CN113720666A - Method for separating cancer cells from human urine sample - Google Patents
Method for separating cancer cells from human urine sample Download PDFInfo
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- CN113720666A CN113720666A CN202111021344.7A CN202111021344A CN113720666A CN 113720666 A CN113720666 A CN 113720666A CN 202111021344 A CN202111021344 A CN 202111021344A CN 113720666 A CN113720666 A CN 113720666A
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- cancer cells
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- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 50
- 201000011510 cancer Diseases 0.000 title claims abstract description 49
- 238000000034 method Methods 0.000 title claims abstract description 33
- 210000002700 urine Anatomy 0.000 title claims abstract description 27
- 230000005291 magnetic effect Effects 0.000 claims abstract description 30
- 239000002086 nanomaterial Substances 0.000 claims abstract description 20
- 238000007885 magnetic separation Methods 0.000 claims abstract description 10
- 239000008188 pellet Substances 0.000 claims abstract description 9
- 239000002244 precipitate Substances 0.000 claims abstract description 8
- 238000003306 harvesting Methods 0.000 claims abstract description 4
- 239000000523 sample Substances 0.000 claims description 16
- 239000002122 magnetic nanoparticle Substances 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 12
- 229920002873 Polyethylenimine Polymers 0.000 claims description 11
- 239000011259 mixed solution Substances 0.000 claims description 11
- 238000002156 mixing Methods 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 10
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 9
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims description 7
- NDLPOXTZKUMGOV-UHFFFAOYSA-N oxo(oxoferriooxy)iron hydrate Chemical group O.O=[Fe]O[Fe]=O NDLPOXTZKUMGOV-UHFFFAOYSA-N 0.000 claims description 7
- 239000002243 precursor Substances 0.000 claims description 6
- 238000011534 incubation Methods 0.000 claims description 5
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 4
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N iron oxide Inorganic materials [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 claims description 4
- 238000000464 low-speed centrifugation Methods 0.000 claims description 4
- FVBUAEGBCNSCDD-UHFFFAOYSA-N silicide(4-) Chemical compound [Si-4] FVBUAEGBCNSCDD-UHFFFAOYSA-N 0.000 claims description 4
- 229910052710 silicon Inorganic materials 0.000 claims description 4
- 239000010703 silicon Substances 0.000 claims description 4
- 239000012488 sample solution Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 229910021332 silicide Inorganic materials 0.000 claims description 2
- 239000000725 suspension Substances 0.000 claims 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 6
- 239000002953 phosphate buffered saline Substances 0.000 description 6
- 238000003745 diagnosis Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 230000007547 defect Effects 0.000 description 2
- 230000005389 magnetism Effects 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 208000008839 Kidney Neoplasms Diseases 0.000 description 1
- 206010038389 Renal cancer Diseases 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical group O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 201000010982 kidney cancer Diseases 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 230000005298 paramagnetic effect Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a method for separating cancer cells from human urine, which comprises the following steps: the first step is as follows: centrifuging the urine sample; the second step is as follows: capturing cancer cells of the centrifuged sample by using positively charged magnetic nano materials; the third step: purifying cancer cells captured from urine by magnetic separation to obtain precipitate; the fourth step: resuspend the pellet with PBS and harvest the desired cancer cells.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a method for separating cancer cells from human urine samples, which is used for separating the cancer cells from the urine.
Background
The diagnosis and prognosis of many cancers (e.g., bladder cancer, colorectal cancer, renal cancer, breast cancer, etc.) requires imaging, microscopy, biopsy, etc. However, these methods have the disadvantages of low sensitivity, poor sensitivity, easy omission and the like, and even have the defect of possibly causing wound which is difficult to recover for patients.
In clinic, the method for separating the cancer cells from the human urine sample, which has the advantages of no wound, high sensitivity and simple operation, has bright application prospect.
Disclosure of Invention
The invention aims to solve the technical problem of providing a noninvasive, high-sensitivity and simple-operation method for separating cancer cells from human urine, aiming at the defects in the prior art.
According to the present invention, there is provided a method for isolating human urine-like cancer cells, comprising:
the first step is as follows: centrifuging the urine sample;
the second step is as follows: capturing cancer cells of the centrifuged sample by using positively charged magnetic nano materials;
the third step: purifying cancer cells captured from urine by magnetic separation to obtain precipitate;
the fourth step: resuspend the pellet with PBS and harvest the desired cancer cells.
Preferably, in the first step, the patient urine sample is treated using low speed centrifugation to obtain a pellet, wherein the centrifugation speed is between 400 and 5000 rpm.
Preferably, in the second step, the pellet is resuspended using PBS, and positively charged magnetic nanoparticles are added to the centrifuged sample solution and incubated.
Preferably, the urine sample of the first step is 10-150 ml; the second step of PBS is 0.5-3ml, the positive charged magnetic nanoparticles is 30-80 μ g, the incubation temperature is 4 ℃, and the incubation time is 3-30 minutes.
Preferably, in the third step, the magnetic separation is performed using a magnetic rack, the time of the magnetic separation process is between 3 and 30 minutes, and a precipitate is obtained.
Preferably, the method for preparing the positively charged magnetic nanomaterial used in the second step comprises:
dissolving a magnetic nanoparticle precursor in ethylene glycol to obtain a mixed solution, and placing the mixed solution in a vacuum reaction kettle for reaction to obtain a ferric oxide core;
mixing and stirring the obtained ferric oxide nuclei and tetraethyl silicate solution to obtain silicon magnetic nuclei;
and mixing and stirring the obtained silicon magnetic cores and the polyethyleneimine water solution to obtain the positively charged magnetic nano material.
Preferably, the concentration of the precursor of the magnetic nano-particles in the mixed solution is 10-50 mg/ml; the tetraethyl silicate solution is 30-150 mu L, the mixing and stirring time of the ferric oxide nucleus and the tetraethyl silicate solution is 8-24 hours, and the mixing and stirring time of the magnetic silicide nucleus and the polyethyleneimine aqueous solution is 0.5-24 hours.
Preferably, the mixed solution is placed in a vacuum reaction kettle to react for 6 to 15 hours at the temperature of 150 ℃ and 250 ℃.
Preferably, the polyethyleneimine is a branched polyethyleneimine.
Preferably, the positive electro-magnetic nanomaterial has a ZETA potential of 30 mV.
In the invention, a positively charged magnetic nano material is adopted, and cancer cells can be specifically enriched by positively charging; has magnetism, and can be enriched by simple magnetic rack. The method can simply, quickly, cheaply and efficiently separate out the cancer cells in the urine of cancer patients and can be used as a powerful basis for cancer diagnosis.
According to the method for separating the cancer cells from the human urine sample, the cancer cells can be sampled noninvasively and conveniently and can be separated and purified efficiently. The method has the advantages of simple experimental conditions and operation, short time consumption, noninvasive sampling, strong specificity, convenience, easiness, low price and the like, and has higher clinical application potential.
Drawings
A more complete understanding of the present invention, and the attendant advantages and features thereof, will be more readily understood by reference to the following detailed description when considered in conjunction with the accompanying drawings wherein:
fig. 1 schematically shows a flowchart of a separation method of human urine-like separated cancer cells according to a preferred embodiment of the present invention.
Fig. 2 schematically shows a transmission electron microscope image of the positively charged magnetic nanomaterial employed in the method for isolating human urine-like isolated cancer cells according to the preferred embodiment of the present invention.
Fig. 3 schematically shows a ZETA potential (potential of shear plane, also called electromotive potential or electromotive potential) of the positively charged magnetic nanomaterial employed in the method for separating human urine-like separated cancer cells according to the preferred embodiment of the present invention.
It is to be noted, however, that the appended drawings illustrate rather than limit the invention. It is noted that the drawings representing structures may not be drawn to scale. Also, in the drawings, the same or similar elements are denoted by the same or similar reference numerals.
Detailed Description
In order that the present disclosure may be more clearly and readily understood, reference will now be made in detail to the present disclosure as illustrated in the accompanying drawings.
The invention adopts the principle of electrostatic adsorption, is designed aiming at the characteristic of negative charge on the surface of cancer cells, and provides a method for separating the cancer cells by using a urine sample.
Fig. 1 schematically shows a flowchart of a separation method of human urine-like separated cancer cells according to a preferred embodiment of the present invention.
As shown in fig. 1, the method for isolating human urine-like isolated cancer cells according to the preferred embodiment of the present invention comprises:
first step S1: centrifuging the urine sample;
preferably, in a first step S1, a patient urine sample (e.g., a 10-150ml patient urine sample) is processed using low speed centrifugation to obtain a pellet.
Preferably, the first step uses low speed centrifugation, wherein the centrifugation rotation speed is between 400-.
Second step S2: capturing cancer cells of the centrifuged sample by using positively charged magnetic nano materials;
preferably, the positively charged magnetic nanomaterial is a positively charged magnetic nanoparticle.
Further, preferably, the positively charged magnetic nanomaterial is a positively charged magnetic particle of a paramagnetic nanoparticle.
Specifically, for example, in the second step S2, the pellet is resuspended using 0.5-3ml PBS (phosphate buffered saline), 30-80 μ g of positively charged magnetic nanoparticles are added to the sample solution after centrifugation, and incubation is performed at 4 ℃ for, for example, 3-30 minutes.
Third step S3: purifying cancer cells captured from urine by magnetic separation to obtain precipitate;
preferably, in the third step S3, the magnetic separation is performed using a magnetic rack, the time of the magnetic separation process is between 3-30 minutes, and a precipitate is obtained.
Fourth step S4: resuspend the pellet with PBS and harvest the desired cancer cells.
Preferably, the method for preparing the positively charged magnetic nanomaterial used in the second step comprises:
dissolving the magnetic nanoparticle precursor in ethylene glycol to obtain a mixed solution, enabling the concentration of the magnetic nanoparticle precursor in the mixed solution to be 10-50mg/ml, and placing the mixed solution in a vacuum reaction kettle to react for 6-15 hours at the temperature of 150 ℃ and 250 ℃ to obtain a ferroferric oxide core;
mixing and stirring the obtained ferric oxide nuclei and 30-150 mu L of tetraethyl silicate solution for 8-24 hours to obtain magnetic silicide nuclei;
mixing the obtained magnetic silicide core with the water solution of polyethylene imine (for example, polyethylene imine is branched polyethylene imine) and stirring for 0.5-24 hours to obtain the positively charged magnetic nano material.
Fig. 2 schematically shows a transmission electron micrograph of a positively charged magnetic nanomaterial, which has a diameter of about 0.4um, obtained by the above-described steps. Fig. 3 schematically shows the ZETA potential (shear plane potential, also called electromotive potential or electromotive potential) of the positive electromagnetic nanomaterial obtained by the above-described steps, and it can be seen that the ZETA potential of the positive electromagnetic nanomaterial is about 30 mV.
In the invention, a positively charged magnetic nano material is adopted, and cancer cells can be specifically enriched by positively charging; has magnetism, and can be enriched by simple magnetic rack. The method can simply, quickly, cheaply and efficiently separate out the cancer cells in the urine of cancer patients and can be used as a powerful basis for cancer diagnosis.
According to the method for separating the cancer cells from the human urine sample, the cancer cells can be sampled noninvasively and conveniently and can be separated and purified efficiently. The method has the advantages of simple experimental conditions and operation, short time consumption, noninvasive sampling, strong specificity, convenience, easiness, low price and the like, and has higher clinical application potential.
It should be noted that the terms "first", "second", "third", and the like in the description are used for distinguishing various components, elements, steps, and the like in the description, and are not used for indicating a logical relationship or a sequential relationship between the various components, elements, steps, and the like, unless otherwise specified.
It is to be understood that while the present invention has been described in conjunction with the preferred embodiments thereof, it is not intended to limit the invention to those embodiments. It will be apparent to those skilled in the art from this disclosure that many changes and modifications can be made, or equivalents modified, in the embodiments of the invention without departing from the scope of the invention. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are still within the scope of the protection of the technical solution of the present invention, unless the contents of the technical solution of the present invention are departed.
Claims (10)
1. A method for isolating human urine-like cancer cells, comprising:
the first step is as follows: centrifuging the urine sample;
the second step is as follows: capturing cancer cells of the centrifuged sample by using positively charged magnetic nano materials;
the third step: purifying cancer cells captured from urine by magnetic separation to obtain precipitate;
the fourth step: resuspend the pellet with PBS and harvest the desired cancer cells.
2. The method for separating human urine-like cancer cells as claimed in claim 1, wherein in the first step, the urine specimen of the patient is processed by low speed centrifugation to obtain the precipitate, wherein the centrifugation speed is between 400-5000 rpm.
3. The method for separating cancer cells from human urine according to claim 1 or 2, wherein in the second step, the suspension of the pellet is resuspended using PBS, and positively charged magnetic nanoparticles are added to the centrifuged sample solution and incubated.
4. The method for isolating human urine-like cancer cells according to claim 1 or 2, wherein the urine sample of the first step is 10 to 150 ml; the second step of PBS is 0.5-3ml, the positive charged magnetic nanoparticles is 30-80 μ g, the incubation temperature is 4 ℃, and the incubation time is 3-30 minutes.
5. The method for separating human urine-like cancer cells according to claim 1 or 2, wherein in the third step, magnetic separation is performed using a magnetic frame, and the time of the magnetic separation treatment is between 3 and 30 minutes to obtain a precipitate.
6. The method for separating human urine-like cancer cells according to claim 1 or 2, wherein the method for preparing the positively charged magnetic nanomaterial used in the second step comprises:
dissolving a magnetic nanoparticle precursor in ethylene glycol to obtain a mixed solution, and placing the mixed solution in a vacuum reaction kettle for reaction to obtain a ferric oxide core;
mixing and stirring the obtained ferric oxide nuclei and tetraethyl silicate solution to obtain silicon magnetic nuclei;
and mixing and stirring the obtained silicon magnetic cores and the polyethyleneimine water solution to obtain the positively charged magnetic nano material.
7. The method for separating human urine-like cancer cells as claimed in claim 6, wherein the concentration of the precursor of magnetic nanoparticles in the mixed solution is 10-50 mg/ml; the tetraethyl silicate solution is 30-150. mu.L.
8. The method for separating human urine-like cancer cells according to claim 6 or 7, wherein the time for mixing and stirring the ferric oxide nuclei and the tetraethyl silicate solution is 8 to 24 hours, and the time for mixing and stirring the magnetic silicide nuclei and the polyethyleneimine aqueous solution is 0.5 to 24 hours.
9. The method for separating human urine-like cancer cells as claimed in claim 6 or 7, wherein the mixed solution is placed in a vacuum reaction vessel and reacted at a temperature of 150 ℃ and 250 ℃ for 6-15 hours.
10. The method for separating human urine-like cancer cells according to claim 1 or 2, wherein the polyethyleneimine is a branched polyethyleneimine.
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| CN202111021344.7A CN113720666A (en) | 2021-09-01 | 2021-09-01 | Method for separating cancer cells from human urine sample |
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| CN103630440A (en) * | 2013-11-28 | 2014-03-12 | 武汉大学 | Enriching method of circulating tumor cells |
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| US20180179515A1 (en) * | 2016-12-23 | 2018-06-28 | National Cancer Center | Magnetic nanostructure for detecting and isolating cell-free dna comprising cationic polymer and magnetic-nanoparticle-containing conductive polymer |
| CN109100511A (en) * | 2018-07-20 | 2018-12-28 | 四川大学 | Capture and the immune magnetic nano particle of release and preparation method thereof are visualized for circulating tumor cell |
| CN109112107A (en) * | 2018-09-11 | 2019-01-01 | 上海浦美医学科技有限公司 | A method of separation CTC is captured based on rgd peptide |
| CN111487404A (en) * | 2019-01-28 | 2020-08-04 | 猎源（上海）生物医药科技有限公司 | Kit for extracting DNA of body fluid tumor cells |
| CN111826351A (en) * | 2020-03-13 | 2020-10-27 | 武汉大学深圳研究院 | Magnetic red blood cell cluster based on magnetic separation method for enriching circulating tumor cells |
-
2021
- 2021-09-01 CN CN202111021344.7A patent/CN113720666A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103630440A (en) * | 2013-11-28 | 2014-03-12 | 武汉大学 | Enriching method of circulating tumor cells |
| CN105388055A (en) * | 2015-12-11 | 2016-03-09 | 浙江省肿瘤医院 | Method for separating tumor cell derived-exosomes from urine |
| US20180179515A1 (en) * | 2016-12-23 | 2018-06-28 | National Cancer Center | Magnetic nanostructure for detecting and isolating cell-free dna comprising cationic polymer and magnetic-nanoparticle-containing conductive polymer |
| CN107389937A (en) * | 2017-07-20 | 2017-11-24 | 上海长海医院 | The kit of tumour cell and its detection method and application in a kind of quick detection body fluid |
| CN109100511A (en) * | 2018-07-20 | 2018-12-28 | 四川大学 | Capture and the immune magnetic nano particle of release and preparation method thereof are visualized for circulating tumor cell |
| CN109112107A (en) * | 2018-09-11 | 2019-01-01 | 上海浦美医学科技有限公司 | A method of separation CTC is captured based on rgd peptide |
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Inventor after: Chen Bingdi Inventor after: Liu Zhongmin Inventor after: Yue Wenjun Inventor after: Cui Zheng Inventor after: Luo Cici Inventor before: Chen Bingdi Inventor before: Liu Zhongmin Inventor before: Yue Wenjun Inventor before: Urge sign Inventor before: Luo Cici |
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