阅读说明：本技术 间皮素检测细胞质控片 (Mesothelin detects cytoplasm control wafer ) 是由 于占娇 毛海燕 王庆波 李壮林 李元浩 于 2018-05-25 设计创作，主要内容包括：本发明涉及一种间皮素免疫组化检测细胞质控片以及相关检测试剂,所述细胞质控片包括切片区；所述切片区粘附有石蜡切片,所述石蜡切片含有4种起对照作用的表达间皮素的阴性、弱阳性、阳性、强阳性细胞团；本发明涉及的免疫组化检测细胞质控片主要用于提高免疫组化检测结果的准确性和一致性,有良好的应用前景。(The present invention relates to a kind of mesothelin immunohistochemistry detection cytoplasm control wafer and coherent detection reagent, the cytoplasm control wafer includes slice area；The slice area is stained with paraffin section, and the paraffin section contains 4 kinds of feminine gender, weakly positive, positives, strong positive cell mass for playing the expression mesothelin of control；Immunohistochemistry detection cytoplasm control wafer of the present invention is mainly used for improving the accuracy and consistency of immunohistochemistry testing result, there is good application prospect.)

1. a kind of mesothelin immunohistochemistry detects cytoplasm control wafer, including slice area；The slice area is stained with paraffin section, institute It states paraffin section and contains 4 kinds of feminine gender, weakly positive, positives, strong positive cell mass for playing the expression mesothelin of control.

2. cytoplasm control wafer described in claim 1,4 kinds of cell masses are the different cells of 4 kinds of mesothelin expression quantity, respectively It is: CFPAC-1 cell, the mesothelin positive expression that 293 cells of mesothelin-negative expression, mesothelin weakly positive are expressed The OVCAR-3-MSLN cell that OVCAR-3 cell, mesothelin strong positive are expressed.

3. cytoplasm control wafer as claimed in claim 2, wherein the OVCAR-3-MSLN cell is genetically modified to express The OVCAR-3 cell of external source MSLN albumen.

4. cytoplasm control wafer described in claim 1, the thickness of the paraffin section are 3-5 μm.

5. cytoplasm control wafer described in claim 1, the diameter of the cell mass is 3-5mm.

6. cytoplasm control wafer described in claim 1-5 any claim is preparing the purposes in mesothelin detection reagent.

7. the OVCAR-3-MSLN cell of mesothelin strong positive expression is preparing mesothelin immunologic combined detection reagent kit Quality Control piece In application, the OVCAR-3-MSLN cell by OVCAR-3 cell be transferred to external source MSLN gene obtain, external source can be expressed MSLN albumen.

8. a kind of object of reference for immunohistochemistry technology's detection mesothelin protein quality control, the object of reference include: Positive control, negative control object；Wherein, positive control are as follows: CFPAC-1 cell, OVCAR-3 cell and OVCAR-3- MSLN cell, wherein the OVCAR-3-MSLN cell is the genetically modified OVCAR-3 cell to express external source MSLN；Yin Property reference material are as follows: 293 cells.

9. a kind of method for immunohistochemical detection of non-diagnostic purpose, the method is made using the cytoplasm control wafer of claim 1-5 For control.

10. a kind of method for being used to prepare mesothelin immunohistochemistry detection cytoplasm control wafer comprising following steps:

(1) cell culture

Exist to the OVCAR-3-MSLN cell of 293 cells, CFPAC-1 cell, OVCAR-3 cell and mesothelin strong positive expression It is cultivated in culture medium；

(2) cell is collected and is fixed

When cultivating above-mentioned cell and being paved with the 80-90% of Tissue Culture Dish bottom to it, cell is collected；Culture medium is removed, phosphoric acid is slow Rush brine, pancreatin digestion when microscopically observation cell rounding, adds 1ml cell culture medium to terminate digestion, uses pipettor By cell processing uniformly, collect and count, phosphate buffered saline solution is added and washs cell, supernatant is abandoned in centrifugation, and cell is with more than 4% Polyformaldehyde or 10% neutral formalin fixer are resuspended, and room temperature is fixed；

(3) agarose supports cell

Ago-Gel is prepared: weighing agarose and phosphate buffered saline solution is added to be configured to the solution of 3-4% concentration, be heated to agar Sugar dissolution, taking-up are placed in heat preservation for standby use in 60 DEG C of water-baths；

Cell is mixed with agarose: passing through fixation procedure, about 100 μ l of natural subsidence to volume carefully removes supernatant with pipettor； The centrifuge tube for filling cell is preheated in 60 DEG C of water-baths, is pipetted with the pipettor of preheating solidifying with the isometric agarose of cell Glue is added in centrifuge tube and mixes with cell, and agarose, cell mixture after mixing are transferred in syringe needle tube, and room temperature is cold But it forms；

(4) cell mass is dehydrated

The cell mass mixed with agarose is taken out, segment is cut into, is placed in the dewatering basket marked；Successively by cell mass It immerses in the ethyl alcohol of various concentration gradient and carries out tissue dewatering；

(5) cell mass is transparent

By step (4) obtain cell mass be successively immersed in dimethylbenzene carry out it is transparent；

(6) cell mass waxdip

Paraffin mass is placed in waxdip cup in 65 DEG C of baking ovens and is melted completely to paraffin for a period of time, after carrying out transparency of organization Cell mass, which is immersed in the paraffin of thawing, carries out tissue waxdip；

(7) cell mass embeds

The paraffin of thawing is poured into organization embedding bed die, the cell mass that waxdip terminates is taken out from corresponding dewatering basket, is put Enter into the paraffin of organization embedding bed die, embedding frame is covered on embedded box, is transferred on cooling bench cooling, acquisition tissue packet It layings of markstone wax stone.

11. method described in any one of claim 10, culture medium used in the culture includes:

The culture medium of 293 cells includes: Gibco RPMI1640 culture medium+10%FBS；

The culture medium of CFPAC-1 cell includes: Gibco IMDM culture medium+10%FBS；

The culture medium of OVCAR-3 cell includes: Gibco RPMI1640-A10491-01 culture medium+20%FBS+0.01mg/ml Insulin；

The culture medium of the OVCAR-3-MSLN cell of mesothelin strong positive expression includes: GibcoRPMI1640-A10491-01 training Support base+20%FBS+0.01mg/ml insulin.

12. method described in any one of claim 10, the condition of the cell culture includes: 37 DEG C, 5%CO₂Stationary culture 2-3 days.

13. method described in any one of claim 10, the dehydration of the step (4) are as follows: 70% ethyl alcohol is stayed overnight；80% ethyl alcohol 1.5h；90% ethyl alcohol 1.5h；95% ethyl alcohol 1.5h；100% alcohol treatment 50min；100% alcohol treatment 50min.

14. a kind of immunohistochemical kit of detection mesothelin expression, it includes cytoplasm control wafers described in claim 1-5.

Technical field

The present invention relates to a kind of mesothelin immunohistochemistry detection relevant cell or tissue sample control Quality Control piece and correlations Detection reagent.

Background technique

Mesothelin (Mesothelin, MSLN) is a memebrane protein, and what mesothelin gene encoded is the preceding egg of a 71KD It is white, become two sections through modification cutting, the glycolsyl-phosphatidylinositol cell membrane anchorin of 40KD and the free segment of 31KD claim For megakaryocyte-potentiating factor.There is research to confirm that the film anchorin of 40KD includes one section and another tumor markers CA125 The combined area of albumen, they may be related with cell adherence, thus conjecture might have and help primary carcinogenesis far-end transfer (example Such as abdominal metastas), and cause prognosis poor.

Although the function of Mesothelin is still inaccurate, either still free area is all proved for cell membrane combined area It can be used as the marker and therapy target of the kinds of tumors such as carcinoma mesothelial, cancer of pancreas, oophoroma.It is reported that cancer of pancreas (86%- 100%) it and the non-mucus cancer of ovary, wherein serous carcinoma (93%-100%), clear cell carcinoma (43%-75%) and migrates There are MSLN positive expressions in cell cancer (100%).About 40% to 50% adenocarcinoma of lung and 15% to 30% lung squamous it is thin Born of the same parents' cancer also has been reported that there are MSLN positive expressions.Accurately identify with great expression MSLN protein tumor patient ( The patient that can be benefited from MSLN targeted therapy), for instructing the medication of patient meaningful.

The cardinal principle of ImmunohistochemistryMethods Methods is: applied immunology basic principle-antigen-antibody reaction, i.e. antigen and anti- The principle of body specific binding, the color developing agent (fluorescein, enzyme, metal ion, isotope) of labelled antibody is made by chemically reacting Colour developing positions it, qualitative and relative quantification research mode to determine histocyte endoantigen (peptide and protein).

Immunohistochemistry technique is mainly characterized by: 1 high specificity: immunologic basic principle determine antigen and antibody it Between combination have high degree of specificity；2 sensibility are high: antibody dilutes thousands of times, up to ten thousand times even more than one hundred million times still can be thin in tissue With antigen binding in born of the same parents, the antibody antigen of such hypersensitivity reacts, and is conveniently used in ImmunohistochemistryMethods Methods more and more often Advise pathological analysis work.3 accurate positionings: the technology by antigen-antibody reaction and color reaction, can in tissue and cell into The accurate positionin of row antigen, thus position observation can be carried out in same tissue or cell to not synantigen simultaneously, thus may be used To carry out the research that form and function combines, to deeply having great importance for pathological research.

Since immunohistochemical experiment process is relative complex, the factor for influencing testing result is more, and ImmunohistochemistryResults Results Interpretation to be sentenced reader's subjective impact relatively large, therefore, it is however generally that, need standardized comparison device, such as: cytoplasm Control wafer.The control cell in the prior art that different expression intensities are not set for the Quality Control piece of immunohistochemical experiment, because This, control effect is to be improved.

It is acted on during the experiment using cytoplasm control wafer as follows:

Whether immunohistochemistry detection system normally can be used as just for the dyeing of cytoplasm control wafer during immunohistochemistry Normal foundation, if experimentation occurs surprisingly assisting searching problem.

Quality Control piece can be used for antibody screening, and the processes such as experiment condition screening save the cost of purchase Hospital Pathological Department slice, Compared to pathological tissue, cytoplasm control wafer consistency with higher is used.

After pathological tissue carries out immunohistochemistry detection, tissue will appear the dyeing of varying strength, tumour cell dye in tissue The height of the intensity of colour percentage shared with dyeing usually can reflect the expression quantity of target antigen in tumor tissues.Cytoplasm There are the cell of MSLN difference expression intensity in control wafer, different staining powers can be shown after immunohistochemical staining, in pathology When histotomy ImmunohistochemistryResults Results interpretation, cytoplasm control wafer can be used as " colorimetric card " effect of staining power, assist pathology It is sliced the interpretation of ImmunohistochemistryResults Results.The precision to immunohistochemical experiment result interpretation is improved, so that it is immune to improve the target The accuracy of groupization testing result.

Summary of the invention

The object of the present invention is to provide a kind of matter for Immunohistochemical Method detection tumor targets MSLN protein expression situation Control slice, the effect of Quality Control slice are to can be improved the confidence level of MSLN target proteins immunohistochemistry testing result and accurate Property.

On the one hand, the present invention provides one to detect tumor targets MSLN protein expression situation using Immunohistochemical Method When can be realized the adherency glass slide of Quality Control function, including frosted area and slice area, it is characterised in that: post mark in the frosted area Label, label substance includes Quality Control piece title and lot number；The slice area is stained with the paraffin section with a thickness of 3-5 μm, every stone Wax slice arranges the cell mass of totally 4 points containing 2 rows 2, and the diameter of each cell mass is 3-5mm, with a thickness of 3-5 μm.It is aforementioned four Four plants of cells of cell mass are that 293 cells, CFPAC-1 cell, OVCAR-3 cell and mesothelin strong positive are expressed respectively OVCAR-3-MSLN cell.On the other hand, four cell masses have different MSLN expression quantity, express magnitude relation are as follows: 293 Cell (not expressing MSLN) < CFPAC-1 cell < OVCAR-3 cell < OVCAR-3-MSLN cell；

On the other hand, this application involves relevant cell Quality Control pieces to prepare the purposes in mesothelin detection reagent.

On the other hand, this application involves the OVCAR-3-MSLN cells of mesothelin strong positive expression to prepare mesothelin immune Application in groupization detection kit.

On the other hand, this application involves the immunohistochemical kits for detecting MSLN, and it includes the thin of the application preparation Cytoplasm control wafer.

On the other hand, this application involves a kind of object of reference for the control of immunohistochemistry technology's quality, feature exists In: the object of reference includes two kinds of reference materials: the negative control object that the positive control of quality control, quality control；Wherein, positive Property reference material are as follows: CFPAC-1 cell, OVCAR-3 cell and mesothelin strong positive expression OVCAR-3-MSLN cell；Yin Property reference material are as follows: 293 cells.

On the other hand, this application involves the cells that a kind of method for immunohistochemical detection, the method use the application to prepare Quality Control piece is as control.

On the other hand, the present invention provides the method and a kind of preparation method of cytoplasm control wafer of a kind of cell paraffin embedding.

Specifically, the present invention provides a kind of method of cell paraffin embedding, step includes:

(1) cell culture

293 cells, CFPAC-1 cell, OVCAR-3 cell and OVCAR-3-MSLN cell are carried out in the medium Culture；

(2) cell is collected and is fixed

When above-mentioned cell culture is to the 80-90% for being paved with Tissue Culture Dish bottom, cell is collected.Culture medium is outwelled, is used Phosphate buffered saline solution washing, adds pancreatin to digest 3-5min, and microscopically observation cell rounding adds 1ml cell culture medium to terminate Cell is blown and beaten uniform collection with pipettor and counted by digestion, and phosphate buffered saline solution is added and washs cell, and supernatant is abandoned in centrifugation, 4% paraformaldehyde of cell or 10% neutral formalin fixer are resuspended, and room temperature is fixed；

(3) agarose supports cell

Ago-Gel is prepared: being weighed the solution that agarose adds phosphate buffered saline solution to be configured to 3-4% concentration, is covered tin Foil paper is heated to agarose in micro-wave oven and is completely dissolved, and taking-up is placed in heat preservation for standby use in 60 DEG C of water-baths；

Cell is mixed with agarose: passing through fixation procedure, natural subsidence to volume to 100 μ l is carefully removed with pipettor Supernatant；The centrifuge tube for filling cell is preheated in 60 DEG C of water-baths, the agar isometric with cell is pipetted with the pipettor of preheating Sugared gel is added in centrifuge tube and mixes with cell, and agarose, cell mixture after mixing move on to the 1ml injection of removal syringe needle In device needle tubing, room temperature is cooled and shaped；

(4) cell mass is dehydrated

The cell mass mixed with agarose is taken out, the segment of 0.5cm thickness is cut into, is placed on the dewatering basket marked It is interior；Cell mass is successively immersed in the ethyl alcohol of various concentration gradient and carries out tissue dewatering:

(5) cell mass is transparent

Carried out the cell mass of graded ethanol dehydration successively block be immersed in dimethylbenzene I and dimethylbenzene II carry out it is transparent, often Secondary progress clearing time is 20min (dimethylbenzene I is consistent with dimethylbenzene II ingredient, and being intended merely to distinguish is to operate twice)；

(6) cell mass waxdip

Paraffin mass is placed in waxdip cup in 65 DEG C of baking ovens to be melted to paraffin completely for a period of time, after carrying out transparency of organization Cell mass be immersed in the paraffin of thawing and carry out tissue waxdip；

(7) cell mass embeds

Manual embedding process carries out in 60 DEG C of baking ovens, and the paraffin of thawing is poured into organization embedding bed die, by waxdip knot The cell mass of beam takes out from corresponding dewatering basket, is put into rapidly in the paraffin of organization embedding bed die, tries not when being put into There is bubble generation, adjust the position of cell, the embedding frame marked up and down will be carried out and be covered on embedded box, cooling bench is transferred to Upper cooling 30min can obtain organization embedding paraffin mass.Before using paraffin mass, by marginal lappet outside 293 cell positions Vertical cut falls a fritter paraffin, is used as slice azimuth mark.

In some embodiments, culture medium used in the culture includes:

The culture medium of 293 cells includes: Gibco RPMI1640 culture medium+10%FBS；

The culture medium of CFPAC-1 cell includes: Gibco IMDM culture medium+10%FBS；

The culture medium of OVCAR-3 cell includes: Gibco RPMI1640-A10491-01 culture medium+20%FBS+ 0.01mg/ml insulin；

The culture medium of OVCAR-3-MSLN cell includes: Gibco RPMI1640-A10491-01 culture medium+20%FBS+ 0.01mg/ml insulin.

In some embodiments, the condition of the cell culture includes: 37 DEG C, 5%CO2 stationary culture 2-3 days, Zhi Daoxi Born of the same parents' proliferation is to accounting for culture dish bottom 80-90%.

In some embodiments, the dehydration of the step (4) are as follows: 70% ethyl alcohol stays overnight -80% ethyl alcohol 1.5h - 90% ethyl alcohol of ethyl alcohol 1.5h -95% ethyl alcohol of 1.5h -100% ethyl alcohol of 50min -100% 50min.

In some embodiments, the mixing step whole process of the step (3) is completed within 3min.

Pancreatin additional amount in some embodiments, in the step (2) are as follows: the pancreatin of 1-2ml 0.25%.

In some embodiments, the additive amount of fixer is 2 × 10 in the step (2)⁷cells/ml。

In some embodiments, the preparation method of cytoplasm control wafer includes: to make different MSLN target proteins expression intensities The neat technical effect for being arranged on cell section, realizing of the sequence that cell is arranged according to 2 rows 2 are as follows: produced in different batches Cytoplasm control wafer out can reach height unanimously, thus the consistency between guaranteeing product batches.

In some embodiments, wherein the FBS is fetal calf serum, Gibco RPMI1640 (ATCC improvement) culture Base is 1640 culture medium of RPMI (the as Gibco RPMI1640-A10491- that U.S.'s Gibco company article No. is A1049101 01)；Gibco IMDM culture medium is the IMDM culture medium that U.S.'s Gibco company article No. is 12440053；Gibco RPMI1640 Ordinary culture medium is the culture medium that Gibco company article No. is 11875135；

Wherein, steps are as follows for the acquisition of OVCAR-3-MSLN cell:

1. the amplification of target gene: design primer obtains amplifying target genes MSLN overall length from target gene carrier；

2. target gene and expression plasmid digestion: the target gene MSLN after amplification carries out cutting glue using plastic recovery kit Recycling, and digestion is carried out with restriction enzyme, while using endonuclease digestion expression vector of the same race；

3. the building of destination gene expression plasmid: target gene fragment and expression vector are attached；By connection product It is transformed into competent cell, spread plate after amplification；Picking single bacterium colony carries out bacterium colony PCR；It is correct to choose amplified production Bacterium colony is sequenced；It is the expression plasmid built that correct recombinant plasmid, which is sequenced,；

4. the packaging of target gene slow virus: inoculation 293T cell replaces serum-free after being incubated overnight in T75 culture bottle 293T is added after mixing again with Lipofectamine2000 after mixing expression plasmid, packaging plasmid and envelope plasmid in culture medium In cell (human renal epithelial cell line of transfection Adenovirus E1A gene), culture 48 was as a child collected supernatant and was dispensed after being concentrated；

5. aim cell infects: logarithmic growth phase aim cell is added in 24 orifice plates, is incubated overnight；Suck cell original Some culture mediums, the polybrene solution that viral dilution is added and has diluted, infection total volume are 250 μ L, are mixed gently； After infection 24 hours, the culture solution containing virus is removed, fresh complete medium is changed and continues to cultivate；For carrying puromycin The virus of resistant gene changes the fresh complete medium of purine-containing mycin, screens the cell strain of stable transfection；

6. cell strain monoclonal: the overexpression MSLN target gene stable cell strain of above-mentioned acquisition is used limiting dilution Method obtains the cell strain individually cloned, and according to MSLN expression intensity in pathological tissue, selects MSLN expression intensity and pathological tissue The middle consistent cell strain of MSLN high expression intensity is are as follows: MSLN strong positive expresses OVCAR-3-MSLN cell.

In addition, phosphate fliud flushing of the PBST (phosphate buffered solution) i.e. containing Tween-20, Neng Gouti For metastable ionic environment and pH buffer capacity, it is the buffer salt solution being commonly used in biology, is mainly used for being immunized Groupization and in situ hybridization, in the experiment such as enzyme linked immunological, cleaning immunoblotting film etc..

Detailed description of the invention

Fig. 1: fluidic cell screening experiment result.

Fig. 2: cell smear immunocyte dyes the selection result.

A figure: 293 cell MSLN specific antibody, 1 coloration result.

B figure: 1 coloration result of CFPAC-1 cell MSLN specific antibody.

C figure: 1 coloration result of OVCAR-3 cell MSLN specific antibody.

D figure: 1 coloration result of OVCAR-3-MSLN cell MSLN specific antibody.

E figure: 293 cell MSLN specific antibody, 2 coloration result.

F figure: 2 coloration result of CFPAC-1 cell MSLN specific antibody.

G figure: 2 coloration result of OVCAR-3 cell MSLN specific antibody.

H figure: 2 coloration result of OVCAR-3-MSLN cell MSLN specific antibody.

I figure: 293 cell blank results of comparison.

J figure: CFPAC-1 cell blank results of comparison.

K figure: OVCAR-3 cell blank results of comparison.

L figure: OVCAR-3-MSLN cell blank results of comparison.

The experimental result of Fig. 3: WB method screening cell.

Wherein, cell pyrolysis liquid loading sequence is: 1.293；2.CFPAC-1；3.OVCAR-3；4.OVCAR-3-MSLN.

Antibody incubation sequence: 1.GAPDH；2.MSLN specific antibody 1；3.MSLN specific antibody 2.

Fig. 4: cell embedding and slicing processes schematic diagram.

Fig. 5: the comparative experiments of cytoplasm control wafer coloration result and pathological tissue coloration result.

A figure: 293 cell dyeing results.

B figure: CFPAC-1 cell dyeing result.

C figure: pathological anatomy coloration result corresponding with 293 staining intensity of cells.

D figure: pathological anatomy coloration result corresponding with CFPAC-1 staining intensity of cells.

E figure: OVCAR-3 cell dyeing result.

F figure: OVCAR-3-MSLN cell dyeing result.

G figure: pathological anatomy coloration result corresponding with OVCAR-3 staining intensity of cells.

H figure: pathological anatomy coloration result corresponding with OVCAR-3-MSLN staining intensity of cells.

Fig. 6: Quality Control chip architecture schematic diagram.

Specific embodiment