CN101013086A - Tissue chip for detecting backbone metastatic carcinoma prognosis related molecule sign - Google Patents
Tissue chip for detecting backbone metastatic carcinoma prognosis related molecule sign Download PDFInfo
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- CN101013086A CN101013086A CN 200710037010 CN200710037010A CN101013086A CN 101013086 A CN101013086 A CN 101013086A CN 200710037010 CN200710037010 CN 200710037010 CN 200710037010 A CN200710037010 A CN 200710037010A CN 101013086 A CN101013086 A CN 101013086A
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- metastatic cancer
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Abstract
The invention relates to the biotechnology area, and the spinal metastatic cancer prognosis judgment is the key to correct determine clinical development strategy, and in the invention, for the present spinal metastatic cancer related molecular biology prognosis judgment evaluation lacking problem, it constructs the spinal metastatic cancer prognosis related molecular marker tissue chip. By screening the clinical data and determining the random data spinal metastatic cancer pathological wax block to produce the spinal metastatic cancer tissue chip; selecting a variety of molecular markers and tissue chips for immunohistochemistry or in-situ hybridization, and through image analysis and quantitative measurement to analyze the expression instance of each molecular marker in the spinal metastatic cancer pathological specimens, and to analyze the relationship between the molecular markers expression and the patients prognosis, and screening the molecular markers which are closely related to the spinal metastatic cancer prognosis. The tissue chip provides a high-throughput, large sample, rapid detection tool for screening the spinal metastatic cancer prognostic related molecular markers, and provides a theoretical basis for the rational determination of the spinal metastatic cancer development strategies.
Description
Technical field:
The present invention relates to biological technical field, be specifically related to a kind of organization chip that is used to detect backbone metastatic carcinoma prognosis related molecule sign.
Background technology:
Backbone metastatic carcinoma is the important illness of a class of serious harm human health, have higher disable, fatal rate.Metastatic carcinoma is destroyed the backbone sclerotin, causes the vertebra joint to subside and the spinal nerve pressurized, causes function of spinal nerves obstacle, paralysis and intractable pain, has a strong impact on patients ' life quality and life cycle.Along with the renewal of surgical intervention theory and technology, surgical intervention becomes the important means of Spinal Metastases treatment day by day.But because the backbone metastatic carcinoma patient is limited life cycle, its operative treatment still is to have the more field of arguement at present.Fact proved not only to be helpless to unsuitable operative treatment the patient and to prolong life cycle, and be unfavorable for the life quality of patient in limited life cycle, also can cause unnecessary financial burden simultaneously to society.It is the key point of correctly formulating the backbone metastatic carcinoma therapeutic strategy that prognosis is judged.And at present existing clinical assessment system is still not accurate enough for patient's prognosis judge, and related index all is cardinal principle indexs of clinical observation, lacks relevant molecular biological judging quota.The screening prognosis related molecule sign will help further accurately backbone metastatic carcinoma patient's prognosis, thereby formulate proper treatment strategy more.
Organization chip (tissue chip) claims micro-array tissue (tissue microarray) technology again, principle is to be arranged on the microslide in good orderly and to make miniature organization chip to up to a hundred even how little organizing tens of, 100～200 of application organizes chip wax stone serial section, like this can be to hundreds of biomolecular labeling (as antibody with same set of organization chip, DNA and RNA) analyze, detect, has high flux, the advantage of large sample and express-analysis, and can save the organizer material, guarantee the experiment condition consistance, reduce experimental error.Appearances such as at present existing normal structure chip, tumor tissues chip, but be not at being used to screen backbone metastatic carcinoma prognosis related molecule sign.Press for a kind of organization chip that is used for large sample, polygenes screening backbone metastatic carcinoma prognosis related molecule clinically.
Summary of the invention:
The object of the present invention is to provide a kind of novel organization chip, be used to detect backbone metastatic carcinoma prognosis related molecule sign, judge so that the backbone metastatic carcinoma patient is carried out the true prognosis of calibration before formulating therapeutic strategy.
Design of the present invention is that screening has the backbone metastatic carcinoma case of clinical data and clear and definite follow up data, adopt corresponding pathology wax stone to make up the backbone metastatic carcinoma organization chip, select multiple molecular marker and organization chip and carry out SABC or in situ hybridization, analyze the expression of each molecular marker in each Pathologic specimen of backbone metastatic carcinoma by graphical analysis and quantitative measurement, each molecular marker expression is entered computer-aided analysis system jointly with corresponding clinical indices and follow up data, analyze the relation between each molecular marker expression and the patient's prognosis, filter out and the closely-related molecular marker of backbone metastatic carcinoma prognosis, as protein molecular, DNA and RNA.
The invention provides a kind of organization chip that is used to detect backbone metastatic carcinoma prognosis related molecule sign, its making comprises the making of making, organization chip paraffin mass and the organization chip section of foundation, specimen collection and preparation, the acceptor paraffin mass in clinical data and follow up data storehouse.Wherein, specimen collection and preparation require the clinical middle tumor of spine sample of gathering to carry out paraffin embedding, select make a definite diagnosis Spinal Metastases and have complete clinical data and follow up a case by regular visits to situation sample carry out routine section HE dye further consultation and typical organization's zone location, as the donor wax stone; The acceptor paraffin mass will and be organized core bar diameter array of designs by the density of making organization chip, is affixed on the acceptor wax block surface with the mould paper of the lattice array of corresponding specification, punches; The making of organization chip wax stone is with tissue array instrument organizing the core bar and move in the acceptor paraffin mass respective aperture at donor wax stone locating area extracting certain diameter.
Each metastatic carcinoma is organized in the backbone metastatic carcinoma organization chip of the present invention all has corresponding pathology further consultation situation, clinical data and follows up a case by regular visits to situation.The structure of this organization chip comprises that clinical data and follow up data storehouse are set up and organization chip is made.
(A) foundation in clinical data and follow up data storehouse
Collecting clinical index comprises: sex, age, excision mode, visceral metastases situation, bone transfer case, tumor type, follow up a case by regular visits to patient's postoperation radiotherapy, chemotherapy situation and survival state (person of dying in heaven writes down date of death, and the survivor writes down whole end and follows up a case by regular visits to the time and be designated censored data).
(B) specimen collection and preparation: gather the Spinal Metastases sample, be divided into 5mm * 15mm * 15mm size, tissue specimen is through routine dehydration, paraffin embedding.Select the backbone metastasis of the common cancers such as liver cancer, lung cancer, breast cancer, kidney, cancer of the stomach, colorectal cancer, prostate cancer that possess complete clinical data and follow up a case by regular visits to situation and each 100 example of not clear primary tumor backbone metastatic carcinoma sample as the donor wax stone, carry out the routine section, HE dyeing, microscopically turn out to be Spinal Metastases and to typical tumor tissues zone location.
(C) making of acceptor paraffin mass: by the density of making organization chip with organize the size of core bar diameter, be affixed on the acceptor wax block surface, punch with the mould paper of the lattice array of corresponding specification;
(D) making of organization chip paraffin mass: organize core according to array design extracting pathology wax stone and be listed on the blank acceptor wax block by designing and arranging in advance with the tissue array instrument, the tissue array piece heats fusion in 52 ℃ of constant temperature roasters, make and organize core closely to link to each other with acceptor wax block;
(E) making of organization chip section: with the feed velocity of 20 microns/commentaries on classics the tissue array piece is repaired wax stone with full-automatic histotome, the core of organizing until 80% exposes to the open air fully; The tissue array piece is cut into slices with the feed velocity of 4 microns/commentaries on classics with full-automatic histotome, section is mounted on the import microslide that is attached to anti-flake processing; Array slice placed the roasting sheet of 60 ℃ of constant temperature roasters 16 hours.
The organization chip quality evaluation: SABC and in situ hybridization detect related gene albumen and the expression of mRNA in organization chip, and to detect the tissue specimen result consistent with the conventional sense method, and organization chip do not have and take off a phenomenon, and cellular morphology is clear.
The white sheet of the experiment of organization chip is kept in the section box, places 5 ℃ of reefers of refrigerator to preserve; Used pathological tissue wax stone sealing wax, and itself and corresponding HE stained playback put back to wax stone cabinet and section box.
The a large amount of tumor proliferation found at present and the correlating markings thing of transfer are as P53, erbB-2, nm23, CDv44, bcl-2 etc.Confirmed that the some of them mark has important correlativity for the prognosis of solid tumor.But these have found whether study with the relevant nothing of prognosis of backbone metastatic carcinoma with the tumor markers that will find.With organization chip of the present invention the MMP-9 gene is detected, carry out statistical analysis in conjunction with the follow up data storehouse.The result shows that the patient of the MMP-9 gene expression positive expresses the negative patient weak point life cycle, P＜0.05, prompting significant difference.Cox multiplicity prompting MMP-9 is a backbone metastatic carcinoma prognosis risk factor.
Whether not only can help us to understand these marks of present discovery by organization chip of the present invention relevant with the backbone metastatic carcinoma prognosis.And adopt organization chip of the present invention, and detect backbone metastatic carcinoma prognosis related molecule sign, thereby take proper treatment strategy more, improve the clinical therapeutic efficacy of backbone metastatic carcinoma.
Embodiment:
Below in conjunction with drawings and Examples the present invention is further described:
Embodiment 1: backbone adenocarcinoma metastatic organization chip and making thereof
Collect clinical backbone adenocarcinoma metastatic patient's sex, age, excision mode, visceral metastases situation, bone transfer case, tumor type, follow up a case by regular visits to patient's postoperation radiotherapy, chemotherapy situation and survival state (person of dying in heaven writes down date of death, and the survivor writes down whole end and follows up a case by regular visits to the time and be designated censored data).Set up clinical data and follow up data storehouse.
Gather the Spinal Metastases sample, cut apart into about 5mm * 15mm * 15mm size, tissue specimen is through routine dehydration, paraffin embedding.Select and possess complete clinical data and follow up a case by regular visits to backbone adenocarcinoma metastatic sample 100 examples of situation as the donor wax stone.
With the full-automatic paraffin slicing machine histopathologic slide that 4 micron thickness are made in section to the donor paraffin mass.
With full-automatic dyeing machine histopathologic slide is carried out HE dyeing.(standard HE dyeing procedure)
Microscopically is diagnosed as the backbone adenocarcinoma metastatic, and mark typical case tumor tissues zone.
The types of organization of design organization chip array and arrangement mode.The embedding again of every routine wax stone paraffin embedding machine, and sampling (the about 1mm of diameter)
In tissue database, select the suitable case of organizing to number, according to organizing the case numbering from tissue bank, to take out paraffin embedded tissues and corresponding HE stained.
Prepare suitable blank acceptor wax block with tissue embedding machine.
Organize core according to array design extracting pathology wax stone and be listed on the blank acceptor wax block with the tissue array instrument by designing and arranging in advance.
The tissue array piece heats fusion in 52 ℃ of constant temperature roasters, make and organize core closely to link to each other with acceptor wax block.
With the feed velocity of 20 microns/commentaries on classics the tissue array piece is repaired wax stone with full-automatic histotome, the core of organizing until 80% exposes to the open air fully.
The tissue array piece is cut into slices with the feed velocity of 4 microns/commentaries on classics with full-automatic histotome, section is mounted on the import microslide that is attached to anti-flake processing
Array slice placed the roasting sheet of 60 ℃ of constant temperature roasters 16 hours.
Organization chip extracts one by numbering every 10 and does HE dyeing (standard HE dyeing procedure), and each tissue samples in the sampling observation chip is carried out the further consultation quality inspection, and the result is by information portion input organization chip database.
Organization chip is tested white sheet and is kept in the section box, places 5 ℃ of reefers of refrigerator to preserve.To used pathological tissue wax stone sealing wax, and itself and corresponding HE stained playback put back to wax stone cabinet and section box.
Embodiment 2: the immunohistochemical analysis of making the MMP-9 gene of this chip
(1) histotomy dewaxing aquation.
Before the dewaxing, organization chip should be placed at room temperature 60 minutes or 60 ℃ of constant temperature ovens in baking 20 minutes.
(A) organization chip places dimethylbenzene to soak 10 minutes, soaks 10 minutes behind the replacing dimethylbenzene again;
(B) soaked 5 minutes in the absolute ethyl alcohol;
(C) soaked 5 minutes in 95% ethanol;
(D) soaked 5 minutes in 70% ethanol;
(2) antigen retrieval (adopting the hot high pressure repairing method)
In boiling water, add 0.01M sodium citrate buffer solution (pH6.0).Cover the lid of stainless steel pressure cooker, but do not lock.Slide is placed on the metallochromy frame, and slowly pressurization was soaked slide 5 minutes in damping fluid, and then with cover lock, minor valve will rise.After 10 minutes, remove thermal source, insert in the cold water uncap after minor valve sinks.
(3) immunohistochemical staining (SP method)
(A) dewaxing, aquation;
(B) PBS washed 2～3 times each 5 minutes;
(C) 3%H202 (80% methyl alcohol) drips on TMA, and room temperature left standstill 10 minutes;
(D) PBS washed 2～3 times each 5 minutes;
(E) antigen retrieval;
(F) PBS washed 2～3 times each 5 minutes;
(G) drip normal goats serum confining liquid, room temperature 20 minutes.Get rid of unnecessary liquid;
(H) drip anti-(MMP-9 antibody) the 50 μ l of I, room temperature leave standstill spent the night in 1 hour or 4 ℃ or 37 ℃ 1 hour;
(I) 4 ℃ need after spending the night 37 ℃ of rewarmings 45 minutes;
(J) PBS washed 3 times each 5 minutes;
(K) drip anti-40～50 μ l of II, room temperature leaves standstill, or 37 ℃ 1 hour;
(L) II can add 0.05% tween-20 in anti-;
(M) PBS washed 3 times each 5 minutes;
(N) the DAB colour developing is 5～10 minutes, grasps dye levels at microscopically;
(O) flushing of PBS or tap water is 10 minutes;
(P) haematoxylin redyeing is 2 minutes, the hydrochloride alcohol differentiation;
(Q) the tap water flushing is 10～15 minutes;
(R) dehydration, transparent, mounting.
Organization chip does not have and takes off a phenomenon, and the microscopically cellular morphology is clear, most case MMP-9 endochylema stained positive, MMP-9 gene expression in the prompting cell.
Claims (3)
1, a kind of organization chip that is used to detect backbone metastatic carcinoma prognosis related molecule sign, its manufacturing process comprises:
(A) set up clinical data and follow up data storehouse: the collection clinical indices is also followed up a case by regular visits to patient's postoperation radiotherapy, chemotherapy situation and survival state and is set up the case database;
(B) specimen collection and preparation: in clinical, gather the tumor of spine sample and carry out paraffin embedding, select make a definite diagnosis Spinal Metastases and have complete clinical data and follow up a case by regular visits to situation sample carry out routine section HE dye further consultation and typical organization's zone location, as the donor wax stone;
(C) making of organization chip paraffin mass: the organizing the core bar and be listed on the blank acceptor wax block by designing and arranging in advance of donor wax stone locating area extracting certain diameter, the heating of tissue array piece is merged, and makes and organizes core closely to link to each other with acceptor wax block with the tissue array instrument;
(D) making of organization chip section: with full-automatic histotome the tissue array piece is repaired wax stone, the core of organizing until 80% exposes to the open air fully.
2, the described organization chip that is used to detect backbone metastatic carcinoma prognosis related molecule sign of claim 1 is characterized in that each interlacing point is all corresponding with the clinical case database on the organization chip.
3, claim 1 is described is used for detecting the organization chip of backbone metastatic carcinoma prognosis related molecule sign in the application of analyzing related molecule sign and backbone metastatic carcinoma prognosis relation.
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| CN 200710037010 CN101013086A (en) | 2007-01-31 | 2007-01-31 | Tissue chip for detecting backbone metastatic carcinoma prognosis related molecule sign |
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| CN 200710037010 CN101013086A (en) | 2007-01-31 | 2007-01-31 | Tissue chip for detecting backbone metastatic carcinoma prognosis related molecule sign |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102439454A (en) * | 2009-02-11 | 2012-05-02 | 卡里斯Mpi公司 | Molecular profiling of tumors |
| CN108037278A (en) * | 2017-12-13 | 2018-05-15 | 广州吉奥生物科技有限责任公司 | The preparation method of immunohistochemistry detection section |
| CN113588375A (en) * | 2021-08-20 | 2021-11-02 | 中科光华(西安)智能生物科技有限公司 | High-stability bone and bone tumor tissue microarray chip and preparation method thereof |
| CN113658209A (en) * | 2021-08-12 | 2021-11-16 | 山东省计算中心（国家超级计算济南中心） | Morphology-based method for judging excellent mounting position of sliced tissue |
-
2007
- 2007-01-31 CN CN 200710037010 patent/CN101013086A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102439454A (en) * | 2009-02-11 | 2012-05-02 | 卡里斯Mpi公司 | Molecular profiling of tumors |
| CN102439454B (en) * | 2009-02-11 | 2015-05-13 | 卡里斯Mpi公司 | Molecular profiling of tumors |
| CN108037278A (en) * | 2017-12-13 | 2018-05-15 | 广州吉奥生物科技有限责任公司 | The preparation method of immunohistochemistry detection section |
| CN108037278B (en) * | 2017-12-13 | 2018-09-25 | 广州吉奥生物科技有限责任公司 | The preparation method of immunohistochemistry detection slice |
| CN113658209A (en) * | 2021-08-12 | 2021-11-16 | 山东省计算中心（国家超级计算济南中心） | Morphology-based method for judging excellent mounting position of sliced tissue |
| CN113658209B (en) * | 2021-08-12 | 2023-06-16 | 山东省计算中心（国家超级计算济南中心） | Morphology-based excellent discriminating method for mounting position of slice tissue |
| CN113588375A (en) * | 2021-08-20 | 2021-11-02 | 中科光华(西安)智能生物科技有限公司 | High-stability bone and bone tumor tissue microarray chip and preparation method thereof |
| CN113588375B (en) * | 2021-08-20 | 2023-09-29 | 中科光华(西安)智能生物科技有限公司 | High-stability bone and bone tumor tissue microarray chip and preparation method thereof |
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