包含二氧化氯的细胞凋亡诱导剂及其在制备化妆品或抗衰老或抗肿瘤药物中的用途Apoptosis inducing agent comprising chlorine dioxide and use thereof in preparing cosmetic or anti-aging or anti-tumor drugs
本发明属于抗衰老和肿瘤治疗领域，具体涉及通过诱导衰老细胞和肿瘤细胞发生凋亡，从而抗衰老和治疗肿瘤的方法，以及二氧化氯在制备用于哺乳动物细胞凋亡诱导剂的化妆品或药物的应用。The invention belongs to the field of anti-aging and tumor treatment, and particularly relates to a method for preventing apoptosis and senescence and treating tumor by inducing apoptosis of senescent cells and tumor cells, and chlorine dioxide for preparing cosmetics for mammalian apoptosis inducer or The application of drugs.
机体的衰老不能等同于细胞的衰老，但是前者是后者累计的结果，因此机体衰老一定程度上可以用衰老细胞的百分比来刻画。机体内总有细胞在不断地衰老与死亡，同时又有细胞增殖产生新生细胞进行补偿。如果细胞增殖产生的新生细胞不仅全部补充死亡的细胞，而且能够将部分的衰老细胞替换，那么机体就表现的更为年轻。有研究表明：终身清除衰老细胞，能够延迟与年龄相关的表现型的最初出现时间。而且，晚年的清除会延缓已经出现的与年龄相关的病症的发展。这表明，衰老细胞的确能引起与年龄相关的表现型，而且它们的清除能够防止或延迟与年龄相关的组织功能丧失(Baker DJ,et al.Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders.Nature,2011,479:232-236)。可以说，清除衰老细胞，降低衰老细胞的比例，机体就能表现的更为年轻，并且可以防止或者延迟与年龄相关的组织功能丧失。衰老细胞的死亡实质上就是细胞凋亡。(惠宏襄等，《自由基与细胞凋亡》，《生物化学与生物物理进展》，1996；23：12)。The aging of the body can not be equated with the aging of the cells, but the former is the cumulative result of the latter, so the body aging can be characterized to a certain extent by the percentage of senescent cells. There are always cells in the body that are constantly aging and dying, and at the same time, cells proliferate to produce new cells to compensate. If the nascent cells produced by cell proliferation not only fully replenish dead cells, but also replace some of the senescent cells, the body will behave younger. Studies have shown that lifetime aging of senescent cells can delay the initial appearance of age-related phenotypes. Moreover, the elimination of later years will delay the development of age-related conditions that have already occurred. This suggests that senescent cells do cause age-related phenotypes, and that their clearance prevents or delays age-related loss of tissue function (Baker DJ, et al. Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature, 2011, 479: 232-236). It can be said that by eliminating aging cells and reducing the proportion of aging cells, the body can be younger and can prevent or delay the loss of age-related tissue function. The death of aging cells is essentially apoptosis. (Hui Hongwei et al., "Free Radicals and Apoptosis", Progress in Biochemistry and Biophysics, 1996; 23:12).
衰老并不意味着濒临死亡,如果定期更换培养液,衰老细胞仍可长期生存(Seshadri T,Campisi J.Repression of c-fos transcription and an altered genetic program in senescent human fibroblasts.Science,1990 Jan 12；247(4939):205-9)。但衰老细胞的增殖抑制是不可逆的,其生长停滞在细胞周期G1期,不能再进入S期。这提示衰老细胞可能比增殖活跃的年轻细胞抗凋亡,从而长期维持生存(黄英、张宗玉童坦君，《人衰老成纤维细胞凋亡的可诱导性》，《中华老年医学杂志》，2000年6月第19卷第3期)。Aging does not mean dying. If the culture medium is changed regularly, aging cells can survive for a long time (Seshadri T, Campisi J. Repression of c-fos transcription and an altered genetic program in senescent human fibroblasts. Science, 1990 Jan 12;247 (4939): 205-9). However, the inhibition of proliferation of senescent cells is irreversible, and its growth is arrested in the G1 phase of the cell cycle, and can no longer enter the S phase. This suggests that senescent cells may be anti-apoptotic compared to proliferating young cells, thus maintaining long-term survival (Huang Ying, Zhang Zongyu, Tong Tanjun, "Inducible Apoptosis of Human Aging Fibroblasts", Chinese Journal of Geriatrics, 2000 June, Vol. 19, No. 3).
Therefore, in theory, an anti-aging idea was found, that is, through the induction of apoptosis of senescent cells, the senescent cells are caused to undergo apoptosis and go to death, so that they can be removed from the body. The body will automatically initiate stem cell regeneration, regenerate new cells to replace the cleared cells, and make the body younger.
有意识的诱导衰老细胞发生凋亡，是清除衰老细胞最适宜的思路。在胚胎发育阶段通过细胞凋亡清除多余的和已完成使命的细胞，保证了胚胎的正常发育；在成年阶段通过细胞凋亡清除衰老和病变的细胞，保证了机体的健康。比如，在小鼠研究中发现，当把年老的干细胞置于一个年轻的微环境中，它们能够返老还童(Rescuing replication and osteogenesis of aged mesenchymal stem cells by exposure to a young extracellular matrix Yun Sun,et al.The FASEB Journal,May 2011,vol.25 no.51474-1485)，即年轻化，也表示再生能力更强。从另一个方面理解，组织中衰老细胞得以清除，由于特异性的诱导空间存在，组织中的干细胞会自动再生，再生的组织更为年轻化，因此机体整体表现为年轻化。Consciously inducing apoptosis in senescent cells is the most appropriate way to eliminate senescent cells. In the embryonic development stage, the excess and completed mission cells are cleared by apoptosis, which ensures the normal development of the embryo; in the adult stage, the aging and diseased cells are cleared by apoptosis to ensure the health of the body. For example, in mouse studies, it was found that when old stem cells were placed in a young microenvironment, they were able to rejuvenate (Rescuing replication and osteogenesis of aged mesenchymal stem cells by exposure to a young extracellular matrix Yun Sun, et al. The FASEB Journal, May 2011, vol. 25 no. 51474-1485), that is, younger, also means more regenerative capacity. On the other hand, it is understood that the senescent cells in the tissue are removed, and the stem cells in the tissue are automatically regenerated due to the specific induction space, and the regenerated tissue is younger, so the whole body is younger.
有较多研究指出细胞凋亡有助于机体延迟与衰老相关的组织功能丧失。比如，DNA断裂也是细胞凋亡的标志特征，DNA双链断裂(DNA double-strand breaks)在老鼠大脑活动的变化关系表明：这种DNA分子的断裂，即使在完全健康的小鼠神经元中也会发生，在适应新环境过程的小鼠大脑中，DNA断裂的速度是那些待在旧环境中不动小鼠的六倍(Elsa Suberbielle,et al.pHysiologic brain activity causes DNA double-strand breaks in neurons,with exacerbation by amyloid-βNature Neuroscience 16,613–621(2013))。显然适应新环境的小鼠记忆和思维能力更强，这表明DNA双链断裂的这种细胞凋亡过程能够促进小鼠的大脑年轻化。More studies have pointed out that apoptosis helps the body to delay the loss of tissue function associated with aging. For example, DNA fragmentation is also a hallmark of apoptosis. The relationship between DNA double-strand breaks in mouse brain activity indicates that this DNA molecule is broken, even in fully healthy mouse neurons. It happens that in the brains of mice adapted to the new environment, the rate of DNA fragmentation is six times that of mice that are still immobile in the old environment (Elsa Suberbielle, et al. pHysiologic brain activity causes DNA double-strand breaks in neurons , with exacerbation by amyloid-βNature Neuroscience 16, 613–621 (2013)). It is clear that mice adapted to the new environment have stronger memory and thinking ability, which indicates that this apoptotic process of DNA double-strand breaks can promote the brain rejuvenation of mice.
自噬促进了凋亡细胞最终的降解，也是衰老细胞清理的最后一步。自噬增多具有抗衰老效应(Louis R.Lapierre，et al.The TFEB orthologue HLH-30 regulates autopHagy and modulates longevity in Caenorhabditis elegans，Nature Communications.4,Article number:2267)。能引起细胞氧化的因素均能引起细胞凋亡。通过对巨噬细胞和单核细胞的研究，Albina等提出NO也是细胞凋亡的诱导剂(Albina JE，Cui S,Mateo RB et al.Nitric oxide-mediated apoptosis in murine peritoneal macropHages.J Immunol,1993 Jun1；150(11):5080-5)。Autophagy promotes the ultimate degradation of apoptotic cells and is the last step in the clean-up of senescent cells. Increased autophagy has an anti-aging effect (Louis R. Lapierre, et al. The TFEB orthologue HLH-30 regulates autopHagy and modulates longevity in Caenorhabditis elegans, Nature Communications. 4, Article number: 2267). Factors that cause cellular oxidation can cause apoptosis. By studying macrophages and monocytes, Albina et al. proposed that NO is also an inducer of apoptosis (Albina JE, Cui S, Mateo RB et al. Nitric oxide-mediated apoptosis in murine peritoneal macropHages.J Immunol, 1993 Jun1 ;150(11):5080-5).
一氧化氮(NO)对诸多衰老性疾病有治疗作用，比如，，一氧化氮具有防止导致帕金森氏症的神经细胞异常的作用，这其中一氧化氮促进了废弃蛋白质的分解，起到了保护神经细胞的作用(Kentaro Ozawa，et al.S-nitrosylation regulates mitochondrial quality control via activation of parkin Scientific Reports 3,Article number:2202(July2013)。一氧化氮的这种缓解衰老性疾病的机理似乎就是促进衰老细胞发生凋亡。Nitric oxide (NO) has a therapeutic effect on many aging diseases. For example, nitric oxide has a role in preventing neuronal abnormalities that cause Parkinson's disease. Nitric oxide promotes the decomposition of waste proteins and protects them. The role of nerve cells (Kentaro Ozawa, et al. S-nitrosylation regulates mitochondrial quality control via activation of parkin Scientific Reports 3, Article number: 2202 (July 2013). The mechanism of nitric oxide to alleviate aging diseases seems to promote aging The cells undergo apoptosis.
However, in reality, there is no technology that can safely and conveniently target the removal of aging cells and rejuvenate the body.
申请号为201110102598.1的中国专利《紫檀提取物与一氧化氮相结合的护肤品》提到：一氧化氮本身对皮肤的促进再生、抵抗衰老的良好功效，还能增加皮肤对传统化妆品有效成分的吸收。The Chinese patent "Pearl Extract Extracted from Nitric Oxide" with the application number 201110102598.1 mentions that nitric oxide itself promotes regeneration and anti-aging effects on the skin, and also increases the skin's effective ingredients for traditional cosmetics. absorb.
申请号为201210499300.X的专利《一种启动哺乳动物干细胞的方法及二氧化氯在制备用于启动哺乳动物干细胞的药物的应用》，有使用二氧化氯用于启动干细胞从而促进再生，但是没有提及如何通过清除衰老细胞使组织年轻化的机理，也没有可用于药物或化妆品中的抗衰老应用。尽管该专利依据干细胞再生机理提到二氧化氯治疗一些老年性疾病，但是并没有在预防这些疾病方面给出更多方案。Patent No. 201210499300.X, "A Method for Initiating Mammalian Stem Cells and Application of Chlorine Dioxide in the Preparation of Drugs for Initiating Mammalian Stem Cells," uses chlorine dioxide to initiate stem cells to promote regeneration, but does not Reference is made to the mechanism of how to rejuvenate tissues by eliminating senescent cells, and there is no anti-aging application that can be used in medicines or cosmetics. Although the patent refers to chlorine dioxide to treat some senile diseases according to the stem cell regeneration mechanism, it does not give more solutions in preventing these diseases.
癌症和衰老的根源是一样的(Carlos López-Otín,et al.The Hallmarks of Aging.Cell,6 June 2013)。诱导肿瘤细胞凋亡已经成为治疗肿瘤的一个重要途径。如果将肿瘤细胞看作衰老细胞，那么通过诱导肿瘤细胞凋亡从而治疗肿瘤的思路和通过诱导衰老细胞凋亡来抗衰老的思路是一致的。The roots of cancer and aging are the same (Carlos López-Otín, et al. The Hallmarks of Aging. Cell, 6 June 2013). Induction of tumor cell apoptosis has become an important way to treat tumors. If tumor cells are regarded as senescent cells, the idea of treating tumors by inducing apoptosis of tumor cells and anti-aging by inducing apoptosis of senescent cells are consistent.
肿瘤细胞通常具有一定的凋亡抵抗机制,利用细胞凋亡机制针对肿瘤的治疗也就是在凋亡调节的各个水平上使促凋亡和抗凋亡的平衡发生改变,诱导肿瘤细胞凋亡。Tumor cells usually have a certain mechanism of apoptosis resistance, and the apoptosis mechanism is used to treat tumors, that is, to change the balance between pro-apoptosis and anti-apoptosis at various levels of apoptosis regulation, and induce tumor cell apoptosis.
尽管有关凋亡机制的研究在近十多年得到了很大的进展，但能够确切详尽解释细胞凋亡,尤其是哺乳动物细胞凋亡的机制并不多,目前普遍接受的经典途径有以下两条：死亡受体途径和线粒体途径。Although the research on apoptosis mechanism has made great progress in the past ten years, it can explain the apoptosis in detail, especially the mechanism of apoptosis in mammals. There are two commonly accepted classical pathways. Bar: Death receptor pathway and mitochondrial pathway.
针对肿瘤细胞特殊的pH环境，有研究表明，碳酸氢钠增加了肿瘤pH值抑制了自发肿瘤转移(Ian F.Robey，et al.Bicarbonate Increases Tumor pH and Inhibits Spontaneous Metastases Cancer Res March 15,200969；2260)，但是其对肿瘤细胞本身并未形成凋亡诱导。In view of the special pH environment of tumor cells, studies have shown that sodium bicarbonate increases tumor pH and inhibits spontaneous tumor metastasis (Ian F. Robey, et al. Bicarbonate Increases Tumor pH and Inhibits Spontaneous Metastases Cancer Res March 15,200969; 2260 ), but it does not form apoptosis induction on tumor cells themselves.
现有的肿瘤细胞凋亡诱导剂有如下几个缺点：靶向性不明确、副作用大、治疗手段复杂且不确定性高、会引起肿瘤细胞的抵抗、只对少数类型肿瘤细胞有效。The existing tumor cell apoptosis inducing agents have the following disadvantages: unclear targeting, large side effects, complicated treatment means and high uncertainty, which may cause resistance of tumor cells, and are effective only for a few types of tumor cells.
如果诱导肿瘤细胞发生凋亡是治疗肿瘤的正确思路，那么需要寻找更具靶向性，更低毒副作用，更具有普适性的细胞凋亡诱导剂。If the induction of apoptosis in tumor cells is the correct way to treat tumors, it is necessary to find more targeted, lower toxic side effects and more universal apoptosis inducers.
(WHO)和世界粮食组织(FAO)也已将二氧化氯列为A1级安全高效消毒剂。为控制饮水中“三致物质”(致癌、致畸、致突变)的产生，欧美发达国家已广泛应用二氧化氯替代氯气进行饮用水的消毒。但二氧化氯作为药品还未被市场所接受。然而，有部分专利涉及到利用二氧化氯用于美容或治疗疾病的用途(例如，CN102137651A、CN101641104A和CN1199633C、US5750108、CN102441006A)，这些专利仍未发现二氧化氯，或者含有二氧化氯的酸性溶液具有清除衰老细胞的潜力。Chlorine dioxide is an internationally recognized new generation of high-efficiency, broad-spectrum, safe sterilization and preservatives. It is an ideal substitute for chlorine preparations and has been widely used in developed countries around the world. Relevant organizations in developed countries such as the United States, Western Europe, Canada, and Japan, such as the US Environmental Protection Agency, the Food and Drug Administration, and the US Department of Agriculture, have approved and recommended the use of chlorine dioxide for disinfection of food, food processing, pharmaceuticals, hospitals, and public environments. Anti-corrosion and preservation of mildew and food. World Health Organization
(WHO) and the World Food Organization (FAO) have also listed chlorine dioxide as a Class A1 safe and effective disinfectant. In order to control the production of “three substances” (carcinogenic, teratogenic and mutagenic) in drinking water, chlorine dioxide has been widely used in developed countries in Europe and America to disinfect drinking water. However, chlorine dioxide has not been accepted by the market as a medicine. However, some patents relate to the use of chlorine dioxide for cosmetic or therapeutic use of diseases (eg, CN102137651A, CN101641104A, and CN1199633C, US5750108, CN102441006A), which have not found chlorine dioxide or acidic solutions containing chlorine dioxide. Has the potential to clear aging cells.
发明概述Summary of invention
第一方面，本发明涉及一种包含二氧化氯的细胞凋亡诱导剂。In a first aspect, the invention relates to an apoptosis inducing agent comprising chlorine dioxide.
在一种优先的实施方案中，该细胞凋亡诱导剂，包含溶解在水中的二氧化氯，其中二氧化氯浓度为500-2900ppm，基于质量计算。In a preferred embodiment, the apoptosis inducing agent comprises chlorine dioxide dissolved in water, wherein the concentration of chlorine dioxide is from 500 to 2900 ppm, based on mass.
在一种更优选的实施方案中，本发明的细胞凋亡诱导剂还含有酸性pH调节剂，使得该细胞凋亡诱导剂的pH＝1.5-6.5，所述酸性pH调节剂选自以下各集合中的至少一种：In a more preferred embodiment, the apoptosis inducing agent of the present invention further comprises an acidic pH adjusting agent such that the pH of the apoptosis inducing agent is 1.5 to 6.5, and the acidic pH adjusting agent is selected from the following collections At least one of:
有机酸或其盐，其选自由甲酸、乙酸、丙酸、丁酸、乳酸、丙酮酸、柠檬酸、苹果酸、酒石酸、葡糖酸、乙醇酸、富马酸、丙二酸、马来酸、草酸、琥珀酸、丙烯酸、巴豆酸、戊二酸和它们的盐组成的集合；An organic acid or a salt thereof selected from the group consisting of formic acid, acetic acid, propionic acid, butyric acid, lactic acid, pyruvic acid, citric acid, malic acid, tartaric acid, gluconic acid, glycolic acid, fumaric acid, malonic acid, maleic acid a collection of oxalic acid, succinic acid, acrylic acid, crotonic acid, glutaric acid, and salts thereof;
无机酸或其盐，其选自由盐酸、磷酸、硼酸、偏磷酸、焦磷酸、氨基磺酸、磷酸二氢盐、磷酸氢盐组成的集合。An inorganic acid or a salt thereof selected from the group consisting of hydrochloric acid, phosphoric acid, boric acid, metaphosphoric acid, pyrophosphoric acid, sulfamic acid, dihydrogen phosphate, and hydrogen phosphate.
最优选地，其中所述酸性pH调节剂选自柠檬酸、乙酸或磷酸二氢盐。Most preferably, wherein the acidic pH adjusting agent is selected from the group consisting of citric acid, acetic acid or dihydrogen phosphate.
第二方面，本发明涉及一种包含二氧化氯的细胞凋亡诱导剂试剂盒，其包含以下两种独立的组分：In a second aspect, the present invention relates to an apoptosis inducing agent kit comprising chlorine dioxide comprising the following two separate components:
第一组分：二氧化氯前体固体，或包含二氧化氯前体的溶液；a first component: a chlorine dioxide precursor solid, or a solution comprising a chlorine dioxide precursor;
第二组分：酸性pH调节剂的水溶液；a second component: an aqueous solution of an acidic pH adjuster;
二者分开存放，且能在使用前进行混合，以现场反应并制得包含二氧化氯的细胞凋亡诱导剂；且第一组分和第二组分的量和浓度能够使得混合后的溶液的pH＝1.5-6.5；The two are stored separately and can be mixed before use to react in situ and prepare an apoptosis inducing agent comprising chlorine dioxide; and the amount and concentration of the first component and the second component can enable the mixed solution pH=1.5-6.5;
其中所述二氧化氯前体选自亚氯酸钠、亚氯酸钾、亚氯酸锂、亚氯酸钙、亚氯酸镁或亚氯酸钡中的至少一种；Wherein the chlorine dioxide precursor is selected from at least one of sodium chlorite, potassium chlorite, lithium chlorite, calcium chlorite, magnesium chlorite or bismuth chlorite;
其中所述酸性pH调节剂选自以下各集合中的至少一种：Wherein the acidic pH adjusting agent is selected from at least one of the following groups:
酒石酸、葡糖酸、乙醇酸、富马酸、丙二酸、马来酸、草酸、琥珀酸、丙烯酸、巴豆酸、戊二酸和它们的盐组成的集合；An organic acid or a salt thereof selected from the group consisting of formic acid, acetic acid, propionic acid, butyric acid, lactic acid, pyruvic acid, citric acid, malic acid,
a collection of tartaric acid, gluconic acid, glycolic acid, fumaric acid, malonic acid, maleic acid, oxalic acid, succinic acid, acrylic acid, crotonic acid, glutaric acid, and salts thereof;
无机酸或其盐，其选自由盐酸、磷酸、硼酸、偏磷酸、焦磷酸、氨基磺酸、磷酸二氢盐、磷酸氢盐组成的集合。An inorganic acid or a salt thereof selected from the group consisting of hydrochloric acid, phosphoric acid, boric acid, metaphosphoric acid, pyrophosphoric acid, sulfamic acid, dihydrogen phosphate, and hydrogen phosphate.
第三方面，本发明涉及上述包含二氧化氯的细胞凋亡诱导剂或上述包含二氧化氯的细胞凋亡诱导剂试剂盒用于制备诱导细胞凋亡的药物的用途。优选地，所述细胞为哺乳动物的细胞。In a third aspect, the present invention relates to the use of the above-described apoptosis inducing agent comprising chlorine dioxide or the above-described apoptosis inducing agent kit comprising chlorine dioxide for preparing a medicament for inducing apoptosis. Preferably, the cell is a mammalian cell.
第四方面，本发明涉及上述包含二氧化氯的细胞凋亡诱导剂或上述包含二氧化氯的细胞凋亡诱导剂试剂盒用于制备治疗肿瘤的药物的用途，或者用于制备哺乳动物目标组织抗衰老药物的用途，或者用作化妆品的用途，或者用于制备化疗药物的用途。In a fourth aspect, the present invention relates to the use of the above-described apoptosis inducing agent comprising chlorine dioxide or the above-mentioned apoptosis inducing agent kit comprising chlorine dioxide for preparing a medicament for treating tumor, or for preparing a mammalian target tissue The use of anti-aging drugs, either for cosmetic purposes or for the preparation of chemotherapeutic drugs.
发明详述Detailed description of the invention
在对现有技术研究的基础上，本发明的一个目的是提供二氧化氯在制备用于哺乳动物细胞凋亡诱导剂的药物或化妆品的应用。根据该应用，用于抗衰老的药物或化妆品中有含二氧化氯的细胞凋亡诱导剂，该药物或化妆品能够清除目标组织中的衰老细胞，促进身体的再生，再生年轻的组织，以达到抗衰老的作用。该应用具有很小的副作用或者无副作用。Based on prior art studies, it is an object of the present invention to provide the use of chlorine dioxide for the preparation of a medicament or cosmetic for use in a mammalian apoptosis inducing agent. According to the application, there is a chlorine dioxide-containing apoptosis inducing agent for anti-aging drugs or cosmetics, which can remove aging cells in target tissues, promote body regeneration, and regenerate young tissues to achieve Anti-aging effect. This application has little side effects or no side effects.
本发明的另一个目的是提供二氧化氯在制备用于哺乳动物细胞凋亡诱导剂的药物的应用。根据该应用，用于促进哺乳动物肿瘤细胞凋亡的药物中有含二氧化氯的细胞凋亡诱导剂，该药物能够靶向明确的诱导肿瘤细胞发生凋亡，达到更好的肿瘤治疗效果，且具有很小的副作用或者无副作用，对患者的负担较轻。Another object of the present invention is to provide an application of chlorine dioxide in the preparation of a medicament for use in a mammalian apoptosis inducing agent. According to the application, a drug for promoting apoptosis of a tumor cell in a mammal has an apoptosis inducer containing chlorine dioxide, and the drug can specifically induce apoptosis of the tumor cell to achieve better tumor treatment effect. With little side effects or no side effects, the burden on patients is lighter.
为达到以上目的，本发明采用的技术方案是：制备含二氧化氯的细胞凋亡诱导剂以及对哺乳动物目标组织给予所述细胞凋亡诱导剂的步骤，其中所述细胞凋亡诱导剂被给予哺乳动物目标组织时提供有效量的二氧化氯。In order to achieve the above object, the present invention adopts a technical solution of preparing a chlorine dioxide-containing apoptosis inducing agent and administering the apoptosis inducing agent to a mammalian target tissue, wherein the apoptosis inducing agent is An effective amount of chlorine dioxide is provided when the mammalian target tissue is administered.
进一步，制备含二氧化氯的细胞凋亡诱导剂的方法为：将二氧化氯气体溶于含酸性pH调节剂的pH值为1.5～6.5的酸性溶液A中，制备500～2900ppm的二氧化氯溶液。Further, the method for preparing an apoptosis inducing agent containing chlorine dioxide is: dissolving chlorine dioxide gas in an acidic solution A having an acidic pH adjuster and having a pH of 1.5 to 6.5 to prepare 500 to 2900 ppm of chlorine dioxide. Solution.
进一步，制备含二氧化氯的细胞凋亡诱导剂的方法为：将二氧化氯前体溶解于水中制备成1％～40％的水溶液，向该水溶液中加入含有酸性pH调节剂的酸性溶液B，调节混合溶液的pH值为1.5～6.5。Further, a chlorine dioxide-containing apoptosis inducing agent is prepared by dissolving a chlorine dioxide precursor in water to prepare an aqueous solution of 1% to 40%, and adding an acidic solution B containing an acidic pH adjuster to the aqueous solution. Adjust the pH of the mixed solution to 1.5 to 6.5.
混合溶液C、D，调节混合溶液的pH值为1.5～6.5。Further, the method for preparing a chlorine dioxide-containing apoptosis inducing agent kit comprises: dissolving a chlorine dioxide precursor in water to prepare an aqueous solution C having a concentration of 1% to 40%; and dissolving the acidic pH adjusting agent in water Prepare acidic solution D; before use
Mix the solutions C and D and adjust the pH of the mixed solution to 1.5 to 6.5.
再进一步，二氧化氯前体为亚氯酸钠、亚氯酸钾、亚氯酸锂、亚氯酸钙、亚氯酸镁和亚氯酸钡。Still further, the chlorine dioxide precursor is sodium chlorite, potassium chlorite, lithium chlorite, calcium chlorite, magnesium chlorite and bismuth chlorite.
再进一步，酸性pH调节剂为柠檬酸、乙酸或磷酸二氢钠。Still further, the acidic pH adjusting agent is citric acid, acetic acid or sodium dihydrogen phosphate.
进一步，细胞凋亡诱导剂通过动脉注射、肌肉注射、皮下注射、心内注射、鞘内注射、关节内注射、穿刺注射、直肠给药、鼻腔给药、经皮给药或吸入给药的方式直接给到损伤的器官或组织或给到其它目标组织中。Further, the apoptosis inducing agent is administered by arterial injection, intramuscular injection, subcutaneous injection, intracardiac injection, intrathecal injection, intra-articular injection, puncture injection, rectal administration, nasal administration, transdermal administration or inhalation. Directly to the damaged organ or tissue or to other target tissues.
进一步，可将细胞凋亡诱导剂制成注射剂、软膏剂、吸入剂、滴鼻剂、洗剂、栓剂、贴剂、糊剂、片剂、口服液、胶囊剂、颗粒剂、冲剂、丸剂或糖浆剂。Further, the apoptosis inducing agent can be prepared as an injection, an ointment, an inhalant, a nasal drop, a lotion, a suppository, a patch, a paste, a tablet, an oral solution, a capsule, a granule, a granule, a pill or Syrup.
本发明提供了二氧化氯在制备用于二氧化氯在制备用于哺乳动物细胞凋亡诱导剂的药物、化妆品的应用，其中，该药物或化妆品用于哺乳动物目标组织的抗衰老，使其年轻化。The present invention provides an application of chlorine dioxide in the preparation of a medicament for the preparation of a mammalian apoptosis inducer for chlorine dioxide, wherein the medicament or cosmetic is used for anti-aging of a mammalian target tissue, Younger.
进一步，这种抗衰老包括，皮肤年轻化即皮肤美容，身体其他组织年轻化，比如增加记忆力、提高睡眠治疗、预防阿尔兹海默症、帕金森综合症、骨质疏松、糖尿病、心脑血管疾病等老年性疾病等，包括但不限于这些。Further, this anti-aging includes skin rejuvenation, skin beauty, and rejuvenation of other tissues in the body, such as increased memory, improved sleep therapy, prevention of Alzheimer's disease, Parkinson's syndrome, osteoporosis, diabetes, cardiovascular and cerebrovascular diseases. Diseases and other senile diseases, etc., including but not limited to these.
更进一步，二氧化氯在制备用于哺乳动物细胞凋亡诱导剂的药物、化妆品的应用，其中，该药物用于人体组织抗衰老，并预防阿尔兹海默症、帕金森综合症、骨质疏松、糖尿病、心脑血管疾病等老年性疾病等；该化妆品用于皮肤美容护肤、祛痘或去除痤疮，也可以用于口腔护理的漱口水，包括但不限于这些。Further, chlorine dioxide is used for the preparation of a medicament for the mammalian apoptosis inducer, which is used for anti-aging of human tissues and for preventing Alzheimer's disease, Parkinson's syndrome, bone Osteoporosis, diabetes, cardiovascular and cerebrovascular diseases, etc.; the cosmetic is used for skin care, acne or acne removal, and can also be used for oral care mouthwashes, including but not limited to these.
本发明还提供了二氧化氯在制备用于诱导哺乳动物细胞凋亡的药物中的应用，其中，该药物用于治疗癌症，具体为诱导哺乳动物肿瘤细胞凋亡。The present invention also provides the use of chlorine dioxide for the preparation of a medicament for inducing apoptosis in a mammal, wherein the medicament is for treating cancer, in particular for inducing tumor cell apoptosis in a mammal.
进一步，通过诱导肿瘤细胞凋亡，其中针对的癌症包括：Further, by inducing apoptosis of tumor cells, the cancers targeted therein include:
Intracranial metastases, meningiomas, skull tumors, brain cancer, pituitary adenomas, auditory nerve sheath tumors, gliomas, brain tumors; maxillary sinus cancer, laryngeal cancer, nasopharyngeal cancer, tongue cancer, thyroid cancer, gingival cancer , lip cancer; thymoma, lung cancer, adenocarcinoma, breast sarcoma, lung metastases, breast fibroma, breast cancer; pancreatic head cancer, stomach cancer, gallbladder cancer, rectal cancer, pancreatic cancer, esophageal cancer, colon cancer, liver cancer; Tumor, penile cancer, urothelial carcinoma, prostate cancer, urethral cancer, testicular cancer, bladder cancer, nephroblastoma, kidney cancer; ovarian cancer, fallopian tube tumor, vulvar cancer, vaginal tumor, uterine cancer, cervical cancer, chorion Cancer, pelvic cancer; skin cancer, liposarcoma, malignant teratoma, fibroids, neurofibromatosis, melanoma, cholangiocarcinoma, squamous cell carcinoma, basal cell carcinoma; chordoma, osteoma, chondroma, osteosarcoma, synovium Sarcoma, giant cell tumor of bone, osteofibrosarcoma; acute leukemia, malignant lymphoma, chronic leukemia; hepatic hemangioma, islet cell carcinoid, blastoma, myxoma, cervical metastases, cardia cancer. These include, but are not limited to, these cancers.
进一步，本发明的二氧化氯在制备用于诱导哺乳动物细胞凋亡的药物中的应用，该应用中的药物可投予存在发展癌症的风险、经诊断患有癌症、处于癌症治疗期间或处于癌症治疗后恢复期间的受检者个体，或该药物可作为预防剂投予受检者个体以阻止或延缓癌症的发展。Further, the chlorine dioxide of the present invention is used in the preparation of a medicament for inducing apoptosis in a mammal, the medicament of which may be administered at risk of developing cancer, diagnosed with cancer, during cancer treatment or at The individual of the subject during recovery from cancer treatment, or the drug can be administered as a prophylactic to the individual of the subject to prevent or delay the progression of the cancer.
下面结合具体实施方式对本发明作进一步描述。The invention is further described below in conjunction with specific embodiments.
本发明将细胞凋亡定义为细胞的死亡、并被降解清除，而不严格区分是否属于凋亡(apoptosis)、坏死(necrosis)或自噬(autophagy)。本发明中的诱导衰老细胞和肿瘤细胞凋亡就是指促使衰老细胞死亡、并被降解清除和肿瘤细胞死亡、并被降解清除，而并不细究死亡的方式和过程。The present invention defines apoptosis as the death of a cell and is eliminated by degradation without strictly distinguishing whether it belongs to apoptosis, necrosis or autophagy. Induction of senescent cells and tumor cell apoptosis in the present invention refers to a method and process for causing death of senescent cells, being degraded by clearing and killing of tumor cells, and being degraded by degradation, without diagnosing death.
在描述本发明的具体实施方式之前，首先对本发明所提供方法的机理进行描述。这些描述再结合具体实施例将有助于本领域技术人员理解本发明的技术方案和保护范围。Before describing a particular embodiment of the invention, the mechanism of the method provided by the invention will first be described. These descriptions, in conjunction with the specific embodiments, will help those skilled in the art to understand the technical scope and the scope of the invention.
有研究表明：终身清除衰老细胞，能够延迟与年龄相关的表现型的最初出现时间。而且，晚年的清除会延缓已经出现的与年龄相关的病症的发展。这表明，衰老细胞的确能引起与年龄相关的表现型，而且它们的清除能够防止或延迟与年龄相关的组织功能丧失。(Baker DJ,et al.Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders.Nature,2011,479:232-236)。可以说，清除衰老细胞，降低衰老细胞的比例，机体就能表现的更为年轻，并且可以防止或者延迟与年龄相关的组织功能丧失。衰老细胞的死亡实质上就是细胞凋亡。(惠宏襄等，《自由基与细胞凋亡》.《生物化学与生物物理进展》，1996；23：12)。Studies have shown that lifetime aging of senescent cells can delay the initial appearance of age-related phenotypes. Moreover, the elimination of later years will delay the development of age-related conditions that have already occurred. This suggests that senescent cells do cause age-related phenotypes and that their clearance prevents or delays age-related loss of tissue function. (Baker DJ, et al. Clearance of p16 Ink4a-positive senescent cells delays ageing-associated disorders. Nature, 2011, 479: 232-236). It can be said that by eliminating aging cells and reducing the proportion of aging cells, the body can be younger and can prevent or delay the loss of age-related tissue function. The death of aging cells is essentially apoptosis. (Hui Hongwei et al., "Free Radicals and Apoptosis". Progress in Biochemistry and Biophysics, 1996; 23:12).
自噬促进凋亡细胞的降解，所以也能起到衰老细胞清除的作用。研究发现，果蝇的记忆缺失能够通过食用富含多胺的食物扭转过来。喂果蝇等吃多胺能够增强生物体的寿命。它似乎通过逆转与年龄相关的自噬的衰退来起作用。自吞噬是指细胞用来清除自身残片的机制。通过基因技术或限制卡路里摄入提高自吞噬，也能够延长果蝇的寿命。(Varun K Gupta,et al.Restoring polyamines protects from age-induced memory impairment in an autopHagy-dependent manner.Nature Neuroscience,01 September 2013)。Autophagy promotes the degradation of apoptotic cells, so it can also play a role in the clearance of senescent cells. The study found that the loss of memory in Drosophila can be reversed by eating foods rich in polyamines. Feeding polyamines such as fruit flies can enhance the lifespan of organisms. It seems to work by reversing the decline in age-related autophagy. Autophagy refers to the mechanism by which cells use to remove their own fragments. Increasing autophagy by genetic technology or limiting calorie intake can also extend the lifespan of fruit flies. (Varun K Gupta, et al. Restoring polyamines protects from age-induced memory impairment in an autopHagy-dependent manner. Nature Neuroscience, 01 September 2013).
高水平的ROS可诱导细胞凋亡(Lau AT,Wang Y,Chiu JF.Reactive oxygen species:current knowledge and applications in cancer research and therapeutic.J Cell Biochem.2008 May 15；104(2):657-67)。研究显示当线虫以生产一氧化氮的细菌为食时，寿命得到显著延长(Ivan Gusarov，et al.Bacterial Nitric Oxide Extends the Lifespan
of C.elegans Cell,Volume 152,Issue 4,818-830,14 February 2013)。这说明氧化物可以促进细胞发生凋亡。High levels of ROS induce apoptosis (Lau AT, Wang Y, Chiu JF. Reactive oxygen species: current knowledge and applications in cancer research and therapeutic. J Cell Biochem. 2008 May 15; 104(2): 657-67) . Studies have shown that lifespan is significantly prolonged when nematodes feed on nitric oxide-producing bacteria (Ivan Gusarov, et al. Bacterial Nitric Oxide Extends the Lifespan
Of C.elegans Cell, Volume 152, Issue 4, 818-830, 14 February 2013). This suggests that oxides can promote apoptosis in cells.
尽管抗氧化作为抗衰老的有效标准一直存在，但是现实证据却很少支持。如果无法及时清除缺陷型的蛋白，就会导致蛋白内稳态的丧失，引发衰老相关疾病。例如在阿尔茨海默症中，无法清除的蛋白形成斑块，导致神经元死亡。虽然大量的自由基可能有害，但它们的存在也会触发保护性的应答。没有任何遗传学证据能说明，增强机体的抗氧化防御能够延缓衰老。(Carlos López-Otín,et al.The Hallmarks of Aging.Cell,6 June 2013)。Although anti-oxidation has always existed as an effective standard for anti-aging, the actual evidence is rarely supported. If the defective protein cannot be removed in time, it will lead to the loss of protein homeostasis and cause aging-related diseases. For example, in Alzheimer's disease, proteins that cannot be cleared form plaques, leading to neuronal death. Although a large number of free radicals may be harmful, their presence also triggers a protective response. There is no genetic evidence to suggest that enhancing the body's antioxidant defenses can delay aging. (Carlos López-Otín, et al. The Hallmarks of Aging. Cell, 6 June 2013).
在一些实施例中，本发明也证明了具有较强氧化性物质(二氧化氯)可以促进细胞发生凋亡，并且有助于机体的年轻化。In some embodiments, the present invention also demonstrates that a more oxidizing substance (chlorine dioxide) can promote cell apoptosis and contribute to the rejuvenation of the body.
-交换器的改变也与细胞凋亡关系密切，细胞浆酸化程度与细胞凋亡发生率存在量效关系；②单纯的碱处理通过减少胞内酸化程度而表现出对抗诱导剂诱导的细胞凋亡；③细胞内存在酸性核酸内切酶，细胞内酸化能激活该酶，触发核小体DNA裂解，该酶是细胞酸化介导细胞凋亡的一个重要物质基础；④许多与细胞凋亡有关的酶和蛋白质的活性在酸性环境中增强(边肖海等，《胞内pH值在细胞凋亡中的研究进展》，《长治医学院学报》2004年4期)。On the other hand, intracellular acidification is prevalent in the growth factor-dependent cell line apoptosis. Intracellular acidification is an intracellular signal change in the process of apoptosis. It promotes apoptosis and has the following facts. Supported: 1 specific Na + /H + exchange inhibitor can induce apoptosis by inhibiting Na + /H + exchanger by intracellular acidification, in addition to Na + /H + exchanger, Na + -HCO 3 - coordinated transport The change of the device and the Cl - /HCO 3 - exchanger is also closely related to apoptosis. There is a dose-effect relationship between the degree of cytoplasmic acidification and the incidence of apoptosis. 2 Simple alkali treatment shows resistance by reducing the degree of intracellular acidification. Inducer-induced apoptosis; there are acidic endonucleases in 3 cells, and intracellular acidification can activate the enzyme to trigger cleavage of nucleosome DNA, which is an important material basis for cell acidification-mediated apoptosis; The activity of many enzymes and proteins involved in apoptosis is enhanced in an acidic environment (Bian Xiaohai et al., "Research progress in intracellular pH in apoptosis", Journal of Changzhi Medical College, 2004, 4).
衰老或者凋亡细胞在酸性的条件下更容易走向死亡。凋亡细胞糖酵解作用加强，酸性代谢产物增多，如果外部施加酸压力(降低pH值)，凋亡细胞将加速走向死亡。比如，心肌肥大是心肌组织对负荷增加的一种防御性反应，以暂时适应总体或局部心肌应力的改变，如果心脏生理机能的需要超过心肌细胞的代偿适应能力，心肌细胞将发生自杀性死亡即细胞凋亡，成为心肌肥大向心力衰竭转化的主要机制之一。在缺氧时，肥大心肌细胞糖酵解率仅轻度下降，但在复氧后糖酵解率迅速升高，呈爆发样达峰值后又逐渐恢复到缺氧前水平。糖酵解率的升高必然引起细胞内乳酸酸中毒。常氧培养时肥大心肌细胞凋亡率即显著高于正常心肌细胞，二者在缺氧后的细胞凋亡均显著增加，复氧后正常心肌细胞凋亡逐渐减少，而肥大心肌细胞在复氧早期细胞凋亡率继续大幅度上升，此后逐渐减少。(冯兵，刘伟，徐静，何作云，杨惠标，《缺氧复氧时肥大心肌细胞凋亡及其与能量代谢途径转换的关系》，Acta pHysiologica Sinica,October 25,2005,57(5):636-642)Aging or apoptotic cells are more likely to die under acidic conditions. Apoptotic cells have enhanced glycolysis and increased acidic metabolites. If externally applied acid pressure (lower pH), apoptotic cells will accelerate toward death. For example, cardiac hypertrophy is a defensive response of myocardial tissue to increased load to temporarily adapt to changes in overall or local myocardial stress. If the need for cardiac physiology exceeds the compensatory capacity of cardiomyocytes, suicide death occurs in cardiomyocytes. That is, apoptosis is one of the main mechanisms for the conversion of cardiac hypertrophy to heart failure. In the absence of oxygen, the rate of glycolysis in hypertrophic cardiomyocytes decreased only slightly, but the rate of glycolysis increased rapidly after reoxygenation, and then reached the peak of the burst and then gradually returned to the level before hypoxia. An increase in the rate of glycolysis inevitably causes intracellular lactic acidosis. The apoptotic rate of hypertrophic cardiomyocytes was significantly higher than that of normal cardiomyocytes during normoxia culture. The apoptosis of both cardiomyocytes was significantly increased after hypoxia. After reoxygenation, the apoptosis of normal cardiomyocytes was gradually reduced, while the hypertrophic cardiomyocytes were reoxygenated. The rate of early apoptosis continued to increase substantially and gradually decreased thereafter. (Feng Bing, Liu Wei, Xu Jing, He Zuoyun, Yang Huibiao, "The relationship between hypertrophic cardiomyocyte apoptosis and energy metabolism pathway transition during hypoxia-reoxygenation", Acta pHysiologica Sinica, October 25, 2005, 57(5): 636-642)
能够辅助为肌肉和神经细胞提供能量。研究发现，口服肌酸能够改善G93A转基因小鼠(肌萎缩性脊髓侧索硬化症)机体性能，并延长寿命，该种作用呈现量效关系，同时还保护小鼠运动神经元和黑质神经元的损伤(Klivenyi P,et al.Neuroprotective effects of creatine in a transgenic animal model of amyotropHic lateral sclerosis..Nature Medicine.1999.mar,5(3):347–350)。研究也发现硫辛酸(lipoic acid)能够减缓衰老过程。In biochemistry, creatine is a nitrogen-containing organic acid naturally present in vertebrates.
Can assist in providing energy to muscles and nerve cells. Studies have found that oral creatine can improve the body performance and prolong life of G93A transgenic mice (muscle atrophic lateral sclerosis), which has a dose-effect relationship and also protects mouse motor neurons and substantia nigra neurons. Klivenyi P, et al. Neuroprotective effects of creatine in a transgenic animal model of amyotrop Hic lateral sclerosis.. Nature Medicine. 1999. mar, 5(3): 347-350). Studies have also found that lipoic acid can slow down the aging process.
普遍认为L-抗坏血酸的抗衰老作用在于其抗氧化能力。但是，2009年，由德国耶拿大学的迈克尔·瑞斯托实验室进行的重要实验表明ROS对于维持肝脏的基本功能必不可少。实验结果表明，体育锻炼通过提高来自线粒体(细胞内产能的微小器官，产能原理部分经由氧化还原链)内的电子传递链体系ROS的产生，阻止Ⅱ型糖尿病的发生。然而如果同时每天服用抗氧化剂维生素C和维生素E，锻炼就没有积极的效果。2009年2月出版的《临床营养》就发表了一篇综述，总结了总参与人数多达十几万的22项公开发表的随机双盲对照研究，结论是没有支持“抗氧化剂防止冠状动脉硬化”的说法。而《美国医学会杂志》2007年2月发表的一篇综述更加打击抗氧化剂保健品。对总共涉及23万多人的68项研究进行总结，分析的几种抗氧化剂对于死亡率没有影响。如果剔除那些质量不高的研究，只对47项总计超过18万人参与的高质量研究进行分析，那么这几种抗氧化剂甚至小幅度增加了死亡率。因此，本发明人认为，L-抗坏血酸的抗衰老作用在于其酸性。It is generally believed that the anti-aging effect of L-ascorbic acid lies in its antioxidant capacity. However, in 2009, important experiments conducted by the Michael Resto laboratory at the University of Jena, Germany, showed that ROS are essential for maintaining the basic functions of the liver. The experimental results show that physical exercise prevents the occurrence of type 2 diabetes by increasing the production of ROS from the electron transport chain system within the mitochondria (the tiny organ of intracellular production, the principle of production through the redox chain). However, if you take the antioxidants vitamin C and vitamin E every day, exercise has no positive effect. A review of Clinical Nutrition published in February 2009 summed up 22 published randomized, double-blind, controlled trials with a total of more than 100,000 participants. The conclusion was that there was no support for "antioxidants to prevent coronary arteriosclerosis." "The saying." A review published in the February 2007 issue of the Journal of the American Medical Association is more resistant to antioxidant health products. A total of 68 studies involving more than 230,000 people were summarized and several antioxidants analyzed had no effect on mortality. If only those low-quality studies were excluded and only 47 high-quality studies involving more than 180,000 people were analyzed, these antioxidants even increased mortality slightly. Therefore, the inventors believe that the anti-aging effect of L-ascorbic acid lies in its acidity.
多种酸性物质有美容的作用。比如，以30％高浓度的水杨酸作为化学换肤的药剂，可达到和70％果酸换肤相同的淡化色素斑、缩小毛孔、去除细小皱纹及改善日晒引起的老化等多项效果。皮肤美容就是让皮肤年轻化，所以大致可以得出结论，酸性物质能够清除衰老细胞，促进年轻皮肤再生。中剂量和高剂量柠檬酸处理组引起了小鼠睾丸组织细胞的凋亡(王艳杰：柠檬酸对小鼠睾丸组织凋亡的影响[D]；河南科技大学：2012年)。A variety of acidic substances have a cosmetic effect. For example, with 30% high concentration of salicylic acid as a chemical peeling agent, it can achieve the same effect as lightening pigmentation spots, shrinking pores, removing fine wrinkles and improving aging caused by sun exposure with 70% acid acid peeling. . Skin beauty is to make the skin younger, so it can be concluded that acidic substances can remove aging cells and promote young skin regeneration. The medium-dose and high-dose citric acid treatment groups caused apoptosis in mouse testicular tissue cells (Wang Yanjie: Effect of citric acid on apoptosis of mouse testis tissue [D]; Henan University of Science and Technology: 2012).
发明人猜想，L-抗坏血酸，肌酸，硫辛酸等酸性物质能够抗衰老的作用，更多的是因为其酸性特征能够加速衰老细胞发生凋亡，从而引起机体年轻化。发明人意外的发现，用有较高氧化电位的二氧化氯液剂并辅以酸性环境能够抗衰老。The inventors suspect that acidic substances such as L-ascorbic acid, creatine, and lipoic acid can resist aging, and more because their acidic characteristics can accelerate the apoptosis of senescent cells, causing the body to rejuvenate. The inventors have unexpectedly discovered that anti-aging can be achieved by using a chlorine dioxide solution having a higher oxidation potential and supplemented by an acidic environment.
在一些实施例中，本发明通过试验证明了，酸性环境有助于提高二氧化氯制剂对细胞的凋亡作用。In some embodiments, the present invention demonstrates through experiments that an acidic environment contributes to an increase in the apoptotic effect of a chlorine dioxide formulation on cells.
在本发明的多个实施例中，我们采用酸性二氧化氯溶液靶向给予目标组织，证明可以使目标组织年轻化。In various embodiments of the invention, we targeted the administration of the target tissue with an acidic chlorine dioxide solution, demonstrating that the target tissue can be rejuvenated.
肿瘤细胞有十大特征：自给自足生长信号(Self-Sufficiency in Growth Signals)；抗生长信号的不敏感(Insensitivity to Antigrowth Signals)；抵抗细胞死亡(Resisting Cell
Death)；潜力无限的复制能力(Limitless Replicative Potential)；持续的血管生成(Sustained Angiogenesis)；组织浸润和转移(Tissue Invasion and Metastasis)；避免免疫摧毁(Avoiding Immune Destruction)；促进肿瘤的炎症(Tumor Promotion Inflammation)；细胞能量异常(Deregulating Cellular Energetics)；基因组不稳定和突变(Genome Instability and Mutation)。Hanahan D,Weinberg RA.Hallmarks of cancer:the next generation.Cell.2011 Mar 4；144(5):646-74)。如果抑制其抵抗细胞死亡，也就是诱导肿瘤细胞凋亡，很大程度抑制了癌症的发生。从逻辑上考虑，由于肿瘤细胞是非正常细胞，机体自身天生应该对其有抑制作用，也就是这些细胞应该自动发生凋亡，之所以不发生凋亡与其自身特征有关。研究发现，一些癌症或许不能完全归因为遗传损伤，而是衰老细胞绕过了告诉它们停止生长的开关所引起，衰老细胞与癌细胞之间的行为存在相似之处表明，如果衰老细胞设法逃脱死亡，它们有潜力变为癌症(Hazel A.Cruickshanks，et al.Senescent cells harbour features of the cancer epigenome，Nature Cell Biology(2013))。新的观点认为，癌症和衰老的根源是一样的(Carlos López-Otín,et al.The Hallmarks of Aging.Cell,6 June 2013)。发明人将肿瘤细胞看作衰老细胞，它理应走向凋亡。因此发明人认为，肿瘤细胞是因为具有的一些特殊能力让其不走向凋亡，而长期滞留在机体内。Tumor cells have ten characteristics: Self-Sufficiency in Growth Signals; Insensitivity to Antigrowth Signals; Resisting Cell
Death); Limitless Replicative Potential; Sustained Angiogenesis; Tissue Invasion and Metastasis; Avoiding Immune Destruction; Promoting Tumor Inflammation (Tumor Promotion) Inflammation); Degegulating Cellular Energetics; Genome Instability and Mutation. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011 Mar 4; 144(5): 646-74). If it inhibits its resistance to cell death, that is, induces tumor cell apoptosis, it greatly inhibits the occurrence of cancer. Logically, since tumor cells are abnormal cells, the body itself should be inhibited by it, that is, these cells should automatically undergo apoptosis, and the reason why apoptosis does not occur is related to its own characteristics. The study found that some cancers may not be completely attributed to genetic damage, but that aging cells bypass the switch that tells them to stop growing. The similarities between behaviors between senescent cells and cancer cells suggest that if aging cells manage to escape death They have the potential to become cancer (Hazel A. Cruickshanks, et al. Senescent cells harbour features of the cancer epigenome, Nature Cell Biology (2013)). The new view is that the root causes of cancer and aging are the same (Carlos López-Otín, et al. The Hallmarks of Aging. Cell, 6 June 2013). The inventors regard tumor cells as senescent cells, which should be directed toward apoptosis. Therefore, the inventors believe that tumor cells are left in the body for a long time because of their special ability to prevent apoptosis.
针对肿瘤细胞的两个特征也许是消灭肿瘤的最佳方式。该两个特征就是，抵抗细胞死亡和细胞能量异常。参考诱导衰老细胞发生凋亡的思路同样适用于肿瘤细胞。尽管肿瘤细胞有自身的特征，一般癌症病人存在是由于肿瘤细胞不在机体环境作用下自动走向死亡，但是相对于正常的细胞，肿瘤细胞本身发生了变化，比如更怕氧化物，也更怕酸性环境。Two features for tumor cells may be the best way to destroy tumors. These two features are resistance to cell death and abnormal cellular energy. The same applies to the induction of apoptosis in senescent cells. Although tumor cells have their own characteristics, the general cancer patients exist because tumor cells do not automatically die under the action of the body environment, but compared with normal cells, the tumor cells themselves have changed, such as being more afraid of oxides and more afraid of acidic environment. .
通过口服西地那非治疗3例患有囊状淋巴管瘤的儿童，通过用药，3位患儿的肿瘤体积均出现明显的缩小(Glenda L.et al.Swetman,Sildenafil for Severe LympHatic Malformations N Engl J Med 2012；366:384-386 January 26,2012)。西地那非是5-磷酸二酯酶抑制剂，磷酸二酯酶是NO-cGMP通路的负调节因子，因此西地那非能够通过释放生物活性物质一氧化氮。因此，本发明人认为一氧化氮这种氧化物起到抑制肿瘤的直接作用。Three children with cystic lymphangioma were treated with oral sildenafil, and the tumor volume of the three children was significantly reduced by medication (Glenda L. et al. Swetman, Sildenafil for Severe LympHatic Malformations N Engl J Med 2012;366:384-386 January 26,2012). Sildenafil is a 5-phosphodiesterase inhibitor, and phosphodiesterase is a negative regulator of the NO-cGMP pathway, so sildenafil is able to release nitric oxide, a biologically active substance. Therefore, the inventors believe that nitric oxide, an oxide, acts as a direct inhibitor of tumors.
在针对乳腺和黑素瘤癌细胞的实验中发现，硝酸甘油通过产生一氧化氮可以加强一般化疗药物杀死癌细胞的作用(Barsoum IB,et al.Hypoxia induces escape from innate immunity in cancer cells via increased expression of ADAM10:role of nitric oxide.Cancer Res.2011 Dec 15；71(24):7433-41)。促一氧化氮释放的前体药物，诱导乳腺癌癌细胞死亡，同时保留正常乳腺上皮细胞(Vanity McMurtry,et al.JS-K,a nitric oxide-releasing pro-drug,induces breast cancer cell death while sparing normal
mammary epithelial cells，International of journal of oncology,Published online on:Tuesday,January 25,2011,Pages:963-971)。普遍认为，高浓度的一氧化氮合酶表达，可以对肿瘤细胞产生抑制其生长的毒性作用(Xu W,et al.The role of nitric oxide in cancer.Cell Res.2002 Dec；12(5-6):311-20)。In experiments with breast and melanoma cancer cells, it was found that nitroglycerin can enhance the action of common chemotherapeutic drugs to kill cancer cells by producing nitric oxide (Barsoum IB, et al. Hypoxia induces escape from innate immunity in cancer cells via increased Expression of ADAM10: role of nitric oxide. Cancer Res. 2011 Dec 15;71(24):7433-41). A prodrug that promotes nitric oxide release, induces death of breast cancer cells while retaining normal mammary epithelial cells (Vanity McMurtry, et al. JS-K, a nitric oxide-releasing pro-drug, induces breast cancer cell death while sparing Normal
Mammary epithelial cells, International of journal of oncology, Published online on: Tuesday, January 25, 2011, Pages: 963-971). It is generally believed that high concentrations of nitric oxide synthase can produce toxic effects on tumor cells that inhibit their growth (Xu W, et al. The role of nitric oxide in cancer. Cell Res. 2002 Dec; 12 (5-6 ): 311-20).
细胞增殖实验显示，400μmol/L、800μmol/L、1600μmol/L对体外培养的Hela细胞有不同程度的细胞毒性，最高达89.0％。过氧化氢处理后，细胞衰老率和活性氧产生水平随过氧化氢浓度升高而增高，呈现量效关系。结论：过氧化氢对Hela细胞有明显的细胞毒性，还能诱导其衰老和影响活性氧的产生水平。Cell proliferation experiments showed that 400μmol/L, 800μmol/L and 1600μmol/L had different degrees of cytotoxicity to Hela cells cultured in vitro, up to 89.0%. After hydrogen peroxide treatment, the cell senescence rate and the level of reactive oxygen species increased with the increase of hydrogen peroxide concentration, showing a dose-effect relationship. Conclusion: Hydrogen peroxide has obvious cytotoxicity to Hela cells, and can induce its senescence and affect the production of reactive oxygen species.
肿瘤细胞和衰老细胞一样，更多的是依靠低氧的糖酵解方式提供能量。早在80多年前就发现肿瘤细胞的糖代谢较正常细胞旺盛，而且即使是在有氧条件下也依赖于糖酵解，该现象称为“Warburg效应”，其机制与肿瘤细胞的细胞膜上的葡萄糖转运蛋白(Glut)功能活跃，且己糖激酶活性增强有关。“Warburg效应”的结果是细胞产生大量糖酵解产物——乳酸。从糖脱下来的大量H+不能像有氧氧化那样经过呼吸链氧化为水，大量的H+聚集将使细胞内面临pH(pHi)酸化和发生凋亡的威胁，而肿瘤细胞增强的泌酸功能使细胞内pHi得以维持，但对周围正常的宿主细胞将产生不利影响。肿瘤细胞的“Warburg效应”使肿瘤细胞比正常细胞产生更多的酸，但对肿瘤细胞pH检测发现细胞内pHi值较正常细胞无明显区别，而细胞外pH(pHe)和细胞内酸性囊泡内的pH(pHv)明显低于正常细胞，提示可能与肿瘤细胞有较强的泌酸功能有关。(Izumi H,Torigoe T,Ishiguchi H,et al.Cellular pH regulators:potentially promising molecular targets for cancer chemotherapy.Cancer Treat Rev,2003,29(6):541-549)。Like aging cells, tumor cells rely more on low-oxygen glycolysis to provide energy. It was discovered more than 80 years ago that the glucose metabolism of tumor cells is stronger than that of normal cells, and even under aerobic conditions, it depends on glycolysis. This phenomenon is called "Warburg effect", and its mechanism is related to the cell membrane of tumor cells. Glucose transporter (Glut) is active and is involved in increased hexokinase activity. The result of the "Warburg effect" is that the cells produce a large amount of glycolysis product, lactic acid. A large amount of H + removed from sugar cannot be oxidized into water through the respiratory chain like aerobic oxidation, and a large amount of H + aggregation will threaten the pH (pHi) acidification and apoptosis in the cells, while the tumor cells enhance the acid secretion. The function allows the pHi in the cell to be maintained, but will have an adverse effect on the surrounding normal host cells. The "Warburg effect" of tumor cells causes tumor cells to produce more acid than normal cells, but the pH value of tumor cells is found to be indistinguishable from normal cells, while extracellular pH (pHe) and intracellular acidic vesicles. The pH inside (pHv) is significantly lower than that of normal cells, suggesting that it may be related to the strong acid function of tumor cells. (Izumi H, Torigoe T, Ishiguchi H, et al. Cellular pH regulators: potentially promising molecular targets for cancer chemotherapy. Cancer Treat Rev, 2003, 29(6): 541-549).
为了躲避酸性微环境的毒性，肿瘤细胞遂向外排出氢离子，最终产生细胞外的酸性环境以及细胞内的碱性环境。肿瘤细胞通过上调细胞质膜的氢离子相关转运蛋白，如钠氢交换蛋白(Na+/H+)、Na+/K+-ATPase、囊泡型H+-ATPases、H+/CI-共输送体和单羧酸转运蛋白(MCT)等，来实现这一机制(Harguindey S，et al.The role of pH dynamics and the Na+/H+antiporter in the etiopathogenesis and treatment of cancer.Two faces of the same coin--one single nature,[J].Biochim BiopHys Acta，2005，1756(1)：1-24)。In order to avoid the toxicity of the acidic microenvironment, the tumor cells excrete hydrogen ions outward, eventually producing an extracellular acidic environment and an alkaline environment inside the cells. Tumor cells regulate hydrogen ion-associated transporters of the plasma membrane, such as sodium-hydrogen exchange protein (Na + /H + ), Na + /K + -ATPase, vesicular H + -ATPases, H + /CI - co-transporters And monocarboxylic acid transporter (MCT), etc., to achieve this mechanism (Harguindey S, et al. The role of pH dynamics and the Na+/H+antiporter in the etiopathogenesis and treatment of cancer. Two faces of the same coin- -one single nature, [J]. Biochim BiopHys Acta, 2005, 1756(1): 1-24).
因此，发明人判断如果通过增加压力让肿瘤细胞泌酸功能降低，那就会引起肿瘤细胞内pHi降低，使其酸中毒，从而诱导肿瘤细胞凋亡。而增加压力，即使肿瘤细胞外环境的pH(pHe)降低，让肿瘤细胞的运输H+离子的质子泵增加压力，并最终崩溃。Therefore, the inventors judged that if the acid secretion function of the tumor cells is lowered by increasing the pressure, the pHi in the tumor cells is lowered, causing acidosis, thereby inducing apoptosis of the tumor cells. While increasing the pressure, even if the pH (pHe) of the tumor's extracellular environment is lowered, the proton pump that transports the H + ions of the tumor cells increases the pressure and eventually collapses.
介导诱导人腺癌和胃癌的细胞株凋亡(A C Williams,et al.An acidic environment leads to p53 dependent induction of apoptosis in human adenoma and carcinoma cell lines:implications for clonal selection during colorectal carcinogenesis，Oncogene.1999May 27；18(21):3199-204)。There is more literature support, and the acidic environment can induce apoptosis in tumor cells. For example, an acidic environment, through p53
A C Williams, et al. An acidic environment leads to p53 induction of apoptosis in human adenoma and carcinoma cell lines:implications for clonal selection during colorectal carcinogenesis,Oncogene.1999May 27;18(21):3199-204).
实验表明：给予体外培养的胃癌AGS细胞一个pH为6.0的酸性环境,这种酸性环境使细胞外H+离子浓度升高，可能通过抑制肿瘤细胞的NHE-1的Na+-H+交换功能使细胞内糖酵解产生的过多H+离子无法排出使本来偏碱性的细胞内环境明显酸化，这样既起到了改变胃癌细胞外环境的作用，同时也改变了细胞内环境。胃癌细胞的这种内外环境的酸化作用改变了适合它生长增殖的细胞内外环境的酸碱度，从而使大部分胃癌细胞停止增殖(高艳等，《胃癌A G S细胞内pH值的测定及酸性环境对其增殖和凋亡的影响》，《现代生物医学进展》，2008年09期)。Experiments have shown that gastric cancer AGS cells cultured in vitro have an acidic environment with a pH of 6.0. This acidic environment increases the concentration of extracellular H + ions, possibly by inhibiting the Na + -H + exchange function of NHE -1 in tumor cells. Excessive H + ions produced by intracellular glycolysis cannot be discharged to significantly acidify the otherwise alkaline environment, which not only changes the extracellular environment of gastric cancer, but also changes the intracellular environment. The acidification of this internal and external environment of gastric cancer cells changes the pH of the cells inside and outside the environment suitable for its growth and proliferation, so that most gastric cancer cells stop proliferating (Gao Yan et al., "Measurement of pH in gastric cancer AGS cells and acidic environment Effects of Proliferation and Apoptosis, Progress in Modern Biomedicine, 2008, 09).
Rich IN等证明了从白血病患者体内获得的白细胞系及外周淋巴血细胞有着比正常造血组织细胞普遍的、具有统计学意义的高的pH值。说明了在胞内pH值与正常造血细胞和白血病细胞的细胞周期调控之间存在一种直接的关系。利用这种关系，他们用Na+/H+交换体的抑制剂5-(N，N-hexamethylene)-amiloride(HMA)处理白血病细胞，降低了胞内pH值，从而诱导了细胞凋亡。(Rich IN，Worthington-White D，Garden OA，Musk P.Apoptosis of leukemic cells accompanies reduction in intracellular pH after targeted inhibition of the Na+/H+exchanger.Blood.2000，15，95(4):1427～1434)。Rich IN et al. demonstrated that leukocyte lines and peripheral lymphocytes obtained from leukemia patients have a statistically high pH value that is more common than normal hematopoietic tissue cells. This illustrates a direct relationship between intracellular pH and cell cycle regulation of normal hematopoietic and leukemia cells. Using this relationship, they treated leukemia cells with the Na + /H + exchanger inhibitor 5-(N,N-hexamethylene)-amiloride (HMA), which reduced intracellular pH and induced apoptosis. (Rich IN, Worthington-White D, Garden OA, Musk P. Apoptosis of leukemic cells accompanies reduction in intracellular pH after targeted inhibition of the Na+/H+exchanger. Blood. 2000, 15, 95(4): 1427~1434) .
在本发明的一个实施方式中，我们采用酸性二氧化氯溶液靶向给予肿瘤细胞，使肿瘤细胞发生凋亡,并且随着酸性环境的加强，肿瘤细胞死亡率相应上升。In one embodiment of the present invention, we use an acidic chlorine dioxide solution to target tumor cells, cause tumor cells to undergo apoptosis, and with the strengthening of the acidic environment, the tumor cell mortality increases accordingly.
本领域技术人员均理解强氧化剂和酸性环境对正常细胞有损伤作用，也就是说，酸性二氧化氯溶液对正常细胞也有加速凋亡作用。但是，本发明人认为，正常细胞的损伤会刺激机体干细胞的再生，因此一般性损伤可以完全被修复；另外，衰老细胞或癌细胞是功能不全的细胞，他们抵御氧化剂或酸性环境的凋亡诱导能力更弱。因此本发明的技术方案是靶向明确而副作用较小的一种抗衰老或抗肿瘤的技术。Those skilled in the art understand that strong oxidants and acidic environments have a damaging effect on normal cells, that is, acidic chlorine dioxide solution also accelerates apoptosis in normal cells. However, the inventors believe that normal cell damage stimulates the regeneration of body stem cells, so that general damage can be completely repaired; in addition, senescent cells or cancer cells are dysfunctional cells that resist apoptosis in oxidants or acidic environments. The ability is weaker. Therefore, the technical solution of the present invention is an anti-aging or anti-tumor technique with targeted and less side effects.
在一些实施例中，本发明证明了酸性二氧化氯制剂对衰老细胞或肿瘤细胞具有更强的凋亡诱导作用，而对正常细胞损伤较小，或者说这种损伤是在可以被弥补的程度。In some embodiments, the present invention demonstrates that an acidic chlorine dioxide formulation has a greater apoptosis-inducing effect on senescent cells or tumor cells, but less damage to normal cells, or that the damage is at a level that can be compensated. .
本发明所述的含二氧化氯的细胞凋亡诱导剂可以单独包含二氧化氯的单一剂型或者包含二氧化氯前体的若干份组合。其中单独包含二氧化氯的单一剂型可以如下地制成：The chlorine dioxide-containing apoptosis inducing agent of the present invention may comprise a single dosage form of chlorine dioxide alone or a combination of several parts comprising a chlorine dioxide precursor. A single dosage form in which chlorine dioxide alone is included can be made as follows:
并且浓度在99.9％以上二氧化氯气体的制备方式(优选为亚氯酸盐与酸反应)产生二氧化氯气体。通过将该二氧化氯气体向上述酸性水溶液中鼓泡并溶解从而制备500～2900ppm的二氧化氯溶液。该溶液在使用于本发明所述用途之前，应当避光密闭保存，并一直保存于4℃～15℃较低温状态下。该制备含二氧化氯制剂的方法并不要求一定是水溶液，唯有保证在施用于目标组织时能够在目标组织中发挥作用的物质主要为二氧化氯，且在一定酸性环境下。Method 1: A pH adjuster is added to water to prepare an acidic aqueous solution having a pH of 1.5 to 6.5. By conformity with the regulations,
Further, the chlorine dioxide gas is produced by a method of preparing a chlorine dioxide gas having a concentration of 99.9% or more (preferably, the chlorite reacts with an acid). A chlorine dioxide solution of 500 to 2900 ppm was prepared by bubbling and dissolving the chlorine dioxide gas into the above acidic aqueous solution. The solution should be stored in the dark and sealed at a temperature of 4 ° C to 15 ° C before being used in the application of the present invention. The method for preparing a chlorine dioxide-containing preparation is not necessarily an aqueous solution, and only the substance capable of functioning in the target tissue when applied to the target tissue is mainly chlorine dioxide, and in a certain acidic environment.
方法2：将二氧化氯前体溶解于水中制备1％～40％的水溶液。向该水溶液中加入含有pH调节剂的酸性溶液(优选为2％～50％的柠檬酸溶液)，调节混合溶液的pH值为1.5～6.5。该溶液在使用于本发明所述用途之前，应当避光密闭保存，并一直保存于4℃～15℃较低温状态下。该制备含二氧化氯制剂的方法并不要求一定是水溶液，唯有保证在施用于目标组织时能够在目标组织中发挥作用的物质主要为二氧化氯，且在一定酸性环境下。Method 2: A chlorine dioxide precursor is dissolved in water to prepare a 1% to 40% aqueous solution. An acidic solution (preferably 2% to 50% citric acid solution) containing a pH adjuster is added to the aqueous solution to adjust the pH of the mixed solution to 1.5 to 6.5. The solution should be stored in the dark and sealed at a temperature of 4 ° C to 15 ° C before being used in the application of the present invention. The method for preparing a chlorine dioxide-containing preparation is not necessarily an aqueous solution, and only the substance capable of functioning in the target tissue when applied to the target tissue is mainly chlorine dioxide, and in a certain acidic environment.
包含二氧化氯前体的若干份组合可以如下地制成：将二氧化氯前体溶解于水中制备1％～40％的水溶液，这是第一份溶液；用pH调节剂制备出酸性溶液(优选为2％～50％的柠檬酸溶液)，这是第二份溶液。在使用于本发明所述用途之前，现场混合以上溶液，最终混合溶液的pH值调整为1.5～6.5，待反应产生二氧化氯后，将该溶液施用于目标组织。该制备含二氧化氯制剂的方法并不要求一定是水溶液，唯有保证在施用于目标组织时能够在目标组织中发挥作用的物质主要为二氧化氯，且在一定酸性环境下。The combination of several parts comprising the chlorine dioxide precursor can be prepared by dissolving the chlorine dioxide precursor in water to prepare a 1% to 40% aqueous solution, which is the first solution; preparing an acidic solution with a pH adjuster ( It is preferably a 2% to 50% citric acid solution), which is a second solution. Before use in the application of the present invention, the above solution is mixed in the field, and the pH of the final mixed solution is adjusted to 1.5 to 6.5. After the chlorine dioxide is reacted to generate chlorine dioxide, the solution is applied to the target tissue. The method for preparing a chlorine dioxide-containing preparation is not necessarily an aqueous solution, and only the substance capable of functioning in the target tissue when applied to the target tissue is mainly chlorine dioxide, and in a certain acidic environment.
作为能在本发明中使用的二氧化氯前体，可以举出例如亚氯酸碱金属盐、亚氯酸碱土类金属盐。作为亚氯酸碱金属盐，可以举出例如亚氯酸钠、亚氯酸钾、亚氯酸锂；作为亚氯酸碱土类金属盐，可以举出例如亚氯酸钙、亚氯酸镁、亚氯酸钡。The chlorine dioxide precursor which can be used in the present invention may, for example, be an alkali metal chlorite or an alkali metal chlorite. Examples of the alkali metal chlorite include sodium chlorite, potassium chlorite, and lithium chlorite. Examples of the alkali metal chlorite include, for example, calcium chlorite, magnesium chlorite, and sub Bismuth chlorate.
其中，不仅从获得容易的理由，而且从二氧化氯活性的持续性优异的观点出发，优选亚氯酸钠、亚氯酸钾，更优选亚氯酸钠。Among them, sodium chlorite and potassium chlorite are preferable, and sodium chlorite is more preferable, from the viewpoint of obtaining an easy reason and being excellent in the sustainability of chlorine dioxide activity.
作为能在本发明中使用的pH调节剂，只要是具有缓冲性的酸就可以很好的使用。The pH adjuster which can be used in the present invention can be preferably used as long as it is a buffering acid.
作为有机酸或其盐可以举出甲酸、乙酸、丙酸、丁酸、乳酸、丙酮酸、柠檬酸、苹果酸、酒石酸、葡糖酸、乙醇酸、富马酸、丙二酸、马来酸、草酸、琥珀酸、丙烯酸、巴豆酸、戊二酸及他们的盐。Examples of the organic acid or a salt thereof include formic acid, acetic acid, propionic acid, butyric acid, lactic acid, pyruvic acid, citric acid, malic acid, tartaric acid, gluconic acid, glycolic acid, fumaric acid, malonic acid, and maleic acid. , oxalic acid, succinic acid, acrylic acid, crotonic acid, glutaric acid and their salts.
作为无机酸可以举出盐酸、磷酸、硼酸、偏磷酸、焦磷酸、氨基磺酸等。作为无机酸盐，例如可以举出磷酸二氢盐(钠盐、钾盐，下同)、磷酸二氢盐与磷酸氢盐的混合物等。pH调节剂可以单独使用1种，也可以并用2种以上。Examples of the inorganic acid include hydrochloric acid, phosphoric acid, boric acid, metaphosphoric acid, pyrophosphoric acid, and sulfamic acid. Examples of the inorganic acid salt include a dihydrogen phosphate salt (sodium salt, potassium salt, the same applies hereinafter), a mixture of a dihydrogen phosphate salt and a hydrogen phosphate salt, and the like. One type of the pH adjuster may be used alone or two or more types may be used in combination.
From the standpoint of use in human safety, the pH adjusting agent is preferably citric acid, acetic acid and sodium dihydrogen phosphate, more preferably citric acid.
另外，二氧化氯液剂的最终pH值优选为1.5～5.5，更优选为1.5～3.5。Further, the final pH of the chlorine dioxide solution is preferably from 1.5 to 5.5, more preferably from 1.5 to 3.5.
本发明中的含二氧化氯的细胞凋亡诱导剂优选为液剂。The chlorine dioxide-containing apoptosis inducing agent in the present invention is preferably a liquid preparation.
本发明中的细胞凋亡诱导剂的药剂量因患者的年龄、体重、疾病性质和状况而变化。但是在成人的情况下，例如为每日1毫克至5000毫克的二氧化氯，优选为每日1毫克至1000毫克的二氧化氯。The dose of the apoptosis inducing agent in the present invention varies depending on the age, body weight, disease properties and condition of the patient. However, in the case of an adult, for example, 1 mg to 5000 mg of chlorine dioxide per day, preferably 1 mg to 1000 mg of chlorine dioxide per day.
本发明中的含二氧化氯的细胞凋亡诱导剂可以通过多种方式给药。含二氧化氯的细胞凋亡诱导剂可以全身性地给药，如通过静脉内、动脉内或腹膜内给药；也可以局部患处的直接给药，该方法可以通过透皮、穿刺等方法直达患处给药。The chlorine dioxide-containing apoptosis inducing agent of the present invention can be administered by various means. The chlorine dioxide-containing apoptosis inducing agent can be administered systemically, such as by intravenous, intra-arterial or intraperitoneal administration; or directly administered to a local affected area, the method can be directly administered by transdermal, puncture, etc. The affected area is administered.
本发明中的含二氧化氯的细胞凋亡诱导剂的给药方式，可以通过任何一种能到达预期组织的途径给药，比如，可以经由静脉点滴、静脉注射、动脉注射、肌肉注射、皮下注射、皮内注射、心内注射、腹腔注射、鞘内注射、关节内注射、穿刺注射、直肠给药、舌下给药、鼻腔给药、经皮给药、吸入或者局部给药到目标的器官或组织等，但不限于这些。The method for administering the chlorine dioxide-containing apoptosis inducing agent in the present invention can be administered by any route which can reach the intended tissue, for example, by intravenous drip, intravenous injection, intraarterial injection, intramuscular injection, subcutaneous Injection, intradermal injection, intracardiac injection, intraperitoneal injection, intrathecal injection, intra-articular injection, puncture injection, rectal administration, sublingual administration, nasal administration, transdermal administration, inhalation or topical administration to the target Organs, tissues, etc., but are not limited to these.
通过以上方式给药时，因目标组织的大小，本发明中的细胞凋亡诱导剂的有效量范围为每日0.1～500mg/kg二氧化氯,有时候目标组织用面积更容易表述，则本发明中的细胞凋亡诱导剂的有效范围为每日0.1～500mg二氧化氯/100cm2，按所述剂量给药时，至少10天后有效。When administered by the above method, the effective amount of the apoptosis inducing agent in the present invention ranges from 0.1 to 500 mg/kg of chlorine dioxide per day due to the size of the target tissue, and sometimes the area of the target tissue is more easily expressed. The effective range of the apoptosis inducing agent in the invention is 0.1 to 500 mg of chlorine dioxide per 100 cm 2 per day, and is effective after at least 10 days when administered at the dose.
无论如何，本发明所述的含二氧化氯的细胞凋亡诱导剂，唯有保证在适用于目标有效剂量的二氧化氯以合适方式进入目标组织中，发挥诱导细胞凋亡作用，而不限于任何的形式、方式和步骤，及其他辅助性物质的帮助。In any case, the chlorine dioxide-containing apoptosis inducing agent of the present invention can only exert the effect of inducing apoptosis when the chlorine dioxide suitable for the target effective dose enters the target tissue in an appropriate manner, and is not limited thereto. Any form, method and procedure, and the help of other auxiliary substances.
《中国药典制剂通则》中的任何制剂都能够作为本发明中的细胞凋亡诱导剂的剂型。本发明方法中的细胞凋亡诱导剂作为药物的剂型的实例包括直接用于体内的注射剂(包括混悬液、乳剂)；软膏剂(包括油脂性软膏、乳剂型软膏(霜)、水溶性软膏等)、吸入剂、液剂制剂(包括滴眼剂、滴鼻剂等)、栓剂、贴剂、糊剂、洗剂等外用剂；或片剂(包括糖衣、薄膜、胶衣)、液体制剂、胶囊剂、颗粒剂、散剂(包括细粒级)、丸剂、糖浆剂、含片等。这些制剂可按照中国药典制剂通则的方法进行制备。Any of the preparations in the "Chinese Pharmacopoeia General Rules" can be used as a dosage form of the apoptosis inducing agent in the present invention. Examples of the dosage form of the apoptosis inducing agent in the method of the present invention include injections (including suspensions, emulsions) for direct use in the body; ointments (including oily ointments, cream ointments (creams), water-soluble ointments Etc., inhalation, liquid preparation (including eye drops, nasal drops, etc.), suppositories, patches, pastes, lotions and other external preparations; or tablets (including sugar coatings, films, gel coats), liquid preparations , capsules, granules, powders (including fine-grain grade), pills, syrups, lozenges, and the like. These preparations can be prepared according to the general principles of Chinese Pharmacopoeia preparations.
此外，本发明方法中的细胞凋亡诱导剂给药时还可以包括可药用的固态或液态载体或介入治疗材料。作为可药用的固态或液态载体，可列举溶剂、稳定剂、助溶解剂、乳化剂、悬浊剂、缓冲剂、等渗剂、着色剂、基质、增稠剂、赋形剂、润滑剂、粘合剂、崩解剂、包衣剂、矫味剂、调理剂、发泡剂、高吸水性树脂、表面活性剂、渗透促进剂和pH调节剂等，但并不限于这些。Furthermore, the apoptosis inducing agent in the method of the present invention may also comprise a pharmaceutically acceptable solid or liquid carrier or interventional therapeutic material when administered. As the pharmaceutically acceptable solid or liquid carrier, there may be mentioned solvents, stabilizers, solubilizers, emulsifiers, suspensions, buffers, isotonic agents, colorants, bases, thickeners, excipients, lubricants. And the like, but not limited to, a binder, a disintegrant, a coating agent, a flavoring agent, a conditioning agent, a foaming agent, a superabsorbent resin, a surfactant, a penetration enhancer, and a pH adjuster.
结晶纤维素、甲基纤维素、乙基纤维素、羟丙基纤维素、低取代羟丙基纤维素、羟丙基甲基纤维素、羟丙基甲基纤维素邻苯二甲酸酯、醋酸羟丙基甲基纤维素琥珀酸酯、羟甲基纤维素、羟甲基纤维素钙、羟甲基纤维素钠、交联羟甲基纤维素钠、羟甲基乙基纤维素、乙酸邻苯二酸纤维素等纤维素及其相关衍生物；玉米淀粉、小麦淀粉、米淀粉、马铃薯淀粉、环糊精、支链淀粉等点功夫及其相关衍生物；琼脂、藻酸钠、阿拉伯胶、明胶、胶原、虫胶、黄薯胶、黄原胶等天热高分子(海藻类、植物粘质、蛋白质等)；聚乙烯吡咯烷酮、氨基烷基甲基丙烯酸共聚物、甲基丙烯酸共聚物、羟基乙烯基共聚物、聚乙烯醇、二甲基聚硅氧烷等合成高分子；橄榄油、可可油、巴西棕榈蜡、牛油、硬化油、大豆油、芝麻油、山茶油、亚麻籽油、石蜡、液体石蜡、黄蜂蜡、白色凡士林、椰子油、微晶蜡等油脂类；硬脂酸、硬脂酸铝、硬脂酸钙、硬脂酸镁、柠檬酸三乙酯、三乙酯甘油酯、中链脂肪酸三甘油酯、硬脂、肉豆蔻酸异丙酯等脂肪酸及其衍生物；乙醇、甘油、硬脂醇、鲸蜡醇、丙二醇、聚乙二醇等醇和多元醇；氧化锌、磷酸氢钙、沉降碳酸钙、合成硅酸铝、硅酸酐、高岭土、干燥氢氧化铝凝胶、合成水滑石、氧化钛、滑石、膨润土、硅酸铝镁、硫酸铝钾、次没食子酸铋、次水杨酸铋、乳酸钙、柠檬酸钠、氯化钠、碳酸氢钠等无机物质和金属盐化合物；蔗糖脂肪酸酯、硬脂酸聚烃氧酯、氢化蓖麻油聚氧乙烯醚、聚氧乙烯聚氧丙烯二醇、倍伴油酸脱水山梨醇酯、三油酸脱水山梨醇酯、单硬脂酸脱水山梨醇酯、但棕榈酸脱水山梨醇酯、单月桂酸脱水山梨醇酯、聚山梨醇酯、单硬脂酸甘油酯、十二烷基硫酸钠、聚桂醇等表面活性剂；二甲基亚砜及其类似物、氮酮类化合物、吡咯酮衍生物、醇类化合物及脂肪酸类化合物等渗透促进剂；色素；香料等。但并不限于这些。Specific examples include deionized water, lactose, white sugar, fructose, glucose, mannose, sorbitol, etc., or sugar alcohols;
Crystalline cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose phthalate, Hydroxypropylmethylcellulose acetate succinate, hydroxymethylcellulose, hydroxymethylcellulose calcium, sodium carboxymethylcellulose, crosslinked hydroxymethylcellulose sodium, hydroxymethylethylcellulose, acetic acid Cellulose and its related derivatives such as cellulose phthalate; corn starch, wheat starch, rice starch, potato starch, cyclodextrin, amylopectin and other related derivatives; agar, sodium alginate, arabic Gelatin, gelatin, collagen, shellac, yellow potato gum, xanthan gum and other natural polymers (seaweed, plant mucilage, protein, etc.); polyvinylpyrrolidone, aminoalkyl methacrylic acid copolymer, methacrylic acid copolymerization Synthetic polymer such as hydroxyvinyl copolymer, polyvinyl alcohol, dimethylpolysiloxane; olive oil, cocoa butter, carnauba wax, butter, hardened oil, soybean oil, sesame oil, camellia oil, flaxseed Oil, paraffin, liquid paraffin, yellow beeswax White petroleum jelly, coconut oil, microcrystalline wax and other oils; stearic acid, aluminum stearate, calcium stearate, magnesium stearate, triethyl citrate, triethyl glyceride, medium chain fatty acid triglyceride Fatty acids such as stearin and isopropyl myristate and their derivatives; alcohols and polyols such as ethanol, glycerin, stearyl alcohol, cetyl alcohol, propylene glycol, polyethylene glycol; zinc oxide, calcium hydrogen phosphate, and precipitated calcium carbonate , synthetic aluminum silicate, silicic anhydride, kaolin, dry aluminum hydroxide gel, synthetic hydrotalcite, titanium oxide, talc, bentonite, magnesium aluminum silicate, potassium aluminum sulfate, bismuth sub-gallate, bismuth subsalicylate, lactic acid Inorganic substances and metal salt compounds such as calcium, sodium citrate, sodium chloride, sodium hydrogencarbonate; sucrose fatty acid esters, polyoxyl stearates, hydrogenated castor oil polyoxyethylene ethers, polyoxyethylene polyoxypropylene diols , sorbitan sorbitan ester, sorbitan trioleate, sorbitan monostearate, but sorbitan palmitate, sorbitan monolaurate, polysorbate, single hard Glyceryl glyceride, sodium lauryl sulfate, poly gui Surfactants such as alcohol; osmotic accelerators such as dimethyl sulfoxide and its analogues, azone compounds, pyrrolidone derivatives, alcohol compounds and fatty acid compounds; pigments; But it is not limited to these.
介入治疗材料的实例包括注射器、支架、人工血管、穿刺注射器、导管、球囊等，但并不限于这些。Examples of the interventional treatment material include, but are not limited to, a syringe, a stent, an artificial blood vessel, a puncture syringe, a catheter, a balloon, and the like.
本发明中的含二氧化氯的细胞凋亡诱导剂在给药前，可以依据给药方式而施用麻醉剂，如巴比妥酸盐等注射型麻醉剂、一氧化二氮等吸入型麻醉剂、利多卡因等表面麻醉剂等，但不限于这些。The chlorine dioxide-containing apoptosis inducing agent of the present invention may be administered an anesthetic according to the administration mode before administration, such as an injection type anesthetic such as barbiturate or an inhaled anesthetic such as nitrous oxide, Lidoca. Because of the surface anesthetic, etc., but not limited to these.
本发明中的细胞凋亡诱导剂应用于哺乳动物，哺乳动物的具体实例包括人、猴、狗、猪、猫、兔、大鼠和小鼠。其中，人是优选者。The apoptosis inducing agent in the present invention is applied to mammals, and specific examples of mammals include humans, monkeys, dogs, pigs, cats, rabbits, rats, and mice. Among them, people are preferred.
提供以下实施例以说明本发明，而不以任何方式限制本发明。The following examples are provided to illustrate the invention and are not intended to limit the invention in any way.
Example 1: Effect of chlorine dioxide-containing preparation on apoptosis of proliferative scar skin fibroblasts
增生性瘢痕作为一种影响器官功能、美观的疾病，病因有多种，但凡损伤累及真皮深层的损伤，都有可能产生增生性瘢痕。普遍认为成纤维细胞凋亡不足是增生性瘢痕产生的原因(Ogawa,R.The most current algorithms for the treatment and prevention of hypertropHic scars and keloids[J].Plast Reconstr Surg,2010,125:557-568)。常识表明，同样大小的伤口，相对比年轻人，老年人的伤口愈合慢且容易留下疤痕。因此，增生性瘢痕的形成可以理解为一种皮肤老化的现象，通过诱导成纤维细胞凋亡可能是治愈瘢痕性皮肤的好方法。As a kind of disease that affects organ function and appearance, hypertrophic scar has many causes, but any damage that affects deep layers of the dermis may cause hypertrophic scars. It is generally believed that insufficient apoptosis of fibroblasts is the cause of hypertrophic scars (Ogawa, R. The most current algorithms for the treatment and prevention of hypertrop Hic scars and keloids [J]. Plast Reconstr Surg, 2010, 125: 557-568) . Common sense shows that wounds of the same size are relatively slower to heal and tend to leave scars than younger people. Therefore, the formation of hypertrophic scar can be understood as a phenomenon of skin aging, which may be a good way to cure scarred skin by inducing apoptosis of fibroblasts.
1)凋亡试验1) Apoptosis test
增生性瘢痕患者共6例，男5例，女1例，平均年龄42岁。实验共分4组：瘢痕中央部(中央2/3半径范围内)、瘢痕边缘部(周边1/3半径范围内)、瘢痕周围皮肤(距瘢痕疙瘩边缘0.5cm范围内)、瘢痕病人其他部位正常皮肤。There were 6 patients with hypertrophic scars, including 5 males and 1 female, with an average age of 42 years. The experiment was divided into 4 groups: the central part of the scar (within the central 2/3 radius), the edge of the scar (within the radius of 1/3 of the periphery), the skin around the scar (within 0.5 cm from the edge of the keloid), and other parts of the scar patient. Normal skin.
成纤维细胞的培养：所取标本按上述方法分组后在无菌条件下切除其表皮及皮下组织，在少量胎牛血清中将标本切成1mm3左右的组织块，在37℃、5％CO2饱和湿度条件下培养4-6h，使组织块粘附于瓶壁上，然后加入含15％胎牛血清的DMEM(dulbecco's modified eagle medium)培养基适量，3-4天换液1次，2-3周后，原代细胞长满并汇合成片，每3-5天传代1次，实验用第6-8代细胞。Culture of fibroblasts: The specimens were grouped according to the above method, and the epidermis and subcutaneous tissues were excised under aseptic conditions. The specimens were cut into tissue pieces of about 1 mm 3 in a small amount of fetal bovine serum at 37 ° C, 5% CO. 2 Incubate for 4-6 hours under saturated humidity conditions, so that the tissue block adheres to the bottle wall, then add appropriate amount of DMEM (dulbecco's modified eagle medium) containing 15% fetal bovine serum, and change the solution once every 3-4 days, 2 - After 3 weeks, the primary cells were overgrown and pooled into pellets, passaged once every 3-5 days, and cells 6-8 were used for the experiment.
用去离子水配置浓度为7.47％亚氯酸钠和1.59％氯化钠的混合溶液，制备出第一份溶液；用去离子水配置浓度为16.7％的柠檬酸溶液，制备出第二份溶液。分别从不同份溶液的容器中取出相同体积的溶液，混合，等待溶液混合静止3～5分钟，再用0.22μm的双层滤膜过滤，用去离子水稀释，制备不同浓度的二氧化氯酸性溶液。The first solution was prepared by dissolving a mixed solution of 7.47% sodium chlorite and 1.59% sodium chloride in deionized water; a citric acid solution having a concentration of 16.7% was prepared with deionized water to prepare a second solution. . Take the same volume of solution from the different parts of the solution, mix, wait for the solution to mix and stand for 3 to 5 minutes, then filter with 0.22μm double-layer filter, dilute with deionized water to prepare different concentrations of chlorine dioxide. Solution.
细胞接种并施加不同处理因素诱导细胞凋亡。各组分别取一瓶处于对数生长期的细胞，接种于6孔细胞培养板中，每组细胞于每块板接种1个孔(1×105个细胞/孔)，共接种6块板24个孔，继续在37℃、5％CO2饱和湿度条件下培养48-72h，至细胞长满孔底，取其中1块板，吸出培养基，用Hank液(一种平衡盐溶液)洗涤后加入不含胎牛血清的DMEM培养基，另取3块板分别加入含二氧化氯制剂，使其终浓度分别达到100ppm、1000ppm、2900ppm，取1块板加入FasMcAb(Fas单抗)，使其终浓度达到1μg/ml，另外1块板不施加任何处理因素，继续培养24h后检测其凋亡率。Cells were seeded and different treatment factors applied to induce apoptosis. Each group was taken from a cell in the logarithmic growth phase and inoculated into a 6-well cell culture plate. Each group of cells was seeded with 1 well (1 × 10 5 cells/well) per plate, and a total of 6 plates were inoculated. 24 wells, continue to culture at 37 ° C, 5% CO 2 saturated humidity for 48-72h, until the cells are full of the bottom of the well, take one of the plates, aspirate the medium, wash with Hank liquid (a balanced salt solution) After adding DMEM medium containing no fetal bovine serum, another three plates were added to the chlorine dioxide-containing preparation to achieve a final concentration of 100 ppm, 1000 ppm, and 2900 ppm, respectively, and one plate was added to FasMcAb (Fas mAb). The final concentration reached 1 μg/ml, and the other plate did not apply any treatment factor, and the apoptosis rate was detected after 24 hours of culture.
入RNase A 2μl(20mg/ml)，37℃孵育30min后立即投入冰浴中停止RNase A作用，再加入500μl PI染液(100μg/ml)避光孵育30min，吹散细胞，300目滤网过滤后在流式细胞仪上进行细胞DNA分析检测细胞凋亡率。PI staining and flow cytometry were used to compare the apoptosis rate of each component fiber cell. The treated cells were digested and collected in a 10 ml centrifuge tube, centrifuged at 1000 r/min for 10 min, washed with pre-cooled PBS (phosphate buffer solution), added with 70% ethanol at 4 ° C overnight, then centrifuged at low speed for 10 min, washed with PBS. Resuspend the cells and add in the remaining 0.5 ml of cell suspension
Into RNase A 2μl (20mg/ml), incubate at 37 °C for 30min, immediately put into the ice bath to stop the action of RNase A, then add 500μl PI staining solution (100μg/ml) and incubate for 30min in the dark, blow off the cells, filter through 300 mesh filter Cellular DNA analysis was performed on a flow cytometer to detect apoptosis rate.
所有各组细胞经无血清培养24h后其细胞凋亡率均有不同程度的增高，然而经过比较各组无血清培养后细胞凋亡的增长率，发现正常皮肤显著高于瘢痕疙瘩边缘部(P＜0.01)，而瘢痕疙瘩周围皮肤和瘢痕疙瘩中央部介于两者之间，与各组比较差异均无显著性意义(P＞0.05，表1)。这说明被看作老化组织的瘢痕处具有明显的抗凋亡能力，所以瘢痕组织不仅不能得以自动清除，反而会增生。The apoptosis rate of all the cells was increased after 24 hours of serum-free culture. However, after comparing the growth rate of apoptosis in each group, the normal skin was found to be significantly higher than the edge of keloid. <0.01), and the skin around the keloid and the central part of the keloid were between the two, and there was no significant difference between the two groups (P>0.05, Table 1). This indicates that the scar that is regarded as an aging tissue has obvious anti-apoptotic ability, so the scar tissue can not only be automatically removed, but will proliferate.
表1 无血清培养24h后瘢痕及其周围皮肤成纤维细胞凋亡率Table 1 Apoptosis rate of scar and its surrounding skin fibroblasts after 24 hours of serum-free culture
注：*与瘢痕边缘部比较，P＜0.01Note: *Compared with the edge of the scar, P<0.01
FasMcAb作用下正常皮肤的成纤维细胞凋亡率明显高于瘢痕及其周围皮肤成纤维细胞凋亡率(P＜0.01，表2)。这也说明瘢痕处组织具有抗凋亡能力。The apoptosis rate of fibroblasts in normal skin under FasMcAb was significantly higher than that in scar and surrounding skin fibroblasts (P<0.01, Table 2). This also indicates that the tissue at the scar has anti-apoptotic ability.
表2 FasMcAb作用下瘢痕及其周围皮肤成纤维细胞凋亡率Table 2 Apoptosis rate of fibroblasts in scar and its surrounding skin under FasMcAb
FasMcAb作用(1μg/ml)FasMcAb action (1μg/ml)
瘢痕周围皮肤Scar around the skin
注：*与瘢痕边缘部比较，P＜0.01Note: *Compared with the edge of the scar, P<0.01
具有明显的促凋亡作用。Under the action of chlorine dioxide preparation, the apoptosis rate of the four-component fiber cells gradually increased with the increase of the chlorine dioxide concentration gradient, and the increase was significant (P<0.01, Table 3). This shows the chlorine dioxide preparation in acidic environment for hypertrophic scar tissue
Has a significant pro-apoptotic effect.
表3 二氧化氯作用下瘢痕及其周围皮肤成纤维细胞凋亡率Table 3 Apoptosis rate of fibroblasts in scars and their surrounding skin under chlorine dioxide
2)对疤痕的治疗2) Treatment of scars
用去离子水配置浓度为7.47％亚氯酸钠和1.59％氯化钠的混合溶液，制备出第一份溶液；用去离子水配置浓度为16.7％的柠檬酸溶液，制备出第二份溶液。分别从不同份溶液的容器中取出相同体积的溶液，混合，等待溶液混合静止3～5分钟，再用0.22μm的双层滤膜过滤。制备出二氧化氯制剂。The first solution was prepared by dissolving a mixed solution of 7.47% sodium chlorite and 1.59% sodium chloride in deionized water; a citric acid solution having a concentration of 16.7% was prepared with deionized water to prepare a second solution. . The same volume of solution was taken from the containers of different solutions, mixed, and the solution was allowed to stand still for 3 to 5 minutes, and then filtered through a 0.22 μm double-layer filter. A chlorine dioxide preparation was prepared.
使用以上二氧化氯制剂分两次对增生性瘢痕进行治疗，一遍直接用该二氧化氯制剂，第二遍再加等量二甲基亚砜。即两遍都直接涂抹瘢痕处，中间间隔30min，连续涂抹15天。The hypertrophic scar was treated twice with the above chlorine dioxide preparation, the chlorine dioxide preparation was directly used once, and the same amount of dimethyl sulfoxide was added for the second time. That is, the scar is directly applied to the two times, with an interval of 30 minutes in between, and continuous application for 15 days.
治疗后即刻增生性瘢痕呈暗红色，瘢痕表面见均匀分布的灰色至微黄的点状气化皮屑，无明显渗血、渗液。治疗后第一天，创面干燥见红色点状痂皮形成，此后至治疗后第五天，治疗的创面呈鲜红色，创面痂皮逐渐脱落。至治疗后第二十天，治疗创面完全愈合，痂皮全部脱落，同时可观察到瘢痕的中央的乳头状突起高度变得低平，瘢痕处皮肤明显光滑一些，有色素沉着。治疗后25天左右，有光滑的新鲜皮肤生成，在40±20天左右的时间，原来疤痕不规则形状完全平坦。Immediately after treatment, the hypertrophic scar was dark red, and the surface of the scar was uniformly distributed with gray to yellowish punctate gasification dander, and there was no obvious bleeding or exudation. On the first day after treatment, the wound surface was dry and red punctate suede was formed. From then on to the fifth day after treatment, the treated wound surface was bright red, and the wound surface gradually fell off. On the twentieth day after the treatment, the treated wounds completely healed, and the suede all fell off. At the same time, the height of the papillary lobes in the center of the scar was observed to be low, and the skin at the scar was smooth and pigmented. About 25 days after the treatment, smooth fresh skin was formed, and the original irregular shape of the scar was completely flat at about 40±20 days.
取治疗前、治疗后1h、治疗后2天、7天、14天、28天瘢痕，切取疤痕处全层组织，切取组织经4％多聚甲醛固定后常规脱水、石蜡包埋，切片后行HE染色观察各组各时期镜下成纤维细胞形态、密度，胶原纤维排列、毛细血管增生情况，TUNEL(Terminal deoxynucleotidyl transferase dUTP nick end labeling)法检测成纤维细胞凋亡率变化。The scar was taken before treatment, 1 hour after treatment, 2 days, 7 days, 14 days, and 28 days after treatment. The whole layer of the scar was cut, and the tissue was removed by 4% paraformaldehyde, then dehydrated and embedded in paraffin. HE staining was used to observe the morphology, density, collagen fiber arrangement and capillary proliferation of microfibrous cells in each group. TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) method was used to detect the apoptosis rate of fibroblasts.
应率(R)＝阳性细胞数/细胞总数，结果取平均数。Using the TUNEL method, the stained area is located in the nucleus. Under normal light microscope, the nuclei of normal fibroblasts were blue, which was a negative reaction. The nucleus of apoptotic cells showed a dark brown color, which was a positive reaction, namely apoptotic cells. Under the observation of 40 times light microscope, each slice was randomly selected to have the highest number of positive cells with the highest number of fibroblast positive cells, and the positive cells were reversed.
Rate (R) = number of positive cells / total number of cells, and the results were averaged.
在成纤维细胞、角朊细胞、血管内皮细胞中均可见凋亡小体，即凋亡细胞。治疗后即刻即有明显的成纤维细胞凋亡增多表现，治疗后1h、2天、7天、14天、28天治疗组成纤维细胞凋亡率逐渐增高，各时期凋亡率均比治疗前高，P值<0.01，说明差异有统计学意义。Apoptotic bodies, ie apoptotic cells, are found in fibroblasts, keratinocytes, and vascular endothelial cells. Immediately after treatment, there was an obvious increase in fibroblast apoptosis. The apoptosis rate of fibroblasts was gradually increased at 1h, 2 days, 7 days, 14 days, and 28 days after treatment, and the apoptosis rate was higher than that before treatment. P value <0.01, indicating that the difference is statistically significant.
表4 瘢痕组织中TUNEL检测成纤维细胞凋亡率的变化Table 4 Changes of apoptotic rate of fibroblasts detected by TUNEL in scar tissue
*代表与治疗前相比P<0.01。* represents P < 0.01 compared with before treatment.
因此不管从体外培养，或是直接治疗，都显示本发明的酸性环境下的二氧化氯制剂对凋亡不足的组织可以诱导其中细胞加速凋亡，从而使皮肤健康并年轻化。Therefore, whether in vitro culture or direct treatment, it is shown that the chlorine dioxide preparation in the acidic environment of the present invention can induce apoptosis of the cells in which the cells are accelerated, thereby making the skin healthy and young.
实施例2：含二氧化氯制剂对皮肤抗衰老的美容试验Example 2: Cosmetic test for anti-aging of skin containing chlorine dioxide preparation
用于皮肤美容的含二氧化氯制剂的化妆品制备：用去离子水配置浓度为7.47％亚氯酸钠和1.59％氯化钠的混合溶液，制备出第一份溶液；用去离子水配置浓度为16.7％的柠檬酸溶液，制备出第二份溶液。分别从不同份溶液的容器中取出相同体积的溶液，混合，等待溶液混合静止3～5分钟，再用0.22μm的双层滤膜过滤。再加入等体积90％的二甲基亚砜，然后装入玻璃瓶密封保存，制成针对皮肤抗衰老或年轻化的化妆品，用于皮肤涂抹。Cosmetic preparation for chlorine-containing preparations for skin cosmetic: a mixed solution of 7.47% sodium chlorite and 1.59% sodium chloride is prepared with deionized water to prepare a first solution; concentration is set with deionized water A second solution was prepared as a 16.7% citric acid solution. The same volume of solution was taken from the containers of different solutions, mixed, and the solution was allowed to stand still for 3 to 5 minutes, and then filtered through a 0.22 μm double-layer filter. An equal volume of 90% dimethyl sulfoxide is added, and then sealed in a glass bottle to prepare an anti-aging or youthful cosmetic for the skin.
通过女性观察员(100名)实际涂抹到皮肤上使用，按照下述的评价区分，评价“黏腻使用感的有无”、“发涩感的有无”、“皮肤上的移动的有无”以及“对皱纹、松弛(皮肤的拉伸感)的效果的有无”。The female observer (100) was actually applied to the skin and evaluated according to the following evaluations. The evaluation of "the presence or absence of sticky feeling", "the presence or absence of the feeling of hairiness", and "the presence or absence of movement on the skin" And the presence or absence of the effect of wrinkles and slacks (skinning of the skin).
从结果看(表5)，本发明的二氧化氯制剂作为化妆品具备非常优异的美容护肤作用。From the results (Table 5), the chlorine dioxide preparation of the present invention has a very excellent cosmetic and skin care function as a cosmetic.
表5：皮肤美容使用效果观察Table 5: Observation of skin cosmetic use effect
采用本发明以上制备的化妆品为例进行疗效观察，在人群中进行功效测试，观察使用者在使用过程中的肌肤水分含量、弹性、细纹、粗糙度等改善情况。选取30名健康女性和10名健康男性，每天涂抹自由选择皮肤位置，共15天，在使用前、1h、7天(使用1h后)、第14天(使用1h后)、第28天对受试者进行皮肤状况的评估，包括临床判断及非创性仪器评估。临床评判包括以下参数：皮肤水分含量、皮肤弹性、皮肤光泽度、皮肤粗糙度/光滑度、肤色均匀度、细纹的改善。非创性仪器评估是利用皮肤水分测定仪Corneometer CM820测定皮肤水分含量，利用皮肤弹性测定仪(Cutometer SEM 575)测定皮肤弹性。实验的数据如表6所示。The cosmetic prepared by the above invention is taken as an example for observation of the therapeutic effect, and the efficacy test is carried out in the human population, and the improvement of the skin moisture content, elasticity, fine lines and roughness during the use of the user is observed. 30 healthy women and 10 healthy men were selected and applied freely to the skin for 15 days. Before use, 1h, 7 days (after 1h use), 14th day (after 1h use), and 28th day The tester performed an assessment of the condition of the skin, including clinical judgment and non-invasive instrument evaluation. Clinical evaluation included the following parameters: skin moisture content, skin elasticity, skin gloss, skin roughness/smoothness, skin tone uniformity, and fine lines. Non-invasive instrument evaluation was performed by measuring the skin moisture content using a skin moisture meter Corneometer CM820, and skin elasticity was measured using a skin elasticity meter (Cutometer SEM 575). The experimental data is shown in Table 6.
由表6可见：使用此化妆品后，皮肤含水量急剧增加，说明产品对改善皮肤干燥效果非常明显，随着时间的推进到第28天时，皮肤的含水量继续得到持续的改善。It can be seen from Table 6 that after using this cosmetic, the moisture content of the skin increases sharply, indicating that the product is very effective in improving the skin drying effect, and the water content of the skin continues to be improved continuously as time advances to the 28th day.
皮肤弹性值越大越好，说明皮肤弹性越好，由表6可看出7天后，皮肤的弹性得到了明显的改善，弹性变得越来越好并与基础值之间有显著差异性。The greater the skin elasticity value, the better, indicating that the skin elasticity is better. It can be seen from Table 6 that after 7 days, the elasticity of the skin is obviously improved, the elasticity becomes better and better, and there is a significant difference from the basic value.
光泽度等四个指标值，数据数值越少，说明改善越好。从上述数据结果可以看出，与基础值相比，各项参数在第7、14、28天均有改善，与基础值之间有显著差异性(表现在各项数值随时间推移的下降趋势)。Four indicators such as glossiness, the smaller the data value, the better the improvement. From the above data, it can be seen that compared with the basic values, the parameters are improved on the 7th, 14th and 28th days, and there is a significant difference from the basic values (expressed in the downward trend of each value over time). ).
表6 含二氧化氯化妆品使用后皮肤改善情况Table 6 Skin improvement after use of chlorine dioxide cosmetics
注：*与基础值相比，P＜0.01。Note: *P<0.01 compared to the base value.
Therefore, the chlorine dioxide-containing cosmetic of the present invention has a cosmetic effect of skin rejuvenation such as skin care, hydration, freckle, whitening, and wrinkles.
实施例3含二氧化氯制剂治疗痤疮的试验Example 3 Test for the treatment of acne with chlorine dioxide preparation
痤疮是青少年经常反复发作的皮肤问题，主要原因是老化角质层不能及时清除，堵塞毛孔，导致油脂堆积，形成肿胀(粉刺)；在痤疮杆菌的侵袭后感染，发红形成脓包。由于二氧化氯本身是高效的杀菌剂，所以只用保证老化角质细胞得到清除，那么二氧化氯就可以治疗痤疮。Acne is a frequent skin problem in adolescents. The main reason is that the aging cuticle cannot be removed in time, clogged pores, causing oil to accumulate and form swelling (acne). After infection by acne bacteria, redness forms pustules. Since chlorine dioxide itself is a highly effective fungicide, it is only used to ensure that aged keratinocytes are removed, so chlorine dioxide can treat acne.
用于祛痘(治疗痤疮)的含二氧化氯制剂的化妆品制备：用实施例2的制备方法，制成祛痘溶液，用于痤疮皮肤涂抹。Cosmetic preparation of chlorine dioxide-containing preparation for acne (treatment of acne): Using the preparation method of Example 2, an acne solution was prepared for acne skin application.
选择30位痤疮问题青年(男20，女10)，平均年龄22岁，每天用以上配置的含二氧化氯制剂涂抹痤疮患处一次，连续5天。具体效果如下表所示。Thirty young people with acne problems (male 20, female 10) were selected, with an average age of 22 years. The acne affected area was applied once daily for 5 days with the above-mentioned chlorine dioxide-containing preparation. The specific effects are shown in the table below.
表7 痤疮治疗试验Table 7 Acne Treatment Test
30人祛痘，连续用5天，90％痤疮痊愈。说明本发明的含二氧化氯制剂治疗痤疮非常有效。30 people with acne, for 5 days in a row, 90% of acne recovered. It is indicated that the chlorine dioxide-containing preparation of the present invention is very effective for treating acne.
实施例4含二氧化氯制剂通过静脉注射对小鼠寿命影响的试验Example 4 Test of the effect of intravenous injection of chlorine dioxide preparation on the lifespan of mice
二氧化氯制剂制备：用去离子水配置浓度为2.49％亚氯酸钠和0.53％氯化钠的混合溶液，制备出第一份溶液；用去离子水配置浓度为5.57％的柠檬酸溶液，制备出第二份溶液。分别从不同份溶液的容器中取出相同体积的溶液，混合，等待溶液混合静止5～10分钟，加入等量50％二甲基亚砜，再用0.22μm的双层滤膜过滤，马上装入注射器。制备一次，使用一次。Preparation of chlorine dioxide preparation: a mixed solution of 2.49% sodium chlorite and 0.53% sodium chloride was prepared with deionized water to prepare a first solution; a citric acid solution having a concentration of 5.57% was prepared with deionized water. A second solution was prepared. Take the same volume of solution from the different parts of the solution, mix, wait for the solution to mix and stand for 5 to 10 minutes, add an equal amount of 50% dimethyl sulfoxide, then filter with 0.22 μm double-layer filter, load immediately syringe. Prepare once and use once.
C57BL/6雄性小鼠，12月龄，每只小鼠单笼饲料。100只小鼠随机分为两组：实验组(50只)和对照组(50只)。两组均充足的饲料和饮水，实验组自15月龄后，每周一次，每次以10ml/kg的剂量通过静脉注射由以上方法制备的二氧化氯制剂。详细记录各组每个小鼠自然死亡的日期，并计算各组小鼠的平均寿命和最大寿命。C57BL/6 male mice, 12 months old, each cage was fed in a single cage. 100 mice were randomly divided into two groups: experimental group (50) and control group (50). Both groups had sufficient feed and drinking water. The experimental group was intravenously injected with the chlorine dioxide preparation prepared by the above method at a dose of 10 ml/kg once a week after 15 months of age. The dates of natural death of each mouse in each group were recorded in detail, and the average lifespan and maximum lifespan of each group of mice were calculated.
结果显示：对照组小鼠的平均寿命为696.5天，试验组小鼠的平均寿命为778.3天，每周注射一次的二氧化氯制剂使小鼠的平均寿命延长11％。说明，注射二氧化氯制剂明显延长小鼠平均寿命(如表8)。The results showed that the average lifespan of the control mice was 696.5 days, and the average lifespan of the mice in the test group was 778.3 days. The weekly chlorine dioxide preparation extended the average lifespan of the mice by 11%. This indicates that the injection of chlorine dioxide preparation significantly prolonged the average lifespan of mice (see Table 8).
结果显示：对照组小鼠的最大寿命是932天，试验组小鼠的最大寿命是987.5天，注射二氧化氯制剂使小鼠的最大寿命延长5.9％，说明：注射二氧化氯制剂具有明显延长小鼠的最大寿命的作用。According to the currently used maximum life calculation method, the average life expectancy of 10% of the mice that died naturally in the group is the maximum life expectancy.
The results showed that the maximum lifespan of the control mice was 932 days, and the maximum lifespan of the mice in the test group was 987.5 days. The injection of chlorine dioxide preparation extended the maximum lifespan of the mice by 5.9%, indicating that the injection of chlorine dioxide was significantly prolonged. The role of the maximum lifespan of mice.
表8 注射二氧化氯制剂对小鼠寿命的影响Table 8 Effect of injection of chlorine dioxide on the lifespan of mice
注：*与对照组相比P＜0.05Note: *P<0.05 compared with the control group
在小鼠月龄19月的时间，从实验组和对照组分别随机选择10只小鼠，进行水迷宫和平衡耐力测试。两测试时间间隔2周。At the time of the mouse's age of 19 months, 10 mice were randomly selected from the experimental group and the control group, respectively, and subjected to a water maze and a balanced endurance test. The two test intervals are 2 weeks.
水迷宫试验：实验进行前连续训练3天,2次/天。方形水迷宫装置为50cm×30cm×15cm的黑色木槽、内设起步区、4盲端曲折回路、安全台。水深控制在12cm,水温为(25±2)℃。小鼠由入口端放入开始计时,自动记录触及盲端的次数,以小鼠爬上安全台为结束时间,称为潜伏期。潜伏期设定最大为120s,筛除超过120s仍找不到出口的动物。Water maze test: continuous training for 3 days, 2 times / day before the experiment. The square water labyrinth device is a black wooden trough 50cm×30cm×15cm, a starting zone, a 4-blind end meandering loop, and a safety platform. The water depth is controlled at 12 cm and the water temperature is (25 ± 2) °C. The mouse is placed from the entrance end to start timing, and the number of times the blind end is touched is automatically recorded. The mouse is climbed up to the safety station as the end time, which is called the incubation period. The incubation period is set to a maximum of 120s, and animals that cannot be exported can be found after screening for more than 120s.
平衡耐力测试：实验进行前连续训练3天，2次/天。在两根固定柱子之间拉一根直径为2mm的铁丝，距离为2m,高度为1m。用绳子捆绑下肢,使小鼠用上肢握紧铁丝，用秒表计算开始握紧到摔下的时间。观察时间设定为30s，筛除30s内摔下的动物。Balanced endurance test: Continuous training for 3 days, 2 times/day before the experiment. A wire with a diameter of 2 mm is pulled between the two fixed columns at a distance of 2 m and a height of 1 m. Bundle the lower limbs with a rope, hold the mouse tightly with the upper limbs, and use a stopwatch to calculate the time to start gripping to the fall. The observation time was set to 30 s, and the animals that fell within 30 s were screened out.
由表9可见，各组动物第1天记忆力无差异；实验第3天、6天和9天,试验组的错误次数及潜伏期明显少于对照组。可见二氧化氯制剂显著提高了小鼠的学习能力和记忆能力。It can be seen from Table 9 that there is no difference in memory on the first day of each group of animals; on the 3rd, 6th and 9th day of the experiment, the number of errors and latency of the test group were significantly less than that of the control group. It can be seen that the chlorine dioxide preparation significantly improves the learning ability and memory ability of the mouse.
表9 各组小鼠在不同时间内记忆力的比较Table 9 Comparison of memory in different groups of mice at different times
Note: *P<0.05 compared with the control group
由表10可见，试验组小鼠在1天、3天、6天和9天的平衡耐力显著高于对照组。说明二氧化氯制剂显著提高了小鼠的机体能力。As can be seen from Table 10, the balance endurance of the test group mice at 1 day, 3 days, 6 days, and 9 days was significantly higher than that of the control group. It is indicated that the chlorine dioxide preparation significantly improves the body capacity of the mouse.
表10 各组小鼠在不同时间内平衡耐力的比较Table 10 Comparison of balanced endurance in different groups of mice at different times
注：*与对照组相比，P＜0.05Note: *P<0.05 compared with the control group
从以上多个实施例的试验数据，我们可以得出，酸性环境下的二氧化氯制剂能够显著提高动物的寿命，并伴随提高记忆力和机体能力，其机理应该是二氧化氯制剂能够诱导衰老细胞发生凋亡从而衰老细胞得以被清除，机体自身的再生能力将恢复年轻化。因此本发明所述的二氧化氯制剂可以被制成化妆品用于皮肤年轻化的美容，也可以制成药品用于使机体年轻化，从而预防老年性疾病的发生。From the experimental data of the above various examples, we can conclude that the chlorine dioxide preparation in an acidic environment can significantly improve the lifespan of the animal, accompanied by the improvement of memory and body capacity, the mechanism should be that the chlorine dioxide preparation can induce senescent cells. Apoptosis occurs and aging cells are cleared, and the body's own ability to regenerate will rejuvenate. Therefore, the chlorine dioxide preparation of the present invention can be used as a cosmetic for the skin rejuvenation, or can be made into a medicine for rejuvenating the body, thereby preventing the occurrence of senile diseases.
实施例5二氧化氯制剂对人非小细胞肺癌细胞A549的杀伤作用。Example 5 The killing effect of chlorine dioxide preparation on human non-small cell lung cancer cell A549.
用去离子水配置浓度为7.47％亚氯酸钠和1.59％氯化钠的混合溶液，制备出第一份溶液；用去离子水配置浓度为16.7％的柠檬酸溶液，制备出第二份溶液。分别从不同份溶液的容器中取出相同体积的溶液，混合，等待溶液混合静止3～5分钟，再用0.22μm的双层滤膜过滤，用去离子水稀释，制备不同浓度的二氧化氯溶液。The first solution was prepared by dissolving a mixed solution of 7.47% sodium chlorite and 1.59% sodium chloride in deionized water; a citric acid solution having a concentration of 16.7% was prepared with deionized water to prepare a second solution. . Remove the same volume of solution from the different parts of the container, mix, wait for the solution to mix and stand for 3 to 5 minutes, then filter with 0.22μm double-layer filter, dilute with deionized water to prepare different concentrations of chlorine dioxide solution. .
1)实验方法1) Experimental method
用含10％胎牛血清的PRMI1640培养液，并置37℃、5％CO2培养箱中培养人非小细胞肺癌细胞A549细胞，每3天用0.25％胰蛋白酶液消化细胞进行传代，并更换培养液。按0.7×104/孔浓度接种于96孔板上，置37℃、5％CO2培养箱中培养24h后，分别加入二氧化氯溶液，每种二氧化氯溶液按照终浓度100ppm、200ppm、400ppm、700ppm、1000ppm、1500ppm、2000ppm、2900ppm加入，阴性对照组细胞加入0.1％(v/v)的DMSO(二甲基亚砜)，另设调零组(即细胞培养孔中没有细胞，只加入细胞培养液)。每个组设四个复孔，然后将96孔板置37℃、5％CO2培养箱再培养24h，然后按下述方法检测化合物对肿瘤细胞的杀伤作用。The human non-small cell lung cancer cell A549 cells were cultured in a culture medium containing 10% fetal bovine serum with PRMI1640 and placed in a 37 ° C, 5% CO 2 incubator. The cells were digested with 0.25% trypsin solution every 3 days, and replaced. liquid. Inoculate 96-well plates at a concentration of 0.7×10 4 /well, and incubate at 37 ° C, 5% CO 2 incubator for 24 h, then add chlorine dioxide solution, each chlorine dioxide solution according to the final concentration of 100ppm, 200ppm, 400ppm , 700ppm, 1000ppm, 1500ppm, 2000ppm, 2900ppm were added, the negative control cells were added with 0.1% (v/v) DMSO (dimethyl sulfoxide), and the other group was adjusted to zero (ie, there were no cells in the cell culture well, only added Cell culture medium). Four replicate wells were set in each group, and then 96-well plates were placed in a 37 ° C, 5% CO 2 incubator for another 24 h, and then the killing effect of the compounds on tumor cells was examined as follows.
2) Detection method:
通过乳酸脱氢酶(LDH)释放实验，采用乳酸脱氢酶检测试剂盒检测肿瘤细胞死亡率，具体操作方法按照所述乳酸脱氢酶检测试剂盒的说明书进行。用检测缓冲液(Assay Buffer)重悬反应底物(Substrate Mix)。取96孔细胞培养板中每个组的3复孔的培养上清液50μl到一个新的96孔板中，各组剩下的一个复孔加入终浓度为0.9％(V/V)的Triton-x100(试剂盒提供)，并置于37℃孵箱中50分钟以裂解细胞后，取50μl上清液加入新的96孔板中，然后在新的96孔板中每孔再加入等体积的重悬好的反应底物液，室温孵育30分钟后，每孔加入50μl终止液即1M的乙酸(试剂盒中提供)终止反应，用酶标仪(490nm波长)测每孔的OD值，裂解细胞孔所检测的OD值记为“细胞最大释放OD值”，阴性对照组的OD值记为“自然释放对照组OD值”。The lactate dehydrogenase (LDH) release test is used to detect tumor cell death rate using a lactate dehydrogenase detection kit, and the specific method is carried out according to the instructions of the lactate dehydrogenase detection kit. Resuspend the reaction substrate (Substrate Mix) with Assay Buffer. 50 μl of the culture supernatant of 3 replicate wells of each group in a 96-well cell culture plate was plated into a new 96-well plate, and the remaining one of each group was added to a Triton with a final concentration of 0.9% (v/v). -x100 (provided in the kit) and placed in a 37 ° C incubator for 50 minutes to lyse the cells, 50 μl of the supernatant was added to the new 96-well plate, and then an equal volume was added to each well in the new 96-well plate. Resuspend the reaction substrate solution, incubate for 30 minutes at room temperature, add 50 μl of stop solution per well, ie 1 M acetic acid (provided in the kit) to stop the reaction, and measure the OD value of each well with a microplate reader (490 nm wavelength). The OD value detected by lysing the cell well was recorded as "the maximum release OD value of the cells", and the OD value of the negative control group was recorded as "the OD value of the natural release control group".
根据检测的OD490值，按照下列公式计算细胞死亡率，结果以均数±标准差表示，并且采用SPSS软件Probit模块计算半数杀细胞浓度，即IC50。Based on the detected OD490 value, the cell death rate was calculated according to the following formula, and the results were expressed as mean ± standard deviation, and the half-cell killing concentration, i.e., IC 50 , was calculated using the SPSS software Probit module.
肿瘤细胞死亡率(％)＝(实验组OD值-自然释放对照组OD值)/(细胞最大释放OD值-自然释放对照组OD值)Tumor cell mortality (%) = (experimental group OD value - natural release control OD value) / (cell maximum release OD value - natural release control OD value)
3)检测结果：3) Test results:
如表10所示，二氧化氯制剂可有效杀死A549细胞，且随着浓度的增加，它们对A549的杀伤作用也剂量依赖性地增加，通过SPSS软件Probit模块计算，二氧化氯制剂导致半数A549细胞死亡的有效剂量(IC50)为495ppm。As shown in Table 10, the chlorine dioxide preparation can effectively kill A549 cells, and their killing effect on A549 increases dose-dependently as the concentration increases, and the chlorine dioxide preparation results in half of the calculation by the SPSS software Probit module. effective dose (IC 50) A549 cell death is 495ppm.
表10 A549细胞的体外杀伤Table 10 In vitro killing of A549 cells
本实施例证实，酸性二氧化氯制剂能够显著地杀死A549肿瘤细胞。This example demonstrates that an acidic chlorine dioxide formulation is capable of significantly killing A549 tumor cells.
实施例6二氧化氯制剂对人宫颈癌细胞HeLa的杀伤作用Example 6 killing effect of chlorine dioxide preparation on human cervical cancer cell line HeLa
按照实施例5的方法制备二氧化氯制剂，按实施例5的细胞培养方法将人宫颈癌细胞HeLa细胞按保种培养并接种于96孔培养板中。按实施例5中的方法设实验组、对照组和调零组，并通过乳酸脱氢酶(LDH)释放实验，检测二氧化氯溶剂所导致的HeLa细胞死亡率，并计算导致半数HeLa细胞死亡的剂量(IC50)。A chlorine dioxide preparation was prepared according to the method of Example 5, and human cervical cancer HeLa cells were cultured in a cell culture method according to Example 5 and seeded in a 96-well culture plate. The experimental group, the control group and the zeroing group were set according to the method in Example 5, and the death rate of HeLa cells caused by the chlorine dioxide solvent was detected by the lactate dehydrogenase (LDH) release test, and the death of half of HeLa cells was calculated. Dosage (IC 50 ).
增加，它们对HeLa的杀伤作用是剂量依赖性地增加，通过SPSS软件Probit模块计算，二氧化氯制剂导致半数HeLa细胞死亡的有效剂量为405ppm。Test results: As shown in Table 11, the chlorine dioxide preparation can effectively kill HeLa cells, and with the concentration of the compound
Increasingly, their killing effect on HeLa was dose-dependently increased, and the effective dose of chlorine dioxide preparation resulting in the death of half of HeLa cells was 405 ppm as calculated by the SPSS software Probit module.
表11：二氧化氯制剂对HeLa细胞的体外杀伤Table 11: In vitro killing of HeLa cells by chlorine dioxide preparation
实施例7：采用乳酸脱氢酶释放实验，检测二氧化氯制剂对多种人类肿瘤细胞，包括乳腺癌、卵巢癌、肝癌、鼻咽癌、胃癌、喉癌、胰腺癌、黑色素瘤、膀胱癌和白血病细胞的杀伤作用。Example 7: Using a lactate dehydrogenase release assay to detect chlorine dioxide preparations against a variety of human tumor cells, including breast, ovarian, liver, nasopharyngeal, gastric, laryngeal, pancreatic, melanoma, bladder cancer And the killing effect of leukemia cells.
按照实施例5的方法制备二氧化氯制剂。按实施例5中的方法保种培养乳腺癌细胞(MCF-7)、卵巢癌细胞(SKOV3)、肝癌细胞(Bel-7402)、鼻咽癌细胞(HNE)、胃癌细胞(MKN-45)、喉癌细胞(Hep-2)、胰腺癌细胞(Pan-1)、黑色素瘤细胞(A375)、膀胱癌细胞(Biu-87)、白血病细胞(Jurkat)同样采用含10％胎牛血清PRMI1640、置于37℃、5％CO2培养箱中培养，并按常规悬浮细胞培养方式进行传代，即在传代时先做离心处理去除旧培养液，然后添加新培养液。然后按实施例5方法设实验组、对照组和调零组，并通过乳酸脱氢酶(LDH)释放实验检测肿瘤细胞死亡率，并计算导致半数肿瘤细胞死亡的剂量(IC50)。A chlorine dioxide preparation was prepared in accordance with the method of Example 5. Breast cancer cells (MCF-7), ovarian cancer cells (SKOV3), liver cancer cells (Bel-7402), nasopharyngeal carcinoma cells (HNE), gastric cancer cells (MKN-45) were preserved as in Example 5. Laryngeal carcinoma cells (Hep-2), pancreatic cancer cells (Pan-1), melanoma cells (A375), bladder cancer cells (Biu-87), and leukemia cells (Jurkat) are also treated with 10% fetal bovine serum PRMI1640. The cells were cultured in a 37 ° C, 5% CO 2 incubator and passaged in a conventional suspension cell culture mode, that is, centrifugation was performed to remove the old culture solution, and then a new culture solution was added. Then, the experimental group, the control group, and the zeroing group were set according to the method of Example 5, and the tumor cell death rate was measured by a lactate dehydrogenase (LDH) release test, and the dose (IC 50 ) which caused half of the tumor cell death was calculated.
检测结果：如表12所示，二氧化氯制剂可有效杀死各种肿瘤细胞，且随着二氧化氯浓度的增加，它们对各肿瘤的杀伤作用也剂量依赖性地增加。As a result, as shown in Table 12, the chlorine dioxide preparation was effective in killing various tumor cells, and as the concentration of chlorine dioxide increased, their killing effect on each tumor also increased in a dose-dependent manner.
说明，本发明提供的酸性二氧化氯制剂对于肿瘤细胞具有广谱的杀伤作用。It is indicated that the acidic chlorine dioxide preparation provided by the present invention has a broad spectrum of killing effect on tumor cells.
表12 多种人肿瘤细胞在不同二氧化氯浓度下的死亡率(％)Table 12 Mortality of various human tumor cells at different concentrations of chlorine dioxide (%)
实施例8二氧化氯制剂诱导肿瘤细胞凋亡的试验Example 8 Test for Inducing Apoptosis of Tumor Cells by Chlorine Dioxide Formulation
对结肠癌细胞系LS174T和乳腺癌细胞系Cama-1这两个细胞系的死亡和细胞周期变化进行了分析。分别在不存在二氧化氯或存在100ppm二氧化氯(通过实施例5中的方法制备)的条件下，培养细胞48小时。用1μg/ml溴脱氧尿苷(BrdU)脉冲30分钟后，细胞在70％乙醇中于4℃固定过夜，然后用FITC偶联的抗BrdU单克隆抗体和3μg/ml碘化丙锭染色。通过流式细胞术(FACS)分析细胞死亡和细胞周期。掺入BrdU是一种增殖测量方法，而碘化丙锭染色则可确定DNA含量，特别是正在经历细胞凋亡的近二倍体细胞群体。The death and cell cycle changes of the colon cancer cell line LS174T and the breast cancer cell line Cama-1 were analyzed. The cells were cultured for 48 hours in the absence of chlorine dioxide or in the presence of 100 ppm chlorine dioxide (prepared by the method of Example 5). After 30 minutes of pulsed with 1 μg/ml bromodeoxyuridine (BrdU), the cells were fixed overnight in 70% ethanol at 4 ° C, and then stained with FITC-conjugated anti-BrdU monoclonal antibody and 3 μg/ml propidium iodide. Cell death and cell cycle were analyzed by flow cytometry (FACS). Incorporation of BrdU is a method of proliferation measurement, while propidium iodide staining determines the DNA content, particularly the population of near diploid cells that are undergoing apoptosis.
数据显示，掺入BrdU的LS174T细胞的百分比从处理前的27％变为100ppm二氧化氯制剂存在下培养48小时后的6％。相反，具有近二倍体DNA含量的LS174T细胞的百分比，从处理前的4％变为100ppm二氧化氯制剂存在下培养48小时后的23％，说明100ppm二氧化氯制剂的细胞凋亡促进能力强。The data showed that the percentage of BrdU-incorporated LS174T cells was changed from 27% before treatment to 6% after 48 hours of culture in the presence of 100 ppm chlorine dioxide preparation. In contrast, the percentage of LS174T cells with near diploid DNA content was changed from 4% before treatment to 23% after 48 hours of culture in the presence of 100 ppm chlorine dioxide preparation, indicating the ability of apoptosis promotion of 100 ppm chlorine dioxide preparation. Strong.
数据还显示，掺入BrdU的Cama-1细胞的百分比，由处理前的15％变为100ppm二氧化氯制剂存在下培养48小时后的2％。相反，具有近二倍体DNA含量的Cama-1细胞的百分比，从处理前的4％变为100ppm二氧化氯制剂存在下培养48小时后的17％，表明100ppm二氧化氯制剂引发细胞凋亡。The data also showed that the percentage of Cmad-1 cells spiked with BrdU was changed from 15% before treatment to 2% after 48 hours of incubation in the presence of 100 ppm chlorine dioxide preparation. In contrast, the percentage of Cama-1 cells with near diploid DNA content was changed from 4% before treatment to 17% after 48 hours of culture in the presence of 100 ppm chlorine dioxide preparation, indicating that 100 ppm chlorine dioxide preparation triggered apoptosis. .
这些数据表明，用100ppm二氧化氯制剂处理48小时，LS 174T和Cama-1细胞系停止分裂，并经历细胞凋亡。These data indicate that the LS 174T and Cama-1 cell lines stopped dividing and were subjected to apoptosis after treatment with a 100 ppm chlorine dioxide formulation for 48 hours.
实施例9不同pH值情况下的二氧化氯制剂对肿瘤细胞的凋亡影响Example 9 Effect of Chlorine Dioxide Formulation on Apoptosis of Tumor Cells at Different pH Values
膜联蛋白V染色对细胞死亡进行了分析。存在或不存在400ppm二氧化氯制剂(通过实施例5中的方法制备)时培养细胞24小时。通过膜联蛋白V染色和流式细胞术检测细胞凋亡。数据显示，超过70％的Cama-1细胞被膜联蛋白V染色，进一步证明400ppm二氧化氯制剂诱导细胞凋亡。其他条件不变，但将400ppm二氧化氯制剂通过pH调节剂，使其pH值由3.5提高值5.5，则只有超过50％的Cama-1细胞被膜联蛋白V染色，这说明酸性环境有助于提高癌细胞凋亡率。To further investigate the effects of chlorine dioxide preparation on breast tumor cell lines, in Cama-1 cells,
Cell death was analyzed by annexin V staining. Cells were cultured for 24 hours with or without the presence of a 400 ppm chlorine dioxide formulation (prepared by the method of Example 5). Apoptosis was detected by annexin V staining and flow cytometry. The data showed that more than 70% of Cama-1 cells were stained with annexin V, further demonstrating that 400 ppm chlorine dioxide preparation induced apoptosis. Other conditions remain the same, but the 400 ppm chlorine dioxide preparation is passed through a pH adjuster to increase the pH from 3.5 to 5.5, and only over 50% of Cama-1 cells are stained with annexin V, indicating that the acidic environment contributes Increase the rate of cancer cell apoptosis.
我们还试图测定二氧化氯诱导细胞凋亡的动力学。500ppm二氧化氯制剂(通过实施例5中的方法制备)存在或不存在时，pH值为3.5，培养Cama-1细胞。30小时后，检测培养物中凋亡细胞(膜联蛋白阳性细胞)的百分比。数据显示，30小时后，未处理细胞显示15％的自发性细胞凋亡。然而，80％用二氧化氯制剂处理的细胞显示细胞死亡。具体地讲，Cama-1细胞中，加入500ppm二氧化氯制剂后9小时便开始二氧化氯引发的细胞凋亡，处理30小时后，凋亡细胞达到80％。其他条件不变，但将500ppm二氧化氯制剂通过pH调节剂使其pH值由3.0提高值6，则只有超过45％的细胞显示死亡，这说明酸性环境有助于提高癌细胞凋亡率。We also attempted to determine the kinetics of chlorine dioxide-induced apoptosis. Cama-1 cells were cultured in the presence or absence of a 500 ppm chlorine dioxide preparation (prepared by the method of Example 5) at a pH of 3.5. After 30 hours, the percentage of apoptotic cells (Annexin positive cells) in the culture was examined. The data showed that after 30 hours, untreated cells showed 15% spontaneous apoptosis. However, 80% of cells treated with the chlorine dioxide formulation showed cell death. Specifically, in Cama-1 cells, chlorine dioxide-induced apoptosis was started 9 hours after the addition of 500 ppm of chlorine dioxide preparation, and after 30 hours of treatment, the apoptotic cells reached 80%. Other conditions were the same, but the 500 ppm chlorine dioxide preparation was adjusted to a pH of 3.0 by a pH adjuster, and only over 45% of the cells showed death, indicating that the acidic environment contributes to an increase in cancer cell apoptosis.
我们于是试图测定二氧化氯制剂对人原代乳腺肿瘤细胞的作用。刚恢复的肿瘤单细胞悬液与PBS或二氧化氯(100ppm)制剂孵育48小时。通过PI染色测定细胞凋亡。百分比表示含有低含量DNA的细胞(subG0/G1细胞)的比例，即凋亡细胞的比例。数据显示，19.5％用PBS处理的细胞具有低含量的DNA，而38.6％用二氧化氯制剂处理的细胞具有低含量的DNA。因此，观察到相似的100ppm二氧化氯制剂对人乳腺原代肿瘤细胞的细胞凋亡作用。其他条件不变，但将100ppm二氧化氯制剂通过pH调节剂，使其pH值由4.5提高值6，则只有25％用二氧化氯制剂处理的细胞具有低含量的DNA。，这说明酸性环境有助于提高癌细胞凋亡率。We then attempted to determine the effect of chlorine dioxide preparations on human primary breast tumor cells. The newly recovered tumor single cell suspension was incubated with PBS or chlorine dioxide (100 ppm) formulation for 48 hours. Apoptosis was determined by PI staining. The percentage indicates the proportion of cells (subG0/G1 cells) containing a low amount of DNA, that is, the proportion of apoptotic cells. The data shows that 19.5% of cells treated with PBS have low levels of DNA, while 38.6% of cells treated with chlorine dioxide preparations have low levels of DNA. Thus, a similar 100 ppm chlorine dioxide formulation was observed to have an apoptotic effect on human breast primary tumor cells. Other conditions were unchanged, but the 100 ppm chlorine dioxide formulation was passed through a pH adjuster to increase the pH from 4.5 to 6, and only 25% of the cells treated with the chlorine dioxide formulation had low levels of DNA. This indicates that the acidic environment helps to increase the apoptosis rate of cancer cells.
结合以上肿瘤体外杀伤试验，本领域技术人员可以判断出，酸性二氧化氯制剂对肿瘤细胞具有明显的杀伤作用，而且这种作用机制是来自于酸性二氧化氯制剂可以显著诱导肿瘤细胞发生凋亡。Combined with the above tumor in vitro killing test, those skilled in the art can judge that the acidic chlorine dioxide preparation has obvious killing effect on tumor cells, and the mechanism of action is that the acidic chlorine dioxide preparation can significantly induce apoptosis of tumor cells. .
实施例10二氧化氯直接对S180荷瘤小鼠抑瘤效果及免疫器官指数的影响Example 10 Effect of Chlorine Dioxide Directly on Tumor Suppressive Effect and Immune Organ Index in S180 Tumor-bearing Mice
用去离子水配置浓度为7.47％亚氯酸钠和1.59％氯化钠的混合溶液，制备出第一份溶液；用去离子水配置浓度为16.7％的柠檬酸溶液，制备出第二份溶液。分别从不同份溶液的容器中取出相同体积的溶液，混合，等待溶液混合静止5～10分钟，再加入等体积90％的二甲基亚砜，再用0.22μm的双层滤膜过滤。定量分别制备出pH＝5和pH＝3.5的100ppm、500ppm和2000ppm的二氧化氯制剂。The first solution was prepared by dissolving a mixed solution of 7.47% sodium chlorite and 1.59% sodium chloride in deionized water; a citric acid solution having a concentration of 16.7% was prepared with deionized water to prepare a second solution. . The same volume of solution was taken from the containers of different solutions, mixed, and the solution was allowed to stand still for 5 to 10 minutes, then an equal volume of 90% dimethyl sulfoxide was added, and then filtered through a 0.22 μm double-layer filter. 100 ppm, 500 ppm, and 2000 ppm chlorine dioxide formulations of pH = 5 and pH = 3.5 were prepared separately.
二氧化氯低、中、高剂量组(pH恒定为5)，二氧化氯低剂量(pH＝3.5)组、中剂量(pH＝3.5)组、高剂量(pH＝3.5)组。环磷酰胺ip给药剂量为20mg/kg，二氧化氯瘤内注射低、中、高剂量分别为100ppm、500ppm和2000ppm，空白对照组给予生理盐水。各组小鼠接种S180实体瘤24h后给药，容积为0.1ml/10g，1次/d，连续14d。于停药5日处死小鼠，剥离瘤块及脾和胸腺并称重，计算抑瘤率及脾和胸腺指数。Kunming mice were randomly divided into 8 groups, 15 in each group, male and female, respectively: blank control group, cyclophosphamide group,
Low, medium and high doses of chlorine dioxide (pH is constant at 5), low dose chlorine dioxide (pH = 3.5), medium dose (pH = 3.5), high dose (pH = 3.5). The dose of Cyclophosphamide ip was 20 mg/kg, and the low, medium and high doses of chloramphenicol injection were 100 ppm, 500 ppm and 2000 ppm, respectively. The blank control group was given normal saline. Each group of mice was inoculated with S180 solid tumors for 24 hours, and the volume was 0.1 ml/10 g once daily for 14 days. The mice were sacrificed on the 5th day of withdrawal, the tumor mass and the spleen and thymus were removed and weighed, and the tumor inhibition rate and spleen and thymus index were calculated.
由表1可以看出，与空白对照组相比，二氧化氯制剂(高pH值)低、中、高剂量组，环磷酰胺化疗组，以及二氧化氯制剂(低pH值)低、中、高剂量组均可显著抑制S180肿瘤的生长(P<0.05、P<0.01)。与高pH值组相比，二氧化氯制剂低pH值组均可显著提高二氧化氯制剂对S180肿瘤生长的抑制作用(P<0.05)。As can be seen from Table 1, compared with the blank control group, the chlorine dioxide preparation (high pH) low, medium and high dose groups, cyclophosphamide chemotherapy group, and chlorine dioxide preparation (low pH) are low, medium High-dose group significantly inhibited the growth of S180 tumors (P<0.05, P<0.01). Compared with the high pH group, the low pH group of chlorine dioxide preparation can significantly increase the inhibitory effect of chlorine dioxide preparation on S180 tumor growth (P<0.05).
表13 二氧化氯制剂对S180荷瘤小鼠的抑瘤作用Table 13 Anti-tumor effect of chlorine dioxide preparation on S180 tumor-bearing mice
注：*P<0.05，**P<0.01与空白对照组比；#P<0.05，##P<0.01与高pH值组比Note: *P<0.05, **P<0.01 vs. blank control group; #P<0.05, ##P<0.01 vs. high pH group
由表14可以看出，与空白对照组相比，环磷酰胺化疗组小鼠的胸腺、脾脏指数显著性降低(P<0.01)，说明化疗手段的副作用很大。二氧化氯制剂中、高剂量组小鼠的胸腺和脾脏指数都显著性增高(P<0.05)。针对高低pH值，二氧化氯的影响不显著。说明二氧化氯促进肿瘤凋亡过程中对免疫器官的副作用微乎其微。As can be seen from Table 14, compared with the blank control group, the thymus and spleen index of the mice in the cyclophosphamide chemotherapy group were significantly decreased (P<0.01), indicating that the side effects of chemotherapy were great. The thymus and spleen index of the mice in the high-dose group of chlorine dioxide were significantly increased (P<0.05). For high and low pH values, the effect of chlorine dioxide is not significant. It shows that the side effects of immune dioxide on the process of apoptosis induced by chlorine dioxide are minimal.
表14 二氧化氯制剂对免疫器官指数的影响Table 14 Effect of chlorine dioxide preparation on immune organ index
注：*P<0.05，**P<0.01与空白对照组比。Note: *P<0.05, **P<0.01 compared with the blank control group.
It is to be noted that the acidic chlorine dioxide preparation provided by the present invention has obvious apoptosis-inducing effect on tumor cells, and has almost no side effects on the body.