CN105748515A - Malaria serum with anti-tumor function and preparation method and application of malaria serum with anti-tumor function - Google Patents

Abstract

The invention discloses a malaria serum with an anti-tumor function and a preparation method and application of the malaria serum with the anti-tumor function.According to long-term arduous scientific experiments, the serum of patients with malaria has a certain anti-tumor function, and particularly the serum of patients with a syndrome of malaria and thrombocytopenia is effective in treatment or prevention of tumors, especially liver cancers, under specific concentration conditions.According to experiments, the serum has an evident apoptosis induction function on hepatoma cell strains (7721) cultured in vitro, and the hepatoma cell apoptosis induction ratio is increased along with increase of serum concentration.The malaria serum with the anti-tumor function and the preparation method and application of the malaria serum with the anti-tumor function provide theoretical and experimental bases for further researches on tumor cell apoptosis induction factors generated by plasmodia, breaks a new path for rediscovery pathogenesis of critical and cerebral malaria cases and epidemiologic researches of various plasmodium species and strains, and make new effort in application of the malaria serum or preparations thereof to preparation of medicines for treatment or prevention of the tumors, thereby having great scientific research values and significances.

Description

Malaria serum with anti-tumor effect and its preparation method and application

technical field

The invention belongs to the technical field of tumor treatment, and in particular relates to a malaria serum with anti-tumor effect and its preparation method and application.

Background technique

In 1927, the Nobel Prize in Medicine was awarded to Austrian psychiatrist Julius Wagner-Jauregg (1857-1940) for his invention of artificially infecting patients with General Paresis of the Insane GPI with Plasmodium. He was cured and relieved of the high fever caused by the patients who were helpless in the treatment of GPI diseases at that time. Therefore, he was called "the father of fever therapy" by the academic circles. Before the 1940s, "fever therapy" was widely used in the treatment of various difficult diseases at that time, such as tumors, syphilis and gonorrhea. It is still used in the treatment of tumors, and various physical heating methods have been developed to treat tumors, such as microwave thermal therapy and nano-thermal therapy in recent years. The treatment mechanism of this method is that tumor cells are easily killed at a temperature above 40°C, and people have a high fever of about 40°C after being infected with malaria parasites, which undoubtedly has a certain killing effect on tumor cells, but in addition to this physical effect outside.

Observation of clinical malaria cases found that about one-third of malaria cases had thrombocytopenia, especially in critically ill malaria patients caused by falciparum malaria infection and cerebral malaria patients, all had thrombocytopenia. Animal experiments have confirmed that when the experimental monkeys were infected with Plasmodium a few days later, the platelets began to decrease. Without drug treatment, the platelets remained at a low level for a long time. , after a few days of treatment with antimalarial drugs, the platelets returned to normal.

Since E.A. Carswell et al. discovered in 1975 that mice inoculated with BCG were injected with bacterial lipopolysaccharide, a substance that could cause hemorrhagic necrosis of various tumors appeared in the serum, and they named it tumor necrosis factor (tumor necrosis factor, TNF). This tumor necrosis factor is mainly produced by the body's activated macrophages, NK cells and T lymphocytes. Similarly, in the mid-1970s, the body of animals and humans infected with Plasmodium also produced tumor necrosis factor in foreign countries, but so far, domestic and foreign data are limited to studies on the toxicity of tumor necrosis factor produced by Plasmodium to Plasmodium However, there are no reports on the effects of tumor necrosis factor induced by general malaria parasites and tumor necrosis factors induced by malaria parasites that cause thrombocytopenia on various tumors.

Contents of the invention

The technical problem to be solved by the present invention is to provide a malaria serum with anti-tumor effect and its preparation method and application.

In order to solve the above technical problems, the present invention adopts the following technical scheme: the malaria serum with anti-tumor effect is derived from the serum of malaria patients.

Malaria patients are patients with malaria and thrombocytopenia.

Malaria is P. vivax or P. falciparum.

In the preparation method of the above-mentioned malaria serum with anti-tumor effect, after obtaining the whole blood of a malaria patient, the serum is obtained through centrifugation.

A serum preparation prepared from the above-mentioned malaria serum with anti-tumor effect.

The concentration of serum in this preparation is 0-16%

Application of the above-mentioned malaria serum or serum preparation in the preparation of medicaments for treating or preventing tumors.

The tumors mentioned above are liver cancer and gastric cancer.

Aiming at the gap in the research on the effect of tumor necrosis factor caused by malaria parasite infection on various tumor cells, the inventors conducted in-depth exploration and found through long-term and arduous scientific experiments that the serum of malaria patients has a certain anti-tumor effect. In particular, serum from patients with malaria and thrombocytopenia has a significant effect on treating or preventing tumors, especially liver cancer, under specific concentration conditions. Experiments have confirmed that the serum has an obvious effect of inducing apoptosis on the liver cancer cell line (7721) cultured in vitro, and the apoptosis rate of liver cancer cells increases with the increase of serum concentration. Accordingly, the inventor speculates that after the Plasmodium infects the body, it produces a biologically active factor that can induce early apoptosis (programmed cell death) of tumor cells in vitro during the development of the human body. This factor is different from tumor necrosis factor. It not only has an inhibitory effect on the white blood cells and platelets of the human body, but also has an obvious effect on inducing apoptosis. Because studies have shown that only in the case of malaria patients with thrombocytopenia can the serum induce tumor cell apoptosis, and the lower the patient's platelets, the higher the rate of tumor cell apoptosis induction. The present invention provides a theoretical and experimental basis for further research on tumor cell apoptosis-inducing factors produced by Plasmodium, and also opens up a new understanding of the pathogenesis of critical malaria and cerebral malaria cases and epidemiological research on various Plasmodium strains. It is a new idea, and at the same time, a new attempt has been made in the preparation of drugs for treating or preventing tumors by using malaria serum or serum preparations, which is of great scientific research value and significance.

Description of drawings

Figure 1 is a diagram of the early apoptosis of 7721 liver cancer cells induced by normal human serum. In the figure, the serum concentrations are 0%, 4%, 8%, 12%, 16%, and 20% from left to right and then from top to bottom.

Figure 2 is the result of early apoptosis of 7721 liver cancer cells induced by serum concentration in the same patient with malaria and thrombocytopenia. In the figure: the serum concentration from left to right and then from top to bottom is 0%, 4%, 8%, 12% , 16%.

Fig. 3 is a graph showing the results of early apoptosis of 7721 liver cancer cells induced by serum concentration in different patients with malaria and thrombocytopenia, in the figure: the serum concentration is 8% and 16% respectively.

Figure 4 is the results of the early apoptosis of 7721 liver cancer cells induced by the serum concentration of the same patient with malaria and normal platelets. In the figure: the serum concentration from left to right and then from top to bottom is 0%, 4%, 8%, 12% , 16%.

Fig. 5 is a graph showing the results of detecting the apoptosis of 7721 hepatocellular carcinoma induced by the serum of patients with malaria and thrombocytopenia by TUNEL method, in the figure: the magnification indicated by the arrow is 200X.

Fig. 6 is a graph showing the results of early apoptosis of 7901 gastric cancer cells induced by serum from patients with malaria and thrombocytopenia. In the figure: the serum concentrations are 0%, 4%, 8%, and 12% from left to right.

Figure 7 is a graph showing the results of early apoptosis of leukemia Jurkat cells induced by the serum of patients with malaria and thrombocytopenia. In the figure: the serum concentration is 0%, 4%, 8%, 12%, and 16% from left to right and then from top to bottom.

detailed description

Example 1 Experiment of Inducing Early Apoptosis of 7721 Liver Cancer Cells by Serum from Malaria Patients

1. Experimental materials

1.1 Materials

1.1.1 Collection of serum from malaria patients: extract whole blood from hospitalized malaria patients (including patients infected with different types of Plasmodium) accompanied by thrombocytopenia in blood routine examination.

1.1.2 Separation and storage of serum from malaria patients: after obtaining whole blood from malaria patients, centrifuge within a short time to sort and aliquot the serum and store it at low temperature for later use.

1.1.3 Malaria patient serum preparation before the experiment: before the experiment, the malaria patient serum stored at low temperature was taken out and warmed up at room temperature before being used for the experiment.

1.2.1 Cell line: 7721 liver cancer cells were provided by Shanghai Cell.

1.2.2 Reagents: 1640 culture medium: the improved 1640 culture medium was provided by Thermo Fisher Biochemicals (Beijing) Co., Ltd. (Thermo); fetal bovine serum: imported from New Zealand by Thermo Fisher Biochemicals ( Beijing) Co., Ltd. (Thermo) provided and inactivated sub-packaging in this laboratory; flow cytometry apoptosis kit was provided by ebioscience; TUNEL apoptosis kit was provided by Roche Reagent Company; trypsin was provided by Nanning Haotai Biotechnology Limited offer.

1.2.3 Equipment: CO2 incubator (HERAEUS, Japan); fluorescent inverted microscope (NIKON, Japan); flow cytometer (EpicsXL, Beckman Coulter, USA, acquisition and Mfa32 software analysis)

2 Experimental methods

2.1 Cell culture: resuscitate 7721 liver cancer cells and put them into the modified 1640 culture medium containing 10% fetal bovine serum for culture (at a temperature of 37°C, containing 5% CO2) for 48 hours, until the cells grow well (90%) and above the cells are Adhesive growth, only 0.25% trypsin was digested in the case of no pollution, divided into 6-well plate and cultured for 48 hours, waited for the cells to cover the entire bottom (more than 80% of the cells were adherent growth), washed twice with PBS, Then add the corresponding reagents according to the table.

Table 1 Serum needs to be added for each serum concentration in cell culture

2.2 Collect all the suspended cells and pour them into centrifuge tubes (10ml) marked with 8% and 16%, centrifuge at 2000r/2min, discard the supernatant, wash twice with PBS, then centrifuge at 2000r/2min, discard the supernatant, Resuspend, centrifuge at 2000r/2min, resuspend again, count cells (1X10 ⁶ )/ml, add corresponding reagents according to Table 2, keep away from light, put in ice bath, put on FACSCalibur within 1h, and perform flow cytometry blocking for 24 hours detection.

Table 2 The results of cell apoptosis detected by flow cytometry

2.3 TUNEL method to detect cell apoptosis experiment: prepare 0.1M citrate buffer solution (PH=6): 21.01g sodium citrate+1000mL distilled water. 0.1M Tris-HCL (PH=7.5): 12.1g Tris+100mL distilled water. Blocking solution: 0.1MTris-HCL8mL+fetal bovine serum 2mL+BSA0.3g. TUNEL reaction solution: Enzymesolution 20μL + Labelsolution 180μL. Aspirate the culture medium of the well plate, wash with PBS three times, immerse in 1ml of acetone at 4°C and fix in the dark for 10min. Wash with PBS, 1 min each time, 3 times. Add the prepared blocking solution dropwise into the well plate, add 200 μL dropwise to each tissue area, and incubate at room temperature for 30 min. Wash 3 times with PBS, 1 min each time. Add 200 μL of TUNEL reaction solution dropwise to each well, and incubate at 37°C for 60 min. Wash 3 times with PBS, 5min each time. The nuclei were stained with 0.5 μg/mL DAPI solution for 30 min at room temperature. Shake off the liquid, add anti-fluorescence quenching mounting medium dropwise to seal the slide. And observed and photographed under a fluorescent microscope, the blue fluorescence is the nucleus, and the green fluorescence is the positive signal of the apoptotic cells.

3 Experimental results

3.1 Quantitative detection of early apoptosis of 7721 liver cancer cells induced by serum from malaria patients by flow cytometry. The results showed that: normal human serum could not induce early apoptosis of 7721 liver cancer cells, as shown in Figure 1; serum from the same malaria patient combined with thrombocytopenia could induce 7721 Early apoptosis of liver cancer cells was more obvious with the increase of serum concentration, as shown in Figure 2; different malaria patients combined with thrombocytopenic serum could induce early apoptosis of 7721 liver cancer cells, as shown in Figure 3, malaria patients infected with Plasmodium and The platelet status is shown in Table 3 (the reference value of normal human platelet is (100-300)X10 ⁹ ); different malaria patients with normal platelet serum cannot induce early apoptosis of 7721 liver cancer cells, as shown in Figure 4 .

Table 3 Different types of malaria combined with thrombocytopenia and information on cell apoptosis ratio at different serum concentrations

3.2 The TUNEL method was used to detect the apoptosis of 7721 hepatocellular carcinoma induced by serum from patients with malaria and thrombocytopenia, as shown in Figure 5 .

Example 2 Serum from Malaria Patients Induced Early Apoptosis of 7901 Gastric Cancer Cells

1. Experimental materials

1.1 Materials

1.1.1 Collection of serum from malaria patients: extract whole blood from hospitalized malaria patients (including patients infected with different types of Plasmodium) accompanied by thrombocytopenia in blood routine examination.

1.1.2 Separation and storage of serum from malaria patients: after obtaining whole blood from malaria patients, centrifuge within a short time to sort and aliquot the serum and store it at low temperature for later use.

1.1.3 Malaria patient serum preparation before the experiment: before the experiment, the malaria patient serum stored at low temperature was taken out and warmed up at room temperature before being used for the experiment.

1.2.1 Cell line: 7901 gastric cancer cells were provided by Shanghai Cell.

1.2.2 Reagents: 1640 culture medium: the improved 1640 culture medium was provided by Thermo Fisher Biochemicals (Beijing) Co., Ltd. (Thermo); fetal bovine serum: imported from New Zealand by Thermo Fisher Biochemicals ( Beijing) Co., Ltd. (Thermo) provided and inactivated subpackaged in our laboratory; flow cytometry apoptosis kit was provided by ebioscience; trypsin was provided by Nanning Haotai Biotechnology Co., Ltd.

1.2.3 Equipment: CO2 incubator (HERAEUS, Japan); inverted microscope (NIKON, Japan); flow cytometer (EpicsXL, Beckman Coulter, USA, acquisition and Mfa32 software analysis)

2 Experimental methods

2.1 Cell culture: resuscitate 7901 gastric cancer cells and put them into modified 1640 culture medium containing 10% fetal bovine serum for culture (at a temperature of 37°C, containing 5% CO2) for 48 hours, until the cells grow well (90%) and above the cells are Adhesive growth, only 0.25% trypsin was digested in the case of no pollution, divided into 6-well plate and cultured for 48 hours, waited for the cells to cover the entire bottom (more than 80% of the cells were adherent growth), washed twice with PBS, Then add the corresponding reagents according to the table.

Table 4 Serum needs to be added for each serum concentration in cell culture

2.2 Collect all the suspended cells and pour them into centrifuge tubes (10ml) marked with 0%, 4%, 8%, and 12%, centrifuge at 2000r/2min, discard the supernatant, wash twice with PBS, and then centrifuge at 2000r/2min. 2min, discard the supernatant, resuspend, centrifuge at 2000r/2min, resuspend, count the cells (1X10 ⁶ )/ml, add the corresponding reagents in the following table, keep away from light, put it in an ice bath, and perform flow cytometry on FACSCalibur within 1h Quantitative detection at 48 hours of surgical blockade.

Table 5 The results of cell apoptosis detected by flow cytometry

3 Experimental results

3.1 Quantitative detection by flow cytometry Serum from malaria patients could not induce early apoptosis of 7901 gastric cancer cells, as shown in FIG. 6 .

Example 3 Serum from Malaria Patients Induced Jurkat Cells (Leukemia Cells) Early Apoptosis Experiment

1. Experimental materials

1.1 Materials

1.1.1 Collection of serum from malaria patients: extract whole blood from hospitalized malaria patients (including patients infected with different types of Plasmodium) accompanied by thrombocytopenia in blood routine examination.

1.1.2 Separation and storage of serum from malaria patients: after obtaining whole blood from malaria patients, centrifuge within a short time to sort and aliquot the serum and store it at low temperature for later use.

1.1.3 Malaria patient serum preparation before the experiment: before the experiment, the malaria patient serum stored at low temperature was taken out and warmed up at room temperature before being used for the experiment.

1.2.1 Cell line: Jurkat cells (leukemia cells) were provided by Shanghai Cell.

1.2.2 Reagents: 1640 culture medium: the improved 1640 culture medium was provided by Thermo Fisher Biochemicals (Beijing) Co., Ltd. (Thermo); fetal bovine serum: imported from New Zealand by Thermo Fisher Biochemicals ( Beijing) Co., Ltd. (Thermo) provided and inactivated sub-packaging in our laboratory; flow cytometry apoptosis kit was provided by ebioscience; trypsin was provided by Nanning Haotai Biotechnology Co., Ltd.

1.2.3 Equipment: CO2 incubator (HERAEUS, Japan); inverted microscope (NIKON, Japan); flow cytometer (EpicsXL, Beckman Coulter, USA, acquisition and Mfa32 software analysis)

2 Experimental methods

2.1 Cell culture: resuscitate Jurkat cells and put them into modified 1640 culture medium containing 10% fetal bovine serum for culture (at a temperature of 37°C, containing 5% CO2) for 48 hours until the cells grow well (90%) and there is no contamination Digest with 0.25% trypsin, divide into 6-well plates and culture for 48 hours, wait for the cells to cover the entire bottom (more than 80% of the cells are adherent growth), wash twice with PBS, and then add the corresponding reagents as shown in the table below .

Table 6 Serum needs to be added for each serum concentration in cell culture

2.2 Collect all the suspended cells and pour them into centrifuge tubes (10ml) marked with 0%, 4%, 8%, 12%, and 16%, centrifuge at 2000r/2min, discard the supernatant, wash twice with PBS, and then Centrifuge at 2000r/2min, discard the supernatant, resuspend, centrifuge at 2000r/2min, resuspend, count the cells (1X10 ⁶ )/ml, add the corresponding reagents in the following table, keep away from light, put it in an ice bath and put it on FACSCalibur within 1h Quantitative detection of 24-hour blocking by flow cytometry.

Table 7 The results of cell apoptosis detected by flow cytometry

3 Experimental results

3.1 Quantitative detection by flow cytometry Serum from malaria patients cannot induce early apoptosis of Jurkat cells, as shown in FIG. 7 .

Claims (8)

1. a malaria serum with antitumor action, it is characterised in that come from the serum of malaria patient.

2. the malaria serum with antitumor action according to claim 1, it is characterised in that: the artificial malaria of described malaria disease merges thrombocytopenic patient.

3. the malaria serum with antitumor action according to claim 2, it is characterised in that: described malaria is tertian malaria or subtertian malaria.

4. the preparation method described in claim 1 with the malaria serum of antitumor action, it is characterised in that after obtaining malaria patients blood, be performing centrifugal separation on obtaining serum.

5. there is described in claim 2 serum preparation that the malaria serum of antitumor action is made.

6. serum preparation according to claim 5, it is characterised in that in said preparation, the concentration of serum is 0-16%.

7. malaria serum described in claim 2 or serum preparation described in claim 5 application in the medicine of preparation treatment or prophylaxis of tumours.

8. application according to claim 7, it is characterised in that: described tumor is hepatocarcinoma, gastric cancer.