阅读说明：本技术 一种功能化磁珠、制备方法及特异性捕获结直肠癌循环肿瘤细胞的方法 (Functionalized magnetic bead, preparation method and method for specifically capturing colorectal cancer circulating tumor cells ) 是由 王振新 杨致亭 田荣荣 马立娜 杨锋斌 范文翠 于 2019-09-23 设计创作，主要内容包括：本发明提供一种功能化磁珠、制备方法及特异性捕获结直肠癌循环肿瘤细胞的方法,属于化学生物学技术领域。该功能化磁珠是利用酰胺化反应,在羧基化Fe<Sub>3</Sub>O<Sub>4</Sub>磁珠MBs表面修饰可特异性结合α-1,2-岩藻糖的凝集素UEA-I而得到的。本发明还提供一种功能化磁珠的制备方法。本发明还提供功能化磁珠用于特异性捕获结直肠癌循环肿瘤细胞的方法。本发明提供的功能化磁珠在外加磁场的作用下,实现了特定循环肿瘤细胞的分离,其在完全培养基及血液中捕获循环肿瘤细胞的效率分别达到94％和89％,被捕获的细胞可正常的贴壁、增殖及传代,其生长速率与正常培养细胞的生长速率之间没有明显的区别,便于后期对被捕获的细胞进行深入研究。(The invention provides a functionalized magnetic bead, a preparation method and a method for specifically capturing colorectal cancer circulating tumor cells, belonging to the technical field of chemical biology. The functionalized magnetic beads are prepared by carboxylating Fe through amidation reaction 3 O 4 The magnetic bead MBs are surface-modified by agglutinin UEA-I which can specifically bind alpha-1, 2-fucose. The invention also provides a preparation method of the functionalized magnetic bead. The invention also provides a method for specifically capturing the colorectal cancer circulating tumor cells by using the functionalized magnetic beads. The invention provides a functionalized magnetThe separation of specific circulating tumor cells is realized by the beads under the action of an external magnetic field, the efficiency of capturing the circulating tumor cells in a complete culture medium and blood respectively reaches 94 percent and 89 percent, the captured cells can be attached to the wall, proliferated and passaged normally, the growth rate of the captured cells is not obviously different from that of the normally cultured cells, and the captured cells can be deeply researched in the later period.)

1. A functionalized magnetic bead, abbreviated as MB-UEA-I, is characterized in that the functionalized magnetic bead is prepared by carboxylating Fe through amidation reaction₃O₄The magnetic bead MBs are surface-modified by agglutinin UEA-I which can specifically bind alpha-1, 2-fucose.

2. The method of claim 1, comprising:

1) cleaning MBs, and then adding EDC and NHS for activation to obtain activated MBs;

2) carrying out magnetic separation on the activated MBs to remove the supernatant, washing the MBs with a HEPES buffer solution, adding a HEPES buffer solution containing UEA-I, and reacting at room temperature;

3) and after the reaction is finished, removing the supernatant through magnetic separation, washing the supernatant by using a HEPES buffer solution, removing unreacted UEA-I, suspending the modified MB-UEA-I in the HEPES buffer solution, and storing the modified MB-UEA-I for later use.

3. The method of claim 2, wherein the ratio of the mass of MBs to the volume of EDC to the volume of NHS is 0.5-1: 500-800.

4. The method of claim 2 or 3, wherein the EDC and the NHS solutions are both at a concentration of 100 mmol/L.

5. The method as claimed in claim 2, wherein the activation temperature in step 1) is room temperature, and the activation time is 30-120 min.

6. The method as claimed in claim 2, wherein the reaction time in step 2) is 1-3 h.

7. The method for preparing functionalized magnetic beads according to claim 2, wherein the mass ratio of UEA-I in the HEPES buffer solution of MBs and UEA-I is 0.5-1: 0.025.

8. The method of claim 1, wherein the functionalized magnetic beads are used for specifically capturing circulating tumor cells of colorectal cancer, for non-disease diagnosis and non-therapeutic purposes, and the method comprises the following steps:

step one, adding SW480 cells into blood of a healthy person to prepare a blood sample for simulating a colorectal cancer patient, adding functional magnetic beads MB-UEA-I into the blood sample to be detected, uniformly mixing, and then putting on a shaking table for incubation;

removing the supernatant through magnetic sorting to obtain MB-UEA-I captured cells;

and step three, staining the captured cells by using an ICC staining method, and accurately identifying the captured colorectal cancer circulating tumor cells through confocal fluorescence imaging.

9. The method of claim 8, wherein the amount of the functionalized magnetic beads used in the first step is 0.05-0.1 mg of functionalized magnetic beads MB-UEA-I per 1mL of the blood sample.

10. The method for capturing circulating tumor cells of colorectal cancer according to claim 8, wherein the incubation temperature in the first step is 25-37 ℃ and the incubation time is 30-60 min.

Technical Field

The invention belongs to the technical field of chemical biology, and particularly relates to a functionalized magnetic bead, a preparation method and a method for specifically capturing colorectal cancer circulating tumor cells.

Background

Circulating Tumor Cells (CTCs) refer to tumor cells that are shed from primary or metastatic foci into the blood circulation during tumor formation or progression, with the potential to develop into metastatic lesions. Therefore, the capture and detection of CTCs are of great medical significance in early diagnosis of major diseases, evaluation of therapeutic effects, and the like. But due to high heterogeneity and rarity (1 ml peripheral blood 10)⁶-10⁹Several to several hundred CTCs in a single blood cell), efficient, high purity enrichment of CTCs remains a significant challenge.

Currently, many techniques and devices based on the physical properties (size, density, deformability, and adhesion preference) or biological properties (antibody antigens, aptamers, peptides, and E-selectin) of CTCs have been developed for efficient enrichment and detection of CTCs. Among them, the immunomagnetic bead separation and enrichment method based on antibodies (such as epithelial cell adhesion molecule (EPCAM), human epidermal growth factor receptor 2(HER2), Epidermal Growth Factor Receptor (EGFR), carcinoembryonic antigen (CEA), etc.) has become the most common method for separating CTCs from complex peripheral blood due to its advantages of easy magnetic manipulation, high-efficiency specific binding with target cells, rapid magnetic response, convenient subsequent identification and analysis (such as Immunocytochemistry (ICC) and raman imaging). However, it is still limited by non-large-scale production, high price, etc., and some tumor (such as melanoma and sarcoma) antibodies have low or almost no expression. Therefore, CTCs enrichment methods based on other targeting molecules (such as DNA, aptamers, recognition peptides or lectins, etc.) are also being gradually applied to enrichment and isolation of CTCs.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a functionalized magnetic bead, a preparation method and a method for specifically capturing colorectal cancer circulating tumor cells, wherein the functionalized magnetic bead can sensitively and specifically identify and separate the circulating tumor cells and has low cost.

The invention firstly provides a functional magnetic bead, which is abbreviated as MB-UEA-I, and the functional magnetic bead is prepared by carboxylating Fe by amidation reaction₃O₄The magnetic bead MBs are surface-modified by agglutinin UEA-I which can specifically bind alpha-1, 2-fucose.

The invention also provides a preparation method of the functionalized magnetic bead, which comprises the following steps:

1) cleaning MBs, and then adding EDC and NHS for activation to obtain activated MBs;

2) carrying out magnetic separation on the activated MBs to remove the supernatant, washing the MBs with a HEPES buffer solution, adding a HEPES buffer solution containing UEA-I, and reacting at room temperature;

3) and after the reaction is finished, removing the supernatant through magnetic separation, washing the supernatant by using a HEPES buffer solution, removing unreacted UEA-I, suspending the modified MB-UEA-I in the HEPES buffer solution, and storing the modified MB-UEA-I for later use.

Preferably, the mass mg of MBs, the volume muL of EDC and the volume muL of NHS are 0.5-1: 500-800.

Preferably, the concentration of the EDC and NHS solutions is 100 mmol/L.

Preferably, the activation temperature in the step 1) is room temperature, and the activation time is 30-120 min.

Preferably, the reaction time of the step 2) is 1-3 h.

Preferably, the mass ratio of UEA-I in the MBs HEPES buffer solution to UEA-I is 0.5-1: 0.025.

The invention provides a method for specifically capturing colorectal cancer circulating tumor cells by using the functionalized magnetic beads, which is used for non-disease diagnosis and non-treatment purposes and comprises the following steps:

step one, adding SW480 cells into blood of a healthy person to prepare a blood sample for simulating a colorectal cancer patient, adding functional magnetic beads MB-UEA-I into the blood sample to be detected, uniformly mixing, and then putting on a shaking table for incubation;

removing the supernatant through magnetic sorting to obtain MB-UEA-I captured cells;

and step three, staining the captured cells by using an ICC staining method, and accurately identifying the captured tumor cells through confocal fluorescence imaging.

Preferably, the amount of the magnetic beads in the first step is 0.05-0.1 mg of the functionalized magnetic beads MB-UEA-I added into each 1mL of blood sample to be detected.

Preferably, the incubation temperature in the first step is 25-37 ℃, and the incubation time is 30-60 min.

Principle of the invention

The functionalized magnetic beads of the invention are obtained by modifying lectin UEA-I on the surface of carboxylated MBs by amidation reaction. Through specific binding of lectin UEA-I and alpha-1, 2-fucose on the surface of a colorectal cancer SW480 cell, the functional magnetic bead MB-UEA-I can efficiently and specifically capture the tumor cell, and the captured cell is separated through magnetic sorting. Meanwhile, the captured tumor cells can be accurately identified by using an ICC staining method and confocal fluorescence imaging.

The invention has the advantages of

(1) The lectin-modified functionalized magnetic beads for capturing the circulating tumor cells have the advantages of simple preparation method and short required time, and the targeted molecular lectin is relatively cheap and easily obtained compared with other antibodies, so that the problem of high cost of the existing capturing method can be solved.

(2) The cell recognition has specificity, UEA-I is a lectin which can be specifically combined with alpha-1, 2-fucose on the surface of a colorectal cancer SW480 cell and is obtained by utilizing a lectin microarray chip through high-throughput analysis and screening, and can efficiently and specifically recognize tumor cells, namely the method for capturing CTCs has better specificity.

(3) The capture efficiency is high, and the capture efficiency of the functional magnetic beads in a complete culture medium and blood reaches 94% and 89% respectively. .

(4) The influence on the captured cells is small, the functionalized magnetic beads hardly have influence on the activity of the captured cells, and along with the sugar metabolism on the cell surface, the functionalized magnetic beads can be gradually separated from the cell surface and are not required to be released through special treatment, the captured cells can be normally attached to the wall, proliferated and passaged, the growth rate of the captured cells is not obviously different from that of the cells cultured normally, and the captured cells are conveniently deeply researched in the later stage.

Drawings

FIG. 1 is a SEM representation before and after MBs modify UEA-I in example 1 of the present invention.

FIG. 2 is a bar graph showing the results of specificity of capturing tumor cells by MB-UEA-I in example 2 of the present invention.

FIG. 3 is a graph showing the capturing efficiency of MB-UEA-I in complete medium and blood in examples 3 and 4 of the present invention;

FIG. 4 is a fluorescent image of captured cells in example 5 of the present invention;

FIG. 5 is a graph showing the comparison of the proliferation rate of normally cultured cells and trapped cells in example 5 of the present invention;

FIG. 6 is a fluorescent image of captured cells after adherence, proliferation and passaging in example 5 of the present invention.

FIG. 7 is a graph showing the result of capturing CTCs (SW480 cells) in a blood sample of a patient with simulated colorectal cancer in example 6 of the present invention.

Detailed Description

The invention firstly provides a functional magnetic bead, which is abbreviated as MB-UEA-I, and the functional magnetic bead is prepared by carboxylating Fe by amidation reaction₃O₄Magnetic Beads (MBs) are surface-modified with a lectin UEA-I that specifically binds to alpha-1, 2-fucose.

According to the invention, the lectin UEA-I is a lectin which can be specifically combined with alpha-1, 2-fucose on the surface of a colorectal cancer SW480 cell and is obtained by utilizing a lectin microarray chip through high-throughput analysis and screening, can efficiently and specifically identify tumor cells, and has better specificity.

The invention also provides a preparation method of the functionalized magnetic bead, which comprises the following steps:

1) cleaning MBs, and then adding EDC and NHS for activation to obtain activated MBs;

2) carrying out magnetic separation on the activated MBs to remove the supernatant, washing the MBs with a HEPES buffer solution, adding a HEPES buffer solution containing UEA-I, and reacting at room temperature;

3) and after the reaction is finished, removing the supernatant through magnetic separation, washing the supernatant by using a HEPES buffer solution, removing unreacted UEA-I, suspending the modified MB-UEA-I in the HEPES buffer solution, and storing the modified MB-UEA-I for later use.

According to the invention, MBs are added into a centrifuge tube, washed for 3-5 times by MES buffer solution, and then EDC and NHS are added for activation; the activation temperature is preferably room temperature, the activation time is preferably 30-120 min, the concentration of the MES buffer solution is preferably 100mmol/L, the pH value is 5.5-6.7, and the mass mg of the MBs, the volume mu L of EDC and the volume mu L of NHS are 0.5-1: 500-800; the concentration of EDC and NHS solutions is preferably both 100 mmol/L;

according to the invention, activated MBs are subjected to magnetic sorting to remove supernatant, and are washed for 3-5 times by using HEPES buffer solution, the concentration of the HEPES buffer solution is preferably 10mmol/L, the pH value is 7.2-8.5, then HEPES buffer solution containing UEA-I is added, and the reaction is carried out at room temperature, and the reaction time is preferably 1-3 h; the mass ratio of the MBs magnetic beads to the UEA-I in the HEPES buffer solution of the UEA-I is preferably 0.5-1: 0.025.

According to the invention, after the reaction is finished, the supernatant is removed by magnetic separation, and is washed for 3-5 times by using HEPES buffer solution, unreacted agglutinin is removed, and the modified MB-UEA-I is suspended in the HEPES buffer solution and is stored for later use at 2-8 ℃.

The invention provides a method for specifically capturing colorectal cancer circulating tumor cells by using the functionalized magnetic beads, which comprises the following steps:

step one, adding SW480 cells into blood of a healthy person to prepare a blood sample for simulating a colorectal cancer patient, adding functional magnetic beads MB-UEA-I into the blood sample to be detected, uniformly mixing, and then putting on a shaking table for incubation; the dosage of the magnetic beads is that 0.05-0.1 mg of functional magnetic beads MB-UEA-I are preferably added into each 1mL of blood sample to be detected; the incubation temperature is preferably 25-37 ℃, and the incubation time is preferably 30-60 min.

Removing the supernatant through magnetic sorting to obtain MB-UEA-I captured cells;

and step three, staining the captured cells by using an ICC staining method, and accurately identifying the captured tumor cells through confocal fluorescence imaging.

In the third step of the invention, the captured cells are stained by an ICC staining method, which specifically comprises the following steps:

1) fixing the cells obtained by magnetic separation with 4% paraformaldehyde for 10-30 min;

2) after removing the supernatant by magnetic separation, adding 0.1% Triton X-100 to permeate the membrane for 10-30 min;

3) after removing the supernatant by magnetic separation, adding 1% BSA and sealing at room temperature for 30-60 min; the BSA was dissolved in 0.05% PBS-Tween.

4) Magnetic separation is carried out to remove supernatant, and 0.05 percent PBS-Tween is used for washing 3-5 times respectively;

5) adding mixed solution of AF568-CK19 and AF488-CD45, keeping away from light and incubating in a refrigerator at 4 ℃; the concentration of the AF568-CK19 and AF488-CD45 is preferably 10 mug/mL, the adding volume is 100-200 mug L, and the incubation time is 2-8 h;

6) magnetic separation is carried out to remove supernatant, and PBS is used for washing for 3-5 times;

7) adding Hoechst 33342, and dyeing in a refrigerator at 4 ℃ in the dark; the concentration of the Hoechst 33342 is 4 mug/mL, the added volume is 100-200 mug L, and the dyeing time is 10-30 min.

8) And magnetically sorting to remove supernatant, and washing with PBS for 3-5 times.

The invention is described in further detail below by means of specific embodiments in conjunction with the attached drawings: