WO2023005232A1 - 一种促进肿瘤细胞凋亡的方法 - Google Patents

一种促进肿瘤细胞凋亡的方法 Download PDF

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WO2023005232A1
WO2023005232A1 PCT/CN2022/081990 CN2022081990W WO2023005232A1 WO 2023005232 A1 WO2023005232 A1 WO 2023005232A1 CN 2022081990 W CN2022081990 W CN 2022081990W WO 2023005232 A1 WO2023005232 A1 WO 2023005232A1
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cells
cancer cells
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cell apoptosis
incubation
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范声芳
周程
陈晓森
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苏州博思得电气有限公司
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Definitions

  • the application relates to a method for promoting tumor cell apoptosis, which belongs to the technical field of biomedicine.
  • Glioma is the most common primary brain malignant tumor caused by the carcinogenesis of glial cells in the brain and spinal cord, and it is one of the deadliest and most stubborn tumors among all malignant solid tumors. Its incidence rate accounts for about 35.2-61.0% of intracranial tumors. It is derived from glial cells and has the characteristics of high incidence, high recurrence rate, high mortality rate and low cure rate. In the United States, there are about 50,000 patients with glioma, and about 10,000 new cases are diagnosed every year. The 5-year survival rate of patients is only 5%.
  • the survival period and quality of life of patients with glioma are closely related to the degree of surgical resection.
  • the treatment of brain glioma has always been one of the difficult problems for neurosurgeons, especially the operation of brain functional areas (cortical and subcortical pathways closely related to language, motor and sensory functions) and low-grade gliomas Treatment is a difficult problem in the clinical work of neurosurgery, and the main contradiction is the choice between the degree of lesion resection and neurological function.
  • the average median survival time of glioma patients worldwide is only 12-14 months.
  • glioma mainly include surgery, radiotherapy, chemotherapy and ablation techniques.
  • surgical treatment is based on the growth characteristics of gliomas.
  • Radiotherapy is almost a routine treatment for various types of glioma, including X-knife and ⁇ -knife, etc.
  • glioma especially malignant astrocytoma grade III-IV or glioblastoma
  • Chemotherapy is limited by the blood-brain barrier (Blood-Brain Barrier, BBB and Blood-Brain Tumor Barrier, BBTB), which hinders the delivery of drugs and the side effects of drugs, making it very difficult to exert a limited therapeutic effect.
  • Ablation techniques mainly include radiofrequency ablation, microwave ablation, cryoablation, and nanoknife (generally at a frequency of 5-10kHz) ablation. These ablation methods cause great damage to important tissues such as blood vessels and nerves. Therefore, ablation techniques are not suitable for treating brain tumors. Glioma.
  • the present application provides a method for promoting apoptosis of tumor cells.
  • the method is to ablate the tumor cells with a nanoknife first, that is, irreversible electroporation (Ireversible Electroporation, IRE), and then place the tumor cells in Incubate in an incubation environment containing antineoplastic drugs.
  • irreversible electroporation Ireversible Electroporation, IRE
  • the method is to first ablate the tumor cells with a nanomaid knife, then place the tumor cells in an incubation environment containing anti-tumor drugs for incubation, and finally place the tumor cells in an incubation environment containing antitumor drugs. Cultured in the incubation environment of antineoplastic drugs.
  • the tumor cells are glioma cells, pancreatic cancer cells, liver cancer cells, cholangiocarcinoma cells, gastric cancer cells, lung cancer cells, breast cancer cells, intestinal cancer cells, bone cancer cells or At least one type of prostate cancer cell.
  • the antineoplastic drug is at least one of temozolomide, gemcitabine, nab-paclitaxel, vincristine or doxorubicin hydrochloride.
  • the drug concentration of the antineoplastic drug is 25 nM-100 ⁇ M.
  • the pH of the incubation environment is 7.0-7.4.
  • the incubation environment is at least one of physiological saline, buffer solution, plasma or interstitial fluid.
  • the incubation temperature is 36-38° C. and the incubation time is 2-8 hours.
  • the voltage amplitude of the Namao knife is 500-10000V
  • the frequency is 50-500kHz
  • the ablation time is 1-30min.
  • the rise time of the pulse edge of the nanomasonry knife is less than 100 ns.
  • the present application also provides the application of the above method in promoting tumor cell apoptosis.
  • the tumor cells are glioma cells, pancreatic cancer cells, liver cancer cells, cholangiocarcinoma cells, gastric cancer cells, lung cancer cells, breast cancer cells, intestinal cancer cells, bone cancer cells or At least one type of prostate cancer cell.
  • the present application provides a method for promoting apoptosis of tumor cells.
  • the method effectively overcomes the traditional chemotherapy and the traditional Namaidao by combining chemotherapy based on antineoplastic drugs and Nanomaidao based on high-frequency irreversible electroporation.
  • the defect of poor apoptosis effect of glioma cells, among them, the effect of combined chemotherapy and Namaidao on promoting the apoptosis of glioma cells is better than that of chemotherapy and Namaidao alone on promoting the apoptosis of glioma cells An increase of 10% and 28%, respectively.
  • the use of low-concentration (temozolomide 25 ⁇ M, paclitaxel 2 ⁇ M) chemotherapeutic drugs combined with Namaikao can achieve far more than that of high-concentration (temozolomide 50 ⁇ M, paclitaxel 5 ⁇ M) chemotherapeutic drugs alone. Therefore, using the method to promote the apoptosis of glioma cells has the advantages of small dosage of chemotherapeutic drugs and relatively low toxic and side effects, and has a very high application prospect in the treatment of glioma.
  • the nanopulse knife used in the method is based on high-frequency irreversible electroporation. Compared with traditional ablation techniques, high-frequency irreversible electroporation has less damage to important tissues such as blood vessels and nerves. Therefore, the method is effective in the treatment of glioma. It has a very high application prospect.
  • Figure 1 Cell viability of U87 cells under different treatment conditions in Experimental Example 1.
  • Figure 2 Apoptosis of U87 cells under different treatment conditions in Experimental Example 1.
  • A blank control
  • B treatment with temozolomide (TMZ 50 ⁇ M)
  • C treatment with Namaknife
  • D treatment with Namaknife combined with temozolomide (TMZ 25 ⁇ M).
  • FIG. 1 The expression of BCL-2 gene in U87 cells under different treatment conditions in Experimental Example 1.
  • FIG. 4 The expression of BAX gene in U87 cells under different treatment conditions in Experimental Example 1.
  • Figure 5 The expression of Caspase-3 gene in U87 cells under different treatment conditions in Experimental Example 1.
  • Figure 6 Cell viability of U87 cells under different treatment conditions in Experimental Example 2.
  • Figure 7 Apoptosis of U87 cells under different treatment conditions in Experimental Example 2.
  • A blank control
  • B treatment with paclitaxel (PTX 5 ⁇ M)
  • C treatment with Namaknife
  • D treatment with Namaknife combined with paclitaxel (PTX 2 ⁇ M).
  • Figure 8 The expression of BCL-2 gene in U87 cells under different treatment conditions in Experimental Example 2.
  • Figure 9 The expression of BAX gene in U87 cells under different treatment conditions in Experimental Example 2.
  • Figure 10 The expression of Caspase-3 gene in U87 cells under different treatment conditions in Experimental Example 2.
  • Example 1 A method for promoting apoptosis of glioma cells
  • the present embodiment provides a method for promoting the apoptosis of glioma cells.
  • the method is as follows: use the Namai Knife (a high-frequency steep pulse tumor therapeutic instrument, recorded in the patent application text with publication number CN107809184A), to Tumor cells were ablated for 15 min under the conditions of a voltage amplitude of 1000 V, a frequency of 500 kHz, and a pulse edge rise time of less than 100 ns.
  • the glioma cells were placed in RPMI medium containing 50 ⁇ M temozolomide (pH 7.4), incubate at 37°C for 4 hours, after the incubation, suck off the RPMI medium (pH 7.4) containing 50 ⁇ M Temozolomide, and place the glioma cells in the RPMI medium (pH 7.4) , and continued culturing for 44 h at 37°C.
  • Example 2 A method for promoting apoptosis of glioma cells
  • the present embodiment provides a method for promoting the apoptosis of glioma cells.
  • the method is as follows: use the Namai Knife (a high-frequency steep pulse tumor therapeutic instrument, recorded in the patent application text with publication number CN107809184A), to Tumor cells were ablated for 15 min under the conditions of voltage amplitude of 1000V, frequency of 500kHz, and pulse edge rise time of less than 100ns.
  • brain glioma cells were placed in RPMI medium (pH 7.4), incubate at 37°C for 4 h, after the incubation, suck off the RPMI medium (pH 7.4) containing 25 ⁇ M temozolomide, and place the glioma cells in the RPMI medium (pH 7.4) , and continued culturing for 44 h at 37°C.
  • Example 3 A method for promoting apoptosis of glioma cells
  • the present embodiment provides a method for promoting the apoptosis of glioma cells.
  • the method is as follows: use the Namai Knife (a high-frequency steep pulse tumor therapeutic instrument, recorded in the patent application text with publication number CN107809184A), to Tumor cells were ablated for 15 minutes under the conditions of voltage amplitude of 1000V, frequency of 500kHz, and pulse edge rise time of less than 100ns.
  • brain glioma cells were placed in RPMI medium containing nab-paclitaxel at a concentration of 5 ⁇ M (pH 7.4), incubate at 37°C for 4 h, after the incubation, suck off the RPMI medium (pH 7.4) containing nab-paclitaxel at a concentration of 5 ⁇ M, and place the glioma cells in the RPMI medium (pH 7.4), continue to culture at 37 ° C for 44 h.
  • RPMI medium containing nab-paclitaxel at a concentration of 5 ⁇ M
  • Example 4 A method for promoting apoptosis of glioma cells
  • the present embodiment provides a method for promoting the apoptosis of glioma cells.
  • the method is as follows: use the Namai Knife (a high-frequency steep pulse tumor therapeutic instrument, recorded in the patent application text with publication number CN107809184A), to Tumor cells were ablated for 15 minutes under the conditions of voltage amplitude of 1000V, frequency of 500kHz, and pulse edge rise time of less than 100ns.
  • brain glioma cells were placed in RPMI medium containing nab-paclitaxel at a concentration of 2 ⁇ M (pH 7.4), incubate at 37°C for 4 h, after the incubation, suck off the RPMI medium (pH 7.4) containing nab-paclitaxel at a concentration of 2 ⁇ M, and place the glioma cells in the RPMI medium (pH 7.4), continue to culture at 37 ° C for 44 h.
  • RPMI medium containing nab-paclitaxel at a concentration of 2 ⁇ M
  • Example 5 A method for promoting apoptosis of glioma cells
  • the present embodiment provides a method for promoting the apoptosis of glioma cells.
  • the method is as follows: use the Namai Knife (a high-frequency steep pulse tumor therapeutic instrument, recorded in the patent application text with publication number CN107809184A), to Tumor cells were ablated for 15 min under the conditions of voltage amplitude of 1000 V, frequency of 500 kHz, and rise time of pulse edge less than 100 ns.
  • brain glioma cells were placed in RPMI medium (pH 7.4), incubate at 37°C for 4 hours, after the incubation, suck off the RPMI medium (pH 7.4) containing gemcitabine at a concentration of 100 ⁇ M, and place the glioma cells in the RPMI medium (pH 7.4) , and continued culturing for 44 h at 37°C.
  • Example 6 A method for promoting apoptosis of glioma cells
  • the present embodiment provides a method for promoting the apoptosis of glioma cells.
  • the method is as follows: use the Namai Knife (a high-frequency steep pulse tumor therapeutic instrument, recorded in the patent application text with publication number CN107809184A), to Tumor cells were ablated for 15 minutes under the conditions of voltage amplitude of 1000V, frequency of 500kHz, and pulse edge rise time of less than 100ns. After ablation, brain glioma cells were cultured in RPMI containing 1 ⁇ M doxorubicin hydrochloride.
  • Example 7 A method for promoting apoptosis of glioma cells
  • the present embodiment provides a method for promoting the apoptosis of glioma cells.
  • the method is as follows: use the Namai Knife (a high-frequency steep pulse tumor therapeutic instrument, recorded in the patent application text with publication number CN107809184A), to Tumor cells were ablated for 15 minutes under the conditions of voltage amplitude of 1000V, frequency of 500kHz, and pulse edge rise time of less than 100ns. After ablation, brain glioma cells were cultured in RPMI containing vincristine at a concentration of 0.5 ⁇ M.
  • Experimental example 1 Apoptosis experiment of brain glioma cells
  • This embodiment provides the apoptosis experiment of brain glioma cells, and the experimental process is as follows:
  • 3D Flo Trix microslides (3D Flo Trix microslides were purchased from Beijing Huakan Biology) and put them into the wells of the dry well plate, and add RPMI medium (pH 7.4, purchased from Gibco), the added RPMI medium (pH 7.4) does not exceed the edge of the gap, and then the orifice plate is placed at a temperature of 37 ° C and contains 5% (v/v) CO in an incubator for 12 h for pretreatment , after the pretreatment, U87 cells (purchased from the Chinese Academy of Sciences Cell Bank) were inoculated on the treated 3D Flo Trix microslides with an inoculation amount of 2*10 ⁇ 5 cells/sheet.
  • RPMI medium pH 7.4, purchased from Gibco
  • the U87 cells were divided into four The four groups were the blank group, the control group 1 (temozolomide treatment), the control group 2 (nomadao treatment) and the experimental group (temozolomide+nomaknife treatment). After the grouping, the U87 cells in each group were treated. in:
  • the treatment method of the blank group is: do not do any treatment
  • control group 1 The treatment method of control group 1 was as follows: U87 cells were placed in RPMI medium (pH 7.4, containing 10% FBS and 1% double antibody, v/v) containing temozolomide at a concentration of 50 ⁇ M, and incubated at 37°C After 4 hours of incubation, the RPMI medium (pH 7.4, containing 10% FBS and 1% double antibody, v/v) containing temozolomide at a concentration of 50 ⁇ M was sucked off, and the U87 cells were placed in RPMI medium (pH 7.4, Containing 10% FBS and 1% double antibody), continue culturing at 37°C for 44h;
  • control group 2 The treatment method of control group 2 was as follows: U87 cells were ablated for 15 min with the Namai Knife under the conditions of voltage amplitude of 1000 V, frequency of 500 kHz, and pulse edge rise time of less than 100 ns. After ablation, U87 cells were placed in RPMI In the medium (pH 7.4, containing 10% FBS and 1% double antibody, v/v), continue to culture at 37°C for 44h;
  • the treatment method of the experimental group was as follows: U87 cells were ablated for 15 minutes under the conditions of voltage amplitude of 1000V, frequency of 500kHz, and pulse edge rise time of less than 100ns with Namai Knife. After ablation, U87 cells were placed in a concentration containing In RPMI medium (pH 7.4, containing 10% FBS and 1% double antibody, v/v) containing 25 ⁇ M temozolomide, incubate at 37°C for 4 h, after the incubation, suck off the medium containing 25 ⁇ M temozolomide RPMI medium (pH 7.4, containing 10% FBS and 1% double antibody, v/v), U87 cells were placed in RPMI medium (pH 7.4, containing 10% FBS and 1% double antibody, v/v), The culture was continued for 44 h at 37°C.
  • the process of MTT cytotoxicity experiment is as follows: After inoculating U87 cells of each group treated according to the experimental conditions in a 96-well plate (1 ⁇ 10 ⁇ 4 cells/well), at 37°C, containing 5% (v/v) CO 2 After culturing in an incubator for 48 hours, add 10 ⁇ L of MTT-containing PBS solution (5.0 mg/mL) to a 96-well plate, and continue to incubate at 37°C in an incubator containing 5% (v/v) CO 2 After 4 hours of incubation, aspirate the supernatant and add 150 ⁇ L DMSO to the 96-well plate to dissolve the purple formazan produced by the living cells.
  • MTT cytotoxicity test can be found in the literature: R.Supino, MTT Assays, in: S.O'Hare, CKAtterwill (Eds.), In Vitro Toxicity Testing Protocols, Humana Press, Totowa, NJ, 1995, pp.137-149 .
  • the changes in apoptosis of U87 cells in each group were detected by flow cytometry, and the detection results are shown in Figure 2.
  • the experimental process of flow cytometry analysis is as follows: After inoculating the U87 cells of each group treated according to the experimental conditions in a 6-well plate (2 ⁇ 10 ⁇ 5 cells/well), at 37°C, containing 5% (v/v) CO 2 in an incubator for 24 hours, after the end of the culture, add 500 ⁇ L of 0.25% (m/v, g/100mL) trypsin to the 6-well plate, collect the cells after centrifugation at 800 rpm for 5 minutes, and wash the collected cells with PBS solution (5.0 mg/mL) washed twice, resuspended in 300 ⁇ L binding buffer, after adding Annexin V-FITC (5 ⁇ L) and PI (5 ⁇ L) to the binding buffer, incubate at 25 °C for 15 min in the dark, After incubation, the stained U87 cells were analyzed by flow cyto
  • the changes in the gene levels of apoptosis (BCL-2)/autophagy (Bax)/pyroptosis (Caspase3) factors in U87 cells in each group were detected by analyzing the expression of apoptosis-related genes.
  • the detection results are shown in Figures 3-5.
  • the experimental process of apoptosis-related gene expression analysis is as follows: firstly extract the RNA of U87 cells in each group after treatment, then use the reverse transcription kit to reverse transcribe the extracted RNA, and finally use Premix Ex TaqTM II Fluorescent Quantitative PCR Kit to process cDNA obtained by reverse transcription, and finally use real-time fluorescent quantitative PCR instrument The processed cDNA was analyzed on a 96-well plate.
  • Fig. 2 It can be seen from Fig. 2 that 65% of the cells were apoptotic after ablation with the single nanoknife, 83% of the cells were apoptotic after incubation with temozolomide (50 ⁇ M) alone, and 93% of the cells were apoptotic after ablation with the ablation of the nanoknife combined with temozolomide (25 ⁇ M) incubation.
  • the therapeutic effect of single nanoknife ablation is equivalent; compared with single nanoknife ablation and single temozolomide (50 ⁇ M) incubation, the pro-apoptotic genes BAX and Caspase in U87 cells after ablation combined with temozolomide (25 ⁇ M) incubation
  • the expression level was higher, among which, the expression level of Bax apoptosis gene was 2.2 times that of the blank control group, and the expression level of Caspase3 gene was 2.5 times that of the blank control group. more obvious.
  • the results showed that Namaikao combined with temozolomide could significantly promote the apoptosis of tumor cells, and it is expected to become a new treatment method for glioma.
  • Experimental example 2 Apoptosis experiment of brain glioma cells
  • This embodiment provides the apoptosis experiment of brain glioma cells, and the experimental process is as follows:
  • 3D Flo Trix microslides (3D Flo Trix microslides were purchased from Beijing Huakan Biology) and put them into the wells of the dry well plate, and add RPMI medium (pH 7.4, purchased from Gibco), the added RPMI medium (pH 7.4) does not exceed the edge of the gap, and then the orifice plate is placed at a temperature of 37 ° C and contains 5% (v/v) CO in an incubator for 12 h for pretreatment , after the pretreatment, U87 cells (purchased from the Chinese Academy of Sciences Cell Bank) were inoculated on the treated 3D Flo Trix microslides with an inoculation amount of 2*10 ⁇ 5 cells/sheet.
  • RPMI medium pH 7.4, purchased from Gibco
  • the U87 cells were divided into four The four groups were the blank group, the control group 1 (paclitaxel treatment), the control group 2 (Nomadao treatment) and the experimental group (paclitaxel + Namaidao treatment). After the grouping, the U87 cells in each group were treated. in:
  • the treatment method of the blank group is: do not do any treatment
  • control group 1 The treatment method of control group 1 was as follows: U87 cells were placed in RPMI medium (pH 7.4, containing 10% FBS and 1% double antibody, v/v) containing paclitaxel at a concentration of 5 ⁇ M, and incubated at 37°C After 4 hours of incubation, the RPMI medium (pH 7.4, containing 10% FBS and 1% double antibody, v/v) containing paclitaxel at a concentration of 5 ⁇ M was sucked off, and the U87 cells were placed in RPMI medium (pH 7.4, containing 10% FBS and 1% double antibody), continue culturing at 37°C for 44h;
  • control group 2 The treatment method of control group 2 was as follows: U87 cells were ablated for 15 min with the Namai Knife under the conditions of voltage amplitude of 1000 V, frequency of 500 kHz, and pulse edge rise time of less than 100 ns. After ablation, U87 cells were placed in RPMI In the culture medium (pH 7.4, containing 10% FBS and 1% double antibody, v/v), continue to culture at 37°C for 44h;
  • the treatment method of the experimental group was as follows: U87 cells were ablated for 15 minutes under the conditions of voltage amplitude of 1000V, frequency of 500kHz, and pulse edge rise time of less than 100ns with Namai Knife. After ablation, U87 cells were placed in a concentration containing In RPMI medium (pH 7.4, containing 10% FBS and 1% double antibody, v/v) containing 2 ⁇ M paclitaxel, incubate at 37°C for 4 h, after the incubation, suck off the medium containing 2 ⁇ M paclitaxel RPMI medium (pH 7.4, containing 10% FBS and 1% double antibody, v/v), U87 cells were placed in RPMI medium (pH 7.4, containing 10% FBS and 1% double antibody, v/v), The culture was continued for 44 h at 37°C.
  • the process of MTT cytotoxicity experiment is as follows: After inoculating U87 cells of each group treated according to the experimental conditions in a 96-well plate (1 ⁇ 10 ⁇ 4 cells/well), at 37°C, containing 5% (v/v) CO 2 After culturing in an incubator for 48 hours, add 10 ⁇ L of MTT-containing PBS solution (5.0 mg/mL) to a 96-well plate, and continue to incubate at 37°C in an incubator containing 5% (v/v) CO 2 After 4 hours of incubation, aspirate the supernatant and add 150 ⁇ L DMSO to the 96-well plate to dissolve the purple formazan produced by the living cells.
  • MTT cytotoxicity test can be found in the literature: R.Supino, MTT Assays, in: S.O'Hare, CKAtterwill (Eds.), In Vitro Toxicity Testing Protocols, Humana Press, Totowa, NJ, 1995, pp.137-149 .
  • the changes of apoptosis of U87 cells in each group were detected by flow cytometric analysis, and the detection results are shown in Fig. 7 .
  • the experimental process of flow cytometry analysis is as follows: After inoculating the U87 cells of each group treated according to the experimental conditions in a 6-well plate (2 ⁇ 10 ⁇ 5 cells/well), at 37°C, containing 5% (v/v) CO 2 in an incubator for 24 hours, after the end of the culture, add 500 ⁇ L of 0.25% (m/v, g/100mL) trypsin to the 6-well plate, collect the cells after centrifugation at 800 rpm for 5 minutes, and wash the collected cells with PBS solution (5.0 mg/mL) washed twice, resuspended in 300 ⁇ L binding buffer, after adding Annexin V-FITC (5 ⁇ L) and PI (5 ⁇ L) to the binding buffer, incubate at 25 °C for 15 min in the dark, After incubation, the stained U87 cells were
  • the changes in the gene levels of apoptosis (BCL-2)/autophagy (Bax)/pyroptosis (Caspase3) factors in U87 cells in each group were detected by analyzing the expression of apoptosis-related genes.
  • the detection results are shown in Figures 8-10.
  • the experimental process of apoptosis-related gene expression analysis is as follows: firstly extract the RNA of U87 cells in each group after treatment, then use the reverse transcription kit to reverse transcribe the extracted RNA, and finally use Premix Ex TaqTM II Fluorescent Quantitative PCR Kit to process cDNA obtained by reverse transcription, and finally use real-time fluorescent quantitative PCR instrument The processed cDNA was analyzed on a 96-well plate.
  • Fig. 7 It can be seen from Fig. 7 that 69% of the cells were apoptotic after ablation with single nanoknife, 53% of cells were apoptotic after incubation with paclitaxel (5 ⁇ M), and 95% of cells were apoptotic after incubation with paclitaxel (2 ⁇ M) after ablation with nanoknife.

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Abstract

提供了一种促进肿瘤细胞凋亡的方法，所述方法通过联用基于抗肿瘤药的化疗和基于高频不可逆电穿孔的纳脉刀，克服了传统化疗和传统纳脉刀促进脑胶质瘤细胞凋亡效果差的缺陷，其中，联用化疗和纳脉刀促进脑胶质瘤细胞凋亡的效果较单独使用化疗和单独使用纳脉刀促进脑胶质瘤细胞凋亡的效果分别提高了10％和28％。

Description

一种促进肿瘤细胞凋亡的方法
本申请要求在2021年7月30日提交中国专利局、申请号为202110872999.9、发明名称为“一种促进肿瘤细胞凋亡的方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本申请涉及一种促进肿瘤细胞凋亡的方法，属于生物医药技术领域。
背景技术
脑胶质瘤是因为大脑和脊髓胶质细胞癌变所产生的最常见的原发性颅脑恶性肿瘤，是所有恶性实体瘤中最致命、最顽固的肿瘤之一。其发病率约占颅内肿瘤的35.2～61.0％，由成胶质细胞衍化而来，具有发病率高、复发率高、死亡率高以及治愈率低的特点。在美国，共有大约5万名脑胶质瘤患者，且每年大约有1万名新确诊病例，患者5年生存率仅为5％。
脑胶质瘤病人的生存期及生存质量与手术切除程度密切相关。脑胶质瘤的治疗一直是困扰神经外科医师的难题之一，尤其是大脑功能区(与语言、运动和感觉功能密切相关的皮质和皮质下通路)胶质瘤及低级别胶质瘤的手术治疗是神经外科临床工作的一个难题，其主要矛盾是病灶切除程度与神经功能的取舍。尽管当前临床已有多种针对脑胶质瘤的治疗策略，但全球脑胶质瘤患者的平均中位生存期仅为12～14个月。
目前，治疗脑胶质瘤的常用方法主要有手术、放疗、化疗和消融技术等。其中，手术治疗基于脑胶质瘤的生长特点，理论上，手术不可能完全切除脑胶质瘤，生长在脑干等重要部位的脑胶质瘤有的则根本不能手术。放疗几乎是各型胶质瘤的常规治疗方法，包括X-刀和γ-刀等，因肿瘤的部位、瘤体大小及瘤 体对射线的敏感程度，放疗的治疗范畴局限性较大，且疗效评价不一，例如，除髓母细胞瘤对放疗高度敏感，室管膜瘤对放疗中度敏感外，其他类型的肿瘤细胞均对放疗均不敏感。有观察发现，针对这部分对放疗不敏感的肿瘤细胞，放疗与非放疗者预后相同。并且，放疗所使用的外射线引起的放射性坏死对于脑功能的影响亦不可低估。因此，现阶段可以认为，胶质瘤，特别是性质恶性的星形Ⅲ-Ⅳ级或胶质母细胞瘤均不适合采用放疗进行治疗。化疗受限于血脑屏障(Blood-Brain Barrier,BBB及Blood-Brain Tumor Barrier,BBTB)阻碍了药物的递送及药物的毒副作用，使其发挥有限的治疗效果变得非常困难。消融技术则主要包括射频消融、微波消融、冷冻消融和纳米刀(频率一般在5～10kHz)消融，这些消融方法对血管、神经等重要组织损伤较大，因此，消融技术也不适用于治疗脑胶质瘤。
亟需找到可有效治疗脑胶质瘤且对血管、神经等重要组织损伤较小的方法。
发明内容
为解决上述问题，本申请提供了一种促进肿瘤细胞凋亡的方法，所述方法为先用纳脉刀对肿瘤细胞进行消融，即不可逆电穿孔(Irreversible Electroporation,IRE)，再将肿瘤细胞置于含有抗肿瘤药的孵育环境中进行孵育。
在本申请的一种实施方式中，所述方法为先用纳脉刀对肿瘤细胞进行消融，再将肿瘤细胞置于含有抗肿瘤药的孵育环境中进行孵育，最后将肿瘤细胞置于不含抗肿瘤药的孵育环境中进行培养。
在本申请的一种实施方式中，所述肿瘤细胞为脑胶质瘤细胞、胰腺癌细胞、肝癌细胞、胆管癌细胞、胃癌细胞、肺癌细胞、乳腺癌细胞、肠癌细胞、骨癌细胞或前列腺癌细胞中的至少一种。
在本申请的一种实施方式中，所述抗肿瘤药为替莫唑胺、吉西他滨、白蛋白紫杉醇、长春新碱或盐酸阿霉素中的至少一种。
在本申请的一种实施方式中，所述孵育环境中，抗肿瘤药的药物浓度为25nM～100μM。
在本申请的一种实施方式中，所述孵育环境的pH为7.0～7.4。
在本申请的一种实施方式中，所述孵育环境为生理盐水、缓冲液、血浆或组织液中的至少一种。
在本申请的一种实施方式中，所述孵育的温度为36～38℃、时间为2～8h。
在本申请的一种实施方式中，所述纳脉刀的电压幅值为500～10000V、频率为50～500kHz、消融时间为1～30min。
在本申请的一种实施方式中，所述纳脉刀的脉冲边沿的上升时间小于100ns。
本申请还提供了上述方法在促进肿瘤细胞凋亡中的应用。
在本申请的一种实施方式中，所述肿瘤细胞为脑胶质瘤细胞、胰腺癌细胞、肝癌细胞、胆管癌细胞、胃癌细胞、肺癌细胞、乳腺癌细胞、肠癌细胞、骨癌细胞或前列腺癌细胞中的至少一种。
本申请技术方案，具有如下优点：
本申请提供了一种促进肿瘤细胞凋亡的方法，所述方法通过联用基于抗肿瘤药的化疗和基于高频不可逆电穿孔的纳脉刀，有效克服了传统化疗和传统纳脉刀促进脑胶质瘤细胞凋亡效果差的缺陷，其中，联用化疗和纳脉刀促进脑胶质瘤细胞凋亡的效果较单独使用化疗和单独使用纳脉刀促进脑胶质瘤细胞凋亡的效果分别提高了10％和28％。
使用所述方法促进脑胶质瘤细胞凋亡时，使用低浓度(替莫唑胺25μM、紫杉醇2μM)化疗药物搭配纳脉刀就可获得远超单独使用高浓度(替莫唑胺50μM、紫杉醇5μM)化疗药物时的效果，因此，使用所述方法促进脑胶质瘤细胞凋亡具有化疗药物用量小以及毒副作用相对低的优势，在治疗脑胶质瘤方面具有极高的应用前景。
所述方法使用的纳脉刀基于的高频不可逆电穿孔，与传统消融技术相比，高频不可逆电穿孔对血管、神经等重要组织损伤较小，因此，所述方法在治疗脑胶质瘤方面具有极高的应用前景。
附图说明
图1：实验例1中不同处理条件下U87细胞的细胞存活率情况。
图2：实验例1中不同处理条件下U87细胞的细胞凋亡情况。图2中，A：空白对照；B：替莫唑胺(TMZ 50μM)处理、C：纳脉刀处理、D：纳脉刀联合替莫唑胺(TMZ 25μM)处理。
图3：实验例1中不同处理条件下U87细胞中BCL-2基因的表达情况。
图4：实验例1中不同处理条件下U87细胞中BAX基因的表达情况。
图5：实验例1中不同处理条件下U87细胞中Caspase-3基因的表达情况。
图6：实验例2中不同处理条件下U87细胞的细胞存活率情况。
图7：实验例2中不同处理条件下U87细胞的细胞凋亡情况。图2中，A：空白对照；B：紫杉醇(PTX 5μM)处理、C：纳脉刀处理、D：纳脉刀联合替紫杉醇(PTX 2μM)处理。
图8：实验例2中不同处理条件下U87细胞中BCL-2基因的表达情况。
图9：实验例2中不同处理条件下U87细胞中BAX基因的表达情况。
图10：实验例2中不同处理条件下U87细胞中Caspase-3基因的表达情况。
具体实施方式
提供下述实施例是为了更好地进一步理解本申请，并不局限于所述最佳实施方式，不对本申请的内容和保护范围构成限制，任何人在本申请的启示下或是将本申请与其他现有技术的特征进行组合而得出的任何与本申请相同或相近似的产品，均落在本申请的保护范围之内。
下述实施例中未注明具体实验步骤或条件者，按照本领域内的文献所描述的常规实验步骤的操作或条件即可进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规试剂产品。
实施例1：一种促进脑胶质瘤细胞凋亡的方法
本实施例提供了一种促进脑胶质瘤细胞凋亡的方法，所述方法为：用纳脉刀(高频陡脉冲肿瘤治疗仪，记载于公开号为CN107809184A的专利申请文本中)，对肿瘤细胞于电压幅值为1000V、频率为500kHz、脉冲边沿的上升时间小于100ns的条件下消融15min，消融结束后，将脑胶质瘤细胞置于含有浓度为50μM的替莫唑胺的RPMI培养基(pH 7.4)中，于37℃的条件下孵育4h，孵育结束后，吸掉含有浓度为50μM的替莫唑胺的RPMI培养基(pH 7.4)，将脑胶质瘤细胞置于RPMI培养基(pH 7.4)中，于37℃的条件下继续培养44h。
实施例2：一种促进脑胶质瘤细胞凋亡的方法
本实施例提供了一种促进脑胶质瘤细胞凋亡的方法，所述方法为：用纳脉刀(高频陡脉冲肿瘤治疗仪，记载于公开号为CN107809184A的专利申请文本中)，对肿瘤细胞于电压幅值为1000V、频率为500kHz、脉冲边沿的上升时间小于100ns的条件下消融15min，消融结束后，将脑胶质瘤细胞置于含有浓度为25μM的替莫唑胺的RPMI培养基(pH 7.4)中，于37℃的条件下孵育4h，孵育结束后，吸掉含有浓度为25μM的替莫唑胺的RPMI培养基(pH 7.4)，将脑胶质瘤细胞置于RPMI培养基(pH 7.4)中，于37℃的条件下继续培养44h。
实施例3：一种促进脑胶质瘤细胞凋亡的方法
本实施例提供了一种促进脑胶质瘤细胞凋亡的方法，所述方法为：用纳脉刀(高频陡脉冲肿瘤治疗仪，记载于公开号为CN107809184A的专利申请文本中)，对肿瘤细胞于电压幅值为1000V、频率为500kHz、脉冲边沿的上升时间小于100ns的条件下消融15min，消融结束后，将脑胶质瘤细胞置于含有浓度为5μM的白蛋白紫杉醇的RPMI培养基(pH 7.4)中，于37℃的条件下孵育4h，孵育结束后，吸掉含有浓度为5μM的白蛋白紫杉醇的RPMI培养基(pH7.4)，将脑胶质瘤细胞置于RPMI培养基(pH 7.4)中，于37℃的条件下继续培养44h。
实施例4：一种促进脑胶质瘤细胞凋亡的方法
本实施例提供了一种促进脑胶质瘤细胞凋亡的方法，所述方法为：用纳脉刀(高频陡脉冲肿瘤治疗仪，记载于公开号为CN107809184A的专利申请文本中)，对肿瘤细胞于电压幅值为1000V、频率为500kHz、脉冲边沿的上升时间小于100ns的条件下消融15min，消融结束后，将脑胶质瘤细胞置于含有浓度为2μM的白蛋白紫杉醇的RPMI培养基(pH 7.4)中，于37℃的条件下孵育4h，孵育结束后，吸掉含有浓度为2μM的白蛋白紫杉醇的RPMI培养基(pH7.4)，将脑胶质瘤细胞置于RPMI培养基(pH 7.4)中，于37℃的条件下继续培养44h。
实施例5：一种促进脑胶质瘤细胞凋亡的方法
本实施例提供了一种促进脑胶质瘤细胞凋亡的方法，所述方法为：用纳脉刀(高频陡脉冲肿瘤治疗仪，记载于公开号为CN107809184A的专利申请文本中)，对肿瘤细胞于电压幅值为1000V、频率为500kHz、脉冲边沿的上升时间小于100ns的条件下消融15min，消融结束后，将脑胶质瘤细胞置于含有浓度为100μM的吉西他滨的RPMI培养基(pH 7.4)中，于37℃的条件下孵育4h，孵育结束后，吸掉含有浓度为100μM的吉西他滨的RPMI培养基(pH 7.4)，将脑胶质瘤细胞置于RPMI培养基(pH 7.4)中，于37℃的条件下继续培养44h。
实施例6：一种促进脑胶质瘤细胞凋亡的方法
本实施例提供了一种促进脑胶质瘤细胞凋亡的方法，所述方法为：用纳脉刀(高频陡脉冲肿瘤治疗仪，记载于公开号为CN107809184A的专利申请文本中)，对肿瘤细胞于电压幅值为1000V、频率为500kHz、脉冲边沿的上升时间小于100ns的条件下消融15min，消融结束后，将脑胶质瘤细胞置于含有浓度为1μM的盐酸阿霉素的RPMI培养基(pH 7.4)中，于37℃的条件下孵育4h，孵育结束后，吸掉含有浓度为1μM的盐酸阿霉素的RPMI培养基(pH 7.4)，将脑胶质瘤细胞置于RPMI培养基(pH 7.4)中，于37℃的条件下继续培养44h。
实施例7：一种促进脑胶质瘤细胞凋亡的方法
本实施例提供了一种促进脑胶质瘤细胞凋亡的方法，所述方法为：用纳脉刀(高频陡脉冲肿瘤治疗仪，记载于公开号为CN107809184A的专利申请文本中)，对肿瘤细胞于电压幅值为1000V、频率为500kHz、脉冲边沿的上升时间小于100ns的条件下消融15min，消融结束后，将脑胶质瘤细胞置于含有浓度为0.5μM的长春新碱的RPMI培养基(pH 7.4)中，于37℃的条件下孵育4h，孵育结束后，吸掉含有浓度为0.5μM的长春新碱的RPMI培养基(pH 7.4)，将脑胶质瘤细胞置于RPMI培养基(pH 7.4)中，于37℃的条件下继续培养44h。
实验例1：脑胶质瘤细胞的凋亡实验
本实施例提供了脑胶质瘤细胞的凋亡实验，实验过程如下：
取3D Flo Trix微载片(3D Flo Trix微载片购自北京华龛生物)放入干燥孔板的孔中，在孔板的孔与孔的间隙中加入RPMI培养基(pH 7.4，购自Gibco)，加入的RPMI培养基(pH 7.4)不超过所述间隙的边沿，然后将孔板放在温度为37℃、含有5％(v/v)CO 2的培养箱中放置12h进行预处理，预处理结束后，将U87细胞(购自中科院细胞库)以2*10^5个/片的接种量接种在经处理的3D Flo Trix微载片上，接种结束后，将U87细胞分为四组，四组分别为空白组、对照组1(替莫唑胺处理)、对照组2(纳脉刀处理)以及实验组(替莫唑胺+纳脉刀处理)，分组结束后，对各组U87细胞进行处理，其中：
空白组的处理方法为：不做任何处理；
对照组1的处理方法为：将U87细胞置于含有浓度为50μM的替莫唑胺的RPMI培养基(pH 7.4，含有10％FBS和1％双抗，v/v)中，于37℃的条件下孵育4h，孵育结束后，吸掉含有浓度为50μM的替莫唑胺的RPMI培养基(pH7.4，含有10％FBS和1％双抗，v/v)，将U87细胞置于RPMI培养基(pH 7.4，含有10％FBS和1％双抗)中，于37℃的条件下继续培养44h；
对照组2的处理方法为：用纳脉刀，对U87细胞于电压幅值为1000V、频率为500kHz、脉冲边沿的上升时间小于100ns的条件下消融15min，消融结束后，将U87细胞置于RPMI培养基(pH 7.4，含有10％FBS和1％双抗，v/v) 中，于37℃的条件下继续培养44h；
实验组的处理方法为：用纳脉刀，对U87细胞于电压幅值为1000V、频率为500kHz、脉冲边沿的上升时间小于100ns的条件下消融15min，消融结束后，将U87细胞置于含有浓度为25μM的替莫唑胺的RPMI培养基(pH 7.4，含有10％FBS和1％双抗，v/v)中，于37℃的条件下孵育4h，孵育结束后，吸掉含有浓度为25μM的替莫唑胺的RPMI培养基(pH 7.4，含有10％FBS和1％双抗，v/v)，将U87细胞置于RPMI培养基(pH 7.4，含有10％FBS和1％双抗，v/v)中，于37℃的条件下继续培养44h。
实验结束后，通过MTT细胞毒性实验检测各组U87细胞的细胞存活率，检测结果见图1。MTT细胞毒性实验过程为：将按照实验条件处理过的各组U87细胞接种在96孔板(1×10^4细胞/孔)中后，于37℃、含有5％(v/v)CO 2的培养箱中培养48h，培养结束后，在96孔板中加入含有10μL MTT的PBS溶液(5.0mg/mL)，于37℃、含有5％(v/v)CO 2的培养箱中继续孵育4h，孵育结束后，吸出上清液并在96孔板中加入150μL DMSO以溶解活细胞产生的紫色甲瓒，加入DMSO 10min后，用酶标仪(Multiskan FC)测量96孔板于570nm处的吸光度，并通过比较PBS孔的吸光度来获得细胞存活率(％)，数据表示为平均值±SD(n＝6)。MTT细胞毒性实验具体可参见文献：R.Supino,MTT Assays,in:S.O’Hare,C.K.Atterwill(Eds.),In Vitro Toxicity Testing Protocols,Humana Press,Totowa,NJ,1995,pp.137-149.
通过流式分析检测各组U87细胞凋亡的变化，检测结果见图2。流式分析实验过程为：将按照实验条件处理过的各组U87细胞接种在6孔板(2×10^5个细胞/孔)中后，于37℃、含有5％(v/v)CO 2的培养箱中培养24h，培养结束后，在6孔板中加入500μL 0.25％(m/v，g/100mL)胰酶，在800rpm下离心5min后收集细胞，将收集得到的细胞用PBS溶液(5.0mg/mL)清洗两遍后，重悬于300μL结合缓冲液中，在结合缓冲液中加入Annexin V-FITC(5μL)和PI(5μL)后，于25℃、黑暗条件下孵育15min，孵育结束后，使用流式细胞 仪对经染色的U87细胞进行分析。流式分析具体可参见文献：S.F.Ibrahim,G.van den Engh,Flow Cytometry and Cell Sorting,in:A.Kumar,I.Y.Galaev,B.Mattiasson(Eds.),Cell Separation:Fundamentals,Analytical and Preparative Methods,Springer Berlin Heidelberg,Berlin,Heidelberg,2007,pp.19-39.
通过细胞凋亡相关基因表达分析检测各组U87细胞凋亡(BCL-2)/自噬(Bax)/焦亡(Caspase3)因子基因水平的变化，检测结果见图3～5。细胞凋亡相关基因表达分析实验过程为：先提取经过处理的各组U87细胞RNA，然后使用反转录试剂盒反转录提取出的RNA，最后使用
Figure PCTCN2022081990-appb-000001
Premix Ex TaqTM II荧光定量PCR试剂盒对反转录得到cDNA进行处理，最后使用实时荧光定量PCR仪
Figure PCTCN2022081990-appb-000002
的96孔板上对经处理的cDNA进行分析。细胞凋亡相关基因表达分析具体可参见文献：A.S.Devonshire,R.Elaswarapu,C.A.Foy,Applicability of RNA standards for evaluating RT-qPCR assays and platforms,BMC Genomics 12(1)(2011)118.
由图1可知，单纳脉刀消融后细胞存活率为35％，单替莫唑胺(50μM)孵育后细胞存活率为22％，纳脉刀消融联合替莫唑胺(25μM)孵育后细胞存活率为14％。结果表明纳脉刀联合替莫唑胺可显著抑制细胞增殖，且联合治疗组替莫唑胺浓度仅是单替莫唑胺的一半就取得了不错的治疗效果。
由图2可知，单纳脉刀消融后细胞凋亡65％，单替莫唑胺(50μM)孵育后细胞凋亡83％，纳脉刀消融联合替莫唑胺(25μM)孵育后细胞凋亡93％。结果表明纳脉刀联合替莫唑胺可显著促进细胞凋亡，且效果远优于单纳脉刀消融和单替莫唑胺(50μM)孵育，且纳脉刀治疗后再用替莫唑胺治疗在取得显著促进凋亡的效果同时，可大大减少替莫唑胺的用量，可极大避免全身系统毒性。
由图3～5可知，经单纳脉刀消融或单替莫唑胺(50μM)孵育后，U87细胞中凋亡相关基因的表达水平发生了变化，其中，单纳脉刀消融后，U87细胞中Bax凋亡基因的表达水平是空白对照组的1.5倍，Caspase3基因的表达水平是空白对照组的1.7倍，同时，抑制凋亡基因BCL-2的表达水平降低，单替莫唑胺(50μM)孵育的治疗效果和单纳脉刀消融的治疗效果相当；与单纳脉刀消融 和单替莫唑胺(50μM)孵育相比，经纳脉刀消融联合替莫唑胺(25μM)孵育后，U87细胞中促凋亡基因BAX和Caspase的表达水平更高，其中，Bax凋亡基因的表达水平是空白对照组的2.2倍，Caspase3基因的表达水平是空白对照组的2.5倍，同时，抑制凋亡基因BCL-2表达水平的降低情况也更为明显。结果表明纳脉刀联合替莫唑胺可显著促进肿瘤细胞凋亡，有望成为一种新的脑胶质瘤治疗方法。
实验例2：脑胶质瘤细胞的凋亡实验
本实施例提供了脑胶质瘤细胞的凋亡实验，实验过程如下：
取3D Flo Trix微载片(3D Flo Trix微载片购自北京华龛生物)放入干燥孔板的孔中，在孔板的孔与孔的间隙中加入RPMI培养基(pH 7.4，购自Gibco)，加入的RPMI培养基(pH 7.4)不超过所述间隙的边沿，然后将孔板放在温度为37℃、含有5％(v/v)CO 2的培养箱中放置12h进行预处理，预处理结束后，将U87细胞(购自中科院细胞库)以2*10^5个/片的接种量接种在经处理的3D Flo Trix微载片上，接种结束后，将U87细胞分为四组，四组分别为空白组、对照组1(紫杉醇处理)、对照组2(纳脉刀处理)以及实验组(紫杉醇+纳脉刀处理)，分组结束后，对各组U87细胞进行处理，其中：
空白组的处理方法为：不做任何处理；
对照组1的处理方法为：将U87细胞置于含有浓度为5μM的紫杉醇的RPMI培养基(pH 7.4，含有10％FBS和1％双抗，v/v)中，于37℃的条件下孵育4h，孵育结束后，吸掉含有浓度为5μM的紫杉醇的RPMI培养基(pH 7.4，含有10％FBS和1％双抗，v/v)，将U87细胞置于RPMI培养基(pH 7.4，含有10％FBS和1％双抗)中，于37℃的条件下继续培养44h；
对照组2的处理方法为：用纳脉刀，对U87细胞于电压幅值为1000V、频率为500kHz、脉冲边沿的上升时间小于100ns的条件下消融15min，消融结束后，将U87细胞置于RPMI培养基(pH 7.4，含有10％FBS和1％双抗，v/v)中，于37℃的条件下继续培养44h；
实验组的处理方法为：用纳脉刀，对U87细胞于电压幅值为1000V、频率为500kHz、脉冲边沿的上升时间小于100ns的条件下消融15min，消融结束后，将U87细胞置于含有浓度为2μM的紫杉醇的RPMI培养基(pH 7.4，含有10％FBS和1％双抗，v/v)中，于37℃的条件下孵育4h，孵育结束后，吸掉含有浓度为2μM的紫杉醇的RPMI培养基(pH 7.4，含有10％FBS和1％双抗，v/v)，将U87细胞置于RPMI培养基(pH 7.4，含有10％FBS和1％双抗，v/v)中，于37℃的条件下继续培养44h。
实验结束后，通过MTT细胞毒性实验检测各组U87细胞的细胞存活率，检测结果见图6。MTT细胞毒性实验过程为：将按照实验条件处理过的各组U87细胞接种在96孔板(1×10^4细胞/孔)中后，于37℃、含有5％(v/v)CO 2的培养箱中培养48h，培养结束后，在96孔板中加入含有10μL MTT的PBS溶液(5.0mg/mL)，于37℃、含有5％(v/v)CO 2的培养箱中继续孵育4h，孵育结束后，吸出上清液并在96孔板中加入150μL DMSO以溶解活细胞产生的紫色甲瓒，加入DMSO 10min后，用酶标仪(Multiskan FC)测量96孔板于570nm处的吸光度，并通过比较PBS孔的吸光度来获得细胞存活率(％)，数据表示为平均值±SD(n＝6)。MTT细胞毒性实验具体可参见文献：R.Supino,MTT Assays,in:S.O’Hare,C.K.Atterwill(Eds.),In Vitro Toxicity Testing Protocols,Humana Press,Totowa,NJ,1995,pp.137-149.
通过流式分析检测各组U87细胞凋亡的变化，检测结果见图7。流式分析实验过程为：将按照实验条件处理过的各组U87细胞接种在6孔板(2×10^5个细胞/孔)中后，于37℃、含有5％(v/v)CO 2的培养箱中培养24h，培养结束后，在6孔板中加入500μL 0.25％(m/v，g/100mL)胰酶，在800rpm下离心5min后收集细胞，将收集得到的细胞用PBS溶液(5.0mg/mL)清洗两遍后，重悬于300μL结合缓冲液中，在结合缓冲液中加入Annexin V-FITC(5μL)和PI(5μL)后，于25℃、黑暗条件下孵育15min，孵育结束后，使用流式细胞仪对经染色的U87细胞进行分析。流式分析具体可参见文献：S.F.Ibrahim,G.van den Engh,Flow Cytometry and Cell Sorting,in:A.Kumar,I.Y.Galaev,B. Mattiasson(Eds.),Cell Separation:Fundamentals,Analytical and Preparative Methods,Springer Berlin Heidelberg,Berlin,Heidelberg,2007,pp.19-39.
通过细胞凋亡相关基因表达分析检测各组U87细胞凋亡(BCL-2)/自噬(Bax)/焦亡(Caspase3)因子基因水平的变化，检测结果见图8～10。细胞凋亡相关基因表达分析实验过程为：先提取经过处理的各组U87细胞RNA，然后使用反转录试剂盒反转录提取出的RNA，最后使用
Figure PCTCN2022081990-appb-000003
Premix Ex TaqTM II荧光定量PCR试剂盒对反转录得到cDNA进行处理，最后使用实时荧光定量PCR仪
Figure PCTCN2022081990-appb-000004
的96孔板上对经处理的cDNA进行分析。细胞凋亡相关基因表达分析具体可参见文献：A.S.Devonshire,R.Elaswarapu,C.A.Foy,Applicability of RNA standards for evaluating RT-qPCR assays and platforms,BMC Genomics 12(1)(2011)118.
由图6可知，单纳脉刀消融后细胞存活率为34％，单紫杉醇(5μM)孵育后细胞存活率为46％，纳脉刀消融联合紫杉醇(2μM)孵育后细胞存活率为14％。结果表明纳脉刀联合紫杉醇可显著抑制细胞增殖，且联合治疗组紫杉醇浓度仅是单紫杉醇的一半就取得了不错的治疗效果。
由图7可知，单纳脉刀消融后细胞凋亡69％，单紫杉醇(5μM)孵育后细胞凋亡53％，纳脉刀消融联合紫杉醇(2μM)孵育后细胞凋亡95％。结果表明纳脉刀联合紫杉醇可显著促进细胞凋亡，且效果远优于单纳脉刀消融和单紫杉醇(5μM)孵育，且纳脉刀治疗后再用紫杉醇治疗在取得显著促进凋亡的效果同时，可大大减少紫杉醇的用量，可极大避免全身系统毒性。
由图8～10可知，经单纳脉刀消融或单紫杉醇(5μM)孵育后，U87细胞中凋亡相关基因的表达水平发生了变化，其中，单纳脉刀消融后，U87细胞中Bax凋亡基因的表达水平是空白对照组的1.4倍，Caspase3基因的表达水平是空白对照组的1.7倍，同时，抑制凋亡基因BCL-2的表达水平降低，单紫杉醇(5μM)孵育的治疗效果和单纳脉刀消融的治疗效果相当；与单纳脉刀消融和单紫杉醇(5μM)孵育相比，经纳脉刀消融联合紫杉醇(2μM)孵育后，U87细胞中促凋亡基因BAX和Caspase的表达水平更高，其中，Bax凋亡基因的表达水平是 空白对照组的1.9倍，Caspase3基因的表达水平是空白对照组的2.8倍，同时，抑制凋亡基因BCL-2表达水平的降低情况也更为明显。结果表明纳脉刀联合紫杉醇可显著促进肿瘤细胞凋亡，有望成为一种新的脑胶质瘤治疗方法。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本申请的保护范围之中。

Claims (11)

  1. 一种促进肿瘤细胞凋亡的方法，其特征在于，所述方法为先用纳脉刀对肿瘤细胞进行消融，再将肿瘤细胞置于含有抗肿瘤药的孵育环境中进行孵育。
  2. 如权利要求1所述的促进肿瘤细胞凋亡的方法，其特征在于，所述肿瘤细胞为脑胶质瘤细胞、胰腺癌细胞、肝癌细胞、胆管癌细胞、胃癌细胞、肺癌细胞、乳腺癌细胞、肠癌细胞、骨癌细胞或前列腺癌细胞中的至少一种。
  3. 如权利要求1或2所述的促进肿瘤细胞凋亡的方法，其特征在于，所述抗肿瘤药为替莫唑胺、吉西他滨、白蛋白紫杉醇、长春新碱或盐酸阿霉素中的至少一种。
  4. 如权利要求1～3任一项所述的促进肿瘤细胞凋亡的方法，其特征在于，所述孵育环境中，抗肿瘤药的药物浓度为25nM～100μM。
  5. 如权利要求1～4任一项所述的促进肿瘤细胞凋亡的方法，其特征在于，所述孵育环境的pH为7.0～7.4。
  6. 如权利要求1～5任一项所述的促进肿瘤细胞凋亡的方法，其特征在于，所述孵育环境为生理盐水、缓冲液、血浆、组织液或培养基中的至少一种。
  7. 如权利要求1～6任一项所述的促进肿瘤细胞凋亡的方法，其特征在于，所述孵育的温度为36～38℃、时间为2～8h。
  8. 如权利要求1～7任一项所述的促进肿瘤细胞凋亡的方法，其特征在于，所述纳脉刀的电压幅值为500～10000V、频率为50～500kHz、消融时间为1～30min。
  9. 如权利要求8所述的促进肿瘤细胞凋亡的方法，其特征在于，所述纳脉刀的脉冲边沿的上升时间小于100ns。
  10. 权利要求1～9任一项所述的方法在促进肿瘤细胞凋亡中的应用。
  11. 如权利要求10所述的应用，其特征在于，所述肿瘤细胞为脑胶质瘤细胞、胰腺癌细胞、肝癌细胞、胆管癌细胞、胃癌细胞、肺癌细胞、乳腺癌细胞、肠癌细胞、骨癌细胞或前列腺癌细胞中的至少一种。
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