WO2022148138A1 - 一种能够预防和/或治疗子宫颈鳞癌的卷曲乳杆菌 - Google Patents

一种能够预防和/或治疗子宫颈鳞癌的卷曲乳杆菌 Download PDF

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WO2022148138A1
WO2022148138A1 PCT/CN2021/131252 CN2021131252W WO2022148138A1 WO 2022148138 A1 WO2022148138 A1 WO 2022148138A1 CN 2021131252 W CN2021131252 W CN 2021131252W WO 2022148138 A1 WO2022148138 A1 WO 2022148138A1
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lactobacillus crispatus
strains
product
lactobacillus
cells
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王慧妍
李若男
马艳
陈曦曦
李明
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安徽省肿瘤医院
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • 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
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus

Definitions

  • the invention relates to a Lactobacillus crispatus capable of preventing and/or treating cervical squamous cell carcinoma, belonging to the technical field of microorganisms.
  • Cervical cancer is the most common malignant tumor in the female reproductive system, which seriously threatens women's life and health. Cervical squamous cell carcinoma is the main pathological type of cervical cancer, accounting for about 70% of all cervical cancers. Human papillomavirus (HPV) infection is the main cause of cervical cancer. However, in recent years, studies have confirmed that the vaginal microecology of women is also closely related to the occurrence and development of HPV infection and cervical cancer.
  • HPV Human papillomavirus
  • the female vagina is a complex micro-ecosystem composed of squamous epithelium and vaginal flora.
  • the vagina of healthy women of reproductive age is usually dominated by Lactobacillus. Although there are more than 200 species of bacteria in the genus Lactobacillus, there are usually only four that dominate the female vagina: L. crispatus, L. gasseri, L. L. iners and L. jensensii.
  • the vaginal microecological environment is divided into five types. Among them, the dominant bacteria in CST I, II, III and V are the above four lactobacilli, respectively, and CST IV is the dominant type of non-lactic bacteria.
  • CST I and III mainly Lactobacillus crispatus and Lactobacillus inerts
  • CST II and V are relatively rare, accounting for less than 2%.
  • the prevalence of Lactobacillus inerts is higher than that of Lactobacillus crispatus, there is no significant difference between healthy and women with reproductive tract infections (including HPV infection); and Lactobacillus inerts allow strictly anaerobic pathogens to coexist with it, Once these anaerobes prevail, CST III is converted to CST IV.
  • Lactobacillus crispatus rarely coexists with other microorganisms, and CST I is more stable than CST III.
  • Lactobacillus crispatus is significantly negatively correlated with HPV infection and cervical lesions, plays a potential protective role in the occurrence and development of cervical cancer, and is expected to become a new technical means for the prevention and treatment of cervical cancer.
  • the present invention provides a Lactobacillus crispatus capable of preventing and/or treating cervical squamous cell carcinoma.
  • the present invention provides the following technical solutions:
  • the present invention provides two strains of Lactobacillus crispatus (Lactobacillus crispatus) Lc31 and Lc83, the Lactobacillus crispatus is preserved in the China General Microorganism Culture Collection Center, and the preservation numbers are respectively CGMCC No.21348 and CGMCC No.21349 , the preservation date is December 10, 2020.
  • the Lactobacillus crispatus Lc31 and Lc83 provided by the present invention are respectively derived from the vaginal secretions of two 32-year-old and 22-year-old healthy women in Anhui.
  • the strains are sequenced and analyzed, and their 16S rDNA sequences are as SEQ ID NO.1 and SEQ ID NO.
  • the sequence obtained by sequencing was compared in GeneBank, and the results showed that its identity with the 16S rDNA sequence of Lactobacillus crispatus was greater than 99%, so the strains were named Lc31 and Lc83 of Lactobacillus crispatus.
  • the colonies of Lc31 and Lc83 of Lactobacillus crispatus provided by the present invention are milky white, round, opaque and stable in size and shape on MRS solid medium.
  • the present invention provides a Lactobacillus crispatus metabolite.
  • the metabolites are obtained by inoculating Lc31 and Lc83 of Lactobacillus crispatus in MRS medium, respectively, and the metabolites are cell-free supernatants after filtration of the culture medium.
  • the present invention also provides applications of the Lactobacillus crispatus Lc31 and Lc83 in the prevention and/or treatment of cervical squamous cell carcinoma, not for the purpose of disease diagnosis and treatment.
  • the Lc31 and Lc83 Lc31 and Lc83 of the present invention can well adhere to the surface of cervical squamous cell carcinoma SiHa cells in vitro; their metabolites can directly inhibit the growth, proliferation, migration and invasion of SiHa cells, and promote the apoptosis of SiHa cells ; It has great application prospects in the prevention and/or treatment of cervical squamous cell carcinoma (not for the purpose of disease diagnosis and treatment).
  • the present invention has obtained two strains of Lactobacillus crispatus Lc31 and Lc83, and this Lactobacillus crispatus and/or this Lactobacillus crispatus metabolite can prevent and/or treat cervical squamous cell carcinoma, and is embodied in: this Lactobacillus crispatus can in vitro It adheres well to the surface of cervical squamous cell carcinoma SiHa cells; its metabolites can directly inhibit the growth, proliferation, migration and invasion of cancer SiHa cells, and promote the apoptosis of SiHa cells. This Lactobacillus crispatus can prevent and/or treat cervical squamous cell carcinoma. Aspects (not for the purpose of disease diagnosis and treatment) have huge application prospects.
  • Lactobacillus crispatus is a kind of probiotic bacteria, which has been included in the "List of Bacteria that Can Be Used in Food" issued by the Ministry of Health, and will not bring any potential safety hazards to the human body. Therefore, the Lactobacillus crispatus obtained by the present invention When Lc31 and Lc83 are used for the prevention and/or treatment of cervical squamous cell carcinoma, the safety is high.
  • the referenced biological material Lc31 the deposit number is CGMCC No. 21348, the proposed classification is named Lactobacillus crispatus, the biological material was received on December 10, 2020 by the General Microbiology Center of the China Microorganism Preservation and Management Committee, and was received in 2020. Tested on December 10, 2009, the result was alive.
  • FIG. 1 is a graph showing the results of morphological identification of two Lactobacillus crispatus strains provided in the embodiment of the present invention, wherein FIG. 1A is a colony of Lactobacillus crispatus Lc31, FIG. 1B is a Gram staining result of a colony of Lactobacillus crispatus Lc31, and FIG. 1C is a colony of Lactobacillus crispatus Lc31.
  • Bacillus Lc83 colony, Figure 1D is the Gram staining result of Lactobacillus crispatus Lc83 colony;
  • Fig. 2 is a graph showing the results of a cell adhesion test provided in the embodiment of the present invention, wherein Fig. 2A shows that Lactobacillus crispatus Lc31 adheres to the surface of cervical squamous cell carcinoma SiHa cells, and Fig. 2B shows that Lactobacillus crispatus Lc83 adheres to cervical squamous cell carcinoma SiHa cells surface;
  • FIG. 3 is a graph showing the results of CCK-8 detection of cell proliferation provided in the embodiment of the present invention.
  • 4 is a graph of the results of 7-day plate cloning provided in the embodiment of the present invention, from top to bottom are PBS, MRS (pH3.5), Lc31 and Lc83 cell-free supernatants, with 4 duplicate wells in each group;
  • FIG. 5 is a graph showing the results of detecting apoptosis by flow cytometry according to an embodiment of the present invention.
  • FIG. 6 is a graph showing the results of detecting apoptosis by the Tunel method provided in the embodiment of the present invention.
  • FIG. 7 is a graph showing the results of cell cycle detection by flow cytometry according to an embodiment of the present invention.
  • FIG. 8 is a graph showing the results of cell scratching provided by the embodiment of the present invention.
  • MRS liquid medium The components of MRS liquid medium are as follows: peptone 10.0g, beef extract 10.0g, yeast powder 5.0g, glucose 20.0g, diammonium hydrogen citrate 2.0g, sodium acetate (CH 3 OONa ⁇ 3H 2 O) 5.0g, dihydrogen phosphate Potassium (K 2 HP 4 ⁇ 3H 2 O) 2.0g, magnesium sulfate (MgSO 4 ⁇ 7H 2 O) 0.58g, manganese sulfate (MnSO 4 ⁇ H 2 O) 0.25g, Tween-801.0mL, distilled water 1L, adjust pH to 6.2-6.6.
  • MRS solid medium The components of MRS solid medium are as follows: peptone 10.0g, beef extract 10.0g, yeast powder 5.0g, glucose 20.0g, diammonium hydrogen citrate 2.0g, sodium acetate (CH 3 OONa ⁇ 3H 2 O) 5.0g, dihydrogen phosphate Potassium (K 2 HP 4 ⁇ 3H 2 O) 2.0g, magnesium sulfate (MgSO 4 ⁇ 7H 2 O) 0.58g, manganese sulfate (MnSO 4 ⁇ H2O) 0.25g, Tween-801.0mL, distilled water 1L, adjust pH to 6.2-6.6; agar powder 13g.
  • Vaginal secretion samples were collected from healthy women of childbearing age with no signs of vaginitis and normal vaginal microecological examination.
  • a single colony with the same morphology was picked from each plate to 10 mL of MRS liquid medium, and cultured at 37°C for 24 hr.
  • the bacterial cells in each tube were collected separately for culture preservation and DNA preparation.
  • the isolated bacteria were identified for bacterial species; acid production, H 2 O 2 production, bacterial self-flocculation, and adhesion to cervical squamous cell carcinoma SiHa cells were detected, and two strains of bacteria with better probiotic properties were screened. Named Lc31 and Lc83.
  • the isolated strain Lc31 showed: opaque, protruding milky white colonies about 1-2 mm in size, and the colony edges were neat and stable in size and shape, as shown in Figure 1A; Gram staining positive, rod , which can be connected into chains, as shown in Figure 1B.
  • the isolated strain Lc83 showed: opaque, protruding milky white colonies about 2-4 mm in size, and the colony edges were neat and stable in size and shape, as shown in Figure 1C; Gram staining was positive, rod-shaped, and could be connected into chains, see Figure 1D.
  • 16s rDNA sequence homology analysis use MRS liquid medium at 37°C to routinely culture the above-isolated strains Lc31 and Lc83, centrifuge to obtain bacterial cells, extract genomic DNA as a gene amplification template, and use universal primer 27F (SEQ ID NO. 3) and 1492R (SEQ ID NO.4) to amplify the conserved region of bacterial 16S rDNA gene.
  • the amplification system (20 ⁇ L) was: 1 ⁇ PCR reaction buffer, 200 ⁇ mol/L dNTPs, 0.2 ⁇ mol/L upstream and downstream primers, 1 U FastPfu DNA polymerase, 1 ⁇ L template DNA. Reaction conditions: pre-denaturation at 95 °C for 2 min; denaturation at 95 °C for 20 s, annealing at 55 °C for 20 s, extension at 72 °C for 60 s, a total of 30 cycles; final extension at 72 °C for 5 min. PCR products were detected by 1% agarose gel electrophoresis, and positive results were sequenced in two directions using universal primers 27F and 1492R.
  • strains Lc31 and Lc83 were deposited in the China General Microorganism Culture Collection on December 10, 2020, with the preservation numbers of CGMCC No. 21348 and CGMCC No. 21349, and the classification names were Lactobacillus crispatus.
  • Example 2 Prebiotic effects of Lactobacillus crispatus Lc31 and Lc83
  • the preparation method of Lactobacillus crispatus cell-free supernatant is as follows: the Lactobacillus crispatus cells are inoculated into the MRS liquid medium with an inoculum amount of 2% of the total volume of the MRS liquid medium, and cultured at 37° C. for 48 hours to obtain a culture solution; The culture solution was centrifuged at 6000 g for 5 min and filtered with a 0.22 ⁇ m sterile filter to obtain a cell-free supernatant of Lactobacillus crispatus.
  • the pH values of the cell-free supernatant of Lc31 and Lc83 were stable between 3.0 and 3.5, indicating that the two strains of L. crispatus have strong acid-producing ability , plays an important role in maintaining the acidic environment of the lower reproductive tract.
  • Nanjing Jiancheng Hydrogen Peroxide Detection Kit was used to detect the H 2 O 2 content in the above cell-free supernatant.
  • the results showed that both Lc31 and Lc83 could produce H 2 O 2 , and the contents were 109 ⁇ 5mg/L and 102 ⁇ 4mg/L, respectively. , indicating that the two strains of Lactobacillus crispatus have strong H 2 O 2 production capacity, which plays an important role in inhibiting pathogenic bacteria in the lower genital tract and maintaining the micro-ecological environment.
  • the Lactobacillus crispatus cells were inoculated into the MRS liquid medium with an inoculum amount of 2% of the total volume of the MRS liquid medium, and cultured at 37 ° C for 48 hr to obtain a culture solution; the culture solution was centrifuged at 6000 g for 5 min and the precipitated cells were collected. Wash three times with PBS buffer, then resuspend in PBS buffer; use a spectrophotometer to detect the OD 600 value of the resuspension, adjust with PBS buffer so that the final OD 600 value is around 1.0 (OD 0 ); after standing at room temperature for 4 hr Measure the OD 600 value (OD 4 ) again, and calculate the self-flocculation rate.
  • the formula is as follows:
  • Self-flocculation rate% [(OD 0 -OD 4 )/OD 0 ] ⁇ 100%
  • Example 3 Effects of Lactobacillus crispatus strains Lc31 and Lc83 on direct inhibition of cervical squamous cell carcinoma SiHa cells
  • Cervical squamous cell carcinoma SiHa cells were stimulated with Lc31 and Lc83 cell-free supernatants of Lactobacillus crispatus strains, respectively, with PBS as blank control, sterile PBS as blank control, and MRS liquid medium (pH adjusted to 3.5 with HCl) as negative control.
  • the cells in the logarithmic growth phase were digested with trypsin, prepared into a cell suspension with a concentration of 1 ⁇ 10 5 cells/mL, and seeded in a 96-well plate at 10,000 cells/well, adding 100 ⁇ L to each well, and placed in CO 2 (5% ) in an incubator at 37°C for 24 hours to adhere to the wall; continue to culture for 24 hours, respectively, replaced with 100 ⁇ L of cell suspension (premixed with 10 ⁇ L of control or cell-free supernatant), and the control group was replaced with solvent-containing medium. Set five repetitions for each set.
  • the cell survival rate of the Lc31 cell-free supernatant group was 83%
  • the cell survival rate of the Lc83 cell-free supernatant group was 73%
  • the blank and control groups were 100% and 99%, respectively. It shows that the above cell-free supernatant has a certain inhibitory effect on the proliferation of cervical squamous cell carcinoma SiHa cells.
  • Each group consists of three cells. After culturing for 24 hours, the supernatant of each well was removed, and 1 mL of culture medium and 100 ⁇ L of the corresponding samples of each group were added to continue to stimulate the cells; after 48 hours, the cells were washed once with PBS, and 0.5 mL of 0.25% trypsin was added to digest the cells.
  • the apoptosis rates of PBS and MRS (pH3.5) groups were 4.4% and 4.3%, and those of Lc31 and Lc83 cell-free supernatants were 15.8% and 24.7%, respectively;
  • the serum significantly promoted the apoptosis of cervical squamous cell carcinoma SiHa cells.

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Abstract

一种能够预防和/或治疗子宫颈鳞癌的卷曲乳杆菌(Lactobacillus crispatus)Lc31和Lc83。卷曲乳杆菌Lc31的保藏编号为CGMCC No.21348，卷曲乳杆菌Lc83的保藏编号为CGMCC No.21349。该卷曲乳杆菌在体外能够很好的粘附在子宫颈鳞癌SiHa细胞表面，其代谢产物可以抑制SiHa细胞的生长增殖和迁移侵袭，促进SiHa细胞的凋亡。

Description

一种能够预防和/或治疗子宫颈鳞癌的卷曲乳杆菌
本申请要求于2021年01月08日提交中国专利局、申请号为2021100256658、发明名称为“一种能够预防和/或治疗子宫颈鳞癌的卷曲乳杆菌”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。
技术领域
本发明涉及一种能够预防和/或治疗子宫颈鳞癌的卷曲乳杆菌(Lactobacillus crispatus)，属于微生物技术领域。
背景技术
子宫颈癌是女性生殖系统最常见的恶性肿瘤，严重威胁女性生命健康。子宫颈鳞癌是子宫颈癌的主要病理类型，约占全部子宫颈癌的70％。人乳头瘤病毒(Human papillomavirus，HPV)感染是子宫颈癌的主要病因。然而，近年来研究证实女性阴道微生态与HPV感染及子宫颈癌的发生发展也密切相关。
女性阴道是一个复杂的微生态系统，由鳞状上皮和阴道菌群共同组成。健康育龄女性阴道通常以乳杆菌为主导优势菌。虽然乳杆菌属的细菌已经超过200种，但在女性阴道中占据主导优势地位的通常只有四种，分别为：卷曲乳杆菌(L.crispatus)、加氏乳杆菌(L.gasseri)、惰性乳杆菌(L.iners)和詹氏乳杆菌(L.jensensii)。根据主导优势菌的种类，阴道微生态环境分为五种类型，其中CST I、II、III和V中优势菌分别为以上四种乳酸杆菌，CST IV为非乳酸杆菌占优势的类型。
在亚洲女性阴道中，以卷曲乳杆菌和惰性乳杆菌为主的CST I和III较为常见，约占29.5％和21.4％；而CST II和V相对罕见，所占比例不足2％。虽然惰性乳杆菌的泛滥度高于卷曲乳杆菌，但在健康及患有生殖道感染性疾病(包括HPV感染)的女性中没有明显差异；并且惰性乳杆菌允许严格厌氧的病原菌与之共存，一旦这些厌氧菌占据优势，CST III即转换为CST IV。相比之下，卷曲乳杆菌很少与其它微生物共存，CST I较CST III更为稳定。此外，卷曲乳杆菌仅在健康女性中泛滥度和丰度较高，在患有生殖道感染性疾病(特别是HPV感染及子宫颈病变)的女性中泛滥度和丰度均显著降低，是潜在的阴道益生菌。此外，与惰性乳杆菌为主的CST III相 比，以卷曲乳杆菌为主的CST I能够更快速地由HPV阳性状态转为阴性；在所有CST类型中，CST I感染HPV及hrHPV的风险最低，进展为子宫颈病变或子宫颈癌的风险也最低。由此可知，卷曲乳杆菌与HPV感染及子宫颈病变显著负相关，在子宫颈癌的发生发展过程中发挥潜在的保护作用，有望成为预防和治疗子宫颈癌新的技术手段。
发明内容
为了解决上述问题，本发明提供了一种能够预防和/或治疗子宫颈鳞癌的卷曲乳杆菌。
为了实现上述目的，本发明提供如下技术方案：
为解决上述问题，本发明提供了两株卷曲乳杆菌(Lactobacillus crispatus)Lc31和Lc83，所述卷曲乳杆菌保藏于中国普通微生物菌种保藏中心，保藏编号分别为CGMCC No.21348和CGMCC No.21349，保藏日期均为2020年12月10日。
本发明提供的卷曲乳杆菌Lc31和Lc83分别来源于安徽地区两名32周岁和22周岁健康女性的阴道分泌物，菌株经测序分析，其16S rDNA序列分别如SEQ ID NO.1和SEQ ID NO.2所示，将测序得到的序列在GeneBank中进行核酸序列比对，结果显示其与卷曲乳杆菌的16S rDNA序列一致性大于99％，因此将菌株命名为卷曲乳杆菌Lc31和Lc83。
本发明提供的卷曲乳杆菌Lc31和Lc83在MRS固体培养基上菌落呈乳白色，圆形，不透明，大小形态稳定。
另一方面，本发明提供了一种卷曲乳杆菌代谢产物。所述代谢产物由卷曲乳杆菌Lc31和Lc83分别接种于MRS培养基中培养获得，所述代谢产物为培养液过滤后的无细胞上清液。
再一方面，本发明还提供了所述卷曲乳杆菌Lc31和Lc83在预防和/或治疗子宫颈鳞癌方面不以疾病的诊断和治疗为目的的应用。
具体地，本发明的卷曲乳杆菌Lc31和Lc83在体外能够很好的粘附在子宫颈鳞癌SiHa细胞表面；其代谢产物可以直接抑制SiHa细胞的生长增殖和迁移侵袭，促进SiHa细胞的凋亡；在预防和/或治疗子宫颈鳞癌方面(不以疾病的诊断和治疗为目的)具有巨大的应用前景。
有益效果：
1、本发明获得了两株卷曲乳杆菌Lc31和Lc83，此卷曲乳杆菌和/或 此卷曲乳杆菌代谢产物能够预防和/或治疗子宫颈鳞癌，具体体现在：此卷曲乳杆菌在体外能够很好的粘附在宫颈鳞癌SiHa细胞表面；其代谢产物可以直接抑制癌SiHa细胞的生长增殖和迁移侵袭，促进SiHa细胞的凋亡，此卷曲乳杆菌在预防和/或治疗子宫颈鳞癌方面(不以疾病的诊断和治疗为目的)具有巨大的应用前景。
2、卷曲乳杆菌是益生菌的一种，已被纳入卫生部下发的《可用于食品的菌种名单》，不会给人体带来任何潜在的安全隐患，因此，本发明获得的卷曲乳杆菌Lc31和Lc83用于预防和/或治疗子宫颈鳞癌时，安全性较高。
生物保藏信息
两株卷曲乳杆菌Lc31和Lc83，分类学名Lactobacillus crispatus，已于2020年12月10日保藏于中国普通微生物菌种保藏中心(简称CGMCC)，保藏地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏编号分别为CGMCC No.21348和CGMCC No.21349。
参据的生物材料Lc31，保藏编号为CGMCC No.21348，建议的分类命名为卷曲乳杆菌Lactobacillus crispatus，该生物材料于2020年12月10日由中国微生物保藏管理委员会普通微生物中心收到，于2020年12月10日检测，结果是存活。
参据的生物材料Lc83，保藏编号为CGMCC No.21349，建议的分类命名为卷曲乳杆菌Lactobacillus crispatus，该生物材料于2020年12月10日由中国微生物保藏管理委员会普通微生物中心收到，于2020年12月10日检测，结果是存活。
附图说明
图1是本发明实施例提供的两株卷曲乳杆菌株的形态学鉴定结果图，其中图1A为卷曲乳杆菌Lc31菌落，图1B为卷曲乳杆菌Lc31菌落革兰染色结果，图1C为卷曲乳杆菌Lc83菌落，图1D为卷曲乳杆菌Lc83菌落革兰染色结果；
图2是本发明实施例提供的细胞黏附试验结果图，其中图2A为卷曲乳杆菌Lc31粘附在子宫颈鳞癌SiHa细胞表面，图2B为卷曲乳杆菌Lc83粘附在子宫颈鳞癌SiHa细胞表面；
图3是本发明实施例提供的CCK-8检测细胞增殖结果图；
图4是本发明实施例提供的7天平板克隆结果图，自上而下分别为PBS、MRS(pH3.5)、Lc31和Lc83无细胞上清液，每组4个复孔；
图5是本发明实施例提供的流式细胞术检测凋亡结果图；
图6是本发明实施例提供的Tunel法检测凋亡结果图；
图7是本发明实施例提供的流式术检测细胞周期结果图；
图8是本发明实施例提供的细胞划痕结果图。
具体实施方式
下面结合具体实施例，对本发明作进一步详细的阐述，下述实施例不用于限制本发明，仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明，实施例中未注明具体条件的实验方法，通常按照常规条件，下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、卷曲乳杆菌Lc31和Lc83的分离与鉴定
MRS液体培养基成分如下：蛋白胨10.0g，牛肉膏10.0g，酵母粉5.0g，葡萄糖20.0g，柠檬酸氢二铵2.0g，乙酸钠(CH 3OONa·3H 2O)5.0g，磷酸氢二钾(K 2HP 4·3H 2O)2.0g，硫酸镁(MgSO 4·7H 2O)0.58g，硫酸锰(MnSO 4·H 2O)0.25g，吐温-801.0mL，蒸馏水1L，调pH至6.2-6.6。
MRS固体培养基成分如下：蛋白胨10.0g，牛肉膏10.0g，酵母粉5.0g，葡萄糖20.0g，柠檬酸氢二铵2.0g，乙酸钠(CH 3OONa·3H 2O)5.0g，磷酸氢二钾(K 2HP 4·3H 2O)2.0g，硫酸镁(MgSO 4·7H 2O)0.58g，硫酸锰(MnSO 4·H2O)0.25g，吐温-801.0mL，蒸馏水1L，调pH至6.2-6.6；琼脂粉13g。
分离步骤如下：
收集无阴道炎体征、阴道微生态检查正常的健康育龄女性的阴道分泌物样品，编号后分别接种于10mLMRS液体培养基，37℃培养24hr。
分别取一环MRS培养物，在MRS固体平板上三区划线，37℃培养48hr。
从各平板上挑取形态一致的单菌落至10mL MRS液体培养基，37℃ 培养24hr。
分别收集各管中细菌菌体用于菌种保藏和DNA制备。将分离到的细菌分别进行菌种鉴定；进行产酸、产H 2O 2、菌体自絮凝、与子宫颈鳞癌SiHa细胞粘附作用的检测，筛选其中两株益生性状较好的菌，命名为Lc31和Lc83。
对上述分离筛选获得的菌株Lc31和Lc83进行以下鉴定：
形态学鉴定：在MRS平板上，分离得到的菌株Lc31表现为：大小约1-2毫米、不透明、突起的乳白色菌落，且菌落边缘整齐，大小形态稳定，见图1A；革兰染色阳性，杆状，可连成链状，见图1B。分离得到的菌株Lc83表现为：大小约2-4毫米、不透明、突起的乳白色菌落，且菌落边缘整齐，大小形态稳定，见图1C；革兰染色阳性，杆状，可连成链状，见图1D。
16s rDNA序列同源性分析：使用MRS液体培养基37℃培养常规培养上述分离得到的菌株Lc31和Lc83，离心获得菌体，提取基因组DNA作为基因扩增模板，采用通用引物27F(SEQ ID NO.3)和1492R(SEQ ID NO.4)扩增细菌16S rDNA基因保守区。
扩增体系(20μL)为：1×PCR反应缓冲液，200μmol/L dNTPs，上游及下游引物各0.2μmol/L，1U FastPfu DNA聚合酶，1μL模板DNA。反应条件：95℃预变性2min；95℃变性20s，55℃退火20s，72℃延伸60s，共30个循环；72℃终延伸5min。PCR产物用1％琼脂糖凝胶电泳检测，阳性结果用通用引物27F和1492R进行双向测序。序列拼接及相似性分析使用DNAMAN软件完成，序列比对通过美国国家生物技术信息中心NCBI数据库(http://www.ncbi.nlm.nih.gov)在线完成，结果显示Lc31和Lc83均与卷曲乳杆菌的16S rDNA序列一致性大于99％，确定为卷曲乳杆菌。菌株Lc31的16s rDNA序列见SEQ ID NO.1，菌株Lc83的16s rDNA序列见SEQ ID NO.2。
经过上述鉴定，菌株Lc31和Lc83于2020年12月10日，保藏于中国普通微生物菌种保藏中心，保藏编号为CGMCC No.21348和CGMCC No.21349，分类命名为卷曲乳杆菌(Lactobacillus crispatus)。
实施例2：卷曲乳杆菌Lc31和Lc83的益生作用
卷曲乳杆菌无细胞上清液制备方法如下：将卷曲乳杆菌菌体以占MRS液体培养基总体积2％的接种量接种至MRS液体培养基中，于37℃培养48hr，得到培养液；将培养液6000g离心5min后以0.22μm的无菌滤膜过滤，得到卷曲乳杆菌无细胞上清液。
(1)卷曲乳杆菌Lc31和Lc83的产酸能力
使用pH计检测上述无细胞上清液pH值，卷曲乳杆菌Lc31和Lc83的无细胞上清液pH值均稳定在在3.0-3.5之间，表明这两株卷曲乳杆菌产酸能力均较强，对维持下生殖道酸性环境有重要作用。
(2)卷曲乳杆菌Lc31和Lc83的产H 2O 2能力
使用南京建成过氧化氢检测试剂盒检测上述无细胞上清液中H 2O 2含量，结果显示Lc31和Lc83均能产生H 2O 2，含量分别为109±5mg/L和102±4mg/L，表明这两株卷曲乳杆菌均有较强的产H 2O 2能力，对抑制下生殖道病原菌、维持微生态环境有重要作用。
(3)卷曲乳杆菌Lc31和Lc83的自絮凝能力
将卷曲乳杆菌菌体以占MRS液体培养基总体积2％的接种量接种至MRS液体培养基中，于37℃培养48hr，得到培养液；将培养液6000g离心5min后收集沉淀菌体，以PBS缓冲液洗涤三次，然后重悬于PBS缓冲液中；使用分光光度计检测重悬液OD 600值，用PBS缓冲液调整使得最终OD 600值在1.0(OD 0)左右；室温静置4hr后再测OD 600值(OD 4)，计算自絮凝率，公式如下：
自絮凝率％＝[(OD 0-OD 4)/OD 0]×100％
计算结果显示卷曲乳杆菌Lc31和Lc83的自絮凝率分别为89％和84％，表明表明这两株卷曲乳杆菌自絮凝能力均较强，对抑制下生殖道病原菌、维持微生态环境有重要作用。
(4)卷曲乳杆菌Lc31和Lc83粘附子宫颈鳞癌SiHa细胞的能力
在6孔板各孔中分别放入无菌盖玻片，每孔接种5×10 5个子宫颈鳞癌SiHa细胞，使用高糖DMEM培养基(含10％胎牛血清和100X青霉素-链霉素溶液)置于CO 2培养箱中37℃培养过夜；换液后继续培养24hr，吸净培养基，PBS洗涤三遍；每孔加入1mL卷曲乳杆菌Lc31或Lc83悬液(预先使用MRS培养48hr，用PBS洗涤三遍后重悬，并用调整菌体浓 度为5×10 7个细菌/mL)，CO 2培养箱中继续放置30min；吸净菌液，PBS洗涤三遍，4％多聚甲醛固定10min，革兰染色，油镜观察。如图2A和图2B所示，卷曲乳杆菌Lc31和Lc83均可大量粘附到子宫颈鳞癌SiHa细胞表面。
实施例3：卷曲乳杆菌菌株Lc31和Lc83直接抑制子宫颈鳞癌SiHa细胞的作用
使用卷曲乳杆菌菌株Lc31和Lc83无细胞上清液分别刺激子宫颈鳞癌SiHa细胞，以PBS作为空白对照，使用无菌PBS作为空白对照，以MRS液体培养基(HCl调pH至3.5)为阴性对照。
子宫颈鳞癌SiHa细胞常规培养条件：高糖DMEM培养基(含10％胎牛血清和100X青霉素-链霉素溶液)；置于5％CO 2培养箱中37℃培养。以下未做特别说明时均使用此常规条件培养。
(1)CCK8法检测子宫颈鳞癌SiHa细胞的增殖
将对数生长期细胞用胰蛋白酶消化，配制成浓度为1×10 5个/mL的细胞悬液，按10000细胞/孔接种于96孔板，每孔加100μL，置于CO 2(5％)培养箱中37℃下培养24hr以贴壁；继续培养24h分别更换为100μL细胞悬液(预混有10μL对照或无细胞上清液)，对照组更换为含溶剂的培养基。每组设五个重复。所有孔中加入10μL CCK-8溶液，培养箱中孵育1-4小时；使用酶标仪测定450nm光吸收值，以溶剂处理的细胞为对照组，不含细胞的培养基为空白组，按公式计算各组成分对细胞的存活率，计算公式如下：
细胞存活率％＝(实验组-空白组)/(对照组-空白组)×100％
原始数据如表1所示：
表1各组别的细胞存活率
分组 1 2 3 4 5 Mean SD 百分比
PBS 0.683 0.725 0.680 0.749 0.686 0.705 0.031 100.000
MRS 0.704 0.687 0.695 0.730 0.674 0.698 0.021 99.063
Lc31 0.589 0.563 0.594 0.607 0.580 0.587 0.016 83.253
Lc83 0.462 0.526 0.551 0.503 0.517 0.512 0.033 72.637
如图3所示，Lc31无细胞上清液组细胞存活率83％，Lc83无细胞上 清液组细胞存活率73％，空白和对照组分别为100％和99％。表明上述无细胞上清液对子宫颈鳞癌SiHa细胞的增殖具有一定的抑制作用。
(2)平板克隆形成实验检测子宫颈鳞癌SiHa细胞的增殖
向24孔板每孔分别接种500μL SiHa细胞悬液(含1000个细胞/孔)，分别加入10μL PBS、MRS(pH3.5)、Lc31和Lc83无细胞上清液，每组设四个复孔；24hr换液后每组继续加入对应液体；96hr换液后每组继续加入对应液体；培养第7天时吸尽孔内液体，PBS洗涤三遍，结晶紫染色观察。如图4所示，MRS(pH3.5)对SiHa细胞增殖有所抑制，但与之相比，Lc31和Lc83无细胞上清液对SiHa细胞增殖的抑制作用更为显著。
(3)流式细胞术检测子宫颈鳞癌SiHa细胞的凋亡
向6孔板每孔分别接种1mL SiHa细胞悬液(含5×10 5个细胞/孔)，分别加入100μL PBS、MRS(pH3.5)、Lc31和Lc83无细胞上清液，每组设三个复孔；培养24hr后吸去每孔的上清液，重新加入1mL培养基及100μL相应的各组样品，继续刺激细胞；48hr用PBS洗涤一次，加入0.5mL0.25％胰酶消化细胞，待细胞变圆且有部分细胞脱落，即加入培养基终止消化；用移液枪轻轻吹打细胞，使细胞悬浮；收集于预处理后的流式管中，300g离心5min，弃上清；加入1mLPBS重新悬浮细胞，300g离心5min，弃上清；沉淀用300μL的Binding Buffer重悬；加入5μL Annexin V-FITC，混匀后避光孵育10min；加入5μL PE，混匀后避光孵育5min；1h内上机检测。如图5所示，PBS和MRS(pH3.5)组细胞凋亡率为4.4％和4.3％，Lc31和Lc83无细胞上清液组分别为为15.8％和24.7％；Lc31和Lc83无细胞上清液显著促进子宫颈鳞癌SiHa细胞发生凋亡。
(4)Tunel法检测子宫颈鳞癌SiHa细胞的凋亡
向6孔板每孔分别放入无菌盖玻片，分别接种1mL SiHa细胞悬液(含5×10 5个细胞/孔)，分别加入100μL PBS、MRS(pH3.5)、Lc31和Lc83无细胞上清液，每组设三个复孔；培养24hr后吸去每孔的上清液，重新加入1mL培养基及100μL相应的各组样品，继续刺激细胞；48hr用PBS洗涤三次，4％多聚甲醛固定30min，PBS洗3次；爬片稍干后用组化笔在盖玻片中间细胞分布均匀的位置画圈(防止抗体流走)，加50-100μL 破膜工作液，室温孵育10min，PBS洗3次；圈内滴加3％过氧化氢溶液，室温避光孵育20min，将玻片置于PBS(pH7.4)中在脱色摇床上晃动洗涤3次；取tunel试剂盒内适量试剂1(TdT)和试剂2(dUTP)按1:9混合，加到爬片上覆盖住细胞，盖上孔板的盖子，用锡纸包住平放于水浴锅内，37℃水浴锅孵育60min；爬片稍甩干后，每孔内加适量试剂3(converter-POD)覆盖细胞，切片平放于湿盒内，37℃孵育30min；将玻片置于PBS(pH7.4)中在脱色摇床上晃动洗涤3次；爬片稍干后在圈内滴加适量DAPI染液到爬片上，室温避光孵育5min；PBS漂洗爬片3次，从6孔板内取出爬片，将有细胞的一面封到滴加有抗荧光淬灭封片剂的载玻片上，荧光显微镜下观察并拍照；如图6所示，PBS和MRS(pH3.5)组细胞凋亡率为6％和5.8％，Lc31和Lc83无细胞上清液组分别为为14.9％和26.6％；Lc31和Lc83无细胞上清液显著促进子宫颈鳞癌SiHa细胞发生凋亡。
(5)流式细胞术检测子宫颈鳞癌SiHa细胞的周期变化
向6孔板每孔分别接种1mL SiHa细胞悬液(含5×10 5个细胞/孔)，分别加入100μL PBS、MRS(pH3.5)、Lc31和Lc83无细胞上清液，每组设三个复孔；培养24hr后吸去每孔的上清液，重新加入1mL培养基及100μL相应的各组样品，继续刺激细胞；48hr用PBS洗涤一次，加入1mL0.25％胰酶消化细胞，待细胞变圆且有部分细胞悬浮，即加入PBS终止消化；用移液枪轻轻吹打细胞，使细胞悬浮；细胞悬液转入离心管中，300g离心5min收集细胞；沉淀用3mL PBS重悬，300g离心5min收集细胞；沉淀中缓慢加入预冷的90％乙醇，重悬细胞，4℃孵育20min，300g离心5min收集细胞；3mL PBS重悬细胞，300g离心5min；加入500μL试剂盒里的RNase/PI重悬细胞，避光染色20min；上机检测，选择相应通道观察；如图7所示，与PBS和MRS(pH3.5)组相比，Lc31和Lc83无细胞上清液组细胞周期受到明显阻滞。
(6)细胞划痕实验检测子宫颈鳞癌SiHa细胞的迁移能力
在6孔板背后均匀画平行的直线，约0.5-1cm一道，横穿过孔，每孔5条；每孔分别接种2mL SiHa细胞悬液(含5×10 5个细胞/孔)，待细胞铺满孔底后用枪头垂直于横线划掉细胞；PBS轻轻漂洗掉划下的细胞， 加入1mL培养基；分别加入100μL PBS、MRS(pH3.5)、Lc31和Lc83无细胞上清液，每组设三个复孔；培养24hr后吸去每孔的上清液，重新加入1mL培养基及100μL相应的各组样品，继续刺激细胞；48hr取出孔板拍照、测量距离并计算伤口愈合；如图8所示，PBS和MRS(pH3.5)组伤口愈合率为72.4％和71.9％，Lc31和Lc83无细胞上清液组分别为为32.89％和22.69％；Lc31和Lc83无细胞上清液明显抑制了子宫颈鳞癌SiHa细胞的迁移能力。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其它实施例，这些实施例都属于本发明保护范围。

Claims (11)

  1. 两株卷曲乳杆菌(Lactobacillus crispatus)，其特征在于，所述卷曲乳杆菌保藏于中国普通微生物菌种保藏中心，其中卷曲乳杆菌(Lactobacillus crispatus)Lc31的保藏编号为CGMCC No.21348，卷曲乳杆菌(Lactobacillus crispatus)Lc83的保藏编号为CGMCC No.21349，保藏日期均为2020年12月10日。
  2. 根据权利要求1所述两株卷曲乳杆菌，其特征在于，所述卷曲乳杆菌Lc31的16s rDNA序列如SEQ ID NO.1所示；所述卷曲乳杆菌Lc83的16s rDNA序列如SEQ ID NO.2所示。
  3. 一种扩增权利要求1或2所述两株卷曲乳杆菌的方法，其特征在于，包括以下步骤：分别以所述两株卷曲乳杆菌的菌体提取基因组DNA作为基因扩增模板，以引物27F和引物1492R扩增所述两株卷曲乳杆菌的16S rDNA基因保守区；
    所述引物27F的核苷酸如SEQ ID NO.3所示，所述引物1492R的核苷酸如SEQ ID NO.4所示。
  4. 根据权利要求3所述的方法，其特征在于，所述扩增的程序包括95℃预变性2min；95℃变性20s，55℃退火20s，72℃延伸60s，共30个循环；72℃终延伸5min。
  5. 权利要求1或2所述的两株卷曲乳杆菌和/或所述的两株卷曲乳杆菌的代谢产物在制备预防和/或治疗子宫颈鳞癌的产品中的应用。
  6. 一种预防和/或治疗子宫颈鳞癌的产品，其特征在于，所述产品含有权利要求1或2所述的两株卷曲乳杆菌和/或权利要求1或2所述的两株卷曲乳杆菌的代谢产物。
  7. 如权利要求6所述的产品，其特征在于，所述产品包含药品、卫生用品和食品。
  8. 如权利要求7所述的产品，其特征在于，所述药品的成分包含权利要求1或2所述卷曲乳杆菌以及药学上可接受的载体；或者，所述药品的成分包含权利要求1或2所述卷曲乳杆菌的代谢产物以及药学上可接受的载体；或者，所述药品的成分包含权利要求1或2所述卷曲乳杆菌、权利要求1或2所述卷曲乳杆菌的代谢产物以及药学上可接受的载体。
  9. 如权利要求7所述的产品，其特征在于，所述卫生用品包括消毒纸巾、卫生护垫和卫生棉条。
  10. 如权利要求7所述的产品，其特征在于，所述食品的成分包含权利要求1或2所述卷曲乳杆菌；或者，所述食品的成分包含权利要求1或2所述卷曲乳杆菌的代谢产物。
  11. 权利要求1或2所述的卷曲乳杆菌或权利要求6-11任一所述的一种用于预防和/或治疗子宫颈鳞癌的产品在抑制子宫颈鳞癌方面不以疾病的诊断和治疗为目的的应用。
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