WO2022011902A1 - 副干酪乳杆菌及其在制备治疗溃疡性结肠炎药物中的应用 - Google Patents

副干酪乳杆菌及其在制备治疗溃疡性结肠炎药物中的应用 Download PDF

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WO2022011902A1
WO2022011902A1 PCT/CN2020/128122 CN2020128122W WO2022011902A1 WO 2022011902 A1 WO2022011902 A1 WO 2022011902A1 CN 2020128122 W CN2020128122 W CN 2020128122W WO 2022011902 A1 WO2022011902 A1 WO 2022011902A1
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ulcerative colitis
lactobacillus paracasei
mice
strain
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PCT/CN2020/128122
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French (fr)
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曾今诚
黄娟
杨维青
张召
杨紫嫣
林碧华
陈涛
雷春燕
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佛山市朗芯生物科技有限公司
广东南芯医疗科技有限公司
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Priority to US17/130,146 priority Critical patent/US20220016187A1/en
Publication of WO2022011902A1 publication Critical patent/WO2022011902A1/zh

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • 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
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants

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  • the invention relates to the field of biotechnology, in particular to a strain of Lactobacillus paracasei L.p R3-10 and its application in preparing a medicine for treating ulcerative colitis.
  • Ulcerative colitis is a chronic non-specific intestinal inflammation whose etiology and pathogenesis have not yet been fully clarified.
  • the pathogenesis of UC may involve the interaction of genetics, microorganisms, immune system and environmental factors, and its treatment lacks specificity.
  • the commonly used drugs in clinical treatment of UC are aminosalicylic acid preparations, glucocorticoids and immunosuppressants. But these three drugs can only temporarily control and relieve clinical symptoms by inhibiting inflammation and immune response.
  • side effects such as long-term medication, large side effects, and easy recurrence after drug withdrawal, and its clinical application is limited. Therefore, it is imperative to find new safe and effective drugs for prevention and treatment.
  • Probiotics are a class of active microorganisms that are beneficial to the host. When ingested in sufficient quantities, they can colonize the host and maintain the balance of the host's intestinal flora, thus playing a beneficial role.
  • the microorganisms that can be used as probiotics are mainly lactic acid bacteria, which are roughly divided into three categories: Lactobacillus, Bifidobacterium and Gram-positive cocci. After years of exploration and development from discovery to clinical application, probiotics have begun to enter the public's field of vision.
  • probiotics for the prevention and treatment of many gastrointestinal diseases, including irritable bowel syndrome (IBS) and inflammatory bowel disease, has become a research hotspot.
  • IBS irritable bowel syndrome
  • probiotics have good prospects in the prevention and treatment of intestinal diseases such as UC, but their effects are strain-specific.
  • the present invention provides a strain of Lactobacillus paracasei L.p R3-10 and its application in preparing a medicine for treating ulcerative colitis.
  • Lactobacillus paracasei L.pR3-10 in the preparation of a drug for the treatment of ulcerative colitis.
  • Lactobacillus paracasei R3-10 (Lactobacillus paracasei R3-10, Lp R3-10) is a strain derived from Lactobacillus paracasei R3 (Lactobacillus paracasei R3, Lp R3) isolated from infant feces earlier in our laboratory. After acclimating Lp R3 for 10 generations by starvation The obtained Lactobacillus strain induced by low nutrient gradient tolerance; Lp R3-10 can be prepared as a medicine for treating ulcerative colitis.
  • Fig. 1 accompanying drawing is the colony morphology of L.p R3-10 of the present invention on MRS agar plate;
  • Fig. 2 accompanying drawing is the colony morphology of L.p R3-10 of the present invention on anaerobic blood agar plate;
  • Fig. 3 is the morphology under the microscope of L.p R3-10 Gram stain of the present invention, 1000 times of mirror;
  • Fig. 4 accompanying drawing is the change percentage of body weight of ulcerative colitis mouse model of the present invention.
  • FIG. 5 is the DAI score of the ulcerative colitis mouse model of the present invention.
  • Fig. 6 accompanying drawing is the colon morphology of each group of mice of the present invention.
  • A is the NS group
  • B is the DSS group
  • C is the MSLZ group
  • D is the L.p R3-10 group
  • E is the L.p R3-10+MSLZ group
  • FIG. 7 accompanying drawing is the colon length of ulcerative colitis mouse model of the present invention.
  • FIG. 8 accompanying drawing is the HE staining result of colon tissue of ulcerative colitis mouse model of the present invention.
  • A is the NS group
  • B is the DSS group
  • C is the MSLZ group
  • D is the L.p R3-10 group
  • E is the L.p R3-10+MSLZ group
  • Fig. 9 accompanying drawing is the histological damage score of each group of mice of the present invention.
  • Healthy infants aged 0-6 months were selected from school staff families as volunteers. Two weeks before sampling, normal diet was required and there was no recent history of intestinal infection and antibiotic use. The morning stool was taken on the day of sampling. After collection, the intelligent microbial separation system of Nanjing Famet Company was used to separate fecal bacteria. After the separation was completed, the crude fecal bacterial liquid was collected quickly, added with cryopreservation protection solution, and then placed in a -80°C ultra-low temperature refrigerator for future use.
  • the pure strains were transformed on MRS agar plates and passaged for 2 generations. A single colony was picked for Gram stain microscopy and catalase test. Gram-positive bacilli with typical Lactobacillus morphological characteristics and negative catalase test were preliminarily identified. for Lactobacillus.
  • the company uses TIANamp Bacteria DNA Kit (Tiangen, Beijing) to extract DNA from bacterial liquid.
  • TIANamp Bacteria DNA Kit (Tiangen, Beijing) to extract DNA from bacterial liquid.
  • bacterial universal primer 27F/1492R Using the bacterial universal primer 27F/1492R, a near-full-length bacterial 16S rDNA fragment was obtained by PCR expansion, and the DNA sequence of the bacterial 16S rDNA fragment was obtained by DNA sequencing. Alignment analysis is performed to obtain the species information with the most similar sequences, and the species information of the identified microorganisms is deduced according to the similarity of the sequences.
  • the identification number YSJ03 strain was identified as Lactobacillus paracasei, named L.p R3.
  • Colonies were inoculated in 6 mL of MRS liquid medium, and cultured under anaerobic conditions at 37 °C for 16-18 h.
  • the activated bacterial liquid was inoculated into 100 mL of MRS broth with a 3% (v/v) inoculum, 120 rpm/min, and shaken at 37 °C for 16-18 h. Centrifuge at 3500rpm/min for 10min, discard the supernatant, resuspend and wash twice with PBS (pH 7.2-7.4), and harvest the bacterial cells.
  • each tube is subpackaged with 1mL bacterial suspension, divided into 15 tubes, and shaken at 37°C for culture. From 0 h, take out an EP tube every 12 h, draw 100 ⁇ L of bacterial liquid, spread it on the surface of MRS medium, and cultivate it under anaerobic conditions at 37 °C for 48 h. The single colony with the longest survival time was picked, and the above process was carried out in the same way for 10 generations to obtain the L.p R3-10 strain induced by low nutrient gradient tolerance.
  • MRS agar plates were cultured under aerobic conditions at 37°C for 48 hours, and anaerobic blood agar plates were cultured under anaerobic conditions at 37°C.
  • the identification was performed using the API 50CHL bacterial biochemical identification system of Mérieux Bio, France. First, according to the instructions of the API 50CHL identification reagent strip, adjust the activated Lp R3-10 bacterial suspension to 2 McFarland turbidity, add it to 50 microbiological wells on the reagent strip, and cover the biochemical wells with sterile liquid paraffin . Incubate at 35°C for 24 hours to observe the results once, continue to culture for 48 hours, and observe the results again. The results were judged: the color of the 25th tube changed from purple to black as positive, the color of other tubes changed from purple to yellow as positive, otherwise it was negative. The reaction results of the strains were analyzed with API identification software to obtain the identification results of the strains. The L.p R3-10 biochemical reaction results are shown in Table 1. The identification result was Lactobacillus paracasei subsp. casein 1, the identification rate was 99.7%, and the T value was 0.79.
  • No. 0 tube is a blank control tube.
  • mice Twenty-five SPF grade female C57BL/6 mice (6-8 weeks old, body weight 16-18 g) were selected. The mice were randomly divided into 5 groups, namely normal group (normal saline, NS group), model group (DSS group), mesalazine group (MSLZ group), Lp R3-10 group (10 9 CFU/mL), Lp R3-10 combined with mesalazine group (Lp R3-10+MSLZ group), 5 mice in each group.
  • normal group normal saline, NS group
  • DSS group model group
  • MSLZ group mesalazine group
  • Lp R3-10 group (10 9 CFU/mL
  • Lp R3-10+MSLZ group 5 mice in each group.
  • the normal group and model group were given an equal volume of normal saline, and the other three groups were given prophylactic mesalazine (52 mg/mL), Lp R3-10 bacterial solution (1 ⁇ 10 9 CFU/mL), Lp R3 -10 bacterial solution (1 ⁇ 10 9 CFU/mL) combined with mesalamine (52 mg/mL) was administered by gavage for 7 days.
  • prophylactic mesalazine 52 mg/mL
  • Lp R3-10 bacterial solution (1 ⁇ 10 9 CFU/mL
  • Lp R3 -10 bacterial solution (1 ⁇ 10 9 CFU/mL
  • mice were sacrificed by neck-folding method, and the colon was taken out. Part of the distal colon tissue was fixed in formaldehyde, embedded in paraffin, sectioned, and stained with HE. During the experiment, the body weight, stool characteristics and occult blood of the mice were recorded every day.
  • mice in each group were observed daily, and the disease activity index (DAI) score (Table 2) was performed to evaluate the degree of colitis disease activity.
  • the weight of each mouse was weighed with an electronic balance every morning and feces were collected, and the percentage of weight loss was calculated.
  • Percentage of body weight loss (body weight on day 0-body weight on day n)/body weight on day 0 ⁇ 100%, according to the standard in the table below, the mice were scored according to the percentage of weight loss of mice, and the experimental results were recorded.
  • Figure 4 shows the results of the body weight percentage of the experimental mice in each group on the 7th day.
  • the results showed that the NS group remained basically unchanged, and the DSS group showed a significant decrease from the 4th day, and its body weight percentage on the 7th day was about 84%; the MSLZ group , Lp R3-10+MSLZ group and Lp R3-10 group body weight began to decrease from the 5th day, and the degree of decrease decreased in turn.
  • the DAI results of colitis in experimental mice in each group are shown in Figure 5.
  • the results show that: DSS group>MSLZ group>L.p R3-10+MSLZ group>L.p R3-10 group>NS group.
  • mice in each group were sacrificed by cervical dislocation, and the colon was taken out.
  • the colon morphology of the mice in each group is shown in Figure 6.
  • Measure the colon length of mice in each group with a ruler record the change of colon length after dissection of mice, calculate the average value and standard deviation of each group, and carry out relevant statistical analysis.
  • the results are shown in Figure 7.
  • the results in Figure 7 show that the average colon length of mice in each group is: NS group>L.pR3-10 group>L.pR3-10+MSLZ group>MSLZ group>DSS group.
  • mice were sacrificed, the colons of the mice in each group were taken out, fixed with 10% formaldehyde solution, embedded in paraffin and sliced.
  • 2 Immerse the paraffin sections in xylene solution and heat in microwave for 5 minutes; soak the paraffin sections in xylene solution again and heat in microwave for 5 minutes; then soak in absolute ethanol, 95% ethanol, 85% ethanol and 75% ethanol solution respectively 1min for a total of two times; then rinse with tap water.
  • 3 After hematoxylin staining for 5 min, rinse with running water, differentiate with 1% hydrochloric acid alcohol, and rinse with running water again; eosin staining for 1 min.
  • the NS group had a normal structure; the DSS group showed decreased mucosal epithelial cells, disordered crypt structure and texture, and inflammatory cell infiltration, manifested as UC; MSLZ group, Lp R3-10 group and Lp R3-10+MSLZ Compared with the DSS group, the histopathology of the group was significantly improved.
  • Histological damage score 0 points, normal and no inflammatory cell infiltration; 1 point, mild inflammatory cell infiltration, no damage to submucosal tissue; 2 points, moderate inflammatory cell infiltration and submucosal tissue destruction (injury range from 10% to 10%) 25%); 3 points, obvious inflammatory cell infiltration, destruction of submucosal tissue, thickening of the colon wall (injury range from 25% to 50%); 4 points, severe inflammatory cell infiltration, large-scale colon tissue damage (injury extent >50%) and colonic wall thickening.
  • the evaluation results are shown in Figure 9, the results show that the L.p R3-10 group and the L.p R3-10+MSLZ group have significantly lower scores of colon tissue damage than the DSS group.

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Abstract

一株副干酪乳杆菌L.p R3-10及其在制备治疗溃疡性结肠炎药物中的应用，属于生物技术领域。该菌是将L.p R3经饥饿驯化10代之后得到的一株低营养梯度耐受诱导的乳杆菌，其保藏编号为CGMCC No.19520。L.p R3-10能够制备成治疗溃疡性结肠炎的药物。

Description

副干酪乳杆菌及其在制备治疗溃疡性结肠炎药物中的应用 技术领域
本发明涉及生物技术领域，更具体的说是涉及一株副干酪乳杆菌L.p R3-10及其在制备治疗溃疡性结肠炎药物中的应用。
背景技术
溃疡性结肠炎(Ulcerative Colitis，UC)是一种病因和发病机制尚未完全明确的慢性非特异性肠道炎症，主要症状有腹泻、脓血便、腹痛和里急后重，常反复发作。近年来，随着人民生活水平的提高和饮食结构的改变，本病的发病率逐年上升。现普遍认为UC的发病可能涉及遗传、微生物、机体免疫系统和环境因素等多方面相互作用的结果，且其治疗缺乏特异性。目前临床治疗UC常用的药物为氨基水杨酸制剂、糖皮质激素及免疫抑制剂。但这三种药物通过抑制炎症和免疫反应，只能达到暂时控制和缓解临床症状的目的。且具有需长期服药，副作用大，停药后易复发等副作用，在临床应用受到限制。因此，寻找安全有效的新型防治药物势在必行。
益生菌(Probiotics)是一类对宿主有益的活性微生物，当摄入足够数量后能在宿主中定植并维持宿主肠道菌群平衡，从而发挥有益作用的活性微生物的总称。目前可以用作益生菌的微生物主要是乳酸菌，大体分为乳杆菌属、双歧杆菌属和革兰式阳性球菌属三大类。益生菌从发现到临床应用经历了多年的探索与开发，已经开始走进了大众的视野。其相关产品的临床应用也越来越广泛，功能主要包括调节胃肠道失调、增强肠道免疫功能、抗过敏反应和保护心血管系统等。益生菌用于预防和治疗包括肠易激综合征(IBS)、炎性肠道在内的许多胃肠道疾病成为研究热点。国内外的多项研究证实，益生菌在防治UC等肠道疾病中有很好的前景，但其作用效果具有菌株特异性。
因此，提供一株副干酪乳杆菌及其在制备治疗溃疡性结肠炎药物中的应用是本领域技术人员亟需解决的问题。
发明内容
有鉴于此，本发明提供了一株副干酪乳杆菌L.p R3-10及其在制备治疗溃疡性结肠炎药物中的应用。
为了实现上述目的，本发明采用如下技术方案：
一株副干酪乳杆菌L.p R3-10，其保藏编号为CGMCC No.19520，已保藏于中国微生物菌种保藏管理委员会普通微生物中心，简称CGMCC，地址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，保藏日期为2020年03月30日，分类命名为副干酪乳杆菌Lactobacillus paracasei。
进一步，所述副干酪乳杆菌L.p R3-10在制备治疗溃疡性结肠炎药物中的应用。
经由上述的技术方案可知，与现有技术相比，本发明公开提供了一株副干酪乳杆菌L.p R3-10及其在制备治疗溃疡性结肠炎药物中的应用，副干酪乳杆菌R3-10(Lactobacillus paracasei R3-10，L.p R3-10)是一株来源于本实验室前期从婴儿粪便中分离的副干酪乳杆菌R3(Lactobacillus paracasei R3，L.p R3)，将L.p R3经饥饿驯化10代之后得到的一株低营养梯度耐受诱导的乳杆菌；L.p R3-10能够制备成治疗溃疡性结肠炎的药物。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1附图为本发明L.p R3-10在MRS琼脂平板上的菌落形态；
图2附图为本发明L.p R3-10在厌氧血琼脂平板上的菌落形态；
图3附图为本发明L.p R3-10革兰染色镜下形态，1000倍镜；
图4附图为本发明溃疡性结肠炎小鼠模型体重变化百分率；
图5附图为本发明溃疡性结肠炎小鼠模型DAI评分；
图6附图为本发明各组小鼠结肠形态；
其中，A为NS组，B为DSS组，C为MSLZ组，D为L.p R3-10组，E为L.p R3-10+MSLZ组；
图7附图为本发明溃疡性结肠炎小鼠模型结肠长度；
其中，*，P<0.5；**，P<0.1；
图8附图为本发明溃疡性结肠炎小鼠模型结肠组织HE染色结果；
其中，A为NS组，B为DSS组，C为MSLZ组，D为L.p R3-10组，E为L.p R3-10+MSLZ组；
图9附图为本发明各组小鼠组织学损伤评分；
其中，*，P<0.5；**，P<0.1。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1 L.p R3-10的前体菌株L.p R3(副干酪乳杆菌)的分离培养及鉴定
(1)样本来源
从学校教职工家庭中挑选出0-6个月的健康婴儿作为志愿者。采样前两周需正常饮食并且近期无肠道感染史与抗生素服用史，采样当天取晨便，收集后迅速使用南京法迈特公司的智能微生物分离系统进行粪菌分离。分离完成后迅速收集粗提粪菌液，加入冻存保护液后置于-80℃超低温冰箱中冻存备用。
(2)L.p R3的分离培养与鉴定
取1mL的粗提粪菌液加入到9mL的生理盐水，充分混匀后进行梯度稀释。吸取稀释浓度为10 -5～10 -7的菌液各100μL，分别密集涂布于MRS培养基、BBL培养基、M17培养基及厌氧血平板，37℃厌氧条件培养48h-72h。初步根据菌落特征和革兰染色镜检，挑选单菌落在上述对应琼脂培养基上进行分区划线纯培养。收获纯培养后的适量菌体细胞置于菌种保藏管，-80℃冰箱保存备用。纯菌株转种于MRS琼脂平板分区划线传代2代后，挑取单个菌 落做革兰染色镜检及触酶试验，将具有典型乳杆菌形态特征同时触酶试验阴性的革兰阳性杆菌初步认定为乳杆菌。
(3)L.p R3的生化鉴定与测序鉴定
送交东莞市美康生物科技有限公司进行菌株测序鉴定。公司采用TIANamp Bacteria DNA Kit(Tiangen,Beijing)提取菌液中的DNA。通过细菌通用引物27F/1492R，利用PCR扩展得到接近全长的细菌16S rDNA片段，通过DNA测序获得该细菌16S rDNA片段的DNA序列，并通过将该序列与GenBank和RDP数据库中已有的DNA序列进行比对分析，得到序列最相似的物种信息，并根据序列的相似性推定所鉴定微生物的物种信息。经鉴定确定编号YSJ03菌株为副干酪乳杆菌，命名为L.p R3。
实施例2 L.p R3-10的诱导与鉴定
(1)低营养梯度耐受法诱导L.p R3成为L.p R3-10
从-80℃冰箱取出冻存的L.p R3，放入37℃温水浴使其快速融化。将融化后的菌液倒入厌氧血琼脂平板中，置于37℃厌氧条件静置培养48h。观察平板中菌落生长情况以及有无溶血环形成，革兰染色镜下观察菌株形态，确认无污染后，转种MRS琼脂平板上，置于37℃厌氧条件培养24h，挑取平板上的单个菌落接种于6mL MRS液体培养基中，37℃厌氧条件培养活化16-18h。将活化后的菌液以3％(v/v)接种量接种至100mL MRS肉汤中，120rpm/min，37℃振荡培养16-18h。于3500rpm/min条件下离心10min，弃上清，用PBS(pH 7.2-7.4)重悬洗涤2次后收获菌细胞。用PBS调整菌液浓度为2麦氏浊度，分装在无菌的2mL EP管中，每管分装1mL菌悬液，分装15管，37℃振荡培养。从0h开始，每隔12h取出1支EP管，吸取100μL菌液，涂布于MRS培养基表面，37℃厌氧条件下培养48h。挑取存活时间最长的单个菌落，再同样进行上述过程，依次循环10代，得到低营养梯度耐受诱导的L.p R3-10菌株。
(2)L.p R3-10培养特性
用接种环挑取L.p R3-10菌落，分区划线接种于MRS琼脂平板和厌氧血琼脂平板，MRS琼脂平板进行37℃需氧条件培养48h，厌氧血琼脂平板进行37℃厌氧条件培养48h；观察MRS平板中菌落生长情况以及厌氧血琼脂平板上有无溶血环形成，结果见图1-图2；结果显示，L.p R3-10为兼性厌氧菌， 在MRS平板上菌落呈圆形、中等大小、凸起、微白色、湿润、边缘整齐，在厌氧血琼脂平板上不溶血。
(3)L.p R3-10革兰染色观察菌体形态
挑取MRS琼脂平板上的L.p R3-10菌落，涂片进行革兰染色，油镜下观察菌细胞的形态，结果见图3；结果显示，L.p R3-10为革兰染色阳性的杆菌，无芽孢、无荚膜，成短链排列。
(4)L.p R3-10生化鉴定
使用法国梅里埃生物公司的API 50CHL细菌生化鉴定系统进行鉴定。首先按照API 50CHL鉴定试剂条说明书，将活化后的L.p R3-10菌悬液调整到2麦氏浊度，分别加到试剂条上的50个微生化孔中，并用无菌液体石蜡覆盖生化孔。35℃条件静置培养24h观察一次结果，继续培养至48h，再次观察结果。结果判定：25号管颜色由紫变黑为阳性，其余各管颜色由紫变黄为阳性，否则为阴性。将菌株的反应结果用API鉴定软件进行分析，获得菌株的鉴定结果。L.p R3-10生化反应结果见表1。鉴定结果为副干酪乳杆菌酪蛋白亚种1，鉴定率99.7％，T值0.79。
表1 L.p R3-10生化反应结果
Figure PCTCN2020128122-appb-000001
其中，0号管为空白对照管。
实施例3 溃疡性结肠炎小鼠模型的建立
选用SPF级雌性C57BL/6小鼠(6～8周龄，体质量16～18g)25只。将小鼠随机分为5组，分别为正常组(生理盐水，NS组)、模型组(DSS组)、美沙拉嗪组(MSLZ组)、L.p R3-10组(10 9CFU/mL)、L.p R3-10联合美沙拉嗪 组(L.p R3-10+MSLZ组)，每组5只。造模前，正常组和模型组灌胃等体积生理盐水，其余三组分别预防性给予美沙拉嗪(52mg/mL)、L.p R3-10菌液(1×10 9CFU/mL)、L.p R3-10菌液(1×10 9CFU/mL)联合美沙拉嗪(52mg/mL)灌胃7d处理。造模开始，正常组自由饮用双蒸馏水，其余4组小鼠自由饮用3％DSS进行7天的急性UC造模。并同时给予无菌生理盐水、美沙拉嗪(52mg/mL)、L.p R3-10菌液(1×10 9CFU/mL)、L.p R3-10菌液(1×10 9CFU/mL)联合美沙拉嗪(52mg/mL)的灌胃，每次0.2ml，1次/天。造模第8天上午折颈法处死小鼠，取出结肠，取部分远端结肠组织甲醛固定、石蜡包埋、切片，HE染色。实验期间每天记录小鼠体重、大便性状与隐血情况。
实施例4 溃疡性结肠炎小鼠模型的评估
(1)一般情况评估
每日观察每组小鼠进食、活动、毛发等一般情况，并进行疾病活动指数(DAI)评分(表2)，评估结肠炎疾病活动程度。每天上午用电子天平称量每只小鼠的体重并收集粪便，计算体重下降百分率。
体重下降百分率＝(第0天的体重-第n天的体重)/第0天的体重×100％，按照下表的标准根据小鼠体重下降的百分率对小鼠打分，并记录实验结果。
根据实验动物的体重下降百分率、大便黏稠度(正常，稀便，水样便)及粪便潜血情况(正常，潜血阳性，潜血强阳性)三种情况进行综合评分，将3项结果的总分除以3即得到结肠炎DAI值，即结肠炎DAI＝(体重下降百分率得分+粪便形态得分+粪便潜血情况得分)/3。
各组实验小鼠造模7天体重百分率结果见图4，结果显示，NS组基本保持不变，DSS组从第4天开始出现明显下降，其第7天体重百分率约为84％；MSLZ组、L.p R3-10+MSLZ组与L.p R3-10组体重从第5天开始下降，下降程度依次减弱。
各组实验小鼠结肠炎的DAI结果见图5，结果显示：DSS组>MSLZ组>L.p R3-10+MSLZ组>L.p R3-10组>NS组。
表2 疾病活动指数评分标准
Figure PCTCN2020128122-appb-000002
(2)结肠长度
颈椎脱臼法分别处死各组动物，取出结肠，各组小鼠结肠形态见图6。用标尺量取各组小鼠结肠长度，记录小鼠解剖后的结肠长度变化，并计算各组平均值及标准偏差，进行相关统计分析，结果见图7。图7结果显示，各组小鼠结肠平均长度为：NS组>L.p R3-10组>L.p R3-10+MSLZ组>MSLZ组>DSS组。
(3)小鼠结肠组织HE染色
①处死小鼠后，取出各组小鼠的结肠，用10％甲醛溶液固定，经石蜡包埋并切片。②将石蜡切片浸泡于二甲苯溶液中，微波炉加热5min；再次将石蜡切片浸泡入二甲苯溶液中，微波炉加热5min；再分别浸泡于无水乙醇、95％乙醇、85％乙醇、75％乙醇溶液1min共两次；然后用自来水进行冲洗。③苏木素染色5min后，用流水冲洗，1％盐酸酒精分化，再次用流水冲洗；伊红染色1min。再次浸泡于75％乙醇、85％乙醇、95％乙醇、无水乙醇溶液1min共两次，于二甲苯溶液中浸泡5min两次后，用中性树脂封片。④显微镜下观察并拍照。光学显微镜下观察比较各组小鼠结肠组织病理学改变，如粘膜上皮细胞变化、隐窝结构纹理、炎性细胞浸润等，结果见图8。图8结果显示，NS组为正常结构；DSS组可见粘膜上皮细胞变少、隐窝结构纹理紊乱、炎性细胞浸润，表现为UC；MSLZ组、L.p R3-10组和L.p R3-10+MSLZ组的组织病理较DSS组有了明显的改善。
(4)组织学损伤的评估
组织学损伤评分：0分，正常且无炎症细胞浸润；1分，轻微炎症细胞浸润，黏膜下组织无损伤；2分，中度炎症细胞浸润和黏膜下组织被破坏(损伤范围在10％～25％)；3分，明显的炎性细胞浸润，黏膜下组织被破坏，结肠 壁增厚(损伤范围在25％～50％)；4分，严重的炎性细胞浸润，大规模结肠组织损伤(损伤范围>50％)和结肠壁增厚。评估结果见图9，结果显示L.p R3-10组和L.p R3-10+MSLZ组小鼠结肠组织损伤程度评分显著低于DSS组。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (2)

  1. 一株副干酪乳杆菌L.p R3-10，其特征在于，其保藏编号为CGMCC No.19520。
  2. 权利要求1所述的副干酪乳杆菌L.p R3-10在制备治疗溃疡性结肠炎药物中的应用。
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CN114854638A (zh) * 2022-05-24 2022-08-05 江南大学 一株高效表达腺苷脱氨酶缓解结肠炎副干酪乳杆菌
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CN116083277A (zh) * 2022-09-17 2023-05-09 浙江大学 具有缓解溃疡性结肠炎功效的乳酸片球菌zjuids13及其应用
CN116083277B (zh) * 2022-09-17 2024-02-13 浙江大学 具有缓解溃疡性结肠炎功效的乳酸片球菌zjuids13及其应用
CN117586926A (zh) * 2024-01-18 2024-02-23 东北农业大学 副干酪乳杆菌lp-116、lp-116后生元组合物及其制备方法与应用
CN117586926B (zh) * 2024-01-18 2024-04-16 东北农业大学 副干酪乳杆菌lp-116、lp-116后生元组合物及其制备方法与应用

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