WO2024087356A1 - Use of mesenchymal stem cells in preparation of drugs for treating persistent hpv infection - Google Patents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Definitions
- the present invention relates to the field of pharmaceutical technology, and in particular to the use of mesenchymal stem cells in the preparation of a medicine for treating persistent HPV infection.
- HPV Human papillomavirus
- HPV infection is closely related to the occurrence and development of precancerous lesions in the cervix and cervical cancer ( Figure 1).
- High-risk HPV is closely related to the occurrence of cervical cancer. Nearly 99% of cervical cancers are related to HPV infection.
- HPV can be divided into low-risk and high-risk types (HR-HPV) according to its ability to transform cells.
- Low-risk types mainly cause a variety of benign skin and mucosal papilloma or warts in humans, such as exophytic condyloma-like lesions, flat condyloma-like lesions, and low-grade cervical intraepithelial neoplasia (CIN) grade 1 lesions in the perianal skin of the genital tract and the lower part of the vagina, and skin warts at the friction sites of children and adolescents.
- High-risk types are related to the occurrence of cancer, especially cervical cancer, and mainly lead to CIN2, CIN3 grade lesions and cervical cancer.
- HR-HPV Human papillomavirus
- HPV-16 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68.
- 99% of cervical cancer is caused by HR-HPV infection (de Martel, C., et al., Worldwide burden of cancer attributable to HPV by site, country and HPV type. Int J Cancer, 2017. 141(4): p. 664-670).
- HPV16 and HPV18 are considered to be the most common types associated with cervical cancer worldwide, accounting for more than 70% of the total cases (Zeng, Z., et al., Prevalence and Genotype Distribution of HPV Infection in China: Analysis of 51,345 HPV Genotyping Results from China's Largest CAP Certified Laboratory. J Cancer, 2016. 7(9): p.1037-43).
- cervical precancerous lesion CIN From HPV infection to cervical cancer, there is a process called cervical precancerous lesion CIN. In the CIN state, cervical cells are in an immortalized state. When there are synergistic factors acting together, cervical cancer will occur.
- Persistent infection with high-risk human papillomavirus (HR-HPV) can increase the risk of high-grade cervical epithelial lesions by 250 times (Dalstein, V., et al., Persistence and load of high-risk HPV are predictors for development of high-grade cervical lesions: a longitudinal French cohort study. Int J Cancer, 2003. 106 (3): p. 396-403). It takes about 10-20 years for persistent HR-HPV infection to progress to cervical cancer. Therefore, effective control of HPV infection, especially elimination of persistent HR-HPV infection, will greatly reduce the incidence of cervical precancerous lesions and cervical cancer.
- HPV vaccine is the most effective way to prevent cervical cancer by preventing HPV infection and reducing the risk of cervical cancer and precancerous lesions caused by HPV.
- HPV vaccine has no therapeutic effect on people who are already infected with HPV virus, and there is currently no effective strategy for treating HPV infection in clinical practice.
- cervical cancer treatment vaccines such as VGX-3100 therapeutic vaccine
- they have not yet been widely used, and their efficacy needs further observation.
- Cervical precancerous lesions caused by HPV infection can be treated with cold knife cone biopsy, cryotherapy or loop electrosurgical excision procedure (LEEP). If it progresses to cervical cancer, surgery or radiotherapy and chemotherapy can be performed. Close follow-up is required after treatment, and related diseases should be treated again if they recur.
- LEEP loop electrosurgical excision procedure
- the present invention surprisingly found that mesenchymal stem cells have a significant effect in treating persistent HPV infection.
- a single application can turn patients with persistent HPV infection negative and improve the patient's reproductive system circulation. It has significant efficacy, is safe and reliable, and has important application value in the development of clinical drugs for the treatment of persistent HPV infection.
- the present invention provides an application of mesenchymal stem cells in preparing a medicine for treating HPV infection.
- the persistent HPV infection includes persistent high-risk HPV infection.
- the HPV persistent infection includes HPV16 and/or HPV18 persistent infection.
- said treating persistent HPV infection comprises improving estrogen levels.
- the mesenchymal stem cells are selected from human umbilical cord mesenchymal stem cells.
- Clinical-grade human umbilical cord mesenchymal stem cells are prepared in a GMP laboratory environment and are safer to be injected into the human body, especially intravenously.
- the content of mesenchymal stem cells in the drug is not less than 106 cells/mL; further preferably, the content of mesenchymal stem cells in the drug is 106-107 cells/mL or more than 6000w.
- the drug is an intravenous injection.
- a drug for treating persistent HPV infection comprising mesenchymal stem cells at a content of not less than 106 cells/mL is prepared, and the drug is an intravenous injection.
- an intravenous injection containing 106-107/mL mesenchymal stem cells is injected into the patient's body. After one administration, the patient is re-examined 3-6 months later. The patient with persistent HPV infection for more than one year can stably maintain HPV negativity and the patient's reproductive system circulation can be improved.
- the present invention has at least the following beneficial effects:
- MSCs mesenchymal stem cells
- the present invention surprisingly found that MSCs have a significant anti-HPV virus inhibitory effect.
- the negative conversion rate of patients with persistent HPV infection for more than one year treated with MSCs reached 100%, and the efficacy was significantly better than the commonly used clinical HPV, cervical precancerous lesions or cervical cancer treatment drugs.
- the present invention also found that MSCs can improve the estrogen level of HPV-infected patients while treating persistent HPV infection. The increase in estrogen levels can improve the treatment microenvironment and help improve the treatment effect. Compared with existing surgical methods, the use of MSCs to treat persistent HPV infection will not physically damage the patient's reproductive function and is safer and more reliable.
- MSCs As a living cell, MSCs have the ability to home in the patient's body and can target the lesion site. Compared with its exosomes (cytokines), they can be more enriched in the lesion site, exerting a strong immune regulation function and achieving the elimination of persistent HPV infection. In addition to clearing the virus through immune regulation, MSCs can express, synthesize, and secrete a variety of growth factors, cytokines, regulatory factors, signal peptides, etc. that reverse the development of the disease and promote healing under the stimulation of the lesion microenvironment. They communicate with the body through the paracrine effect, provide a stable internal environment for anti-HPV treatment, and repair infected cells.
- FIG1 is a schematic diagram of the natural history of HPV virus-induced cervical lesion infection
- Figure 2 is a schematic diagram of the management of simple HR-HPV infection
- FIG3 is a graph showing the changes in protein levels of E6 in Example 1; wherein A is a protein blot of E6, and B is a quantification graph;
- FIG4 is a graph showing the changes in protein levels of E6 in Example 1; wherein A is a protein blot of E7, and B is a quantification graph;
- FIG5 is a graph showing the results of the changes in the gene level of E6 in Example 1;
- FIG6 is a graph showing the result of the changes in the gene level of E7 in Example 1;
- FIG. 7 is a graph showing changes in estrogen levels in patients in Example 2.
- the "persistent HPV infection” refers to the detection of HPV DNA of the same subtype in cervical vaginal specimens collected during more than two consecutive follow-up visits at an interval of 6 to 12 months in women who have not been infected with the relevant HPV subtype before.
- HPV16 pseudovirus acquisition 293T17 cells were transfected with plasmids containing HPV16 L1 and L2 for 48 hours. After 48 hours, the transfected cells were collected and lysed with cell lysis buffer for 24 hours. The desired pseudovirus was obtained by salt extraction.
- the protein level detection results in the anti-HPV detection step (3) are shown in Figures 3 and 4. It can be seen that the expression of HPV E6 and E7 proteins decreased after the intervention of mesenchymal stem cells, and the relationship was dose-dependent with mesenchymal stem cells.
- the mRNA detection results in the anti-HPV detection step (4) are shown in Figures 5 and 6. It can be seen that the mRNA expression of HPV E6 and E7 decreased after the intervention of mesenchymal stem cells, and the relationship was dose-dependent with mesenchymal stem cells.
- the experiments showed that mesenchymal stem cells had a significant inhibitory effect on the expression of HPV16 E6 and E7 proteins and mRNA in Caski cells in a dose-dependent manner, and had a significant HPV inhibitory effect.
Abstract
The present invention provides a use of mesenchymal stem cells in preparation of drugs for treating persistent HPV infection. It is surprisingly found in the present invention that mesenchymal stem cells are independently administered, such that patients persistently infected with HPV for more than one year can be turned negative, the negative conversion rate is high, the estrogen level of the patients can be improved, the curative effect is remarkable, safety and reliability are achieved, and the mesenchymal stem cells have important application value in research and development of drugs for clinically treating persistent HPV infection.
Description
本申请要求于2022年10月28日提交中国专利局、申请号为202211330564.2、发明名称为“间充质干细胞在制备治疗HPV持续感染的药物中的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application claims the priority of the Chinese patent application filed with the China Patent Office on October 28, 2022, with application number 202211330564.2 and invention name "Application of mesenchymal stem cells in the preparation of drugs for the treatment of persistent HPV infection", the entire contents of which are incorporated by reference into this application.
本发明涉及药物技术领域，尤其涉及间充质干细胞在制备治疗HPV持续感染的药物中的应用。The present invention relates to the field of pharmaceutical technology, and in particular to the use of mesenchymal stem cells in the preparation of a medicine for treating persistent HPV infection.
人乳头瘤病毒(Human Papilloma Virus,HPV)是一种常见的性传播病毒，人乳头瘤病毒的感染与宫颈部位的癌前病变及宫颈癌的发生发展有着密切关系(图1)，且高危型HPV与宫颈癌的发生密切相关，接近99％的宫颈癌都与HPV感染有关。根据HPV对细胞的转化能力可分为低危型和高危型(HR-HPV)。低危型主要引起人类多种良性皮肤和黏膜乳头状瘤或疣，如生殖道肛周皮肤和阴道下部的外生性湿疣类病变、扁平湿疣类病变和低度子宫颈上皮内瘤样(cervical intraepithelial neoplasia，CIN)1级病变，儿童和青少年手足摩擦部位的皮肤疣。高危型则与癌症尤其是宫颈癌的发生有关，主要导致CIN2、CIN3级病变和宫颈癌的发生。常见的高危型人乳头瘤病毒(HR-HPV)包括HPV-16、18、31、33、35、39、45、51、52、56、58、59、66、68，99％的宫颈癌由HR-HPV感染所致(de Martel,C.,et al.,Worldwide burden of cancer attributable to HPV by site,country and HPV type.Int J Cancer,2017.141(4):p.664-670)。HPV16型和HPV18型别被认为是全球宫颈癌相关的最普遍类型，占病例总数的70％以上(Zeng,Z.,et al.,Prevalence and Genotype Distribution of HPV Infection in China:Analysis of 51,345 HPV Genotyping Results from China's Largest CAP Certified Laboratory.J Cancer,2016.7(9):p.1037-43)。Human papillomavirus (HPV) is a common sexually transmitted virus. HPV infection is closely related to the occurrence and development of precancerous lesions in the cervix and cervical cancer (Figure 1). High-risk HPV is closely related to the occurrence of cervical cancer. Nearly 99% of cervical cancers are related to HPV infection. HPV can be divided into low-risk and high-risk types (HR-HPV) according to its ability to transform cells. Low-risk types mainly cause a variety of benign skin and mucosal papilloma or warts in humans, such as exophytic condyloma-like lesions, flat condyloma-like lesions, and low-grade cervical intraepithelial neoplasia (CIN) grade 1 lesions in the perianal skin of the genital tract and the lower part of the vagina, and skin warts at the friction sites of children and adolescents. High-risk types are related to the occurrence of cancer, especially cervical cancer, and mainly lead to CIN2, CIN3 grade lesions and cervical cancer. Common high-risk human papillomavirus (HR-HPV) includes HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. 99% of cervical cancer is caused by HR-HPV infection (de Martel, C., et al., Worldwide burden of cancer attributable to HPV by site, country and HPV type. Int J Cancer, 2017. 141(4): p. 664-670). HPV16 and HPV18 are considered to be the most common types associated with cervical cancer worldwide, accounting for more than 70% of the total cases (Zeng, Z., et al., Prevalence and Genotype Distribution of HPV Infection in China: Analysis of 51,345 HPV Genotyping Results from China's Largest CAP Certified Laboratory. J Cancer, 2016. 7(9): p.1037-43).
从HPV感染发展到宫颈癌中间会经历宫颈癌癌前病变CIN，CIN状态下宫颈细胞处于永生化状态，当有协同因子共同作用时就会导致宫颈癌的发生。高危型人乳头瘤病毒(HR-HPV)的持续感染可导致患有宫颈上皮高度病变的风险增加250倍(Dalstein,V.,et al.,Persistence and load of high-risk HPV are predictors for development of high-grade cervical lesions:a longitudinal French cohort study.Int J Cancer,2003.106(3):p.396-403)。从HR-HPV的持续感染进展为子宫颈癌大约需要10-20年的时间，因此，有效控制HPV的感染尤其是清除HR-HPV的持续感染将大大降低子宫颈癌前病变和宫颈癌的发生率。From HPV infection to cervical cancer, there is a process called cervical precancerous lesion CIN. In the CIN state, cervical cells are in an immortalized state. When there are synergistic factors acting together, cervical cancer will occur. Persistent infection with high-risk human papillomavirus (HR-HPV) can increase the risk of high-grade cervical epithelial lesions by 250 times (Dalstein, V., et al., Persistence and load of high-risk HPV are predictors for development of high-grade cervical lesions: a longitudinal French cohort study. Int J Cancer, 2003. 106 (3): p. 396-403). It takes about 10-20 years for persistent HR-HPV infection to progress to cervical cancer. Therefore, effective control of HPV infection, especially elimination of persistent HR-HPV infection, will greatly reduce the incidence of cervical precancerous lesions and cervical cancer.
目前关于HPV持续感染的定义尚未统一明确，即同一患者间隔至少6个月连续2次或2次以上检测出同一型别HPV即可诊断为HPV的持续感染(Kirschner,B.,et al.,HPV genotypes in invasive cervical cancer in Danish women.Acta Obstet Gynecol Scand,2013.92(9):p.1023-31)。但在临床工作中，普遍将间隔时间为1年持续2次或2次以上检测出同一型别HPV诊断为HPV的持续感染(Xiao,S.,et al.,Analysis of human papillomavirus infection in 16 320 patients from gynecologic clinic.Zhong Nan Da Xue Xue Bao Yi Xue Ban,2015.40(4):p.373-9.；Lei,X.,et al.,Association between life style,diet intake and high risk-human papillomavirus persistent infection among rural women in Xinmi City,Henan Province.Wei Sheng Yan Jiu,2016.45(1):p.45-50.)。At present, there is no unified and clear definition of persistent HPV infection. That is, if the same type of HPV is detected twice or more in the same patient with an interval of at least 6 months, it can be diagnosed as persistent HPV infection (Kirschner, B., et al., HPV genotypes in invasive cervical cancer in Danish women. Acta Obstet Gynecol Scand, 2013. 92(9): p.1023-31). However, in clinical work, the detection of the same HPV type twice or more at an interval of one year is generally diagnosed as persistent HPV infection (Xiao, S., et al., Analysis of human papillomavirus infection in 16 320 patients from gynecologic clinic. Zhong Nan Da Xue Xue Bao Yi Xue Ban, 2015. 40(4): p. 373-9.; Lei, X., et al., Association between lifestyle, diet intake and high risk-human papillomavirus persistent infection among rural women in Xinmi City, Henan Province. Wei Sheng Yan Jiu, 2016. 45(1): p. 45-50.).
HPV疫苗是预防HPV感染，降低HPV导致的宫颈癌和癌前病变的发生风险，是宫颈癌预防最有效的方式。不幸的是，HPV疫苗对已经存在感染HPV病毒的人群是没有治疗功效的，且目前临床上没有治疗HPV感染的有效策略。目前虽已出现宫颈癌治疗疫苗(如VGX-3100治疗性疫苗)，但尚未广泛应用，疗效有待进一步观察。HPV vaccine is the most effective way to prevent cervical cancer by preventing HPV infection and reducing the risk of cervical cancer and precancerous lesions caused by HPV. Unfortunately, HPV vaccine has no therapeutic effect on people who are already infected with HPV virus, and there is currently no effective strategy for treating HPV infection in clinical practice. Although there are cervical cancer treatment vaccines (such as VGX-3100 therapeutic vaccine), they have not yet been widely used, and their efficacy needs further observation.
目前还没有明确的治疗HPV感染的方法，绝大部分都是针对HPV相关疾病，并不是针对病毒本身，包括生殖器疣、癌前病变和癌症有明确的治疗 方法，推荐的治疗方法取决于诊断、病灶大小和位置而不同(Massad,L.S.,et al.,2012 updated consensus guidelines for the management of abnormal cervical cancer screening tests and cancer precursors.Obstet Gynecol,2013.121(4):p.829-846.；Workowski,K.A.and S.M.Berman,Centers for Disease Control and Prevention Sexually Transmitted Disease Treatment Guidelines.Clin Infect Dis,2011.53 Suppl 3:p.S59-63.)。There is currently no clear treatment for HPV infection. Most of them are aimed at HPV-related diseases, not the virus itself. There are clear treatments for genital warts, precancerous lesions and cancers. The recommended treatment depends on the diagnosis, size and location of the lesion (Massad, L.S., et al., 2012 updated consensus guidelines for the management of abnormal cervical cancer screening tests and cancer precursors. Obstet Gynecol, 2013. 121(4): p.829-846.; Workowski, K.A. and S.M. Berman, Centers for Disease Control and Prevention Sexually Transmitted Disease Treatment Guidelines. Clin Infect Dis, 2011. 53 Suppl 3: p.S59-63.).
如图2所示，临床上对于单纯HPV感染患者的管理(宫颈细胞学检测阴性)主要为临床随访观察，期待自然消除(妇科肿瘤协会(Society of Gynecologic Oncology，SGO)和ASCCP联合发布，Huh,W.K.,et al.,Use of primary high-risk human papillomavirus testing for cervical cancer screening:interim clinical guidance.Gynecol Oncol,2015.136(2):p.178-82.)。目前临床缺乏清除HPV感染的特效药物，主要干预HPV感染的药物包括抗病毒药物(如西多福韦)、免疫增强剂(如干扰素)、细胞毒性药物(如5-氟尿嘧啶)、中药(如保妇康栓)和乳酸杆菌制剂等。这些药物主要通过增强宫颈局部或全身免疫力达到抗HPV感染作用，其短期内临床疗效欠佳，且存在一定副作用和安全问题。但是如果病毒不能清除，持续感染则会导致HPV相关的疾病。HPV感染导致的宫颈相关癌前病变，可采用冷刀锥切、冷冻疗法或子宫颈环形电切除术(LEEP)等方式治疗，如果进展为宫颈癌，可进行手术或者放化疗。在治疗之后密切随访，如果复发再次处理相关疾病。As shown in Figure 2, clinical management of patients with simple HPV infection (negative cervical cytology test) is mainly clinical follow-up observation, expecting natural elimination (Huh, W.K., et al., Use of primary high-risk human papillomavirus testing for cervical cancer screening: interim clinical guidance. Gynecol Oncol, 2015. 136 (2): p. 178-82.). Currently, there is a lack of specific drugs to eliminate HPV infection in clinical practice. The main drugs for intervention of HPV infection include antiviral drugs (such as cidofovir), immunopotentiators (such as interferon), cytotoxic drugs (such as 5-fluorouracil), traditional Chinese medicine (such as Baofukang suppository) and lactobacillus preparations. These drugs mainly achieve anti-HPV infection by enhancing local or systemic immunity of the cervix. Their short-term clinical efficacy is poor, and there are certain side effects and safety issues. However, if the virus cannot be eliminated, persistent infection will lead to HPV-related diseases. Cervical precancerous lesions caused by HPV infection can be treated with cold knife cone biopsy, cryotherapy or loop electrosurgical excision procedure (LEEP). If it progresses to cervical cancer, surgery or radiotherapy and chemotherapy can be performed. Close follow-up is required after treatment, and related diseases should be treated again if they recur.
临床上部分患者对HPV的感染比较恐慌，尤其是HR-HPV的持续感染会引起很大的焦虑，故对于未发生宫颈病变的HR-HPV持续感染表现出强烈的治疗的愿望，要求积极治疗。对于HR-HPV持续感染的患者进行预防性治疗，有望减少宫颈病变的发生。但目前国内外对HPV感染治疗方式不统一，且针对HPV感染没有特效药。虽然临床上应用于治疗子宫颈HPV感染的药物有许多种类，但大部分是增强局部或全身的免疫力来达到干预HPV感染的目的，不是针对病毒本身，且疗效是否确切还有待于更多的研究来验证,同时存在一定副作用和安全问题。因此，开发高效清除HPV感染且对宫颈有保护作用的新型治疗策略是当前临床的迫切需求。Clinically, some patients are quite panic about HPV infection, especially persistent HR-HPV infection, which can cause great anxiety. Therefore, they show a strong desire for treatment for persistent HR-HPV infection without cervical lesions and require active treatment. Preventive treatment for patients with persistent HR-HPV infection is expected to reduce the occurrence of cervical lesions. However, there is no unified treatment for HPV infection at home and abroad, and there is no specific drug for HPV infection. Although there are many types of drugs used in the treatment of cervical HPV infection in clinical practice, most of them enhance local or systemic immunity to achieve the purpose of intervening in HPV infection, not targeting the virus itself, and whether the efficacy is accurate still needs to be verified by more research, and there are certain side effects and safety issues. Therefore, the development of a new treatment strategy that can effectively eliminate HPV infection and protect the cervix is an urgent need in current clinical practice.
发明内容Summary of the invention
本发明惊奇的发现间充质干细胞具有显著的治疗HPV持续感染作用，单独施用即可使HPV持续感染患者转阴，且能够改善患者生殖系统循环，疗效显著、安全可靠，在临床治疗HPV持续感染的药物研发中具有重要应用价值。The present invention surprisingly found that mesenchymal stem cells have a significant effect in treating persistent HPV infection. A single application can turn patients with persistent HPV infection negative and improve the patient's reproductive system circulation. It has significant efficacy, is safe and reliable, and has important application value in the development of clinical drugs for the treatment of persistent HPV infection.
本发明提供了一种间充质干细胞在制备治疗HPV感染的药物中的应用。The present invention provides an application of mesenchymal stem cells in preparing a medicine for treating HPV infection.
优选地，所述的HPV持续感染包括高危型HPV持续感染。Preferably, the persistent HPV infection includes persistent high-risk HPV infection.
优选地，所述的HPV持续感染包括HPV16和/或HPV18型持续感染。Preferably, the HPV persistent infection includes HPV16 and/or HPV18 persistent infection.
优选地，所述治疗的HPV持续感染包括改善雌激素水平。Preferably, said treating persistent HPV infection comprises improving estrogen levels.
优选地，所述的间充质干细胞选自人脐带间充质干细胞。临床级人脐带间充质干细胞在GMP实验室环境中制备，输入人体尤其是静脉注射时更安全。Preferably, the mesenchymal stem cells are selected from human umbilical cord mesenchymal stem cells. Clinical-grade human umbilical cord mesenchymal stem cells are prepared in a GMP laboratory environment and are safer to be injected into the human body, especially intravenously.
优选地，所述药物中的间充质干细胞含量不低于106个/mL；进一步优选地，所述药物中的间充质干细胞含量为106-107个/mL或6000w以上。Preferably, the content of mesenchymal stem cells in the drug is not less than 106 cells/mL; further preferably, the content of mesenchymal stem cells in the drug is 106-107 cells/mL or more than 6000w.
优选地，所述的药物为静脉注射剂。Preferably, the drug is an intravenous injection.
在本发明的一些具体实施方式中，制备了一种包括含量不低于106个/mL的间充质干细胞的HPV持续感染治疗药物，该药物为静脉注射剂。In some specific embodiments of the present invention, a drug for treating persistent HPV infection comprising mesenchymal stem cells at a content of not less than 106 cells/mL is prepared, and the drug is an intravenous injection.
在本发明的一些具体实施方式中，将含有106-107个/mL间充质干细胞的静脉注射剂注射至患者体内，1次给药后，3-6个月复查，HPV持续感染一年以上的患者能够稳定保持HPV阴性，且能改善患者的生殖系统循环。In some specific embodiments of the present invention, an intravenous injection containing 106-107/mL mesenchymal stem cells is injected into the patient's body. After one administration, the patient is re-examined 3-6 months later. The patient with persistent HPV infection for more than one year can stably maintain HPV negativity and the patient's reproductive system circulation can be improved.
与现有技术相比，本发明至少具备以下有益效果：Compared with the prior art, the present invention has at least the following beneficial effects:
干细胞是一类具有自我更新和多向分化潜能的细胞，且具有优良的免疫调节作用和旁分泌功能。间充质干细胞(MSCs)对不同肿瘤的影响可能截然相反，MSCs对胃癌、结直肠癌等具有抑制肿瘤生长的作用，但MSCs对肺癌、胰腺癌等则表现为促进肿瘤进展(崔舒悦,汤帅,丁晓玲,等.间充质干细胞及其外泌体对肿瘤影响的研究进展[J].中国肺癌杂 志,2022,25(5):7.)目前MSCs对某一癌症具有抑制作用亦或促进作用尚无法预测。Stem cells are a type of cell with the potential for self-renewal and multidirectional differentiation, and have excellent immunomodulatory and paracrine functions. The effects of mesenchymal stem cells (MSCs) on different tumors may be completely opposite. MSCs have an inhibitory effect on gastric cancer, colorectal cancer, etc., but MSCs promote tumor progression in lung cancer, pancreatic cancer, etc. (Cui Shuyue, Tang Shuai, Ding Xiaoling, et al. Research progress on the effects of mesenchymal stem cells and their exosomes on tumors [J]. Chinese Journal of Lung Cancer, 2022, 25(5):7.) At present, it is still impossible to predict whether MSCs have an inhibitory or promoting effect on a certain cancer.
本发明研究中惊奇的发现，MSCs具有显著的抗HPV病毒抑制作用，应用MSCs治疗持续感染HPV一年以上的患者转阴率达到100％，疗效显著优于临床常用HPV、宫颈癌癌前病变或宫颈癌治疗药物。本发明还发现MSCs在治疗HPV持续感染的同时还能提高HPV感染患者的雌性激素水平，雌激素水平的提升可改善治疗微环境，有利于提升治疗效果。相较于现有的手术方法，应用MSCs治疗HPV持续感染不会物理上损害患者生育功能，更加安全可靠。The present invention surprisingly found that MSCs have a significant anti-HPV virus inhibitory effect. The negative conversion rate of patients with persistent HPV infection for more than one year treated with MSCs reached 100%, and the efficacy was significantly better than the commonly used clinical HPV, cervical precancerous lesions or cervical cancer treatment drugs. The present invention also found that MSCs can improve the estrogen level of HPV-infected patients while treating persistent HPV infection. The increase in estrogen levels can improve the treatment microenvironment and help improve the treatment effect. Compared with existing surgical methods, the use of MSCs to treat persistent HPV infection will not physically damage the patient's reproductive function and is safer and more reliable.
MSCs作为一种活细胞，其在患者体内具有归巢能力，可靶向病变部位，相较于其外泌体(细胞因子)能够更多的富集于病变部位，发挥强效免疫调节功能，实现HPV持续感染的清除。除了通过免疫调节清除病毒外，MSCs在病变微环境刺激下能够表达、合成、分泌多种逆转疾病发展促进愈合的生长因子、细胞因子、调节因子、信号肽等，通过旁分泌效应与体内进行信息交流，为抗HPV治疗提供稳定的内环境，并修复感染的细胞。As a living cell, MSCs have the ability to home in the patient's body and can target the lesion site. Compared with its exosomes (cytokines), they can be more enriched in the lesion site, exerting a strong immune regulation function and achieving the elimination of persistent HPV infection. In addition to clearing the virus through immune regulation, MSCs can express, synthesize, and secrete a variety of growth factors, cytokines, regulatory factors, signal peptides, etc. that reverse the development of the disease and promote healing under the stimulation of the lesion microenvironment. They communicate with the body through the paracrine effect, provide a stable internal environment for anti-HPV treatment, and repair infected cells.
结合附图，并通过参考下面的详细描述，将会更容易地对本发明有更完整的理解并且更容易地理解其伴随的优点和特征，其中：A more complete understanding of the present invention and its attendant advantages and features will be more readily appreciated by referring to the following detailed description taken in conjunction with the accompanying drawings, in which:
图1为HPV病毒致宫颈病变感染自然史示意图；FIG1 is a schematic diagram of the natural history of HPV virus-induced cervical lesion infection;
图2为单纯HR-HPV感染的管理示意图；Figure 2 is a schematic diagram of the management of simple HR-HPV infection;
图3为实施例1中E6在蛋白水平变化的结果图；其中，A为E6的蛋白印迹图，B为量化图；FIG3 is a graph showing the changes in protein levels of E6 in Example 1; wherein A is a protein blot of E6, and B is a quantification graph;
图4为实施例1中E6在蛋白水平变化的结果图；其中，A为E7的蛋白印迹图，B为量化图；FIG4 is a graph showing the changes in protein levels of E6 in Example 1; wherein A is a protein blot of E7, and B is a quantification graph;
图5为实施例1中E6在基因水平变化的结果图；FIG5 is a graph showing the results of the changes in the gene level of E6 in Example 1;
图6为实施例1中E7在基因水平变化的结果图；FIG6 is a graph showing the result of the changes in the gene level of E7 in Example 1;
图7为实施例2中患者雌激素水平变化图。FIG. 7 is a graph showing changes in estrogen levels in patients in Example 2.
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。所有原料未注明合成方法的均购自探索平台、阿拉丁、Sigma-Aldrich等厂家，均为分析纯。The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present invention. The experimental methods in the following embodiments that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. All raw materials that do not specify the synthesis method are purchased from manufacturers such as Exploration Platform, Aladdin, and Sigma-Aldrich, and are all analytically pure.
在本发明中，所述的“HPV持续感染”是指，在既往未感染过相关HPV亚型的女性中，在6～12个月的间隔时间内连续2次以上随访时采集的宫颈阴道部标本中检测到相同亚型的HPV DNA。In the present invention, the "persistent HPV infection" refers to the detection of HPV DNA of the same subtype in cervical vaginal specimens collected during more than two consecutive follow-up visits at an interval of 6 to 12 months in women who have not been infected with the relevant HPV subtype before.
实施例1 体外细胞水平验证MSCs抑制HPV作用Example 1 Verification of the inhibitory effect of MSCs on HPV at the in vitro cell level
实验方法：HPV16假病毒感染的Caski细胞用于疗效评估Experimental Methods: HPV16 pseudovirus-infected Caski cells for efficacy evaluation
1.HPV16假病毒获取：293T17细胞转染含有HPV16 L1和L2的质粒48小时，48小时后收集转染的细胞，用细胞裂解液裂解细胞24小时，采用盐抽提得到所需的假病毒。1. HPV16 pseudovirus acquisition: 293T17 cells were transfected with plasmids containing HPV16 L1 and L2 for 48 hours. After 48 hours, the transfected cells were collected and lysed with cell lysis buffer for 24 hours. The desired pseudovirus was obtained by salt extraction.
2.假病毒细胞模型构建：将步骤1获取的HPV16假病毒与Caski细胞共孵育48小时，即可得到HPV感染的细胞。2. Construction of pseudovirus cell model: The HPV16 pseudovirus obtained in step 1 was incubated with Caski cells for 48 hours to obtain HPV-infected cells.
3.抗HPV检测：3. Anti-HPV detection:
(1)将步骤2构建的HPV感染细胞加入6孔板，每孔2mL(5×10
4个)，培养24小时；
(1) Add the HPV-infected cells constructed in step 2 to a 6-well plate, 2 mL (5 × 10 4 cells) per well, and culture for 24 hours;
(2)去掉上清，以每孔2mL含0、1×10
4、2.5×10
4、5×10
4、1×10
5、5×10
5个临床级人脐带间充质干细胞的培养基加入上述HPV感染的Caski细胞中共培养72小时；
(2) The supernatant was removed, and 2 mL of culture medium containing 0, 1×10 4 , 2.5×10 4 , 5×10 4 , 1×10 5 , and 5×10 5 clinical-grade human umbilical cord mesenchymal stem cells per well was added to the above-mentioned HPV-infected Caski cells and co-cultured for 72 hours;
(3)收集6孔板中各组细胞，提取细胞内的总蛋白，以E6、E7为目的蛋白，β-actin为内参，通过Western Blotting检测E6、E7在蛋白水平的变化。(3) Collect cells from each group in a 6-well plate, extract total intracellular protein, use E6 and E7 as target proteins and β-actin as an internal reference, and detect changes in E6 and E7 protein levels by Western blotting.
(4)收集6孔板中各组细胞，提取细胞内的总RNA，Oligo(dT)15为引物反转录，然后通过Real-time PCR检测E6、E7在基因水平上的变化。(4) Collect cells from each group in a 6-well plate, extract total RNA from the cells, perform reverse transcription using Oligo(dT)15 as a primer, and then use Real-time PCR to detect changes in E6 and E7 at the gene level.
实验结果：抗HPV检测步骤(3)中蛋白水平检测结果如图3、图4所示，可以看出通过间充质干细胞干预治疗后HPV的E6、E7蛋白表达下降，且与间充质干细胞呈剂量依赖关系。抗HPV检测步骤(4)中mRNA 检测结果如图5、图6所示，可以看出通过间充质干细胞干预治疗后HPV的E6、E7的mRNA表达下降，且与间充质干细胞呈剂量依赖关系。Experimental results: The protein level detection results in the anti-HPV detection step (3) are shown in Figures 3 and 4. It can be seen that the expression of HPV E6 and E7 proteins decreased after the intervention of mesenchymal stem cells, and the relationship was dose-dependent with mesenchymal stem cells. The mRNA detection results in the anti-HPV detection step (4) are shown in Figures 5 and 6. It can be seen that the mRNA expression of HPV E6 and E7 decreased after the intervention of mesenchymal stem cells, and the relationship was dose-dependent with mesenchymal stem cells.
综上，实验表明间充质干细胞对Caski细胞HPV16 E6、E7蛋白和mRNA的表达有明显的抑制作用，且呈剂量依赖效应，具有显著的HPV抑制作用。In summary, the experiments showed that mesenchymal stem cells had a significant inhibitory effect on the expression of HPV16 E6 and E7 proteins and mRNA in Caski cells in a dose-dependent manner, and had a significant HPV inhibitory effect.
实施例2 MSCs对HPV的临床治疗效果Example 2 Clinical therapeutic effect of MSCs on HPV
实验方法：给HPV感染1年及以上的患者静脉输注含临床级人脐带间充质干细胞6000W的葡萄糖或生理盐水等等渗液200mL，输注时间0.5-3h。Experimental method: Patients infected with HPV for 1 year or more were intravenously infused with 200 mL of isotonic solution such as glucose or saline containing 6000W clinical-grade human umbilical cord mesenchymal stem cells for 0.5-3 hours.
实验结果：6个月后复查，如表1所示，全部HPV持续感染的患者HPV均为阴性，HPV转阴率为100％。同时本实验还监测了其中4位患者治疗前后雌二醇激素的变化，如表2、图7所示，干细胞治疗后患者雌二醇激素升高，生殖系统得以改善。Experimental results: After 6 months of reexamination, as shown in Table 1, all patients with persistent HPV infection were HPV negative, and the HPV negative conversion rate was 100%. At the same time, this experiment also monitored the changes in estradiol hormones of 4 patients before and after treatment, as shown in Table 2 and Figure 7. After stem cell treatment, the estradiol hormone of the patients increased and the reproductive system improved.
表1Table 1
表2Table 2
可以理解的是，虽然本发明已以较佳实施例披露如上，然而上述实施例并非用以限定本发明。对于任何熟悉本领域的技术人员而言，在不脱离本发明技术方案范围情况下，都可利用上述揭示的技术内容对本发明技术方案作出许多可能的变动和修饰，或修改为等同变化的等效实施例。因此，凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所做的任何简单修改、等同变化及修饰，均仍属于本发明技术方案保护的范围内。It is to be understood that, although the present invention has been disclosed as a preferred embodiment, the above embodiment is not intended to limit the present invention. For any technician familiar with the art, without departing from the scope of the technical solution of the present invention, the technical content disclosed above can be used to make many possible changes and modifications to the technical solution of the present invention, or modified into equivalent embodiments of equivalent changes. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.
Claims (7)
- 间充质干细胞在制备治疗HPV持续感染的药物中的应用。Application of mesenchymal stem cells in the preparation of drugs for the treatment of persistent HPV infection.
- 根据权利要求1所述的应用，其特征在于，所述的HPV持续感染包括高危型HPV持续感染。The use according to claim 1 is characterized in that the persistent HPV infection includes persistent high-risk HPV infection.
- 根据权利要求1或2所述的应用，其特征在于，所述的HPV持续感染包括HPV16和/或HPV18型持续感染。The use according to claim 1 or 2 is characterized in that the HPV persistent infection includes HPV16 and/or HPV18 persistent infection.
- 根据权利要求1所述的应用，其特征在于，所述治疗的HPV持续感染包括改善雌激素水平。The use according to claim 1 is characterized in that the treatment of persistent HPV infection includes improving estrogen levels.
- 根据权利要求1所述的应用，其特征在于，所述的间充质干细胞选自人脐带间充质干细胞。The use according to claim 1, characterized in that the mesenchymal stem cells are selected from human umbilical cord mesenchymal stem cells.
- 根据权利要求1所述的应用，其特征在于，所述药物中的间充质干细胞含量不低于106个/mL。The use according to claim 1, characterized in that the content of mesenchymal stem cells in the drug is not less than 106/mL.
- 根据权利要求1所述的应用，其特征在于，所述药物为静脉注射剂。The use according to claim 1 is characterized in that the drug is an intravenous injection.
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