WO2022165794A2 - 一种抑制肿瘤干细胞的方法及调控肿瘤血管正常化的方法 - Google Patents

一种抑制肿瘤干细胞的方法及调控肿瘤血管正常化的方法 Download PDF

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WO2022165794A2
WO2022165794A2 PCT/CN2021/075770 CN2021075770W WO2022165794A2 WO 2022165794 A2 WO2022165794 A2 WO 2022165794A2 CN 2021075770 W CN2021075770 W CN 2021075770W WO 2022165794 A2 WO2022165794 A2 WO 2022165794A2
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tumor
group
hyperbaric oxygen
mice
nano
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PCT/CN2021/075770
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French (fr)
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李子福
杨祥良
刘欣
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华中科技大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61G—TRANSPORT, PERSONAL CONVEYANCES, OR ACCOMMODATION SPECIALLY ADAPTED FOR PATIENTS OR DISABLED PERSONS; OPERATING TABLES OR CHAIRS; CHAIRS FOR DENTISTRY; FUNERAL DEVICES
    • A61G10/00—Treatment rooms or enclosures for medical purposes
    • A61G10/02—Treatment rooms or enclosures for medical purposes with artificial climate; with means to maintain a desired pressure, e.g. for germ-free rooms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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  • the present invention relates to the field of tumor treatment, and more particularly, to a method for inhibiting tumor stem cells and a method for regulating the normalization of tumor blood vessels.
  • Cancer stem cells are a kind of cells with self-renewal ability and multi-directional differentiation potential. They are the root of tumor growth and the "initiating cells" of tumor occurrence, metastasis and recurrence. The formation of tumor stem cells is one of the reasons for the failure of tumor cells to treat chemotherapy drugs.
  • tumors develop their own functional blood vessels that differ from the normal vasculature.
  • Tumors are highly vascular, characterized by abnormal vascular density, irregular vascular contours, incomplete or absent basement membrane and endothelial layers, and a lack of pericytes and smooth muscle.
  • tumor vascular function is highly abnormal, including unstable blood flow velocity and direction, high vascular resistance, high vascular fragility, red blood cell stasis, leukocyte adhesion, platelet aggregation, and tumor cell occlusion of blood vessels. These abnormal functions make it difficult for drugs to be delivered to the interior of the tumor, thereby protecting cells deep in the tumor and cancer stem cells from being killed by the drug.
  • the present invention provides a method for inhibiting tumor stem cells and a method for regulating the normalization of tumor blood vessels. It can improve the efficacy of tumor chemotherapy and solve the problem of poor efficacy of clinical chemotherapy drugs.
  • the present invention provides a method for reducing the proportion of stem cells in tumor tissue, and hyperbaric oxygen therapy is performed on tumor patients to reduce the proportion of stem cells in the tumor tissue of the tumor patient; the hyperbaric oxygen therapy is specifically: The patient was placed in a hyperbaric oxygen environment for treatment.
  • the present invention also provides a method for regulating the normalization of tumor tissue blood vessels, and performing hyperbaric oxygen therapy on tumor patients to regulate and promote the normalization of tumor tissue blood vessels in tumor patients; the hyperbaric oxygen therapy specifically includes: placing tumor patients under high pressure treatment in an oxygen environment.
  • hyperbaric oxygen therapy is performed on tumor patients, that is, tumor patients are placed in a hyperbaric oxygen environment for treatment.
  • tumor patients are placed in a hyperbaric oxygen environment for treatment.
  • hyperbaric oxygen therapy on tumor patients can significantly reduce the proportion of stem cells in tumor tissue.
  • a certain embodiment In 4T1 breast cancer in situ mice treated with hyperbaric oxygen, the ratio of tumor stem cells in the mice was reduced by 57% compared with the control group without hyperbaric oxygen treatment.
  • the present invention combines hyperbaric oxygen therapy with intravenous injection of nano-chemotherapy drugs to perform anti-tumor therapy on tumor patients.
  • hyperbaric oxygen therapy on tumor patients can further reduce the proportion of stem cells in tumor tissue.
  • hyperbaric oxygen combined with nano-chemotherapy drugs Abraxane and Doxil reduce the proportion of stem cells in tumors by 53% and 97% respectively without increasing the toxic and side effects.
  • the present invention performs hyperbaric oxygen therapy on tumor patients, that is, placing tumor patients in a hyperbaric oxygen environment for treatment.
  • hyperbaric oxygen therapy on tumor patients can significantly improve tumor tissue blood vessels.
  • Breast cancer in situ tumor mice were treated with hyperbaric oxygen.
  • the blood vessels in the Saline group were curved and thickened, while the blood vessels in the HBO group had normal morphology.
  • the present invention combines hyperbaric oxygen therapy with intravenous injection of nano-chemotherapy drugs to perform anti-tumor therapy on tumor patients.
  • hyperbaric oxygen therapy on tumor patients can significantly regulate tumor blood vessels, reduce the overall density of tumor blood vessels, and promote new tumors.
  • the blood vessels are reduced, the tortuosity of tumor blood vessels is reduced, and the blood perfusion in the tumor blood vessels is increased, which promotes the penetration and enrichment of nano-chemotherapy drugs at the tumor site, and at the same time improves the uptake of drugs by tumor cells.
  • hyperbaric oxygen combined with nano-chemotherapy drugs increase the enrichment amount of the drugs in the treatment site by 1.38 times and 1.26 times, respectively, without increasing the toxic and side effects.
  • the present invention combines hyperbaric oxygen therapy with intravenous injection of nano-chemotherapy drugs to perform anti-tumor therapy on tumor patients.
  • hyperbaric oxygen therapy on tumor patients can significantly reduce the proportion of stem cells in tumor tissue, while regulating tumor blood vessels. It reduces the overall density of tumor blood vessels, reduces the number of new tumor blood vessels, reduces the degree of tortuosity of tumor blood vessels, increases blood perfusion in tumor blood vessels, promotes the penetration and enrichment of nano-chemotherapy drugs at tumor sites, and improves the uptake of drugs by tumor cells. , and ultimately improve the efficacy of tumor chemotherapy, and has a good safety.
  • HBO combined with Doxil increased the survival of mice by 11 days and 14.5 days, respectively, compared with Doxil alone.
  • Fig. 1 is the stem cell ratio of tumor in the stem cell ratio experiment carried out in Example 8 of the present invention.
  • Example 2 is a photo of stem cell cloning spheres in the stem cell ratio experiment carried out in Example 8 of the present invention
  • Figure 3 is the number of stem cell clone spheres in the stem cell ratio experiment carried out in Example 8 of the present invention.
  • Fig. 4 is the stem cell cloning sphere size in the stem cell ratio experiment carried out in Example 8 of the present invention.
  • Example 9 is a pie chart of the distribution of stem cell cycle in the stem cell cycle experiment performed in Example 9 of the present invention.
  • Example 7 is a photo of blood vessel immunofluorescence staining in the blood vessel morphology detection experiment carried out in Example 9 of the present invention.
  • Fig. 8 is the statistical result of blood vessel density in the blood vessel morphology detection experiment carried out in Example 9 of the present invention.
  • Fig. 9 is the statistical result of blood vessel tortuosity in the blood vessel shape detection experiment carried out in Embodiment 9 of the present invention.
  • FIG. 10 is an imaging diagram of blood perfusion in the blood perfusion detection experiment performed in Example 11 of the present invention.
  • FIG. 11 is the statistical result of blood perfusion in the blood perfusion detection experiment performed in Example 11 of the present invention.
  • FIG. 12 is a fluorescent imaging diagram of mice in vivo in each experimental group of small animal imaging detection in Example 12 of the present invention.
  • Example 13 is a fluorescence imaging diagram of in vitro tissue imaging detection of small animals in each experimental group in Example 12 of the present invention.
  • Figure 14 is a graph of the enrichment amount-time curve of nano-chemotherapy drug in living tumor tissue detected by imaging of small animals in each experimental group in Example 12 of the present invention
  • Example 15 is a quantitative diagram of tissue distribution of nano-chemotherapy drugs detected by imaging of small animals in each experimental group in Example 12 of the present invention.
  • Figure 16 is the quantitative result of the drug intake of each experimental group in Example 13 of the present invention.
  • Figure 17 is the tumor volume-time curve of mice in the anti-tumor activity experiment carried out in Example 14 of the present invention.
  • Figure 18 is the tumor weight results of mice in the anti-tumor activity experiment carried out in Example 14 of the present invention.
  • Figure 19 is the body weight-time curve of mice in the anti-tumor activity experiment carried out in Example 14 of the present invention.
  • Fig. 20 is the result of blood cell index of each experimental group in Example 14 of the present invention.
  • Figure 21 is the results of the blood biochemical indicators of each experimental group in Example 14 of the present invention.
  • Example 22 is a microscopic observation picture of tissue sections of each experimental group in Example 14 of the present invention.
  • Figure 23 shows the results of the survival period of mice in each group in Example 15 of the present invention.
  • Figure 24 is the tumor weight results of mice in the anti-tumor activity experiment carried out in Example 16 of the present invention.
  • Figure 25 is the body weight-time curve of mice in the anti-tumor activity experiment carried out in Example 16 of the present invention.
  • Figure 26 is the result of blood cell index of each experimental group in Example 16 of the present invention.
  • Figure 27 is the result of the blood biochemical indexes of each experimental group in Example 16 of the present invention.
  • Example 28 is a microscopic observation picture of tissue sections of each experimental group in Example 16 of the present invention.
  • Figure 29 shows the results of the survival period of mice in each group in Example 17 of the present invention.
  • Figure 30 is the number of lung metastases in mice in the anti-tumor activity experiment carried out in Example 18 of the present invention.
  • Figure 31 shows the results of the survival period of mice in each group in Example 19 of the present invention.
  • the present invention provides a method for reducing the proportion of stem cells in tumor tissue and/or regulating the normalization of blood vessels in tumor tissue.
  • the proportion of stem cells in tumor tissue of tumor patients can be reduced, and the proportion of stem cells in tumor patients can be regulated and promoted.
  • the blood vessels in the tumor tissue are normalized; the hyperbaric oxygen therapy is specifically: placing the tumor patient in a hyperbaric oxygen environment for treatment.
  • performing hyperbaric oxygen therapy on a tumor patient includes the following steps: placing the tumor patient in a cabin, gradually introducing pure oxygen to increase the air pressure in the cabin to 2-5 times atmospheric pressure, maintaining for a period of time, and releasing The air reduces the pressure in the cabin to atmospheric pressure.
  • the methods for reducing the proportion of stem cells in tumor tissue and/or regulating the normalization of blood vessels in tumor tissue provided in some embodiments of the present invention further reduce the proportion of stem cells in tumor tissue of tumor patients by combining hyperbaric oxygen therapy with nano-chemotherapy drug therapy. ratio, and regulate and promote the normalization of blood vessels in tumor tissue of tumor patients, including the following steps:
  • Hyperbaric oxygen therapy is performed on the tumor patient; the hyperbaric oxygen therapy increases the amount of oxygen dissolved in the blood of the tumor patient by increasing the air pressure and the concentration of inhaled oxygen in the environment where the tumor patient is located, thereby improving the perfusion of oxygen in each tissue ;
  • the present invention does not limit the sequence of hyperbaric oxygen therapy and intravenous injection of nano-chemotherapy.
  • the nano-chemotherapy drug is intravenously injected into the tumor patient.
  • hyperbaric oxygen therapy for tumor patients can improve the hypoxic environment of the tumor, reduce the proportion of stem cells in the tumor tissue, and most importantly, can significantly regulate tumor blood vessels and promote the penetration and enrichment of nano-chemotherapy drugs in the tumor site. to enhance the antitumor efficacy of nanochemotherapy drugs.
  • Hyperbaric oxygen (HBO) therapy refers to the intermittent administration of pure oxygen to patients under conditions of higher than normal atmospheric pressure.
  • the principle of hyperbaric oxygen therapy is to increase the amount of oxygen dissolved in the blood by increasing the air pressure and the concentration of inhaled oxygen in the patient's environment, thereby improving the perfusion of oxygen in various tissues.
  • Hyperbaric oxygen can effectively increase the partial pressure of oxygen in tissues, relieve tissue hypoxia, reduce edema, activate angiogenesis and collagen synthesis, so it is widely used in the prevention and treatment of various diseases, such as CO poisoning, decompression sickness and air pressure injury etc.
  • the present invention combines hyperbaric oxygen with nano-chemotherapy drugs, improves tumor tissue hypoxia through hyperbaric oxygen, reduces the proportion of stem cells in tumor tissue, and particularly importantly, can significantly regulate the normalization of tumor blood vessels and promote the nano-chemotherapy drugs in the tumor site. Penetration and enrichment to enhance the antitumor efficacy of nano-chemotherapy.
  • the hyperbaric oxygen therapy for tumor patients can be performed according to the conventional method of hyperbaric oxygen therapy in the prior art.
  • the hyperbaric oxygen therapy for tumor patients is specifically: placing the tumor patient in an airtight chamber, and gradually introducing pure Oxygen increases the air pressure in the cabin to 2-5 times atmospheric pressure. After maintaining for a period of time, deflation reduces the air pressure in the cabin to atmospheric pressure.
  • the nano-chemotherapy drug treatment method is: intravenous injection of nano-chemotherapy drugs.
  • the tumor patient is placed in a closed cabin, and pure oxygen is gradually introduced to increase the pressure in the cabin to 2-5 times atmospheric pressure, preferably 2-2.5 times atmospheric pressure, and maintain for 1-4 hours (preferably 1- After 2 hours), slowly deflate to bring the cabin pressure down to atmospheric pressure.
  • the order of administration of hyperbaric oxygen therapy and nano-chemotherapeutic drugs is variable, preferably, hyperbaric oxygen therapy is administered first, followed by intravenous injection of nano-chemotherapy drugs.
  • the interval between the hyperbaric oxygen therapy of the present invention and the intravenous injection of nano-chemotherapy drugs is 0-12 hours.
  • the interval time is preferably 1-12 hours, more preferably 1-3 hours.
  • hyperbaric oxygen therapy is administered to tumor patients 1-10 times, and in a preferred embodiment, 2-5 times are administered.
  • steps (1) and (2) are repeated to complete one treatment, and the treatment is continued for no less than one time.
  • the present invention can adjust the dose of the nano-chemotherapy drug according to the actual application.
  • the tumor patient is intravenously injected with the nano-chemotherapy drug once every to three weeks, and the single injection dose is 100-300 mg/m 2 .
  • hyperbaric oxygen therapy is performed on tumor patients at least once, and the frequency of hyperbaric oxygen therapy is not limited. Hyperbaric oxygen therapy every two days, every three days, or every four days. In a preferred embodiment, hyperbaric oxygen therapy is administered 1-5 times per day, preferably 1-2 times per day.
  • the injection dose of nanochemotherapy drugs can be based on individual differences, such as intravenous injection every 2 weeks or every 3 weeks, until disease progression or intolerable toxicity.
  • hyperbaric oxygen therapy is performed 1-7 times per week.
  • the nanometer chemotherapeutic drugs used in the present invention include various nanometer drugs that have been applied in the clinic or have not been applied in the clinic, including drugs such as Abraxane and Doxil.
  • drugs such as Abraxane and Doxil.
  • the intravenous injection dose of the nano-chemotherapy drug of the present invention reference may be made to the clinically used injection dose of different nano-chemotherapy drug types and the corresponding injection frequency.
  • the clinically recommended dose of Abraxane is 260 mg/m 2 , administered once every 3 weeks.
  • the nano-chemotherapy drug administration method, including reconstitution and dilution, etc., can also be the same as the above-mentioned current marketed drug administration method.
  • the treatment method of the present invention comprises the following steps:
  • Steps (1) and (2) are repeated to complete one treatment, and the continuous treatment is not less than one time.
  • the tumor treatment method proposed in the present invention can be applied to various tumor types, not only non-solid tumors such as melanoma, small cell lung cancer, etc., but also various solid tumors, including head and neck tumors, chest tumors , digestive system tumors, urogenital system tumors, bone tumors, central nervous system tumors, soft tissue tumors, skin and adnexal tumors, etc., combined with hyperbaric oxygen therapy can significantly improve the enrichment and response rate of nano-chemotherapy drugs in solid tumor sites, and significantly inhibit the tumor growth rate.
  • non-solid tumors such as melanoma, small cell lung cancer, etc.
  • solid tumors including head and neck tumors, chest tumors , digestive system tumors, urogenital system tumors, bone tumors, central nervous system tumors, soft tissue tumors, skin and adnexal tumors, etc.
  • hyperbaric oxygen therapy can significantly improve the enrichment and response rate of nano-chemotherapy drugs in solid tumor sites, and significantly inhibit the tumor growth rate.
  • the hyperbaric oxygen combined with the nano-chemotherapy drug of the present invention improves tumor tissue hypoxia through hyperbaric oxygen without increasing toxic and side effects, reduces the proportion of stem cells in tumor tissue, significantly regulates the normalization of tumor blood vessels and promotes
  • the penetration and enrichment of nano-chemotherapy drugs in tumor sites can improve the uptake of nano-chemotherapy drugs by tumor cells and enhance the anti-tumor efficacy of nano-chemotherapy drugs.
  • the invention provides a new anti-tumor combined treatment mode and expands the new application of hyperbaric oxygen.
  • nano-chemotherapy drugs used in specific examples 3 to 7 are Abraxane of CSPC
  • nano-chemotherapy drugs used in specific examples 8 to 19 are Abraxane and Doxil of CSPC.
  • This embodiment provides a method for reducing the proportion of stem cells in tumor tissue and/or promoting the normalization of tumor blood vessels with hyperbaric oxygen.
  • the treated individual is placed in a closed cabin, and pure oxygen is gradually introduced to increase the pressure in the cabin to 2.5 times the atmospheric pressure. After maintaining for 1.5 hours, the air pressure in the chamber was reduced to atmospheric pressure by slowly deflating it.
  • the above hyperbaric oxygen therapy was performed once a day for 14 consecutive days.
  • This embodiment provides a method for reducing the proportion of stem cells in tumor tissue and/or promoting the normalization of tumor blood vessels with hyperbaric oxygen.
  • the treated individual is placed in a closed cabin, and pure oxygen is gradually introduced to increase the pressure in the cabin to 2.5 times the atmospheric pressure. After maintaining for 1.5 hours, the air pressure in the chamber was reduced to atmospheric pressure by slowly deflating it. Hyperbaric oxygen therapy was administered once a day for three consecutive days.
  • This embodiment provides a method of hyperbaric oxygen combined with nano-chemotherapy drugs for tumor treatment, and the method includes two parts: hyperbaric oxygen therapy and nano-chemotherapy drug injection.
  • the hyperbaric oxygen therapy method is: place the treated individual in a closed cabin, gradually introduce pure oxygen to increase the pressure in the cabin to 2.5 times the atmospheric pressure, maintain it for 1.5 hours, and slowly deflate the cabin to reduce the pressure to the atmospheric pressure.
  • the above-mentioned hyperbaric oxygen therapy was performed once a day for 14 consecutive days; after the end of the hyperbaric oxygen therapy on the 14th day, the nano-chemotherapy drug treatment was performed.
  • the drug Abraxane.
  • This embodiment provides a method of hyperbaric oxygen combined with nano-chemotherapy drugs for tumor treatment, and the method includes two parts: hyperbaric oxygen therapy and nano-chemotherapy drug injection.
  • the hyperbaric oxygen therapy method is: place the treated individual in a closed cabin, gradually introduce pure oxygen to increase the pressure in the cabin to 2.5 times the atmospheric pressure, maintain it for 1.5 hours, and slowly deflate the cabin to reduce the pressure to the atmospheric pressure.
  • 260 mg/ m2 of the nano-chemotherapy drug Abraxane was injected intravenously for the first time. Subsequently, hyperbaric oxygen was given every other day, and two weeks later, a second dose of 260 mg/m2 of the nanochemotherapy drug Abraxane was administered intravenously at two-hour intervals after the end of the hyperbaric oxygen treatment. Hyperbaric oxygen was then given every other day, and two weeks later, a third dose of 260 mg/m2 of the nanochemotherapy drug Abraxane was administered intravenously at two -hour intervals after the end of the hyperbaric oxygen treatment.
  • This embodiment provides a method of hyperbaric oxygen combined with nano-chemotherapy drugs for tumor treatment, and the method includes two parts: hyperbaric oxygen therapy and nano-chemotherapy drug injection.
  • the hyperbaric oxygen therapy method is as follows: place the treated individual in a closed cabin, gradually introduce pure oxygen to increase the pressure in the cabin to 2 times the atmospheric pressure, and after maintaining for 2 hours, slowly deflate to reduce the pressure in the cabin to atmospheric pressure.
  • Hyperbaric oxygen therapy was performed every two days, and after 3 weeks, the nano-chemotherapy drug treatment was performed.
  • the specific method was as follows: 260 mg/m 2 nano-chemotherapy drug Abraxane was intravenously injected at an interval of two hours after the end of hyperbaric oxygen therapy.
  • This embodiment provides a method of hyperbaric oxygen combined with nano-chemotherapy drugs for tumor treatment, and the method includes two parts: hyperbaric oxygen therapy and nano-chemotherapy drug injection.
  • the hyperbaric oxygen therapy method is: place the treated individual in a closed cabin, gradually introduce pure oxygen to increase the pressure in the cabin to 2.5 times the atmospheric pressure, maintain it for 1.5 hours, and slowly deflate the cabin to reduce the pressure to the atmospheric pressure.
  • the nano-chemotherapy drug treatment method is as follows: 260 mg/m 2 nano-chemotherapy drug is injected intravenously at an interval of 12 hours after the end of hyperbaric oxygen therapy.
  • Hyperbaric oxygen therapy was performed every other day, and 260 mg/ m2 of the nano-chemotherapy drug Abraxane was injected intravenously for the first time at 12-hour intervals after the end of the hyperbaric oxygen therapy after 2 weeks. Then, hyperbaric oxygen therapy was performed every other day, and 260 mg/ m2 of the nano-chemotherapy drug Abraxane was injected intravenously for a second time at 12-hour intervals after the end of the hyperbaric oxygen therapy after 2 weeks.
  • This embodiment provides a method of hyperbaric oxygen combined with nano-chemotherapy drugs for tumor treatment, and the method includes two parts: hyperbaric oxygen therapy and nano-chemotherapy drug injection.
  • the nano-chemotherapy drug treatment method is: intravenous injection of 260 mg/m 2 nano-chemotherapy drug Abraxane.
  • the hyperbaric oxygen treatment method is: after intravenous injection of nano-chemotherapy drugs, the treated individual is placed in a closed cabin immediately, and pure oxygen is gradually introduced to increase the pressure in the cabin to 2.5 times the atmospheric pressure. After maintaining for 1.5 hours, slowly deflate the cabin to make the cabin. The internal pressure drops to atmospheric pressure.
  • nano-chemotherapy drug Abraxane was dissolved in physiological saline to prepare a 2 mg/mL solution
  • nano-chemotherapy drug Doxil was dissolved in physiological saline to prepare a 0.8 mg/mL solution.
  • a mouse breast cancer 4T1 orthotopic tumor model was established.
  • the tumor - bearing mice were randomly divided into 6 groups (Saline group, HBO group, Abraxane group, HBO+Abraxane group, Doxil group and HBO+Doxil group) and recorded as the first day, on the 1st, 2nd, 5th, 8th and 11th days, the mice in the HBO group, HBO+Abraxane group and HBO+Doxil group were treated with hyperbaric oxygen therapy.
  • the rats were placed in a closed cabin, and pure oxygen was gradually introduced to increase the pressure in the cabin to 2.5 times the atmospheric pressure.
  • mice in the HBO+Abraxane group were administered 0.2 mg/0.1 mL of Abraxane by tail vein injection, and 0.08 mg/0.1 mL of Doxil was administered to the mice in the HBO+Doxil group.
  • Physiological saline, Abraxane and Doxil were injected into the corresponding experimental group mice through tail vein at doses of 0.1 mL/mice, 0.1 mg/0.1 mL and 0.08 mg/0.1 mL, respectively.
  • the mice were sacrificed on the 11th day, and the tumor tissues of the mice were isolated. After the tumor tissue was minced with scissors, collagenase and DNase were added for digestion for 40 minutes, then ground with a 50 mL syringe rubber stopper and filtered through a 70 ⁇ m cell mesh to obtain a single cell suspension.
  • the ratio of CD133 positive cells to all tumor cells was analyzed by flow cytometry after co-incubation with APC-CD1331 flow antibody and cell suspension.
  • the single cell suspension was counted and mixed with 2mg/mL salmon fibrinogen solution to obtain 40000cells/mL mixed cell suspension, which was then mixed with 200ul, 0.1U/ ⁇ L of thrombin and then planted in a 96-well plate, the volume of each well was to 50 ⁇ L. After solidification, add 200 ⁇ L of cell culture medium and place in a 37 °C, 5% CO 2 incubator for culture. Photographs were taken every other day, and the number of formed stem cell clones and the size of clone spheres were observed and counted on the seventh day.
  • Figure 1 shows the ratio of CD133-positive cancer stem cells to all tumor cells in the tumor tissues of each group after the treatment of 4T1 breast cancer in situ tumor was detected by flow cytometry.
  • HBO alone reduced the ratio of cancer stem cells by 57% compared with the control group, and the results were significantly different.
  • the ratio of cancer stem cells in the HBO+Abraxane and HBO+Doxil groups decreased by 52% and 94%, respectively, compared with the respective monotherapy groups, and the results were also significantly different.
  • hyperbaric oxygen treatment alone can reduce the ratio of stem cells in tumor tissue
  • hyperbaric oxygen combined with nanochemotherapy drugs can also reduce the ratio of stem cells in tumor tissue.
  • Figure 2 is a photograph of clonal spheres obtained by fibrin gel screening of stem cells in tumor tissues of each group after the treatment of 4T1 breast cancer in situ tumor. It can be seen from the figure that the size of clonal spheres in HBO, HBO+Abraxane, and HBO+Doxil groups were smaller than their respective control groups.
  • Figure 3 shows the statistical results of the number of clonal spheres obtained by fibrin gel screening of stem cells in tumor tissues of each group after the treatment of 4T1 breast cancer in situ tumors.
  • HBO alone reduced the number of cancer stem cells by 45% compared with the control group, and the results were significantly different.
  • the number of cancer stem cells in the HBO+Abraxane and HBO+Doxil groups decreased by 49% and 75%, respectively, compared with the respective monotherapy groups, and the results were also significantly different. This result indicates that hyperbaric oxygen treatment can significantly reduce the number of stem cells in tumor tissue.
  • Figure 4 shows the statistical results of clonal sphere size obtained by fibrin gel screening of stem cells in tumor tissues of each group after the treatment of 4T1 breast cancer in situ tumor. It can be seen from the figure that the average size of clonal spheres in HBO, HBO+Abraxane, and HBO+Doxil groups were 121 ⁇ m 3 , 74 ⁇ m 3 and 8 ⁇ m 3 , which were significantly smaller than the respective control groups (average sizes were 265, 179, and 36 ⁇ m, respectively). 3 ). This result indicates that hyperbaric oxygen therapy can reduce the stemness and self-renewal capacity of cancer stem cells in tumor tissue.
  • nano-chemotherapy drug Abraxane was dissolved in physiological saline to prepare a 2 mg/mL solution
  • nano-chemotherapy drug Doxil was dissolved in physiological saline to prepare a 0.8 mg/mL solution.
  • a mouse breast cancer 4T1 orthotopic tumor model was established.
  • the tumor-bearing mice were randomly divided into 6 groups (Saline group, HBO group, Abraxane group, HBO+Abraxane group, Doxil group and HBO group). +Doxil group) and recorded as the first day, on the 1st, 2nd, 5th, 8th and 11th days, the mice in the HBO group, the HBO+Abraxane group and the HBO+Doxil group were treated with hyperbaric oxygen. It was placed in a closed cabin, and pure oxygen was gradually introduced to increase the pressure in the cabin to 2.5 times the atmospheric pressure. After maintaining for 1.5 hours, slowly deflate to reduce the pressure in the cabin to atmospheric pressure.
  • mice in the HBO+Abraxane group were given 0.2 mg/0.1 mL of Abraxane by tail vein injection, and mice in the HBO+Doxil group were given 0.08 mg/0.1 mL of Doxil before hyperbaric oxygen.
  • Physiological saline, Abraxane and Doxil were injected into the corresponding experimental group mice through tail vein at doses of 0.1 mL/mice, 0.1 mg/0.1 mL and 0.08 mg/0.1 mL, respectively.
  • the mice were sacrificed on the 11th day, and the tumor tissues of the mice were isolated. After the tumor tissue was minced with scissors, collagenase and DNase were added for digestion for 40 minutes, then ground with a 50 mL syringe rubber stopper and filtered through a 70 ⁇ m cell mesh to obtain a single cell suspension.
  • the cell cycle of CD133-positive cells and the proportion of G0-G1 cells were analyzed by flow cytometry using APC-CD1331 flow antibody, Hoechst33342 and EDU dye after co-incubating with the cell suspension.
  • Figure 5 shows the cell cycle distribution of tumor stem cells in the tumor tissue of each group of mice after the treatment of 4T1 breast cancer. The ratio has increased.
  • Figure 6 shows the ratios of tumor stem cells in G0/G1 cycle arrest in tumor tissues of mice in each group after 4T1 breast cancer treatment. It can be seen from the figure that the ratio of stem cells in the G0/G1 phase in the HBO group, the HBO+Abraxane group and the HBO+Doxil group was significantly decreased compared with the respective control groups. Cells in the G0/G1 phase cannot enter the DNA replication and division phase due to cycle arrest, so they are not sensitive to the killing of Abraxane and Doxil, resulting in chemoresistance. This result suggests that hyperbaric oxygen treatment can reduce the ratio of stem cells in cycle arrest and increase the sensitivity of cancer stem cells to nanochemotherapy drugs.
  • a mouse breast cancer 4T1 orthotopic tumor model was established.
  • the tumor - bearing mice were randomly divided into 2 groups (Saline group, HBO group) and recorded as the first day, respectively.
  • 2, 5, 8, and 11 days the mice in the HBO group were given hyperbaric oxygen therapy.
  • the treatment method was to place the mice in a closed cabin, and gradually introduce pure oxygen to increase the pressure in the cabin to 2.5 times the atmospheric pressure, and maintain it for 1.5 hours. Then, slowly deflate to bring the cabin pressure down to atmospheric pressure.
  • mice in the Saline group were given 0.1 mL of normal saline by tail vein injection. After the treatment, the mice were sacrificed on the 11th day, and the tumor tissues of the mice were isolated. After paraformaldehyde-fixed and embedded sections, the morphology and density of blood vessels in the tumor tissues were detected by CD31 staining.
  • Fig. 7 is the result of photographing the section of CD31 immunofluorescence staining.
  • the shape of the blood vessels can be seen from the figure.
  • the blood vessels in the Saline group are curved and thickened, while the blood vessels in the HBO group have normal shapes.
  • Figure 8 is the statistical result of the blood vessel density in the slices, it can be seen from the figure that the hyperbaric oxygen treatment can significantly reduce the blood vessel density in the tumor tissue.
  • Figure 9 is the statistical result of the degree of tortuosity of blood vessels in slices. It can be seen from the figure that the degree of tortuosity of blood vessels is significantly reduced after hyperbaric oxygen treatment, which indicates that HBO treatment can promote the normalization of tumor blood vessels.
  • the mouse breast cancer 4T1 orthotopic tumor model was established.
  • the mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution, and the tumor blood vessels were detected by laser speckle flow imaging.
  • the blood perfusion situation and then the mice were treated with hyperbaric oxygen.
  • the treatment method was to place the mice in a closed cabin, and gradually introduce pure oxygen to increase the pressure in the cabin to 2.5 times the atmospheric pressure. After maintaining for 1.5 hours, slowly deflate Reduce the cabin pressure to atmospheric pressure. After the treatment, the mice were anesthetized again and the blood perfusion of the tumor blood vessels of the mice was detected by laser speckle blood flow imaging.
  • Figure 10 is the blood perfusion diagram before and after hyperbaric oxygen in mice. As can be seen from the figure, the blood perfusion of tumor vessels in mice was enhanced after hyperbaric oxygen treatment.
  • Figure 11 shows the quantitative statistics of blood perfusion in mice before and after hyperbaric oxygenation. As can be seen from the figure, blood perfusion was enhanced by 7%, 3.7% and 2.9% after hyperbaric oxygen treatment in three different mice, respectively. This result suggests that hyperbaric oxygen treatment can improve blood perfusion in tumor vessels.
  • Abraxane was labeled with Cy5-NHS ester, and excess Cy5 molecules were removed by dialysis. The labeled Abraxane was then dissolved in physiological saline to prepare a 2 mg/mL solution.
  • a mouse breast cancer 4T1 orthotopic tumor model was established.
  • the tumor - bearing mice were randomly divided into 2 groups (Abraxane group and HBO+Abraxane group).
  • 0.3 mL of Cy5 fluorescently labeled Abraxane was administered to mice in Abraxane group and Abraxane group by tail vein injection.
  • the mice in the HBO+Abraxane inhibitor group were treated with hyperbaric oxygen.
  • the treatment method was as follows: the mice were placed in a closed cabin, and pure oxygen was gradually introduced to increase the pressure in the cabin to 2.5 times the atmospheric pressure. After maintaining for 1.5 hours, the air was slowly deflated. Reduce the cabin pressure to atmospheric pressure.
  • mice were imaged at 0, 4, 8, 12, 16, and 24 hours to detect the distribution of Abraxane in the mice, and the mice were sacrificed at the 24th hour, and the heart, liver, spleen, lung and kidney were separated. Tumors, the distribution of Abraxane in various tissues of mice was detected using a small animal imaging system.
  • Figure 12 shows the time-dependent changes in the distribution of nano-chemotherapy drugs in living mice and the distribution in isolated tissues detected by small animal imaging. It can be seen from the figure that at the 0th hour, there was no background fluorescence signal in the two groups of mice. After the injection of fluorescently labeled Abraxane, different intensities of fluorescence signals gradually appeared in the mice. HBO combined The fluorescence signal of Abraxane in the tumor site of the mice in the Abraxane group was stronger than that in the Abraxane alone group at different time points.
  • FIG 13 shows the tissue distribution map at 24 hours. The results also show that the fluorescence signal of Abraxane in the tumor tissue of the mice in the HBO combined with Abraxane group is stronger than that in the Abraxane alone group.
  • Figure 14 shows the quantitative results of the time-dependent changes in the distribution of Abraxane in the tumor tissue of living mice detected by small animal imaging. It can be seen from the figure that the enrichment of Abraxane in the tumor tissues of each group of mice gradually increased with the prolongation of time. The 0 to 24 hours were the rapid enrichment stage, and then the enrichment speed gradually slowed down. During the whole process, the enrichment of Abraxane in the tumor site in the HBO combined with Abraxane treatment group was significantly higher than that in the Abraxane alone group, and the HBO treatment increased the average enrichment of Abraxane in the tumor by about 47%.
  • Figure 15 shows the quantitative results of Abraxane tissue distribution detected by small animal imaging. It can be seen from the figure that compared with the Araxane group alone, the enrichment of Abraxane in the tumor site of the combined treatment group was significantly increased, and there was no significant difference in the enrichment of other organs. site enrichment.
  • Abraxane was labeled with Cy5-NHS ester, and excess Cy5 molecules were removed by dialysis. The labeled Abraxane was then dissolved in physiological saline to prepare a 2 mg/mL solution.
  • a mouse breast cancer 4T1 orthotopic tumor model was established.
  • the tumor - bearing mice were randomly divided into 2 groups (Abraxane group and HBO+Abraxane group) and recorded as the first day.
  • the mice in the HBO+Abraxane group were treated with hyperbaric oxygen.
  • the treatment method was to place the mice in a closed cabin, and gradually introduce pure oxygen to increase the pressure in the cabin to 2.5 times atmospheric pressure. , after maintaining for 1.5 hours, slowly deflate to reduce the pressure in the cabin to atmospheric pressure.
  • mice in the HBO+Abraxane group were given 0.2 mg/0.1 mL of Abraxane via tail vein injection before hyperbaric oxygen, and Abraxane was injected into the mice in the Abraxane group at a dose of 0.1 mg/0.1 mL via tail vein injection. in mice.
  • the mice were sacrificed on the 11th day, and the tumor tissues of the mice were isolated. After the tumor tissue was minced with scissors, collagenase and DNase were added for digestion for 40 minutes, then ground with a 50 mL syringe rubber stopper and filtered through a 70 ⁇ m cell mesh to obtain a single cell suspension.
  • the uptake of Abraxane-Cy5 by tumor cells was detected by flow cytometry.
  • Figure 16 shows the quantitative statistics of the fluorescence intensity of the uptake of Abraxane-Cy5 by tumor cells in each group after 4T1 breast cancer orthotopic tumor treatment. It can be seen from the figure that the uptake of Abraxane-Cy5 by tumor cells in the HBO+Abraxane group Compared with the Abraxane group, it increased by 90%, which indicates that hyperbaric oxygen treatment can improve the uptake of nano-chemotherapy by tumor cells.
  • nano-chemotherapy drug Abraxane was dissolved in physiological saline to prepare a 2 mg/mL solution
  • nano-chemotherapy drug Doxil was dissolved in physiological saline to prepare a 0.8 mg/mL solution.
  • a mouse breast cancer 4T1 orthotopic tumor model was established.
  • the tumor - bearing mice were randomly divided into 6 groups (Saline group, HBO group, Abraxane group, HBO+Abraxane group, Doxil group and HBO+Doxil group) and recorded as the first day, on the 1st, 2nd, 5th, 8th and 11th days, the mice in the HBO group, HBO+Abraxane group and HBO+Doxil group were treated with hyperbaric oxygen therapy.
  • the rats were placed in a closed cabin, and pure oxygen was gradually introduced to increase the pressure in the cabin to 2.5 times the atmospheric pressure.
  • mice in the HBO+Abraxane group were given 0.2 mg/0.1 mL Abraxane by tail vein injection before hyperbaric oxygen, and 0.08 mg/0.1 mL Doxil was given to the mice in the HBO+Doxil inhibitor group.
  • PBS, Abraxane and Doxil were injected into the corresponding experimental group mice through tail vein at doses of 0.1mL/mice, 0.1mg/0.1mL and 0.08mg/0.1mL respectively.
  • the mice in each group were sacrificed on the 23rd day, and the subcutaneous tumors were excised and weighed. Heavy. At the same time as tumor volume was measured, the body weight of the mice was measured every two days and recorded.
  • mice were sacrificed, the whole blood was taken out to detect the composition and content of blood cells, and the blood was collected to separate the serum, and the blood biochemical indexes were analyzed. index.
  • the intact lungs of the mice were removed, fixed and stained with Bouin's stain, washed with 95% ethanol 24 hours later, and the number of metastatic nodules in the lungs was recorded and photographed.
  • the five main organs of the mouse heart, liver, spleen, lung and kidney were taken out, fixed with 4% paraformaldehyde, then routinely embedded in paraffin, and stained with HE, and the tissue structure was observed under a microscope.
  • Figure 17 is the growth-time curve of tumor volume in mice during the treatment process.
  • the tumor growth of the mice in the HBO group was not significantly inhibited, which indicated that HBO alone had no obvious tumor inhibitory effect.
  • the Abraxane group and Doxil group both showed the effect of inhibiting tumor growth compared with the Saline group.
  • the tumor growth rate of the HBO+Abraxane and HBO+Doxil treatment group was significantly slower than that of the nanochemotherapy group.
  • the average tumor volume of the HBO+Abraxane and HBO+Doxil treatment group was 1633 and 255 mm . , significantly lower than the 1949 and 451mm 3 of the Abraxane and Doxil groups alone.
  • the above analysis shows that HBO combined with nano-chemotherapy drugs can significantly enhance the inhibitory effect of nano-chemo drugs on tumor growth.
  • Figure 18 shows the statistical results of tumor weighing after the mice were treated. As can be seen from Figure 18, the tumor weights of the Abraxane and Doxil groups were significantly lower than those of the Saline group, and the tumor weights of the combined treatment group were significantly lower than those of the nanochemotherapy drug alone treatment group, which indicated that HBO combined with nanochemotherapy could enhance the antitumor effect of nanochemotherapy.
  • Figure 19 is a graph of the body weight changes of the above 6 groups during the administration period. It can be seen from Figure 19 that compared with the Saline group, the body weight of the mice in the five experimental groups has no obvious decrease or increase trend, which indicates that the use of HBO or Abraxane alone, Doxil treatment, as well as the combination treatment, did not affect the health of the mice.
  • Figure 20 shows the results of blood cell detection in each group after treatment. As can be seen from Figure 20, compared with the Saline group, there were no significant differences in the content of white blood cells, red blood cells, platelets and average hemoglobin in the blood of mice in the five experimental groups after treatment, which indicated that HBO or nano-chemotherapy drugs were used alone, and Combination therapy has good safety.
  • Figure 21 shows the results of blood biochemical analysis after the treatment in each group, in which alanine aminotransferase and aspartate aminotransferase are indicators of liver function, and the increase of these indicators indicates that the drug has serious liver toxicity.
  • Cardiac creatine kinase is an indicator of cardiac function, which Elevated values indicate pathological changes in the heart, and total urea nitrogen is an indicator of renal function, and an increase in this index indicates a problem with renal function.
  • Figure 22 is a structural diagram of a tissue section at a magnification of 200 times. As can be seen from Figure 22, the structures of the heart, liver, spleen, lung and kidney in the Saline group and the five experimental groups were clear, with no obvious pathological changes, no bleeding and inflammatory infiltration. It will cause damage to the main organs of mice and has good safety.
  • nano-chemotherapy drug Abraxane was dissolved in physiological saline to prepare a 2 mg/mL solution
  • nano-chemotherapy drug Doxil was dissolved in physiological saline to prepare a 0.8 mg/mL solution.
  • the mouse breast cancer 4T1 orthotopic tumor model was established and recorded as the first day.
  • the tumor - bearing mice were randomly divided into 6 groups (Saline group, HBO group, Abraxane group, HBO+ group) Abraxane group, Doxil group and HBO+Doxil group) were recorded as day 1, and mice in HBO group, HBO+Abraxane group and HBO+Doxil group were treated with hyperbaric oxygen on days 1, 2, 5, 8, and 11, respectively.
  • the treatment method is to place the mice in an airtight chamber, gradually introduce pure oxygen to increase the air pressure in the chamber to 2.5 times the atmospheric pressure, maintain it for 1.5 hours, and slowly deflate the air pressure to reduce the air pressure in the chamber to atmospheric pressure.
  • mice in the HBO+Abraxane group were given 0.2 mg/0.1 mL Abraxane by tail vein injection before hyperbaric oxygen, and 0.08 mg/0.1 mL Doxil was given to the mice in the HBO+Doxil inhibitor group. After the treatment, the survival status of the mice was observed and recorded every day.
  • Figure 23 is the Kaplan-Meier chart of the survival time of mice in each group, the median survival time of Saline group, HBO group, Abraxane group, HBO+Abraxane group, Doxil group and HBO+Doxil group were 37, 38.5, 41.5, 43.5, 47 and 58 days.
  • the survival time of HBO + Doxil was significantly prolonged compared with Doxil alone.
  • the above analysis of the results showed that HBO combined with nano-chemotherapy could significantly prolong the survival of mice bearing 4T1 breast cancer in situ.
  • nano-chemotherapy drug Abraxane was dissolved in physiological saline to prepare a 2 mg/mL solution
  • nano-chemotherapy drug Doxil was dissolved in physiological saline to prepare a 0.8 mg/mL solution.
  • the Panc02 orthotopic tumor model of mouse pancreatic cancer was established and recorded as the first day.
  • the tumor-bearing mice were randomly divided into 6 groups (Saline group, HBO group, Abraxane group, HBO+Abraxane group, Doxil group and HBO+ group).
  • Doxil group and recorded as the first day, on the 1st, 2nd, 5th, 8th and 11th days, the mice in the HBO group, the HBO+Abraxane group and the HBO+Doxil group were treated with hyperbaric oxygen therapy.
  • pure oxygen was gradually introduced to increase the pressure in the cabin to 2.5 times the atmospheric pressure. After maintaining for 1.5 hours, the air pressure in the cabin was slowly released to reduce the pressure in the cabin to atmospheric pressure.
  • mice in the HBO+Abraxane group were given 0.2 mg/0.1 mL of Abraxane by tail vein injection before hyperbaric oxygen, and 0.08 mg/0.1 mL of Doxil was given to the mice in the HBO+Doxil inhibitor group.
  • PBS, Abraxane and Doxil were injected into the corresponding experimental group mice through tail vein at doses of 0.1mL/mice, 0.1mg/0.1mL and 0.08mg/0.1mL respectively.
  • the mice were sacrificed, the tumors were stripped and weighed, the whole blood of the mice was taken out to detect the composition and content of blood cells, and the blood was collected to separate the serum, and the blood biochemical indexes were analyzed.
  • Figure 24 shows the statistical results of tumor weighing after the mice were treated. As can be seen from Figure 7, the tumor weights of the Abraxane and Doxil groups were significantly lower than those of the Saline group, and the tumor weights of the combined treatment group were significantly lower than those of the nanochemotherapy drug alone treatment group, which indicated that HBO combined with nanochemotherapy could enhance the antitumor effect of nanochemotherapy.
  • Figure 25 is a graph of the body weight changes of the above 6 groups during the administration period. It can be seen from Figure 25 that compared with the Saline group, the body weight of the mice in the five experimental groups has no obvious decrease or increase trend, which indicates that the use of HBO or Abraxane, Doxil treatment, as well as the combination treatment, did not affect the health of the mice.
  • Figure 26 shows the results of blood cell detection in each group after treatment. As can be seen from Figure 26, compared with the Saline group, there were no significant differences in the content of white blood cells, red blood cells, platelets and average hemoglobin in the blood of mice in the five experimental groups after treatment, which indicated that HBO or nano-chemotherapy drugs were used alone, and Combination therapy has good safety.
  • Figure 27 is the blood biochemical analysis results after the treatment in each group, wherein alanine aminotransferase and aspartate aminotransferase are indicators of liver function, and the increase of this indicator represents that the drug has serious liver toxicity, and myocardial creatine kinase is an indicator of cardiac function. Elevated values indicate pathological changes in the heart, and total urea nitrogen is an indicator of renal function, and an increase in this index indicates a problem with renal function. As can be seen from Figure 27, there were no significant changes in the four blood biochemical indexes of the five experimental groups compared with the control group. The above results show that the use of HBO or nano-chemotherapy drugs alone and combined treatment will not affect the main symptoms of mice. Organs are damaged, and the treatment has good safety
  • Figure 28 is a structural diagram of a tissue section at a magnification of 200 times. As can be seen from Figure 28, the structures of the heart, liver, spleen, lung and kidney in the Saline group and the five experimental groups were clear, with no obvious pathological changes, no bleeding and inflammatory infiltration. It will cause damage to the main organs of mice and has good safety.
  • nano-chemotherapy drug Abraxane was dissolved in physiological saline to prepare a 2 mg/mL solution
  • nano-chemotherapy drug Doxil was dissolved in physiological saline to prepare a 0.8 mg/mL solution.
  • Panc02 orthotopic tumor model of mouse pancreatic cancer was established.
  • the tumor-bearing mice were randomly divided into 6 groups (Saline group, HBO group, Abraxane group, HBO+Abraxane group, Doxil group and HBO+Doxil group) and recorded as On the first day, the mice in the HBO group, the HBO+Abraxane group and the HBO+Doxil group were treated with hyperbaric oxygen on the 1st, 2nd, 5th, 8th and 11th days respectively. Introduce pure oxygen to increase the pressure in the cabin to 2.5 times atmospheric pressure, and after maintaining for 1.5 hours, slowly deflate to reduce the pressure in the cabin to atmospheric pressure.
  • mice in the HBO+Abraxane group were given 0.2 mg/0.1 mL of Abraxane by tail vein injection before hyperbaric oxygen, and 0.08 mg/0.1 mL of Doxil was given to the mice in the HBO+Doxil inhibitor group.
  • PBS, Abraxane and Doxil were injected into the corresponding experimental group mice through tail vein at doses of 0.1mL/mice, 0.1mg/0.1mL and 0.08mg/0.1mL respectively. After the treatment, the survival status of the mice was observed and recorded every day.
  • Figure 29 shows the Kaplan-Meier chart of the survival time of mice in each group.
  • the median survival time of Saline group, HBO group, Abraxane group, and HBO+Abraxane group were 40, 43, 57.5 and 64 days, while Doxil and HBO+Doxil groups Due to the good efficacy, less than half of the mice died, and the median survival period could not be calculated.
  • the survival of HBO+Abraxane was significantly prolonged compared to Abraxane alone.
  • the above analysis of the results showed that HBO combined with nano-chemotherapy drug treatment can significantly prolong the survival of Panc02 pancreatic cancer in situ tumor-bearing mice.
  • nano-chemotherapy drug Abraxane was dissolved in physiological saline to prepare a 2 mg/mL solution
  • nano-chemotherapy drug Doxil was dissolved in physiological saline to prepare a 0.8 mg/mL solution.
  • a mouse breast cancer metastasis model was established by injecting 4T1 cells into the tail vein.
  • the tumor-bearing mice were randomly divided into 6 groups (Saline group, HBO group, Abraxane group, HBO+Abraxane group, Doxil group and HBO+Doxil group). And recorded as the first day, on the 1st, 2nd, 5th, 8th and 11th days, the mice in the HBO group, the HBO+Abraxane group and the HBO+Doxil group were treated with hyperbaric oxygen.
  • the treatment method was to place the mice in a closed cabin. In the body, pure oxygen was gradually introduced to increase the air pressure in the cabin to 2.5 times the atmospheric pressure.
  • mice in the HBO+Abraxane group were given 0.2 mg/0.1 mL of Abraxane by tail vein injection before hyperbaric oxygen, and 0.08 mg/0.1 mL of Doxil was given to the mice in the HBO+Doxil inhibitor group.
  • PBS, Abraxane and Doxil were injected into the corresponding experimental group mice through tail vein at doses of 0.1mL/mice, 0.1mg/0.1mL and 0.08mg/0.1mL respectively.
  • the mice in each group were sacrificed on the 20th day, and the lungs were excised and weighed. They were fixed and stained with Bouin's staining solution. After 24 hours of washing with 95% ethanol, the number of lung tumor metastases was recorded and photographed.
  • Figure 30 shows the statistical results of the number of metastatic nodules in the lungs of the four groups of mice after treatment. From Figure 30, it can be seen that the number of metastatic nodules in the lungs of the mice in the Abraxane and Doxil groups decreased significantly compared with the Saline group, while the lung metastases in the combined treatment group were significantly reduced. The number of metastatic nodules was significantly less than that in the group treated with nano-chemotherapy alone, which indicated that HBO combined with nano-chemotherapy had a significant inhibitory effect on tumor metastasis.
  • nano-chemotherapy drug Abraxane was dissolved in physiological saline to prepare a 2 mg/mL solution
  • nano-chemotherapy drug Doxil was dissolved in physiological saline to prepare a 0.8 mg/mL solution.
  • a mouse breast cancer metastasis model was established by injecting 4T1 cells into the tail vein.
  • the tumor-bearing mice were randomly divided into 6 groups (Saline group, HBO group, Abraxane group, HBO+Abraxane group, Doxil group and HBO+Doxil group). And recorded as the first day, on the 1st, 2nd, 5th, 8th and 11th days, the mice in the HBO group, the HBO+Abraxane group and the HBO+Doxil group were treated with hyperbaric oxygen.
  • the treatment method was to place the mice in a closed cabin. In the body, pure oxygen was gradually introduced to increase the air pressure in the cabin to 2.5 times the atmospheric pressure.
  • mice in the HBO+Abraxane group were given 0.2 mg/0.1 mL of Abraxane by tail vein injection before hyperbaric oxygen, and 0.08 mg/0.1 mL of Doxil was given to the mice in the HBO+Doxil inhibitor group.
  • PBS, Abraxane and Doxil were injected into the corresponding experimental group mice through tail vein at doses of 0.1mL/mice, 0.1mg/0.1mL and 0.08mg/0.1mL respectively. After the treatment, the survival status of the mice was observed and recorded every day.
  • Figure 31 is the Kaplan-Meier chart of the survival time of mice in each group.
  • the median survival time of Saline group, HBO group, Abraxane group, HBO+Abraxane group, Doxil group and HBO+Doxil group was 24.5, 26.5, 27.5, 30.5, 39.5 and 45 days.
  • the survival time of HBO + Doxil was significantly prolonged compared with Doxil alone.
  • the above analysis shows that HBO combined with nano-chemotherapy can significantly prolong the survival time of mice with breast cancer metastases.
  • chemotherapeutic drugs can improve tumor tissue hypoxia through hyperbaric oxygen, reduce the proportion of stem cells in tumor tissue, significantly regulate the normalization of tumor blood vessels, and promote the penetration and enrichment of nano-chemotherapy drugs in the tumor site, and improve tumor cells' resistance to nano-chemotherapy drugs. uptake and enhance the antitumor efficacy of nano-chemotherapy drugs.
  • the method of the present invention is only a preferred embodiment, and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

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Description

一种抑制肿瘤干细胞的方法及调控肿瘤血管正常化的方法 【技术领域】
本发明涉及肿瘤治疗领域，更具体的，涉及一种抑制肿瘤干细胞的方法及调控肿瘤血管正常化的方法。
【背景技术】
肿瘤干细胞是一类具有自我更新能力与多向分化潜能的细胞，是肿瘤不断生长的根源，同时也是肿瘤发生、转移与复发的“起始细胞”。肿瘤干细胞的形成是导致肿瘤细胞对化疗药物治疗失败的原因之一。
另一方面，肿瘤发展出了与正常血管系统不同的自身功能性血管。肿瘤血管结构高度异常，具体表现为血管密度异常，血管轮廓不规则，基底膜和内皮层不完整或缺失，并且缺乏周细胞和平滑肌。此外，肿瘤血管功能高度异常，包括不稳定的血流速度和方向，高血管阻力，高血管脆性，红细胞淤塞，白血球粘连，血小板聚集和肿瘤细胞阻塞血管。这些异常功能导致药物很难递送到肿瘤内部，从而使肿瘤深部细胞与肿瘤干细胞免于被药物杀伤。
【发明内容】
针对目前肿瘤化疗疗效差这一问题，本发明提供一种抑制肿瘤干细胞的方法及调控肿瘤血管正常化的方法，其目的在于通过高压氧治疗降低患者肿瘤组织中干细胞的比例，同时调控肿瘤血管正常化，提高肿瘤化疗的疗效，解决临床上化疗药物疗效差这一问题。
为实现上述目的，本发明提供了一种降低肿瘤组织中干细胞比例的方法，对肿瘤患者进行高压氧治疗，以降低肿瘤患者其肿瘤组织中干细胞的比例；所述高压氧治疗具体为：将肿瘤患者置于高压氧环境中进行治疗。
本发明还提供了一种调控肿瘤组织血管正常化的方法，对肿瘤患者进行高压氧治疗，以调控促进肿瘤患者其肿瘤组织血管正常化；所述高压氧治疗具体为：将肿瘤患者置于高压氧环境中进行治疗。
总体而言，通过本发明所构思的以上技术方案与现有技术相比，能够取得以下有益效果：
(1)本发明通过对肿瘤患者进行高压氧治疗，即将肿瘤患者置于高压氧环境中进行治疗，实验发现，对肿瘤患者进行高压氧治疗能够显著降低肿瘤组织中干细胞 的比例，比如某实施例中对4T1乳腺癌原位瘤小鼠进行高压氧处理，与不进行高压氧处理的对照组相比，小鼠肿瘤干细胞的比率降低了57％。
(2)本发明将高压氧治疗与静脉注射纳米化疗药物相结合，对肿瘤患者进行抗肿瘤治疗，实验发现对肿瘤患者进行高压氧治疗能够进一步降低肿瘤组织中干细胞的比例。优选实施例中，与单用纳米化疗药物相比，高压氧联合纳米化疗药物Abraxane和Doxil在不增加毒副作用的同时，将肿瘤内部干细胞的占比分别降低了53％和97％。
(3)本发明通过对肿瘤患者进行高压氧治疗，即将肿瘤患者置于高压氧环境中进行治疗，实验发现，对肿瘤患者进行高压氧治疗能够显著改善肿瘤组织血管，比如某实施例中对4T1乳腺癌原位瘤小鼠进行高压氧处理，与不进行高压氧处理的对照组相比，Saline组血管呈现弯曲增粗的形态，而HBO组的血管形态正常。
(4)本发明将高压氧治疗与静脉注射纳米化疗药物相结合，对肿瘤患者进行抗肿瘤治疗，实验发现对肿瘤患者进行高压氧治疗能够显著调控肿瘤血管，使肿瘤血管整体密度降低，新生肿瘤血管减少，肿瘤血管弯曲程度降低，并增加肿瘤血管中血液的灌注量，促进纳米化疗药物在肿瘤部位的穿透与富集，同时提高肿瘤细胞对药物的摄取。优选实施例中，与单用纳米化疗药物相比，高压氧联合纳米化疗药物Abraxane和Doxil在不增加毒副作用的同时，将药物在治疗部位的富集量分别提高了1.38倍和1.26倍。
(5)本发明将高压氧治疗与静脉注射纳米化疗药物相结合，对肿瘤患者进行抗肿瘤治疗，实验发现对肿瘤患者进行高压氧治疗能够显著降低肿瘤组织中干细胞的比例，同时调控肿瘤血管，使肿瘤血管整体密度降低，新生肿瘤血管减少，肿瘤血管弯曲程度降低，并增加肿瘤血管中血液的灌注量，促进纳米化疗药物在肿瘤部位的穿透与富集，同时提高肿瘤细胞对药物的摄取，最终提高肿瘤化疗的疗效，并且具有良好的安全性。在4T1原位模型和转移模型的实施例中，HBO联合Doxil与单用Doxil相比，小鼠生存期分别延长了11天和14.5天。
(6)本发明提出的高压氧联合纳米化疗药物用于肿瘤治疗的方法简单易于操作，安全可靠，有利于临床转化。
【附图说明】
图1为本发明实施例8进行的干细胞比率实验中肿瘤的干细胞比率；
图2为本发明实施例8进行的干细胞比率实验中干细胞克隆球照片；
图3为本发明实施例8进行的干细胞比率实验中干细胞克隆球数目；
图4为本发明实施例8进行的干细胞比率实验中干细胞克隆球大小；
图5为本发明实施例9进行的干细胞周期实验中干细胞周期分布饼图；
图6为本发明实施例9进行的干细胞周期实验中干细胞G0/G1周期比率；
图7为本发明实施例9进行的血管形态检测实验中血管免疫荧光染色照片；
图8为本发明实施例9进行的血管形态检测实验中血管密度统计结果；
图9为本发明实施例9进行的血管形态检测实验中血管弯曲度统计结果；
图10为本发明实施例11进行的血流灌注检测实验中血流灌注成像图；
图11为本发明实施例11进行的血流灌注检测实验中血流灌注统计结果；
图12为本发明实施例12各实验组小动物成像检测小鼠活体荧光成像图；
图13为本发明实施例12各实验组小动物成像检测离体组织荧光成像图；
图14为本发明实施例12各实验组小动物成像检测活体肿瘤组织纳米化疗药物富集量-时间曲线；
图15为本发明实施例12各实验组小动物成像检测纳米化疗药物组织分布定量图；
图16是本发明实施例13各实验组对药物摄取的定量结果；
图17为本发明实施例14进行的抗肿瘤活性实验中小鼠的瘤体积-时间曲线；
图18为本发明实施例14进行的抗肿瘤活性实验中小鼠的瘤重结果；
图19为本发明实施例14进行的抗肿瘤活性实验中小鼠的体重-时间曲线；
图20为本发明实施例14各实验组血细胞指标的结果；
图21为本发明实施例14各实验组血生化指标的结果；
图22为本发明实施例14各实验组组织切片的显微观察图片；
图23为本发明实施例15各组小鼠生存期的结果；
图24为本发明实施例16进行的抗肿瘤活性实验中小鼠的瘤重结果；
图25为本发明实施例16进行的抗肿瘤活性实验中小鼠的体重-时间曲线；
图26为本发明实施例16各实验组血细胞指标的结果；
图27为本发明实施例16各实验组血生化指标的结果；
图28为本发明实施例16各实验组组织切片的显微观察图片；
图29为本发明实施例17各组小鼠生存期的结果；
[根据细则91更正 05.03.2021]　
图30为本发明实施例18进行的抗肿瘤活性实验中小鼠的肺转移节结数；
图31为本发明实施例19各组小鼠生存期的结果。
【具体实施方式】
下面结合实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
本发明提供的一种降低肿瘤组织中干细胞比例和/或调控肿瘤组织血管正常化的方法，通过对肿瘤患者进行高压氧治疗，以降低肿瘤患者其肿瘤组织中干细胞的比例，并调控促进肿瘤患者其肿瘤组织中的血管正常化；所述高压氧治疗具体为：将肿瘤患者置于高压氧环境中进行治疗。
本发明一些实施例中，对肿瘤患者进行高压氧治疗包括如下步骤：将肿瘤患者置于舱体内，逐渐通入纯氧使舱内气压升高到2-5倍大气压，维持一段时间后，放气使舱内气压降至大气压。实验证明对肿瘤患者进行高压氧治疗，能够显著降低肿瘤组织中干细胞的比例，并有效改善肿瘤组织中的血管形态。
本发明一些实施例中提供的降低肿瘤组织中干细胞比例和/或调控肿瘤组织血管正常化的方法，通过将高压氧治疗与纳米化疗药物治疗相结合，以进一步降低肿瘤患者其肿瘤组织中干细胞的比例，并调控促进肿瘤患者其肿瘤组织中的血管正常化，具体包括如下步骤：
(1)对肿瘤患者进行高压氧治疗；所述高压氧治疗通过增加肿瘤患者所处环境的气压及吸入氧气的浓度来提高溶解于肿瘤患者血液中氧气的量，从而提高各组织中氧的灌注；
(2)对肿瘤患者静脉注射纳米化疗药物。
原则上本发明不限定高压氧治疗和静脉注射纳米化疗药物治疗的先后顺序。
优选实施例中，对肿瘤患者进行单次或多次高压氧治疗后，再对肿瘤患者静脉注射纳米化疗药物。实验发现首先对肿瘤患者进行高压氧治疗，可以改善肿瘤的乏氧环境，降低肿瘤组织中干细胞的占比，尤为重要地，能够显著调控肿瘤血管并促进纳米化疗药物在肿瘤部位的穿透和富集，增强纳米化疗药物的抗肿瘤疗效。
高压氧(Hyperbaric oxygen，HBO)治疗是指间歇性地在高于正常大气压条件下给予病人纯氧的治疗手段。目前通常采用的治疗压力是2-2.5个大气压(2-2.5ATA,1ATA＝101.32KPa)。高压氧治疗的原理是通过增加病人所处环境的气压及吸入氧气的 浓度来提高溶解于血液中氧气的量，从而提高各组织中氧的灌注。高压氧能够有效提高组织中的氧分压、缓解组织缺氧、减少水肿、激活血管新生和胶原合成，因此广泛应用于各种疾病的预防与治疗，比如用于CO中毒，减压病和气压伤等。然而本发明将高压氧与纳米化疗药物联合，通过高压氧改善肿瘤组织乏氧，降低肿瘤组织中干细胞的占比，尤为重要地，能够显著调控肿瘤血管正常化并促进纳米化疗药物在肿瘤部位的穿透和富集，增强纳米化疗药物的抗肿瘤疗效。
本发明对肿瘤患者进行高压氧治疗可以按照现有技术高压氧治疗的常规方式进行，一些实施例中，对肿瘤患者进行高压氧治疗具体为：将肿瘤患者置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2-5倍大气压，维持一段时间后，放气使舱内气压降至大气压。纳米化疗药物治疗方法为：通过静脉注射纳米化疗药物。
一些实施例中，将肿瘤患者置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2-5倍大气压，优选为2-2.5倍大气压，维持1-4小时(优选为1-2小时)后，缓慢放气使舱内气压降至大气压。
在一个优选实施方式中，高压氧治疗与纳米化疗药物给药的先后次序可变，优选为先高压氧治疗，随后静脉注射纳米化疗药物。
本发明所述高压氧治疗与静脉注射纳米化疗药物之间的间隔时间为0-12小时。优选间隔时间为1-12小时，进一步优选为1-3小时。
本发明对肿瘤患者进行静脉注射纳米化疗药物之前或之后，对肿瘤患者给予1-10次高压氧治疗，优选实施例中给予2-5次。
本发明一些实施例中，重复步骤(1)和步骤(2)以完成一次治疗，持续治疗不少于1次。
本发明可根据实际应用情况调整纳米化疗药物的剂量，一些实施例中，每一至三周对肿瘤患者静脉注射纳米化疗药物一次，其单次注射剂量为100-300mg/m 2。
本发明优选实施例中，每一次静脉注射纳米化疗药物之前，对肿瘤患者进行至少一次的高压氧治疗，其高压氧治疗频率不限，可根据实际需要对肿瘤患者进行每天、每隔一天、每隔两天、每隔三天或每隔四天等的高压氧治疗。在一个优选实施方式中，每天给予高压氧治疗1-5次，优选为1-2次。
纳米化疗药物的注射剂量可依据个体差异而定，比如静脉注射每2周或每3周一次，直至疾病进展或出现不可耐受的毒性。一些实施例中，根据确定的纳米化疗药物的用量和用法，在每一次注射纳米化疗药物之前，进行每周1-7次的高压氧治疗。
本发明采用的纳米化疗药物包括已应用于临床或未应用于临床的各种纳米药物，包括Abraxane、Doxil等药物。本发明纳米化疗药物的静脉注射剂量可参考不同的纳米化疗药物类型其临床上采用的注射剂量，以及相应的注射频次。比如Abraxane在临床上的推荐剂量为260mg/m 2，每3周给一次药。纳米化疗药物给药方法，包括复溶和稀释等，也可与上述现行上市药物给药方法相同。
本发明所述的治疗方法，一些实施例中，包括如下步骤：
(1)将肿瘤患者置于密闭舱体内，逐渐通入纯氧使舱体内气压升高到2-5倍大气压，维持1-4小时后，缓慢放气使舱体内气压降至大气压；
(2)舱体内气压降至大气压0-12小时后，对肿瘤患者静脉注射纳米化疗药物，注射剂量为100-300mg/m 2；
(3)重复步骤(1)和步骤(2)以完成一次治疗，持续治疗不少于1次。
本发明提出的肿瘤治疗方法可以适用于各种肿瘤类型，不仅能够适用于诸如黑色素瘤、小细胞肺癌肿瘤等的非实体瘤，同样也适用于各种实体瘤，包括头颈部肿瘤、胸部肿瘤、消化系统肿瘤、泌尿生殖系统肿瘤、骨肿瘤、中枢神经系统肿瘤、软组织肿瘤、皮肤及附件肿瘤等，联合高压氧治疗能够显著提高纳米化疗药物在实体瘤部位的富集和响应率，显著抑制肿瘤生长速度。
与单用纳米化疗药物相比，本发明高压氧联合纳米化疗药物在不增加毒副作用的同时通过高压氧改善肿瘤组织乏氧，降低肿瘤组织中干细胞的占比，显著调控肿瘤血管正常化并促进纳米化疗药物在肿瘤部位的穿透和富集，提高肿瘤细胞对纳米化疗药物的摄取，增强纳米化疗药物的抗肿瘤疗效。本发明提供了一种新的抗肿瘤联合治疗方式，拓展了高压氧的新用途。
以下为具体实施例，具体实施例3至实施例7中采用的纳米化疗药物为石药集团的Abraxane，具体实施例8至实施例19中采用的纳米化疗药物为石药集团的Abraxane和Doxil。
实施例1
本实施例提供一种高压氧降低肿瘤组织中干细胞比例和/或促进肿瘤血管正常化的方法，将治疗个体置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。每天进行一次上述高压氧治疗，连续14天。
实施例2
本实施例提供一种高压氧降低肿瘤组织中干细胞比例和/或促进肿瘤血管正常化的方法，将治疗个体置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。连续三天每天给予一次高压氧治疗。
实施例3
本实施例提供一种高压氧联合纳米化疗药物用于肿瘤治疗的方法，该方法包括高压氧治疗和纳米化疗药物注射两部分。高压氧治疗方法为：将治疗个体置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。
每天进行一次上述高压氧治疗，连续14天；在第14天的高压氧治疗结束后进行纳米化疗药物治疗，具体方法为：高压氧治疗结束后间隔两小时，通过静脉注射260mg/m 2纳米化疗药物Abraxane。
实施例4
本实施例提供一种高压氧联合纳米化疗药物用于肿瘤治疗的方法，该方法包括高压氧治疗和纳米化疗药物注射两部分。高压氧治疗方法为：将治疗个体置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。
连续三天每天给予一次高压氧治疗后，在第三天高压氧结束后间隔两小时，通过静脉注射260mg/m 2纳米化疗药物Abraxane第一次。随后每间隔一天给予一次高压氧，两周后在高压氧治疗结束后间隔两小时通过静脉注射260mg/m 2纳米化疗药物Abraxane第二次。然后每间隔一天给予一次高压氧，两周后在高压氧治疗结束后间隔两小时通过静脉注射260mg/m 2纳米化疗药物Abraxane第三次。
实施例5
本实施例提供一种高压氧联合纳米化疗药物用于肿瘤治疗的方法，该方法包括高压氧治疗和纳米化疗药物注射两部分。高压氧治疗方法为：将治疗个体置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2倍大气压，维持2小时后，缓慢放气使舱内气压降至大气压。每隔两天进行一次高压氧治疗，持续3周后，进行纳米化疗药物治疗，具体方法为：高压氧治疗结束后间隔两小时，通过静脉注射260mg/m 2纳米化疗药物Abraxane一次。
实施例6
本实施例提供一种高压氧联合纳米化疗药物用于肿瘤治疗的方法，该方法包括高压氧治疗和纳米化疗药物注射两部分。高压氧治疗方法为：将治疗个体置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。纳米化疗药物治疗方法为：高压氧治疗结束后间隔12小时，通过静脉注射260mg/m 2纳米化疗药物。
每间隔一天进行一次高压氧治疗，在2周后的高压氧治疗结束后间隔12小时静脉注射260mg/m 2纳米化疗药物Abraxane第一次。然后再每间隔一天进行一次高压氧治疗，在2周后的高压氧治疗结束后间隔12小时静脉注射260mg/m 2纳米化疗药物Abraxane第二次。
实施例7
本实施例提供一种高压氧联合纳米化疗药物用于肿瘤治疗的方法，该方法包括高压氧治疗和纳米化疗药物注射两部分。纳米化疗药物治疗方法为：通过静脉注射260mg/m 2纳米化疗药物Abraxane。高压氧治疗方法为：在静脉注射纳米化疗药物后，立即将治疗个体置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。
实施例8
基于高压氧联合纳米化疗药物对荷乳腺癌原位瘤小鼠肿瘤干细胞比例的测试。
实验药物的配制：将纳米化疗药物Abraxane溶解在生理盐水中配制成2mg/mL的溶液，将纳米化疗药物Doxil溶解在生理盐水中配制成0.8mg/mL的溶液。
建立小鼠乳腺癌4T1原位瘤模型，当肿瘤长到体积为100mm 3时，随机将荷瘤小鼠分为6个组(Saline组、HBO组、Abraxane组、HBO+Abraxane组、Doxil组和HBO+Doxil组)并记为第1天，分别在第1、2、5、8、11天给予HBO组、HBO+Abraxane组和HBO+Doxil组小鼠高压氧治疗，治疗方式为，将小鼠置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。第2、5、8、11天在高压氧之前通过尾静脉注射给予HBO+Abraxane组小鼠0.2mg/0.1mL Abraxane，给予HBO+Doxil组小鼠0.08mg/0.1mL Doxil。并分别将生理盐水、Abraxane、Doxil以0.1mL/只、0.1mg/0.1mL、0.08mg/0.1mL剂量通过尾静脉注射到对应实验组小鼠。治疗结束后，在第11天处死小鼠，分离小鼠肿瘤组织。用剪刀将肿瘤组织剪碎后，加入胶原蛋白酶和DNA酶消化40分钟后，使用50mL注射器橡胶塞子研磨并通过70μm细胞筛网过滤得到单细胞悬液。
使用APC-CD1331流式抗体与细胞悬液共孵育后流式细胞仪分析CD133阳性细胞占所有肿瘤细胞的比率。
将单细胞悬液计数后与2mg/mL三文鱼纤维蛋白原溶液混合后，得到40000cells/mL混合细胞悬液，再与200ul，0.1U/μL的凝血酶混合后种植于96孔板，每孔体积为50μL。凝固后加入200μL细胞培养基，放置于37℃，5％CO 2培养箱中培养。每间隔一天拍照，并在第七天对形成的干细胞克隆数和克隆球大小进行观测与统计。
图1为流式检测4T1乳腺癌原位瘤治疗结束后各组肿瘤组织中CD133阳性的肿瘤干细胞占所有肿瘤细胞的比率。从图中可以看出，单用HBO与对照组相比，肿瘤干细胞的比率降低了57％，结果具有显著性差异。同时，HBO+Abraxane与HBO+Doxil组相比于各自的单药治疗组，肿瘤干细胞的比率分别下降了52％和94％，结果同样具有显著性差异。这个结果表明高压氧单独处理能降低肿瘤组织中干细胞的比率，而高压氧联合纳米化疗药物同样可以降低肿瘤组织中干细胞的比率。
图2为4T1乳腺癌原位瘤治疗结束后各组肿瘤组织中干细胞通过纤维蛋白凝胶筛选得到的克隆球照片。从图中可以看出，HBO以及HBO+Abraxane、HBO+Doxil组的克隆球大小均小于各自的对照组。
图3为4T1乳腺癌原位瘤治疗结束后各组肿瘤组织中干细胞通过纤维蛋白凝胶筛选得到的克隆球数量统计结果。从图中可以看出，单用HBO与对照组相比，肿瘤干细胞的数量降低了45％，结果具有显著性差异。同时，HBO+Abraxane与HBO+Doxil组相比于各自的单药治疗组，肿瘤干细胞的数量分别下降了49％和75％，结果同样具有显著性差异。这个结果表明高压氧处理可以显著降低肿瘤组织中干细胞的数量。
图4为4T1乳腺癌原位瘤治疗结束后各组肿瘤组织中干细胞通过纤维蛋白凝胶筛选得到的克隆球大小统计结果。从图中可以看出，HBO以及HBO+Abraxane、HBO+Doxil组的克隆球平均大小分别为121μm 3、74μm 3、8μm 3，均显著小于各自的对照组(平均大小分别为265、179、36μm 3)。这个结果表明，高压氧治疗可以降低肿瘤组织中肿瘤干细胞的干性与自我更新能力。
实施例9
基于高压氧联合纳米化疗药物对荷乳腺癌原位瘤小鼠肿瘤干细胞细胞周期的测试。
实验药物的配制：将纳米化疗药物Abraxane溶解在生理盐水中配制成2mg/mL 的溶液，将纳米化疗药物Doxil溶解在生理盐水中配制成0.8mg/mL的溶液。
建立小鼠乳腺癌4T1原位瘤模型，当肿瘤长到体积为100mm3时，随机将荷瘤小鼠分为6个组(Saline组、HBO组、Abraxane组、HBO+Abraxane组、Doxil组和HBO+Doxil组)并记为第1天，分别在第1、2、5、8、11天给予HBO组、HBO+Abraxane组和HBO+Doxil组小鼠高压氧治疗，治疗方式为，将小鼠置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。第2、5、8、11天在高压氧之前通过尾静脉注射给予HBO+Abraxane组小鼠0.2mg/0.1mL Abraxane，给予HBO+Doxil组小鼠0.08mg/0.1mL Doxil。并分别将生理盐水、Abraxane、Doxil以0.1mL/只、0.1mg/0.1mL、0.08mg/0.1mL剂量通过尾静脉注射到对应实验组小鼠。治疗结束后，在第11天处死小鼠，分离小鼠肿瘤组织。用剪刀将肿瘤组织剪碎后，加入胶原蛋白酶和DNA酶消化40分钟后，使用50mL注射器橡胶塞子研磨并通过70μm细胞筛网过滤得到单细胞悬液。
使用APC-CD1331流式抗体、Hoechst33342以及EDU染料与细胞悬液共孵育后流式细胞仪分析CD133阳性细胞的细胞周期及G0-G1细胞占比。
图5为4T1乳腺癌治疗结束后各组小鼠肿瘤组织中肿瘤干细胞的细胞周期分布，从图中可以看出，经过高压氧处理的三个组相比于对照组，处于S分裂期的细胞比率提高了。
图6为4T1乳腺癌治疗结束后各组小鼠肿瘤组织中肿瘤干细胞处于G0/G1周期阻滞的细胞比率。从图中可以看出HBO组、HBO+Abraxane组和HBO+Doxil组处于G0/G1期的干细胞比率相较于各自的对照组显著下降了。而处于G0/G1期的细胞由于周期阻滞，不能进入DNA的复制分裂期，因此对Abraxane和Doxil的药物杀伤不敏感，从而导致化疗耐药。这个结果表明，高压氧处理能降低处于周期阻滞的干细胞比率，增加肿瘤干细胞对纳米化疗药物的敏感性。
实施例10
基于高压氧治疗对荷乳腺癌原位瘤小鼠肿瘤血管形态影响的测试。
建立小鼠乳腺癌4T1原位瘤模型，当肿瘤长到体积为100mm 3时，随机将荷瘤小鼠分为2个组(Saline组、HBO组)并记为第1天，分别在第1、2、5、8、11天给予HBO组小鼠高压氧治疗，治疗方式为，将小鼠置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。第2、5、8、11天通过尾静脉注射给予Saline组小鼠0.1mL生理盐水。治疗结束后， 在第11天处死小鼠，分离小鼠肿瘤组织，多聚甲醛固定包埋切片后，通过CD31染色检测肿瘤组织中血管的形态与密度。
图7是CD31免疫荧光染色的切片拍照结果。从图中可以看出血管的形态，Saline组血管呈现弯曲增粗的形态，而HBO组的血管形态正常。
图8是对切片中血管密度的统计结果，从图中可以看出，高压氧处理会显著降低肿瘤组织中血管的密度。
图9是对切片中血管弯曲程度的统计结果，从图中可以看出，高压氧处理后血管弯曲程度显著降低，这表明HBO处理可以促进肿瘤血管正常化。
实施例11
基于高压氧治疗对荷乳腺癌原位瘤小鼠肿瘤血流灌注影响的测试。
建立小鼠乳腺癌4T1原位瘤模型，当肿瘤长到体积为1500mm 3时，通过腹腔注射1％戊巴比妥钠溶液麻醉小鼠，使用激光散斑血流成像仪检测小鼠肿瘤血管的血流灌注情况，随后给予小鼠高压氧治疗，治疗方式为，将小鼠置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。在治疗结束后再次麻醉小鼠并通过激光散斑血流成像仪检测小鼠肿瘤血管的血流灌注情况。
图10是小鼠高压氧前后血流灌注图。从图中可以看出，高压氧处理后小鼠肿瘤血管的血流灌注量增强了。
图11是对小鼠高压氧前后血流灌注量的定量统计结果。从图中可以看出，三只不同的小鼠在高压氧处理之后，血流灌注分别增强了7％、3.7％和2.9％。这个结果表明，高压氧处理可以提高肿瘤血管中血流灌注量。
实施例12
基于高压氧联合纳米化疗药物对荷乳腺癌原位瘤小鼠药物递送的测试。
实验药物的配制：使用Cy5-NHS酯标记Abraxane，并通过透析除去多余的Cy5分子。再将标记后的Abraxane溶解在生理盐水中配制成2mg/mL的溶液。
建立小鼠乳腺癌4T1原位瘤模型，当皮下瘤长到体积为250mm 3时，随机将荷瘤小鼠分为2个组(Abraxane组和HBO+Abraxane组)分组后记为第0小时，立即通过尾静脉注射给予Abraxane组和Abraxane组小鼠0.3mL Cy5荧光标记的Abraxane。给予HBO+Abraxane抑制剂组小鼠高压氧治疗，治疗方式为，将小鼠置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱 内气压降至大气压。使用小动物成像系统在第0、4、8、12、16、24小时分别对小鼠进行成像，检测Abraxane在小鼠体内的分布，并在第24小时处死小鼠，分离心肝脾肺肾和肿瘤，使用小动物成像系统检测Abraxane在小鼠各组织中的分布情况。
图12为小动物成像检测到纳米化疗药物在活体小鼠体内分布随时间的变化以及离体组织中的分布情况。从图中可以看出，在第0小时，两个组小鼠均无本底荧光信号，在注射荧光标记的Abraxane之后，随时间变化，小鼠体内逐渐出现不同强弱的荧光信号，HBO联合Abraxane组小鼠肿瘤部位Abraxane的荧光信号在不同时间点均强与单用Abraxane组。
图13位24小时的组织分布图，结果同样显示HBO联合Abraxane组小鼠肿瘤组织中Abraxane的荧光信号强于单用Abraxane组。
图14为小动物成像检测到Abraxane在活体小鼠肿瘤组织分布随时间的变化的定量结果。从图中可以看出，随着时间的延长，Abraxane在各组小鼠肿瘤组织中的富集逐渐增多，其中，第0到24小时为快速富集阶段，随后富集速度逐渐减慢。而在整个过程中，HBO联合Abraxane治疗组Abraxane在肿瘤部位的富集量均显著高于单用Abraxane组，HBO处理将Abraxane在肿瘤的平均富集量提高了47％左右。
图15为小动物成像检测的Abraxane组织分布定量结果。从图中可以看出，与单用Araxane组相比，联合治疗组肿瘤部位Abraxane的富集量显著增多，其余各器官富集量无显著性差异，结果表明HBO可以显著提高Abraxane在小鼠肿瘤部位的富集。
实施例13
基于高压氧联合纳米化疗药物对荷乳腺癌原位瘤小鼠肿瘤细胞药物摄取的测试。
实验药物的配制：使用Cy5-NHS酯标记Abraxane，并通过透析除去多余的Cy5分子。再将标记后的Abraxane溶解在生理盐水中配制成2mg/mL的溶液。
建立小鼠乳腺癌4T1原位瘤模型，当肿瘤长到体积为100mm 3时，随机将荷瘤小鼠分为2个组(Abraxane组和HBO+Abraxane组)并记为第1天，分别在第1、2、5、8、11天给予HBO+Abraxane组小鼠高压氧治疗，治疗方式为，将小鼠置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。第2、5、8、11天在高压氧之前通过尾静脉注射给予HBO+Abraxane组小鼠0.2mg/0.1mL Abraxane，并将Abraxane以0.1mg/0.1mL的剂量 通过尾静脉注射到Abraxane组小鼠体内。治疗结束后，在第11天处死小鼠，分离小鼠肿瘤组织。用剪刀将肿瘤组织剪碎后，加入胶原蛋白酶和DNA酶消化40分钟后，使用50mL注射器橡胶塞子研磨并通过70μm细胞筛网过滤得到单细胞悬液。通过流式细胞仪检测肿瘤细胞对Abraxane-Cy5的摄取量。
图16是4T1乳腺癌原位瘤治疗后各组肿瘤细胞对Abraxane-Cy5的摄取量荧光强度定量统计结果，从图中可以看出，HBO+Abraxane组肿瘤细胞对Abraxane-Cy5的摄取量相较于单用Abraxane组提高了90％，这个结果表明高压氧处理可以提高肿瘤细胞对纳米化疗药物的摄取。
实施例14
基于高压氧联合纳米化疗药物对荷乳腺癌原位瘤小鼠抗肿瘤活性的测试。
实验药物的配制：将纳米化疗药物Abraxane溶解在生理盐水中配制成2mg/mL的溶液，将纳米化疗药物Doxil溶解在生理盐水中配制成0.8mg/mL的溶液。
建立小鼠乳腺癌4T1原位瘤模型，当肿瘤长到体积为100mm 3时，随机将荷瘤小鼠分为6个组(Saline组、HBO组、Abraxane组、HBO+Abraxane组、Doxil组和HBO+Doxil组)并记为第1天，分别在第1、2、5、8、11天给予HBO组、HBO+Abraxane组和HBO+Doxil组小鼠高压氧治疗，治疗方式为，将小鼠置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。第2、5、8、11天在高压氧之前通过尾静脉注射给予HBO+Abraxane组小鼠0.2mg/0.1mL Abraxane，给予HBO+Doxil抑制剂组小鼠0.08mg/0.1mL Doxil。并分别将PBS、Abraxane、Doxil以0.1mL/只、0.1mg/0.1mL、0.08mg/0.1mL剂量通过尾静脉注射到对应实验组小鼠。每两天用游标卡尺测量肿瘤的最长处(L)和最宽处(W)，计算肿瘤体积V＝L*W 2/2，在第23天将各组小鼠处死，剥出皮下肿瘤并称重。在检测肿瘤体积的同时，每两天测量小鼠的体重，并记录。
在实验结束时，处死小鼠，取出全血检测血细胞成分及含量，并采血分离出血清，分析其中的血生化指标，共检测谷丙转氨酶、谷草转氨酶、总尿素氮和心肌肌酸激酶四项指标。同时取出小鼠完整的肺，使用Bouin’s染液对其进行固定染色，24小时后用95％乙醇洗涤后，记录肺部肿瘤转移节结数并拍照。同时将小鼠心、肝、脾、肺、肾五种主要脏器取出，用4％多聚甲醛固定，之后进行常规石蜡包埋包埋切片、HE染色，在显微镜下观察组织结构。
图17是治疗过程中小鼠肿瘤体积增长-时间曲线，与Saline组相比，HBO组小 鼠肿瘤生长没有明显的抑制，这说明单独使用HBO治疗并没有明显的抑瘤效果。而Abraxane组和Doxil组相比于Saline组均表现出了抑制肿瘤生长的作用。HBO+Abraxane与HBO+Doxil治疗组肿瘤生长速度明显慢于单用纳米化疗药物治疗组，在第31天，HBO+Abraxane与HBO+Doxil治疗组联合治疗组小鼠平均瘤体积为1633和255mm 3，显著低于单用Abraxane和Doxil组的1949和451mm 3。以上分析表明，HBO联合纳米化疗药物可以显著增强纳米化疗药物对肿瘤生长的抑制效果。
图18为小鼠治疗结束后取出肿瘤称重的统计结果，由图18可知，Abraxane和Doxil组的肿瘤重量明显低于Saline组，同时，联合治疗组的瘤重显著低于单用纳米化疗药物治疗组，这表明HBO联合纳米化疗药物可以增强纳米化疗药物的抗肿瘤效果。
图19为以上6个组在给药期间的体重变化图，由图19可知，相比于Saline组，五个实验组小鼠体重没有明显的下降或上涨趋势，这表明单独使用HBO或Abraxane、Doxil治疗，以及联合治疗均不会影响小鼠的健康状况。
图20为各组治疗结束后血细胞检测结果。由图20可知，与Saline组相比，五个实验组小鼠在治疗结束后血液中白细胞、红细胞、血小板含量以及平均血红蛋白含量均没有明显差异，这表明单独使用HBO或纳米化疗药物治疗，以及联合治疗均具有良好的安全性。
图21为各组治疗结束后的血生化分析结果，其中谷丙转氨酶和谷草转氨酶为肝功能指标，该指标的升高代表着药物具有严重的肝脏毒性，心肌肌酸激酶为心脏功能指标，其值升高意味着心脏发生了病理变化，总尿素氮为肾脏功能指标，该指标的升高意味着肾脏功能出现问题。由图21可知，五个实验组的四项血生化指标与对照组相比都没有显著性变化，以上结果表明，单独使用HBO或纳米化疗药物治疗，以及联合治疗均不会对小鼠的主要脏器产生损伤，治疗具有良好的安全性
图22为组织切片的结构图，放大倍数为200倍。由图22可知，Saline组与五个实验组的心肝脾肺肾结构清晰，没有明显的病理改变，无出血与炎症浸润，以上结果表明，单独使用HBO或纳米化疗药物治疗，以及联合治疗均不会对小鼠的主要脏器产生损伤，具有良好的安全性。
实施例15
基于高压氧联合纳米化疗药物对荷乳腺癌原位瘤小鼠生存期影响的测试。
实验药物的配制：将纳米化疗药物Abraxane溶解在生理盐水中配制成2mg/mL 的溶液，将纳米化疗药物Doxil溶解在生理盐水中配制成0.8mg/mL的溶液。
建立小鼠乳腺癌4T1原位瘤模型并记为第1天，当肿瘤长到体积为100mm 3时，随机将荷瘤小鼠分为6个组(Saline组、HBO组、Abraxane组、HBO+Abraxane组、Doxil组和HBO+Doxil组)并记为第1天，分别在第1、2、5、8、11天给予HBO组、HBO+Abraxane组和HBO+Doxil组小鼠高压氧治疗，治疗方式为，将小鼠置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。第2、5、8、11天在高压氧之前通过尾静脉注射给予HBO+Abraxane组小鼠0.2mg/0.1mL Abraxane，给予HBO+Doxil抑制剂组小鼠0.08mg/0.1mL Doxil。治疗结束后每天观察小鼠的存活状态，并记录。
图23为各组小鼠生存期Kaplan-Meier图，Saline组、HBO组、Abraxane组、HBO+Abraxane组、Doxil组和HBO+Doxil组的中位生存期依次为37、38.5、41.5、43.5、47和58天。HBO+Doxil的生存期相较于单用Doxil显著延长了。以上结果分析表明，HBO联合纳米化疗药物治疗可以显著延长荷4T1乳腺癌原位瘤小鼠生存期。
实施例16
基于高压氧联合纳米化疗药物对荷胰腺癌原位瘤小鼠抗肿瘤效果的测试。
实验药物的配制：将纳米化疗药物Abraxane溶解在生理盐水中配制成2mg/mL的溶液，将纳米化疗药物Doxil溶解在生理盐水中配制成0.8mg/mL的溶液。
建立小鼠胰腺癌Panc02原位瘤模型并记为第1天，一周后随机将荷瘤小鼠分为6个组(Saline组、HBO组、Abraxane组、HBO+Abraxane组、Doxil组和HBO+Doxil组)并记为第1天，分别在第1、2、5、8、11天给予HBO组、HBO+Abraxane组和HBO+Doxil组小鼠高压氧治疗，治疗方式为，将小鼠置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。第2、5、8、11天在高压氧之前通过尾静脉注射给予HBO+Abraxane组小鼠0.2mg/0.1mL Abraxane，给予HBO+Doxil抑制剂组小鼠0.08mg/0.1mL Doxil。并分别将PBS、Abraxane、Doxil以0.1mL/只、0.1mg/0.1mL、0.08mg/0.1mL剂量通过尾静脉注射到对应实验组小鼠。在第21天处死小鼠，剥离肿瘤并称重，取出小鼠全血检测血细胞成分及含量，并采血分离出血清，分析其中的血生化指标，共检测谷丙转氨酶、谷草转氨酶、总尿素氮和心肌肌酸激酶四项指标。同时将小鼠心、肝、脾、肺、肾五种主要脏器取出，用4％多聚甲醛固定，之后进行常规石蜡包埋切片、HE 染色，在显微镜下观察组织结构。
图24为小鼠治疗结束后取出肿瘤称重的统计结果，由图7可知，Abraxane和Doxil组的肿瘤重量明显低于Saline组，同时，联合治疗组的瘤重显著低于单用纳米化疗药物治疗组，这表明HBO联合纳米化疗药物可以增强纳米化疗药物的抗肿瘤效果。
图25为以上6个组在给药期间的体重变化图，由图25可知，相比于Saline组，五个实验组小鼠体重没有明显的下降或上涨趋势，这表明单独使用HBO或Abraxane、Doxil治疗，以及联合治疗均不会影响小鼠的健康状况。
图26为各组治疗结束后血细胞检测结果。由图26可知，与Saline组相比，五个实验组小鼠在治疗结束后血液中白细胞、红细胞、血小板含量以及平均血红蛋白含量均没有明显差异，这表明单独使用HBO或纳米化疗药物治疗，以及联合治疗均具有良好的安全性。
图27为各组治疗结束后的血生化分析结果，其中谷丙转氨酶和谷草转氨酶为肝功能指标，该指标的升高代表着药物具有严重的肝脏毒性，心肌肌酸激酶为心脏功能指标，其值升高意味着心脏发生了病理变化，总尿素氮为肾脏功能指标，该指标的升高意味着肾脏功能出现问题。由图27可知，五个实验组的四项血生化指标与对照组相比都没有显著性变化，以上结果表明，单独使用HBO或纳米化疗药物治疗，以及联合治疗均不会对小鼠的主要脏器产生损伤，治疗具有良好的安全性
图28为组织切片的结构图，放大倍数为200倍。由图28可知，Saline组与五个实验组的心肝脾肺肾结构清晰，没有明显的病理改变，无出血与炎症浸润，以上结果表明，单独使用HBO或纳米化疗药物治疗，以及联合治疗均不会对小鼠的主要脏器产生损伤，具有良好的安全性。
实施例17
基于高压氧联合纳米化疗药物对荷胰腺癌原位瘤小鼠生存期影响的测试。
实验药物的配制：将纳米化疗药物Abraxane溶解在生理盐水中配制成2mg/mL的溶液，将纳米化疗药物Doxil溶解在生理盐水中配制成0.8mg/mL的溶液。
建立小鼠胰腺癌Panc02原位瘤模型，一周后随机将荷瘤小鼠分为6个组(Saline组、HBO组、Abraxane组、HBO+Abraxane组、Doxil组和HBO+Doxil组)并记为第1天，分别在第1、2、5、8、11天给予HBO组、HBO+Abraxane组和HBO+Doxil组小鼠高压氧治疗，治疗方式为，将小鼠置于密闭舱体内，逐渐通入纯氧使舱内气 压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。第2、5、8、11天在高压氧之前通过尾静脉注射给予HBO+Abraxane组小鼠0.2mg/0.1mL Abraxane，给予HBO+Doxil抑制剂组小鼠0.08mg/0.1mL Doxil。并分别将PBS、Abraxane、Doxil以0.1mL/只、0.1mg/0.1mL、0.08mg/0.1mL剂量通过尾静脉注射到对应实验组小鼠。治疗结束后每天观察小鼠的存活状态，并记录。
图29为各组小鼠生存期Kaplan-Meier图，Saline组、HBO组、Abraxane组、HBO+Abraxane组的中位生存期依次为40、43、57.5和64天，而Doxil和HBO+Doxil组由于疗效好，死亡小鼠均未过半，无法计算中位生存期。HBO+Abraxane的生存期相较于单用Abraxane显著延长了。以上结果分析表明，HBO联合纳米化疗药物治疗可以显著延长荷Panc02胰腺癌原位瘤小鼠生存期。
实施例18
基于高压氧联合纳米化疗药物对乳腺癌转移瘤小鼠抗肿瘤活性的测试。
实验药物的配制：将纳米化疗药物Abraxane溶解在生理盐水中配制成2mg/mL的溶液，将纳米化疗药物Doxil溶解在生理盐水中配制成0.8mg/mL的溶液。
通过尾静脉注射4T1细胞建立小鼠乳腺癌转移模型，一周后随机将荷瘤小鼠分为6个组(Saline组、HBO组、Abraxane组、HBO+Abraxane组、Doxil组和HBO+Doxil组)并记为第1天，分别在第1、2、5、8、11天给予HBO组、HBO+Abraxane组和HBO+Doxil组小鼠高压氧治疗，治疗方式为，将小鼠置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。第2、5、8、11天在高压氧之前通过尾静脉注射给予HBO+Abraxane组小鼠0.2mg/0.1mL Abraxane，给予HBO+Doxil抑制剂组小鼠0.08mg/0.1mL Doxil。并分别将PBS、Abraxane、Doxil以0.1mL/只、0.1mg/0.1mL、0.08mg/0.1mL剂量通过尾静脉注射到对应实验组小鼠。第20天将各组小鼠处死，剥出肺并称重，通过使用Bouin’s染液对其进行固定染色，24小时后用95％乙醇洗涤后，记录肺部肿瘤转移节结数并拍照。
图30为四组小鼠治疗结束后肺部转移节结数统计结果，由图30可知，Abraxane和Doxil组相比于Saline组，小鼠肺部转移节结数明显减少，而联合治疗组肺部转移节结数显著少于单用纳米化疗药物治疗组，这表明HBO联合纳米化疗药物治疗对肿瘤转移具有明显的抑制效果。
实施例19
基于高压氧联合纳米化疗药物对乳腺癌转移瘤小鼠生存期影响的测试。
实验药物的配制：将纳米化疗药物Abraxane溶解在生理盐水中配制成2mg/mL的溶液，将纳米化疗药物Doxil溶解在生理盐水中配制成0.8mg/mL的溶液。
通过尾静脉注射4T1细胞建立小鼠乳腺癌转移模型，一周后随机将荷瘤小鼠分为6个组(Saline组、HBO组、Abraxane组、HBO+Abraxane组、Doxil组和HBO+Doxil组)并记为第1天，分别在第1、2、5、8、11天给予HBO组、HBO+Abraxane组和HBO+Doxil组小鼠高压氧治疗，治疗方式为，将小鼠置于密闭舱体内，逐渐通入纯氧使舱内气压升高到2.5倍大气压，维持1.5小时后，缓慢放气使舱内气压降至大气压。第2、5、8、11天在高压氧之前通过尾静脉注射给予HBO+Abraxane组小鼠0.2mg/0.1mL Abraxane，给予HBO+Doxil抑制剂组小鼠0.08mg/0.1mL Doxil。并分别将PBS、Abraxane、Doxil以0.1mL/只、0.1mg/0.1mL、0.08mg/0.1mL剂量通过尾静脉注射到对应实验组小鼠。治疗结束后每天观察小鼠的存活状态，并记录。
图31为各组小鼠生存期Kaplan-Meier图，Saline组、HBO组、Abraxane组、HBO+Abraxane组、Doxil组和HBO+Doxil组的中位生存期依次为24.5、26.5、27.5、30.5、39.5和45天。HBO+Doxil的生存期相较于单用Doxil显著延长了。以上结果分析表明，HBO联合纳米化疗药物治疗可以显著延长乳腺癌转移瘤小鼠生存期。
结合以上动物实验、干细胞与细胞周期检测、血管形态与血流灌注分析、药效与生存期检测、血生化分析、组织切片和药物体内递送与分布的实验结果，可以看出，高压氧与纳米化疗药物联合，通过高压氧改善肿瘤组织乏氧，降低肿瘤组织中干细胞的占比，显著调控肿瘤血管正常化并促进纳米化疗药物在肿瘤部位的穿透和富集，提高肿瘤细胞对纳米化疗药物的摄取，增强纳米化疗药物的抗肿瘤疗效，。最后，本发明的方法仅为较佳的实施方案，并非用于限定本发明的保护范围。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种降低肿瘤组织中干细胞比例的方法，其特征在于，对肿瘤患者进行高压氧治疗，以降低肿瘤患者其肿瘤组织中干细胞的比例；
    所述高压氧治疗具体为：将肿瘤患者置于高压氧环境中进行治疗。
  2. 一种调控肿瘤组织血管正常化的方法，其特征在于，对肿瘤患者进行高压氧治疗，以调控促进肿瘤患者其肿瘤组织血管正常化；
    所述高压氧治疗具体为：将肿瘤患者置于高压氧环境中进行治疗。
  3. 如权利要求1或2所述的方法，其特征在于，对肿瘤患者进行高压氧治疗包括如下步骤：将肿瘤患者置于舱体内，逐渐通入纯氧使舱内气压升高到2-5倍大气压，维持一段时间后，放气使舱内气压降至大气压。
  4. 如权利要求1或2所述的方法，其特征在于，将高压氧治疗与纳米化疗药物治疗相结合，具体包括如下步骤：
    (1)对肿瘤患者进行高压氧治疗；所述高压氧治疗通过增加肿瘤患者所处环境的气压及吸入氧气的浓度来提高溶解于血液中氧气的量，从而提高各组织中氧的含量；
    (2)对肿瘤患者静脉注射纳米化疗药物。
  5. 如权利要求4所述的方法，其特征在于，对肿瘤患者进行单次或多次高压氧治疗后，再对肿瘤患者静脉注射纳米化疗药物。
  6. 如权利要求4所述的方法，其特征在于，所述高压氧治疗与静脉注射纳米化疗药物之间的间隔时间为0-12小时。
  7. 如权利要求4所述的方法，其特征在于，静脉注射纳米化疗药物之前或之后，对肿瘤患者给予1-10次高压氧治疗。
  8. 如权利要求4所述的方法，其特征在于，每一至三周对肿瘤患者静脉注射纳米化疗药物一次，其单次注射剂量为100-300mg/m 2。
  9. 如权利要求4所述的方法，其特征在于，重复步骤(1)和步骤(2)以完成一次治疗，持续治疗不少于1次。
  10. 如权利要求1或2所述的方法，其特征在于，所述肿瘤为实体瘤。
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