技术领域technical field
本发明属于医药技术领域。更具体地，涉及维生素C和奥沙利铂联用在抗肿瘤中的作用。The invention belongs to the technical field of medicine. More specifically, it relates to the role of vitamin C and oxaliplatin in combination in antitumor.
背景技术Background technique
联合用药（Concomitant drugs ）是指为了达到治疗目的或更好的治疗目的而采用的两种或两种以上药物同时或先后应用。但是，药物品种偏多，不同药物之间的相互作用也是各不相同的，配合不好甚至可能影响药物疗效或毒性增加。因此，联合用药药物的筛选研究具有重要的意义。Concomitant drugs refer to the simultaneous or sequential application of two or more drugs to achieve therapeutic purposes or better therapeutic purposes. However, there are many kinds of drugs, and the interactions between different drugs are also different, and poor coordination may even affect the efficacy of drugs or increase toxicity. Therefore, the screening research of drug combination is of great significance.
奥沙利铂是一类新的铂类抗癌药，在多种肿瘤模型系统，包括在人胃癌模型中，都表现出广谱的体外细胞毒性及体内抗肿瘤活性作用。体内、体外试验也证实在顺铂耐药的肿瘤模型中，它仍然有效。维生素C主要存在于水果和蔬菜中，是一种抗氧化剂，在临床上可以用于治疗急性病毒性肝炎，大剂量维生素C 还具有抗肿瘤作用。有学者认为，生理状态下维生素C可以在胃液中停留相对较长的一段时间，且保护其不受破坏。同时维生素C可以清除胃液局部的活性氧代谢产物。但当胃受幽门螺杆菌感染或有残胃、慢性萎缩性胃炎、肠上皮化生等癌前病变或疾病时，胃液中的维生素C水平明显下降，并失去其抗氧化作用。但是在其他肿瘤包括结直肠癌和胰腺癌中，维生素C却可以升高细胞内活性氧水平从而发挥抗肿瘤作用。Oxaliplatin is a new class of platinum-based anticancer drugs. It exhibits broad-spectrum cytotoxicity in vitro and antitumor activity in vivo in a variety of tumor model systems, including human gastric cancer models. In vivo and in vitro experiments also confirmed that it is still effective in cisplatin-resistant tumor models. Vitamin C mainly exists in fruits and vegetables, is an antioxidant, and can be used clinically to treat acute viral hepatitis. Large doses of vitamin C also have anti-tumor effects. Some scholars believe that under physiological conditions, vitamin C can stay in the gastric juice for a relatively long period of time and protect it from destruction. At the same time, vitamin C can remove the active oxygen metabolites in the gastric juice. However, when the stomach is infected by Helicobacter pylori or there are precancerous lesions or diseases such as remnant stomach, chronic atrophic gastritis, intestinal metaplasia, etc., the vitamin C level in gastric juice will drop significantly and lose its antioxidant effect. But in other tumors, including colorectal cancer and pancreatic cancer, vitamin C can increase the level of reactive oxygen species in cells to exert anti-tumor effects.
因此，目前维生素C对胃癌的作用及确切作用机制仍不明确，限制了其在抗肿瘤方面的应用。而且尚未有维生素C与奥沙利铂联合用药实现更好的抗肿瘤作用的相关研究和报道。Therefore, the role and exact mechanism of vitamin C on gastric cancer are still unclear, which limits its application in antitumor. Moreover, there are no relevant studies and reports on the combination of vitamin C and oxaliplatin to achieve a better anti-tumor effect.
发明内容Contents of the invention
本发明要解决的技术问题是克服上述现有技术的缺陷和不足，提供维生素C在提高胃癌细胞对奥沙利铂敏感性方面的应用。本发明研究显示维生素C可以显著抑制胃癌细胞的生长和增殖，诱导肿瘤细胞凋亡，同时还能显著抑制裸鼠皮下模型以及病人来源的移植瘤模型中肿瘤的生长和体积增大。同时维生素C可以提高胃癌细胞对奥沙利铂敏感性，发挥协同抗肿瘤作用。The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the above-mentioned prior art, and provide the application of vitamin C in improving the sensitivity of gastric cancer cells to oxaliplatin. The study of the present invention shows that vitamin C can significantly inhibit the growth and proliferation of gastric cancer cells, induce tumor cell apoptosis, and at the same time significantly inhibit the growth and volume increase of tumors in subcutaneous models of nude mice and transplanted tumor models derived from patients. At the same time, vitamin C can increase the sensitivity of gastric cancer cells to oxaliplatin and play a synergistic anti-tumor effect.
本发明的目的是提供维生素C在制备能够提高奥沙利铂抗肿瘤效果的药物方面的应用，以及维生素C和奥沙利铂联用在制备抗肿瘤药物方面的应用。The purpose of the present invention is to provide the application of vitamin C in the preparation of drugs capable of improving the anti-tumor effect of oxaliplatin, and the application of vitamin C and oxaliplatin in combination in the preparation of anti-tumor drugs.
本发明另一目的是提供维生素C在制备抗胃癌药物方面的应用，以及Glut1基因表达水平检测在制备能够提高维生素C对胃癌治疗效果的药物方面的应用。Another object of the present invention is to provide the application of vitamin C in the preparation of anti-gastric cancer drugs, and the application of Glut1 gene expression level detection in the preparation of drugs capable of improving the therapeutic effect of vitamin C on gastric cancer.
本发明上述目的通过以下技术方案实现：The above object of the present invention is achieved through the following technical solutions:
本发明提供了维生素C在制备能够提高奥沙利铂抗肿瘤效果的药物方面的应用。The invention provides an application of vitamin C in the preparation of a drug capable of improving the antitumor effect of oxaliplatin.
因此，维生素C在制备抗肿瘤药物方面的应用，维生素C和奥沙利铂联用在制备抗肿瘤药物方面的应用，以及一种同时含有维生素C和奥沙利铂的抗肿瘤药物，都在本发明的保护范围之内。Therefore, the application of vitamin C in the preparation of anti-tumor drugs, the combination of vitamin C and oxaliplatin in the preparation of anti-tumor drugs, and an anti-tumor drug containing both vitamin C and oxaliplatin are all in within the protection scope of the present invention.
优选地，所述肿瘤为消化道肿瘤。Preferably, the tumor is a tumor of the digestive tract.
更优选地，所述消化道肿瘤为胃癌。More preferably, the digestive tract tumor is gastric cancer.
更优选地，所述胃癌为人胃癌细胞AGS、SGC7901或MGC803。More preferably, the gastric cancer is human gastric cancer cells AGS, SGC7901 or MGC803.
另外，优选地，所述维生素C和奥沙利铂的用量比例为300-500：1。In addition, preferably, the dosage ratio of the vitamin C and oxaliplatin is 300-500:1.
更优选地，所述维生素C和奥沙利铂的用量比例为400：1。More preferably, the dosage ratio of vitamin C and oxaliplatin is 400:1.
另外，本发明研究发现，下调葡萄糖转运蛋白1（Glut1）的表达，胃癌细胞AGS，SGC7901和MGC803经过维生素C处理之后的凋亡细胞比例显著下调；升高Glut1的表达，胃癌细胞对维生素C更加敏感；表明Glut1的表达可以影响胃癌细胞对维生素C处理的敏感性。In addition, the present study found that down-regulating the expression of glucose transporter 1 (Glut1), the proportion of apoptotic cells in gastric cancer cells AGS, SGC7901 and MGC803 after vitamin C treatment was significantly down-regulated; increasing the expression of Glut1, gastric cancer cells are more sensitive to vitamin C Sensitive; indicating that the expression of Glut1 can affect the sensitivity of gastric cancer cells to vitamin C treatment.
因此，实际临床用药时，可以检测Glut1基因的表达水平指导用药。当患者的癌组织中Glut1基因的表达水平较高时，表明该患者胃癌细胞对维生素C处理较敏感，更加适用于维生素C和和奥沙利铂联合用药的方案。Therefore, in actual clinical medication, the expression level of Glut1 gene can be detected to guide medication. When the expression level of Glut1 gene in the patient's cancer tissue is high, it indicates that the gastric cancer cell in the patient is more sensitive to vitamin C treatment, and it is more suitable for the combination of vitamin C and oxaliplatin.
因此，Glut1基因在指导维生素C抗胃癌用药方案方面的应用，以及Glut1基因表达水平检测试剂在制备能够指导维生素C抗胃癌用药方案的制剂方面的应用，Glut1基因表达水平检测试剂在制备能够指导维生素C和奥沙利铂联用的抗胃癌用药方案的制剂方面的应用，也都应在本发明的保护范围之内。Therefore, the application of Glut1 gene in guiding vitamin C anti-gastric cancer drug regimen, and the application of Glut1 gene expression level detection reagent in the preparation of preparations that can guide vitamin C anti-gastric cancer drug regimen, Glut1 gene expression level detection reagent in the preparation of vitamin C The application of C in combination with oxaliplatin in the formulation of the anti-gastric cancer regimen should also be within the protection scope of the present invention.
本发明具有以下有益效果：The present invention has the following beneficial effects:
本发明首次公开了维生素C在提高胃癌细胞对奥沙利铂敏感性方面的作用，可以实现协同增效的作用，实现抗肿瘤效果的显著提高，对维生素C和奥沙利铂联合用药抗肿瘤的应用具有显著的意义。The present invention discloses for the first time the effect of vitamin C on improving the sensitivity of gastric cancer cells to oxaliplatin, which can realize the synergistic effect and significantly improve the anti-tumor effect, and is effective for the combination of vitamin C and oxaliplatin to treat tumors. application is significant.
同时，本发明明确了维生素C可以显著抑制胃癌细胞的生长和增殖，诱导肿瘤细胞凋亡，同时还能显著抑制裸鼠皮下模型以及病人来源的移植瘤模型中肿瘤的生长和体积增大；以及相关的作用机制，为维生素C的抗胃癌应用提供了更有利的证据和支持。At the same time, the present invention clarifies that vitamin C can significantly inhibit the growth and proliferation of gastric cancer cells, induce tumor cell apoptosis, and at the same time significantly inhibit the growth and volume increase of tumors in subcutaneous models of nude mice and transplanted tumor models derived from patients; and The relevant mechanism of action provides more favorable evidence and support for the anti-gastric cancer application of vitamin C.
本发明还明确了Glut1的表达可以影响胃癌细胞对维生素C处理的敏感性，对胃癌的防治和临床用药指导具有重要的意义。The present invention also clarifies that the expression of Glut1 can affect the sensitivity of gastric cancer cells to vitamin C treatment, which is of great significance to the prevention and treatment of gastric cancer and the guidance of clinical medication.
附图说明Description of drawings
图1为维生素C对胃癌细胞凋亡的影响。Figure 1 shows the effect of vitamin C on the apoptosis of gastric cancer cells.
图2为维生素C对成瘤的影响。Figure 2 shows the effect of vitamin C on tumor formation.
图3为蛋白印迹及实时定量PCR检测Glut1在胃癌细胞中的表达情况。Figure 3 shows the expression of Glut1 in gastric cancer cells detected by Western blot and real-time quantitative PCR.
图4为siRNA下调Glut1表达对胃癌细胞对维生素C的敏感性的影响。Figure 4 shows the effect of siRNA down-regulation of Glut1 expression on the sensitivity of gastric cancer cells to vitamin C.
图5为体内动物模型研究下调Glut1表达对胃癌细胞对维生素C的敏感性的影响。Figure 5 is an in vivo animal model study on the effect of down-regulating Glut1 expression on the sensitivity of gastric cancer cells to vitamin C.
图6为流式细胞术检测维生素C对胃癌细胞凋亡的影响。Figure 6 shows the effect of vitamin C on the apoptosis of gastric cancer cells detected by flow cytometry.
图7为细胞活性试剂盒检测维生素C对胃癌细胞活性的影响。Figure 7 shows the effect of the cell viability kit on the viability of gastric cancer cells detected by vitamin C.
图8为维生素C和奥沙利铂联用对胃癌细胞成瘤的影响。Fig. 8 is the effect of the combination of vitamin C and oxaliplatin on tumorigenesis of gastric cancer cells.
图9为流式细胞术检测维生素C对胃癌细胞活性氧的影响。Figure 9 shows the effect of vitamin C on reactive oxygen species in gastric cancer cells detected by flow cytometry.
图10为流式细胞术检测谷胱甘肽前体NAC对维生素C抗肿瘤作用的影响。Fig. 10 is flow cytometry detection of the effect of glutathione precursor NAC on the anti-tumor effect of vitamin C.
图11为维生素C联合奥沙利铂在病人来源的移植瘤模型中的治疗作用。Figure 11 shows the therapeutic effect of vitamin C combined with oxaliplatin in the patient-derived xenograft tumor model.
具体实施方式detailed description
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
除非特别说明，以下实施例所用试剂和材料均为市购。Unless otherwise specified, the reagents and materials used in the following examples are commercially available.
实施例1 维生素C对胃癌细胞及成瘤的影响Example 1 Effect of Vitamin C on Gastric Cancer Cells and Tumor Formation
1、实验材料1. Experimental materials
（1）药物：维生素C。(1) Drugs: Vitamin C.
（2）胃癌细胞：人胃癌细胞AGS，SGC7901和MGC803。(2) Gastric cancer cells: human gastric cancer cells AGS, SGC7901 and MGC803.
（3）凋亡试剂盒。(3) Apoptosis kit.
（4）雌性裸鼠，4-5周。(4) Female nude mice, 4-5 weeks old.
2、实验分组2. Experimental grouping
（1）对照组：空白对照，即胃癌细胞不经过任何药物处理。(1) Control group: blank control, that is, gastric cancer cells were not treated with any drugs.
（2）实验组：使用维生素C处理胃癌细胞：维生素C处理组：（Vit.(4g/kg)）以及对照组。(2) Experimental group: use vitamin C to treat gastric cancer cells: vitamin C treatment group: (Vit. (4g/kg)) and the control group.
3、流式细胞术检测维生素C对胃癌细胞凋亡的影响3. The effect of vitamin C on the apoptosis of gastric cancer cells detected by flow cytometry
（1）我们在六孔板中铺好胃癌细胞，待其贴壁后，加入2mM和4mM的维生素C处理细胞24h，然后通过流式细胞术检测细胞凋亡情况。(1) We paved gastric cancer cells in a six-well plate, and after they adhered to the wall, added 2mM and 4mM vitamin C to treat the cells for 24 hours, and then detected cell apoptosis by flow cytometry.
具体方法如下：The specific method is as follows:
1）对数生长期的AGS，SGC7901和MGC803细胞用胰酶消化后，取4×105细胞均匀铺到六孔板中。1) After the AGS, SGC7901 and MGC803 cells in the logarithmic growth phase were digested with trypsin, 4×105 cells were spread evenly in a six-well plate.
2）12h后细胞贴壁，更换成含有2mM和4mM维生素C的培养基。2) After 12 hours, the cells adhered to the wall, and replaced with the medium containing 2mM and 4mM vitamin C.
3）消化细胞之后，根据凋亡检测试剂盒的方法处理细胞，Annexin V和PI染色完成之后通过流式细胞仪检测细胞凋亡情况。3) After the cells were digested, the cells were processed according to the method of the apoptosis detection kit, and the apoptosis of the cells was detected by flow cytometry after Annexin V and PI staining.
（2）实验结果(2) Experimental results
结果如图1所示，图1中显示的Annexin V和PI双阴性（即存活细胞）比例，使用维生素C（4mM）处理后，胃癌细胞的死亡数接近一半，其中AGS细胞比SGC7901和MGC803细胞对维生素C更敏感。表明维生素C能够诱导胃癌细胞凋亡。The results are shown in Figure 1. The proportion of Annexin V and PI double-negative (ie surviving cells) shown in Figure 1, after treatment with vitamin C (4mM), the death of gastric cancer cells is close to half, and AGS cells are more than SGC7901 and MGC803 cells more sensitive to vitamin C. It shows that vitamin C can induce the apoptosis of gastric cancer cells.
4、维生素C对成瘤的影响4. The effect of vitamin C on tumor formation
（1）在体内动物模型中，我们给裸鼠皮下注射胃癌细胞（1×107/只），待肿瘤体积达到100mm3后，给予维生素C（4g/kg，每天两次，腹腔注射）治疗，观察肿瘤的变化情况。(1) In the in vivo animal model, we subcutaneously injected gastric cancer cells (1×107 /mouse) into nude mice. After the tumor volume reached 100 mm3 , vitamin C (4 g/kg, twice a day, intraperitoneal injection) was given for treatment. , to observe the changes in the tumor.
具体方法如下：The specific method is as follows:
1）培养对数生长期的胃癌AGS细胞，胰酶消化后，计数成1×108/ml浓度，用PBS重悬之后通过冰盒运输至动物实验室。1) Culture gastric cancer AGS cells in the logarithmic growth phase, digest with trypsin, count to a concentration of 1×108 /ml, resuspend in PBS, and transport to the animal laboratory by ice box.
2）选取4-5周龄的裸鼠，在每只裸鼠的皮下接种100μl上述细胞悬液。2) Select nude mice aged 4-5 weeks, and inoculate 100 μl of the above cell suspension subcutaneously in each nude mouse.
3）观察裸鼠成瘤情况，并测量其最长径（L）和最短径（S），待肿瘤体积（V=L×S2/2）达到100 mm3后，随机分为两组，分别给于注射用水，维生素C（4g/kg，每天两次，腹腔注射）治疗腹腔注射。3) Observe the tumor formation in nude mice, and measure their longest diameter (L) and shortest diameter (S). After the tumor volume (V=L×S2 /2) reaches 100 mm3 , they are randomly divided into two groups, Water for injection and vitamin C (4g/kg, twice a day, intraperitoneal injection) were given respectively for intraperitoneal injection.
4）每三天测量一次裸鼠肿瘤体积，3周后处死动物，统计结果。4) The tumor volume of nude mice was measured every three days, and the animals were sacrificed after 3 weeks, and the results were counted.
（2）实验结果(2) Experimental results
结果如附图2所示，治疗组（n=6）肿瘤体积比对照组明显缩小，表明维生素C能够显著的抑制胃癌的生长和体积增大，对胃癌具有显著的疗效。同时裸鼠体重未见明显下降，表明维生素C无明显副作用。The results are shown in Figure 2, the tumor volume of the treatment group (n=6) was significantly smaller than that of the control group, indicating that vitamin C can significantly inhibit the growth and volume increase of gastric cancer, and has a significant curative effect on gastric cancer. At the same time, the body weight of the nude mice did not decrease significantly, indicating that vitamin C had no obvious side effects.
实施例2 Glut1表达高低可以影响胃癌细胞对维生素C的敏感性Example 2 The expression level of Glut1 can affect the sensitivity of gastric cancer cells to vitamin C
1、实验材料1. Experimental materials
（1）药物：维生素C。(1) Drugs: Vitamin C.
（2）胃癌细胞：人胃癌细胞AGS，HGC27，SGC7901和MGC803以及胃黏膜上皮细胞GES1。(2) Gastric cancer cells: human gastric cancer cells AGS, HGC27, SGC7901 and MGC803 and gastric mucosal epithelial cells GES1.
（3）凋亡试剂盒，Lipofectamine 2000转染试剂，针对Glut1的siRNA，Glut1抗体及引物，实时定量PCR试剂盒，RNA逆转录试剂盒。(3) Apoptosis kit, Lipofectamine 2000 transfection reagent, siRNA against Glut1, Glut1 antibody and primers, real-time quantitative PCR kit, RNA reverse transcription kit.
2、实验分组2. Experimental grouping
（1）对照组：阴性对照，转染了阴性对照siRNA的胃癌细胞。(1) Control group: negative control, gastric cancer cells transfected with negative control siRNA.
（2）实验组：转染了针对Glut1的siRNA的胃癌细胞。(2) Experimental group: gastric cancer cells transfected with siRNA against Glut1.
3、蛋白印迹及实时定量PCR检测Glut1在胃癌细胞中的表达情况3. Detection of Glut1 expression in gastric cancer cells by Western blot and real-time quantitative PCR
（1）具体方法如下：(1) The specific method is as follows:
1）收取对数生长期的AGS，SGC7901和MGC803细胞1) Collect AGS, SGC7901 and MGC803 cells in logarithmic growth phase
2）分别通过RIPA裂解液和Trizol试剂提取细胞总蛋白和总RNA，再通过逆转录试剂盒获得cDNA。2) Extract the total protein and total RNA of cells by RIPA lysate and Trizol reagent respectively, and then obtain cDNA by reverse transcription kit.
3）通过Glut1抗体和引物分别检测胃癌细胞中Glut1的表达情况3) Detection of Glut1 expression in gastric cancer cells by Glut1 antibody and primers
（2）实验结果：(2) Experimental results:
结果如图3所示，AGS细胞中Glut1表达显著高于SGC7901和MGC803细胞，而图1显示AGS比SGC7901和MGC803细胞对维生素更敏感。The results were shown in Figure 3, Glut1 expression was significantly higher in AGS cells than in SGC7901 and MGC803 cells, while Figure 1 showed that AGS was more sensitive to vitamins than SGC7901 and MGC803 cells.
4、通过siRNA敲降Glut1表达后检测胃癌细胞对维生素C敏感性4. Detection of sensitivity of gastric cancer cells to vitamin C after knockdown of Glut1 expression by siRNA
（1）我们在六孔板中铺好胃癌细胞，待其贴壁后，加入10nM针对Glut1的siRNA或者阴性对照siRNA，28h后加入4mM的维生素C处理细胞24h，然后通过流式细胞术检测细胞凋亡情况。(1) We paved gastric cancer cells in six-well plates, and after they adhered to the wall, we added 10nM siRNA against Glut1 or negative control siRNA, and after 28h, added 4mM vitamin C to treat the cells for 24h, and then detected the cells by flow cytometry. Apoptosis.
具体方法如下：The specific method is as follows:
1）对数生长期的AGS，SGC7901和MGC803细胞用胰酶消化后，取1×105细胞均匀铺到六孔板中。1) After the AGS, SGC7901 and MGC803 cells in the logarithmic growth phase were digested with trypsin, 1×105 cells were spread evenly in a six-well plate.
2）12h后细胞贴壁，根据操作手册利用Lipofectamine 2000转染试剂转入10nm针对Glut1的siRNA或者阴性对照siRNA2) After 12 hours, the cells adhered to the wall, and transferred 10nm siRNA against Glut1 or negative control siRNA using Lipofectamine 2000 transfection reagent according to the operation manual
3）48h后更换成含有4mM维生素C的培养基。3) After 48 hours, replace with the medium containing 4mM vitamin C.
4）维生素C处理细胞24h后，消化细胞，根据凋亡检测试剂盒的方法处理细胞，Annexin V和PI染色完成之后通过流式细胞仪检测细胞凋亡情况。4) After the cells were treated with vitamin C for 24 hours, the cells were digested, and the cells were treated according to the method of the apoptosis detection kit. After Annexin V and PI staining, the apoptosis of the cells was detected by flow cytometry.
（2）实验结果：(2) Experimental results:
结果如图4所示，siRNA可以显著下调Glut1表达。转染Glut1的siRNA后，AGS，SGC7901和MGC803细胞经过维生素C处理之后的凋亡细胞比例显著下调，表明Glut1的表达可以影响胃癌细胞对维生素C处理的敏感性。The results are shown in Figure 4, siRNA can significantly down-regulate Glut1 expression. After transfection of Glut1 siRNA, the proportion of apoptotic cells in AGS, SGC7901 and MGC803 cells treated with vitamin C was significantly down-regulated, indicating that the expression of Glut1 can affect the sensitivity of gastric cancer cells to vitamin C treatment.
5、Glut1表达对维生素C治疗效果的影响5. The effect of Glut1 expression on the therapeutic effect of vitamin C
（1）在体内动物模型中，我们首先通过慢病毒方法构建Glut1稳定敲降的AGS细胞系，然后给裸鼠皮下分别注射对照及敲降胃癌细胞（1×107/只），待肿瘤体积达到100mm3后，给予维生素C（4g/kg，每天两次，腹腔注射）治疗，观察肿瘤的变化情况。(1) In the in vivo animal model, we first constructed the Glut1 stable knockdown AGS cell line by the lentivirus method, and then subcutaneously injected the control and knockdown gastric cancer cells (1×107 /mouse) into nude mice respectively. After reaching 100mm3 , vitamin C (4g/kg, twice a day, intraperitoneal injection) was given to treat the tumor, and the changes of the tumor were observed.
具体方法如下：The specific method is as follows:
1）培养对数生长期的胃癌AGS对照及敲降细胞，胰酶消化后，计数成1×108/ml浓度，用PBS重悬之后通过冰盒运输至动物实验室。1) Culture gastric cancer AGS control and knockdown cells in the logarithmic growth phase, digest with trypsin, count to a concentration of 1×108 /ml, resuspend in PBS, and transport to the animal laboratory by ice box.
2）选取4-5周龄的裸鼠，在每只裸鼠的皮下接种100μl上述细胞悬液。2) Select nude mice aged 4-5 weeks, and inoculate 100 μl of the above cell suspension subcutaneously in each nude mouse.
3）观察裸鼠成瘤情况，并测量其最长径（L）和最短径（S），待肿瘤体积（V=L×S2/2）达到100 mm3后，随机分组，分别给于注射用水，维生素C（4g/kg，每天两次，腹腔注射）治疗腹腔注射。3) Observe the tumor formation in nude mice, and measure their longest diameter (L) and shortest diameter (S). After the tumor volume (V=L×S2 /2) reaches 100 mm3 , they are randomly divided into groups and given to Water for injection, vitamin C (4g/kg, twice a day, intraperitoneal injection) treatment intraperitoneal injection.
4）每三天测量一次裸鼠肿瘤体积，3周后处死动物，统计结果。4) The tumor volume of nude mice was measured every three days, and the animals were sacrificed after 3 weeks, and the results were counted.
（2）实验结果(2) Experimental results
结果如附图5所示，下调Glut1表达水平后，胃癌细胞对维生素C处理的敏感性显著降低。即在对照细胞成瘤裸鼠中，维生素C可以显著抑制肿瘤生长，而在Glut1敲降细胞成瘤裸鼠中，维生素C未见明显抑制肿瘤生长作用。The results are shown in Figure 5, after down-regulating the expression level of Glut1, the sensitivity of gastric cancer cells to vitamin C treatment was significantly reduced. That is, in nude mice with control cell tumors, vitamin C can significantly inhibit tumor growth, but in nude mice with Glut1 knockdown cell tumors, vitamin C has no obvious inhibitory effect on tumor growth.
实施例3 维生素C可以提高胃癌细胞对奥沙利铂的敏感性Example 3 Vitamin C can increase the sensitivity of gastric cancer cells to oxaliplatin
1、实验材料1. Experimental materials
（1）药物：维生素C，奥沙利铂。(1) Drugs: vitamin C, oxaliplatin.
（2）胃癌细胞：人胃癌细胞AGS，SGC7901和MGC803。(2) Gastric cancer cells: human gastric cancer cells AGS, SGC7901 and MGC803.
（3）凋亡试剂盒，细胞活性检测试剂盒。(3) Apoptosis kit, cell viability detection kit.
（4）雌性裸鼠，4-5周。(4) Female nude mice, 4-5 weeks old.
2、实验分组2. Experimental grouping
（1）对照组：空白对照，即胃癌细胞不经过任何药物处理。(1) Control group: blank control, that is, gastric cancer cells were not treated with any drugs.
（2）实验组：分别使用维生素C、奥沙利铂或两者合用处理胃癌细胞。(2) Experimental group: Gastric cancer cells were treated with vitamin C, oxaliplatin or both.
3、流式细胞术检测维生素C对胃癌细胞凋亡的影响3. The effect of vitamin C on the apoptosis of gastric cancer cells detected by flow cytometry
（1）我们在六孔板中铺好胃癌细胞，待其贴壁后，加入2mM的维生素C，50μM奥沙利铂，或者两者联合使用处理细胞24h，然后通过流式细胞术检测细胞凋亡情况。(1) We paved gastric cancer cells in six-well plates, and after they adhered to the wall, we added 2 mM vitamin C, 50 μM oxaliplatin, or both to treat the cells for 24 hours, and then detected cell apoptosis by flow cytometry. death situation.
具体方法如下：The specific method is as follows:
1）对数生长期的AGS和SGC7901细胞用胰酶消化后，取4×105细胞均匀铺到六孔板中。1) After the AGS and SGC7901 cells in the logarithmic growth phase were digested with trypsin, 4×105 cells were spread evenly in a six-well plate.
2）12h后细胞贴壁，更换成新鲜培养基，分别加入2mM的维生素C，50μM奥沙利铂，或者两者联合使用。2) After 12 hours, the cells adhered to the wall, replaced with fresh medium, and added 2mM vitamin C, 50μM oxaliplatin, or a combination of both.
3）消化细胞之后，根据凋亡检测试剂盒的方法处理细胞，Annexin V和PI染色完成之后通过流式细胞仪检测细胞凋亡情况。3) After the cells were digested, the cells were processed according to the method of the apoptosis detection kit, and the apoptosis of the cells was detected by flow cytometry after Annexin V and PI staining.
（2）实验结果(2) Experimental results
结果如图6所示，图6中显示的Annexin V和PI双阴性（即存活细胞）比例，使用维生素C（2mM）或者奥沙利铂（50μM）单药处理后，胃癌细胞有所死亡，但是两者联合使用之后凋亡细胞比例明显增加，表明维生素C可以增强奥沙利铂的抗胃癌作用。The results are shown in Figure 6. The ratio of Annexin V and PI double-negative (ie, surviving cells) shown in Figure 6 shows that gastric cancer cells died after single-drug treatment with vitamin C (2mM) or oxaliplatin (50μM). However, the proportion of apoptotic cells increased significantly after the combination of the two, indicating that vitamin C can enhance the anti-gastric cancer effect of oxaliplatin.
4、细胞活性试剂盒检测维生素C对胃癌细胞活性的影响4. The cell viability kit detects the effect of vitamin C on the viability of gastric cancer cells
（1）我们在96孔板中铺好胃癌细胞，待其贴壁后，加入不同浓度的奥沙利铂（0-20μM），加或不加维生素C（0.1mM）处理细胞72h，然后检测细胞活性。(1) We paved gastric cancer cells in a 96-well plate. After they adhered to the wall, we added different concentrations of oxaliplatin (0-20 μM) and treated the cells with or without vitamin C (0.1 mM) for 72 hours, and then detected cell activity.
具体方法如下：The specific method is as follows:
1）对数生长期的AGS和SGC7901细胞用胰酶消化后，取2×103细胞均匀铺到96孔板中。1) After the AGS and SGC7901 cells in the logarithmic growth phase were digested with trypsin, 2×103 cells were spread evenly in a 96-well plate.
2）12h后细胞贴壁，更换成新鲜培养基，分别加入不同浓度的奥沙利铂（0-20μM），加或不加维生素C（0.1mM）处理细胞72h2) After 12 hours, the cells were adhered to the wall, replaced with fresh medium, and different concentrations of oxaliplatin (0-20 μM) were added, and the cells were treated with or without vitamin C (0.1mM) for 72 hours
3）加入细胞活性检测试剂后，通过酶标仪检测每个孔在490nm波长的吸光值。3) After adding the cell viability detection reagent, detect the absorbance value of each well at a wavelength of 490nm by a microplate reader.
4）通过Calcusyn软件计算维生素C和奥沙利铂的相互作用。4) Calculate the interaction between vitamin C and oxaliplatin by Calcusyn software.
（2）实验结果(2) Experimental results
结果如图7所示，图7中显示联用维生素C之后，细胞活性明显低于未加维生素C组。软件计算出两者的联合指数均小于1，表明维生素C和奥沙利铂具有协同作用。The results are shown in Figure 7. Figure 7 shows that after vitamin C was used in combination, the cell viability was significantly lower than that in the group without vitamin C. The combination index calculated by the software is less than 1, indicating that vitamin C and oxaliplatin have a synergistic effect.
5、维生素C和奥沙利铂对胃癌细胞成瘤的影响5. Effects of vitamin C and oxaliplatin on tumorigenesis of gastric cancer cells
（1）在体内动物模型中，我们给裸鼠皮下注射胃癌细胞（1×107/只），待肿瘤体积达到100mm3后，给予维生素C（4g/kg，每天两次，腹腔注射），奥沙利铂（10mg/kg，每周一次，腹腔注射），或者两者联合使用治疗，观察肿瘤的变化情况。(1) In the in vivo animal model, we subcutaneously injected gastric cancer cells (1×107 /mouse) into nude mice, and gave vitamin C (4 g/kg, twice a day, intraperitoneal injection) after the tumor volume reached 100 mm3 , Oxaliplatin (10mg/kg, once a week, intraperitoneal injection), or a combination of the two, was used to observe the changes in the tumor.
具体方法如下：The specific method is as follows:
1）培养对数生长期的胃癌AGS细胞，胰酶消化后，计数成1×108/ml浓度，用PBS重悬之后通过冰盒运输至动物实验室。1) Culture gastric cancer AGS cells in the logarithmic growth phase, digest with trypsin, count to a concentration of 1×108 /ml, resuspend in PBS, and transport to the animal laboratory by ice box.
2）选取4-5周龄的裸鼠，在每只裸鼠的皮下接种100μl上述细胞悬液。2) Select nude mice aged 4-5 weeks, and inoculate 100 μl of the above cell suspension subcutaneously in each nude mouse.
3）观察裸鼠成瘤情况，并测量其最长径（L）和最短径（S），待肿瘤体积（V=L×S2/2）达到100 mm3后，随机分为三组，分别给于注射用水，维生素C（4g/kg，每天两次，腹腔注射），奥沙利铂（10mg/kg，每周一次，腹腔注射），或者两者联合使用治疗。3) Observe the tumor formation in nude mice, and measure their longest diameter (L) and shortest diameter (S). After the tumor volume (V=L×S2 /2) reaches 100 mm3 , they are randomly divided into three groups, They were given water for injection, vitamin C (4g/kg, twice a day, intraperitoneal injection), oxaliplatin (10mg/kg, once a week, intraperitoneal injection), or a combination of the two.
4）每三天测量一次裸鼠肿瘤体积，3周后处死动物，统计结果。4) The tumor volume of nude mice was measured every three days, and the animals were sacrificed after 3 weeks, and the results were counted.
（2）实验结果(2) Experimental results
结果如附图8所示，与对照组相比，维生素C组和奥沙利铂组肿瘤体积均缩小，但是两者联用之后肿瘤体积缩小更明显，表明维生素C能够显著提高胃癌细胞对奥沙利铂敏感性，可以发挥协同抗肿瘤作用。The results are shown in Figure 8. Compared with the control group, the tumor volume of the vitamin C group and the oxaliplatin group decreased, but the tumor volume decreased more significantly after the combination of the two, indicating that vitamin C can significantly improve the resistance of gastric cancer cells to Oxaliplatin. Saliplatin is sensitive and can exert a synergistic antitumor effect.
实施例4 维生素C通过升高胃癌细胞内的活性氧发挥抗肿瘤作用Example 4 Vitamin C exerts anti-tumor effect by increasing reactive oxygen species in gastric cancer cells
1、实验材料1. Experimental materials
（1）药物：维生素C，活性氧探针DCFDA，谷胱甘肽前体NAC。(1) Drugs: vitamin C, active oxygen probe DCFDA, glutathione precursor NAC.
（2）胃癌细胞：人胃癌细胞AGS，SGC7901和MGC803。(2) Gastric cancer cells: human gastric cancer cells AGS, SGC7901 and MGC803.
（3）凋亡试剂盒。(3) Apoptosis kit.
2、实验分组2. Experimental grouping
（1）对照组：空白对照，即胃癌细胞不经过任何药物处理。(1) Control group: blank control, that is, gastric cancer cells were not treated with any drugs.
（2）实验组：使用维生素C处理胃癌细胞。(2) Experimental group: Gastric cancer cells were treated with vitamin C.
3、流式细胞术检测维生素C对胃癌细胞活性氧的影响3. Flow cytometry detection of the effect of vitamin C on reactive oxygen species in gastric cancer cells
（1）我们在六孔板中铺好胃癌细胞，待其贴壁后，加入1mM或2mM的维生素C处理细胞24h，然后通过流式细胞术检测细胞凋亡情况。(1) We paved gastric cancer cells in a six-well plate, and after they adhered to the wall, added 1 mM or 2 mM vitamin C to treat the cells for 24 hours, and then detected cell apoptosis by flow cytometry.
具体方法如下：The specific method is as follows:
1）对数生长期的AGS，SGC7901和MGC803细胞用胰酶消化后，取4×105细胞均匀铺到六孔板中。1) After the AGS, SGC7901 and MGC803 cells in the logarithmic growth phase were digested with trypsin, 4×105 cells were spread evenly in a six-well plate.
2）12h后细胞贴壁，更换成新鲜培养基，分别加入1mM，2mM的维生素C。2) After 12 hours, the cells adhered to the wall, replaced with fresh medium, and added 1mM and 2mM vitamin C respectively.
3）药物处理24h后，加入DCFDA探针，37℃孵育30min后，消化细胞通过流式细胞仪检测胞内活性氧含量。3) After 24 hours of drug treatment, DCFDA probe was added, and after incubation at 37°C for 30 minutes, the digested cells were detected by flow cytometry for intracellular active oxygen content.
（2）实验结果(2) Experimental results
结果如图9所示，图9中显示经过维生素C处理之后，AGS，SGC7901和MGC803细胞内活性氧水平显著增高。The results are shown in Figure 9, which shows that after vitamin C treatment, the levels of reactive oxygen species in AGS, SGC7901 and MGC803 cells were significantly increased.
4、流式细胞术检测谷胱甘肽前体NAC对维生素C抗肿瘤作用的影响4. The effect of glutathione precursor NAC on the anti-tumor effect of vitamin C detected by flow cytometry
（1）我们在六孔板中铺好胃癌细胞，待其贴壁后，在加入4mM的维生素C处理细胞24h之前，加或不加NAC（3mM）孵育2h，然后通过流式细胞术检测细胞凋亡情况。(1) We paved gastric cancer cells in a six-well plate, and after they adhered to the wall, before adding 4mM vitamin C to treat the cells for 24h, they were incubated with or without NAC (3mM) for 2h, and then the cells were detected by flow cytometry Apoptosis.
具体方法如下：The specific method is as follows:
1）对数生长期的AGS，SGC7901和MGC803细胞用胰酶消化后，取4×105细胞均匀铺到六孔板中。1) After the AGS, SGC7901 and MGC803 cells in the logarithmic growth phase were digested with trypsin, 4×105 cells were spread evenly in a six-well plate.
2）12h后细胞贴壁，更换成新鲜培养基，在加入4mM的维生素C之前，加或不加NAC（3mM）孵育2h。2) After 12h, the cells adhered to the wall, replaced with fresh medium, and incubated with or without NAC (3mM) for 2h before adding 4mM vitamin C.
3）药物处理24h后，消化细胞，根据凋亡检测试剂盒的方法处理细胞，Annexin V和PI染色完成之后通过流式细胞仪检测细胞凋亡情况。3) After 24 hours of drug treatment, the cells were digested, and the cells were processed according to the method of the apoptosis detection kit. After Annexin V and PI staining, the apoptosis of the cells was detected by flow cytometry.
（2）实验结果(2) Experimental results
结果如图10所示，图10中显示经过NAC预处理之后，维生素C诱导的凋亡细胞比例显著下调。表明细胞内活性氧参与介导了维生素C的抗肿瘤作用。The results are shown in Figure 10, which shows that after NAC pretreatment, the proportion of apoptotic cells induced by vitamin C was significantly down-regulated. It indicated that intracellular reactive oxygen species participated in mediating the anti-tumor effect of vitamin C.
实施例5 维生素C联合奥沙利铂在病人来源的移植瘤模型中的治疗作用Example 5 The therapeutic effect of vitamin C combined with oxaliplatin in a patient-derived xenograft model
1、实验材料1. Experimental materials
（1）药物：维生素C，奥沙利铂。(1) Drugs: vitamin C, oxaliplatin.
（2）病人来源的移植瘤模型。(2) Patient-derived xenograft tumor model.
（4）雌性裸鼠，4-5周。(4) Female nude mice, 4-5 weeks old.
2、实验分组2. Experimental grouping
（1）对照组：空白对照，即给予注射用水治疗。(1) Control group: blank control, that is, treated with water for injection.
（2）实验组：分别使用维生素C，奥沙利铂或者两者联合治疗。(2) Experimental group: vitamin C, oxaliplatin, or a combination of both.
3、实验方法3. Experimental method
我们选取胃癌病人的新鲜手术标本，接种到裸鼠皮下，观察肿瘤生长，并在裸鼠模型中进行稳定传代。在P2代中，待肿瘤体积达到100mm3后，给予维生素C（4g/kg，每天两次，腹腔注射），奥沙利铂（10mg/kg，每周一次，腹腔注射），或者两者联合使用治疗，观察肿瘤的变化情况。We selected fresh surgical specimens from patients with gastric cancer, inoculated them subcutaneously into nude mice, observed tumor growth, and carried out stable passage in the nude mouse model. In the P2 generation, after the tumor volume reached100mm3 , vitamin C (4g/kg, twice a day, intraperitoneal injection), oxaliplatin (10mg/kg, once a week, intraperitoneal injection), or a combination of both Use the treatment and watch how the tumor changes.
具体方法如下：The specific method is as follows:
（1）取得新鲜标本后，置于冰冻培养基中转运到动物房。将组织分割成约2mm的小粒。(1) After obtaining fresh specimens, place them in frozen medium and transfer them to the animal room. The tissue was divided into pellets of approximately 2 mm.
（2）选取4-5周龄的裸鼠，异氟烷麻醉后利用接种针将组织片接种到裸鼠双侧腋下。(2) Nude mice aged 4-5 weeks were selected, and tissue slices were inoculated into the bilateral axillae of the nude mice with an inoculation needle after isoflurane anesthesia.
（3）观察裸鼠成瘤情况，并测量其最长径（L）和最短径（S），待肿瘤体积（V=L×S2/2）达到100 mm3后，随机分为三组，分别给于注射用水，维生素C（4g/kg，每天两次，腹腔注射），奥沙利铂（10mg/kg，每周一次，腹腔注射），或者两者联合使用治疗。(3) Observe the tumor formation in nude mice, and measure the longest diameter (L) and shortest diameter (S). After the tumor volume (V=L×S2 /2) reaches 100 mm3 , they are randomly divided into three groups , were given water for injection, vitamin C (4g/kg, twice a day, intraperitoneal injection), oxaliplatin (10mg/kg, once a week, intraperitoneal injection), or a combination of both.
（4）每三天测量一次裸鼠肿瘤体积，3周后处死动物，统计结果。(4) The tumor volume of nude mice was measured every three days, and the animals were sacrificed after 3 weeks, and the results were counted.
4、实验结果4. Experimental results
结果如附图11所示，与对照组相比，维生素C组和奥沙利铂组肿瘤体积均缩小，但是两者联用之后肿瘤体积缩小更明显，表明维生素C联合奥沙利铂能够显著可以在病人来源的移植瘤模型中发挥协同抗肿瘤作用。同时裸鼠体重未见明显下降，提示维生素C治疗无显著副作用。The results are shown in Figure 11. Compared with the control group, the tumor volume of the vitamin C group and the oxaliplatin group both decreased, but the tumor volume decreased more significantly after the combination of the two, indicating that vitamin C combined with oxaliplatin can significantly reduce the tumor volume. Can exert synergistic antitumor effect in patient-derived xenograft model. At the same time, the body weight of the nude mice did not decrease significantly, suggesting that vitamin C treatment had no significant side effects.
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|---|---|---|---|---|
| CN109557317A (en)* | 2019-01-10 | 2019-04-02 | 南方医科大学南方医院 | Application of the ATXN2L as the marker of aided assessment gastric cancer oxaliplatin secondary resistance |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109557317A (en)* | 2019-01-10 | 2019-04-02 | 南方医科大学南方医院 | Application of the ATXN2L as the marker of aided assessment gastric cancer oxaliplatin secondary resistance |
| CN109557317B (en)* | 2019-01-10 | 2021-11-30 | 南方医科大学南方医院 | Application of ATXN2L as marker for assisting in evaluating oxaliplatin secondary drug resistance of gastric cancer |
| CN110193021A (en)* | 2019-07-14 | 2019-09-03 | 清华大学 | The purposes of the combination of Astragaloside IV and oxaliplatin in the preparation of antitumor drugs |
| WO2021213455A1 (en)* | 2020-04-23 | 2021-10-28 | 浙江养生堂天然药物研究所有限公司 | Drug combination and use thereof |
| CN115397411A (en)* | 2020-04-23 | 2022-11-25 | 浙江养生堂天然药物研究所有限公司 | Pharmaceutical combination and use thereof |
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| Date | Code | Title | Description |
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| SE01 | Entry into force of request for substantive examination | ||
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| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication | Application publication date:20170804 |