WO2023221324A1 - 一种非编码rna snhg17作为标志物和治疗靶点的用途 - Google Patents

一种非编码rna snhg17作为标志物和治疗靶点的用途 Download PDF

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WO2023221324A1
WO2023221324A1 PCT/CN2022/115048 CN2022115048W WO2023221324A1 WO 2023221324 A1 WO2023221324 A1 WO 2023221324A1 CN 2022115048 W CN2022115048 W CN 2022115048W WO 2023221324 A1 WO2023221324 A1 WO 2023221324A1
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snhg17
group
seq
cells
breast cancer
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雷蕾
王晓稼
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浙江省肿瘤医院
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Definitions

  • the invention belongs to the field of tumor biological therapy, and specifically relates to the use of a non-coding RNA SNHG17 as a marker and therapeutic target.
  • Breast cancer is the female malignant tumor with the highest incidence rate worldwide, with approximately 304,000 cases occurring every year. The incidence rate continues to rise, seriously affecting women's health. Nearly 70% of breast cancers are hormone receptor-positive breast cancer, so treatments and drugs based on hormone receptor-positive breast cancer are crucial. Endocrine therapy is a common method for treating this type of breast cancer in the prior art. Although it is a more effective treatment method in the prior art, it still has major shortcomings, that is, primary or secondary endocrine resistance in endocrine therapy. Drugs, this type of drug resistance will seriously affect the patient's treatment effect, making the treatment effect unable to meet expectations. However, there are currently no effective biomarkers that can be used to predict fulvestrant sensitivity or to reverse drug resistance with targeted therapy.
  • Long non-coding RNA is a type of non-coding RNA molecule with a transcript length of more than 200nt, which is generally considered to have no protein-coding function.
  • Long non-coding RNA is highly specific in different tissues. Not only is its expression level closely related to different stages of tumor development and different histological types, it can be used as a specific marker for tumor detection; The important regulatory role of long non-coding RNA in tumor development also shows its possibility as a target for tumor treatment.
  • the purpose of the present invention is to provide a use of non-coding RNA SNHG17 as a marker and therapeutic target.
  • By detecting the expression of non-coding RNA SNHG17 in breast cancer it can predict the fulvestrant resistance of breast cancer.
  • By knocking down the non-coding RNA SNHG17, Expression of coding RNA SNHG17 can increase the sensitivity of drug-resistant breast cancer cells to fulvestrant.
  • shRNA that inhibits the expression of non-coding RNA SNHG17 in the preparation of drugs that reverse fulvestrant resistance.
  • the coding sequence of the above-mentioned shRNA is as shown in SEQ ID NO.1 and SEQ ID NO.2, or SEQ ID NO. 3 and SEQ ID NO.4.
  • the construct is obtained by inserting the above-mentioned shRNA into the lentiviral vector plasmid pLKO.1-GFP-Puro.
  • the constructs described above are stored in a lyophilized formulation.
  • RNA SNHG17 in the preparation of detection agents for predicting fulvestrant resistance in breast cancer.
  • the above-mentioned detection agent includes a primer pair for detecting the expression of non-coding RNA SNHG17.
  • the above primer pair is as shown in SEQ ID NO.5 and SEQ ID NO.6.
  • shRNA that inhibits the expression of non-coding RNA SNHG17 and the use of fulvestrant in the preparation of drugs for the treatment of breast cancer.
  • the coding sequence of the above-mentioned shRNA is as shown in SEQ ID NO.1 and SEQ ID NO.2, or SEQ ID NO. 3 and SEQ ID NO.4.
  • a pharmaceutical composition including fulvestrant, also includes one of the above-mentioned shRNA or one of the above-mentioned constructs, and pharmaceutically acceptable carriers and/or excipients.
  • the dosage form of the above-described construct is a lyophilized dosage form.
  • the beneficial effects of the present invention are: based on the abnormal expression of non-coding RNA SNHG17 in different breast cancer tissues, detecting non-coding RNA SNHG17 in the sample can help determine whether breast cancer cells in the sample are resistant to Fulvest group drug resistance, thereby formulating a more effective treatment plan and improving the therapeutic effect; in addition, the present invention also provides an shRNA that knocks down the non-coding RNA SNHG17, and achieves fulvestrant resistance in breast cancer by knocking down the expression of the non-coding RNA SNHG17. The reversal of the drug can increase the sensitivity of breast cancer to fulvestrant and improve the therapeutic effect.
  • Figure 1 shows the relative expression of SNHG17 in MCF-7 and MCF-7R cells in Example 1 of the present invention
  • Figure 2 is a fluorescence in situ hybridization diagram of SNHG17 in cells in Example 1 of the present invention
  • Figure 3 shows the relative expression of SNHG17 in SNHG17 overexpressing breast cancer cells in Example 2 of the present invention
  • Figure 4 shows the colony formation of cells in Example 2 of the present invention
  • Figure 5 is the growth inhibition rate of fulvestrant on breast cancer cells in Example 2 of the present invention.
  • Figure 6 shows the relative expression of SNHG17 in cells in Example 3 of the present invention
  • Figure 7 shows the colony formation of cells in Example 3 of the present invention.
  • Figure 8 is a comparison chart of the biological titers of the lentiviral vectors in the K1 group, K2 group, K3 group, and K4 group after freeze-drying in Example 4 of the present invention
  • Figure 9 is a comparison chart of the freeze-drying survival rates of the K1 group, K2 group, K3 group, and K4 group in Example 4 of the present invention.
  • Figure 10 shows the titer recovery rate of the lentiviral vector stored at -20°C for 3 months in Example 4 of the present invention
  • Figure 11 shows the titer recovery rate of lentiviral vectors stored at 23°C for 20 days in Example 4 of the present invention
  • Figure 12 shows the titer recovery rate of the lentiviral vector stored at 4°C for 30 days in Example 4 of the present invention.
  • MCF-7 is a fulvestrant-sensitive breast cancer cell line
  • MCF-7R is a fulvestrant-resistant breast cancer cell line.
  • MCF-7 cells were obtained from the ATCC cell bank. MCF-7 cells were cultured in DMEM complete medium containing 10wt% fetal calf serum. The medium was added with 200U/mL penicillin and 200U/mL streptomycin.
  • MCF-7R 1.2 Construction of MCF-7R cell line: Take 1 ⁇ 10 7 MCF-7 cells in the logarithmic growth phase and inoculate them into a 10cm diameter culture dish (containing 10wt% fetal bovine serum, penicillin 200U/mL, streptomycin DMEM complete culture medium (200 U/mL) is used as the culture medium). After the cell growth is stable, 1 ⁇ mol/L fulvestrant is added to the culture medium. After changing the medium every 48 hours, the same concentration of fulvestrant is added. After 12 months, it passes The breast cancer MCF-7 fulvestrant-resistant monoclonal cell line (called MCF-7R) was obtained by the linear dilution method, and then expanded without drug intervention. In order to maintain the drug resistance of MCF-7R, later culture in culture medium containing 0.5 ⁇ mol/L fulvestrant.
  • MCF-7R breast cancer MCF-7 fulvestrant-resistant monoclonal cell line
  • MCF-7 and MCF-7R cells into a 1.5mL centrifuge tube respectively, wash twice with 2mL PBS, add 1mL Trizol, pipette repeatedly for 20 times to ensure that the cells are completely lysed, and then pipet them into a 1.5mL centrifuge tube; Leave it at room temperature for 5 minutes to fully lyse the sample. Add 0.2 mL of chloroform, shake vigorously for 10 seconds, and leave it at room temperature for 2 minutes. Centrifuge at 12000g and 4°C for 15 minutes. Absorb the upper colorless aqueous phase and move it to a new centrifuge tube. Add 0.5 ml of isopropyl alcohol and invert.
  • the reaction conditions were: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, amplification for 40 cycles, and then full extension at 72°C for 10 min.
  • GAPDH GAPDH as the internal reference gene
  • the relative expression of SNHG17 was calculated using the 2 - ⁇ CT method.
  • the relative expression of SNHG17 in MCF-7 and MCF-7R cells is shown in Figure 1, where A is the MCF-7 parental cell and B is the MCF-7R drug-resistant cell. **P ⁇ 0.01 indicates statistical significance.
  • the expression level of long non-coding RNA SNHG17 in MCF-7 parental cells is significantly lower than that in MCF-7R drug-resistant cells, indicating that the expression level of long non-coding RNA SNHG17 in drug-resistant fulvestrant-sensitive breast cancer cell lines Expression levels were higher than in fulvestrant-resistant breast cancer cell lines.
  • the probe sequence is 5'-AGTCTCCCATGTCTGGACCCCGAATCTTG-3', and FITC (fluorescein isothiocyanate) fluorophore is added to its 5' end as a universal fluorescent labeling sequence for FISH probes. It is provided by Shanghai Sangon Co., Ltd. synthesis.
  • FITC fluorescent dye emits green fluorescence under green excitation light
  • DAPI fluorescent dye emits blue fluorescence under UV light
  • Two channels are used for observation and photo recording of results.
  • the fluorescence in situ hybridization diagram of SNHG17 in cells is shown in Figure 2, where A is MCF-7 parental cells, B is MCF-7R drug-resistant cells, a is a photo using DAPI channel, b is a photo using FITC channel, c For photos using merged channels.
  • Upstream primer 5'-AGGA GCTAGC TGCTTGTAAGGCAGGGTCTC-3'
  • Downstream primer 5'-ATGC GAATTC ACAGCCACTGAAAGCATGTG-3';
  • the underlined parts of the upstream and downstream primers are NheI and EcoRI enzyme cleavage sites.
  • RNA of breast cancer cell MCF-7 was extracted using the Trizol method and reverse transcribed into cDNA (the method is the same as 1.3.1 and 1.3.2 in Example 1).
  • the PCR reaction system is shown in Table 3 (20 ⁇ L):
  • Reaction steps Pre-denaturation at 94°C for 5 minutes; denaturation at 95°C for 10 seconds, annealing at 62°C for 30 seconds, extension at 72°C for 60 seconds, 39 cycles of amplification, and extension at 72°C for 10 minutes.
  • the PCR product is electrophoresed and gel is used to recover the target fragment.
  • the ligation reaction system (10 ⁇ L) is shown in Table 5:
  • DMEM complete medium containing 300 ⁇ g/mL G418 to monoclonalize the selected cells, expand the culture of the monoclonal cells, and divide the experiment into the control group (empty system lentivirus) and the pcDNA3.1-SNHG17 group to obtain SNHG17 Overexpression control breast cancer stable cell line, SNHG17 overexpression breast cancer stable cell line.
  • the relative expression of SNHG17 in SNHG17-overexpressing breast cancer cells is shown in Figure 3, where C is SNHG17-overexpressing control breast cancer cells, and B is SNHG17-overexpressing breast cancer cells. **P ⁇ 0.01 indicates statistical significance.
  • the number of colonies formed by SNHG17-overexpressing breast cancer cells is greater than that of SNHG17-overexpressing control breast cancer cells, indicating that overexpression of SNHG17 in sensitive strains will enhance the resistance of breast cancer cells to fulvestrant.
  • MCF-7 and SNHG17 overexpressing breast cancer cells in the logarithmic growth phase were seeded into a 96-well plate (with DMEM complete medium) at a density of 3000 cells per well. ), culture it in a 37°C 5v/v% CO2 incubator until the cells adhere, and then add fulvestrant respectively.
  • the final concentrations are 0 (cell control group), 2, 4, 8 and 16 ⁇ mol/L, each Set 4 duplicate wells at each concentration and set up a blank control group. After placing it in a 37°C, 5v/v% CO2 incubator for 48 hours, add 20 ⁇ L MTT (5g/L) solution to each well.
  • the growth inhibition rate of fulvestrant on SNHG17-overexpressing breast cancer cells is significantly greater than that of SNHG17-overexpressing control breast cancer cells. This shows that overexpression of SNHG17 in sensitive strains will reduce the response of breast cancer cells to fulvestrant. Si Qun's sensitivity.
  • shRNA oligonucleotide chain was synthesized by Shanghai Sangon Bioengineering Technology Services Co., Ltd., see Table 6.
  • reaction conditions were set to 95°C for 5 minutes, and the subsequent systems were 85°C for 5 minutes, 75°C for 5 minutes, and 70°C for 5 minutes. Finally, the reaction system was stored at 4°C. The final products were 10 ⁇ M shRNA1 template and 10 ⁇ M shRNA2. template.
  • DMEM complete culture medium 300 ⁇ L DMEM complete culture medium and mixed well to obtain system X2; add system X2 to system Cultivate overnight in a 37°C 5v/v% CO2 incubator; 18h after transfection, replace the medium with 5 mL of DMEM complete medium containing 10wt% fetal calf serum. Collect the supernatant (viral stock solution) 48h after transfection. Centrifuge the virus stock solution at 2500 rpm for 15 minutes, filter the supernatant with a 0.45 ⁇ m filter, and store the filtrate at -80°C to obtain pLKO.1-shRNA lentivirus.
  • pLKO.1-shRNA1 lentivirus is obtained from the above different recombinant plasmids. , pLKO.1-shRNA2 lentivirus. Empty system lentivirus was prepared using the same method.
  • the experiment was divided into control group (empty system lentivirus), pLKO.1-shRNA1 group, and pLKO.1-shRNA2 group.
  • Detection of knockdown effect The lentiviral vector contains GFP green fluorescent protein. 96 hours after virus infection, use an inverted fluorescence microscope to observe the GFP green fluorescence to observe the infection of the target cells by the virus; use BD influx cell sorter to sort cells. The knockdown cells were sorted using an instrument to obtain 100% GFP-selected cells.
  • E is the control group (empty system lentivirus silencing control cells)
  • F is pLKO.1-shRNA1 silencing cells
  • G is pLKO.1-shRNA2 silencing cells
  • * indicates the same as Compared with the control group, **P ⁇ 0.01 indicates statistical significance.
  • Plate colony formation experiment Use empty system lentivirus silencing control cells and pLKO.1-shRNA2 silencing cells to conduct experiments to detect resistance to fulvestrant. Inoculate 1,000 cells in good growth status into a culture dish (diameter 60mm) containing DMEM complete culture medium. Gently shake the culture dish in the cross direction to disperse the cells evenly; culture it at 37°C and 5v/v% CO2. For 15 days, replace the complete culture medium every 3 days; when clones are visible to the naked eye in the culture dish, terminate the culture, discard the culture medium, carefully soak twice in PBS solution, and air dry.
  • the lentiviral vector filtrate was prepared according to the preparation method of Example 1, and concentrated at 1:50 times using ultrafiltration to obtain a lentiviral vector concentrate.
  • the titer of the lentiviral vector concentrate is 4.13 ⁇ 10 7 TU/mL.
  • the preparation method of the K1 group lyophilizing protectant solution is: adding 15wt% trehalose, 0.009wt% L-alanine, 0.016wt% histidine, 0.001wt% CaCl 2 , 0.0008wt% MgSO 4 to PBS; K2
  • the preparation method of the freeze-drying protectant solution is as follows: adding 15wt% trehalose, 1wt% glycerol diacetate, 0.2wt% glyceryl phosphorylcholine, 0.009wt% L-alanine, and 0.016wt% histamine to PBS acid, 0.001wt% CaCl 2 , 0.0008wt% MgSO 4 ;
  • the preparation method of the K3 group lyophilizing protectant solution is: add 15wt% trehalose, 1wt% glyceryl diacetate, and 0.009wt% L-alanine to PBS , 0.016wt% histidine,
  • Detection of lentiviral vector biological titer Take a 24-well plate as an example, add 400 ⁇ L of 293FT cell suspension to each well, the cell density is 1.0 ⁇ 10 5 cells/mL, take the lentiviral vector freeze-dried preparation and add 500 ⁇ L of bacterial endotoxin Check the redissolution with water. After the dissolution is complete, add DMEM complete medium for serial dilutions of 10 times, 100 times, and 1000 times. After gently pipetting evenly, add 200 ⁇ L lentiviral vector diluent to each well, and place the well plate in the incubator.
  • Cultivate for 24 hours replenish the cells the next day, continue culturing for 48 hours, take out the 24-well lentivirus titer detection cell plate, digest it with trypsin, collect the cells in each well, and transfer the cells to a 1.5 mL centrifuge tube; centrifuge at 4°C, 1500 rpm 5min, collect the cell pellet; discard the supernatant, resuspend the cell pellet in 1mL of pre-cooled PBS in each tube; centrifuge for 5 minutes at 4°C, 1500rpm and wash twice, discard the PBS in the centrifuge tube, and resuspend with 500ul of pre-cooled PBS.
  • Lentivirus titer calculation data selection Select the group with a positive rate between 1 and 20% for titer calculation; the titer calculation formula is as follows:
  • N is the number of cells before lentivirus infection
  • P is the cell positivity rate (1 ⁇ 20%)
  • V is the lentivirus infection volume of cells in each well
  • TU infectious titer.
  • Lyophilization survival rate (virus titer after freeze-drying/virus titer before freeze-drying) ⁇ 100%.
  • the average titers of lentiviral vectors in each group before and after lyophilization are shown in Table 9.
  • the comparison chart of biological titers of lentiviral vectors after freeze-drying in K1 group, K2 group, K3 group and K4 group is shown in Figure 8, where H is K1 group, I is K2 group, J is K3 group, K is K4 group, * indicates Compared with the K1 group, **P ⁇ 0.01 indicates statistical significance; # indicates that compared with the K2 group, #P ⁇ 0.05, ##P ⁇ 0.01 indicates statistical significance.
  • Group Exterior Resolubility (redispersion time) K1 Loose, honeycomb-like Good, within 30s K2 Loose, honeycomb-like Good, within 30s K3 Loose, honeycomb-like Good, within 30s K4 Loose, honeycomb-like Good, within 30s
  • the lentiviral vector lyophilized preparations of groups K1, K2, K3, and K4 have a fixed shape and are in good condition after being reconstituted with water.
  • the redispersion times are all within 30 seconds.
  • the titer recovery rate of lentiviral vectors stored at –20°C for 3 months is shown in Figure 10, where H is the K1 group, I is the K2 group, J is the K3 group, and K is the K4 group, * indicates compared with the K1 group, * *P ⁇ 0.01 indicates statistical significance; # indicates compared with K2 group, ##P ⁇ 0.01 indicates statistical significance.
  • the titer recovery rate of the lentiviral vector stored at 23°C for 20 days is shown in Figure 11, where H is the K1 group, I is the K2 group, J is the K3 group, and K is the K4 group.
  • the titer recovery rate of the lentiviral vector freeze-dried preparation prepared by the K2 group was 94.2% when stored at –20°C for 3 months, which was greater than that of the K1 group, K3 group, and K4 group. ;
  • the titer recovery rate of the lentiviral vector freeze-dried preparation prepared by the K2 group was 92.1% when stored at 23°C for 20 days, which was greater than that of the K1 group, K3 group, and K4 group; the lentiviral vector freeze-dried preparation prepared by the K2 group was stored at 23°C for 20 days.
  • the titer recovery rate stored at °C for 30 days was 92.4%, which was greater than that of the K1 group, K3 group, and K4 group; indicating that compared with the K1 group, K3 group, and K4 group, the lyophilized protective agent of the K2 group can effectively reduce the lentivirus titer. It has good storage stability against loss and damage during long-term storage.
  • the inventor analyzed this: it may be that the hydroxyl groups on trehalose replace water molecules and are partially bound to the protein surface, but due to steric hindrance and other effects, part of the protein surface is still bound to water molecules, while glycerophosphorylcholine and diacetate Glyceride molecules are small and can enter the gaps to supplement the surface of this part of the protein, and fill these gaps through the entanglement of flexible molecular chains, reducing the contact between the protein and water molecules, thereby reducing the mechanical damage caused by ice crystals to the protein, and at the same time Protein is lost and destroyed during storage of lyophilized preparations.

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Abstract

本发明提供一种非编码RNA SNHG17作为标志物和治疗靶点的用途，属于肿瘤生物治疗领域。本发明公开了一种非编码RNA SNHG17在制备预测乳腺癌氟维司群耐药的检测剂中的用途，该非编码RNA SNHG17的核苷酸序列如SEQ ID NO.1所示；还公开了抑制非编码RNA SNHG17表达的shRNA的用途，该shRNA的编码序列如SEQ ID NO.2和SEQ ID NO.3所示，或SEQ ID NO.4和SEQ ID NO.5所示，所述shRNA能够敲低非编码RNA SNHG17的表达，从而提高耐药乳腺癌细胞对氟维司群的敏感性。

Description

一种非编码RNA SNHG17作为标志物和治疗靶点的用途 技术领域
本发明属于肿瘤生物治疗领域，具体涉及一种非编码RNA SNHG17作为标志物和治疗靶点的用途。
背景技术
乳腺癌是当今全球范围内发病率最高的女性恶性肿瘤，每年发病约为30.4万，发病率呈现持续上升的趋势，严重影响女性健康。而乳腺癌中又有将近70％为激素受体阳性乳腺癌，所以基于激素受体阳性乳腺癌的治疗方法和药物显得至关重要。内分泌治疗就是现有技术中常见的治疗该类乳腺癌的方法，其虽然是现有技术中较有效的治疗方法，但其仍存在较大缺陷，即内分泌治疗中的原发或继发内分泌耐药，该类耐药特性会严重影响患者的治疗效果，使得治疗效果无法达到预期。而目前尚无有效的生物标志物可用于预测氟维司群敏感性或用于靶向治疗逆转耐药。
近年来以非编码RNA为核心的表观遗传调控机制在生命活动中的作用越来越受到重视。长非编码RNA是一类转录本长度在200nt以上的非编码RNA分子，通常被认为不具备编码蛋白功能。但越来越多研究显示长非编码RNA的表达在不同的组织具有高度特异性，不仅其表达水平与肿瘤发展的不同阶段、不同组织学类型密切相关，可以作为肿瘤检测的特异标志物；而且长非编码RNA在肿瘤发生发展中的重要的调控作用也显示其作为肿瘤治疗靶点的可能性。
发明内容
本发明的目的在于提供一种非编码RNA SNHG17作为标志物和治疗靶点的用途，通过检测非编码RNA SNHG17在乳腺癌中的表达，能够预测乳腺癌氟维司群耐药，通过敲低非编码RNA SNHG17的表达能够提高耐药乳腺癌细胞对氟维司群的敏感性。
本发明为实现上述目的所采取的技术方案为：
一种抑制非编码RNA SNHG17表达的shRNA在制备逆转氟维司群耐药的药物中的用途，上述shRNA的编码序列如SEQ ID NO.1和SEQ ID NO.2所示，或SEQ ID NO.3和SEQ ID NO.4所示。
一种构建物在制备增加乳腺癌细胞对氟维司群敏感性的药物中的用途，该构建物由上述shRNA插入慢病毒载体质粒pLKO.1-GFP-Puro所得。
在一些实施方式中，上述构建物以冻干制剂形式保存。
一种非编码RNA SNHG17在制备预测乳腺癌氟维司群耐药的检测剂中的用途。
在一些实施方式中，上述检测剂包括用于检测非编码RNA SNHG17的表达情况的引物对。
在一些实施方式中，上述引物对如SEQ ID NO.5和SEQ ID NO.6所示。
一种抑制非编码RNA SNHG17表达的shRNA和氟维司群在制备治疗乳腺癌药物中的用途，上述shRNA的编码序列如SEQ ID NO.1和SEQ ID NO.2所示，或SEQ ID NO.3和SEQ ID NO.4所示。
一种药物组合物，包括氟维司群，还包括上述的一种shRNA或上述的一种构建物，以及药学上可接受的载体和/或辅料。
在某些实施方案中，上述构建物的剂型为冻干剂型。
与现有技术相比，本发明的有益效果为：基于非编码RNA SNHG17在不同乳腺癌组织中的异常表达，通过检测样本中非编码RNA SNHG17能够帮助判断样本中乳腺癌细胞是否对氟维司群耐药，从而制定更为有效的治疗方案，提高治疗效果；此外，本发明还提供一种敲低非编码RNA SNHG17的shRNA，通过敲低非编码RNA SNHG17表达实现乳腺癌氟维司群耐药的逆转，提高乳腺癌对氟维司群的敏感性，提高治疗效果。
附图说明
图1为本发明实施例1中MCF-7及MCF-7R细胞中SNHG17的相对表达量；
图2为本发明实施例1中细胞中SNHG17的荧光原位杂交图；
图3为本发明实施例2中SNHG17过表达乳腺癌细胞中SNHG17的相对表达量；
图4为本发明实施例2中细胞的集落形成；
图5为本发明实施例2中氟维司群对乳腺癌细胞的生长抑制率；
图6为本发明实施例3中细胞中SNHG17的相对表达量；
图7为本发明实施例3中细胞的集落形成；
图8为本发明实施例4中K1组、K2组、K3组、K4组慢病毒载体冻干后的生物滴度对比图；
图9为本发明实施例4中K1组、K2组、K3组、K4组的冻干存活率对比图；
图10为本发明实施例4中–20℃保存3个月的慢病毒载体的滴度回收率；
图11为本发明实施例4中23℃保存20天的慢病毒载体的滴度回收率；
图12为本发明实施例4中4℃保存30天的慢病毒载体的滴度回收率。
具体实施方式
应该指出，以下详细说明都是示例性的，旨在对本发明提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本发明的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式。
为了使得本领域技术人员能够更加清楚地了解本发明的技术方案，以下结合实施例对本发明作进一步详细描述：
实施例1：
1、检测在氟维司群敏感乳腺癌细胞系、氟维司群耐药乳腺癌细胞系中长非编码RNA SNHG17的表达水平。MCF-7为氟维司群敏感乳腺癌细胞系；MCF-7R为氟维司群耐药乳腺癌细胞系。
1.1 MCF-7细胞从ATCC细胞库获得，将MCF-7细胞培养于含10wt％胎牛血清的DMEM完全培养基，培养基加入200U/mL青霉素及200U/mL链霉素。
1.2 MCF-7R细胞系的构建：取对数生长期的MCF-7细胞1×10 7个，接种于直径为10cm的培养皿(以含10wt％胎牛血清、青霉素200U/mL、链霉素200U/mL的DMEM完全培养基为培养液)，待细胞生长稳定后于培养液中加入1μmol/L氟维司群，每48h换液后加入等浓度的氟维司群，12个月后通过有线稀释法获得乳腺癌MCF-7氟维司群耐药的单克隆细胞株(称为MCF-7R)，进而在无药物干预下进行扩增，为了维持MCF-7R的耐药性，后期培养于含有0.5μmol/L的氟维司群培养液中。
1.3 RT-PCR检测MCF-7、MCF-7R细胞中的SNHG17表达水平：
1.3.1用Trizol法分别提取总RNA：
分别取MCF-7、MCF-7R细胞放入1.5mL离心管中，2mL PBS洗涤2次，加入1mL Trizol，移液枪反复吹打20下，确保细胞全部裂解，然后吸至1.5mL离心管中；室温放置5分钟，使样品充分裂解，加入0.2mL氯仿，猛烈晃动10s，室温放置2min；12000g 4℃离心15min，吸取上层无色水相移至新的离心管，加入0.5ml异丙醇，颠倒混匀，冰上放置10分钟；12000g 4℃离心10min，可见管底的RNA沉淀，小心吸去上清；加入1mL 75v/v％乙醇(DEPC水配制)，颠倒混匀，7500g 4℃离心5min，小心吸去上清；加入20μL DEPC水溶解，Nanodrop分光光度计定量并质检，-80℃保存待用。
1.3.2反转录：加入2μg总RNA，1μL随机引物，加入DEPC水至12μL，在68℃ 孵育5min。取出产物后立即放置冰上4min，分别加入4μL Reaction Buffer，2μL 10mM dNTP Mix，1μL Reverse Transcriptase，M-MLV-Reverse和1μL RNase Inhibitor。在42℃水浴60min，再置于70℃水浴15min，立即放在冰上，静置5min，得cDNA模板，于-20℃下保存。
1.3.3实时荧光定量PCR检测SNHG17表达量：SNHG17和β-acting的引物如表1所示。
表1荧光定量PCR引物序列
Figure PCTCN2022115048-appb-000001
取各组细胞的cDNA 1μL为模板，进行PCR反应，PCR反应体系(10μL)见表2：
表2 PCR反应体系(10μL)
模板cDNA 1μL
2×SYBR Premix Ex Taq 5μL
上游引物(10μM) 0.2μL
下游引物(10μM) 0.2μL
ddH 2O 3.6μL
反应条件为：95℃预变性3min；以95℃变性15s，60℃退火30s，72℃延伸30s，扩增40个循环，再以72℃充分延伸10min。以GAPDH为内参基因，用2 -ΔΔCT法计算SNHG17的相对表达量。MCF-7及MCF-7R细胞中SNHG17的相对表达量见图1，其中A为MCF-7亲本细胞，B为MCF-7R耐药细胞，**P＜0.01表示有统计学意义。
由图1可以看出，MCF-7亲本细胞中长非编码RNA SNHG17的表达水平明显小于MCF-7R耐药细胞，说明耐药性氟维司群敏感乳腺癌细胞系中长非编码RNA SNHG17的表达水平比氟维司群耐药乳腺癌细胞系更高。
1.4荧光原位杂交(FISH)实验：
1.4.1分别取MCF-7、MCF-7R细胞1×10 3接种于6孔板的玻璃盖板上，37℃培养过夜，用1 x PBS清洗细胞两次，加入1mL 4g/mL％多聚甲醛固定15min，将悬液涂在粘附性玻片上，每片约50μL的悬液；60℃恒温干燥箱烘片10min，得到细胞涂片。
1.4.2探针序列为5’-AGTCTCCCATGTCTGGACCCCGAATCTTG-3’，并在其5’端添加FITC(异硫氰酸荧光素)荧光基团作为FISH探针的通用荧光标记序列，由上海生工有限公司合成。
1.4.3取细胞涂片，样品用胃蛋白酶溶液(10v/v％胃蛋白酶水溶液100mL+40mL纯水+1mol/L盐酸400μL)37℃消化10分钟，然后依次在70v/v％、90v/v％、100v/v％的梯度乙醇溶液中脱水1min，风干涂片；暗室避光操作，样品上滴加荧光标记探针10μL，荧光浓度为10ng/μL，使用盖玻片覆盖样本，放入预热的FISH专用湿盒中，90℃变性处理10min，将装有切片的湿盒放入37℃的恒温箱中杂交4h；暗室避光操作，涂片浸泡在2×SSC中2min，小心除去盖玻片后，2×SSC冲洗3次，每次3min；DAPI复染：暗室避光操作，在样品上滴加DAPI 10μL，使用盖玻片覆盖样品，放置于暗盒中，25℃孵育5min，缓慢除去盖玻片后，2×SSC冲洗3次，每次3min，后依次在70v/v％、90v/v％、100v/v％的梯度乙醇溶液中脱水1min；在盖玻片上滴加抗荧光猝灭封片剂封片，使用激光共聚焦镜检，由于FITC荧光染料在绿色激发光下法绿色荧光，DAPI荧光染料在UV光下发蓝色荧光，因此，拍照过程中个选用FITC、DAPI两种通道进行结果观察和照片记录。细胞中SNHG17的荧光原位杂交图见图2，其中，其中A为MCF-7亲本细胞，B为MCF-7R耐药细胞，a为用DAPI通道的照片，b为用FITC通道的照片，c为用合并通道的照片。
图2可以看出，MCF-7R耐药细胞中的SNHG17的表达高于MCF-7亲本细胞，即FISH实验证明了SNHG17在耐药细胞中表达更高。
实施例2：
1、SNHG17过表达乳腺癌细胞系的构建：
1.1扩增SNHG17的引物：
上游引物：5’-AGGA GCTAGCTGCTTGTAAGGCAGGGTCTC-3’，
下游引物：5’-ATGC GAATTCACAGCCACTGAAAGCATGTG-3’；
其中，上、下游引物的下划线部分为NheΙ、EcoRΙ酶切位点。
1.2PCR扩增人的SNHG17基因片段：
利用Trizol法提取乳腺癌细胞MCF-7的总RNA，逆转录为cDNA(方法同实施例1中1.3.1及1.3.2)。PCR反应体系见表3(20μL)：
表3 PCR反应体系(20μL)
Figure PCTCN2022115048-appb-000002
反应步骤：94℃预变性5min；95℃变性10s，62℃退火30s，72℃延伸60s，扩增39个循 环，72℃延伸10min。将PCR产物电泳、胶回收目的片段。
1.3分别对pcDNA3.1质粒和SNHG17 PCR产物进行双酶切，酶切反应体系见表4：表4酶切反应体系
  SNHG17 PCR产物 pcDNA3.1质粒
10×Buffer H 5μL 5μL
EcoR I 2μL 2μL
Nhe I 2μL 2μL
SNHG17 RCR产物 41μL -
pcDNA3.1质粒 - 5μL
ddH 2O - 36μL
反应条件：37℃水浴60min。
1.4分别对双酶切后的pcDNA3.1质粒和SNHG17 PCR产物电泳，胶回收目的片段，将酶切后的pcDNA3.1质粒和SNHG17 PCR产物进行连接，连接反应体系(10μL)见表5：
表5连接反应体系(10μL)
酶切SNHG17 PCR产物 6.5μL
酶切pcDNA3.1质粒 2.5μL
10X T4 DNA Ligase Buffer 1.0μL
T4 DNA Ligase 0.5μL
反应条件：25℃水浴60min。
1.5将SNHG17的表达载体质粒转化到大肠杆菌E.coli DH 5α中，经LB(100μg/mL氨苄青霉素)平板筛选过夜后，挑取阳性克隆子进行扩大培养，利用质粒提取试剂盒提取大肠杆菌中的质粒测序验证。测序结果验证重组质粒pcDNA3.1-SNHG17构建成功。
1.6细胞转染：
1.6.1将生长状态良好的乳腺癌细胞MCF-7按2×10 5个细胞/孔接种于24孔板中，每孔加入500μL DMEM完全培养基培养至细胞融合度达到60％时，开始转染。
1.6.2将1μg pcDNA3.1-SNHG17重组质粒加入50μL DMEM完全培养基，混合均匀，得到体系N1；将2.5μL Lipofectamine 2000加入50μL DMEM完全培养基，混合均匀，静置5min，得到体系N2；将体系N1和体系N2混合均匀，静置25min，得到体系N。
1.6.3弃取细胞培养板内原有的培养基，每孔加入400μL DMEM完全培养基及100μL体系N，6h后，每孔用2mL含10wt％胎牛血清的DMEM完全培养基换液，48h后用含600μg/mL G418的DMEM完全培养基，筛选14d，每隔3d换一次培养基。挑选单克隆细胞群维持G418(遗传霉素)抗性培养，G418的维持浓度为300μg/mL，得到稳定转染的 SNHG17细胞株，将筛选后的细胞采用有限稀释法传代于96孔板中，采用含有300μg/mL G418的DMEM完全培养基单克隆化筛选后的细胞，将单克隆化的细胞扩大培养，实验分为对照组(空载系统慢病毒)、pcDNA3.1-SNHG17组，得到SNHG17过表达对照乳腺癌稳定细胞系、SNHG17过表达乳腺癌稳定细胞系。SNHG17过表达乳腺癌细胞中SNHG17的相对表达量见图3，其中C为SNHG17过表达对照乳腺癌细胞，B为SNHG17过表达乳腺癌细胞，**P<0.01表示有统计学意义。
由图3可以看出，乳腺癌细胞MCF-7转染后，SNHG17过表达乳腺癌细胞的SNHG17相对表达量明显大于SNHG17过表达对照乳腺癌细胞。
2、平板克隆形成实验：分别取上述的SNHG17过表达对照乳腺癌细胞、SNHG17过表达乳腺癌细胞，进行实验，检测对氟维司群的耐药性。向含有DMEM完全培养基的培养皿(直径60mm)中，接种1000个生长状态良好的细胞，以十字方向轻轻晃动培养皿，使细胞分散均匀；置37℃、5v/v％CO 2下培养15d，每隔3d更换一次完全培养基；当培养皿中出现肉眼可见克隆时，终止培养，弃去培养液，PBS液小心浸洗2次，空气干燥。甲醇固定15min，弃甲醇后空气干燥，用Giemsa染液(购自默克)染色10min，流水缓慢洗去染液，空气干燥。实验分为空白组(SNHG17过表达对照乳腺癌稳定细胞+培养基中含有6μmol/L DMSO)、对照组(SNHG17过表达对照乳腺癌细胞+培养基中含有6μmol/L氟维司群)、过表达组(SNHG17过表达乳腺癌细胞+培养基中含有6μmol/L氟维司群)3组。细胞的集落形成见图4，其中O为空白组，P为对照组，Q为过表达组。
由图4可以看出，SNHG17过表达乳腺癌细胞形成的集落数大于SNHG17过表达对照乳腺癌细胞，说明敏感株中过表达SNHG17会增强乳腺癌细胞对氟维司群的耐药性。
3、乳腺癌细胞对氟维司群的敏感性检测：分别取对数生长期MCF-7和SNHG17过表达乳腺癌细胞以每孔3000个细胞的密度接种到96孔板(用DMEM完全培养基)中，在37℃5v/v％CO 2培养箱中培养至细胞贴壁后分别加氟维司群，最终浓度分别为0(细胞对照组)，2，4，8和16μmol/L，每个浓度设置4个复孔，同时设空白对照组，置37℃、5v/v％CO 2培养箱48h后，每孔加20μL MTT(5g/L)溶液，继续培养4h后，吸出培养基，每孔加200μL三联液(10g SDS，异丁醇5mL，0.1mL 10M盐酸与ddH 2O配置成100mL溶液)，继续培养过夜，酶标仪570nm条件下测定吸光度(A570nm)值，以三联液作为空白对照组，检测细胞存活率。细胞生长抑制率(％)＝(对照组A 570nm-实验组 A570nm)/细胞对照组A 570nm×100％。氟维司群对乳腺癌细胞的生长抑制率见图5，其中C为SNHG17过表达对照乳腺癌细胞，D为SNHG17过表达乳腺癌细胞，*P<0.05，**P<0.01表示有统计 学意义。
由图5可以看出，氟维司群对SNHG17过表达乳腺癌细胞的生长抑制率明显大于SNHG17过表达对照乳腺癌细胞，这说明，敏感株中SNHG17的过表达会降低乳腺癌细胞对氟维司群的敏感性。
实施例3：
1、根据人类非编码RNA SNHG17基因的mRNA序列(NR_015366.5)设计2个靶序列，分别为靶点1GGATTGTCAGCTGACCTCTGT，靶点2GTGACGTGTCTTCAAGAAGAG。
2、慢病毒干扰载体的构建
2.1选择PLKO.1-GFP-Puro作为慢病毒shRNA敲除质粒载体，购自上海圻明生物科技有限公司；
2.2对于每个位点合成相应的shRNA，设计的shRNA寡核苷酸链由上海生工生物工程技术服务有限公司合成，见表6。
表6 SNHG17基因shRNA寡核苷酸链序列
Figure PCTCN2022115048-appb-000003
2.3由质粒提取试剂盒提取PLKO.1-GFP-Puro载体，通过双酶切体系：1μL EcoRΙ+1μL AgeΙ+5μL 10×NEB Cutsmart Buffer+1μg PLKO.1-GFP-Puro+ddH 2O(补充至50μL)，37℃，1h，双酶切后琼脂糖凝胶电泳回收双酶切后载体。
2.4用pH调至8.0的TE溶液对DNA oligo(寡核苷酸)将shRNA引物溶解至浓度为100μM，再分别取相应的两条链进行退火，退火体系(50μL)见表7：
表7退火体系(50μL)
ddH 2O 35
10×shRNA annealing buffer 5
shRNA正义链 5
shRNA反义链 5
反应条件设置为95℃5min，随后进行的体系分别为85℃5min，75℃5min，70℃5min，最后将反应完的体系在4℃下保存，最终产物分别为10μM的shRNA1模板、10μM的shRNA2模板。
2.5将shRNA与质粒连接，连接反应体系(10μL)见表8：
表8连接反应体系(10μL)
shRNA模板 2μL
PLKO.1-EGFP-Puro双酶切后载体 2μL
T4DNA连接酶(10U/μL) 1μL
10×T4 Buffer 1μL
ddH 2O 4μL
混匀后瞬时离心，22℃连接2h。
取连接反应产物转化到大肠杆菌E.coli DH 5α中，经LB(100μg/mL氨苄青霉素)平板筛选过夜后，挑取阳性克隆子进行扩大培养，利用质粒提取试剂盒提取大肠杆菌中的质粒测序验证。测序结果验证重组质粒构建成功。即构建了靶向性沉默SNHG17基因的重组质粒PLKO.1-shRNA1、PLKO.1-shRNA2。
2.6慢病毒包装：293FT细胞在6cm培养皿(用DMEM完全培养基)中培养至70％融合时，用DMEM完全培养基换液；将2μg重组质粒PLKO.1-shRNA、2μg包膜载体psPAX2(购自上海圻明生物科技有限公司)以及2μg包装载体pMD2.G(购自上海圻明生物科技有限公司)溶于300μL DMEM完全培养基中混匀，得到体系X1，将18μL Lipofectamine 2000转染试剂(购自赛默飞)溶于300μL DMEM完全培养基中混匀，得到体系X2；将体系X2加入体系X1中混匀，静置10min后，加入293FT细胞的培养皿中，轻轻摇匀，在37℃5v/v％CO 2培养箱中培养过夜；转染18h后，用5mL含10wt％胎牛血清的DMEM完全培养基换液，在转染后48h收集上清液(病毒原液)，将病毒原液在2500rpm离心15min，取上清用0.45μm滤膜过滤后，滤液保存于-80℃，得到pLKO.1-shRNA慢病毒，由上述不同的重组质粒分别得到pLKO.1-shRNA1慢病毒、pLKO.1-shRNA2慢病毒。空载系统慢病毒采用同样的方法制备。
2.7取生长状态良好的MCF-7细胞接种到24孔板中，每孔4×10 5个细胞，每孔加入400μL含有10wt％胎牛血清的DMEM完全培养基，在37℃5v/v％CO 2培养箱中培养24h后，去24孔板中的培养基，加入500μL含有10v/v％病毒液、5μg/mL聚凝胺、10wt％胎牛血清的DMEM完全培养基，继续培养24h后用含有10wt％胎牛血清的DMEM完全培养基换液，继续培养48h，检测敲低效果。实验分为对照组(空载系统慢病毒)、pLKO.1-shRNA1组、pLKO.1-shRNA2组。敲低效果的检测：慢病毒载体上带有GFP绿色荧光蛋 白，在病毒感染96小时后用倒置荧光显微镜观察GFP绿色荧光，以观察病毒对目的细胞的感染情况；用BD influx cell sorter细胞分选仪对敲低的细胞分选，得到100％GFP的筛选细胞。
2.8分别将空载系统慢病毒沉默对照细胞、pLKO.1-shRNA1沉默细胞、pLKO.1-shRNA2沉默细胞按照每孔4×10 5个细胞铺板在24孔板，培养至细胞融合至85％时，采用RT-PCR法检测各组细胞中的SNHG17基因的表达量。细胞中SNHG17的相对表达量见图6，其中E为对照组(空载系统慢病毒沉默对照细胞)，F为pLKO.1-shRNA1沉默细胞，G为pLKO.1-shRNA2沉默细胞，*表示与对照组相比，**P<0.01表示有统计学意义。
由图6可以看出，pLKO.1-shRNA1沉默细胞、pLKO.1-shRNA2沉默细胞中SNHG17的相对表达量显著低于空载系统慢病毒沉默对照细胞，这说明本实施例提供的pLKO.1-shRNA1慢病毒、pLKO.1-shRNA2慢病毒能够显著敲低SNHG17基因。
3、平板克隆形成实验：空载系统慢病毒沉默对照细胞、pLKO.1-shRNA2沉默细胞，进行实验，检测对氟维司群的耐药性。向含有DMEM完全培养基的培养皿(直径60mm)中，接种1000个生长状态良好的细胞，以十字方向轻轻晃动培养皿，使细胞分散均匀；置37℃、5v/v％CO 2下培养15d，每隔3d更换一次完全培养基；当培养皿中出现肉眼可见克隆时，终止培养，弃去培养液，PBS液小心浸洗2次，空气干燥。甲醇固定15min，弃甲醇后空气干燥，用Giemsa染液(购自默克)染色10min，流水缓慢洗去染液，空气干燥。实验分为空白组(空载系统慢病毒沉默对照细胞+培养基中含有6μmol/L DMSO)、对照组(空载系统慢病毒沉默对照细胞+培养基中含有6μmol/L氟维司群)、敲除组(pLKO.1-shRNA2沉默细胞+培养基中含有6μmol/L氟维司群)3组。细胞的集落形成见图7，其中R为空白组、S为对照组、T为敲除组。
由图7可以看出，敲除组细胞的集落数明显小于对照组，这说明，SNHG17的敲低能够提高乳腺癌细胞对氟维司群的敏感性。
实施例4：
1、慢病毒载体冻干制剂的制备：
按实施例1的制备方法制得慢病毒载体滤液，采用超滤法按1∶50倍浓缩，得到慢病毒载体浓缩液。慢病毒载体浓缩液的滴度为4.13×10 7TU/mL。
配制慢病毒载体冻干保护剂溶液，用1mol/L PB调节混合溶液的pH值至7.4，将配制好的冻干保护剂溶液按照冻干保护剂：慢病毒载体浓缩液为3∶2的体积比加入浓缩液中，混合均匀后进行冻干，冻干程序如下：-60℃预冻3h；-40℃25h，真空度1Pa；-20℃ 10h，真空度1Pa；0℃30h，真空度1Pa。实验分为K1组、K2组、K3组、K4组、对照组5组，每组6个重复。K1组冻干保护剂溶液的制备方法为：向PBS中加入15wt％海藻糖、0.009wt％L-丙氨酸、0.016wt％组氨酸、0.001wt％CaCl 2、0.0008wt％MgSO 4；K2组冻干保护剂溶液的制备方法为：向PBS中加入15wt％海藻糖、1wt％二乙酸甘油酯、0.2wt％甘油磷酰胆碱、0.009wt％L-丙氨酸、0.016wt％组氨酸、0.001wt％CaCl 2、0.0008wt％MgSO 4；K3组冻干保护剂溶液的制备方法为：向PBS中加入15wt％海藻糖、1wt％二乙酸甘油酯、0.009wt％L-丙氨酸、0.016wt％组氨酸、0.001wt％CaCl 2、0.0008wt％MgSO 4；K4组冻干保护剂溶液的制备方法为：向PBS中加入15wt％海藻糖、0.2wt％甘油磷酰胆碱、0.009wt％L-丙氨酸、0.016wt％组氨酸、0.001wt％CaCl 2、0.0008wt％MgSO 4；对照组冻干保护剂溶液为：PBS。
2、慢病毒载体生物滴度的检测：以24孔板为例，每孔加入293FT细胞悬液400μL，细胞密度为1.0×10 5个/mL，取慢病毒载体冻干制剂加入500μL细菌内毒素检查用水复溶，待溶解完全后加入DMEM完全培养基进行10倍、100倍、1000倍的系列稀释，轻柔吹打均匀后取200μL慢病毒载体稀释液加入每孔中，将孔板放置培养箱内培养24h，第二天细胞补液，继续培养48h，取出24孔慢病毒滴度检测细胞板，使用胰酶消化，收集各孔细胞，将细胞转移至1.5mL离心管中；4℃，1500rpm，离心5min,收集细胞沉淀；弃去上清，每管使用1mL预冷的PBS重悬细胞沉淀；4℃，1500rpm，离心5min洗涤2次，弃去离心管内PBS，再用500ul预冷的PBS重悬细胞沉淀，并转移至流式管中；流式上机检测，检测GFP表达阳性的细胞，收集数据并分析结果。其中，流式细胞术检测慢病毒滴度的方法如下：
慢病毒滴度计算数据选择：选择阳性率介于1～20％间的组别用于滴度计算；滴度计算公式如下：
Titer(TU/mL)＝DF×N×P/V
其中：N为慢病毒感染前细胞数目；P为细胞阳性率(1～20％)；V为每孔细胞慢病毒感染体积；DF＝稀释倍数，稀释10倍，DF＝10 -1，稀释100倍，DF＝10 -2，稀释1000倍，DF＝10 -3；TU＝感染滴度。冻干存活率＝(冻干后病毒滴度/冻干前病毒滴度)×100％。各组慢病毒载体冻干前后的平均滴度见表9。K1组、K2组、K3组、K4组慢病毒载体冻干后的生物滴度对比图见图8，其中H为K1组、I为K2组、J为K3组、K为K4组，*表示与K1组相比，**P<0.01表示有统计学意义；#表示与K2组相比，#P<0.05，##P<0.01表示有统计学意义。K1组、K2组、K3组、K4组的冻干存活率对比图见图9，其中H为K1组、I为K2组、J为K3组、K为K4组，*表示与K1组相比，**P<0.01表示有统计学意义；#表示与K2组相比，#P<0.05，##P<0.01表示有统计学意义。
表9各组慢病毒载体冻干前的生物滴度
Figure PCTCN2022115048-appb-000004
由图8、图9可以看出，K2组慢病毒载体冻干后的生物滴度、冻干存活率大于K1组、K3组、K4组，说明与K1组、K3组、K4组相比，K2组的冻干保护剂能够有效降低慢病毒载体在冻干过程中受到的损伤。
2、慢病毒载体冻干制剂的外观、复溶性：不同慢病毒载体冻干制剂的外观和复溶性(再分散时间)见表10。
表10不同慢病毒载体冻干制剂的外观和复溶性(再分散时间)
组别 外观 复溶性(再分散时间)
K1 疏松，呈蜂窝状 良好，30s以内
K2 疏松，呈蜂窝状 良好，30s以内
K3 疏松，呈蜂窝状 良好，30s以内
K4 疏松，呈蜂窝状 良好，30s以内
由表10可以看出，K1、K2、K3、K4组慢病毒载体冻干制剂具有固定的形态，加水复溶后状态良好，考察再分散时间均在30s以内。
3、慢病毒载体冻干制剂的稳定性：分别取适量慢病毒载体冻干制剂，置于–20℃保存3个月、23℃保存20天、4℃保存30天，考察慢病毒载体冻干制剂的外观、复溶性(再分散时间)和生物滴度，计算滴度回收率＝(储存一段时间的慢病毒载体冻干制剂的病毒滴度/冻干前病毒滴度)×100％。
观察K1、K2、K3、K4组慢病毒载体冻干制剂在–20℃保存3个月、23℃保存20天、4℃保存30天过程中外观效果均较好，且复溶后状态也较好。
–20℃保存3个月的慢病毒载体的滴度回收率见图10，其中H为K1组、I为K2组、J为K3组、K为K4组，*表示与K1组相比，**P<0.01表示有统计学意义；#表示与K2组相比，##P<0.01表示有统计学意义。23℃保存20天的慢病毒载体的滴度回收率见图11，其中H为K1组、I为K2组、J为K3组、K为K4组，*表示与K1组相比，**P<0.01表示有统计学意义；#表示与K2组相比，##P<0.01表示有统计学意义。4℃保存30天的慢病毒载体的滴度回收率见图12，其中H为K1组、I为K2组、J为K3组、K为K4组，*表示与K1组相比，**P<0.01表示有统计学意义；#表示与K2组相比，##P<0.01表示有统计 学意义。
由图10、图11、图12可以看出，K2组制得的慢病毒载体冻干制剂在–20℃保存3个月的滴度回收率为94.2％，大于K1组、K3组、K4组；K2组制得的慢病毒载体冻干制剂在23℃保存20天的滴度回收率为92.1％，大于K1组、K3组、K4组；K2组制得的慢病毒载体冻干制剂在4℃保存30天的滴度回收率为92.4％，大于K1组、K3组、K4组；说明与K1组、K3组、K4组相比，K2组的冻干保护剂能够有效减少慢病毒滴度在长期保存过程中受到的损失和破坏，具有较好的储存稳定性。发明人对此进行了分析：可能是海藻糖上的羟基替代水分子与蛋白质表面部分结合，但由于空间位阻等作用使得部分蛋白质表面仍与水分子结合，而甘油磷酰胆碱和二乙酸甘油酯分子小，能够进入空隙补充到这部分蛋白质表面，并通过柔性分子链相互缠结填补这部分空隙，减少蛋白质与水分子的接触，从而减少了因冰晶对蛋白质造成的机械损伤，同时能够冻干制剂保存过程中蛋白质受到损失和破坏。
上述实施例中的常规技术为本领域技术人员所知晓的现有技术，故在此不再详细赘述。
以上实施方式仅用于说明本发明，而并非对本发明的限制，本领域的普通技术人员，在不脱离本发明的精神和范围的情况下，还可以做出各种变化和变型。因此，所有等同的技术方案也属于本发明的范畴，本发明的专利保护范围应由权利要求限定。

Claims (7)

  1. 一种抑制非编码RNA SNHG17表达的shRNA在制备逆转氟维司群耐药的药物中的用途，其特征在于：所述shRNA的编码序列如SEQ ID NO.1和SEQ ID NO.2所示，或SEQ ID NO.3和SEQ ID NO.4所示。
  2. 一种构建物在制备增加乳腺癌细胞对氟维司群敏感性的药物中的用途，其特征在于：所述构建物由权利要求1所述的shRNA插入慢病毒载体质粒pLKO.1-GFP-Puro所得。
  3. 一种非编码RNA SNHG17在制备预测乳腺癌氟维司群耐药的检测剂中的用途。
  4. 根据权利要求3所述的用途，其特征在于：所述检测剂包括用于检测非编码RNA SNHG17的表达情况的引物对。
  5. 根据权利要求4所述的用途，其特征在于：所述引物对如SEQ ID NO.5和SEQ ID NO.6所示。
  6. 一种抑制非编码RNA SNHG17表达的shRNA和氟维司群在制备治疗乳腺癌药物中的用途，其特征在于：所述shRNA的编码序列如SEQ ID NO.1和SEQ ID NO.2所示，或SEQ ID NO.3和SEQ ID NO.4所示。
  7. 一种药物组合物，包括氟维司群，其特征在于：还包括权利要求1所述的shRNA或权利要求2所述的构建物，以及药学上可接受的载体和/或辅料。
PCT/CN2022/115048 2022-05-18 2022-08-26 一种非编码rna snhg17作为标志物和治疗靶点的用途 WO2023221324A1 (zh)

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