CN107988161A - A kind of method for promoting tumour cell reprogramming - Google Patents
A kind of method for promoting tumour cell reprogramming Download PDFInfo
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- 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
- C12N5/0693—Tumour cells; Cancer cells
- C12N5/0695—Stem cells; Progenitor cells; Precursor cells
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- C12N2500/24—Iron; Fe chelators; Transferrin
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Abstract
The invention discloses a kind of method for promoting tumour cell reprogramming, it carries out in vitro culture using serum free medium to tumour cell, the seralbumin that concentration is 1~55g/L is added in serum free medium, the serum free medium is using DMEM/F12 as basic culture medium, and it is the β mercaptoethanols of 0.52~0.88mg/L, the transferrins of 3.6~5.8mg/L, the sodium selenite of 0.0017~0.002mg/L, the insulin of 1.6~12.8mg/L and the ethylaminoethanol of 0.48~1.1mg/L to add ultimate density wherein.The present invention, by promoting the CSC that tumour cell reprogramming obtains more efficient, is embodied in shorter time used, the CSC ratios bigger of acquisition, obtains having higher success rate for CSC, and the CSC properties obtained are stablized relative to traditional serum free suspension method.The present invention is by adjusting various concentrations seralbumin, energy high degree simulation tumour cell is transferred to the change of residing tumor vessel microenvironment during the circulatory system by primary tumor, and the research to circulating tumor cell in metastasis models and blood plasma microenvironment provides more suitably tumor models.
Description
Technical field
The present invention relates to RESEARCH ON CELL-BIOLOGY field, and in particular to a kind of method for promoting cell reprogramming.
Background technology
Tumour is a kind of one of common disease of serious threat human health, and causes tracing it to its cause for tumour high mortality
It is the complexity of tumour itself.It is a variety of difference differentiation degrees and not isophenic tumour cell often with tumours of chemotherapeutic medicine
The clinical pictures such as the tolerance of thing and radiation cure, postoperative recurrence are related.In recent years, with the continuous depth to tumor research
Enter, the theoretical proposition of tumor stem cell (Cancer Stem Cells, CSC) brings new thinking for the treatment of tumour.Tumour
Stem cell is that a kind of quantity is few in cancer colonies and exists with self-renewing, a group cell of the of self-replication capacity, this group
The cell in quiescent condition can be divided into not isophenic tumour cell under specific microenvironment induction in vivo.So it is directed to
The research of CSC, can help to fundamentally inquire into tumor development, transfer and prognosis mala equimolecular mechanism, to tumour
Clinical treatment has great importance.
Authoritative cell bank on the market can only be provided into the non-dryness tumor cell line for being or only minority class at present
The tumor stem cell strain of type.If the and immunology separating method or side group (side for passing through CSC surface molecular markers
Population, SP) separation of the sorting to tumor cell line or primary tumor cell progress CSC, it can only often obtain content pole
Few CSC, it is difficult to meet the cell number of increasingly demand increase, in addition by the process of selected by flow cytometry apoptosis cell to cell
It can cause inevitably to damage, and it is not convenient enough to operate.So on this basis, without animal blood serum and its
He is developed the serum free medium of animal extract component, by serum free medium, most of common malignant tumours
Stem cell can be enriched to, and these are proved by stem cell surface mark, drug resistance, the foundation such as culture and stable passage that suspends
The really CSC for the tumour sacculus cell being enriched to.However, in most report, these are obtained by serum free medium
There is problems with by CSC：Success rate is unstable, the required reprogramming time is long and lasting passage seldom can exceed that for 10 generations
More than.And commercialized serum free medium even more with the addition of the growth factor of substantial amounts of various composition wherein, it is unfavorable for
Subsequently it is directed to the CSC correlative studys of these paths.
In order to obtain the CSC that maximum ratio is used to study in adherent tumour cell, except in enriched cell population
Outside the CSC of existing only a few, reprogramming is carried out to adherent tumour cell in vitro and obtains substantial amounts of CSC, then can guarantee that every
The amount of secondary enrichment, it is ensured that the repeatability and reliability of experiment, and substantial amounts of enrichment time can be saved.Blood vessel microenvironment is swollen
The starting of knurl stem cell, maintenance etc. are respectively provided with consequence, and blood leakage involved in tumor vessel microenvironment with
And containing substantial amounts of seralbumin in blood plasma microenvironment, it is CSC that this, which prompts our seralbumins to be reprogrammed in tumour cell,
Have the function that important.
Seralbumin is the albumen that content is most abundant in vertebrate blood plasma, it is synthesized by liver cell and discharges into door
Arteries and veins circulates, and seralbumin concentration is about in 35g/L-50g/L in adult human body, and the seralbumin concentration in child's body
About in 29g/L-55g/L.The sero-abluminous amino acid sequence and its space structure of separate sources are all very conservative, it
In vivo just like maintenance blood colloid osmotic pressure, combination and transport endogenous and exogenous material, removing free radical and enhancing enzyme activity
The multiple functions such as property.In previously reported nutrient solution, addition seralbumin is will also tend to as offer nutrition and maintenance
The function of cytoactive, but be all in the majority with the seralbumin of low concentration (1-30g/L).
Publication No. CN102634482A, entitled " a kind of Serum-free complete medium of mescenchymal stem cell " and
Publication No. CN104694470A, entitled " a kind of stem cell serum-free culture medium " Chinese invention patent application in
A kind of Serum-free complete medium in vitro culture mescenchymal stem cell is referred to, wherein containing human seralbumin egg respectively
White 1-30g/L and 5-30g/L.The point that two patent applications are focused on is ready-made mescenchymal stem cell cultivate simultaneously big
Amount amplification, and wherein seralbumin role simply maintains osmotic pressure and provides the basic physiology functions such as nutrition, and
It is difficult to largely obtain from the seralbumin of people, and it is expensive, for needing a large amount of experiments for obtaining CSC then difficult
To realize；Publication No. CN103898056B, entitled " cell culture medium and its in people's primary tumor cell is cultivated
Using " and Publication No. CN101984051A, the entitled " serum-free cell for being adapted to tumor stem cell enrichment and cultivating
The Chinese invention patent application of nutrient solution " all refers to a kind of method for being used for tumor stem cell enrichment and in vitro culture.It is logical
Cross and tumor cell line or people's primary tumor cell are placed in the serum free medium referred in invention, can be enriched with to a certain degree
Tumor stem cell after to reprogramming, respectively comprising plant origin recombination human serum albumin 3-5g/L and bovine serum albumin(BSA)
0.45-0.6g/L, is the seralbumin of low concentration.And the former is except seralbumin is in addition to, also added such as more
Kind of hormone such as hydrocortisone, the growth factor such as additive such as hEGF EFG, substantially increase obtain CSC into
This, and cause great interference for the research related with the parahormone or growth factor.
The present invention is enriched with the serum-free cell culture medium with cultivating existing for CSC, establishes a kind of applicable model
Enclose wider, experimental period is shorter, and cost is more cheap and the method for tumour cell reprogramming that has higher success rate.Pass through the present invention
The CSC of acquisition can stablize, and it is more than generation to be passaged to 10, and has good experimental repeatability and uniformity, micro- with blood vessel for CSC
The correlative study of environment provides a kind of applied extremely strong operating method.
The content of the invention
The purpose of invention is to provide a kind of method for promoting tumour cell reprogramming.
To achieve the above object, the technical solution used in the present invention is：Addition is one or more in cell culture medium
Seralbumin.
The seralbumin is human serum albumins or bovine serum albumin(BSA).
The method is to add the bovine serum albumin(BSA) or human serum albumins of 1~55g/L in cell culture medium.
The method of the promotion tumour cell reprogramming is suitable for most of malignant tumour cell, including the overwhelming majority
The tumour cell of mesenchymal derivation.Source and transfer characteristic for CSC, to primary tumor under simulation tumor vessel oozing of blood environment
The region of CSC, the reprogramming efficiency of tumour cell can be significantly improved by adding the seralbumin of 1g-8g/L；To simulate blood
The CSC in microenvironment is starched, the reprogramming effect of tumour cell can farthest be improved by adding the seralbumin of 35-55g/L
Rate, and depressed in this colloid osmotic, the non-CSC tumour cells for not tolerating the environment can be removed, final obtain has more
The tumour initiator cell of strong transfer ability and Anoikis resistance ability.
It is of the present invention promote tumour cell reprogramming the concrete operation method of method be：Tumour cell is simply gone
After serum, it is digested to unicellular, inoculates in serum-free cell culture medium, adds requirement of experiment and different cell types
The seralbumin of institute's suitable concentration, then after suitable (37 DEG C, 5%~10%CO2) culture 48-72h of condition of culture,
Can be seen in nutrient solution suspension into bulk or spherical CSC.
The good effect of CSC is obtained by the present invention is：
(1) relative to traditional serum free suspension method, the CSC by promoting tumour cell reprogramming acquisition is more efficient,
It is embodied in having higher success rate for the CSC ratios bigger, acquisition CSC that the time used is shorter, obtains；(2) by promoting tumour cell
The CSC properties that reprogramming obtains are stablized, and can stablize passage in the state of the culture that suspends, and can farthest maintain Tumor Stem
The physiological activity and cytoactive of cell.(3) it is thin by adjusting various concentrations seralbumin, energy high degree simulation tumour
Born of the same parents are transferred to the change of residing tumor vessel microenvironment during the circulatory system by primary tumor, to metastasis models and blood plasma
The research of circulating tumor cell provides more suitably tumor models in microenvironment.
Brief description of the drawings
Fig. 1 a~1c are the CSC obtained using method provided by the invention from osteosarcoma cell line, colon carcinoma cell line
Mirror under cytological map, wherein Fig. 1 a are MNNG osteosarcoma stem cells, and Fig. 1 b are MG63 osteosarcoma stem cells, and Fig. 1 c are Sw620 knots
Intestinal cancer stem cell.
Fig. 2 a, Fig. 2 b, Fig. 2 c are respectively to add 0g/L respectively in serum free medium using method provided by the invention
Under conditions of the bovine serum albumin(BSA) of (not adding group), 2.5g/L and 50g/L, osteosarcoma stem cell is cultivated to the weight of the 3rd day
Programming situation.
Fig. 3 is to add 0g/L (not adding group) respectively in serum free medium using method provided by the invention contrast
And under conditions of the bovine serum albumin(BSA) of 2.5g/L, weight of the osteosarcoma cell in culture to first day, the 3rd day and the 5th day
Programming situation.After from figure as it can be seen that with the addition of seralbumin, the reprogramming time of tumour cell is shifted to an earlier date, and is finally obtained
The CSC numbers obtained are more also relative to traditional serum free medium.
Fig. 4 a~4c are the CSC energy self-renewings obtained using method provided by the invention, and in the situation of culture that suspends
The result of lower stable passage.Wherein Fig. 4 a, Fig. 4 b, Fig. 4 c are respectively the MNNG osteosarcoma stem cells second generation, the 17th generation, the 3rd
The cell mirror figure below in 14 generations.
Fig. 5 a~Fig. 5 b are to detect it using the CSC progress immunofluorescent stainings of method provided by the invention acquisition to do
The significant gene of cell, wherein Fig. 5 a are that the CD133 molecular cells of the non-CSC of MNNG (bulk) and CSC (Sphere) are immunized
Luciferase expression situation；The cell that Fig. 5 b are the C-kit and TRA-1-81 of the non-CSC of MNNG (bulk) and CSC (Sphere) is exempted from
Epidemic disease luciferase expression situation.
Specific embodiment
With reference to specific embodiment, the present invention is further explained.It is to be understood that these embodiments are merely to illustrate the present invention
Rather than the application range of the limitation present invention.
Embodiment 1
This example is serum free medium and sero-abluminous formulation example.
Bovine serum albumin(BSA) storing solution is configured, is 100g/ with distilled water dissolving bovine serum albumin(BSA) powder to ultimate density
L, it is spare that 0.2 μM of filter filtering is placed in 4 degree of refrigerators.
Exemplified by serum free medium is using another disclosed invention in this laboratory, Publication No. CN101984051A is public
It is on March 9th, 2011 to open day, entitled " serum-free cell culture medium for being adapted to tumor stem cell enrichment and culture ", should
Culture medium using DMEM/F12 as basic culture medium, and add wherein beta -mercaptoethanol that ultimate density is 0.52mg/L,
The transferrins of 3.6mg/L, the sodium selenite of 0.0017mg/L, the ethylaminoethanol of the insulin of 1.6mg/L and 0.48mg/L.
Added in above-mentioned serum free medium bovine serum albumin(BSA) storing solution to ultimate density for 2.5g/L, 5g/L and
50g/L.Specifically, more seralbumin concentrations are by depending on the source for the tumour cell to be reprogrammed and spy
Property, for the true environment in fitting body, seralbumin concentration is to be fluctuated in the range of 1g/L-55g/L.
Embodiment 2
This example is culture example when culture is human osteosarcoma cell line MNNG, MG63 and colon carcinoma cell line Sw620.
Using the culture medium that the embodiment of the present invention 1 provides in the case where seralbumin concentration is 2.5g/L, people is cultivated
Osteosarcoma cell line MNNG, cultivates human osteosarcoma cell line Mg63 and colon in the case of being 5g/L with seralbumin concentration
Cancerous cell line Sw620, after cultivating five days, if Fig. 1 a~Fig. 1 c are the CSC after the reprogramming obtained.From Fig. 1 a~Fig. 1 c as it can be seen that
The culture medium provided using this patent, which to answer the tumour cell of adherent growth to shrink originally and assemble formation, departs from culture dish
Sacculus shape form, illustrate that we obtain the CSC after the reprogramming with stronger dryness.
Embodiment 3
This example is culture when being human osteosarcoma cell line MNNG, adds the seralbumin of different content in same culture
Comparative example under time.
For 2.5g/L and 50g/L and do not add in seralbumin concentration using the culture medium that the embodiment of the present invention 1 provides
In the case of increase serum albumin, human osteosarcoma cell line MNNG is cultivated, the cell after contrast is cultivated three days reprograms situation.Such as
Shown in Fig. 2 a, in the case where not adding seralbumin culture three days, adherent form is still presented in MNNG cells, only a small amount of
Cell starts to assemble；As shown in Figure 2 b, in the case where with the addition of the culture of 2.5g/L seralbumins three days, MNNG cell masses
Occur significantly assembling and there is a small amount of sacculus to be formed；Enter shown in Fig. 2 c, with the addition of the seralbumin culture of 50g/L
In the case of three days, almost all of cell is all separately aggregate to form the CSC sacculus with stronger dryness.Illustrate that osteosarcoma is thin
Born of the same parents also improve therewith with the raising of seralbumin concentration, the efficiency of reprogramming.
Embodiment 4
This example is culture when being human osteosarcoma cell line MNNG, addition and does not add seralbumin in multiple identical trainings
Support the comparative example under the time.
Using the culture medium that the embodiment of the present invention 1 provides seralbumin concentration is 2.5g/L and not add serum white
In the case of albumen, human osteosarcoma cell line MNNG is cultivated, respectively first day, the 3rd day and the 5th day thin after contrast culture
Born of the same parents reprogram situation.It can be seen from figure 3 that the sero-abluminous MNNG cells that with the addition of 2.5g/L concentration are being cultivated first day, its
The form of adherent growth is that the form of full extension is not presented, and has arrived culture the 3rd day, and MNNG cells group energy occurs obvious
Aggregation and sacculus, until culture the 5th day, obvious CSC sacculus can have been seen in culture dish.It is white that serum is not added in contrast
The MNNG groups of albumen, then by the 5th day still with the presence of substantial amounts of attached cell.It is hereby understood that compared to not adding the white egg of serum
White control group, addition seralbumin can significantly improve tumour cell reprogramming speed and efficiency.
Embodiment 5
This example is culture when being human osteosarcoma cell line MNNG, the embodiment persistently cultivated.
Obtained using the culture medium that the embodiment of the present invention 1 provides in the case where seralbumin concentration is 2.5g/L
MNNG tumor stem cells, digest them into unicellular, and continue to cultivate under this condition.As shown in figure 4, CSC cells are in this feelings
Under condition, without by low absorption or it is anti-it is adherent handle and the growth of suspended state can be presented in Nostoc commune Vanch ware, and biography can be stablized
In generation, there is powerful self-renewal capacity.
The MNNG tumor stem cells and MNNG attached cells that the method provided using the embodiment of the present invention 2 obtains, to it
Carry out immunofluorescence dyeing, detect the expression of their stem-cell marker genes.As shown in Fig. 5 a and Fig. 5 b, by the present invention
The CSC obtained is reprogrammed relative to the dryness marker gene such as attached cell, expression C-Kit, CD133, TRA-1-81, is had stronger
Cellular immunofluorescence signal, truly having for the MNNG tumor stem cells that the culture medium for illustrating to provide by this patent obtains are stronger dry
Property.
Claims (5)
- A kind of 1. method for promoting tumour cell reprogramming, it is characterised in that：Tumour cell is carried out using serum free medium In vitro culture, adds one or more seralbumins that concentration is 1~55g/L, the serum-free in serum free medium Culture medium adds β-sulfydryl second that ultimate density is 0.52~0.88mg/L wherein using DMEM/F12 as basic culture medium Alcohol, the transferrins of 3.6~5.8mg/L, the sodium selenite of 0.0017~0.002mg/L, 1.6~12.8mg/L insulin with And the ethylaminoethanol of 0.48~1.1mg/L.
- A kind of 2. method for promoting tumour cell reprogramming according to claim 1, it is characterised in that：The white egg of serum White is human serum albumins or bovine serum albumin(BSA).
- A kind of 3. method for promoting tumour cell reprogramming according to claim 1, it is characterised in that：Tumour cell refers to Tumor cell line or shredded by the tissue block of tumor tissues or digest formed it is unicellular after be seeded in the serum free medium Culture.
- 4. the method reprogrammed according to a kind of any promotion tumour cell of claims 1 to 3, it is characterised in that：It is described Tumour cell include one kind in human osteosarcoma cell or colon cancer cell.
- A kind of 5. method for promoting tumour cell reprogramming according to claim 1, it is characterised in that：It is thin for Tumor Stem The source of born of the same parents and transfer characteristic, the region to simulate primary tumor tumor stem cell under tumor vessel oozing of blood environment, addition Seralbumin concentration is 1g-8g/L；To the tumor stem cell in simulating blood plasma microenvironment, the seralbumin concentration of addition For 35-55g/L.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101984051A (en) * | 2010-11-19 | 2011-03-09 | 中山大学 | Serum-free cell culture fluid suitable for enriching and culturing tumour stem cells |
| CN102719393A (en) * | 2012-06-05 | 2012-10-10 | 中山大学 | Serum-free medium capable of inducing tumor stem cells for differentiation towards lymphatic endothelial cells, and method for inducing tumor stem cells for differentiation towards lymphatic endothelial cells |
| US20160010060A1 (en) * | 2011-12-05 | 2016-01-14 | Factor Bioscience Inc. | Methods and products for transfection |
-
2017
- 2017-10-20 CN CN201710997134.9A patent/CN107988161A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101984051A (en) * | 2010-11-19 | 2011-03-09 | 中山大学 | Serum-free cell culture fluid suitable for enriching and culturing tumour stem cells |
| US20160010060A1 (en) * | 2011-12-05 | 2016-01-14 | Factor Bioscience Inc. | Methods and products for transfection |
| CN102719393A (en) * | 2012-06-05 | 2012-10-10 | 中山大学 | Serum-free medium capable of inducing tumor stem cells for differentiation towards lymphatic endothelial cells, and method for inducing tumor stem cells for differentiation towards lymphatic endothelial cells |
Non-Patent Citations (2)
| Title |
|---|
| KOIZUME, SHIRO等: "Potential Coagulation Factor-Driven Pro-Inflammatory Responses in Ovarian Cancer Tissues Associated with Insufficient O-2 and Plasma Supply", 《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》 * |
| 张雁: "肿瘤干细胞理论的演变", 《中山大学学报（医学科学版）》 * |
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