WO2021104215A1 - 蜕膜nk细胞及其细胞亚群在制备不孕不育相关疾病治疗药物中的用途 - Google Patents

蜕膜nk细胞及其细胞亚群在制备不孕不育相关疾病治疗药物中的用途 Download PDF

Info

Publication number
WO2021104215A1
WO2021104215A1 PCT/CN2020/130939 CN2020130939W WO2021104215A1 WO 2021104215 A1 WO2021104215 A1 WO 2021104215A1 CN 2020130939 W CN2020130939 W CN 2020130939W WO 2021104215 A1 WO2021104215 A1 WO 2021104215A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
decidual
cell
drugs
endometrial
Prior art date
Application number
PCT/CN2020/130939
Other languages
English (en)
French (fr)
Inventor
胡适
Original Assignee
沣潮医药科技（上海）有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 沣潮医药科技（上海）有限公司 filed Critical 沣潮医药科技（上海）有限公司
Publication of WO2021104215A1 publication Critical patent/WO2021104215A1/zh

Links

Classifications

    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • 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
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • 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
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • 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
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

  • the invention belongs to the field of biomedicine, and relates to the use of decidual NK cells and their cell subgroups in the preparation of drugs for treating infertility-related diseases, methods for separating and culturing cells, and pharmaceutical compositions containing them.
  • Endometrial injury is mainly the injury of the basal layer of the endometrium.
  • the main reason is related to curettage during pregnancy. Compared with the normal endometrium, the basal layer of the endometrium during pregnancy is loose and more susceptible to injury. Based on the current situation of my country's national conditions and the increasing rate of induced abortion, the occurrence of endometrial damage cannot be ignored. Damage to the basal layer of the endometrium may result in damage or loss of endometrial stem cells; at the same time, infection and aseptic inflammation in the damaged endometrium will destroy the niche microenvironment of stem cells and cause epithelial and mesenchymal cell regeneration Obstacles to repair occur, blood vessel formation is blocked, and dense fibrous tissue is formed.
  • decidual NK cell population itself is extremely heterogeneous, that is , the NK cells in the decidual tissue with the CD56 bright CD16 - KIR + CD9 + CD49a + phenotype can also be based on the expression of their surface markers. Different groups are divided into groups with different functions. The prior art does not disclose or suggest a technical solution for preparing products of decidual NK cell subsets expressing specific markers for disease treatment.
  • the present invention was made to solve the above technical problems, and aims to provide a use, preparation method and pharmaceutical composition containing NK cells and cell subgroups thereof.
  • the first aspect of the present invention is to provide the use of decidual NK cells and cell subgroups in the preparation of drugs for treating infertility-related diseases.
  • the decidual NK cells of the present invention refer to the NK cells in the decidual tissue whose surface markers are CD56 bright CD16 - KIR + CD9 + CD49a + phenotype.
  • Decidual NK cell subpopulation refers to the above-mentioned NK cell characteristics and further screening through cell markers CD39 + , CD27 + , CD160 + or TIGIT + ; decidual NK cell subpopulation includes the marker CD39 + , Any one or a combination of CD27 + , CD160 + or TIGIT + decidual NK sub-cells.
  • the infertility-related diseases of the present invention include endometrial growth disorders and maternal-fetal immune tolerance disorders.
  • Endometrial growth disorders related diseases include endometrial injury, premature ovarian failure, sex hormone disorders, polycystic ovary syndrome, pelvic inflammatory disease, decreased endometrial receptivity, endometritis, endometrial polyps, intrauterine adhesions , Endometrial gland reduction, endometrial fibrosis, amenorrhea, abnormal uterine bleeding, adenomyosis and endometriosis, reproductive system infection, uterine fibroids, etc.; diseases related to maternal-fetal immune tolerance disorders Including recurrent spontaneous abortion, threatened abortion or failure of assisted reproductive technology treatment.
  • the treatment drugs for infertility-related diseases are drugs for the treatment of endometrial growth disorders, drugs for the treatment of diseases related to maternal-fetal immune tolerance disorders, or drugs that increase the pregnancy rate when the endometrium is damaged.
  • the drugs for treating endometrial growth disorders are drugs that promote the increase of endometrial thickness, drugs that enhance the viability of endometrial stromal cells, drugs that reduce endometrial cell damage, and promote endometrial stromal cells. Any one or a combination of VEGF-expressing drugs, drugs that maintain the stemness of endometrial stromal cells and stimulate proliferation; the drugs for treating diseases related to maternal-fetal immune tolerance disorders are drugs that exert immune tolerance, Any one or more combinations of drugs that reduce spontaneous abortion and drugs that increase the level of T-helper lymphocytes; the drug that increases the pregnancy rate when the endometrium is injured is to reduce the degree of endometrial fibrosis or increase secretory glands The number of drugs.
  • the second aspect of the present invention is to provide a method for preparing the decidual NK cells and cell subgroups, which are prepared by a method including the following steps:
  • NK cells or cell subpopulations to CTS AIM-V medium, 1640 medium or DMEM medium, and incubate at 37°C and 5% CO 2 for 12-48 hours.
  • the pharmaceutical composition includes tablets, pills, powders, injections, tinctures, solutions, extracts, ointments, etc. commonly used in the pharmaceutical field; it also includes preparations for uterine mucosal administration, such as membranes, suppositories, and tablets.
  • Drugs, effervescent tablets, gels, and stents and other intrauterine drug delivery systems, etc.; can also include mucosal absorption enhancers, such as surfactants, chelating agents, fatty acids, fatty alcohols, fatty acid esters, cyclodextrin-derived Substances, protease inhibitors, etc.
  • the invention provides the use of decidual NK cells and their cell subgroups in the preparation of drugs for treating infertility-related diseases.
  • the decidual NK cells and their cell subsets promote the increase of endometrial thickness, promote endometrial cell viability, reduce endometrial cell damage, promote VEGF expression, maintain endometrial stromal cell stemness and stimulate proliferation
  • the treatment of endometrial growth disorder disease has increased the pregnancy success rate of endometrial injury model mice from 30% to 60 ⁇ 70%; by exerting immune tolerance, reducing spontaneous abortion rate and increasing T-assistance Lymphocyte level treatment of maternal-fetal immune tolerance disorders related diseases provides a new approach for the treatment of infertility.
  • the statistical calculations in this embodiment and the following embodiments select the different statistical modes of software SPSS 22.0 version to calculate the p value according to the comparison between two groups, multiple groups, and the comparison requirements of rates. P value less than 0.05 is considered statistically significant.
  • First screening uses CD56 antibody, CD3 antibody, CD14 antibody to preliminarily sort NK cells, and then use CD16 antibody, CD49a antibody to further screen decidual NK cells, that is, decidual NK cells with a phenotype of CD56 bright CD16 - CD49a + (CD56 bright CD16 - CD49a + CD3 - CD14 - ).
  • the NK cells obtained in the normal group and the abortion group were lysed.
  • Example 1 Use healthy non-medical reasons to terminate the early pregnancy decidual tissue to prepare decidual NK cells, and implement the NK cell isolation method described in Example 1, briefly as follows: After 1 mg/mL collagenase IV (Sigma-Aldrich) and 0.01 mg/mL After digestion with DNase I (Shanghai Sangon) for 1 h, the lymphocytes were obtained by Percoll (GE Healthcare) density gradient centrifugation. Incubate at 37°C for 2h in a petri dish to remove stromal cells and macrophages, and then isolate NK cells using flow cytometry. The decidual NK cells with the phenotype of CD56 bright CD16 - CD49a + are obtained.
  • Example 3 Decidual NK cells and decidual NK cell subsets enhance endometrial cell viability, reduce endometrial cell damage, and increase VEGF expression
  • NK cells non-pathological endometrial stromal cells
  • each group of NK cells described in Example 2 was added, and the ratio of the number of stromal cells: NK cells was 10:1 and 5:1, respectively.
  • the control group used the control NK cells described in Example 2. After 48 hours of treatment, NK cells were removed, and stromal cells and culture medium were sampled for analysis.
  • the cell viability of the stromal cells was detected by the PrestoBlue method (Thermo Fisher Scientific), and the measurement was performed 48 hours after the treatment, and the value was expressed as the mean% of the normalized control (Table 1 and Table 2).
  • lactate dehydrogenase detection method to measure cell damage by colorimetric method, so that cell damage can be quantified based on the measurement of lactate dehydrogenase activity in damaged cells in the culture medium.
  • Increased cell membrane damage and cell lysis lead to an increase in lactate dehydrogenase activity, which is proportional to the number of lysed cells.
  • lactate dehydrogenase activity was measured in the culture medium (Table 3).
  • NK cells non-pathological endometrial stromal cells
  • each group of NK cells described in Example 2 was added, and the ratio of the number of stromal cells: NK cells was 10:1 and 5:1, respectively.
  • the control group used the control NK cells described in Example 2.
  • the NK cells were removed, and then the ALDH positive rate and Ki67 positive rate of stromal cells were detected by flow cytometry. The results are shown in Table 5.
  • the patient's endometrial thickness is less than 8mm due to factors such as induced abortion and curettage, infection, and the clinical diagnosis is that the endometrium is thin.
  • the treatment of anti-infection and other treatments to patients is ineffective.
  • the active ingredient administered is a composition prepared using the decidual NK cells and decidual NK cell subsets prepared in Example 2, such as intravenous infusion, containing cellular active ingredients such as 2 ⁇ 10 9 .
  • Example 6 The effect of decidual NK cells and decidual NK cell subsets on decidual DC cells
  • Example 7 Therapeutic effects of decidual NK cells and decidual NK cell subsets on spontaneous abortion models
  • CBA/J female mice and DBA/2J male mice were used to establish a stress abortion model.
  • the abortion model is a classic research model of maternal-fetal immune tolerance.
  • the establishment methods, experimental methods and observation time points are equivalent to the literature (Blois SM, et al.. Nature Medicine, 2007, 13(12): 1450-1457.).
  • CBA/J female mice were divided into negative control group, model group, stress pressure + control cell group, and treatment group before being caged.
  • the treatment group was administered 1 ⁇ 10 5 decidual NK cells and cell subgroups of the present invention via the veins, once every 3 days, for a total of 3 administrations.
  • the cages were closed 3 days after the first application.
  • Example 8 The effect of decidual NK cells and decidual NK cell subsets on T helper cells
  • an animal endometrial injury model (C57 mice) was constructed. Eight-week-old female mice were divided into groups, each with 10 mice, and the double (infection + mechanical) injury method was used to construct the endometrial injury model. That is, after the mouse is anesthetized, a longitudinal incision of about 2cm in the middle of the abdomen is taken into the abdomen, a 0.5cm longitudinal incision is made in the middle and lower 1/3 of the uterus, and the middle and upper uterine cavity is scraped with an endometrial spatula.
  • a blank control group (sham operation group) is set up, with only saline injection (model) group; control group, cell treatment group.
  • the treatment group was administered 1 ⁇ 10 5 decidual NK cells and decidual NK cell subgroups of the present invention via the veins, once every 3 days, for a total of 3 administrations.
  • the mice were mated with male mice after 3 cycles of estrus.
  • Group Pregnancy rate p value (compared to control cells) Negative control group 100% To Model group (no cells) 30% To Model group + control cells 30% To CD56 bright CD16 - CD49a + decidual NK cells 60% p ⁇ 0.05 CD56 bright CD16 - CD49a + CD39 + decidual NK cell population 60% p ⁇ 0.05 CD56 bright CD16 - CD49a + CD27 + decidual NK cell population 70% p ⁇ 0.05 CD56 bright CD16 - CD49a + CD160 + decidual NK cell population 70% p ⁇ 0.05 CD56 bright CD16 - CD49a + TIGIT + decidual NK cell population 70% p ⁇ 0.05 CD56 bright CD16 - CD49a + CD39 + TIGIT + decidual NK cell population 70% p ⁇ 0.05 CD56 bright CD16 - CD49a + CD39 + TIGIT + decidual NK cell population 70% p ⁇ 0.05 CD56 bright CD16 - CD49a + CD39 + TIGIT + decidual NK cell population 70% p ⁇ 0.05

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Hematology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Reproductive Health (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Virology (AREA)
  • Endocrinology (AREA)
  • Epidemiology (AREA)
  • Pregnancy & Childbirth (AREA)
  • Microbiology (AREA)
  • Gynecology & Obstetrics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

提供了蜕膜NK细胞及其细胞亚群在制备不孕不育相关疾病治疗药物中的用途。通过实验证实，该蜕膜NK细胞及其细胞亚群通过促进子宫内膜厚度增加、增强子宫内膜细胞活力、降低内膜细胞损伤、促进VEGF表达、维持子宫内膜基质细胞干性和刺激增殖的方式治疗子宫内膜生长障碍性疾病。

Description

蜕膜NK细胞及其细胞亚群在制备不孕不育相关疾病治疗药物中的用途 技术领域
本发明属于生物医药领域，涉及蜕膜NK细胞及其细胞亚群在制备不孕不育相关疾病治疗药物中的用途、细胞的分离培养方法以及含有其的药物组合物。
背景技术
子宫内膜损伤主要为内膜基底层的损伤，其主要原因与妊娠期刮宫有关，与正常子宫内膜相比，妊娠期子宫内膜基底层疏松，更容易受到损伤。基于我国国情以及人工流产率逐渐增加的现状，子宫内膜受损的发生情况不容忽视。子宫内膜基底层受损，可能导致子宫内膜干细胞受损或缺失；与此同时，损伤内膜局部的感染和无菌性炎症会破坏干细胞的壁龛微环境，造成上皮和间质细胞再生修复发生障碍，血管形成受阻，致密纤维组织形成，最终导致宫腔仅覆盖少量内膜，甚至无内膜，腺体萎缩，宫腔失去正常形态及功能。进一步的，宫腔内的免疫情况紊乱进一步介导子宫内膜免疫性流产的风险。
NK细胞是存在子宫内膜一种固有免疫细胞。在妊娠早期，NK细胞占蜕膜中淋巴细胞总数的40-70％，是正常蜕膜中含量较高的一类的淋巴细胞。现代生殖医学研究认为，人类蜕膜NK(dNK)细胞具有独特的CD56 brightCD16 -KIR +CD9 +CD49a +表型，与外周NK细胞不同，几乎没有细胞毒性。蜕膜NK细胞在蜕膜组织局部的参与促进新生血管生成、组织重塑、免疫调节和胎盘形成。
根据现有探究，未公开或提示利用蜕膜NK细胞制备产品以用于疾病的治疗的技术方案，这是未知的和需要证实的。另一方面，蜕膜NK细胞群本身是具有极强的异质性的，即具有CD56 brightCD16 -KIR +CD9 +CD49a +表型的蜕膜组织内NK细胞还可以根据其表面标志物的表达不同分为多群，且功能各异。现有技术没有公开或提示表达特异性标志物的蜕膜NK细胞亚群制备产品用于疾病治疗的技术方案。
发明内容
本发明是为解决上述技术问题进行的，目的在于提供一种NK细胞及其细胞亚群的用途、制备方法以及含有其的药物组合物。
本发明的第一方面在于提供蜕膜NK细胞及其细胞亚群在制备不孕不育相关疾病治疗药物中的用途。
本发明所述的蜕膜NK细胞，指的是表面标志物为CD56 brightCD16 -KIR +CD9 +CD49a +表型的蜕膜组织内NK细胞。蜕膜NK细胞亚群，指在上述NK细胞特征基础上，进一步通过细胞标志物CD39 +，CD27 +，CD160 +或TIGIT +进行筛选得到的；蜕膜NK细胞亚群包括标志物为CD39 +，CD27 +，CD160 +或TIGIT +的蜕膜NK亚细胞中的任意一种或多种的组合。
本发明所述的不孕不育相关疾病包括子宫内膜生长障碍性疾病以及母胎免疫耐受障碍相关性疾病两类。子宫内膜生长障碍相关疾病包括子宫内膜损伤、卵巢早衰、性激素紊乱、多囊卵巢综合症、盆腔炎性疾病、子宫内膜容受性下降、子宫内膜炎、内膜息肉、宫腔粘连、子宫内膜腺体减少、内膜性纤维化、闭经、异常性子宫出血、子宫腺肌症和子宫内膜异位症、生殖系统感染、子宫肌瘤等；母胎免疫耐受障碍相关性疾病包括反复性自发流产、先兆流产或辅助生殖技术治疗失败等。
因此，不孕不育相关疾病治疗药物为治疗子宫内膜生长障碍性疾病的药物、治疗母胎免疫耐受障碍相关性疾病的药物或子宫内膜损伤时提高受孕率的药物。
具体的，所述治疗子宫内膜生长障碍性疾病的药物为促进子宫内膜厚度增加的药物、增强子宫内膜基质细胞活力的药物、降低子宫内膜细胞损伤的药物、促进子宫内膜基质细胞VEGF表达的药物、维持子宫内膜基质细胞干性和刺激增殖药物中的任意一种或多种组合；所述治疗母胎免疫耐受障碍相关性疾病的药物为发挥免疫耐受性作用的药物、降低自发性流产的药物、以及提高T辅助淋巴细胞水平的药物的任意一种或多种组合；所述子宫内膜损伤时提高受孕率的药物为降低子宫内膜纤维化程度或提高分泌腺体数量的药物。
更具体的，维持子宫内膜基质细胞干性和刺激增殖药物为提高基质细胞ALDH和Ki67阳性率的药物；发挥免疫耐受性作用的药物为介导DC细胞分泌IL-10的药物。
本发明的第二方面在于，提供了该蜕膜NK细胞及细胞亚群的制备方法，由包括如下步骤的方法制备得到：
A、分离蜕膜NK细胞和蜕膜NK细胞亚群
(i)蜕膜组织细胞分离：蜕膜组织经胶原酶Ⅳ(Sigma-Aldrich)以及DNase Ⅰ(Shanghai Sangon)消化获取细胞悬液
(ii)淋巴细胞获取：Percoll(GE Healthcare)密度梯度离心得到。
(iii)以流式细胞仪分离蜕膜NK细胞，首先利用通用技术分离一些杂质细胞，然后利用anti-CD56抗体、anti-CD16抗体和anti-CD49a抗体，实施流式细胞仪法或磁珠法 分离蜕膜NK细胞，蜕膜NK细胞的标志物为CD56 brightCD16 -CD49a +。
(iv)根据需要进一步利用anti-CD39抗体，anti-CD27抗体，anti-CD160抗体，和anti-TIGIT抗体，实施流式细胞仪法或磁珠法分离蜕膜NK细胞亚群。蜕膜NK细胞亚群标志物为CD39 +，CD27 +，CD160 +，和TIGIT +中的任一种和或至少两种的组合。
B、NK细胞体外培养
将NK细胞或细胞亚群转移至CTS AIM-V培养基，1640培养基或DMEM培养基中，37℃、5％CO 2环境中培养时间为12-48小时。
本发明的第三方面在于，提供了一种不孕不育相关疾病的药物组合物，由蜕膜NK细胞或其细胞亚群以及药学上可接受的辅料组成。辅料有助于细胞更稳定地发挥疗效，这些制剂可以保证本发明公开的细胞的结构完整性和稳定性，同时还要保护的细胞的活性官能团，防止其活性变化。
通常情况下，液体制剂可以在2℃-8℃条件下保存至少稳定三个月，冷冻制剂在-30℃至少六个月保持稳定。
在药物形式上，该药物组合物包括制药领域常用的片剂、丸剂、散剂、注射剂、酊剂、溶液剂、浸膏剂、软膏剂等；还包括子宫黏膜给药制剂，例如膜剂、栓剂、片剂、泡腾片剂、凝胶剂、及支架等子宫内药物释放系统等；还可包括黏膜吸收促进剂，例如表面活性剂、螯合剂、脂肪酸、脂醇、脂肪酸酯、环糊精衍生物、蛋白酶抑制剂等。
相比现有技术，本发明的技术效果如下：
本发明提供了蜕膜NK细胞及其细胞亚群在制备不孕不育相关疾病治疗药物中的用途。通过实验证实，该蜕膜NK细胞及其细胞亚群通过促进子宫内膜厚度增加、促进子宫内膜细胞活力、降低内膜细胞损伤、促进VEGF表达、维持子宫内膜基质细胞干性和刺激增殖的方式治疗子宫内膜生长障碍性疾病，使得子宫内膜损伤模型小鼠的受孕成功率由30％提高至60～70％；通过发挥免疫耐受性作用、降低自发性流产率以及提高T辅助淋巴细胞水平治疗母胎免疫耐受障碍相关性疾病，为不孕不育的治疗提供了一种新的途径。
具体实施方式
以下实施例、实验例对本发明进行进一步的说明，不应理解为对本发明的限制，同时实施例不包括对传统方法的详细描述。
实施例1 子宫蜕膜NK细胞候选标志物筛选
本实施例及后述实施例中的统计学计算根据两组比较、多组比较、率的比较需求选择软件SPSS 22.0版本的不同统计模式计算p值。P值小于0.05认为有统计学显著性。
首先利用10例健康非医学原因终止早期妊娠蜕膜组织(正常组)和5例自然流产早期妊娠蜕膜组织(流产组)分离NK细胞，NK细胞的分离和流式细胞分选可以参考文献[Fu B,et al.Immunity,2017,47(6):1100-1113.e6.]。举例如下：经1mg/mL胶原酶Ⅳ(Sigma-Aldrich)和0.01mg/mL DNaseⅠ(Shanghai Sangon)消化1h,再经Percoll(GE Healthcare)密度梯度离心获取淋巴细胞。利用培养皿37℃培养2h以去除基质细胞和巨噬细胞，然后利用流式细胞仪分离NK细胞。首先筛选利用CD56抗体，CD3抗体，CD14抗体初步分选NK细胞，然后利用CD16抗体，CD49a抗体进一步筛选蜕膜NK细胞，即得到表型为CD56 brightCD16 -CD49a +的蜕膜NK细胞(CD56 brightCD16 -CD49a +CD3 -CD14 -)。裂解正常组和流产组获得的NK细胞，Bradford法测定蛋白质浓度后两组裂解物以iTRAQ-nano-HPLC-MS/MS方法进行蛋白质表达分析，确认两组NK细胞内含量不同的膜表面标志物。方法参考文献[Jiang,Hong-Lin,et al.Cancer research 76.4(2016):952-964.]经聚类统计分析膜表面标志物，正常组蜕膜NK细胞中CD39(UniProtKB:P49961)、CD27(UniProtKB:P26842)、CD160(UniProtKB:O95971)、TIGIT(UniProtKB:Q495A1)表达均显著高于流产组蜕膜细胞(p＜0.05)。
实施例2 子宫蜕膜NK细胞及蜕膜NK细胞亚群制备
利用健康非医学原因终止早期妊娠蜕膜组织制备蜕膜NK细胞，按照实施例1所述NK细胞分离方法实施，简述如下：经1mg/mL胶原酶Ⅳ(Sigma-Aldrich)和0.01mg/mL DNase Ⅰ(Shanghai Sangon)消化1h，再经Percoll(GE Healthcare)密度梯度离心获取淋巴细胞。利用培养皿37℃培养2h以去除基质细胞和巨噬细胞，然后利用流式细胞仪分离NK细胞。即得到表型为CD56 brightCD16 -CD49a +的蜕膜NK细胞。进一步利用抗体磁珠分选CD56 brightCD16 -CD49a +CD39 +蜕膜NK细胞群、CD56 brightCD16 -CD49a +CD27 +蜕膜NK细胞群、CD56 brightCD16 -CD49a +CD160 +蜕膜NK细胞群、CD56 brightCD16 -CD49a +TIGIT +蜕膜NK细胞群、和CD56 brightCD16 -CD49a +CD39 +TIGIT +蜕膜NK细胞群。获得的细胞可以直接进行实验、使用或冻存。采集同一名志愿者的外周血，按照通用方法采集分离NK细胞作为对照细胞。
实施例3 蜕膜NK细胞及蜕膜NK细胞亚群增强子宫内膜细胞活力、降低内膜细胞 损伤、增加VEGF表达
将非病理性子宫内膜基质细胞(stroma cell)培养24小时后，加入实施例2所述各群NK细胞，基质细胞：NK细胞个数比例分别为10:1和5:1。对照组采用实施例2所述对照NK细胞。处理48小时后清除NK细胞，对基质细胞和培养基采样用于分析。
通过PrestoBlue方法(Thermo Fisher Scientific)检测基质细胞的细胞存活力，处理48小时后进行测定，值表示为对照进行标准化后的平均值％(表1和表2)。
表1 10:1培养条件时基质细胞相对细胞活力
  平均值 SD p值
空白(单独培养，无NK细胞) 100 6.72  
对照细胞 97.45 8.34  
CD56 brightCD16 -CD49a +的蜕膜NK细胞 139.7 17.6 p＜0.05
CD56 brightCD16 -CD49a +CD39 +蜕膜NK细胞群 160 17.1 p＜0.05
CD56 brightCD16 -CD49a +CD27 +蜕膜NK细胞群 142.5 12.2 p＜0.05
CD56 brightCD16 -CD49a +CD160 +蜕膜NK细胞群 168.5 10.2 p＜0.01
CD56 brightCD16 -CD49a +TIGIT +蜕膜NK细胞群 173.3 20.7 p＜0.01
CD56 brightCD16 -CD49a +CD39 +TIGIT +蜕膜NK细胞群 162.7 13.5 p＜0.01
表2 5:1培养条件时基质细胞相对细胞活力
  平均值 SD p值
空白(单独培养，无NK细胞) 100 11.3  
对照细胞 94.36 6.5  
CD56 brightCD16 -CD49a +的蜕膜NK细胞 184.3 15.5 p＜0.05
CD56 brightCD16 -CD49a +CD39 +蜕膜NK细胞群 213.9 17.5 p＜0.05
CD56 brightCD16 -CD49a +CD27 +蜕膜NK细胞群 175.6 11.5 p＜0.05
CD56 brightCD16 -CD49a +CD160 +蜕膜NK细胞群 211.5 15.3 p＜0.05
CD56 brightCD16 -CD49a +TIGIT +蜕膜NK细胞群 265.5 28.4 p＜0.01
CD56 brightCD16 -CD49a +CD39 +TIGIT +蜕膜NK细胞群 232.3 20.5 p＜0.05
结论：本发明所述蜕膜NK细胞及其细胞亚群具有增加子宫基质细胞存活力的能力，能作为增强子宫内膜增殖的产品使用
进一步，评估基质细胞水平上的细胞损伤：利用乳酸脱氢酶检测法，通过比色法测量细胞损伤，使得能够基于培养基中受损细胞中乳酸脱氢酶活性的测量来定量细胞损伤。细胞膜损伤的增加和细胞溶解导致乳酸脱氢酶活性增加，其与裂解细胞的数量成比例。细胞处理48小时后，在培养基中进行了乳酸脱氢酶活性测量(表3)。
表3 10:1培养条件时相对细胞损伤
  平均值 SD p值
空白(单独培养，无NK细胞) 100 9.15  
对照细胞 95.35 4.16  
CD56 brightCD16 -CD49a +的蜕膜NK细胞 61.50 4.55 p＜0.05
CD56 brightCD16 -CD49a +CD39 +蜕膜NK细胞群 72.21 5.22 p＜0.05
CD56 brightCD16 -CD49a +CD27 +蜕膜NK细胞群 59.83 8.95 p＜0.05
CD56 brightCD16 -CD49a +CD160 +蜕膜NK细胞群 49.72 3.32 p＜0.05
CD56 brightCD16 -CD49a +TIGIT +蜕膜NK细胞群 37.7 4.9 p＜0.05
CD56 brightCD16 -CD49a +CD39 +TIGIT +蜕膜NK细胞群 35.53 2.52 p＜0.01
结论：本发明的蜕膜NK细胞及其细胞亚群具有减轻膜损伤的能力，能作为增强基质细胞活性的产品使用。
进一步，研究了蜕膜NK细胞及其细胞亚群对子宫内膜基质细胞VEGF的潜在增强影响。结果呈现于表4中。细胞处理48小时后，在培养基中进行了VEGF浓度的ELISA检测。
表4 10:1培养条件时是VEGF表达
  VEGF(pg/ml) SD p值
空白(单独培养，无NK细胞) 412.59 35.21  
对照细胞 400.87 26.33  
CD56 brightCD16 -CD49a +的蜕膜NK细胞 682.98 55.14 p＜0.05
CD56 brightCD16 -CD49a +CD39 +蜕膜NK细胞群 778.70 40.66 p＜0.05
CD56 brightCD16 -CD49a +CD27 +蜕膜NK细胞群 553.07 37.81 p＜0.05
CD56 brightCD16 -CD49a +CD160 +蜕膜NK细胞群 840.90 67.11 p＜0.05
CD56 brightCD16 -CD49a +TIGIT +蜕膜NK细胞群 1091.98 87.85 p＜0.01
CD56 brightCD16 -CD49a +CD39 +TIGIT +蜕膜NK细胞群 972.87 75.32 p＜0.05
结论：本发明的蜕膜NK细胞及其细胞亚群具有促进VEGF表达，其具有增强子宫内膜血管新生的作用。
实施例4 蜕膜NK细胞和蜕膜NK细胞亚群进子宫内膜基质细胞标志物表达
将非病理性子宫内膜基质细胞(stroma cell)培养24小时后，加入实施例2所述各群NK细胞，基质细胞：NK细胞个数比例分别为10:1和5:1。对照组采用实施例2所述对照NK细胞。在37℃、5％CO2条件的培养箱中培养24小时后，后清除NK细胞，然后利用流式细胞术检测基质细胞的ALDH阳性率和Ki67阳性率，其结果如表5所示。
表5.ALDH阳性率和Ki67阳性率分析
Figure PCTCN2020130939-appb-000001
结果显示蜕膜NK细胞及其亚群体具有非常强的维持基质细胞干性和刺激增殖的作 用。
实施例5 蜕膜NK细胞和蜕膜NK细胞亚群治疗子宫内膜损伤
患者因人工流产刮宫后、感染等因素子宫内膜厚度低于8mm，临床诊断为子宫内膜薄。给与患者抗感染等治疗无效。给与活性成分为用实施例2所制备的蜕膜NK细胞和蜕膜NK细胞亚群制备组合物，如静脉输入含有细胞活性成分如2×10 9。一次或多次静脉施用于患者，以促进子宫内膜厚度增加。
实施例6.蜕膜NK细胞和蜕膜NK细胞亚群对蜕膜DC细胞的作用
从人非医学原因终止妊娠的蜕膜组织分离树突状细胞DC(CD1c阳性)，分离及筛选方法等同文献(Guo P F,et al.Blood,2010,116(12):2061-2069.)对DC细胞分为阴性对照组(采用实施例2所述对照NK细胞共培养，基质细胞：NK细胞比例为10:1)，处理组(采用实施例2所述蜕膜NK细胞和蜕膜NK细胞亚群，基质细胞：NK细胞比例为10:1)，LPS处理组(100纳克/毫升)，空白组为单独培养，48小时后检测培养体系中白介素10(IL-10)和肿瘤坏死因子α(TNFα)水平，检测方法同文献(Guo P F,et al.Blood,2010,116(12):2061-2069.)结果显示蜕膜NK细胞和蜕膜NK细胞亚群可以显著增加IL-10分泌水平而不增加TNFα水平(表6-7)。这些结果证实蜕膜NK细胞和蜕膜NK细胞亚群可以通过DC发挥免疫耐受作用。
表6.IL-10含量分析
  IL-10(pg/ml) SD p值
空白(单独培养，无NK细胞) 79.69 7.60  
对照细胞 88.35 7.15  
CD56 brightCD16 -CD49a +的蜕膜NK细胞 437.52 47.95 p＜0.05
CD56 brightCD16 -CD49a +CD39 +蜕膜NK细胞群 469.58 24.50 p＜0.05
CD56 brightCD16 -CD49a +CD27 +蜕膜NK细胞群 380.04 44.93 p＜0.05
CD56 brightCD16 -CD49a +CD160 +蜕膜NK细胞群 371.64 55.36 p＜0.05
CD56 brightCD16 -CD49a +TIGIT +蜕膜NK细胞群 365.73 45.33 p＜0.05
CD56 brightCD16 -CD49a +CD39 +TIGIT +蜕膜NK细胞群 597.77 49.54 p＜0.05
LPS 468.49 48.94 p＜0.05
表7.TNFα含量分析
  IL-10(pg/ml) SD p值
空白(单独培养，无NK细胞) 20.97 1.45  
对照细胞 15.38 2.22  
CD56 brightCD16 -CD49a +的蜕膜NK细胞 19.56 2.06 p＞0.05
CD56 brightCD16 -CD49a +CD39 +蜕膜NK细胞群 28.48 3.08 p＞0.05
CD56 brightCD16 -CD49a +CD27 +蜕膜NK细胞群 18.32 1.18 p＞0.05
CD56 brightCD16 -CD49a +CD160 +蜕膜NK细胞群 20.14 2.73 p＞0.05
CD56 brightCD16 -CD49a +TIGIT +蜕膜NK细胞群 29.42 3.71 p＞0.05
CD56 brightCD16 -CD49a +CD39 +TIGIT +蜕膜NK细胞群 15.04 1.43 p＞0.05
LPS 1506.51 209.69 p＜0.01
实施例7.蜕膜NK细胞和蜕膜NK细胞亚群对自发性流产模型的治疗作用
利用CBA/J雌性小鼠和DBA/2J雄性小鼠建立应激流产模型，该流产模型为经典的母胎免疫耐受障碍的研究模型，建立方法、实验方法和观察时间点等同文献(Blois S M,et al..Nature Medicine,2007,13(12):1450-1457.)。CBA/J雌性小鼠合笼前分为阴性对照组，模型组，应激压力+对照细胞组，处理组。处理组分别经脉施用1×10 5本发明所述蜕膜NK细胞及其细胞亚群，3天一次，共施用3次。第一次施用后3天后合笼。确定阴栓怀孕后立即将小鼠分笼(有效n＝10)。
实验结果显示(表8)，治疗组的流产率显著低于应激压力流产组，说明采用蜕膜NK细胞和蜕膜NK细胞亚群具有良好的治疗效果。
表8 各组小鼠胚胎吸收率(流产)分析
Figure PCTCN2020130939-appb-000002
Figure PCTCN2020130939-appb-000003
实施例8.蜕膜NK细胞和蜕膜NK细胞亚群对T辅助细胞影响
将实施例8所述阴性对照组、模型组、对照组和处理组的小鼠主动脉旁淋巴结分离，检测其中的Foxp3阳性T辅助淋巴细胞水平。分离和检测的方法同文献(Kim B J,et al..Proceedings of the National Academy of Sciences,2015,112(5):1559-1564。结果显示蜕膜NK细胞和蜕膜NK细胞亚群处理可以有效增加Foxp3阳性T辅助淋巴细胞水平(表9)。
表9 各组小鼠Foxp3 +％表达分析
Figure PCTCN2020130939-appb-000004
实施例9 蜕膜NK细胞和蜕膜NK细胞亚群治疗小鼠子宫内膜损伤模型
首先构建动物子宫内膜损伤模型(C57小鼠)，取8周龄大的雌性小鼠分为组，每组10只小鼠，采用双重(感染+机械)损伤法构建子宫内膜损伤模型，即小鼠麻醉后，取下腹部正中长约2cm纵切口进腹，于子宫中下1/3处作0.5cm纵行切口，采用子宫内膜刮勺 搔刮中上段子宫腔，当刮勺进出子宫凹凸感消失，四壁感觉粗糙时，停止刮宫，刮宫后宫腔留置脂多糖棉线，缝合腹部切口，48h后取出脂多糖棉线。建模完成后，设置空白对照组(假手术组)，仅注射生理盐水(模型)组；对照组，细胞处理组。处理组分别经脉施用1×10 5本发明所述蜕膜NK细胞和蜕膜NK细胞亚群，3天一次，共施用3次。在小鼠动情3个周期后将小鼠与雄性小鼠进行交配。1个月后取材行HE染色和Masson染色进行子宫内膜组织功能学评估，3个月后行小鼠妊娠结果评估。结果：术后1个月组织功能学评估显示，与各对照组相比，蜕膜NK细胞和蜕膜NK细胞亚群组的纤维化程度明显减少；与各对照组相比，分泌腺体数量均高于各对照组。妊娠结果评估显示，蜕膜NK细胞和蜕膜NK细胞亚群组的妊娠率高于各对照组，结果如表10所示。
表10 各组小鼠妊娠结果分析
组别 怀孕率 p值(比对照细胞)
阴性对照组 100％  
模型组(无细胞) 30％  
模型组+对照细胞 30％  
CD56 brightCD16 -CD49a +的蜕膜NK细胞 60％ p＜0.05
CD56 brightCD16 -CD49a +CD39 +蜕膜NK细胞群 60％ p＜0.05
CD56 brightCD16 -CD49a +CD27 +蜕膜NK细胞群 70％ p＜0.05
CD56 brightCD16 -CD49a +CD160 +蜕膜NK细胞群 70％ p＜0.05
CD56 brightCD16 -CD49a +TIGIT +蜕膜NK细胞群 70％ p＜0.05
CD56 brightCD16 -CD49a +CD39 +TIGIT +蜕膜NK细胞群 70％ p＜0.05
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等同物界定。

Claims (9)

  1. 蜕膜NK细胞及其细胞亚群在制备不孕不育相关疾病治疗药物中的用途。
  2. 根据权利要求1所述的蜕膜NK细胞及其细胞亚群在制备不孕不育相关疾病治疗药物中的用途，其特征在于：
    其中，所述蜕膜NK细胞的表面标志物为CD56 brightCD16 -CD49a +，所述细胞亚群是表面标志物为CD39 +，CD27 +，CD160 +及TIGIT +中的任意一种或多种的组合的蜕膜NK细胞。
  3. 根据权利要求1所述的蜕膜NK细胞及其细胞亚群在制备不孕不育相关疾病治疗药物中的用途，其特征在于：
    其中，所述不孕不育相关疾病治疗药物为治疗子宫内膜生长障碍性疾病的药物、治疗母胎免疫耐受障碍相关性疾病的药物或子宫内膜损伤时提高受孕率的药物。
  4. 根据权利要求3所述的蜕膜NK细胞及其细胞亚群在制备不孕不育相关疾病治疗药物中的用途，其特征在于：
    其中，所述治疗子宫内膜生长障碍性疾病的药物为促进子宫内膜厚度增加的药物、增强子宫内膜基质细胞活力的药物、降低子宫内膜细胞损伤的药物、促进子宫内膜基质细胞VEGF表达的药物、维持子宫内膜基质细胞干性和刺激增殖药物中的任意一种或多种组合；
    所述治疗母胎免疫耐受障碍相关性疾病的药物为维持或增强免疫耐受性作用的药物、降低自发性流产的药物、以及提高T辅助淋巴细胞水平的药物的任意一种或多种组合；
    所述子宫内膜损伤时提高受孕率的药物为降低子宫内膜纤维化程度或提高分泌腺体数量的药物。
  5. 根据权利要求4所述的蜕膜NK细胞及其细胞亚群在制备不孕不育相关疾病治疗药物中的用途，其特征在于：
    其中，所述维持子宫内膜基质细胞干性和刺激增殖药物为提高基质细胞ALDH和Ki67阳性率的药物；
    所述发挥免疫耐受性作用的药物为介导DC细胞分泌IL-10的药物。
  6. 根据权利要求1～5任一项所述的蜕膜NK细胞及其细胞亚群在制备不孕不育相关疾病治疗药物中的用途，其特征在于，该细胞及细胞亚群的制备方法如下：
    A、分离蜕膜NK细胞和蜕膜NK细胞亚群
    将蜕膜组织经胶原酶Ⅳ以及DNaseⅠ酶解消化后获取细胞悬液，而后通过密度梯度离心法得到淋巴细胞，然后利用anti-CD56抗体、anti-CD16抗体和anti-CD49a抗体，从中分离标志物为CD56 brightCD16 -CD49a +的蜕膜NK细胞；再根据需要利用anti-CD39抗体，anti-CD27抗体，anti-CD160抗体和anti-TIGIT抗体，分离蜕膜NK细胞亚群；
    B、NK细胞体外培养
    将NK细胞或细胞亚群转移至CTS AIM-V培养基，1640培养基或DMEM培养基中，37℃、5％CO 2环境中培养时间为12-24小时。
  7. 根据权利要求1所述的蜕膜NK细胞及其细胞亚群在制备不孕不育相关疾病治疗药物中的用途，其特征在于：
    其中，所述药物是以蜕膜NK细胞或其细胞亚群作为唯一活性成分或者是包含蜕膜NK细胞或其细胞亚群的药物组合物。
  8. 根据权利要求7所述的蜕膜NK细胞及其细胞亚群在制备治疗不孕不育相关疾病药物的用途，其特征在于：
    其中，所述药物组合物为片剂、丸剂、散剂、注射剂、酊剂、溶液剂、浸膏剂、软膏剂、膜剂、栓剂、片剂、泡腾片剂、凝胶剂、黏膜吸收促进剂或子宫内药物释放系统。
  9. 一种治疗不孕不育相关疾病的药物组合物，其特征在于，由蜕膜NK细胞或其细胞亚群以及药学上可接受的辅料组成。
PCT/CN2020/130939 2019-11-27 2020-11-23 蜕膜nk细胞及其细胞亚群在制备不孕不育相关疾病治疗药物中的用途 WO2021104215A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201911178836.XA CN110862963B (zh) 2019-11-27 2019-11-27 蜕膜nk细胞及其细胞亚群在制备不孕不育相关疾病治疗药物中的用途
CN201911178836.X 2019-11-27

Publications (1)

Publication Number Publication Date
WO2021104215A1 true WO2021104215A1 (zh) 2021-06-03

Family

ID=69656650

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/130939 WO2021104215A1 (zh) 2019-11-27 2020-11-23 蜕膜nk细胞及其细胞亚群在制备不孕不育相关疾病治疗药物中的用途

Country Status (2)

Country Link
CN (1) CN110862963B (zh)
WO (1) WO2021104215A1 (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110862963B (zh) * 2019-11-27 2021-08-27 沣潮医药科技（上海）有限公司 蜕膜nk细胞及其细胞亚群在制备不孕不育相关疾病治疗药物中的用途
CN111607564A (zh) * 2020-04-30 2020-09-01 浙江大学 一种评估蜕膜nk细胞分泌功能的方法
CN112708653B (zh) * 2020-11-26 2023-06-16 中国科学技术大学 一种用月经血预测反复流产和/或诊断反复流产的原因的检测方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106701677A (zh) * 2015-11-18 2017-05-24 深圳爱生再生医学科技有限公司 Nk细胞的体外扩增培养方法
CN107144687A (zh) * 2017-05-03 2017-09-08 首都医科大学附属北京妇产医院 蜕膜淋巴细胞亚型的检测方法、试剂盒及其应用
CN110721196A (zh) * 2019-11-27 2020-01-24 沣潮医药科技（上海）有限公司 蜕膜nk细胞及其细胞亚群来源外泌体在制备不孕不育相关疾病药物及辅助治疗剂中的用途
CN110862963A (zh) * 2019-11-27 2020-03-06 沣潮医药科技（上海）有限公司 蜕膜nk细胞及其细胞亚群在制备不孕不育相关疾病治疗药物中的用途

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3325967B1 (en) * 2015-07-24 2019-12-04 Innate Pharma Methods for detecting tissue infiltrating nk cells
EP3368902A1 (en) * 2015-10-30 2018-09-05 INSERM (Institut National de la Santé et de la Recherche Médicale) Method for monitoring the immunological profile of a subject
CN107029233A (zh) * 2016-02-04 2017-08-11 复旦大学附属妇产科医院 白细胞介素抗体在制备子宫内膜异位症药物组合物中的用途

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106701677A (zh) * 2015-11-18 2017-05-24 深圳爱生再生医学科技有限公司 Nk细胞的体外扩增培养方法
CN107144687A (zh) * 2017-05-03 2017-09-08 首都医科大学附属北京妇产医院 蜕膜淋巴细胞亚型的检测方法、试剂盒及其应用
CN110721196A (zh) * 2019-11-27 2020-01-24 沣潮医药科技（上海）有限公司 蜕膜nk细胞及其细胞亚群来源外泌体在制备不孕不育相关疾病药物及辅助治疗剂中的用途
CN110862963A (zh) * 2019-11-27 2020-03-06 沣潮医药科技（上海）有限公司 蜕膜nk细胞及其细胞亚群在制备不孕不育相关疾病治疗药物中的用途

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GUO, WENWEI: "Research Progress on Impact of Decidual NK Cells on Unexplained Recurrent Spontaneous Miscarriage", CHINESE JOURNAL OF CELLULAR AND MOLECULAR IMMUNOLOGY, FOURTH MILITARY MEDICAL UNIVERSITY, XI'AN, CN, 18 April 2017 (2017-04-18), CN, pages 571 - 57, XP055814775, ISSN: 1007-8738, DOI: 10.13423/j.cnki.cjcmi.008123 *
QUILLAY H., EL COSTA H., DURIEZ M., MARLIN R., CANNOU C., MADEC Y., DE TRUCHIS C., RAHMATI M., BARRÉ-SINOUSSI F., NUGEYRE M. T., M: "NK cells control HIV‐1 infection of macrophages through soluble factors and cellular contacts in the human decidua", RETROVIROLOGY, vol. 13, no. 39, 1 December 2016 (2016-12-01), pages 1 - 11, XP055814778, DOI: 10.1186/s12977-016-0271-z *
THIRUCHELVAM UMA, WINGFIELD MARY, O'FARRELLY CLIONA: "Increased uNK Progenitor Cells in Women With Endometriosis and Infertility are Associated With Low Levels of Endometrial Stem Cell Factor", AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, WILEY-BLACKWELL PUBLISHING, INC., US, vol. 75, no. 4, 1 April 2016 (2016-04-01), US, pages 493 - 502, XP055814777, ISSN: 1046-7408, DOI: 10.1111/aji.12486 *

Also Published As

Publication number Publication date
CN110862963A (zh) 2020-03-06
CN110862963B (zh) 2021-08-27

Similar Documents

Publication Publication Date Title
WO2021052503A1 (zh) 一种多能干细胞、药物组合物及其制备方法与用途
Ebrahim et al. Human mesenchymal stem cell-derived extracellular vesicles/estrogen combined therapy safely ameliorates experimentally induced intrauterine adhesions in a female rat model
WO2021104215A1 (zh) 蜕膜nk细胞及其细胞亚群在制备不孕不育相关疾病治疗药物中的用途
CN110721196B (zh) 蜕膜nk细胞及其细胞亚群来源外泌体在制备不孕不育相关疾病药物及辅助治疗剂中的用途
Zhao et al. Exosomes derived from adipose mesenchymal stem cells restore functional endometrium in a rat model of intrauterine adhesions
Lee et al. Effect of autologous adipose-derived stromal vascular fraction transplantation on endometrial regeneration in patients of Asherman’s syndrome: a pilot study
WO2021103816A1 (zh) 子宫腔液来源外泌体在制备不孕不育相关疾病治疗药物及辅助治疗剂中的用途
Cameron et al. Delayed post-treatment with bone marrow-derived mesenchymal stem cells is neurorestorative of striatal medium-spiny projection neurons and improves motor function after neonatal rat hypoxia–ischemia
KR20080106332A (ko) 인간 배아 줄기 세포 및 그의 유도체를 포함하는 조성물, 그의 사용 방법 및 제조 방법
WO2020147547A1 (zh) 分离的赤红球菌细胞壁骨架在制备治疗宫颈糜烂的药物中的用途
US20230256021A1 (en) Compositions for preventing or treating diabetic skin disease comprising exosome-derived from thrombin-treated stem cell
Zhou et al. Inhibition by Medroxyprogesterone Acetate of Interleukin-1β–Induced Collagen Degradation by Corneal Fibroblasts
Hou et al. Endometrial regeneration in Asherman's syndrome: clinical and translational evidence of stem cell therapies
Chiappalupi et al. Intraperitoneal injection of microencapsulated Sertoli cells restores muscle morphology and performance in dystrophic mice
Shi et al. Biological mechanisms and applied prospects of mesenchymal stem cells in premature ovarian failure
Chen et al. Effect of human umbilical cord mesenchymal stem cells transplantation on nerve fibers of a rat model of endometriosis
KR102477288B1 (ko) 혈관외피줄기세포 배양액 또는 사이클로필린 a를 포함하는 아셔만 증후군 치료용 조성물
KR101617912B1 (ko) 말초혈액 단핵세포를 유효성분으로 포함하는 예방 또는 치료용 약제학적 조성물
CN114984028A (zh) 黄芪甲苷的用途
WO2022037362A1 (zh) 一种治疗带状疱疹的注射剂
CN111135193A (zh) 一种应用干细胞恢复子宫内膜的方法
US20160367602A1 (en) Methods for treating hidradenitis suppurativa
EP3146975A1 (en) Agent for prevention and treatment of chlamydia infection
RU2428173C1 (ru) Фармацевтическая композиция для лечения заболеваний нижнего отдела мочеполовой системы
Mingxia et al. Efficacy of gamma-irradiated adipose-derived stem cells for treatment of thin endometrium in rats

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20893981

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20893981

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 26/09/2022)

122 Ep: pct application non-entry in european phase

Ref document number: 20893981

Country of ref document: EP

Kind code of ref document: A1