CN113970633A - 一种综合评价人体免疫功能的检测方法 - Google Patents
一种综合评价人体免疫功能的检测方法 Download PDFInfo
- Publication number
- CN113970633A CN113970633A CN202010721273.0A CN202010721273A CN113970633A CN 113970633 A CN113970633 A CN 113970633A CN 202010721273 A CN202010721273 A CN 202010721273A CN 113970633 A CN113970633 A CN 113970633A
- Authority
- CN
- China
- Prior art keywords
- cells
- cell
- immune
- detection
- peripheral blood
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 29
- 230000036039 immunity Effects 0.000 title claims description 8
- 210000004027 cell Anatomy 0.000 claims abstract description 67
- 210000004881 tumor cell Anatomy 0.000 claims abstract description 21
- 210000002865 immune cell Anatomy 0.000 claims abstract description 20
- 230000036737 immune function Effects 0.000 claims abstract description 16
- 238000011156 evaluation Methods 0.000 claims abstract description 12
- 230000007651 self-proliferation Effects 0.000 claims abstract description 10
- 230000000694 effects Effects 0.000 claims abstract description 9
- 210000003289 regulatory T cell Anatomy 0.000 claims abstract description 9
- 210000004479 myeloid suppressor cell Anatomy 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims abstract description 4
- 210000005259 peripheral blood Anatomy 0.000 claims description 11
- 239000011886 peripheral blood Substances 0.000 claims description 11
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims description 9
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 7
- 210000000822 natural killer cell Anatomy 0.000 claims description 7
- 210000004698 lymphocyte Anatomy 0.000 claims description 5
- 238000003556 assay Methods 0.000 claims description 4
- 210000004970 cd4 cell Anatomy 0.000 claims description 4
- 238000007747 plating Methods 0.000 claims description 3
- 230000010100 anticoagulation Effects 0.000 claims description 2
- 230000017531 blood circulation Effects 0.000 claims description 2
- 210000004405 cytokine-induced killer cell Anatomy 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 230000000007 visual effect Effects 0.000 claims description 2
- 208000008839 Kidney Neoplasms Diseases 0.000 claims 1
- 206010038389 Renal cancer Diseases 0.000 claims 1
- 238000004220 aggregation Methods 0.000 claims 1
- 230000002776 aggregation Effects 0.000 claims 1
- 210000004700 fetal blood Anatomy 0.000 claims 1
- 201000010982 kidney cancer Diseases 0.000 claims 1
- 210000004976 peripheral blood cell Anatomy 0.000 claims 1
- 238000000926 separation method Methods 0.000 claims 1
- 230000002147 killing effect Effects 0.000 abstract description 9
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 abstract description 4
- 239000002609 medium Substances 0.000 description 7
- 230000007423 decrease Effects 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 208000023275 Autoimmune disease Diseases 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 4
- 210000000987 immune system Anatomy 0.000 description 4
- 210000000207 lymphocyte subset Anatomy 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 210000000662 T-lymphocyte subset Anatomy 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 210000000649 b-lymphocyte subset Anatomy 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 238000000432 density-gradient centrifugation Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 230000004727 humoral immunity Effects 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 208000030507 AIDS Diseases 0.000 description 2
- 230000033289 adaptive immune response Effects 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 239000012888 bovine serum Substances 0.000 description 2
- 230000022534 cell killing Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000001506 immunosuppresive effect Effects 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 210000004180 plasmocyte Anatomy 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 206010039073 rheumatoid arthritis Diseases 0.000 description 2
- 230000000630 rising effect Effects 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 206010008909 Chronic Hepatitis Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 208000005176 Hepatitis C Diseases 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 208000025747 Rheumatic disease Diseases 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 238000010322 bone marrow transplantation Methods 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 230000009610 hypersensitivity Effects 0.000 description 1
- 238000011502 immune monitoring Methods 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 230000008629 immune suppression Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 230000006054 immunological memory Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 206010028417 myasthenia gravis Diseases 0.000 description 1
- 210000004985 myeloid-derived suppressor cell Anatomy 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000000552 rheumatic effect Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 1
- 201000008827 tuberculosis Diseases 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 231100000747 viability assay Toxicity 0.000 description 1
- 238000003026 viability measurement method Methods 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5047—Cells of the immune system
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
- G01N33/5026—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on cell morphology
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5047—Cells of the immune system
- G01N33/505—Cells of the immune system involving T-cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5047—Cells of the immune system
- G01N33/5052—Cells of the immune system involving B-cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5047—Cells of the immune system
- G01N33/5055—Cells of the immune system involving macrophages
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1006—Investigating individual particles for cytology
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Cell Biology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Microbiology (AREA)
- Physiology (AREA)
- Dispersion Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明涉及医疗领域，公开了一种通过联合外周血淋巴细胞亚群的流式检测以及外周血淋巴细胞活力检测的方法，用以综合评价人体免疫功能的情况。本项发明在检测方法上增加了调节性T细胞和髓样抑制细胞的流式检测，同时联合外周血淋巴细胞的活力检测方法；在评价方法上，综合免疫细胞亚群的评价结果、受检细胞和免疫细胞自我增殖活性、受检细胞和免疫细胞对肿瘤细胞的杀伤能力的结果，更加学准确、全面的评估机体的免疫功能状态。
Description
技术领域
本发明涉及医疗领域，特别提供一种综合评价人体免疫功能的检测方法。这一检测方法更加科学准确、全面的评估机体的免疫功能状态，也使结果更具有参考价值。
背景技术
免疫系统是机体执行免疫应答以及免疫功能的重要系统，由免疫器官、免疫细胞和免疫分子组成。免疫系统具有识别和排除抗原性异物、与机体其他系统相互协调.共同维持机体内环境稳定和生理平衡的功能。近年来肿瘤发病率的不断增高，其中免疫系统的失衡和衰退占有主要因素。
本项发明公开一种综合评价人体免疫功能的检测方法，通过机体对肿瘤细胞的识别杀伤作用，并联合流式检测结果对免疫功能进行综合评价，能够更加科学全面的评估机体状态和肿瘤的发病风险。
发明内容
为提高人体免疫功能检测方法的准确性和全面性，本发明提供了一种综合评价人体免疫功能的检测方法，主要包括以下检测步骤：
一、抗凝外周血的流式检测
提取10-30 uL受检者抗凝外周血，应该用BD公司的流式细胞检测仪（仪器编号：FACSVia）以及BD公司生产的淋巴细胞亚群检测试剂盒（NO:340503)来检测淋巴细胞亚群:总T细胞亚群、CD8细胞亚群、CD4细胞亚群、B细胞亚群、NK细胞亚群、调节性T细胞亚群、髓样抑制细胞亚群；并计算CD4/CD8的比值。
二、提取抗凝外周血的PBMC
1.提取10-30 mL受检者抗凝外周血，以1：1的比例加入密度梯度离心液
10-30 mL。
2.密度梯度离心：将加好密度梯度离心液的样本进行密度梯度离心：
2051 rpm,离心25 min；梯度离心结束后提取中间白膜层，白膜层即为受检者的PBMC。
3.洗涤:向受检者PBMC内加入15-25 mL PBS进行洗涤，应用离心机1000 rpm，
5 min；离心结束后将液体弃去，只留下底层细胞。
4.二次洗涤：向步骤3中的细胞内加入15-25 mL PBS，应用离心机1000 rpm
5 min进行二次洗涤。
5.向步骤4中得到的细胞加入1-3mL CIK培养基，留以使用。
三、铺板
1.肿瘤细胞116：116细胞为本实验室储存，用含有10%的新生牛血清RPM1640培养基进行培养。在活力检测前4-6小时将肿瘤116细胞接种到96孔板，其中每孔加入培养液体积为50-100 uL，细胞接种数量为2×103-4×103个。共需要接种9孔，其中3孔为和受检细胞共培养，3孔为和激活的免疫细胞共培养，3孔为阴性对照。
2.激活的免疫细胞：激活的免疫细胞CIK为本实验室储存，在接种时与受检
细胞同时提前4-6小时内接种到96孔板，其中每孔加入培养液体积为50-100 uL，细胞接种数量为8×104-105个。共需要接种6孔，其中3孔为独立孔，3孔为和肿瘤细胞116共培养。
3.受检者的PBMC细胞：使用步骤2-5中得到的受检者的PBMC细胞进行铺板，用含有10%新生牛血清CIK培养基进行培养，其中每孔加入培养液体积为50-100 uL，细胞接种数量为8×104-105个。共需要接种6孔，其中3孔为独立孔，3孔为和肿瘤细胞116共培养。
四、培养及拍照
铺板后立即在倒置显微镜下采集细胞图像，采集结束后放入孵箱内进行培养，在48 h后再次采集细胞的增殖状况以及免疫细胞和受检细胞分别对肿瘤细胞的识别杀伤图像，每孔内不同视野采集图像数量不少于10张。
五、免疫功能评价的说明
1.与正常范围内的参考区间进行比较，对应用流式检测的淋巴细胞亚
群进行评价
1）总T细胞亚群：T淋巴细胞来源于骨髓的多能干细胞，有利于广泛接触
进入体内的抗原物质，加强免疫应答，较长期保持免疫记忆。总T细胞比例上升，表明机体的适应性免疫应答存在过度活跃状态，提示机体可能存在感染性疾病、自身免疫性疾病等状态；总T细胞比列下降，表明机体整体的适应性免疫应答能力下降，可能会引发恶性肿瘤、结核病等。
2）CD8细胞亚群：CD8细胞是T淋巴细胞其中的一个亚群。在正常的T
淋巴细胞亚群检测中，CD8淋巴细胞应该占到15%-44%左右。CD8细胞代表细胞毒性T细胞，其可以抵制外来病原体的入侵，具有杀伤靶细胞的重要作用，因此对机体是有一定保护作用的。CD8比例上升表明机体可能存在炎症反应，如乙肝、丙肝等慢性肝炎、病毒感染等；CD8比例下降表明机体对靶细胞杀伤能力下降，一般可以见于风湿免疫系统，例如类风湿性关节炎、系统性红斑狼疮等。
3）CD4细胞亚群：CD8细胞是T淋巴细胞其中的一个亚群。在正常的T
淋巴细胞亚群检测中，CD4淋巴细胞应该占到21%-51%左右。CD4细胞代表着辅助性T细胞，其可以对细胞免疫、体液免疫起到辅助的作用。CD4比例上升表明机体免疫抑制占优势，表明机体可能存在免疫衰退、脏器炎性微环境异常以及长期亚健康状态；CD4比例下降表明调控机体免疫功能下降，常见于艾滋病、重大手术预后、恶性肿瘤等。
4）CD4/CD8的比值：CD4/CD8的比值时监测机体内免疫功能能状态的重要
指标，比值升高表明机体处于“过度免疫活跃”的状态，常见于I型糖尿病、类风湿性关节炎的活动期等；比值降低说明机体处于“免疫抑制”状态，常见艾滋病、骨髓移植的恢复期、恶性肿瘤复发期等。
5）B细胞亚群：B细胞在抗原刺激下可分化成浆细胞，浆细胞可合成和分
泌抗体免疫球蛋白，主要执行机体的体液免疫。B细胞数值上升说明机体内近期可发生细菌或病毒感染；B细胞数值下降说明机内体液免疫功能下降，常见于淋巴瘤等。
6）NK细胞亚群：NK细胞是自然杀伤细胞，是机体重要的免疫细胞，不仅与抗肿瘤、抗病毒和免疫调节有关，而且还在某些情况下参与超敏反应和自身免疫性疾病的发生。NK细胞亚群数量下降，表明机体自然杀伤能力下降，进一步提升机体固有免疫功能下降。
7）调节性T细胞亚群：调节性T细胞是一类控制体内自身免疫反应性的T细胞亚群，与自身免疫性疾病的发生关系密切，其异常表达可导致自身免疫性疾病。调节性T细胞亚群的失常常见于重症肌无力、多发性硬化、自身溶血性贫血等。
8）髓样抑制细胞：是一群具有免疫抑制功能细胞的统称，由未成熟的巨噬细胞、粒细胞、树突细胞和其他处于早期分化的骨髓细胞组成，他对机体的平衡状态具有维持作用，并且能够对肿瘤的产生起到免疫监视作用。
2．外周血淋巴细胞活力检测结果：
1）受检细胞、免疫细胞自我增殖的活性检测结果评价：根据采集到的图片分为六个等级：
良好：细胞呈现圆形，每孔内细胞数量大于5×105个。
轻度：细胞呈现圆形，每孔内细胞数量4×105 - 5×105个。
轻中度：细胞呈现圆形，每孔内细胞数量3×105 -4×105个。
中度：细胞呈现椭圆形，每孔内细胞数量2×105 -3×105个。
中重度：细胞呈现椭圆形，每孔内细胞数量105 -2×105个。
重度：细胞呈现不规则图形，每孔内细胞数量少于105个。
2）受检细胞、免疫细胞杀伤肿瘤细胞结果评价：根据采集到的图片分为六个等级：良好：呈现6个以上的细胞聚集群落，每个细胞聚集群落直径大于100μm以上。
轻度：呈现4-6个细胞聚集群落，每个细胞聚集群落直径在80 μm-100μm。
轻中度：呈现3-4个细胞聚集群落，每个细胞聚集群落直径在60 μm-80μm。
中度：呈现2-3个细胞聚集群落，每个细胞聚集群落直径在40 μm-60 μm。
中重度：只有1-2个细胞聚集群落，每个细胞聚集群落直径20 μm-40 μm。
重度：几乎没有细胞聚集群落。
3.免疫功能检测结果评价：综合以上检测，分为六个等级：
4、附图说明
图1为为96孔板铺板示意图
图2为总T细胞亚群流式结果图
图3为CD4亚群流式结果图
图4为CD8亚群流式结果图
图5为NK细胞亚群流式结果图
图6为B细胞亚群流式结果图
图7、8为髓样抑制细胞亚群流式结果图
图9为调节性T细胞亚群流式结果图
图10为阳性对照：激活的免疫细胞CIK自我增殖情况 0h
图11为阳性对照：激活的免疫细胞CIK自我增殖情况 48h
图12为阴性对照：肿瘤细胞116 自我增殖情况0h
图13为阴性对照：肿瘤细胞116自我增殖情况 48h
图14为受检细胞自我增殖情况 0h
图15为受检细胞自我增殖情况 48h
图16为激活的免疫细胞杀伤肿瘤细胞的阳性对照 0h
图17为激活的免疫细胞杀伤肿瘤细胞的阳性对照 48h
图18为受检细胞杀伤肿瘤细胞情况 0h
图19为受检细胞杀伤肿瘤细胞情况 48h。
具体实施方式
一、抗凝外周血流式检测：
提取20 uL受检者抗凝外周血，分别加入相应抗体，用流式检测仪进行流
检测。
二、提取受检细胞PBMC
取20 mL受检者抗凝外周血，以1：1的比例加入密度梯度离心液20 mL
进行密度梯度离心，提取中间层并用20mL PBS进行洗涤，得到受检细胞的PBMC。
三、铺板
提前4 h内对肿瘤细胞116进行铺板，每孔内加入100 uL培养基，肿瘤116细胞个数为3×103个，共设9孔，每孔内加入100 uL培养基；同时间内对激活的免疫细胞CIK进行铺板，每孔内CIK细胞数量为9×104，共加6孔，每孔内加入培养基100 uL，其中三孔与肿瘤细胞共培养。得到受检细胞PBMC之后，对受检细胞进行铺板，每孔内受检细胞数量为9×104，共加6孔，每孔内加入培养基100 uL，其中三孔与肿瘤细胞共培养。
四、图像采集
分别在0 h、48 h时对细胞形态进行采集。
五、免疫功能评价
1.与正常范围内的参考区间进行比较，对应用流式检测的淋巴细胞亚
群进行评价，所有结果均在正常范围内。
2.免疫细胞增殖的活性检测结果分析：根据受检细胞的自我增殖图像图像
评价为：良好。
3.激活的免疫细胞识别并杀伤肿瘤细胞的活力检测：根据受检细胞采集的
图像评价为：良好。
4.免疫功能评价:综合以上评价，该机体免疫功能状态：良好。
Claims (4)
1.一种综合评价人体免疫功能的检测方法，其特征在于包括以下检测步骤：
1）抗凝外周血流式检测：提取受检者抗凝外周血10-30 μL,加入相应抗体，对总T细胞、CD4细胞亚群、CD8细胞亚群、NK细胞亚群、调节性T细胞、髓样抑制细胞进行流式检测；
2）提取PBMC:取受检者抗凝外周血10-20 mL，以1:1的比例加入密度梯度分离液10-20mL；
3）铺板：肿瘤细胞每孔铺板数量为：2×103 - 4×103个，免疫细胞每孔铺板数量为：8×104 - 105个，肿瘤细胞除自我增殖外，需同受检细胞和免疫细胞共培养，在铺板后0h、48h使用倒置显微镜进行图像采集，每孔不同视野拍照数量大于10张；
4）免疫功能检测评价：综合淋巴细胞亚群检测与外周血细胞活力检测结果，对人体免疫功能进行综合评价。
2.根据权利要求1所述的检测方法，其特征在于增加了调节性T细胞和髓样抑制细胞的流式检测。
3.根据权利要求1所述的评价方法，其特征在于通过细胞形态、细胞数量、细胞群落聚集情况以及群落直径进行综合分级评价。
4.根据权利要求1所述的肿瘤细胞，其特征在于为HCT116细胞（来源于肾癌细胞）；如步骤3所述的免疫细胞，其特征在于为CIK细胞（来源于脐带血细胞）。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010721273.0A CN113970633A (zh) | 2020-07-24 | 2020-07-24 | 一种综合评价人体免疫功能的检测方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010721273.0A CN113970633A (zh) | 2020-07-24 | 2020-07-24 | 一种综合评价人体免疫功能的检测方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113970633A true CN113970633A (zh) | 2022-01-25 |
Family
ID=79585811
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010721273.0A Pending CN113970633A (zh) | 2020-07-24 | 2020-07-24 | 一种综合评价人体免疫功能的检测方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113970633A (zh) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114791411A (zh) * | 2022-04-21 | 2022-07-26 | 广州先康达生物科技有限公司 | 评估人体免疫功能的指标组合、试剂盒和方法 |
| TWI812274B (zh) * | 2022-06-10 | 2023-08-11 | 何鈞軒 | 檢測免疫力效能的方法及其系統 |
-
2020
- 2020-07-24 CN CN202010721273.0A patent/CN113970633A/zh active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114791411A (zh) * | 2022-04-21 | 2022-07-26 | 广州先康达生物科技有限公司 | 评估人体免疫功能的指标组合、试剂盒和方法 |
| TWI812274B (zh) * | 2022-06-10 | 2023-08-11 | 何鈞軒 | 檢測免疫力效能的方法及其系統 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Timonen et al. | Characteristics of human large granular lymphocytes and relationship to natural killer and K cells | |
| JP2769156B2 (ja) | 単核白血球免疫システムの免疫調節状態の自動的評価のための方法及び装置 | |
| US5100777A (en) | Antibody matrix device and method for evaluating immune status | |
| US5766944A (en) | T cell differentiation of CD34+ stem cells in cultured thymic epithelial fragments | |
| EP0289896B1 (en) | A simplified method for the preparation of human lymphokine activated killer cells | |
| Silvers et al. | Mixed leukocyte reactions and histocompatibility in rats | |
| CN113970633A (zh) | 一种综合评价人体免疫功能的检测方法 | |
| US7442390B2 (en) | Method for enhancing engraftment of cells using mesenchymal progenitor cells | |
| CN106566806B (zh) | 体外培养富集cd8+t细胞的方法 | |
| Gladwin et al. | Identification of mRNA for PDGF B‐chain in human megakaryocytes isolated using a novel immunomagnetic separation method | |
| CN111394310A (zh) | 一种强免疫调节，强杀伤肿瘤细胞和病毒感染细胞的增强nk细胞和其制备方法与试剂盒 | |
| CN106932576A (zh) | 一种人调节性t细胞的免疫抑制功能的检测方法 | |
| CN111235209A (zh) | 一种评估干细胞免疫调控功能的方法 | |
| METCALF | Detection and analysis of human granulocyte–monocyte precursors using semi-solid cultures | |
| CN114791411A (zh) | 评估人体免疫功能的指标组合、试剂盒和方法 | |
| CN113549595A (zh) | 基于外周血的nk细胞体外培养方法 | |
| CN112852731A (zh) | 一种体外诱导造血干细胞向调节性t细胞分化的方法 | |
| Thorsby | HL—A Antigens and Genes-I. A Study of Unrelated Norwegians | |
| CN110055219B (zh) | 一种利用非动员外周血制备异质性造血干祖细胞的方法 | |
| Glass et al. | Use of cell separation and short-term culture techniques to study erythroid cell development | |
| Barrena et al. | Imbalance of the CD4+ subpopulations expressing CD45RA and CD29 antigens in the peripheral blood of adults and children with Down syndrome | |
| EP3203237B1 (en) | Urine-derived epithelial cell lines for diagnosis and therapy of an anti-bk-virus or anti-graft immune response | |
| JP5791095B2 (ja) | Ｐｎｈ型白血球の検出方法 | |
| CN115094034B (zh) | 一种人nkt细胞系及其应用 | |
| CN111172110A (zh) | 一种脐带血cik细胞的培养方法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |