CN102178959B - Combination and application of siRNA and its oligonucleotide for inhibiting growth of lung metastatic tumor - Google Patents
Combination and application of siRNA and its oligonucleotide for inhibiting growth of lung metastatic tumorDownload PDFInfo
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Abstract
Translated fromChineseDescription
Translated fromChinese技术领域technical field
本发明涉及抑制肺转移肿瘤生长的siRNA及其寡聚核酸组合与应用。The invention relates to the combination and application of siRNA for inhibiting the growth of lung metastasis tumor and its oligonucleotide.
背景技术Background technique
微小核酸(miRNA)是一类高度保守的非编码小核酸，广泛存在于动物植物和病毒中。miRNA通过转录后调控基因的表达，进而调节机体重要的生理与病理过程。微小核酸与肿瘤的发生密切相关。近年来的研究发现，miRNA在肿瘤组织和与正常组织间的表达明显不同。miRNA既可以作为原癌基因促进肿瘤的发生发展，又能作为抑癌基因抑制肿瘤的发生发展，根据miRNA调节肿瘤发生发展的不同机理，将它们组成不同的配方或将它们与其它抗肿瘤的小干扰RNA(siRNA)组成不同的配方，将比单一的miRNA更能有效地治疗肿瘤性疾病。Micronucleic acid (miRNA) is a kind of highly conserved small non-coding nucleic acid, which widely exists in animals, plants and viruses. miRNA regulates the expression of genes through post-transcriptional regulation, and then regulates the important physiological and pathological processes of the body. Micronucleic acid is closely related to the occurrence of tumors. In recent years, studies have found that the expression of miRNA is significantly different between tumor tissue and normal tissue. miRNA can not only promote the occurrence and development of tumors as proto-oncogenes, but also inhibit the occurrence and development of tumors as tumor suppressor genes. Different formulations of interfering RNA (siRNA) will be more effective in treating tumor diseases than single miRNA.
发明内容Contents of the invention
本发明的目的在于提供一种寡聚核酸在制备抑制肺转移肿瘤生长的试剂中的应用。The object of the present invention is to provide an application of an oligonucleotide in the preparation of a reagent for inhibiting the growth of lung metastatic tumors.
本发明所述的寡聚核酸是如下1)-4)中任一所述的双链RNA：The oligomeric nucleic acid of the present invention is the double-stranded RNA described in any of the following 1)-4):
1)序列表中序列1和序列2组成的寡聚核酸；1) The oligonucleotide composed of sequence 1 and sequence 2 in the sequence listing;
2)所述1)的寡聚核酸与PTN-siRNA组成的双寡聚核酸，所述PTN-siRNA是序列表中序列3和序列4组成的寡聚核酸；2) a double oligonucleotide composed of the oligonucleotide of 1) and PTN-siRNA, wherein the PTN-siRNA is an oligonucleotide composed of sequence 3 and sequence 4 in the sequence listing;
3)所述1)的寡聚核酸与miR-34a组成的双寡聚核酸，所述miR-34a是序列表中序列5和序列6组成的寡聚核酸；3) a double oligonucleotide composed of the oligonucleotide of 1) and miR-34a, wherein the miR-34a is an oligonucleotide composed of sequence 5 and sequence 6 in the sequence listing;
4)所述1)的寡聚核酸与VEGF-siRNA组成的双寡聚核酸，所述VEGF-siRNA是序列表中序列7和序列8组成的寡聚核酸。4) A double oligonucleotide consisting of the oligonucleotide of 1) and VEGF-siRNA, wherein the VEGF-siRNA is an oligonucleotide composed of sequence 7 and sequence 8 in the sequence listing.
上述2)的双寡聚核酸中，所述1)的寡聚核酸与PTN-siRNA的质量比是(0.5-2)∶(0.5-2)，优选是1∶1。In the above-mentioned duplex oligonucleotide in 2), the mass ratio of the oligonucleotide in 1) to PTN-siRNA is (0.5-2):(0.5-2), preferably 1:1.
上述3)的双寡聚核酸中，所述1)的寡聚核酸与miR-34a的质量比是(0.5-2)∶(0.5-2)，优选是1∶1。In the above-mentioned duplex oligonucleotide in 3), the mass ratio of the oligonucleotide in 1) to miR-34a is (0.5-2):(0.5-2), preferably 1:1.
上述4)的双寡聚核酸中，所述1)的寡聚核酸与VEGF-siRNA的质量比是(0.5-2)∶(0.5-2)，优选是1∶1。In the double oligonucleotide of 4) above, the mass ratio of the oligonucleotide of 1) to VEGF-siRNA is (0.5-2):(0.5-2), preferably 1:1.
进一步，上述1)-4)中的寡聚核酸均是经过如下a)或b)或c)修饰得到的寡聚核酸：Further, the oligonucleotides in the above-mentioned 1)-4) are oligonucleotides modified by the following a) or b) or c):
a)对所述寡聚核酸的连接核苷酸的磷酸二酯键进行修饰，优选将所述磷酸二酯键的氧用硫取代；a) modifying the phosphodiester bond connecting the nucleotides of the oligonucleotide, preferably replacing the oxygen of the phosphodiester bond with sulfur;
b)对所述寡聚核酸的核糖的2’-OH的修饰，优选将所述2’-OH用甲氧基或氟取代或者对所述2’-OH进行脱氧修饰；b) modification of the 2'-OH of the ribose of the oligonucleotide, preferably replacing the 2'-OH with methoxy or fluorine or deoxygenating the 2'-OH;
c)对所述寡聚核酸5’端连上胆固醇的修饰。c) the modification of linking cholesterol to the 5' end of the oligonucleotide.
上述序列表中序列1、3、5、7、9从左至右方向为5’-3’，序列表中序列2、4、6、8、10从左至右方向为3’-5’。The sequences 1, 3, 5, 7, and 9 in the above sequence listing are 5'-3' from left to right, and the
上述肿瘤是肠癌、鼻咽癌、宫颈癌、肝癌、乳腺癌或肺癌。The aforementioned tumor is colon cancer, nasopharyngeal cancer, cervical cancer, liver cancer, breast cancer or lung cancer.
上述试剂可以包含上述寡聚核酸分子中的至少一种以及至少一种药学上可接受的载体。本文所用的“药学上可接受的载体”应当与本发明试剂中的双链RNA分子相容。在一个优选实施例中，所述“药学上可接受的载体”是指体内转染试剂，如聚乙烯亚胺(PEI)，jetPEI(线性聚乙烯亚胺)，TF-PEI(转铁蛋白聚乙烯亚胺)脂质体，转铁蛋白，叶酸等。The above reagent may contain at least one of the above oligonucleotide molecules and at least one pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" should be compatible with the double-stranded RNA molecule in the reagent of the present invention. In a preferred embodiment, the "pharmaceutically acceptable carrier" refers to in vivo transfection reagents, such as polyethyleneimine (PEI), jetPEI (linear polyethyleneimine), TF-PEI (transferrin poly Ethyleneimine) liposomes, transferrin, folic acid, etc.
所述寡聚核酸为化学合成，可以进行2′氟代(2′-F)、2′甲氧基(2′-OMe)、硫代(PS)和2′脱氧(2′-deoxy)化学修饰，也可以在5’端连上胆固醇修饰。The oligonucleotide is chemically synthesized and can carry out 2' fluoro (2'-F), 2' methoxy (2'-OMe), thio (PS) and 2' deoxy (2'-deoxy) chemistry Modifications can also be modified with cholesterol at the 5' end.
用化学合成并修饰的上述寡聚核酸分子转染鼻咽癌细胞株CNE形成的肺转移瘤肿瘤细胞的实验结果表明miR-210、siPTN、miR-210+siVEGF、miR-34a+miR-210、miR-210+siPTN这几组均可以有效的抑制肺转移瘤的生长。Experimental results of transfection of the above-mentioned oligomeric nucleic acid molecules that were chemically synthesized and modified into lung metastatic tumor cells formed by the nasopharyngeal carcinoma cell line CNE showed that miR-210, siPTN, miR-210+siVEGF, miR-34a+miR-210, These groups of miR-210+siPTN can effectively inhibit the growth of lung metastases.
作为新型小核酸类的抗肿瘤药物，化学合成并修饰的所述寡聚核酸不易被降解，有较长的效应半衰期，能用于体外实验，更能用于体内治疗。As a novel small nucleic acid antitumor drug, the chemically synthesized and modified oligomeric nucleic acid is not easy to be degraded, has a longer half-life of effect, can be used for in vitro experiments, and can be used for in vivo treatment.
附图说明Description of drawings
图1为2′-F-siPTN及其寡聚核酸治疗抑制肺转移瘤形成的肺部压片效果图。Fig. 1 is a lung compression effect diagram of 2'-F-siPTN and its oligonucleotide treatment inhibiting the formation of lung metastases.
图2为2′-F-siPTN及其寡聚核酸组合治疗鼻炎癌肺转移6次以后肺部转移灶统计柱形图。Fig. 2 is a histogram of statistics of lung metastases after 6 treatments of 2'-F-siPTN and its oligomeric nucleic acid combination.
图3为2′-F-siPTN及其寡聚核酸组合治疗鼻炎癌肺转移组织切片图。Fig. 3 is a diagram of tissue slices of lung metastases of rhinitis carcinoma treated with 2'-F-siPTN and its oligonucleotide combination.
具体实施方式Detailed ways
下面结合具体实施例对本发明作进一步说明，但本发明并不限于以下实施例。The present invention will be further described below in conjunction with specific examples, but the present invention is not limited to the following examples.
下述实施例中，如无特殊说明，均为常规方法。In the following examples, unless otherwise specified, all are conventional methods.
实施例1、寡聚核酸的制备Embodiment 1, the preparation of oligomeric nucleic acid
本发明中所述寡聚核酸miR-210正义链为5’-CUGUGCGUGUGACAGCGGCUGA-3’(SEQ ID No.1)；反义链为3’-UUGACACGCACACUGUCGCCGA-5’序列(SEQ ID No.2)；PTN-siRNA序列正义链为：5’-GCCCAAACCUCAAGCAGAATT-3’(SEQ ID No.3)；反义链为3’-TTCGGGUUUGGAGUUCGUCUU-5’(SEQ ID No.4)；本发明中miR-34a正义链为5’-UGGCAGUGUCUUAGCUGGUUGU-3’(SEQ ID No.5)；反义链为3’-UUACCGUCACAGAAUCGACCAA-5’(SEQ ID No.6)；本发明中VEGF-siRNA正义链为5’-GGAGUACCCUGAUGAGAUCTT-3’(SEQ ID No.7)；反义链为3’-TTCCUCAUGGGACUACUCUAG-5’(SEQ ID No.8)；本发明中所述随机序列作为阴性对照(NC)，NC的正义链序列为：5’-UUCUCCGAACGUGUCACGUTT-3’(SEQ ID No.9)，反义链序列为：3’-TTAAGAGGCUUGCACAGUGCA-5’(SEQ IDNo.10)。所述寡聚核酸和NC序列中的A、G、C、U表示腺嘌呤核糖核苷酸、鸟嘌呤核糖核苷酸、胞嘧啶核糖核苷酸和尿嘧啶核糖核苷酸，T表示胸腺嘧啶脱氧核苷酸。The sense strand of oligomeric nucleic acid miR-210 described in the present invention is 5'-CUGUGCGUGUGACAGCGGCUGA-3' (SEQ ID No.1); the antisense strand is 3'-UUGACACGCACACUGUCGCCGA-5' sequence (SEQ ID No.2); PTN - The sense strand of the siRNA sequence is: 5'-GCCCAAACCUCAAGCAGAATT-3' (SEQ ID No.3); the antisense strand is 3'-TTCGGGUUUGGAGUUCGUCUU-5' (SEQ ID No.4); the sense strand of miR-34a in the present invention is 5'-UGGCAGUGUCUUAGCUGGUUGU-3'(SEQ ID No.5); the antisense strand is 3'-UUACCGUCACAGAAAUCGACCAA-5'(SEQ ID No.6); the sense strand of VEGF-siRNA in the present invention is 5'-GGAGUACCCUGAUGAGAUCTT-3' (SEQ ID No.7); the antisense strand is 3'-TTCCUCAUGGGACUACUCUAG-5' (SEQ ID No.8); the random sequence described in the present invention is used as a negative control (NC), and the sense strand sequence of NC is: 5' -UUCUCCGAACGUGUCACGUTT-3' (SEQ ID No.9), the antisense strand sequence is: 3'-TTAAGAGGCUUGCACAGUGCA-5' (SEQ ID No.10). A, G, C, and U in the oligonucleotide and NC sequences represent adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides and uracil ribonucleotides, and T represents thymine deoxynucleotides.
所述寡聚核酸和NC委托上海吉玛(GenePharma)制药技术有限公司合成并进行如下任意一种化学修饰：2′甲氧基(2′-OMe)、2′氟代(2′-F)、硫代和2′-脱氧(2’-deoxy)，5′胆固醇(5′-Cholesterol)。然后经冻干处理得到寡聚核酸粉末，用于实施例2中的实验。The oligonucleotide and NC were commissioned by Shanghai GenePharma Pharmaceutical Technology Co., Ltd. to synthesize and perform any of the following chemical modifications: 2'methoxy (2'-OMe), 2'fluoro (2'-F) , Thio and 2'-deoxy (2'-deoxy), 5' cholesterol (5'-Cholesterol). Then the oligonucleotide powder was obtained by freeze-drying, which was used in the experiment in Example 2.
其中：miR-210经2′氟代修饰后命名为2′-F-miR-210；miR-34a经2′氟代修饰后命名为2′-F-miR-34a；PTN-siRNA经2′甲氧基修饰后命名为2′-F-siPTN。VEGF-siRNA经2′甲氧基修饰后命名为2′-F-siVEGF；NC经2′氟代修饰后命名为2′-F-NC。Among them: miR-210 was named 2′-F-miR-210 after 2′-fluoro modification; miR-34a was named 2′-F-miR-34a after 2′-fluoro modification; PTN-siRNA was named after 2′-fluoro It was named 2'-F-siPTN after methoxy modification. VEGF-siRNA was named 2'-F-siVEGF after 2'methoxy modification; NC was named 2'-F-NC after 2'fluoro modification.
上述合成方法源自一篇1995年公开的文献：Wincott F，DiRenzo A，Shaffer C，GrimmS，Tracz D，Workman C，Sweedler D，Gonzalez C，Scaringe S and Usman N.Synthesis，deprotection，analysis and purification of RNA and ribozymes.Nucleic Acids Res.1995，23：2677-84。整个化学合成可大致分成四个的过程(1)寡聚核糖核酸的合成；(2)脱保护；(3)纯化分离；(4)脱盐退火无菌消毒。The above synthesis method is derived from a document published in 1995: Wincott F, DiRenzo A, Shaffer C, Grimm S, Tracz D, Workman C, Sweedler D, Gonzalez C, Scaringe S and Usman N. Synthesis, degradation, analysis and purification of RNA and ribozymes. Nucleic Acids Res. 1995, 23: 2677-84. The whole chemical synthesis can be roughly divided into four processes (1) synthesis of oligoribonucleic acid; (2) deprotection; (3) purification and separation; (4) desalination annealing aseptic disinfection.
(1)寡聚核糖核苷酸的合成(1) Synthesis of oligoribonucleotides
在自动DNA/RNA合成仪(例如，AppliedBiosystems EXPEDITE8909)上设定合成1毫摩尔的RNA，同时设定每个循环的偶联时间为10-15分钟，起始物为固相连接的5’-0-对二甲氧基-胸苷支持物，第一个循环在固相支持物上连接一个碱基，然后在第n次(19≥n≥2)循环中，在第n-1次循环所连接的碱基上连接一个碱基，重复此循环直至完成全部核酸序列的合成。On an automatic DNA/RNA synthesizer (for example, AppliedBiosystems EXPEDITE8909), set to synthesize 1 millimolar RNA, and set the coupling time of each cycle to be 10-15 minutes, and the starting material is solid phase-linked 5'- 0-p-dimethoxy-thymidine support, the first cycle connects a base on the solid support, and then in the nth (19≥n≥2) cycle, the n-1 cycle A base is attached to the attached base, and this cycle is repeated until the synthesis of the entire nucleic acid sequence is completed.
(2)脱保护(2) Deprotection
将连接有RNA的固相支持物加入到试管中，并在此试管中加入1毫升的乙醇/乙胺(体积比为1∶3)，然后密封，置于55-70℃温箱中，孵育2-30小时，取出连接有siRNA的固相支持物并用双蒸水淋洗2次(每次1毫升)，收集洗脱液，并在室温下干燥30分钟。然后，加入1毫升四丁基氟化铵的四氢呋喃溶液(1M)，室温放置4-12小时，再加入2毫升乙醇，收集沉淀即得到小RNA的粗产物。Add the solid phase support connected with RNA to the test tube, and add 1 ml of ethanol/ethylamine (volume ratio: 1:3) to the test tube, then seal it, place it in a 55-70°C incubator, and incubate After 2-30 hours, the solid phase support connected with siRNA was taken out and rinsed twice with double distilled water (1 ml each time), and the eluate was collected and dried at room temperature for 30 minutes. Then, 1 ml of tetrabutylammonium fluoride tetrahydrofuran solution (1M) was added, left at room temperature for 4-12 hours, and then 2 ml of ethanol was added, and the precipitate was collected to obtain a crude small RNA product.
(3)纯化分离(3) Purification and separation
将得到的RNA的粗产物溶解于2毫升浓度为1摩尔/毫升的乙酸铵水溶液中，然后通过C18高压液相色谱进行分离，得到纯化的RNA产物。The obtained crude RNA product was dissolved in 2 ml of aqueous ammonium acetate solution with a concentration of 1 mol/ml, and then separated by C18 high-pressure liquid chromatography to obtain a purified RNA product.
(4)脱盐退火(4) Desalting annealing
用浓度为75重量％的乙醇水溶液洗涤纯化的RNA产物2-4次(每次2毫升)，以除去盐份，并室温下干燥。然后将正义链和反义链的寡聚核糖核酸混合溶解在1-2毫升的缓冲液中(10mM Tris，pH＝7.5-8.0，50mM NaCl)，将此溶液加热至95℃，然后缓缓将此溶液冷却至室温，并维持室温16-22小时，得到含有双链微小RNA的溶液。The purified RNA product was washed 2-4 times (2 ml each time) with 75 wt% ethanol aqueous solution to remove salt, and dried at room temperature. Then the oligoribonucleic acid of the sense strand and the antisense strand is mixed and dissolved in 1-2 ml of buffer solution (10mM Tris, pH=7.5-8.0, 50mM NaCl), the solution is heated to 95°C, and then slowly The solution was cooled to room temperature and maintained at room temperature for 16-22 hours to obtain a solution containing double-stranded microRNA.
实施例2、2′-F寡聚核酸对肺转移肿瘤生长的抑制作用Example 2, 2'-F oligonucleotides inhibit the growth of lung metastatic tumors
用含10％胎牛血清的DMEM培养液培养鼻咽癌CNE细胞株，取对数生长期的细胞，制成单细胞悬液，调节细胞浓度1.5×106个细胞/ml。在5周龄的雄性BALB/C裸鼠的尾静脉注射0.35ml肿瘤细胞悬液，15天左右可形成肺转移瘤。将裸鼠分为6组(7只/组)，分别为2′-F-NC(简称NC组)，2′-F-miR-210加2′-F-siVEGFA(简称miR-210+siVEGF组)，2′-F-miR-34a加2′-F-miR-210(简称miR-34a+miR-210组)，2′-F-miR-210加2′-F-siPTN(简称miR-210+siPTN组)，2′-F-siPTN(简称siPTN组)，2′-F-miR-210(简称miR-210组)，接种肿瘤细胞以后，先后进行6次尾静脉注射。然后处死小鼠，取左肺部压片计数肺转移瘤个数，并用10％的福尔马林固定样本进行病理切片检查。CNE cell lines of nasopharyngeal carcinoma were cultured with DMEM medium containing 10% fetal bovine serum, and the cells in the logarithmic growth phase were collected to make a single cell suspension, and the cell concentration was adjusted to 1.5×106 cells/ml. Inject 0.35ml of tumor cell suspension into the tail vein of 5-week-old male BALB/C nude mice, and lung metastases can form in about 15 days. The nude mice were divided into 6 groups (7 mice/group), respectively 2′-F-NC (referred to as NC group), 2′-F-miR-210 plus 2′-F-siVEGFA (referred to as miR-210+siVEGF group), 2′-F-miR-34a plus 2′-F-miR-210 (referred to as miR-34a+miR-210 group), 2′-F-miR-210 plus 2′-F-siPTN (referred to as miR -210+siPTN group), 2′-F-siPTN (abbreviated as siPTN group), 2′-F-miR-210 (abbreviated as miR-210 group), after inoculation of tumor cells, 6 tail vein injections were performed successively. Then the mice were sacrificed, and the number of pulmonary metastases was counted by pressing pieces of the left lung, and 10% formalin fixed samples were used for pathological examination.
各组的尾静脉注射用的寡聚核酸制剂的配制方法是：单寡聚核酸组(NC组、miR-210a组和siPTN组)是用不含RNA酶的DEPC水稀释20×HBS至1×HBS(试剂盒HBS原始浓度为20×HBS)取50μl分别溶解40μg每组相应的寡聚核酸；双寡聚核酸组(miR-210+siVEGF组、miR-210+siPTN组、miR-210+miR-34a)是用两份不含RNA酶的DEPC水稀释的1×HBS无菌水25μl分别溶解每组内的两种20μg寡聚核酸后混合(总容积50ul)。The preparation method of the oligonucleotide preparation for tail vein injection of each group is as follows: the single oligonucleotide group (NC group, miR-210a group and siPTN group) is diluted with RNase-free DEPC water to 1× Take 50 μl of HBS (the original concentration of HBS in the kit is 20×HBS), respectively dissolve 40 μg of the corresponding oligonucleotides of each group; double oligonucleotide groups (miR-210+siVEGF group, miR-210+siPTN group, -34a) Dissolve two 20 μg oligonucleotides in each group with 25 μl of 1×HBS sterile water diluted with two portions of RNase-free DEPC water and mix (total volume 50 μl).
然后各组再加转染剂TF-PEI(加入DEPC水120μl/瓶溶解混匀)6μl(购自Bander公司)，与294μl 1×HBS溶液混合起来，总体积350ul，在室温孵育15分钟后进行尾静脉注射。一次注射的寡核苷酸用量为40μg/只。每隔2天注射一次，尾静脉注射共6次。Then each group was added transfection agent TF-PEI (add 120 μl/bottle of DEPC water to dissolve and mix) 6 μl (purchased from Bander Company), mixed with 294 μl 1×HBS solution, the total volume was 350ul, and incubated at room temperature for 15 minutes. Tail vein injection. The dosage of oligonucleotide for one injection is 40 μg/mouse. Injected once every 2 days, a total of 6 injections into the tail vein.
图1为寡聚核酸治疗抑制肺转移瘤形成的效果图，NC为对照组，只注射转染剂。与NC比较，可以观察寡聚核酸治疗组的肺部压片肿瘤转移灶个数明显少于对照组。图中透光度高的白色点状病灶经组织切片检查为肿瘤转移灶(图1)。Figure 1 is the effect of oligomeric nucleic acid treatment to inhibit the formation of lung metastases, NC is the control group, and only the transfection agent is injected. Compared with NC, it can be observed that the number of lung compressed tumor metastases in the oligomeric nucleic acid treatment group is significantly less than that in the control group. The white dot-like lesions with high light transmittance in the picture were tumor metastases after tissue biopsy (Figure 1).
图2为寡聚核酸治疗抑制肺转移瘤形成的统计图。与肺压片对比图相似，siPTN单独处理组(肿瘤转移灶平均值23.67)、miR-210+siVEGF组(肿瘤转移灶平均值14.71)、miR-210+siPTN组(肿瘤转移灶平均23.66)、miR-34a+miR-210(肿瘤转移灶平均值21.85)与对照组(NC)(肿瘤转移灶平均值43.86)肿瘤转移灶个数明显减少，且有显著性差异。Fig. 2 is a statistical graph showing that oligonucleotide treatment inhibits the formation of lung metastases. Similar to the comparison chart of lung compression, siPTN treatment group alone (mean of tumor metastases 23.67), miR-210+siVEGF group (mean of tumor metastases 14.71), miR-210+siPTN group (mean of tumor metastases 23.66), The number of tumor metastases decreased significantly between miR-34a+miR-210 (average tumor metastases 21.85) and the control group (NC) (average tumor metastases 43.86), and there was a significant difference.
图3为肺组织切片结果，可以观察到正常鼠(normal)的肺部组织均一，而模型组(NC)肺部有明显的转移灶，与肺压片中透明点的转移灶相对应。因此可以用计算肺压片中转移灶的个数来确定肿瘤的治疗情况。Figure 3 shows the results of lung tissue sections. It can be observed that the lung tissue of normal mice (normal) is uniform, while the model group (NC) has obvious metastases in the lungs, corresponding to the metastases of clear spots in the lung compression. Therefore, the number of metastases in the lung compression can be used to determine the treatment of the tumor.
本实施例中，各组寡聚核酸经2′甲氧基(2′-OMe)、硫代(PS)、2′脱氧和5′胆固醇(5′-Cholesterol)修饰对肺转移肿瘤生长的抑制作用与经2′氟代(2′-F)修饰后的作用无显著性差异。In this example, each group of oligonucleotides modified by 2'methoxyl (2'-OMe), thiol (PS), 2'deoxygenation and 5'cholesterol (5'-Cholesterol) inhibits the growth of lung metastatic tumors There is no significant difference between the effect and the effect modified by 2' fluoro (2'-F).
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