet onto the PVDF membrane for imaging.

[0026] Experimental results are as follows Figure 4 As shown in the figure, hypoxia-reoxygenation reduces the protein expression of the invasion-related protein MMP-2, while transfection with si-LOC101929800-3 can reverse the decrease in MMP-2 protein expression caused by hypoxia-reoxygenation to a certain extent. sequence list <110> Qingdao Sitop New Source Cell Medicine Co., Ltd. <120> Application of a biological agent in the treatment of placental dysfunction <160> 13 <170> SIPOSequenceListing 1.0 <210> 1 <211> 2274 <212> DNA <213> Human Source <400> 1 atgggggtcc taagagccgg gggggggaaga ggggctggct ctcagtcccc gcctcgcggg 60 aggtgcctcc ccccactgcg atgggggtcc caagagccag ggagggaaga ggggctggct 120 ctcagtcccc gcctcgcggg aggtgcctcc gccccttccg atgggggtcc caagacccag 180 ggaggggaga ggggctggct ctcagtcccc tcctcccggg ggtacctcct ccgcctgcga 240 tgggggtccc aagagacagg gggggaagag gggctggctc tcagtccacg cctcgtgggg 300 gatgcctccc ccccctgcga ttggggtccc aacagccagc gggggaagag gggctggttc 360 tcagtccccg cctcgcgggg gatgcctccc cgtgctgcga tgggggtcct aaaagccagg 420 gggtgaaaag gggctggctc tcagtcccca cctcgcgagg ggtgcctctc ccccctgcga 480 taggtgtcct aatagccagg gggggaagag gggctggctg tcagtcccca cctcgcgagg 540 ggtgcctctc ccccctgcga caggtgtcct aatagccagg gggggaagag gggctggctg 600 tcagtccctg cctcgcgggt ggtccttcac cccgctgcga tgggggtcct aagagcaagg 660 gggggaagtg gggctggctc tctgtccccg cctcgccggg ggtgcctcgc acccttgcga 720 tgggggtcct aagagccagt aggggaagag gagctggctc tcagtcacct cagcatggag 780 ggcctttctg ttctggtttt gcccaagagt aagcttattt gcatctggtt ctagcaaggg 840 aattgctgcg aaggccctca aacagggggg ccatcctttc gaatccctat ctagttgttt 900 agagatgtag gccaccggcc tcagccaagg ccccacagtt tgggttaaaa gtccagctgc 960 catcttttct ctctgacgca tacaatggaa aaggctttgt gagatcgggt agcccctcgg 1020 ctggggcttt cagaagtttt tcctttaagt catgaaagac ttgctgttgt tggaatcccc 1080 attccaaaag ttcccggtcc ccgcccccat tgtgacctca tacaaaggct tggctaatac 1140 tgcaaagttt gggattccca gtctacaaaa ccccacagct cctaggaatt ctctcaccac 1200 ccttgtgcct ttaggcttcg gtagattgca aatgacctgc tttctttcgg atcccgggct 1260 gctttcggac acctgtcgaa tagtaaatcc caagtaaggt acctgcggtc gtcggcagat 1320 ctgaatttc ttcttggaca cttaataccc acagccctcc agagaccgag aggttgatgt 1380 cactcccaat atcggaggaa gtatacaccc ccgtgtgaga tggtccttaa tatattcca 1440 cggcggaggg ggtgatatga ctacatacat ggcagaaagt ggaaacctcc cagggatatt 1500 gttcccacga tcctggaggg aagaagatga tgttactttc aatatgacag aaggtggatg 1560 aagtggtgga ctgcccctcc acacctgtgg acacgccccc actgatattc cttctaattg 1620 cagcgtggga gaggaga tgacacgcga tatcgcaggg agtagaaaca cccctgtgat actgttctta atattcaggg aggagagga tgatattact cccaatacag acgggtgtac accctctgta caccgagggt gtacacctgt ctgtgaaaga atggacacgc ccccactgat attgttttta atgcagcgtg ggaggggagg atatgacacg cgatatcgca gctgagtgcc 1860. acgtgctgac gtcactaaga tcaatacagc aaactctgaa agatggacag aggacagga gatggtcctt fathergcag tgtgatctgt gctgcaatag agttttggaa gctagaagtt caaaacgagg tgttggcagc accatgctct ctctgaagat gctaggaaga atctgctcca tgccttcca ttcgctcctg gggtttcctg caagccctga cattccttgg cttgtagatg 2160. catcacccca gtttccgccc ccatcatcac atggcctcct ctctgtgtgt gcctctgcgt tccctctatt cttcttctaa ggacaccgac accagtcata gtggattag ggtccactcc snow catctgcaac aaccctattt snow tcacattcta agat <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 tggatgaagt ggtggactgc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 agcgaatgga aaggcatgga 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 tcaccaactg ggacgacatg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 ccaggaagga aggctggaag 20 <210> 6 <211> 19 <212> RNA <213> Artificial Sequence <400> 6 cuaguuguuu agagaugua 19 <210> 7 <211> 19 <212> RNA <213> Artificial Sequence <400> 7 uacaucucua aacaacuag 19 <210> 8 <211> 19 <212> RNA <213> Artificial Sequence <400> 8 cgugugagau gguccuuaa 19 <210> 9 <211> 19 <212> RNA <213> Artificial Sequence <400> 9 uuaaggacca ucucacacg 19 <210> 10 <211> 19 <212> RNA <213> Artificial Sequence <400> 10 ggcuaauacu gcaaaguuu 19 <210> 11 <211> 19 <212> RNA <213> Artificial Sequence <400> 11 aaacuuugca guauuagcc 19 <210> 12 <211> 19 <212> RNA <213> Artificial Sequence <400> 12 cucuccguac gugucacga 19 <210> 13 <211> 19 <212> RNA <213> Artificial Sequence <400> 13 ucgugacacg uacggagag 19

Claims

1. The application of siRNA inhibiting LOC101929800 gene expression in the preparation of a biological agent for treating placental dysfunction caused by decreased trophoblast cell invasion due to hypoxia-reoxygenation, characterized in that... The transcript sequence of the LOC101929800 gene is SEQ ID NO.1 in the sequence listing; The siRNA sequence is SEQ ID NO.10 and SEQ ID NO.11 in the sequence listing.

Images