CN103257229A - Method for evaluating prognosis of colorectal cancer patient through positive expression quantity of colorectal cancer immunological index CD3 - Google Patents
Method for evaluating prognosis of colorectal cancer patient through positive expression quantity of colorectal cancer immunological index CD3 Download PDFInfo
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Abstract
The invention discloses a method for evaluating prognosis of a colorectal cancer patient through positive expression quantity of colorectal cancer immunological index CD3. The method comprises the following steps of: firstly preparing a tissue wax block of the colorectal cancer patient into a tissue chip; then carrying out an immunohistochemical experiment and immune scoring; grouping according to immune score; drawing a graph by taking survival periods of patients, obtained by follow-up visit, with one group of low CD3 positive expression quantity and one group of high CD3 positive expression quantity as an x-coordinate, and taking the overall survival rate of the patients as a y-coordinate; and analyzing according to a relation between the survival period and the overall survival rate in the obtained graph to judge the CD3 positive expression quantity and a prognosis effect of the colorectal cancer patient. Under the condition of high CD3 positive expression quantity, the prognosis of the patient is relatively good; and under the condition of low CD3 positive expression quantity, the prognosis of the patient is relatively poor. The evaluation method is capable of predicting the prognosis of the colorectal cancer patient accurately and rapidly.
Description
Technical field
The present invention relates to a kind of method of colorectal cancer amynologic index prognostic evaluation.Be specifically related to a kind of method of the prognosis of colorectal cancer patients being estimated by colorectal cancer amynologic index CD3 positive expression amount.
Background technology
Research prompting T cellular infiltration position, type and a degree that the Jerome Galon of national health Medicine Research Academy of France etc. is published in the science magazine in 2006 are the important indicators of colorectal cancer prognosis judgement, and the CD8+ cell reaches 65.3% at the infiltration degree at tumor tissues central authorities and edge for the accuracy of prognosis prediction, the 50-60% by stages higher than TNM, and the scoring of ROC curve prompting immunology has a better operability clinical.They have set up take CD8+ and CD45RO+ cell quantity and infiltrates a set of colorectal cancer tumour immunity environment standards of grading of position as basis, CD8+ and CD45RO+ cell quantity that two positions of tumour infiltrate are all 0 minute less, CD8+ and CD45RO+ cell quantity that two positions of tumour infiltrate all mostly are 4 minutes, only has 4.8% Patients on Recurrence in the case group that the immunity scoring is 4 minutes, five year survival rate reaches 86.2%, and in the case group that immunity scoring is 0 minute and 1 minute, 72% Patients on Recurrence is arranged, five year survival rate only has 27.5%, after this immunocyte of colorectal cancer infiltrates with prognosis judge effect and causes extensive concern.
Can provide at present the molecular target of the information of colorectal cancer patients prognosis accurately and reliably considerably less, between 1998-2012, the research about the effect of colorectal cancer amynologic index prognostic evaluation of foreign literature report has 26 more than, judgment criteria is also uneven in a jumble, thereby never standard research is reported.
Summary of the invention
The objective of the invention is the defect in order to solve above-mentioned assessment colorectal cancer patients prognostic evaluation and a kind of method of the prognosis of colorectal cancer patients being estimated by colorectal cancer amynologic index CD3 positive expression amount is provided, first by the CD3 positive cell number, carry out immunity scoring, then by CD3 positive expression amount, determine the relation with patient's prognosis.
Technical scheme of the present invention
A kind of method of the prognosis of colorectal cancer patients being estimated by colorectal cancer amynologic index CD3 positive expression amount, specifically comprise the steps:
(1), at first, the 64-180 of excision colorectal cancer tumor tissues sample carried out to paraffin embedding, make paraffin embedded tissues;
(2), then,, according to the position of tumor tissues in the wax stone of HE dyeing determining step (1) gained, choose the central part of tumor tissues wherein and make the organization chip that contains 64-180 some position;
(3), then, immunohistochemical staining (Wang Guanghui Identification of MXRA5 as a novel tissue biomarker for colorectal cancer progression in 2013) is carried out in organization chip, positive control and the negative control of step (2) gained;
Wherein positive control is used and has turned out to be the organization chip that the positive tumor cell tissue organized makes;
Negative control is used identical organization chip;
(4), the conventional dewaxing of organization chip, aquation;
(5), microwave antigen retrieval;
Histotomy is dipped in the antigen retrieval liquid of sodium citrate, trisodium citrate and pure water preparation, and in micro-wave oven, high fire screen heating is 3 minutes, then in low fire screen heating 4 minutes * 2 times, cooling under room temperature after taking out, with the washing of PBS damping fluid, 5 minutes * 3 times;
(6), deactivating endogenous peroxydase:
The deactivation liquid be made into 30% hydrogen peroxide and methyl alcohol, be immersed in deactivation liquid 30 minutes by histotomy, after deactivating endogenous peroxydase, with the washing of PBS damping fluid, 5 minutes * 3 times;
(7), sealing nonspecific proteins:
By the histotomy scrub, the confining liquid that drips 50 microlitres on every histotomy is 5% BSA aqueous solution, hatches the sealing heterogenetic antigen 30 minutes;
(8), drip primary antibodie:
CD3 primary antibodie (YH112318, EPITOMICS) is pressed the 1:50 dilution by 5% Bovine Serum Albumin in Aqueous Solution, as for 4 ℃ of overnight incubation in wet box; Negative control replaces primary antibodie with the PBS damping fluid;
(9), rewarming is 45 minutes;
(10), dripping two resists
The universal immunologic combined detection reagent kit of anti-rabbit/mouse is hereinafter to be referred as two anti-kits, section is inserted in wet box, moisture with filter paper around will tissue sucks, and the A liquid that every histotomy drips in two anti-kits is one, horseradish peroxidase polymkeric substance, under room temperature, hatches half an hour;
(11), DAB colour developing:
The B liquid of two anti-kits in is DAB stoste is the DBA damping fluid according to B liquid with C liquid: the proportional arrangement that C liquid is 1:50 becomes nitrite ion, every histotomy adds the nitrite ion of 70ul, control under the microscope developing time, observe and positive findings stopped reaction when obviously background is painted again occurs, with distilled water flushing 10 minutes;
(12), haematoxylin is redyed;
(13), dehydration and transparent;
(14), mounting;
(15), interpretation of result:
Positive signal is brown yellow granule, under 200 times of enlargement factors, two pictures are taken to the Olympus optical microscope by each tissue site of above-mentioned gained, the number of counting picture middle-jiao yang, function of the spleen and stomach staining cell, if the positive staining cell number is 0, the immunity scoring is 0 minute; If the positive staining cell number is 1-19, the immunity scoring is 1 minute; If the positive staining cell number is 20-49, the immunity scoring is 2 minutes; If the positive staining cell number is greater than 49, the immunity scoring is 3 minutes;
(16), according to the immunity of step (15) gained, divided into groups, the immunity scoring be 0,1 to be one group be low one group of CD3 positive expression amount, the immunity scoring be 2,3 to be one group be high one group of CD3 positive expression amount;
(17), utilize Graph Pad prism 5 mapping softwares, using CD3 positive expression amount in step (16), low one group and CD3 positive expression amount high one group follows up a case by regular visits to survival of patients phase of obtaining as horizontal ordinate, patient's overall survival is mapped as ordinate, judge the prognosis effect of CD3 positive expression amount and colorectal cancer patients according to the relation analysis of figure life cycle of gained and overall survival, from figure, can judge, the survival of patients phase that CD3 positive expression amount is high is long, good prognosis; The survival of patients phase that CD3 positive expression amount is low is short, poor prognosis.
Beneficial effect of the present invention
A kind of method of being estimated by the prognosis of colorectal cancer amynologic index CD3 positive expression amount colorectal cancer patients of the present invention, by the positive expression amount of the CD3 of tumor tissues central authorities, predicted accurately and rapidly the relation between the positive variable expression situation of CD3 and patient's prognosis.
The accompanying drawing explanation
Fig. 1 a, under 50 times of enlargement factors tumor tissues middle section CD3 positive expression amount;
Fig. 1 b, under 100 times of enlargement factors tumor tissues middle section CD3 positive expression amount;
Fig. 1 c, under 200 times of enlargement factors tumor tissues middle section CD3 positive expression amount;
Fig. 2 a, under 50 times of enlargement factors positive expression amount a little less than tumor tissues middle section CD3;
Fig. 2 b, under 100 times of enlargement factors positive expression amount a little less than tumor tissues middle section CD3;
Fig. 2 c, under 200 times of enlargement factors positive expression amount a little less than tumor tissues middle section CD3;
Fig. 3 a, under 50 times of enlargement factors tumor tissues and normal structure handover region CD3 positive expression amount;
Fig. 3 b, under 100 times of enlargement factors tumor tissues and normal structure handover region CD3 positive expression amount;
Fig. 3 c, under 200 times of enlargement factors tumor tissues and normal structure handover region CD3 positive expression amount;
The positive expression amount of Fig. 4, CD3 and the relation of patient's prognosis, the life cycle that wherein horizontal ordinate is the patient, ordinate patient's overall survival
.
Embodiment
Also by reference to the accompanying drawings the present invention is further set forth below by specific embodiment, but do not limit the present invention.
In the embodiment of the present invention, the wax stone of 156 Colorectal Carcinomas used is all from Shanghai Communications University's attached Xinhua Hospital knot anorectum disease surgery.Xinhua Hospital Ethics Committee has been passed through in this experiment, and every patient has signed Informed Consent Form.
Immunohistochemical method, with reference to Wang Guanghui Identification of MXRA5 as a novel tissue biomarker for colorectal cancer progression in 2013.
Mapping software used is Graph Pad prism 5.
In embodiments of the invention, reagent used is in Table 1, and main experimental apparatus and consumptive material be in Table 2, test reagent preparation used in Table 3.
table 1 experiment reagent
table 2 experimental apparatus and consumptive material
the preparation of table 3 reagent
embodiment 1
A kind of method of the prognosis of colorectal cancer patients being estimated by colorectal cancer amynologic index CD3 positive expression amount, specifically comprise the steps:
(1), at first, 156 colorectal cancer tumor tissues samples of excision are carried out to paraffin embedding, make the wax stone of 156 colorectal cancer tumor tissues;
(2), then, according to HE, organization type is determined in dyeing, chooses tumor tissues and is made into two organization chips by Shanghai Biochip Co., Ltd, and 78 tumor tissues sites are arranged on every chip;
By the organization chip of above-mentioned steps (2) immunohistochemical experiment gained at Olympus optical microphotograph Microscopic observation, choosing the tumour middle position is taken, under 50,100 and 200 times, observed respectively, can find out that CD3 is at Partial tumors central authorities positive expression, and expression is higher, specifically as shown in Fig. 1 a, Fig. 1 b and Fig. 1 c; CD3 is weak positive expression in Partial tumors central authorities, and expression is lower, specifically as shown in Fig. 2 a, Fig. 2 b and Fig. 2 c; Intersection in Partial tumors and normal structure also can be observed the positive expression of CD3, concrete as Fig. 3 a, Fig. 3 b and Fig. 3 c.
In sum, the positive expression of CD3 is very special, all express among the interstitial between tumour, any tumor tissues dyeing does not appear, thereby this experiment can obtain the positive expression situation of CD3 more accurately, thereby carry out following survival Analysis, result is accurately credible, the positive staining that is CD3 mainly concentrates in the tumour cell interstitial, CD3 positive expression amount difference in different tumor tissues interstitials and the invasion and attack edge of tumour;
(3), the wax stone of organization chip is made to the serial section of 4 μ m, the histotomy of gained is placed on the metallochromy frame, be positioned in 60 ℃ of baking ovens roasting sheet 60 minutes to surperficial paraffin and melt;
(4), the conventional dewaxing of section aquation:
Histotomy is successively through dimethylbenzene washing 2 times, each 10 minutes, absolute ethanol washing 2 times, each 5 minutes, 95% alcohol washing 2 times, each 5 minutes, 75% alcohol washing 2 times, each 5 minutes, the paraffin on histotomy surface is taken off and abundant aquation, rinsed section 1min with tap water afterwards, wash 1 minute * 3 times with the PBS damping fluid;
(5), microwave antigen retrieval:
Histotomy is dipped in the antigen retrieval liquid of sodium citrate, trisodium citrate and pure water preparation, and in micro-wave oven, high fire screen heating is 3 minutes, then in low fire screen heating 4 minutes * 2 times, cooling under room temperature after taking out, with the washing of PBS damping fluid, 5 minutes * 3 times;
(6), deactivating endogenous peroxydase:
The deactivation liquid be made into 30% hydrogen peroxide and methyl alcohol, be immersed in deactivation liquid 30 minutes by histotomy, after deactivating endogenous peroxydase, with the washing of PBS damping fluid, 5 minutes * 3 times;
(7), sealing nonspecific proteins:
By the histotomy scrub, the confining liquid that drips 50 microlitres on every histotomy is 5% BSA aqueous solution, hatches the sealing heterogenetic antigen 30 minutes;
(8), drip primary antibodie:
The primary antibodie dilution ratio of recommending according to the antibody instructions is pressed 1:50 dilution antibody by 5% Bovine Serum Albumin in Aqueous Solution, as for 4 ℃ of overnight incubation in wet box, using PBS damping fluid replacement primary antibodie as negative control, with known positive setup action positive control by experimental design;
(9), second day by histotomy as for rewarming in 37 ℃ of baking ovens 45 minutes; The washing of PBS damping fluid, 10 minutes * 3 times;
(10), dripping two resists:
The universal immunologic combined detection reagent kit of anti-rabbit/mouse is hereinafter to be referred as two anti-kits, section is inserted in wet box, moisture with filter paper around will tissue sucks, and the A liquid that every histotomy drips in two anti-kits is one, horseradish peroxidase polymkeric substance, under room temperature, hatches half an hour;
(11), DAB colour developing:
The B liquid of two anti-kits in is DAB stoste is the DBA damping fluid according to B liquid with C liquid: the proportional arrangement that C liquid is 1:50 becomes nitrite ion, every histotomy adds the nitrite ion of 70ul, control under the microscope developing time, observe and positive findings stopped reaction when obviously background is painted again occurs, with distilled water flushing 10 minutes;
(12), redye:
Haematoxylin is redyed 30 seconds, distilled water flushing 10 minutes;
(13), dehydration and transparent:
By histotomy successively through 75% alcohol washing 2 times, each 5 minutes, 95% alcohol washing 2 times, each 5 minutes, absolute ethanol washing 2 times, each 5 minutes, dimethylbenzene washing 2 times, each 10 minutes;
(14), place 5min in fuming cupboard, make the dimethylbenzene volatilization on histotomy, then use the neutral gum mounting, after mounting, in baking oven, place 1 hour, natural gum is solidified as early as possible;
(15), each tissue site of step (14) gained is carried out to picture shooting under the Olympus optical microscope, enlargement factor is selected 200 times, and two pictures are taken by each tissue site, the number of counting picture middle-jiao yang, function of the spleen and stomach staining cell;
According to the positive staining cell number, be 0, the immunity scoring is 0 minute;
The positive staining cell number is 1-19, and the immunity scoring is 1 minute;
The positive staining cell number is 20-49, and the immunity scoring is 2 minutes;
The positive staining cell number is greater than 49, and the immunity scoring is 3 minutes;
Finally the scoring distribution situation of 156 above-mentioned Colorectal Carcinomas sees the following form:
| The immunity scoring | Number of cases | Number percent |
| 0 minute | 0/156 | 0 |
| 1 minute | 24/156 | 15.4% |
| 2 minutes | 38/156 | 24.4% |
| 3 minutes | 94/156 | 60.2% |
(16), according to the immunity of step (15) scoring, by the immunity scoring be 0 minute and 1 minute to be classified as one group be low one group of CD3 positive expression amount, immunity mark be 2,3 to be classified as one group be high one group of CD3 positive expression amount;
(17), analyze the relation of the how many and patient's overall survival of CD3 positive expression amount
Utilize Graph Pad prism 5 mapping softwares, using CD3 positive expression amount in step (16), low one group of survival of patients phase obtained with following up a case by regular visits to of CD3 positive expression amount high a group is as horizontal ordinate, overall survival is mapped as ordinate, the results are shown in Figure 4, in figure, the low expression of CD3 means low one group of CD3 positive expression amount, the CD3 high expressed means high one group of CD3 positive expression amount, judges the prognosis effect of CD3 positive expression amount and colorectal cancer patients according to the relation analysis of figure life cycle of gained and overall survival.
As can be seen from Figure 4, the patient's that CD3 positive expression amount is high prognosis is significantly better than the low patient of CD3 positive expression amount, and higher CD3 positive expression amount and patient's prognosis preferably are closely related.
In sum, a kind of method of the prognosis of colorectal cancer patients being estimated by colorectal cancer amynologic index CD3 positive expression amount of the present invention, can directly determine patient's prognosis information according to the height of CD3 positive expression rate in the colorectal cancer patients tumor tissues.
The above is only giving an example of embodiments of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the technology of the present invention principle; can also make some improvement, these improvement also should be considered as protection scope of the present invention.
Claims (6)
1. a method of the prognosis of colorectal cancer patients being estimated by colorectal cancer amynologic index CD3 positive expression amount, is characterized in that specifically comprising the steps:
(1), at first, the 64-180 of excision colorectal cancer tumor tissues sample carried out to paraffin embedding, make paraffin embedded tissues;
(2), then,, according to the position of tumor tissues in the wax stone of HE dyeing determining step (1) gained, choose the central part of tumor tissues wherein and make the organization chip that contains 64-180 some position;
(3), then, organization chip, positive control and the negative control of step (2) gained are carried out to immunohistochemical staining;
Wherein said positive control is used and has turned out to be the organization chip that the positive tumor cell tissue organized makes;
Described negative control is used identical organization chip;
(4), the conventional dewaxing of organization chip, aquation;
(5), microwave antigen retrieval;
(6), deactivating endogenous peroxydase;
(7), sealing nonspecific proteins;
(8), drip primary antibodie
The CD3 primary antibodie is pressed the 1:50 dilution by 5% Bovine Serum Albumin in Aqueous Solution, as for 4 ℃ of overnight incubation in wet box;
Replace primary antibodie with the PBS damping fluid in negative control;
(9), rewarming is 45 minutes;
(10), dripping two resists;
(11), DAB colour developing;
(12), haematoxylin is redyed;
(13), dehydration and transparent;
(14), mounting;
(15), interpretation of result:
Positive signal is brown yellow granule, with the Olympus optical microscope, under 200 times of enlargement factors, two pictures is taken to by each tissue site of above-mentioned gained, the number of counting picture middle-jiao yang, function of the spleen and stomach staining cell;
If the positive staining cell number is 0, the immunity scoring is 0 minute;
If the positive staining cell number is 1-19, the immunity scoring is 1 minute;
If the positive staining cell number is 20-49, the immunity scoring is 2 minutes;
If the positive staining cell number is greater than 49, the immunity scoring is 3 minutes;
(16), according to the immunity of step (15) gained, divided into groups, the immunity scoring is 0,1 is classified as one group and is low one group of CD3 positive expression amount, and the immunity scoring is 2,3 is classified as one group and is high one group of CD3 positive expression amount;
(17), utilize Graph Pad prism 5 mapping softwares, using CD3 positive expression amount in step (16), low one group and CD3 positive expression amount high one group follows up a case by regular visits to survival of patients phase of obtaining as horizontal ordinate, patient's overall survival is mapped as ordinate, judged the prognosis effect of CD3 positive expression amount and colorectal cancer patients according to the relation analysis of figure life cycle of gained and overall survival.
2. a kind of method of the prognosis of colorectal cancer patients being estimated by colorectal cancer amynologic index CD3 positive expression amount as claimed in claim 1, it is characterized in that the described microwave antigen retrieval of step (5), being about to histotomy is dipped in the antigen retrieval liquid of sodium citrate, trisodium citrate and pure water preparation, in micro-wave oven, high fire screen heating is 3 minutes, then in, low fire screen heating is 4 minutes * 2 times, cooling under room temperature after taking out, with the washing of PBS damping fluid, 5 minutes * 3 times.
3. a kind of method of the prognosis of colorectal cancer patients being estimated by colorectal cancer amynologic index CD3 positive expression amount as claimed in claim 2, it is characterized in that the described deactivating endogenous peroxydase of step (6), the deactivation liquid be made into 30% hydrogen peroxide and methyl alcohol, histotomy is immersed in deactivation liquid to 30 minutes, after deactivating endogenous peroxydase, with the washing of PBS damping fluid, 5 minutes * 3 times.
4. a kind of method of the prognosis of colorectal cancer patients being estimated by colorectal cancer amynologic index CD3 positive expression amount as claimed in claim 3, it is characterized in that the described sealing nonspecific proteins of step (7) is about to the histotomy scrub, the confining liquid that drips 50 microlitres on every histotomy is 5% BSA aqueous solution, hatch the sealing heterogenetic antigen 30 minutes.
5. a kind of method of the prognosis of colorectal cancer patients being estimated by colorectal cancer amynologic index CD3 positive expression amount as claimed in claim 4, it is characterized in that the described dropping two of step (10) resists soon section to insert in wet box, with filter paper, the moisture around tissue is sucked, the A liquid that every histotomy drips in the universal immunologic combined detection reagent kit of anti-rabbit/mouse is one, horseradish peroxidase polymkeric substance, under room temperature, hatches half an hour.
6. a kind of method of the prognosis of colorectal cancer patients being estimated by colorectal cancer amynologic index CD3 positive expression amount as claimed in claim 5, it is characterized in that the described DAB colour developing of step (11), be about to B liquid in the universal immunologic combined detection reagent kit of anti-rabbit/mouse and be DAB stoste and C liquid and be the DBA damping fluid according to B liquid: the proportional arrangement that C liquid is 1:50 becomes nitrite ion, every histotomy adds the nitrite ion of 70ul, control under the microscope developing time, observe and positive findings stopped reaction when obviously background is painted again occurs, with distilled water flushing 10 minutes.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103487583A (en) * | 2013-10-14 | 2014-01-01 | 中南大学 | Immunohistochemical diagnosis kit for colorectal cancer malignancy and metastasis of colorectal cancer |
| CN107976442A (en) * | 2017-11-21 | 2018-05-01 | 陈素贤 | A kind of tumor prognosis predicting marker and its detection method |
| CN109738640A (en) * | 2019-01-18 | 2019-05-10 | 上海交通大学医学院附属第九人民医院 | Composition and its kit, biochip and application |
| CN110646271A (en) * | 2019-09-20 | 2020-01-03 | 四川大学华西医院 | Preparation method of paraffin section of mouse or rat acute pancreatitis tissue |
| CN115612742A (en) * | 2022-10-31 | 2023-01-17 | 南方医科大学 | Molecular marker model for predicting new adjuvant therapy curative effect of middle and late rectal cancer and application thereof |
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2013
- 2013-05-29 CN CN 201310206356 patent/CN103257229A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103487583A (en) * | 2013-10-14 | 2014-01-01 | 中南大学 | Immunohistochemical diagnosis kit for colorectal cancer malignancy and metastasis of colorectal cancer |
| CN107976442A (en) * | 2017-11-21 | 2018-05-01 | 陈素贤 | A kind of tumor prognosis predicting marker and its detection method |
| CN109738640A (en) * | 2019-01-18 | 2019-05-10 | 上海交通大学医学院附属第九人民医院 | Composition and its kit, biochip and application |
| CN109738640B (en) * | 2019-01-18 | 2022-04-19 | 上海交通大学医学院附属第九人民医院 | Composition, kit, biochip and application thereof |
| CN110646271A (en) * | 2019-09-20 | 2020-01-03 | 四川大学华西医院 | Preparation method of paraffin section of mouse or rat acute pancreatitis tissue |
| CN115612742A (en) * | 2022-10-31 | 2023-01-17 | 南方医科大学 | Molecular marker model for predicting new adjuvant therapy curative effect of middle and late rectal cancer and application thereof |
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Application publication date: 20130821 |