CN115381857B

CN115381857B - Application of mesenchymal stem cells in preparation of medicine for treating HPV persistent infection

Info

Publication number: CN115381857B
Application number: CN202211330564.2A
Authority: CN
Inventors: 刘中民; 刘为廷; 贾文文; 吴萍; 乐文俊
Original assignee: Shanghai East Hospital Tongji University Affiliated East Hospital
Current assignee: Shanghai East Hospital Tongji University Affiliated East Hospital
Priority date: 2022-10-28
Filing date: 2022-10-28
Publication date: 2023-02-03
Anticipated expiration: 2042-10-28
Also published as: WO2024087356A1; CN115381857A

Abstract

The invention provides application of mesenchymal stem cells in preparation of a medicament for treating HPV persistent infection. The invention surprisingly discovers that the mesenchymal stem cells can convert the patients continuously infected with HPV for more than one year into negative after being singly applied, have high negative conversion rate, can improve the estrogen level of the patients, have obvious curative effect, safety and reliability, and have important application value in the research and development of medicaments for clinically treating the HPV continuous infection.

Description

Application of mesenchymal stem cells in preparation of medicine for treating HPV persistent infection

Technical Field

The invention relates to the technical field of medicines, in particular to application of mesenchymal stem cells in preparing a medicine for treating HPV persistent infection.

Background

Human Papilloma Virus (HPV) is a common sexually transmitted Virus, the infection of the Human Papilloma Virus is closely related to precancerous lesion of cervical part and the occurrence and development of cervical cancer (figure 1), high-risk HPV is closely related to the occurrence of cervical cancer, and nearly 99 percent of cervical cancer is related to HPV infection. The transformation capability of HPV on cells can be divided into low-risk type and high-risk type (HR-HPV). The low-risk type mainly causes various benign cutaneous and mucosal papillomas or warts of human beings, such as ectogenic condyloma lesions, flat condyloma lesions and low-grade Cervical Intraepithelial Neoplasia (CIN) grade 1 lesions of the perianal skin of the genital tract and the lower part of the vagina, and cutaneous warts of the friction parts of hands and feet of children and teenagers. The high-risk type is related to the occurrence of cancers, particularly cervical cancer, and mainly causes CIN2 and CIN3 grade lesions and the occurrence of cervical cancer. Common high risk types of human papillomaviruses (HR-HPV) include HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 99% of cervical cancers caused by HR-HPV infection (de Martel, C., et al, worldwide garden of Cancer acceptable to HPV by site, countryand HPV type. Int J Cancer,2017.141 (4): p.664-670). HPV type 16 and HPV type 18 are considered the most prevalent type of cervical cancer worldwide and account for over 70% of the total number of cases (Zeng, Z., et al, presence and Genotype Distribution of HPV Infection in China: analysis of 51,345 HPV Genotyping Results from China's target CAP verified laboratory. J cancer,2016.7 (9): p.1037-43).

Cervical cancer precancerous lesion CIN is experienced from HPV infection to cervical cancer, cervical cells are in an immortalized state under the CIN state, and the cervical cancer can be caused when synergistic factors act together. Persistent infection with high-risk human papillomaviruses (HR-HPV) can result in a 250-fold increase in the risk of high-grade lesions of the cervical epithelium (Dalstein, V., et al., persistence and load of high-rise HPV area predictors for the reduction of high-grade nuclear dimensions: a longitudinal free code temperature student. Int J Cancer, 2003.106 (3): p. 396-403). Progression from persistent infection with HR-HPV to cervical cancer takes approximately 10-20 years and, therefore, effective HPV infection control, especially clearance of persistent infection with HR-HPV, will greatly reduce the incidence of pre-cervical lesions and cervical cancer.

There is currently no well-defined definition of persistent HPV infection, i.e., HPV infection can be diagnosed by detecting the same type of HPV 2 or more times consecutively in the same patient at intervals of at least 6 months (Kirschner, B., et al., HPV genotypes in an innovative scientific cancer in Danish white. Acta Obstet Gynecol Scan, 2013.92 (9): p.1023-31). However, in clinical work, HPV of the same type is commonly diagnosed as persistent infection of HPV at intervals of 1 year for 2 or more times (Xiao, S., et al, analysis of human papillomavir infection in 16 320 tissues from genomic clinic, zhong Nan Da Xue Xue Bai Yi Bai, 2015.40 (4): p.373-9.; lei, X., et al, association life style, di-inter and high-human papillomavir persistent infection in neurological tissue City, hean Provision, weii Sheng n Yaiu, 2016.45 (1 p.45-50): 1).

The HPV vaccine is used for preventing HPV infection and reducing the risk of cervical cancer and precancerous lesion caused by HPV, and is the most effective mode for preventing cervical cancer. Unfortunately, HPV vaccines are not therapeutically effective for people already with an existing infection with HPV virus, and there is currently no clinically effective strategy for treating HPV infection. At present, although a vaccine for treating cervical cancer (such as a VGX-3100 therapeutic vaccine) appears, the vaccine is not widely applied, and the curative effect needs to be further observed.

There are currently no clear treatments for HPV infection, most of which are directed to HPV related diseases, not to the virus itself, including genital warts, precancerous lesions and cancer, and the recommended treatments vary depending on diagnosis, lesion size and location (Massad, l.s., et al, 2012 updated consensus screening tests and cancer precessors. Objective gynecol,2013.121 (4): p.829-846.; workowski, k.a.and s.m.benman, center for Disease Control and preservation search delivery guide guidelines, clean infection, sample infection, 2011.53.59-59).

As shown in FIG. 2, clinical management of patients with HPV infection alone (negative cervical cytology) is primarily a clinical follow-up observation, with natural elimination expected (Society of clinical Oncology Oncology, SGO) and ASCCP published in combination, huh, W.K., et al, use of primary high-rise human cervical cancer testing for clinical cancer screening. Intrinsic clinical identification. Gynecol Oncol, 2015.136 (2): p.178-82.). Currently, specific drugs for clearing HPV infection are lacking clinically, and the drugs mainly intervening HPV infection comprise antiviral drugs (such as cidofovir), immunopotentiators (such as interferon), cytotoxic drugs (such as 5-fluorouracil), traditional Chinese medicines (such as Baofukang suppository), lactobacillus preparations and the like. The medicines achieve the effect of resisting HPV infection mainly by enhancing the local or systemic immunity of the cervix, have poor clinical curative effect in a short period, and have certain side effect and safety problem. However, if the virus cannot be cleared, persistent infection can lead to HPV-related diseases. Cervical-related precancerous lesions caused by HPV infection can be treated by cold knife conization, cryotherapy or cervical ring-like electrosurgery (LEEP), and if cervical cancer is developed, operation or radiotherapy and chemotherapy can be performed. Follow-up closely after treatment, and re-treat the associated disease if a relapse occurs.

Clinical partial patients are panic in infection with HPV, and especially persistent infection with HR-HPV causes great anxiety, so that strong treatment is required for persistent infection with HR-HPV without cervical lesion. The preventive treatment of patients with continuous HR-HPV infection is expected to reduce the occurrence of cervical lesions. However, the treatment modes for HPV infection are not unified at home and abroad at present, and no specific medicine is provided for HPV infection. Although there are many kinds of drugs clinically used for treating cervical HPV infection, most of them are aimed at enhancing local or systemic immunity to intervene in HPV infection, and are not aimed at viruses per se, and whether the curative effect is exact or not is still to be verified by more researches, and there are certain side effects and safety problems. Therefore, the development of new therapeutic strategies that efficiently clear HPV infection and have a protective effect on the cervix is an urgent clinical need at present.

Disclosure of Invention

The mesenchymal stem cells are surprisingly found to have an obvious HPV persistent infection treatment effect, the HPV persistent infection patient can turn negative after being singly applied, the reproductive system circulation of the patient can be improved, the curative effect is obvious, and the mesenchymal stem cells are safe and reliable and have important application value in the research and development of clinical HPV persistent infection treatment medicines.

The invention provides an application of mesenchymal stem cells in preparing a medicament for treating HPV infection.

Preferably, the persistent infection by HPV comprises persistent infection by HPV of high-risk type.

Preferably, the HPV persistent infection comprises persistent infection of HPV type 16 and/or HPV type 18.

Preferably, the HPV persistent infection treated comprises an improvement in estrogen levels.

Preferably, the mesenchymal stem cells are selected from human umbilical cord mesenchymal stem cells. The clinical-grade human umbilical cord mesenchymal stem cells are prepared in a GMP laboratory environment, and are safer when being input into a human body, particularly when being injected into a vein.

Preferably, the content of the mesenchymal stem cells in the medicament is not less than 10 ⁶ Per mL; further preferably, the content of the mesenchymal stem cells in the medicament is 10 ⁶ -10 ⁷ The dosage is more than 6000 w/mL or 6000 w.

Preferably, the medicament is an intravenous injection.

In some embodiments of the invention, a composition comprising an amount of not less than 10% is prepared ⁶ The medicine is an intravenous injection and is used for treating HPV persistent infection of mesenchymal stem cells per mL.

In some embodiments of the invention, will contain 10 ⁶ -10 ⁷ The intravenous injection of mesenchymal stem cells per mL is injected into a patient body, after 1 time of administration, the patient is rechecked for 3-6 months, and the patient with HPV continuous infection for more than one year can stably keep HPV negative and improve the reproductive system circulation of the patient.

Compared with the prior art, the invention has the beneficial effects that:

stem cells are a class of cells with self-renewal and multi-differentiation potential, and have excellent immunomodulatory and paracrine functions. The influence of Mesenchymal Stem Cells (MSCs) on different tumors may be quite opposite, MSCs have the effect of inhibiting tumor growth on gastric cancer, colorectal cancer and the like, but MSCs have the effect of promoting tumor progression on lung cancer, pancreatic cancer and the like (treshuyue, marshal, ding ling, and the like. The research progress of the influence of mesenchymal stem cells and exosomes on tumors [ J ] in journal of lung cancer in china, 2022,25 (5): 7.) at present, MSCs have the effect of inhibiting or promoting a certain cancer, which is not predicted yet.

The research of the invention surprisingly discovers that the MSCs have obvious HPV virus inhibition resistance, the negative conversion rate of patients who continuously infect HPV for more than one year by applying the MSCs to treat the patients reaches 100 percent, and the curative effect of the MSCs is obviously superior to that of clinical common HPV, cervical cancer precancerous lesion or cervical cancer treatment drugs. The invention also finds that the MSCs can improve the estrogen level of the HPV infected patient while treating the HPV persistent infection, and the improvement of the estrogen level can improve the treatment microenvironment and is beneficial to improving the treatment effect. Compared with the existing operation method, the application of the MSCs to treat HPV continuous infection does not physically damage the fertility function of a patient, and is safer and more reliable.

The MSCs are used as living cells, have homing capability in a patient body, can target a diseased part, can be more enriched in the diseased part compared with exosomes (cytokines) thereof, play a strong immunoregulation function and realize the elimination of HPV continuous infection. Except for removing viruses through immune regulation, the MSCs can express, synthesize and secrete various growth factors, cytokines, regulatory factors, signal peptides and the like which reverse the development of diseases and promote healing under the stimulation of a pathological microenvironment, exchange information with the body through a paracrine effect, provide a stable internal environment for anti-HPV treatment, and repair infected cells.

Drawings

FIG. 1 is a schematic representation of the natural history of cervical lesion infection by HPV virus;

FIG. 2 is a schematic diagram of the management of pure HR-HPV infection;

FIG. 3 is a graph showing the results of changes in E6 at the protein level in example 1; wherein A is a Western blot of E6, and B is a quantification map;

FIG. 4 is a graph showing the results of changes at the protein level of E6 in example 1; wherein A is a Western blot of E7, and B is a quantification map;

FIG. 5 is a graph showing the results of changes in E6 at the gene level in example 1;

FIG. 6 is a graph showing the results of changes in E7 at the gene level in example 1;

FIG. 7 is a graph of the change in estrogen levels in the patients of example 2.

Detailed Description

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally according to conventional conditions, or according to conditions recommended by the manufacturer. All raw materials without the synthesis method are purchased from manufacturers such as exploration platforms, aladdin, sigma-Aldrich and the like, and are all analytically pure.

In the invention, the term "HPV persistent infection" refers to the detection of HPV DNA of the same subtype in a cervicovaginal specimen collected at an interval of 6 to 12 months for more than 2 consecutive follow-up visits in a woman who has not been infected with the relevant HPV subtype.

Example 1 in vitro cellular level validation of HPV inhibition by MSCs

The experimental method comprises the following steps: caski cells infected with HPV16 pseudovirus for efficacy assessment

1. HPV16 pseudovirus acquisition: 293T17 cells were transfected with plasmids containing HPV 16L 1 and L2 for 48 hours, and after 48 hours the transfected cells were collected, lysed with cell lysate for 24 hours, and extracted with salt to obtain the desired pseudovirus.

2. Pseudo virus cell model construction: and (3) incubating the HPV16 pseudovirus obtained in the step (1) and Caski cells for 48 hours to obtain HPV infected cells.

3. anti-HPV detection:

(1) HPV-infected cells constructed in step 2 were added to 6-well plates at 2mL per well (5X 10) ⁴ One), culturing for 24 hours;

(2) The supernatant was removed to give 2mL of a solution containing 0, 1X 10 ⁴ 、2.5×10 ⁴ 、5×10 ⁴ 、1×10 ⁵ 、5×10 ⁵ Adding the culture medium of the individual clinical grade human umbilical cord mesenchymal stem cells into the HPV infected Caski cells for co-culture for 72 hours;

(3) And collecting cells of each group in a 6-well plate, extracting total protein in the cells, taking E6 and E7 as target proteins and beta-actin as an internal reference, and detecting changes of the E6 and E7 in the protein level by Western Blotting.

(4) Collecting each group of cells in the 6-well plate, extracting total RNA in the cells, carrying out reverse transcription by using Oligo (dT) 15 as a primer, and detecting changes of E6 and E7 on a gene level by Real-time PCR.

The experimental results are as follows: the detection results of protein level in the step (3) of anti-HPV detection are shown in fig. 3 and fig. 4, and it can be seen that the expression of E6 and E7 proteins of HPV is reduced after the intervention treatment of mesenchymal stem cells, and the protein level and the mesenchymal stem cells are in a dose-dependent relationship. The detection result of mRNA in the anti-HPV detection step (4) is shown in FIGS. 5 and 6, and it can be seen that the mRNA expression of E6 and E7 of HPV is reduced after intervention and treatment of mesenchymal stem cells, and the mRNA expression and the mesenchymal stem cells are in a dose-dependent relationship.

In conclusion, experiments show that the mesenchymal stem cells have obvious inhibition effect on the expression of Caski cell HPV 16E 6, E7 protein and mRNA, are in a dose-dependent effect and have obvious HPV inhibition effect.

Example 2 clinical therapeutic Effect of MSCs on HPV

The experimental method comprises the following steps: 200mL of a seepage containing 6000W of glucose or physiological saline and the like of clinical-grade human umbilical cord mesenchymal stem cells is intravenously infused into a patient infected with HPV for 1 year or more, and the infusion time is 0.5-3h.

The experimental results are as follows: after 6 months of review, all patients with persistent HPV infection were negative and the HPV negative rate was 100%, as shown in Table 1. Meanwhile, the change of the estradiol hormones of 4 patients before and after treatment is monitored in the experiment, as shown in the table 2 and the figure 7, the estradiol hormones of the patients after the stem cell treatment are increased, and the reproductive system is improved.

TABLE 1

TABLE 2

The foregoing is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be construed as the protection scope of the present invention.

Claims

1. Application of human umbilical cord mesenchymal stem cells in preparing a medicament for treating HPV persistent infection.

2. The use according to claim 1, wherein the persistent infection with HPV comprises persistent infection with HPV of the high risk type.

3. Use according to claim 1 or 2, wherein the persistent infection by HPV comprises persistent infection by HPV16 and/or HPV18 types.

4. The use of claim 1, wherein the medicament for treating HPV persistent infection comprises a medicament that increases estrogen levels in a patient suffering from HPV persistent infection.

5. The use of claim 1, wherein the medicament has a mesenchymal stem cell content of not less than 10 ⁶ one/mL.

6. The use of claim 1, wherein the medicament is an intravenous injection.