CN1131319C - Detection equipment for cell contact inhibition, its preparation method and application - Google Patents
Detection equipment for cell contact inhibition, its preparation method and application Download PDFInfo
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- CN1131319C CN1131319C CN 01135204 CN01135204A CN1131319C CN 1131319 C CN1131319 C CN 1131319C CN 01135204 CN01135204 CN 01135204 CN 01135204 A CN01135204 A CN 01135204A CN 1131319 C CN1131319 C CN 1131319C
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- cell
- adhesive tape
- claire
- paraffin
- proofing unit
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Abstract
The present invention discloses a detecting device for cell contact inhibition and a preparing method and the application thereof. The device mainly comprises a coverglass placed into a cultivating vessel, a non-dry adhesive transparent tape adhered to the coverglass, paraffin coated on the adhesive tape, and an observing tank of cell cultivation surrounded by the paraffin on the coverglass. The device can be used for the accurate detection of the contact inhibition, the non-contact inhibition and the moving contact inhibition of cell growth, and carrying out the observation of the release phenomenon of the cell contact inhibition. The device has the advantages of simple operation and visual and accurate result.
Description
(1) technical field
The present invention relates to a kind of device that detects the cell proliferation regulation and control, relate to proofing unit of a kind of cells contacting inhibition and its production and application specifically.
(2) background technology
Cell is the important substance basis of forming the nature biotechnology body, the growing invariably with the propagation of cell and be divided into prerequisite of people and animal.Aspect medical science, the generation of numerous disease is all relevant unusually with the multiplication regulatory of human body cell, particularly Dai Biao malignant tumour---the cancer of being described as " incurable disease " by people.
Contact inhibition is the characteristic that normal cell has, it is meant that normal cell is in the certain culture environment, be attached at the culture vessel surface and carry out adherent growth, after cell growth reaches converging state and certain density, the state that the growing multiplication that cell occurs stops.
Malignant cell is a kind of unrestricted grown cell that is not subjected to normal cell proliferation regulatory mechanism constraint.One of most important difference feature of the tumour cell of vitro culture and normal cell growth is exactly the forfeiture of growth of tumour cell contact inhibition.Research in this respect, for the Normocellular canceration process of understanding, i.e. the genesis mechanism of cancer, and then further investigation is explored the treatment method for cancer and is had crucial meaning.
Pluralistic theory is thought at present, the oncogene relevant with cell proliferation, the change of cancer suppressor gene or the slit that is considered to belong to the important syndeton of iuntercellular and can exercises the cell-cell communication function connect (Gap junction) dysfunction alone, all can cause the misgrowth of cell, as the forfeiture of contact inhibition.It is believed that it is the important structure that cell transmits the adjusting and controlling growth signal that the slit connects, the cell slit connects can be by being delivered to the cell of active growth with growth-inhibiting information from growth immobilized cell, and when reaching certain threshold value, the latter is stopped growing.And the forfeiture of cell growth contact inhibition appears, and mainly be the unusual of slit linkage function, cause the semiochemicals transmission generation obstacle that connects by the slit, thereby cause the unusual of cell growth; Its detection method mainly is by traditional adherent culture cell H3-TDR labelling method at present, promptly detects the method for proliferative activity.Its principle is, through the cell that certain mode is handled, when growth reaches certain density of converging state, uses H
3-Thymine deoxyriboside (H
3-Thymidine) mark detects through radioautograph again; As if cell proliferate is arranged, then H
3-Thymine deoxyriboside (H
3-Thymidine) be incorporated among the intracellular DNA and produce mark; Otherwise, H then
3-Thymine deoxyriboside (H
3-Thymidine) can not be incorporated into and carry out mark among the intracellular DNA.Therefore, according to the proliferate state of label index decidable cell.Can the soft agar culture methods that adopt be about to cell inoculation in the soft agar of interlayer more when whether the detection cell transforms, observe it and form the clone, and what can form the clone then is transformant or tumour formation cell.Aforesaid method is for detecting the method for proliferative activity, the method for all non-direct research cell proliferation Regulation Mechanism; And utilize proofing unit, and especially utilize the contact inhibition of single-minded vessel cell growth to study, yet there are no report so far.
(3) summary of the invention
The objective of the invention is deficiency at prior art, provide a kind of cells contacting of making by cover glass, paraffin, culture dish to suppress proofing unit, this device effectively test experience cell growth contact inhibition existence whether, cells contacting suppresses release phenomenon and differentiates tumour cell and Normocellular growing multiplication state, reaches the purpose of differentiation normal cell and conversion or cancerous tumor cell.
Another object of the present invention provides preparation method and the application of this device in detecting the cells contacting inhibition that cells contacting suppresses proofing unit.
The objective of the invention is to realize by following technical scheme.
Form by culture dish, cover glass, non-setting adhesive scotch tape, paraffin coating, cell cultures claire.Place the cover glass central authorities in the culture dish to be stained with rectangle non-setting adhesive scotch tape, the little adhesive tape that has several to be cut off on the adhesive tape but be closely adjacent to each other, one end of every little adhesive tape has the termination that can uncover, the surface of adhesive tape scribbles one deck paraffin except that the termination, adhesive tape on cover glass has one to be uncovered the cell cultures claire that is surrounded by paraffin because of little adhesive tape and the paraffin on it on one side.
One of geometricdrawing sample adhesive tape that described little adhesive tape is at least two next-door neighbours.
Described little adhesive tape is meant and is cut off into one of 2～5 rectangle adhesive tapes that are closely adjacent to each other.
The shape of described cell cultures claire changes with the shape of little adhesive tape.
The volume of described cell cultures claire is uncovered and is increased with adjacent little adhesive tape.
The bottom surface of described cell cultures claire and the angle of its edge wall are 45～100 degree.
The preparation method of the proofing unit that cells contacting suppresses, the sequence of steps of this method is as follows:
(1) gets a clean cover glass, the smooth cover glass central authorities that bubble-freely are affixed on of non-setting adhesive scotch tape that will be more smaller than cover glass area;
(2) carve a rectangle frame with blade at above-mentioned adhesive tape middle part, and be cut to the little adhesive tape more than two;
(3) with the end of point of a knife, provoke the termination that can take off respectively at two next-door neighbours' little adhesive tape, standby;
(4) with the paraffin of above-mentioned adhesive tape and the even coating of periphery one melting layer thereof, cooling is solidified paraffin then;
(5) clamp in the little adhesive tape termination of step (3) preparation one, throw off little adhesive tape and apply thereon paraffin, make the cover glass surface form the pan of an area with little adhesive tape to adhesive tape the other end direction;
(6) repair above-mentioned pan, the angle that makes its bottom surface and its edge wall is 45～100 degree, then its sterilization, sterilization promptly is made into the cell cultures claire;
(7) according to the experiment demand, but the operation of repeating step (5), (6) constantly enlarges the volume of cell cultures claire;
(8) the above-mentioned cover glass that has the cell cultures claire is put in the clean culture dish with cover, forms cells contacting jointly and suppress proofing unit.
Application cell contact inhibition proofing unit carries out the method that cells contacting suppresses detection, and its concrete steps are as follows;
(1) get the logarithmic phase experimental cell of adherent growth, make single cell suspension, and to adjust cell concn is 1 * 10
8～1 * 10
13Individual/ml;
(2) get above-mentioned experimental cell suspension, in the cell cultures claire in proofing unit of instiling, suspension amount 10～30 μ l are advisable;
(3) above-mentioned detection device is built, placed 5%CO
2, 37 ℃, in the incubator of 100% relative humidity, be cultured to cell attachment;
(4) take out above-mentioned detection device from incubator, under the aseptic condition, avoid the cell cultures claire, add nutrient solution in the ware body of proofing unit, its liquid measure is advisable to exceed cell cultures claire 1mm;
(5) build proofing unit again, put back to former culture condition continuation and cultivate after 8～20 hours, observation of cell is cultivated the cell growth in the claire, with the situation of test experience cells contacting inhibition;
(6) if experimental cell growth reach converging state and with stop growing after pool wall contact closely, be preliminary contact inhibition, continue cultivation again through 2～5 cell cycles, experimental cell still stops to divide, and promptly is defined as experimental cell and contact inhibition occurs; Otherwise after continuing cultivation, experimental cell still can divide, and presents accumulated growth, and pool wall growth phenomenon can occur crossing over, then is that noncontact suppresses;
(7) in the step (6), confirm to have occurred the experimental cell of contact inhibition, can increase the volume that it cultivates claire by throwing off and its cell cultures claire next-door neighbour's the little adhesive tape and the method for its surperficial paraffin, carry out the observation that cells contacting suppresses release phenomenon.
Proofing unit of the present invention and application method are the direct method of research cell proliferation Regulation Mechanism, have the superiority of more convenient, the objective and "dead" pollution of more traditional soft-agar cloning and isotopic labeling method.The foundation of this device and method, changed traditional contact inhibition theory, the generation that has proved cell growth contact inhibition is closely related with the geometric space structure of cell and extracellular matrix, and any factor (comprising that unusual slit is connected) that influences stress relieving between cell and extracellular matrix all may cause the grow forfeiture of contact inhibition of cell.This discovery has biological significance widely for natural zooblast multiplication regulatory, and for the mechanism that further further investigation cell proliferation is regulated and control, discloses biological phenomena and canceration inducement and have important theoretical and practical significance.
The experiment that utilizes above-mentioned detection device and application method to carry out shows: under the condition that does not have the slit connection, or do not exist semiochemicals to be connected under the situation of iuntercellular transmission by the slit, the normal cell of cultivation still contact inhibition can occur.When the attached cell of cultivating reaches the contact inhibition state, under the condition of not upgrading cell culture fluid, if will with cell cultures claire next-door neighbour's little adhesive tape and on paraffin remove, can be observed formerly contact closely with the paraffin wall of claire, dormant cell recovers propagation again, and along being uncovered because of little adhesive tape and paraffin the new exposed glass surface that forms attaches growth on cover glass, show as " release of contact inhibition ".
For attached cell (comprising normal cell, tumour cell and transformant), utilize above-mentioned detection device and application method to cultivate, when not reaching converging state density, cell can present along the locomotory movement on cell cultures claire surface, but when cell migration closely contacts to cell cultures observation pool wall and with its paraffin wall, the former motion towards paraffin wall direction of cell stops at once, even the phenomenon in paraffin wall zone does not appear crossing in tumour cell yet, show as the mobility contact inhibition of cell.But the cell that the mobility contact inhibition occurs still can move to other directions beyond the paraffin wall.Only build the tumour cell and the transformant that are, when its growth reaches individual layer converging state density and with after paraffin wall that cell cultures is observed pool wall closely contacts, under the situation that does not change culture condition, cell still can occur continuing propagation, and is accumulation property growth conditions; Proliferating cells can be crossed the paraffin wall, and is attached at paraffin surface to the growth of periphery diffustivity, shows as noncontact and suppresses.
(4) description of drawings
Fig. 1 is the state graph that cells contacting suppresses proofing unit
Fig. 2 is that cells contacting suppresses the state graph after volume gain of cell cultures claire in the proofing unit
(5) embodiment
Embodiment 1 gets 15 * 15mm of a cleaning
21 one of cover glasses are cut a 10 * 12mm
2The non-setting adhesive scotch tape 2 smooth cover glass central authorities that bubble-freely are affixed on; With blade above-mentioned adhesive tape is cut into 2 * 10mm more than two
2The little adhesive tape 4 of rectangle, and, provoke the termination that can take off 3 respectively with the end of point of a knife at two little adhesive tapes, standby; It is an amount of to get medical paraffin, and heating and melting is dipped in the paraffin that takes a morsel and melt with small brushes in beaker, evenly coats above-mentioned tape surface (except the termination) and periphery 2～4mm scope thereof, and cooling is solidified paraffin 6 then; Clamp in the above-mentioned standby little adhesive tape termination 3 one with tweezers, throw off little adhesive tape 4 and apply thereon paraffin 6, make the cover glass surface form the pan 5 of an area with little adhesive tape to adhesive tape the other end direction; Repair above-mentioned pan, the angle that makes its bottom surface and its edge wall is 90 degree, use 75% medical alcohol soaking disinfection more than 2 hours then, tri-distilled water flushing three times, be put in the clean culture dish with cover, uviolizing 30 minutes is promptly made cells contacting shown in Figure 1 and is suppressed proofing unit after drying.
Get the logarithmic phase cell of adherent growth, as human embryonic lung fibroblast, the BEL-7402 liver cancer cell, one of SGC stomach cancer cell is digested to single cell suspension with the pancreatin ordinary method; Adjusting cell concn is 1 * 10
12Individual/ml, get 20 μ l and be instilled in the cell cultures claire 5 in the proofing unit, along the inoculating surfaces long axis direction cell suspension inoculation is become a ribbon, avoid cell suspension to contact with the paraffin wall on its both sides; After inoculation finished, the cap seal with proofing unit was good immediately, places 5%CO
2, 37 ℃, in the incubator of 100% relative humidity, be cultured to cell attachment; Take out above-mentioned detection device, under aseptic condition, avoid cell cultures claire 5, in the ware body of proofing unit, add the nutrient solution that exceeds cell cultures claire 1mm amount, seal proofing unit and put back to former culture condition continuation cultivation, observation of cell is cultivated the cell growth in the claire 5 after 12 hours, to determine having or not of experimental cell contact inhibition.
If experimental cell growth reach converging state and with stop growing after pool wall contact closely, be preliminary contact inhibition, continue cultivation again through 4 cell cycles, experimental cell still stops to divide, and promptly is defined as experimental cell and contact inhibition occurs; Otherwise after continuing cultivation, experimental cell still can divide, and presents accumulated growth, and pool wall growth phenomenon can occur crossing over, then is that noncontact suppresses.
For the experimental cell that confirms to occur contact inhibition, can increase the volume that it cultivates claire by throwing off and its cell cultures claire next-door neighbour's the little adhesive tape and the method for its surperficial paraffin, carry out the observation that cells contacting suppresses release phenomenon.
Claims (8)
1. the proofing unit that suppresses of a cells contacting, form by culture dish, cover glass, non-setting adhesive scotch tape, paraffin coating, cell cultures claire, it is characterized in that: place the cover glass central authorities in the culture dish to be stained with rectangle non-setting adhesive scotch tape, this adhesive tape is that several are cut off but the little adhesive tape that is closely adjacent to each other, one end of every little adhesive tape has the termination that can uncover, the surface of adhesive tape scribbles one deck paraffin except that the termination, adhesive tape on cover glass has one to be uncovered the cell cultures claire that is surrounded by paraffin because of little adhesive tape and the paraffin on it on one side.
2. the proofing unit that cells contacting as claimed in claim 1 suppresses is characterized in that: one of geometricdrawing sample adhesive tape that described little adhesive tape is two next-door neighbours at least.
3. the proofing unit that cells contacting as claimed in claim 2 suppresses, it is characterized in that: described little adhesive tape is meant and is cut off into one of 2～5 rectangle adhesive tapes that are closely adjacent to each other.
4. the proofing unit that cells contacting as claimed in claim 1 suppresses, it is characterized in that: the shape of described cell cultures claire changes with the shape of little adhesive tape.
5. the proofing unit that cells contacting as claimed in claim 1 suppresses is characterized in that: the volume of described cell cultures claire is uncovered and is increased with adjacent little adhesive tape.
6. the proofing unit that cells contacting as claimed in claim 1 suppresses is characterized in that: the bottom surface of described cell cultures claire and the angle of its edge wall are 45～100 degree.
7. the preparation method of the proofing unit that cells contacting as claimed in claim 1 suppresses, the sequence of steps of this method is as follows:
(1) gets a clean cover glass, the smooth cover glass central authorities that bubble-freely are affixed on of non-setting adhesive scotch tape that will be more smaller than cover glass area;
(2) carve a rectangle frame with blade at above-mentioned adhesive tape middle part, and adhesive tape in the frame is cut into little adhesive tape more than two;
(3) with the end of point of a knife, provoke the termination that can take off respectively at two next-door neighbours' little adhesive tape, standby;
(4) with the paraffin of above-mentioned adhesive tape and the even coating of periphery one melting layer thereof, cooling is solidified paraffin then;
(5) clamp in the little adhesive tape termination of step (3) preparation one, throw off little adhesive tape and apply thereon paraffin, make the cover glass surface form the pan of an area with little adhesive tape to adhesive tape the other end direction;
(6) repair above-mentioned pan, the angle that makes its bottom surface and its edge wall is 45～100 degree, then its sterilization, sterilization promptly is made into the cell cultures claire;
(7) according to the experiment demand, but the operation of repeating step (5), (6) constantly enlarges the volume of cell cultures claire;
(8) the above-mentioned cover glass that has the cell cultures claire is put in the clean culture dish with cover, forms cells contacting jointly and suppress proofing unit.
8. use cells contacting as claimed in claim 1 and suppress the method that proofing unit carries out cells contacting inhibition detection, its concrete steps are as follows:
(1) get the logarithmic phase experimental cell of adherent growth, make single cell suspension, and to adjust cell concn is 1 * 10
8～1 * 10
13Individual/ml;
(2) get above-mentioned experimental cell suspension, in the cell cultures claire in proofing unit of instiling, suspension amount 10～30 μ l are advisable;
(3) above-mentioned detection device is built, placed 5%CO
2, 37 ℃, in the incubator of 100% relative humidity, be cultured to cell attachment;
(4) take out above-mentioned detection device from incubator, under the aseptic condition, avoid the cell cultures claire, add nutrient solution in the ware body of proofing unit, its liquid measure is advisable to exceed cell cultures claire 1mm;
(5) build proofing unit again, put back to former culture condition continuation and cultivate after 8～20 hours, observation of cell is cultivated the cell growth in the claire, with the situation of test experience cells contacting inhibition;
(6) if experimental cell growth reach converging state and with stop growing after pool wall contact closely, be preliminary contact inhibition, continue cultivation again through 2～5 cell cycles, experimental cell still stops to divide, and promptly is defined as experimental cell and contact inhibition occurs; Otherwise after continuing cultivation, experimental cell still can divide, and presents accumulated growth, and pool wall growth phenomenon can occur crossing over, then is that noncontact suppresses;
(7) in the step (6), confirm to have occurred the experimental cell of contact inhibition, can increase the volume that it cultivates claire by throwing off and its cell cultures claire next-door neighbour's the little adhesive tape and the method for its surperficial paraffin, carry out the observation that cells contacting suppresses release phenomenon.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01135204 CN1131319C (en) | 2001-11-30 | 2001-11-30 | Detection equipment for cell contact inhibition, its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01135204 CN1131319C (en) | 2001-11-30 | 2001-11-30 | Detection equipment for cell contact inhibition, its preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1354256A CN1354256A (en) | 2002-06-19 |
| CN1131319C true CN1131319C (en) | 2003-12-17 |
Family
ID=4673034
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 01135204 Expired - Fee Related CN1131319C (en) | 2001-11-30 | 2001-11-30 | Detection equipment for cell contact inhibition, its preparation method and application |
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|---|---|
| CN (1) | CN1131319C (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101541943B (en) * | 2007-02-28 | 2014-01-29 | 株式会社尼康 | Incubator, schedule management method, and program |
| CN104531517B (en) * | 2014-12-18 | 2016-08-17 | 山东大学 | Cellular density-dependence growth inhibited detection device and application |
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2001
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