CN105717104B - A kind of Hepatocellular Carcinoma GPC3 detection methods - Google Patents

Abstract

The invention discloses a kind of Hepatocellular Carcinoma GPC3 detection methods：The CTC in the Peripheral Blood of Patients with Hepatocellular Carcinoma that can not obtain tissue specimen is obtained using film filter separation, cuts into slices with cell block fabrication techniques thin layer, and then detects GPC3 expressions.By the technical method, without drawing materials, liver cancer tissue can detect GPC3 expressions in Peripheral Blood of Patients with Hepatocellular Carcinoma, and foundation is provided for diagnosis of hepatoma.The technology belongs to minimally invasive or even noninvasive, and can detect in real time.

Description

A method for detecting peripheral blood GPC3 in patients with hepatocellular carcinoma

technical field

The invention relates to a method for detecting GPC3 in peripheral blood, especially for patients with hepatocellular carcinoma who have not been pathologically diagnosed, by detecting GPC3 in peripheral blood, the diagnosis of hepatocellular carcinoma can be further confirmed.

Background technique

GPC3 (Glypican-3, glypican-3) is a membrane heparan sulfate proteoglycan (HSPG), with a relative molecular mass of about 66kDa, and its basic structure includes core protein, heparan sulfate (heparin sulfate, HS) chain and glycosylphosphatidylinositol (GPI), anchored on the cell surface through GPI, can bind to a variety of growth factors and regulate their activity, and then play a key role in the process of growth, development and carcinogenesis. GPC3 mainly acts through the Wnts signaling pathway, and the expression and distribution of GPC3 in normal tissues have obvious tissue specificity and phase specificity of tissue differentiation. Studies have shown that in the embryonic period of the human body, GPC3 mRNA is rich in placenta and fetal tissues, and GPC3 is highly expressed in the gastrointestinal tract, liver, kidney and lung, and the liver has the highest expression level. In adults, GPC3 is highly expressed in the placenta tissue, expressed at a low level in a few tissues such as the ovary, kidney, lung, and heart, and not expressed in normal liver tissue. Studies have confirmed that GPC3 protein is highly expressed in liver cancer tissues, while there is no expression or very low expression in non-cancer tissues, indicating that GPC3 has a specific relationship with liver cancer. GPC3 can inhibit the growth of tumors, which further illustrates the specificity and The specificity of the mechanism of action in liver cancer; at the same time, studies have shown that the expression of GPC3 in lung cancer, breast cancer, mesothelioma, kidney cancer and ovarian cancer tissues is missing. Studies have shown that the expression of GPC3 is significantly up-regulated in 80%-90% of liver cancer tissues, and it is expressed at a low level or not in normal adult tissues, hepatitis, liver cirrhosis patient tissues and other tumors, and it is also slightly expressed in AFP-negative HCC. High expression. Immunohistochemical detection was performed on liver tissues of liver cancer, hepatocellular carcinoma, cirrhotic regenerative tumors, and nodular hyperplasia. It was found that the positive rate of GPC3 in liver cancer tissues was 75.7%, while GPC3 was negative in benign tumors. GPC3 is positive in 90% of HCC with a specificity as high as 100%, but negative in metastatic liver cancer and benign liver disease. Therefore, GPC3 can be used not only to differentiate benign and malignant tumors, but also to differentiate HCC from metastatic liver cancer.

Currently, the diagnosis of hepatocellular carcinoma includes pathological diagnosis and clinical diagnosis. Pathological diagnosis is the gold standard for the diagnosis of liver cancer. Clinical diagnosis criteria mainly include chronic liver disease background, imaging examination results and serum tumor marker levels. However, for some liver cancer patients who cannot obtain pathological diagnosis or have insufficient basis for clinical diagnosis, it becomes very difficult to detect the expression of GPC3.

Contents of the invention

Circulating tumor cells (Circulating tumor cells, CTCs) are tumor cells that come off from solid tumors and enter the peripheral blood circulation. Detection has important clinical significance for evaluating the prognosis of cancer patients, especially advanced cancer patients, and selecting appropriate individualized treatment. Because CTC detection is minimally invasive and real-time, it is called "liquid biopsy".

In order to overcome the problem that liver cancer patients with insufficient clinical diagnosis basis are not suitable for taking tissue samples or avoid tumor dissemination caused by needle biopsy, and cannot clearly diagnose liver cancer, the present invention provides a method for detecting peripheral blood GPC3 in patients with hepatocellular carcinoma: using membrane The filter device was used to separate and obtain CTCs in the peripheral blood of liver cancer patients whose tissue samples could not be obtained, and the cell wax block technique was used to make thin-layer slices, and then the expression of GPC3 was detected.

The technical scheme adopted in the present invention is as follows:

1. Using a membrane filtration device to separate and obtain CTCs in the peripheral blood of liver cancer patients whose tissue samples cannot be obtained:

1. Collect peripheral blood from patients with advanced liver cancer: 5ml from median cubital vein;

2. Peripheral blood sample pretreatment: Dilute the collected peripheral blood sample 10 times, the dilution liquid composition: 1mmol/l EDTA+0.1%BSA, after dilution, add paraformaldehyde to fix the peripheral blood sample for 10 minutes, and fix the final concentration to 0.25%;

3. Use the membrane filtration tumor cell separation device to filter peripheral blood samples to separate and obtain peripheral blood circulating tumor cells: add the pretreated peripheral blood sample to the blood sample container of the membrane filtration tumor cell separation device, and let it naturally filter by gravity;

4. After the filtration, remove the filter from the membrane filtration device for separating tumor cells, add circulating tumor cell staining solution to the filter, stain for 2 minutes, rinse with PBS buffer, remove the filter membrane, place it on a glass slide, and dry Then observe under a microscope to determine the presence or absence of CTCs.

2. Using cell wax block technology to make thin-layer slices:

1. Decolorization: Remove the filter membrane with CTC from the glass slide, soak in the decolorization solution for 4-6 hours, and remove the CTC staining solution. The decolorization solution is: 95% alcohol and 100% xylene by volume Mixed at a ratio of 1:1.

2. Wrapping: After soaking, take out the filter membrane and wrap it with absorbent paper;

3. Fixing: Put the wrapping in 10% neutral formalin and fix it for 4-6 hours;

4. Embedding in wax: After the fixation, take out the wrapper, put it in a paraffin embedding box after dehydration, and embed in wax to make a paraffin block of cells;

5. Thin-layer section: Use a microtome to slice the cell paraffin block continuously to make a thin-layer section with a thickness of 4 μm.

3. Detection of GPC3 expression:

1. Baked slices: Thin-layer slices of CTC wax blocks were baked in a constant temperature oven at 60°C for 2 hours;

2. Dewaxing: Place the slices in two bottles of xylene for 5 minutes respectively;

3. Hydration: Place the slices in absolute ethanol, 95% ethanol, and 85% ethanol for 5 minutes respectively (adjust slightly according to the room temperature), rinse with tap water and distilled water twice, 3 minutes each time;

4. Antigen repair: Boil the repair solution EDTA in the pressure cooker, put the slices into the repair solution, cover the pressure cooker cover and pressure valve, heat until 1.5 minutes after the pressure cooker is sprayed, remove from the heat, cool the pressure cooker slightly, open the cover, and slice Natural cooling in repair fluid;

5. Rinse with distilled water;

6. Remove endogenous peroxidase: use 3% H ₂ O ₂ to act on slices for 10 minutes;

7. Rinse with distilled water;

8. Rinse with PBS 5 times for 1.5 minutes each;

9. Add GPC3 primary antibody dropwise;

10. Overnight in the refrigerator at 4°C;

11. PBS soaking 5 times for 1.5 minutes each;

12. Add general-purpose IgG antibody-HRP polymer dropwise;

13. Incubate in a water bath at 37°C for 20 minutes;

14. Rinse with PBS 5 times for 1.5 minutes each;

15. Control under the microscope, DAB color development: add 2 drops or 100 μl of freshly prepared DAB solution to each section, and observe under the microscope for 3-10 minutes;

16. Rinse twice with distilled water, 3 minutes each time; counterstain with hematoxylin for 1 minute;

17. Alcohol differentiation with 0.1% hydrochloric acid;

18. 0.1% ammonia solution turns blue;

19. 50%, 70%, 85%, 95% gradient anhydrous ethanol dehydration and drying;

20. Xylene transparent, neutral resin glue sealing film;

21. Cytopathology experts read the slides and judge the expression of GPC3 according to the degree of staining of the cell membrane and cytoplasm.

The device for separating tumor cells by membrane filtration described in the technical steps of the present invention includes a filter, a blood sample container, a waste liquid tank, and an iron stand. The iron stand is provided with a base, a stand, and a bracket. On the upper part of the stand, below the blood sample container is a filter, which is connected to the waste liquid tank through the infusion set, and the waste liquid tank is arranged on the base.

The filter includes an upper port of the filter, a filter membrane, a platform for carrying the filter membrane and a lower port of the filter, the filter membrane is placed on the platform for carrying the filter membrane; the upper port of the filter is connected to the blood sample container, and the lower port of the filter is connected to the waste liquid tank through the infusion set.

The filter membrane is made of hydrophobic material, and is evenly covered with filter holes with a diameter of 10 microns.

The beneficial effect of the present invention is that: through the technical method, the expression of GPC3 in the peripheral blood of patients with liver cancer can be detected without taking liver cancer tissue, providing a basis for the diagnosis of liver cell carcinoma. The technology is minimally invasive or even non-invasive, and can detect in real time.

Description of drawings

Fig. 1 is the structural representation of membrane filtration device of the present invention;

Fig. 2 is the schematic sectional view of the structure of the filter of the membrane filtration device of the present invention;

Fig. 3 is the structural representation of the filter membrane of membrane filtration device of the present invention;

Figure 4 is a mirror image of circulating tumor cells in peripheral blood of liver cancer;

Figure 5 is a mirror image of GPC3 expression in peripheral blood circulating tumor cells of liver cancer.

In the figure: 1 iron stand, 2 blood sample container, 3 filter, 4 infusion set, 5 waste liquid cylinder, 6 filter upper port, 7 filter membrane, 8 filter membrane platform, 9 filter lower port, 10 filter hole, 11 base, 12 stands, 13 brackets.

Detailed ways

The present invention will be described below in conjunction with the accompanying drawings and embodiments.

Example

This technique was used to isolate and identify circulating tumor cells in the peripheral blood of 8 patients with liver cancer (while detecting 8 normal samples as negative controls), and to detect the expression of GPC3.

1. Using a membrane filtration device to separate and obtain CTCs in the peripheral blood of liver cancer patients whose tissue samples cannot be obtained:

Collect 5ml of peripheral blood from each sample, dilute the peripheral blood with 45ml diluent (ingredient: 1mmol/l EDTA+0.1%BSA), and then add 3ml of 4% paraformaldehyde to fix the diluted blood sample for 10 minutes.

At a fixed interval, assemble the membrane filter device: as shown in Figure 1, Figure 2, and Figure 3, the filter device consists of a filter 3, a filter membrane 7, a blood sample container 2, a waste liquid tank 5, and an iron stand 1.

Wet the filter 3 with 10ml of PBS, then add the fixed peripheral blood sample into the blood sample container 2 of the membrane filtration device, let it filter naturally by gravity, and CTCs are trapped on the filter membrane 7 .

The diameter of tumor cells is generally greater than 15 microns, while the diameter of blood cells (including red blood cells and white blood cells) is generally less than 10 microns. Therefore, when the peripheral blood containing CTCs is filtered, blood cells can be filtered because the diameter is smaller than filter hole 10, while CTCs can be filtered because the diameter is larger than The filter pores 10 are trapped on the filter membrane 7 .

After the filtration, remove the filter 3 from the filter device, open and remove the upper port 6 of the filter, add circulating tumor cell Diff staining solution to the filter 3, and stain for 2 minutes.

Rinse the filter 3 with PBS buffer solution, remove the filter membrane 7 with fine tweezers, and place it on a glass slide with the cell facing up.

After the filter membrane 7 was properly dried, it was observed under a microscope to confirm whether there were CTCs.

Test results: None of the 8 healthy volunteers detected circulating tumor cells; in the peripheral blood of 8 patients diagnosed with liver cancer, 2 cases did not detect CTC or CTM, 6 cases detected CTC or CTM, and the detection rate was 75%.

2. Using cell wax block technology to make thin-layer slices:

Remove the filter membrane with CTC on the above glass slide, soak it in the decolorization solution for 4-6 hours, and remove the CTC staining solution (the decolorization solution is: 95% alcohol and 100% xylene mixed at a volume ratio of 1:1) After soaking, take out the filter membrane 7 and wrap it with absorbent paper; place the wrapping in 10% neutral formalin and fix it for 4-6h; Dip in wax and embed to make paraffin blocks of cells; slice the paraffin blocks of cells serially with a microtome to make thin slices with a thickness of 2-4 μm.

3. Detection of GPC3 expression:

Thin-layer slices of CTC wax blocks were baked in a constant temperature oven at 60°C for 2 hours; the slices were dewaxed in two bottles of xylene for 5 minutes; Place in ethanol for 5 minutes (adjust slightly depending on the room temperature), rinse with distilled water twice, 3 minutes each time;

Antigen repair: Boil the repair solution EDTA in the pressure cooker, put the slices into the repair solution completely, cover the pressure cooker cover and pressure valve, heat until 1.5 minutes after the pressure cooker is sprayed, remove from the heat, cool the pressure cooker slightly and open the lid, the slices are repaired Natural cooling in liquid;

After rinsing with distilled water, slice with 3% H ₂ O ₂ for 10 minutes to remove endogenous peroxidase; rinse with distilled water, soak in PBS 5 times, 1.5 minutes each time;

Add GPC3 primary antibody dropwise, overnight at 4°C, wash with PBS 5 times, 1.5 minutes each time; add universal IgG antibody-HRP polymer dropwise, incubate in a water bath at 37°C for 20 minutes; soak in PBS 5 times for 1.5 minutes each;

Control under the microscope, DAB color development: shake off the PBS solution, add 100 μl of freshly prepared DAB solution to each section, and observe under the microscope for 5 minutes;

Rinse with distilled water, counterstain, dehydrate, transparent mount: rinse with distilled water twice, 3 minutes each time; counterstain with hematoxylin for 1 minute; 0.1% hydrochloric acid alcohol differentiation; 0.1% ammonia solution to turn blue; 50%, 70%, 85%, Dehydration and drying with 95% gradient anhydrous ethanol; transparent xylene, and neutral resin glue sealing;

Cytopathology experts read the slides and judged the expression of GPC3 according to the degree of staining of the cell membrane and cytoplasm.

Detection results: In the peripheral blood of 8 patients with confirmed liver cancer, CTCs were detected in 6 cases, and GPC3 expression was detected in 5 cases of CTCs by immunohistochemistry, with a positive rate of 62.5% (Table 1).

Figure 4 shows the images of circulating tumor cells obtained by separating peripheral blood from patients with liver cancer. The nuclei are large, irregularly shaped, and have a high nuclear-to-cytoplasmic ratio.

Figure 5 shows the GPC3 immunohistochemical staining images of circulating tumor cells in the peripheral blood of patients with liver cancer, with yellow staining of the cell membrane and cytoplasm. According to the staining distribution and intensity, the positive degree is judged. The staining of less than 10% of the cells is (-), the staining of 10%-25% is (+), the staining of 25%-50% is (++), and the staining is greater than 50%. (+++).

Table 1 embodiment detection result

Claims (4)

1. A method for detecting GPC3 in the peripheral blood of patients with hepatocellular carcinoma, characterized in that the detection method steps are as follows: use a membrane filtration device to separate and obtain CTCs in the peripheral blood of patients with liver cancer who cannot obtain tissue samples; use cell wax block technology to make a thin layer slice; and then detect the expression of GPC3; The method for separating and obtaining CTCs in the peripheral blood of liver cancer patients whose tissue samples cannot be obtained by using a membrane filtration device is as follows: 1) Collect peripheral blood from patients with advanced liver cancer: 5ml from median cubital vein; 2) Peripheral blood sample pretreatment: Dilute the collected peripheral blood sample 10 times, the diluent composition: 1mmol/l EDTA+0.1%BSA, after dilution, add paraformaldehyde to fix the peripheral blood sample for 10 minutes, and fix the final concentration to 0.25%; 3) Using the membrane filtration tumor cell separation device to filter peripheral blood samples to separate and obtain peripheral blood circulating tumor cells: add the pretreated peripheral blood sample to the blood sample container (2) of the membrane filtration tumor cell separation device, allowing it to naturally filter by gravity; 4) After the filtration, remove the filter (3) from the device for separating tumor cells by membrane filtration, add circulating tumor cell staining solution to the filter (3), stain for 2 min, rinse with PBS buffer, and remove the filter membrane (7 ), placed on a glass slide, observed under a microscope after drying, to determine whether there are CTCs; Described use cell wax block technique to make thin-layer section, its method step is as follows: 1) Decolorization: Remove the filter membrane (7) with CTC from the glass slide, soak in the decolorization solution for 4-6 hours, and remove the CTC staining solution. The decolorization solution is: 95% alcohol and 100% bismuth Mix toluene at a volume ratio of 1:1; 2) Wrapping: Take out the filter membrane (7) after soaking and wrap it with absorbent paper; 3) Fixing: place the wrapping in 10% neutral formalin, and fix it for 4-6 hours; 4) Embedding in wax: take out the wrapper after fixation, dehydrate and place in a paraffin embedding box, and embedding in wax to make a paraffin block of cells; 5) Thin-layer sectioning: use a microtome to slice the cell paraffin blocks continuously to make thin-layer sections with a thickness of 4 μm; The method for detecting the expression of GPC3 is as follows: 1) Baking slices: Thin-layer slices of CTC wax blocks were baked in a constant temperature oven at 60°C for 2 hours; 2) Dewaxing: place the slices in two bottles of xylene for 5 minutes respectively; 3) Hydration: place the slices in absolute ethanol, 95% ethanol, and 85% ethanol for 1 minute respectively, and rinse with tap water and distilled water; 4) Antigen repair: Boil the repair solution EDTA in the pressure cooker, put the slices into the repair solution completely, cover the pressure cooker lid and pressure valve, heat until 1.5 minutes after the pressure cooker is sprayed, remove from the heat, cool the pressure cooker slightly, open the lid, and slice Natural cooling in repair fluid; 5) Rinse with distilled water; 6) Remove endogenous peroxidase: slice with 3% H2O2 for 10 minutes; 7) Rinse with distilled water; 8) PBS soaking 5 times for 1.5 minutes each; 9) Add GPC3 primary antibody dropwise; 10) Overnight in the refrigerator at 4°C; 11) PBS soaking 5 times for 1.5 minutes each; 12) Add the universal IgG antibody-HRP multimer dropwise; 13) Incubate in a water bath at 37°C for 20 minutes; 14) PBS soaking 5 times for 1.5 minutes each; 15) Control under the microscope, DAB color development; 16) Rinse with tap water, counterstain with hematoxylin staining solution for 2 minutes, differentiate with hydrochloric acid and alcohol, and turn blue with ammonia water; 17) Rinse with tap water for 5 minutes; 18) 50%, 70%, 85%, 95%, 100% gradient alcohol dehydration, transparent xylene, and neutral quick-drying glue for sealing; 19) Cytopathologists read the slides and interpret the expression of GPC3. 2. the peripheral blood GPC3 detection method of hepatocellular carcinoma patients according to claim 1, is characterized in that, described membrane filtration device comprises filter (3), blood sample container (2), waste liquid tank (5) and iron frame platform (1), the iron stand (1) is provided with a base (11), a stand (12) and a support (13), and the blood sample container (2) is arranged on the top of the iron stand (1) by the support (13), Below the blood sample container (2) is a filter (3), and the filter (3) is connected to the waste liquid tank (5) through the infusion set (4), and the waste liquid tank (5) is arranged on the base (11). 3. The method for detecting GPC3 in peripheral blood of patients with hepatocellular carcinoma according to claim 2, characterized in that: the filter (3) comprises a filter top (6), a filter membrane (7), and a filter membrane platform (8) and the lower port of the filter (9), the filter membrane (7) is placed on the filter membrane platform (8); the upper port of the filter (6) is connected to the blood sample container (2), and the lower port of the filter (9) is connected to the blood sample container (2) through the infusion set (4) Waste tank (5). 4. the method for detecting peripheral blood GPC3 of patients with hepatocellular carcinoma according to claim 3, characterized in that: the filter membrane (7) is made of a hydrophobic material, and it is evenly covered with filter holes (10 microns) with a diameter of 10 microns. ).