CN109266716A - A method of detection cell migration quantity - Google Patents
A method of detection cell migration quantity Download PDFInfo
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- CN109266716A CN109266716A CN201811071113.5A CN201811071113A CN109266716A CN 109266716 A CN109266716 A CN 109266716A CN 201811071113 A CN201811071113 A CN 201811071113A CN 109266716 A CN109266716 A CN 109266716A
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- cell
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- mescenchymal stem
- cancer cell
- cancer
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- 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
Abstract
The invention discloses a kind of methods for detecting cell migration quantity comprising following steps: mescenchymal stem cell and cancer cell co-culture, and mescenchymal stem cell are inoculated in room under Transwell plate, cancer cell inoculation is incubated for for 24 hours in small interior, 37 DEG C；Non- migrating cell is wiped, 0.4% Trypan Blue wears theca cell, and micro- sem observation is taken pictures；The for statistical analysis and computation migration cell number using 19.0 statistical software of SPSS.The present invention can be compared by migrating quantity to the cancer cell of different mescenchymal stem cell cultures, it is any more properly for the theoretical foundation of cancer cell progress cell therapy as determining, which obviate the operating procedures of cell experiment complexity, and have many advantages, such as to prepare in advance, easy to operate, testing result accurate and effective, can in high volume use.
Description
Technical field
The present invention relates to technical field of bioengineering, in particular to a kind of method for detecting cell migration quantity.
Background technique
Cell therapy is possible to provide new therapeutic modality for many diseases including cancer, between stem cell is especially
Mesenchymal stem cells (mesenchymal stem cell, MSC) are tested for delivering Therapeutic Transgenes.However, mutually
On the one hand discovery mankind MSCs can promote to shift conflicting reports, and on the other hand other are research shows that MSCs can prevent to shift
The growth of stove.
It is well known that the interaction between tumour cell and matrix has important meaning for the growth and invasion of tumour
Justice, and microenvironment locating for tumour cell plays an important role in growth and metastasis of tumours.Tumour in tumor microenvironment
Cell, stroma cell and soluble factor coordinate composition reticular structure, have significant interaction: tumour cell between each component
It can change the property of tumor microenvironment；Meanwhile tumor microenvironment may also lead to tumour cell and generate during cancer development
Different biological behaviours, and then influence the growth and diffusion of tumour.Mescenchymal stem cell is the important component of microenvironment,
It is widely present in adult connective tissue and organ interstitial, can also be obtained from corresponding fetal tissue.
MSC is easily isolated culture, and the MSC of purifying can lose with massive amplification and to multiple types cell differentiation in vitro
Background is passed to stablize.MSC can go back to the nest to the tissue and organ (tumour including being referred to as " disunion wound ") of damage, participate in tumour
The composition of microenvironment all has important function in tumor development each stage, is to influence tumour to generate and be transferred to it
Key factor during hetero-organization.MSC passes through transformation tumor microenvironment, adjusting angiogenesis, inhibition tumor immune response etc.
Influence the occurrence and development of tumour.How quickly and efficiently to detect cancer cell and migrate quantity, there is weight to research cell therapy cancer
Want meaning.
Summary of the invention
The purpose of the present invention is to provide it is a kind of detect cell migration quantity method, have quickly and effectively, save at
Originally, the accurate advantage of result lays a good foundation for later clinical use, has a extensive future.
The technical solution used in the present invention is: a kind of method for detecting cell migration quantity comprising following steps:
Mescenchymal stem cell and cancer cell co-culture, and mescenchymal stem cell is inoculated in room under Transwell plate, cancer cell
It is inoculated in small interior, 37 DEG C are incubated for for 24 hours；
Non- migrating cell is wiped, 0.4% Trypan Blue wears theca cell, and micro- sem observation is taken pictures；
The for statistical analysis and computation migration cell number using 19.0 statistical software of SPSS.
As a further improvement of the foregoing solution, the measurement data of the statistical analysis withIt indicates, comparison among groups are adopted
It is examined with t, is that difference is statistically significant with p < 0.05.
As a further improvement of the foregoing solution, 0.4% Trypan Blue is worn to carry out before theca cell being inverted and be air-dried
10min。
As a further improvement of the foregoing solution, the preprocess method before the mescenchymal stem cell and cancer cell co-culture
Are as follows: mescenchymal stem cell reach 80~90% converge when discard cell culture fluid, PBS washing, with the tryptose containing EDTA
Enzymic digestion dissociated cell, with the complete training containing 10% fetal calf serum when cell retraction is rounded and starts shedding off from culture dish
It supporting base and terminates digestion, blow and beat mixed liquor repeatedly and form cell suspension, 1200rpm is centrifuged 5min, culture medium is added after discarding liquid,
30 μ l cell liquid are taken, are mixed with the same dose of 0.4% Trypan Blue liquid.
The beneficial effects of the present invention are: the present invention can be by migrating number to the cancer cell of different mescenchymal stem cell cultures
Amount compares, and as any theoretical foundation for more properly carrying out cell therapy for the cancer cell is determined, which obviate thin
Born of the same parents test complicated operating procedure, and have can prepare in advance, is easy to operate, testing result accurate and effective, can high-volume
The advantages that use.
Detailed description of the invention
Fig. 1 is that (wherein Figure 1A is between amniotic fluid for HELA cancer cell form before being unstained in embodiment 1 on traswell plate film
Mesenchymal stem cells and HELA cancer cell co-cultivation group；Figure 1B is umbilical cord mesenchymal stem cells and HELA cancer cell co-cultivation group；Figure
1C is placenta mesenchyma stem cell and HELA cancer cell co-cultivation group)；
Fig. 2 is that (wherein Fig. 2A is that amniotic fluid mesenchyma is dry for HELA cellular morphology after embodiment 2 dyes on traswell plate film
Cell and HELA cancer cell co-cultivation group；Fig. 2 B is umbilical cord mesenchymal stem cells and HELA cancer cell co-cultivation group；Fig. 2 C is tire
Disk mescenchymal stem cell and HELA cancer cell co-cultivation group)；
Fig. 3 is the HELA cancer cell number that the calculated each group of embodiment 3 is migrated (p < 0.05 * is significant difference).
Specific embodiment
The present invention is specifically described below with reference to embodiment, in order to technical field personnel to of the invention
Understand.It is necessary to it is emphasized that embodiment is only intended to, the present invention will be further described herein, should not be understood as to this
The limitation of invention protection scope, fields person skilled in the art, the non-intrinsically safe that the present invention is made according to foregoing invention content
The modifications and adaptations of property, should still fall within protection scope of the present invention.Mentioned raw materials following simultaneously are unspecified, are
Commercial product；The processing step or preparation method not referred in detail be processing step known to a person skilled in the art or
Preparation method.Serum described in the embodiment of the present invention is purchased from Hyclone company；The Transwell plate is purchased in Corning
Company；The pancreatin is purchased from Gibco company.
Co-cultivation of 1: the three kind of mescenchymal stem cell of embodiment to HELA cancer cell
(1) Amniotic Fluid-derived Mesenchymal Stem Cells
When Amniotic Fluid-derived Mesenchymal Stem Cells, which reach 80~90%, to be converged, cell culture fluid is discarded；It is washed three times with PBS；With
0.25% trypsase covering cell containing 0.02%EDTA dissociates cell；It is rounded and to cell retraction from culture dish
On terminate digestion with the complete medium containing 10% fetal calf serum when starting shedding off；Mixed liquor is blown and beaten repeatedly until adherent thin
Born of the same parents, which fall off to be formed under cell suspension 1200rpm from culture dish, is centrifuged 5min；Addition culture medium softly blows even make after discarding liquid
Cell scatters, and takes 30 μ l cell liquid, mixes with the same dose of 0.4% Trypan Blue liquid, Amniotic Fluid-derived Mesenchymal Stem Cells are connect
Kind room under Transwell plate, HELA cancer cell inoculation are incubated for for 24 hours in small interior, 37 DEG C.
(2) umbilical cord mesenchymal stem cells
When umbilical cord mesenchymal stem cells, which reach 80~90%, to be converged, cell culture fluid is discarded；It is washed three times with PBS；With
0.25% trypsase covering cell containing 0.02%EDTA dissociates cell；It is rounded and to cell retraction from culture dish
On terminate digestion with the complete medium containing 10% fetal calf serum when starting shedding off；Mixed liquor is blown and beaten repeatedly until adherent thin
Born of the same parents, which fall off to be formed under cell suspension 1000rpm from culture dish, is centrifuged 5min；Addition culture medium softly blows even make after discarding liquid
Cell scatters, and takes 30 μ l cell liquid, mixes with the same dose of 0.4% Trypan Blue liquid, umbilical cord mesenchymal stem cells are connect
Kind room under Transwell plate, HELA cancer cell inoculation are incubated for for 24 hours in small interior, 37 DEG C.
(3) placenta mesenchyma stem cell
When placenta mesenchyma stem cell, which reaches 80~90%, to be converged, cell culture fluid is discarded；It is washed three times with PBS；With
0.25% trypsase covering cell containing 0.02%EDTA dissociates cell；It is rounded and to cell retraction from culture dish
On terminate digestion with the complete medium containing 10% fetal calf serum when starting shedding off；Mixed liquor is blown and beaten repeatedly until adherent thin
Born of the same parents, which fall off to be formed under cell suspension 1000rpm from culture dish, is centrifuged 5min；Addition culture medium softly blows even make after discarding liquid
Cell scatters, and takes 30 μ l cell liquid, expects that blue dyeing liquor mixes with the same dose of 0.4%, placenta mesenchyma stem cell is connect
Kind room under Transwell plate, HELA cancer cell inoculation are incubated for for 24 hours in small interior, 37 DEG C.
Embodiment 2: detection cell migration
HELA cancer cell after three kinds of mescenchymal stem cells and HELA cancer cell co-culture in embodiment 1 can migrate to
Outside the film of Transwell upper chamber, the non-migrating cell of Transwell upper chamber first is wiped with cotton swab, then upper chamber is inverted and is air-dried
10min after killing to liquid, wears theca cell using 0.4% Trypan Blue, cleans by PBS primary.
Period is thin by the HELA cancer on the traswell plate film before phase contrast microscope observation Trypan Blue and after dyeing
Born of the same parents' form, observation result is respectively as shown in attached drawing 1 (1A, 1B, 1C) and attached drawing 2 (2A, 2B, 2C).
Embodiment 3: computation migration cell number
5 views are taken at random in the cell that three kinds of mescenchymal stem cells and HELA cancer cell co-culture in example 2 respectively
Open country takes pictures and counts migrating cell number, calculates average value, specifically for statistical analysis using 19.0 statistical software of SPSS,
Measurement data withIt indicating, it is significant difference with p < 0.05 * that the comparison among groups of every kind of mescenchymal stem cell are examined using t,
With statistical significance, then by software cell quantity analyzed, mapped, result is as shown in Fig. 3.
Above-described embodiment is the preferred embodiment of the present invention, all with similar technique of the invention and made equivalence changes,
It should belong to protection category of the invention.
Claims (4)
1. a kind of method for detecting cell migration quantity, it is characterised in that include the following steps:
Mescenchymal stem cell and cancer cell co-culture, and mescenchymal stem cell is inoculated in room under Transwell plate, cancer cell inoculation
In small interior, 37 DEG C are incubated for for 24 hours；
Non- migrating cell is wiped, 0.4% Trypan Blue wears theca cell, and micro- sem observation is taken pictures；
The for statistical analysis and computation migration cell number using 19.0 statistical software of SPSS.
2. a kind of method for detecting cell migration quantity according to claim 1, it is characterised in that: the statistical analysis
Measurement data withIt indicates, comparison among groups are examined using t, are that difference is statistically significant with p < 0.05.
3. a kind of method for detecting cell migration quantity according to claim 1, it is characterised in that: described 0.4% is expected
Indigo plant dyeing carries out being inverted air-dried 10min before wearing theca cell.
4. a kind of method for detecting cell migration quantity according to claim 1, it is characterised in that: the mesenchyma is dry thin
Born of the same parents and cancer cell co-culture before preprocess method are as follows: mescenchymal stem cell reach 80~90% converge when discard cell culture
Liquid, PBS washing are rounded to cell retraction and are taken off since on culture dish with the trypsin digestion dissociated cell containing EDTA
Digestion is terminated with the complete medium containing 10% fetal calf serum when falling, mixed liquor is blown and beaten repeatedly and forms cell suspension, 1200rpm
It is centrifuged 5min, culture medium is added after discarding liquid, takes 30 μ l cell liquid, is mixed with the same dose of 0.4% Trypan Blue liquid.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113106059A (en) * | 2021-04-07 | 2021-07-13 | 清华大学深圳国际研究生院 | High-migration mesenchymal stem cell and preparation method and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101186899A (en) * | 2007-10-18 | 2008-05-28 | 天津医科大学 | Method applied for tumour cell and stem cell co-culture |
| CN103074300A (en) * | 2012-11-30 | 2013-05-01 | 甘肃中医学院 | Co-culture system establishment of mesenchymal stem cells and tumor cells as well as mesenchymal stem cells heredity stability change characteristic in tumor microenvironment |
| CN103087912A (en) * | 2011-10-27 | 2013-05-08 | 中国科学院大连化学物理研究所 | Micro-fluidic chip capable of producing stable concentration gradient and cell co-culture method |
| CN103146642A (en) * | 2013-02-07 | 2013-06-12 | 中国人民解放军总医院第一附属医院 | Method for directional induction of differentiation of mesenchymal stem cells (MSCs) |
| CN103477222A (en) * | 2010-09-29 | 2013-12-25 | 麻省理工学院 | Device for high throughput investigations of cellular interactions |
-
2018
- 2018-09-13 CN CN201811071113.5A patent/CN109266716A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101186899A (en) * | 2007-10-18 | 2008-05-28 | 天津医科大学 | Method applied for tumour cell and stem cell co-culture |
| CN103477222A (en) * | 2010-09-29 | 2013-12-25 | 麻省理工学院 | Device for high throughput investigations of cellular interactions |
| CN103087912A (en) * | 2011-10-27 | 2013-05-08 | 中国科学院大连化学物理研究所 | Micro-fluidic chip capable of producing stable concentration gradient and cell co-culture method |
| CN103074300A (en) * | 2012-11-30 | 2013-05-01 | 甘肃中医学院 | Co-culture system establishment of mesenchymal stem cells and tumor cells as well as mesenchymal stem cells heredity stability change characteristic in tumor microenvironment |
| CN103146642A (en) * | 2013-02-07 | 2013-06-12 | 中国人民解放军总医院第一附属医院 | Method for directional induction of differentiation of mesenchymal stem cells (MSCs) |
Non-Patent Citations (2)
| Title |
|---|
| 刘雯: "《医学细胞与遗传学实验教程》", 31 October 2008, 复旦大学出版社 * |
| 李燕等: "《分子生物学实用用实验技术》", 31 December 2011, 第四军医大学出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113106059A (en) * | 2021-04-07 | 2021-07-13 | 清华大学深圳国际研究生院 | High-migration mesenchymal stem cell and preparation method and application thereof |
| CN113106059B (en) * | 2021-04-07 | 2023-08-15 | 清华大学深圳国际研究生院 | High-migration mesenchymal stem cells, and preparation method and application thereof |
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