CN115678850A - Method for promoting tumor cell apoptosis - Google Patents
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Abstract
The invention relates to a method for promoting tumor cell apoptosis, and belongs to the technical field of biological medicines. The invention provides a method for promoting tumor cell apoptosis, which effectively overcomes the defect of poor effect of promoting the apoptosis of glioma cells by combining chemotherapy based on an antitumor drug and a naught knife based on high-frequency irreversible electroporation, wherein the effect of promoting the apoptosis of glioma cells by combining the chemotherapy and the naught knife is respectively improved by 10 percent and 28 percent compared with the effect of promoting the apoptosis of glioma cells by using the chemotherapy and the naught knife alone.
Description
Technical Field
The invention relates to a method for promoting tumor cell apoptosis, and belongs to the technical field of biological medicines.
Background
Brain gliomas are the most common primary brain malignancies arising from brain and spinal glia canceration, and are among the most lethal and refractory tumors of all malignant solid tumors. The incidence rate of the medicine accounts for 35.2 to 61.0 percent of intracranial tumors, is derived from the glioblasts, and has the characteristics of high incidence rate, high recurrence rate, high death rate and low cure rate. In the united states, there are a total of about 5 million patients with glioma and about 1 million newly diagnosed cases per year, with a 5-year survival rate of only 5% for the patient.
The life cycle and the quality of life of the patient with the brain glioma are closely related to the degree of surgical excision. The treatment of brain glioma is always one of the problems troubling neurosurgeons, and especially the surgical treatment of glioma in brain functional regions (cortical and subcortical pathways closely related to speech, motor and sensory functions) and low-grade glioma is one of the problems of neurosurgical clinical work, and the main contradiction is the choice between the degree of lesion excision and nerve function. Although there are currently a number of clinical treatment strategies for brain gliomas, the mean median survival of brain glioma patients worldwide is only 12-14 months.
At present, the common methods for treating brain glioma mainly comprise surgery, radiotherapy, chemotherapy, ablation technology and the like. The operation treatment is based on the growth characteristics of brain glioma, theoretically, the brain glioma cannot be completely resected by the operation, and some brain gliomas growing in important parts such as brainstem cannot be operated at all. Radiotherapy is almost a conventional treatment method for various types of gliomas, including an X-knife, a gamma-knife and the like, and the treatment scope of radiotherapy is relatively limited due to the positions and the sizes of tumors and the sensitivity of the tumors to rays, and the curative effect evaluation is different, for example, except that medulloblastoma is highly sensitive to radiotherapy and ependymoma is moderately sensitive to radiotherapy, other types of tumor cells are not sensitive to radiotherapy. It was observed that the prognosis for this fraction of tumor cells insensitive to radiotherapy was the same for those with non-radiotherapy. Furthermore, the effect of the necrosis caused by external radiation used in radiotherapy on brain function cannot be underestimated. Thus, it is presently believed that gliomas, particularly those of the malignant grade-III-IV or glioblastoma type, are not amenable to treatment with radiation therapy. Chemotherapy is limited by the blood-brain barrier (BBB and BBTB) which prevents drug delivery and drug toxicity and side effects, making it very difficult to exert limited therapeutic effects. The ablation technology mainly comprises radio frequency ablation, microwave ablation, cryoablation and nanoprobe (the frequency is generally between 5 and 10 kHz) ablation, and the ablation methods have great damage to important tissues such as blood vessels, nerves and the like, so the ablation technology is not suitable for treating brain glioma.
There is a great need to find a method for effectively treating brain glioma with less damage to important tissues such as blood vessels, nerves and the like.
Disclosure of Invention
In order to solve the problems, the invention provides a method for promoting tumor cell apoptosis, which comprises the steps of firstly ablating (IRE) tumor cells by a namai knife and then placing the tumor cells in an incubation environment containing an antitumor drug for incubation.
In one embodiment of the invention, the method comprises the steps of firstly ablating tumor cells by using a namai knife, then placing the tumor cells in an incubation environment containing an anti-tumor drug for incubation, and finally placing the tumor cells in an incubation environment without the anti-tumor drug for culture.
In one embodiment of the present invention, the tumor cell is at least one of a brain glioma cell, a pancreatic cancer cell, a liver cancer cell, a bile duct cancer cell, a stomach cancer cell, a lung cancer cell, a breast cancer cell, an intestinal cancer cell, a bone cancer cell, or a prostate cancer cell.
In one embodiment of the invention, the antineoplastic agent is at least one of temozolomide, gemcitabine, albumin paclitaxel, vincristine, or doxorubicin hydrochloride.
In one embodiment of the present invention, the concentration of the antineoplastic agent in the incubation environment is 25 nM-100 μ M.
In one embodiment of the invention, the pH of the incubation environment is between 7.0 and 7.4.
In one embodiment of the invention, the incubation environment is at least one of saline, buffer, plasma, or interstitial fluid.
In one embodiment of the invention, the incubation temperature is 36-38 ℃ and the incubation time is 2-8 h.
In one embodiment of the invention, the voltage of the nanometerial knife is 500-10000V, the frequency is 50-500 kHz, and the ablation time is 1-30 min.
The invention also provides application of the method in promoting tumor cell apoptosis.
In one embodiment of the present invention, the tumor cell is at least one of a brain glioma cell, a pancreatic cancer cell, a liver cancer cell, a bile duct cancer cell, a stomach cancer cell, a lung cancer cell, a breast cancer cell, an intestinal cancer cell, a bone cancer cell, or a prostate cancer cell.
The technical scheme of the invention has the following advantages:
the invention provides a method for promoting tumor cell apoptosis, which effectively overcomes the defect of poor effect of promoting the apoptosis of glioma cells by combining chemotherapy based on an antitumor drug and a naught knife based on high-frequency irreversible electroporation, wherein the effect of promoting the apoptosis of glioma cells by combining the chemotherapy and the naught knife is respectively improved by 10 percent and 28 percent compared with the effect of promoting the apoptosis of glioma cells by using the chemotherapy and the naught knife alone.
When the method is used for promoting the apoptosis of the glioma cells, the effect far superior to that when the chemotherapy drugs with high concentration (50 mu M of temozolomide and 5 mu M of paclitaxel) are singly used can be obtained by using the chemotherapy drugs with low concentration (25 mu M of temozolomide and 2 mu M of paclitaxel) and matching the naematome, so that the method for promoting the apoptosis of the glioma cells has the advantages of small dosage of the chemotherapy drugs and relatively low toxic and side effects, and has extremely high application prospect in the aspect of treating the glioma.
Compared with the traditional ablation technology, the high-frequency irreversible electroporation based on the nano-pulse knife used in the method has less damage to important tissues such as blood vessels, nerves and the like, so the method has extremely high application prospect in the aspect of treating brain glioma.
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FIG. 1: cell viability of U87 cells under different treatment conditions in experimental example 1.
FIG. 2: apoptosis of U87 cells under different treatment conditions in experimental example 1. In fig. 2, a: blank control; b: temozolomide (TMZ 50 μ M) treatment, C: vein receiving knife treatment and D: naematome was treated in combination with temozolomide (TMZ 25 μ M).
FIG. 3: expression of the BCL-2 gene in U87 cells under different treatment conditions in Experimental example 1.
FIG. 4: expression of BAX Gene in U87 cells under different treatment conditions in Experimental example 1.
FIG. 5: the expression of Caspase-3 gene in U87 cells under different treatment conditions in Experimental example 1.
FIG. 6: cell viability of U87 cells under different treatment conditions in experimental example 2.
FIG. 7: apoptosis of U87 cells under different treatment conditions in experimental example 2. In fig. 2, a: blank control; b: paclitaxel (PTX 5 μ M) treatment, C: vein receiving knife treatment and D: nanmaidsets were treated with paclitaxel (PTX 2. Mu.M).
FIG. 8: expression of the BCL-2 gene in U87 cells under different treatment conditions in Experimental example 2.
FIG. 9: expression of BAX Gene in U87 cells under different treatment conditions in Experimental example 2.
FIG. 10: the expression of Caspase-3 gene in U87 cells under different treatment conditions in Experimental example 2.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The following examples do not show specific experimental procedures or conditions, and can be performed according to the procedures or conditions of the conventional experimental procedures described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
Example 1: method for promoting brain glioma cell apoptosis
The embodiment provides a method for promoting apoptosis of brain glioma cells, which comprises the following steps: a namai knife (high-frequency steep pulse tumor therapeutic apparatus, which is described in patent application text with the publication number of CN 107809184A) is used for ablating tumor cells for 15min under the conditions of 1000V voltage and 500kHz frequency, after the ablation is finished, the brain glioma cells are placed in an RPMI culture medium (pH 7.4) containing temozolomide with the concentration of 50 mu M and incubated for 4h under the condition of 37 ℃, after the incubation is finished, the RPMI culture medium (pH 7.4) containing the temozolomide with the concentration of 50 mu M is sucked off, the brain glioma cells are placed in the RPMI culture medium (pH 7.4) and cultured for 44h under the condition of 37 ℃.
Example 2: method for promoting brain glioma cell apoptosis
The embodiment provides a method for promoting apoptosis of brain glioma cells, which comprises the following steps: a tumor cell is ablated for 15min under the conditions of 1000V voltage and 500kHz by using a namai knife (high-frequency steep pulse tumor therapeutic apparatus which is described in the patent application text with the publication number of CN 107809184A), after the ablation is finished, the brain glioma cell is placed in an RPMI culture medium (pH 7.4) containing temozolomide with the concentration of 25 mu M, incubated for 4h under the condition of 37 ℃, after the incubation is finished, the RPMI culture medium (pH 7.4) containing the temozolomide with the concentration of 25 mu M is sucked off, the brain glioma cell is placed in the RPMI culture medium (pH 7.4), and cultured for 44h under the condition of 37 ℃.
Example 3: method for promoting brain glioma cell apoptosis
This example provides a method for promoting apoptosis of glioma cells, the method comprising: a vein knife (high-frequency steep pulse tumor therapeutic apparatus, which is described in patent application text with publication number CN 107809184A) is used for ablating tumor cells for 15min under the conditions of voltage of 1000V and frequency of 500kHz, after the ablation is finished, the brain glioma cells are placed in an RPMI culture medium (pH 7.4) containing albumin paclitaxel with the concentration of 5 mu M, the brain glioma cells are incubated for 4h under the condition of 37 ℃, after the incubation is finished, the RPMI culture medium (pH 7.4) containing albumin paclitaxel with the concentration of 5 mu M is sucked off, the brain glioma cells are placed in the RPMI culture medium (pH 7.4), and the brain glioma cells are cultured for 44h under the condition of 37 ℃.
Example 4: method for promoting brain glioma cell apoptosis
The embodiment provides a method for promoting apoptosis of brain glioma cells, which comprises the following steps: a vein knife (high-frequency steep pulse tumor therapeutic apparatus, which is described in patent application text with publication number CN 107809184A) is used for ablating tumor cells for 15min under the conditions of voltage of 1000V and frequency of 500kHz, after the ablation is finished, the brain glioma cells are placed in an RPMI culture medium (pH 7.4) containing albumin paclitaxel with the concentration of 2 mu M, the brain glioma cells are incubated for 4h under the condition of 37 ℃, after the incubation is finished, the RPMI culture medium (pH 7.4) containing albumin paclitaxel with the concentration of 2 mu M is sucked off, the brain glioma cells are placed in the RPMI culture medium (pH 7.4), and the brain glioma cells are cultured for 44h under the condition of 37 ℃.
Example 5: method for promoting brain glioma cell apoptosis
The embodiment provides a method for promoting apoptosis of brain glioma cells, which comprises the following steps: a tumor cell is ablated for 15min under the conditions of 1000V voltage and 500kHz by using a namai knife (high-frequency steep pulse tumor therapeutic apparatus, which is described in the patent application text with the publication number of CN 107809184A), after the ablation is finished, the brain glioma cell is placed in an RPMI culture medium (pH 7.4) containing gemcitabine with the concentration of 100 mu M, the brain glioma cell is incubated for 4h under the condition of 37 ℃, after the incubation is finished, the RPMI culture medium (pH 7.4) containing gemcitabine with the concentration of 100 mu M is sucked off, the brain glioma cell is placed in the RPMI culture medium (pH 7.4), and the brain glioma cell is continuously cultured for 44h under the condition of 37 ℃.
Example 6: method for promoting brain glioma cell apoptosis
The embodiment provides a method for promoting apoptosis of brain glioma cells, which comprises the following steps: a tumor cell is ablated for 15min under the conditions of 1000V voltage and 500kHz by using a namai knife (high-frequency steep pulse tumor therapeutic apparatus, which is described in the patent application text with the publication number of CN 107809184A), after the ablation is finished, the brain glioma cell is placed in an RPMI culture medium (pH 7.4) containing adriamycin hydrochloride with the concentration of 1 mu M, incubated for 4h under the condition of 37 ℃, after the incubation is finished, the RPMI culture medium (pH 7.4) containing the adriamycin hydrochloride with the concentration of 1 mu M is sucked off, the brain glioma cell is placed in the RPMI culture medium (pH 7.4), and the brain glioma cell is cultured for 44h under the condition of 37 ℃.
Example 7: method for promoting brain glioma cell apoptosis
The embodiment provides a method for promoting apoptosis of brain glioma cells, which comprises the following steps: a namai knife (high-frequency steep pulse tumor therapeutic apparatus, which is described in patent application text with publication number CN 107809184A) is used for ablating tumor cells for 15min under the conditions of voltage of 1000V and frequency of 500kHz, after the ablation is finished, placing brain glioma cells in an RPMI culture medium (pH 7.4) containing vincristine with concentration of 0.5 mu M, incubating for 4h under the condition of 37 ℃, after the incubation is finished, absorbing the RPMI culture medium (pH 7.4) containing vincristine with concentration of 0.5 mu M, placing the brain glioma cells in the RPMI culture medium (pH 7.4), and continuously culturing for 44h under the condition of 37 ℃.
Experimental example 1: apoptosis assay for brain glioma cells
This example provides an apoptosis assay for brain glioma cells, the experimental procedure is as follows:
placing 3D Flo Trix microchips (3D Flo Trix microchips are obtained from Beijing Hua niche organisms) into the wells of a dry well plate, adding RPMI medium (pH 7.4, obtained from Gibco) into the gap between the wells of the plate, the added RPMI medium (pH 7.4) not exceeding the edge of the gap, and placing the plate at 37 deg.C and 5% (v/v) CO 2 The culture box is placed for 12 hours for pretreatment, after the pretreatment is finished, the U87 cells (purchased from a Chinese academy cell bank) are inoculated on the treated 3D Flo Trix micro-slide glass in the inoculation amount of 2 x 10^5 cells/slide, after the inoculation is finished, the U87 cells are divided into four groups, wherein the four groups are respectively a blank group, a control group 1 (temozolomide treatment), a control group 2 (naematome treatment) and an experimental group (temozolomide + naematome treatment), and after the grouping is finished, the U87 cells of each group are treated, wherein:
the blank group processing method comprises the following steps: no treatment is carried out;
the treatment method of the control group 1 was: placing U87 cells in RPMI medium (pH 7.4, containing 10% FBS and 1% diabody, v/v) containing temozolomide at a concentration of 50 μ M, incubating for 4h at 37 ℃, after the incubation is finished, blotting the RPMI medium (pH 7.4, containing 10% FBS and 1% diabody, v/v) containing temozolomide at a concentration of 50 μ M, placing the U87 cells in RPMI medium (pH 7.4, containing 10% FBS and 1% diabody), and continuing the incubation for 44h at 37 ℃;
the control group 2 was treated by the following method: ablating U87 cells with a namai knife under the conditions of 1000V voltage and 500kHz for 15min, placing the U87 cells in RPMI medium (pH 7.4, containing 10% FBS and 1% double antibody, V/V) after ablation, and continuing culturing at 37 ℃ for 44h;
the treatment method of the experimental group is as follows: the U87 cells were ablated for 15min with a nanomicer at a voltage of 1000V and a frequency of 500kHz, after the ablation, the U87 cells were placed in RPMI medium (pH 7.4 containing 10% FBS and 1% diabody, V/V) containing temozolomide at a concentration of 25 μ M, incubated for 4h at 37 ℃, after the incubation was completed, the RPMI medium (pH 7.4 containing 10% FBS and 1% diabody, V/V) containing temozolomide at a concentration of 25 μ M was aspirated, the U87 cells were placed in RPMI medium (pH 7.4 containing 10% FBS and 1% diabody, V/V), and the culture was continued for 44h at 37 ℃.
After the experiment is finished, the cell survival rate of each group of U87 cells is detected through an MTT cytotoxicity experiment, and the detection result is shown in figure 1. The MTT cytotoxicity experimental process is as follows: each group of U87 cells treated according to the experimental conditions was seeded in 96-well plates (1X 10^4 cells/well) and contained 5% (v/v) CO at 37 ℃ 2 After the culture was completed, a PBS solution (5.0 mg/mL) containing 10. Mu.L of MTT was added to a 96-well plate and the mixture was incubated at 37 ℃ with 5% (v/v) CO 2 After the incubation was completed, the supernatant was aspirated and 150 μ L of DMSO was added to the 96-well plate to dissolve purple formazan produced from living cells, and after 10min of DMSO addition, absorbance at 570nm of the 96-well plate was measured using a microplate reader (Multiskan FC), and cell survival rate (%) was obtained by comparing absorbance of PBS wells, and data were expressed as mean ± SD (n = 6). MTT cytotoxicity assays can be found in particular in the literature: R.Supino, MTT Assays, in: S.O' Hare, C.K.Atterwire (eds.), in Vitro sensitivity Testing Protocols, humanaPress, totowa, NJ,1995, pp.137-149.
The change of apoptosis of the U87 cells in each group is detected by flow analysis, and the detection result is shown in figure 2. The flow analysis experimental process is as follows: each group of U87 cells treated according to the experimental conditions was seeded in 6-well plates (2X 10^5 cells/well) containing 5% (v/v) at 37 ℃)CO 2 After the culture was completed, 500. Mu.L of 0.25% (m/v, g/100 mL) pancreatin was added to a 6-well plate, the cells were collected after centrifugation at 800rpm for 5min, the collected cells were washed twice with PBS solution (5.0 mg/mL), and then resuspended in 300. Mu.L of binding buffer, annexinV-FITC (5. Mu.L) and PI (5. Mu.L) were added to the binding buffer, and then incubated at 25 ℃ in the dark for 15min, and after the incubation was completed, the stained U87 cells were analyzed using a flow cytometer. Flow analysis can be found in particular in the literature: ibrahim, G.van den Engh, flow Cytometry and Cell Sorting, in A.Kumar, I.Y.Galaev, B.Mattisson (eds.), cell Separation, fundamentals, analytical and Preparative Methods, springer Berlin Heidelberg, berlin, heidelberg,2007, pp.19-39.
The change of gene level of U87 apoptosis (BCL-2)/autophagy (Bax)/apoptosis (Caspase 3) factors of each group is detected by analyzing the expression of apoptosis related genes, and the detection result is shown in figures 3-5. The experimental process of apoptosis related gene expression analysis is as follows: extracting the processed group of U87 cell RNA, then reverse transcribing the extracted RNA by using a reverse transcription kit, and finally using
Treating cDNA obtained by reverse transcription with Premix Ex TaqTM II fluorescent quantitative PCR kit, and finally using real-time fluorescent quantitative PCR instrument
The processed cDNA was analyzed on 96-well plates of 480 Sytem. The expression analysis of apoptosis-related genes can be found in the literature: A.S. Devonshire, R.Elaswarapu, C.A.Foy, application of RNA standards for evaluating RT-qPCR assays and platforms, BMC Genomics 12 (1) (2011) 118.
As shown in FIG. 1, the cell survival rate of Shan Na after vein-knife ablation is 35%, the cell survival rate of single temozolomide (50 μ M) after incubation is 22%, and the cell survival rate of vein-knife ablation combined with temozolomide (25 μ M) after incubation is 14%. The results show that the naemamectin benzoate and temozolomide can obviously inhibit cell proliferation, and good treatment effect can be achieved when the concentration of the temozolomide in a combined treatment group is only half of that of the temozolomide.
As shown in FIG. 2, shan Na were apoptotic 65% after vein-knife ablation, 83% after single temozolomide (50 μ M) incubation, and 93% after vein-knife ablation and temozolomide (25 μ M) incubation. The results show that the combination of the naematolite and the temozolomide can obviously promote apoptosis, the effect is far superior to Shan Na pulse ablation and mono-temozolomide (50 mu M) incubation, and the temozolomide treatment is carried out after the naematolite treatment, so that the effect of obviously promoting apoptosis is achieved, the using amount of the temozolomide can be greatly reduced, and the systemic toxicity of the whole body can be greatly avoided.
As can be seen from fig. 3 to 5, after ablation with a monad knife or incubation with single temozolomide (50 μ M), the expression level of the apoptosis-related gene in the U87 cell changed, wherein after ablation with a Shan Na knife, the expression level of the Bax apoptosis gene in the U87 cell was 1.5 times that of the blank control group, and the expression level of the Caspase3 gene was 1.7 times that of the blank control group, and at the same time, the expression level of the apoptosis-inhibiting gene BCL-2 was reduced, and the therapeutic effect of incubation with single temozolomide (50 μ M) was equivalent to that of Shan Na pulse knife ablation; compared with single namai ablation and single temozolomide (50 mu M) incubation, the expression levels of the pro-apoptotic genes BAX and Caspase in the U87 cells are higher after the namai ablation and temozolomide (25 mu M) incubation, wherein the expression level of the Bax apoptotic genes is 2.2 times of that of a blank control group, the expression level of the Caspase3 genes is 2.5 times of that of the blank control group, and meanwhile, the reduction of the expression level of the apoptosis inhibiting gene BCL-2 is more obvious. The results show that the naematole and the temozolomide can obviously promote the apoptosis of tumor cells, and the naematole and the temozolomide are expected to become a new treatment method for the brain glioma.
Experimental example 2: apoptosis assay for brain glioma cells
This example provides an apoptosis assay for brain glioma cells, the procedure being as follows:
placing 3D Flo Trix microchips (3D Flo Trix microchips are obtained from Beijing Hua niche organisms) into the wells of a dry well plate, adding RPMI medium (pH 7.4, obtained from Gibco) into the gap between the wells of the plate, the added RPMI medium (pH 7.4) not exceeding the edge of the gap, and placing the plate in the plateAt a temperature of 37 ℃ and containing 5% (v/v) CO 2 The incubator is placed for 12 hours for pretreatment, after the pretreatment is finished, the U87 cells (purchased from a cell bank of a Chinese academy) are inoculated on the treated 3D Flo Trix micro-slide by the inoculation amount of 2 x 10^5 cells/slide, after the inoculation is finished, the U87 cells are divided into four groups, wherein the four groups are respectively a blank group, a control group 1 (paclitaxel treatment), a control group 2 (vein occlusion knife treatment) and an experimental group (paclitaxel + vein occlusion knife treatment), and after the grouping is finished, the U87 cells of each group are treated, wherein:
the blank group processing method comprises the following steps: no treatment is carried out;
the treatment method of the control group 1 was: placing U87 cells in RPMI medium (pH 7.4, containing 10 FBS and 1% diabody, v/v) containing 5 μ M paclitaxel, incubating for 4h at 37 deg.C, after the incubation is finished, blotting the RPMI medium (pH 7.4, containing 10 FBS and 1% diabody, v/v) containing 5 μ M paclitaxel, placing U87 cells in RPMI medium (pH 7.4, containing 10 FBS and 1% diabody), and continuing culturing for 44h at 37 deg.C;
the control group 2 was treated by the following method: ablating U87 cells with a naughty knife under the conditions of 1000V voltage and 500kHz for 15min, placing the U87 cells in RPMI medium (pH7.4, containing 10% FBS and 1% double antibody, V/V) after ablation, and continuing culturing at 37 ℃ for 44h;
the treatment method of the experimental group is as follows: the U87 cells were ablated with a nanometer at a frequency of 500kHz and a voltage of 1000V for 15min, after the ablation, the U87 cells were placed in RPMI medium (pH 7.4 containing 10% FBS and 1% diabody, V/V) containing paclitaxel at a concentration of 2. Mu.M, incubated at 37 ℃ for 4h, after the incubation was completed, the RPMI medium (pH 7.4 containing 10% FBS and 1% diabody, V/V) containing paclitaxel at a concentration of 2. Mu.M was aspirated, the U87 cells were placed in RPMI medium (pH 7.4 containing 10% FBS and 1% diabody, V/V), and the culture was continued at 37 ℃ for 44h.
After the experiment is finished, the cell survival rate of each group of U87 cells is detected through an MTT cytotoxicity experiment, and the detection result is shown in figure 6. The MTT cytotoxicity experimental process is as follows: groups treated according to the experimental conditionsU87 cells seeded in 96-well plates (1X 10^4 cells/well) at 37 ℃ with 5% (v/v) CO 2 After the culture was completed, a PBS solution (5.0 mg/mL) containing 10. Mu.L of MTT was added to a 96-well plate and the mixture was incubated at 37 ℃ with 5% (v/v) CO 2 After the incubation was completed, the supernatant was aspirated and 150 μ L of DMSO was added to the 96-well plate to dissolve purple formazan produced from living cells, and after 10min of DMSO addition, absorbance at 570nm of the 96-well plate was measured using a microplate reader (Multiskan FC), and cell survival rate (%) was obtained by comparing absorbance of PBS wells, and data were expressed as mean ± SD (n = 6). MTT cytotoxicity assays can be found in particular in the literature: R.Supino, MTT Assays, in: S.O' Hare, C.K.Atterwire (eds.), in Vitro sensitivity Testing Protocols, humanaPress, totowa, NJ,1995, pp.137-149.
The change of apoptosis of each group of U87 cells was detected by flow analysis, and the detection results are shown in FIG. 7. The flow analysis experimental process is as follows: each group of U87 cells treated according to the experimental conditions was seeded in 6-well plates (2X 10^5 cells/well) containing 5% (v/v) CO at 37 ℃ 2 After the culture was completed, 500. Mu.L of 0.25% (m/v, g/100 mL) pancreatin was added to a 6-well plate, the cells were collected after centrifugation at 800rpm for 5min, the collected cells were washed twice with PBS solution (5.0 mg/mL), and then resuspended in 300. Mu.L of binding buffer, annexinV-FITC (5. Mu.L) and PI (5. Mu.L) were added to the binding buffer, and then incubated at 25 ℃ in the dark for 15min, and after the incubation was completed, the stained U87 cells were analyzed using a flow cytometer. Flow analysis can be found in particular in the literature: ibrahim, G.van den Engh, flow Cytometry and Cell Sorting, in A.Kumar, I.Y.Galaev, B.Mattisson (eds.), cell Separation, fundamentals, analytical and Preparative Methods, springer Berlin Heidelberg, berlin, heidelberg,2007, pp.19-39.
The change of gene level of U87 apoptosis (BCL-2)/autophagy (Bax)/apoptosis (Caspase 3) factors of each group is detected by analyzing and analyzing the expression of apoptosis related genes, and the detection result is shown in figures 8-10. The experimental process of apoptosis related gene expression analysis is as follows: extracting the processed RNA of each group of U87 cells, and then performing reverse transcription by using a reverse transcription kit to extractThe RNA of (1), and final use
Treating cDNA obtained by reverse transcription with Premix Ex TaqTM II fluorescent quantitative PCR kit, and finally using real-time fluorescent quantitative PCR instrument
The treated cDNA was analyzed in a 96-well plate. The expression analysis of apoptosis-related genes can be found in the literature: A.S. Devonshire, R.Elaswarapu, C.A.Foy, application of RNA standards for evaluating RT-qPCR assays and platforms, BMC genetics 12 (1) (2011) 118.
As shown in FIG. 6, the cell survival rate of Shan Na after vein-knife ablation is 34%, the cell survival rate of single paclitaxel (5 μ M) after incubation is 46%, and the cell survival rate of nano vein-knife ablation combined paclitaxel (2 μ M) after incubation is 14%. The results show that the namai knife and the paclitaxel can obviously inhibit cell proliferation, and the paclitaxel concentration of the combined treatment group is only half of that of the single paclitaxel, so that good treatment effect is achieved.
As shown in FIG. 7, the apoptosis of Shan Na after vein-knife ablation is 69%, the apoptosis of 53% after single paclitaxel (5 μ M) incubation, and the apoptosis of 95% after vein-knife ablation and paclitaxel (2 μ M) incubation. The result shows that the combination of the naematome and the paclitaxel can obviously promote the apoptosis, the effect is far superior to Shan Na vein knife ablation and single paclitaxel (5 mu M) incubation, and the paclitaxel treatment after the naematome treatment can obtain the effect of obviously promoting the apoptosis, simultaneously greatly reduce the dosage of the paclitaxel and greatly avoid the systemic toxicity of the whole body.
As can be seen from fig. 8 to 10, after ablation with a single nanomesh knife or incubation with single paclitaxel (5 μ M), the expression level of the apoptosis-related gene in the U87 cells changed, wherein after ablation with a Shan Na pulesh knife, the expression level of Bax apoptosis gene in the U87 cells was 1.4 times that of the blank control group, and the expression level of Caspase3 gene was 1.7 times that of the blank control group, and at the same time, the expression level of the apoptosis-inhibiting gene BCL-2 was reduced, and the therapeutic effect of incubation with single paclitaxel (5 μ M) was equivalent to that of Shan Na pulesh ablation; compared with single namai knife ablation and single paclitaxel (5 mu M) incubation, the expression levels of the apoptosis-promoting genes BAX and Caspase in the U87 cells are higher after the namai knife ablation and paclitaxel (2 mu M) incubation, wherein the expression level of the Bax apoptosis gene is 1.9 times of that of a blank control group, the expression level of the Caspase3 gene is 2.8 times of that of the blank control group, and meanwhile, the reduction of the expression level of the apoptosis-inhibiting gene BCL-2 is more obvious. The results show that the combination of the naematology scalpel and the paclitaxel can obviously promote the apoptosis of tumor cells, and is expected to become a new treatment method for the brain glioma.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. This need not be, nor should it be exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. A method for promoting tumor cell apoptosis is characterized in that a nanometerknife is used for ablating tumor cells, and then the tumor cells are placed in an incubation environment containing anti-tumor drugs for incubation.
2. The method according to claim 1, wherein the tumor cell is at least one of a brain glioma cell, a pancreatic cancer cell, a liver cancer cell, a bile duct cancer cell, a stomach cancer cell, a lung cancer cell, a breast cancer cell, an intestinal cancer cell, a bone cancer cell, or a prostate cancer cell.
3. The method of promoting apoptosis in tumor cells of claim 1 or 2, wherein said antineoplastic agent is at least one of temozolomide, gemcitabine, albumin paclitaxel, vincristine or doxorubicin hydrochloride.
4. The method of promoting apoptosis in tumor cells according to any one of claims 1 to 3, wherein the concentration of the antineoplastic agent in the incubation environment is from 25nM to 100. Mu.M.
5. The method of promoting apoptosis in tumor cells of any one of claims 1 to 4, wherein the incubation environment has a pH of from 7.0 to 7.4.
6. The method of promoting apoptosis in tumor cells according to any one of claims 1 to 5, wherein the incubation environment is at least one of saline, buffer, plasma, interstitial fluid or culture medium.
7. The method for promoting apoptosis of tumor cells according to any one of claims 1 to 6, wherein the incubation is carried out at a temperature of 36 to 38 ℃ for a period of 2 to 8 hours.
8. The method for promoting apoptosis of tumor cells according to any one of claims 1 to 7, wherein the nanoartery knife has a voltage of 500 to 10000V, a frequency of 50 to 500kHz, and an ablation time of 1 to 30min.
9. Use of the method of any one of claims 1 to 8 for promoting apoptosis in tumor cells.
10. The use of claim 9, wherein the tumor cell is at least one of a brain glioma cell, a pancreatic cancer cell, a liver cancer cell, a bile duct cancer cell, a stomach cancer cell, a lung cancer cell, a breast cancer cell, an intestinal cancer cell, a bone cancer cell, or a prostate cancer cell.
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