CN109266714A

CN109266714A - A kind of detection method of circulating tumor cell

Info

Publication number: CN109266714A
Application number: CN201811191492.1A
Authority: CN
Inventors: 杜晶春
Original assignee: Guangzhou Medical University
Current assignee: Guangzhou Medical University
Priority date: 2018-10-12
Filing date: 2018-10-12
Publication date: 2019-01-25

Abstract

The present invention utilizes Apoptin albumen in the localization characteristics of normal cell and tumour cell, in conjunction with the visuality of fluorescent material, Apoptin gene and fluorescence protein gene are fused together, give expression to the Apoptin albumen with fluorescence, and the localization characteristics according to Apoptin albumen in tumour cell and normal cell core, it is applied to the detection of circulating tumor cell, and successfully is detected circulating tumor cell.

Description

A kind of detection method of circulating tumor cell

Technical field

The invention belongs to oncology and field of medical examination, are related to a kind of detection method of circulating tumor cell, specifically It is related to a kind of detecting circulating tumor cell from human peripheral in the position disparity of normal cell and tumour cell by apoptosis element Method.

Background technique

Circulating tumor cell (Circulating tumor cells, CTCs) refers to be discharged into outside by primary tumors Tumour cell in all circulatory systems has key effect in cancer metastasis.The trouble of CTCs is not detected relative to peripheral blood For person, detect that patient's prognosis of CTCs is poor；For the raised patient of CTCs number after treatment, CTCs number after treatment Reduced patient's prognosis is obviously improved.Therefore, antidiastole, life cycle prediction, therapeutic effect of the CTCs detection in tumor patient Monitoring etc. has certain application value.

Currently, method there are two main classes the technology of detection CTCs, one kind is the physical property based on CTCs, including cell is big Small, density etc. distinguishes tumour cell and normal cell, second is that based on specific surface marker object by tumour cell and peripheral blood Other cells separation in circulation.Separated by the physical characteristic of tumour cell and normal cell, lack specificity, exist compared with High false positive；The circulating tumor cell in peripheral blood is detected by specific surface marker's object, is only applicable to that there is uniqueness The CTCs of surface marker can not detect the CTCs of shortage unique surface marker, and therefore, one species specificity of exploitation is high, The detection technique of wide adaptation range is very necessary.

Apoptosis element (Apoptin) be it is a kind of from chicken anaemia virus (chicken anemia virus, CAV) can Promote the small molecular protein [i] of apoptosis of tumor cells.Apoptin is by 121 Amino acid profiles, molecular weight 13.6Kd, C-terminal Go out core sequence (NES) with two nuclear localization sequences (NLS) and one.These identification sequences driving Apoptin shuttle enter and Leave nucleus.Experimental data shows that the positioning of Apoptin in the cell is adjusted by two NLS and NES, and NLS is normal It is all active in cell and tumour cell, but NES is only active in normal cell, and activity is not present in tumour cell, To which Apoptin can be located in the cytoplasm of normal cell by NES, cause Apoptin fixed in tumour cell and normal cell Position is different, and Apoptin is primarily located within nucleus in tumour cell, and is primarily located within cytoplasm in normal cell.This is special Property for detection circulating tumor cell provide theoretical foundation.

Summary of the invention

In view of the deficiencies of the prior art, the present invention proposes that a kind of new circulating tumor cell detection method, this method utilize Apoptin is in the localization characteristics of circulating tumor cell and peripheral blood other cells and the visuality of fluorescent material, by Apoptin DNA sequence dna is directly expressed in the cell after merging with fluorescin DNA sequence dna, is reflected by the expression area differentiation of Apoptin Other circulating tumor cell and peripheral blood remaining monocyte, this method can effectively detect the circulating tumor cell in blood, With specific high, the advantages such as applied widely.

Technical solution of the present invention mainly comprises the steps that building containing the true of Apoptin and fluorescence protein gene

Nuclear expression plasmid；Separate the peripheral blood mononuclear cells of cancer patient；Apoptin and fluorescence protein gene will be contained Eukaryon expression plasmid import cancer patient peripheral blood mononuclear cells, cultivate 24-72 hours；Nucleus is dyed, Observation identifies circulating tumor cell.

Preferred embodiment of the invention first is that by Overlap extension PCR method, the threonine of No. 108 positions of Apoptin is dashed forward Become glycine, constructs pEGFP-N1-Apoptin-1T plasmid, the hydroxyl group of threonine structure end can be with phosphate group In conjunction with to be phosphorylated, and glycine cannot be phosphorylated, therefore after substituting threonine with glycine, above-mentioned amino acid position Point will lose phosphorylation abilities, to reduce the activity of Apoptin albumen, extend the time of apoptosis of tumor cells, obtain The sufficiently long time carrys out resolution cycle tumour cell.

One of preferred embodiment of the invention is by Overlap extension PCR method, by 3 Soviet Union's ammonia of the position 106-108 of Apoptin Acid mutation constructs pEGFP-N1-Apoptin-3T plasmid, the hydroxyl of threonine structure end at glycine, proline, glycine Group can be in conjunction with phosphate group, to be phosphorylated, and glycine, proline cannot be phosphorylated, therefore with sweet ammonia After acid, proline substitution threonine, above-mentioned amino acid sites will lose phosphorylation abilities, to reduce Apoptin albumen Activity extends the time of apoptosis of tumor cells, obtains the sufficiently long time and carrys out resolution cycle tumour cell.

The fluorescin that the present invention uses can be green fluorescent protein, red fluorescent protein or other type fluorescence eggs It is white, it is therefore an objective to carry out fluorescence detection or be located separately.

The introduction method that the present invention uses is that electricity turns or by the methods of lipo2000 transfection reagent.

The present invention using Apoptin albumen normal cell and tumour cell localization characteristics, in conjunction with fluorescent material can Depending on property, Apoptin gene and fluorescence protein gene are fused together, give expression to the Apoptin albumen with fluorescence, and according to Localization characteristics of the Apoptin albumen in tumour cell and normal cell core, are applied to the detection of circulating tumor cell, and successfully Detect circulating tumor cell.This method blood using amount is 3ml, is known detection method (such as cell Search system needs 7.5ml) the half of blood using amount improves efficiency, meanwhile, this method specificity is good, overcomes and detects presence based on physical characteristic The not high problem of specificity, also overcome based on specific surface marker's object and detect the circulating tumor cell side in peripheral blood Limitation (for lack the CTCs of unique surface marker, can not detect) of the method to certain tumours.This method testing principle is base In the expression of Apoptin albumen in the cell, as long as, all there is point of cytoplasm and nucleus, not by tumour in tumour cell The limitation of cell type, theoretically, this method can detecte any kind of tumour cell, have the advantages such as applied widely. Therefore, the present invention is good, the applied widely and high-efficient circulating tumor cell detection method of a species specificity, is had good Application prospect.

Detailed description of the invention

Fig. 1 is that Apoptin and Apoptin-EGFP promotees Saos-2 apoptosis result schematic diagram；

Fig. 2 is that Apoptin and Apoptin-EGFP promotees H460 apoptosis result schematic diagram；

Fig. 3-1 is positioning schematic diagram of the Apoptin-EGFP in H460 after transfection 48h；

Fig. 3-2 is positioning schematic diagram of the Apoptin-EGFP in Saos-2 after transfection 48h；

Fig. 3-3 is positioning schematic diagram of the Apoptin-EGFP in MSC after transfection 48h；

Fig. 4 is positioning schematic diagram of the Apoptin-EGFP-3T in the PBMC of separate sources；

"+": Apoptin-EGFP-3T is located in nucleus, is judged as CTCs

"-": Apoptin-EGFP-3T is located in cytoplasm, is judged as normal cell

Embodiment

Embodiment one:

Construct plasmid pEGFP-N1-Apoptin and pcDNA3.1 (+)-Apoptin

PCR amplification Apoptin genetic fragment

Digestion Apoptin genetic fragment and plasmid pEGFP-N1, pcDNA3.1 (+)

Apoptin genetic fragment and plasmid pEGFP-N1, pcDNA3.1 (+) are connected,

Connection product transformed competence colibacillus DH5 α, with ammonia benzyl plate, 37 DEG C of insulating box overnight incubations.

Select single plasmid, extract recombinant plasmid, digestion, sequencing, screen satisfactory pEGFP-N1-Apoptin and pcDNA3.1(+)-Apoptin。

Embodiment two:

Construct mutant pEGFP-N1-Apoptin-1T and pEGFP-N1-Apoptin-3T

PCR amplification primer is designed by Overlap extension PCR method, wherein pEGFP-N1-Apoptin-1T is by the Soviet Union of No. 108 positions Histidine mutations are glycine, and three threonines of the position 106-108 are sported glycine, dried meat by pEGFP-N1-Apoptin-3T Propylhomoserin, glycine.

PCR amplification, digestion, connection construct mutant plasmid pEGFP-N1-Apoptin-1T and pEGFP-N1- Apoptin-3T。

Transformed competence colibacillus DH5 α, with plate containing antibiotic, 37 DEG C of insulating box overnight incubations.

Single bacterium colony is selected, recombinant plasmid, digestion are extracted, satisfactory pEGFP-N1-Apoptin-1T is screened in sequencing And pEGFP-N1-Apoptin-3T.

Embodiment three

Apoptin and Apoptin-EGFP influences (Fig. 1) to human osteosarcoma cell Saos-2

By plasmid pcDNA3.1 (+), pcDNA3.1 (+)-Apoptin, pEGFP-N1, pEGFP-N1-Apoptin, PEGFP-N1-Apoptin-IT, pEGFP-N1-Apoptin-3T pass through lipo2000 transfection reagent respectively, and to be transferred to Saos-2 thin Born of the same parents,

After transfection 48 hours, cell activity is detected by CCK8 reagent, measures OD value,

Testing result shows that Apoptin albumen has apparent apoptotic effect to Saos-2 cell；Apoptin lotus root joins EGFP It will not influence the rush apoptosis capacity of itself；The pEGFP-N1-Apoptin-3T of mutation has bright the apoptosis capacity of tumour cell Aobvious inhibiting effect.

Example IV

Apoptin and Apoptin-EGFP influences (Fig. 2) to human lung carcinoma cell H460

Embodiment five

Positioning (Fig. 3) of the Apoptin-EGFP in tumour cell and normal cell

By plasmid pEGFP-N1-Apoptin, pEGFP-N1-Apoptin-IT, pEGFP-N1-

Apoptin-3T turns mode by electricity respectively and imports people's bone meat cell Saos-2, human lung carcinoma cell H460 and the human world In mesenchymal stem cells MSC；

Transfection for 24 hours, 48h, 72 hours, carries out core dye with Hoechst33342, is observed after continuing culture 30min after core dye；

With cell imaging instrument observe green fluorescence and blue-fluorescence respectively, green fluorescence represents Apoptin in the cell Distribution, blue-fluorescence represent the nucleus dyed by Hoechst33342；

The optimal viewing time is 48 hours or so, in human mesenchymal stem cell MSC, Apoptin- as the result is shown for observation EGFP albumen is distributed in cytoplasm, main in people's bone meat cell Saos-2, human lung carcinoma cell H460, Apoptin-EGFP albumen It concentrates in nucleus.Therefore, differentiation normal cell and tumour cell that Apoptin-EGFP can be specific.

Embodiment six

Detection (Fig. 4) of the Apoptin-EGFP to human peripheral circulating tumor cell

3 patients with lung cancer and each 3ml of normal person's whole blood sample are taken, peripheral blood mononuclear cells is isolated.

Turn mode by electricity and transfects pEGFP-N1-Apoptin-3T, 1640 culture medium cultures.

After transfecting 48h, core dye is carried out with Hoechst33342, continues to observe after cultivating 30min after core dye.

Observation in the peripheral blood of patients with lung cancer, detects the good circulating tumor cell of specificity as the result is shown.

Claims

1. a kind of detection method of circulating tumor cell, it is characterised in that:

(1) eukaryon expression plasmid containing Apoptin gene and fluorescence protein gene is constructed；

(2) peripheral blood mononuclear cells of cancer patient is separated；

(3) the single core of peripheral blood for importing cancer patient containing the eukaryon expression plasmid of Apoptin and fluorescence protein gene is thin Born of the same parents cultivate 24-72 hours；

(4) nucleus is dyed, is observed, identify circulating tumor cell.

2. detection method as described in claim 1, it is characterised in that the fluorescin includes green fluorescent protein, red glimmering Photoprotein or other type fluorescins.

3. detection method as described in claim 1, it is characterised in that the Apoptin gene is the gene of mutation, No. 108 positions Threonine be mutated into glycine.

4. detection method as described in claim 1, it is characterised in that the Apoptin gene is the gene of mutation, 106-108 3 threonines of number position are mutated into glycine, proline, glycine.