WO2022246595A2 - 一种抑制肿瘤相关成纤维细胞及调控肿瘤基质正常化的方法 - Google Patents

一种抑制肿瘤相关成纤维细胞及调控肿瘤基质正常化的方法 Download PDF

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WO2022246595A2
WO2022246595A2 PCT/CN2021/095491 CN2021095491W WO2022246595A2 WO 2022246595 A2 WO2022246595 A2 WO 2022246595A2 CN 2021095491 W CN2021095491 W CN 2021095491W WO 2022246595 A2 WO2022246595 A2 WO 2022246595A2
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tumor
hyperbaric oxygen
group
abraxane
oxygen therapy
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PCT/CN2021/095491
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English (en)
French (fr)
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李子福
杨祥良
王小贤
叶凝冰
张智杰
邓青源
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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
    • A61G10/04—Oxygen tents ; Oxygen hoods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution

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  • the invention belongs to the field of tumor treatment, and more specifically relates to a method for inhibiting tumor-associated fibroblasts and regulating the normalization of tumor stroma.
  • Tumor-associated fibroblasts are fibroblasts in an activated state and are one of the important components in the tumor microenvironment. They interact with tumor cells to regulate biological processes such as cancer cell proliferation, migration, invasion, and drug resistance. .
  • the extracellular matrix is a complex network structure composed of macromolecular substances synthesized by cells and secreted into the extracellular matrix. It is an important part of the tumor microenvironment, mainly including collagen, fibronectin, and hyaluronic acid. It plays an important role in the process of growth, invasion and metastasis.
  • the deposition of tumor extracellular matrix can not only squeeze tumor blood vessels, reduce the blood supply to the tumor site, thereby reducing drug delivery; at the same time, the dense extracellular matrix also acts as a physical barrier to further hinder the penetration of drugs, greatly reducing the efficacy of drugs.
  • the present invention provides a method for inhibiting tumor-associated fibroblasts and regulating the normalization of tumor stroma, the purpose of which is to reduce tumor-associated fibroblasts and extracellular matrix in tumor tissues of patients through hyperbaric oxygen therapy.
  • the proportion of points can enhance the efficiency of drug delivery to improve the curative effect of tumor chemotherapy, and solve the problem of poor curative effect of chemotherapy drugs in clinical practice.
  • the present invention provides a method for inhibiting the proportion of tumor-associated fibroblasts in tumor tissue, performing hyperbaric oxygen therapy on tumor patients to reduce the proportion of tumor-associated fibroblasts in tumor tissues of tumor patients;
  • the hyperbaric oxygen therapy specifically includes placing tumor patients in a hyperbaric oxygen environment for treatment.
  • the present invention also provides a method for regulating the normalization of tumor stroma, performing hyperbaric oxygen therapy on tumor patients to regulate the normalization of tumor stroma in tumor patients; the hyperbaric oxygen therapy specifically includes: placing tumor patients in a hyperbaric oxygen environment for treatment.
  • the present invention treats tumor patients with hyperbaric oxygen therapy, that is, placing tumor patients in a hyperbaric oxygen environment for treatment.
  • hyperbaric oxygen therapy that is, placing tumor patients in a hyperbaric oxygen environment for treatment.
  • ⁇ -SMA tumor-associated fibroblasts
  • Panc02 pancreatic cancer orthotopic tumor mice are treated with hyperbaric oxygen in a certain embodiment, compared with the control group that does not carry out hyperbaric oxygen treatment, tumor-associated fibroblasts ( ⁇ -SMA) in mouse tumor tissue ) decreased by 28%.
  • the present invention combines hyperbaric oxygen therapy with intravenous injection of chemotherapeutic drugs to perform anti-tumor treatment on tumor patients.
  • performing hyperbaric oxygen therapy on tumor patients can further reduce the number of tumor-associated fibroblasts ( ⁇ -SMA) in tumor tissues. )proportion.
  • hyperbaric oxygen combined with chemotherapy drugs GEM, Abraxane and GEM+Abraxane can reduce the proportion of tumor-associated fibroblasts ( ⁇ -SMA) by 29% without increasing the toxic and side effects. %.
  • the present invention treats tumor patients with hyperbaric oxygen therapy, that is, places tumor patients in a hyperbaric oxygen environment for treatment.
  • hyperbaric oxygen therapy that is, places tumor patients in a hyperbaric oxygen environment for treatment.
  • performing hyperbaric oxygen therapy on tumor patients can significantly reduce tumor extracellular matrix deposition, such as in a certain embodiment
  • the proportion of Collagen I and Fibronectin in the tumor tissue of the mice decreased by 42% and 40%, respectively.
  • the present invention combines hyperbaric oxygen therapy with intravenous injection of chemotherapy drugs to perform anti-tumor treatment on tumor patients.
  • hyperbaric oxygen therapy on tumor patients can significantly normalize tumor extracellular matrix and reduce the overall density and curvature of tumor blood vessels. Increase the concentration and penetration of nano-drugs in tumor sites, and at the same time increase the uptake of drugs by tumor cells.
  • hyperbaric oxygen combined with chemotherapy drugs GEM, Abraxane and GEM+Abraxane can reduce the proportion of Collagen I in tumor tissue by 41%, 42% and 46%, respectively, without increasing toxic and side effects. %, the proportion of Fibronectin decreased by 44%, 48% and 49%, respectively. Therefore, the enrichment amount and penetration depth of the nanomedicine Abraxane at the treatment site and the uptake of the chemotherapeutic nanodrug by tumor cells were significantly increased by 1.91 times, 4.78 times and 2.08 times, respectively.
  • the present invention combines hyperbaric oxygen therapy with intravenous injection of chemotherapy drugs to perform anti-tumor treatment on tumor patients.
  • performing hyperbaric oxygen therapy on tumor patients can significantly reduce the proportion of tumor-associated fibroblasts in tumor tissues, while regulating Tumor extracellular matrix reduces the overall density and curvature of blood vessels, increases the enrichment and penetration of chemotherapeutic nano-drugs in tumor sites, and at the same time increases the uptake of tumor cells to drugs, ultimately enhancing the efficacy of tumor chemotherapy, and has good safety.
  • the median survival of mice in the HBO combined with GEM+Abraxane clinical first-line treatment for pancreatic cancer group was at least 10 days longer than other groups.
  • Fig. 1 is the ratio of tumor-associated fibroblasts ( ⁇ -SMA) in the tumor-associated fibroblasts ratio experiment carried out in Example 5 of the present invention
  • Figure 2 is the ratio of tumor-associated fibroblasts (CD45 - Ter-119 - Ep-CAM - CD31- ) in the tumor-associated fibroblast ratio experiment carried out in Example 5 of the present invention
  • Figure 4 is the ratio of Fibronectin in the tumor extracellular matrix ratio experiment carried out in Example 6 of the present invention.
  • Fig. 5 is the statistical result of blood vessel density in the tumor blood vessel morphology detection experiment carried out in Example 7 of the present invention.
  • Fig. 6 is the statistical result of the curvature of blood vessels in the tumor blood vessel shape detection experiment carried out in Example 7 of the present invention.
  • Fig. 7 is the in vivo fluorescence imaging diagram of the small animal imaging detection mouse of each experimental group in Example 8 of the present invention.
  • Fig. 8 is the enrichment amount-time curve of nano-drugs for chemotherapeutics in living tumor tissue detected by imaging of small animals in each experimental group in Example 8 of the present invention
  • Fig. 9 is a fluorescence imaging diagram of isolated tissues detected by imaging of small animals of each experimental group in Example 8 of the present invention.
  • Figure 10 is a quantitative diagram of the enrichment of chemotherapeutic nano-drugs in tumor tissues detected by imaging of small animals in each experimental group in Example 8 of the present invention
  • Example 11 is a quantitative diagram of the distribution of chemotherapeutic nano-drugs in various tissues detected by imaging of small animals in each experimental group in Example 8 of the present invention.
  • Figure 12 is the quantitative result of tumor drug accumulation in each experimental group in Example 9 of the present invention.
  • Figure 13 is the quantitative result of drug penetration depth of tumor blood vessels in each experimental group in Example 9 of the present invention.
  • Figure 14 is the quantitative result of drug uptake by each experimental group in Example 10 of the present invention.
  • Figure 15 is the result of the tumor weight of the mice in the antitumor activity experiment carried out in Example 11 of the present invention.
  • Figure 16 is the number of intestinal metastatic nodules in mice in the antitumor activity experiment carried out in Example 11 of the present invention.
  • Figure 17 is the body weight-time curve of mice in the antitumor activity experiment carried out in Example 11 of the present invention.
  • Figure 18 is the result of the blood cell index of each experimental group in Example 11 of the present invention.
  • Fig. 19 is the result of the blood biochemical indexes of each experimental group in Example 11 of the present invention.
  • Fig. 20 is a microscopic observation picture of tissue sections of each experimental group in Example 11 of the present invention.
  • Fig. 21 is the result of the survival period of mice in each group in Example 12 of the present invention.
  • the present invention provides a method for reducing the proportion of tumor-associated fibroblasts in tumor tissues and/or regulating the normalization of tumor stroma, by performing hyperbaric oxygen therapy on tumor patients to reduce tumor-associated fibroblasts in tumor tissues of tumor patients ratio, and regulate and promote the normalization of the tumor stroma in the tumor tissue of the tumor patient;
  • the hyperbaric oxygen therapy specifically includes: placing the tumor patient in a hyperbaric oxygen environment for treatment.
  • performing a hyperbaric oxygen therapy on a tumor patient includes the following steps: placing the tumor patient in the cabin, gradually introducing pure oxygen to increase the pressure in the cabin to 2-5 times the atmospheric pressure, and after maintaining for a period of time, Deflation reduces the pressure in the cabin to atmospheric pressure.
  • the methods for reducing the proportion of tumor-associated fibroblasts in tumor tissue and/or regulating the normalization of tumor stroma combine hyperbaric oxygen therapy with chemotherapeutic drug treatment to further reduce tumor tissue in tumor patients.
  • the proportion of tumor-associated fibroblasts in the tumor, and regulating and promoting the normalization of the extracellular matrix in the tumor tissue of the tumor patient specifically includes the following steps:
  • hyperbaric oxygen therapy increases the amount of oxygen dissolved in the blood of tumor patients by increasing the air pressure of the tumor patient's environment and the concentration of inhaled oxygen, thereby increasing the perfusion of oxygen in each tissue .
  • the present invention does not limit the sequence of hyperbaric oxygen therapy and intravenous chemotherapy drug therapy.
  • chemotherapy drugs are injected intravenously to tumor patients first, and then single or multiple hyperbaric oxygen therapy is performed on tumor patients.
  • hyperbaric oxygen therapy for tumor patients can improve the hypoxic environment of the tumor, reduce the proportion of tumor-associated fibroblasts in the tumor tissue, and most importantly, it can significantly regulate the extracellular matrix of the tumor and increase the concentration of chemotherapeutic nano-drugs on the tumor site.
  • the enrichment and penetration of nano-drugs can improve the anti-tumor efficacy of chemotherapeutic nano-drugs.
  • hyperbaric oxygen (HBO) therapy refers to intermittent breathing of 100% oxygen at a pressure higher than one atmosphere.
  • the principle of hyperbaric oxygen therapy is to increase the amount of dissolved oxygen in the blood and tissues by ingesting pure oxygen at a pressure higher than normal pressure (1ATA).
  • hyperbaric oxygen therapy can reduce edema, activate angiogenesis, and increase collagen synthesis. Therefore, hyperbaric oxygen is used as the primary treatment for conditions such as decompression sickness (DCS), arterial gas embolism, hemorrhagic anemia, and severe carbon monoxide poisoning.
  • DCS decompression sickness
  • arterial gas embolism hemorrhagic anemia
  • severe carbon monoxide poisoning severe carbon monoxide poisoning.
  • the present invention combines hyperbaric oxygen with chemotherapeutic drugs, and experiments have found that it can significantly reduce the proportion of tumor-associated fibroblasts in tumor tissue, and most importantly, it can significantly regulate the normalization of tumor extracellular matrix and increase the concentration of chemotherapeutic nano-drugs on tumor sites.
  • the enrichment and penetration of nano-drugs can improve the anti-tumor efficacy of chemotherapeutic nano-drugs.
  • the hyperbaric oxygen therapy for tumor patients can be carried out according to the conventional method of hyperbaric oxygen therapy in the prior art.
  • Pure oxygen makes the air pressure in the cabin rise to 2-5 times atmospheric pressure, and after maintaining for a period of time, deflation makes the air pressure in the cabin drop to atmospheric pressure.
  • the chemotherapy drug treatment method is: intravenous injection of chemotherapy drugs.
  • tumor patients are placed in a closed cabin, and pure oxygen is gradually introduced to increase the air pressure in the cabin to 2-5 times the atmospheric pressure (preferably 2-2.5 times the atmospheric pressure), and maintain it for 1-4 hours (preferably 1 -2 hours), slowly deflate to reduce the cabin pressure to atmospheric pressure.
  • the present invention administers chemotherapeutic drugs to tumor patients in the form of intravenous injection.
  • hyperbaric oxygen therapy and chemotherapeutic drugs are variable, and in a preferred embodiment, intravenous chemotherapy drugs are injected first, followed by hyperbaric oxygen therapy.
  • the interval between administering chemotherapeutic drugs and hyperbaric oxygen therapy to patients is 0-12 hours.
  • Hyperbaric oxygen therapy can be given immediately after the chemotherapy drugs are administered, or at a later time.
  • the present invention gives tumor patients 3-4 times of hyperbaric oxygen therapy before or after intravenous injection of chemotherapeutic drugs to tumor patients.
  • step (1) and step (2) are repeated to complete one treatment, and the treatment is repeated no less than once.
  • the present invention can adjust the dosage of chemotherapeutic drugs according to actual application conditions, and can also set appropriate drug dosages according to different types of tumor chemotherapeutic drugs.
  • the chemotherapeutic drug Abraxane or GEM is used to inject the chemotherapeutic drug intravenously to tumor patients once every to three weeks, and the single injection dose is 100-1000 mg/m 2 .
  • the nanometer chemotherapeutic drug Abraxane is intravenously injected once every to three weeks, and the single injection dose is 125 mg/m 2 .
  • the chemotherapy drug GEM is intravenously injected once every to three weeks to tumor patients, and the single injection dose is 1000 mg/m 2 .
  • hyperbaric oxygen therapy is performed on tumor patients at least once, and the frequency of hyperbaric oxygen therapy is not limited, and hyperbaric oxygen therapy can be performed on tumor patients every day or every two days according to actual needs. oxygen therapy. In a preferred embodiment, hyperbaric oxygen therapy is given once a day.
  • the injection dose of chemotherapy drugs can be determined according to individual differences, such as intravenous injection once every week or every 2 weeks, until the disease progresses or unacceptable toxicity occurs.
  • hyperbaric oxygen therapy is performed 1-3 times a week after each injection of chemotherapy drugs.
  • the corresponding treatment plan is set according to the patient's condition and tolerance.
  • the chemotherapeutic drugs used in the present invention are clinically applicable various drugs, including Abraxane and GEM.
  • the intravenous injection dose of the chemotherapeutic drug of the present invention can refer to the clinically used injection dose of different chemotherapeutic drug types, and the corresponding injection frequency.
  • the clinically recommended dose of Abraxane is 125mg/m 2 , administered once a week.
  • the clinically recommended dosage of GEM is 1000mg/m 2 , administered once a week.
  • the method of administration of chemotherapy drugs, including reconstitution and dilution, can also be the same as the method of administration of the above-mentioned currently marketed drugs.
  • the therapeutic method of the present invention comprises the following steps:
  • the tumor treatment method proposed by the present invention can be applied to various tumor types, including non-small cell lung cancer, breast cancer, pancreatic cancer, ovarian cancer, bladder cancer, cervical cancer, liver cancer, biliary tract cancer, nasopharyngeal cancer, testicular tumor, lymphoid Hyperbaric oxygen combined therapy can significantly improve the enrichment and response rate of chemotherapeutic nano-drugs in solid tumors, and significantly inhibit tumor growth.
  • the hyperbaric oxygen combined with chemotherapy drugs of the present invention improves tumor tissue hypoxia through hyperbaric oxygen without increasing toxic side effects, reduces the proportion of tumor-associated fibroblasts in tumor tissues, and significantly regulates tumor extracellular matrix.
  • the hyperbaric oxygen combined with chemotherapy drugs of the present invention improves tumor tissue hypoxia through hyperbaric oxygen without increasing toxic side effects, reduces the proportion of tumor-associated fibroblasts in tumor tissues, and significantly regulates tumor extracellular matrix.
  • Normalize and increase the enrichment and penetration of chemotherapeutic nano-drugs in tumor sites increase the uptake of chemotherapeutic nano-drugs by tumor cells, and enhance the anti-tumor efficacy of chemotherapeutic nano-drugs.
  • the invention provides a new anti-tumor combined treatment mode, and expands the new application of hyperbaric oxygen.
  • chemotherapeutic drugs used in Example 2 to Example 12 are Abraxane of CSPC and GEM of Qilu Pharmaceutical.
  • This embodiment provides a method for reducing the proportion of tumor-associated fibroblasts in tumor tissue and/or promoting the normalization of tumor extracellular matrix by hyperbaric oxygen.
  • the individual to be treated is placed in a closed cabin, and pure oxygen is gradually introduced to reduce the air pressure in the cabin. Increase to 2.5 times the atmospheric pressure, and after maintaining for 1.5 hours, slowly deflate to reduce the air pressure in the cabin to atmospheric pressure.
  • the above-mentioned hyperbaric oxygen therapy was performed once a day for 4 consecutive days.
  • This embodiment provides a method for treating tumors with hyperbaric oxygen combined with chemotherapeutic drugs.
  • the method includes two parts: chemotherapy drug injection and hyperbaric oxygen therapy.
  • the method of hyperbaric oxygen therapy 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 then slowly release the air to reduce the pressure in the cabin to atmospheric pressure.
  • the above-mentioned hyperbaric oxygen therapy was carried out once a day for 4 consecutive days; on the 4th day, immediately after the end of the hyperbaric oxygen therapy, the chemotherapy nano-drug was injected intravenously, and the specific method was: intravenous injection of 125mg/ m2 chemotherapy nano-drug Abraxane once.
  • This embodiment provides a method for treating tumors with hyperbaric oxygen combined with nano-chemotherapy drugs, which includes chemotherapy drug injection and hyperbaric oxygen therapy.
  • the method of hyperbaric oxygen therapy 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 then slowly release the air to reduce the pressure in the cabin to atmospheric pressure.
  • hyperbaric oxygen therapy was given once a day for three consecutive days, on the fourth day, hyperbaric oxygen therapy was performed immediately after intravenous injection of 125 mg/m 2 chemotherapy nano-drug Abraxane and 1000 mg/m 2 chemotherapy drug GEM and the combination of the two. Chemotherapy drugs were injected intravenously every two days, and hyperbaric oxygen therapy was given immediately after the injection, a total of three times.
  • This embodiment provides a method for treating tumors with hyperbaric oxygen combined with nano-chemotherapy drugs, which includes chemotherapy drug injection and hyperbaric oxygen therapy.
  • Chemotherapeutic drug treatment methods are: intravenous injection of 125 mg/m 2 chemotherapeutic nanometer drug Abraxane, intravenous injection of 1000 mg/m 2 chemotherapeutic drug GEM, and combined intravenous injection of both.
  • the method of hyperbaric oxygen therapy is as follows: after intravenous injection of chemotherapy nano-drugs, immediately 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, and after maintaining it for 1.5 hours, slowly deflate the cabin. The internal pressure drops to atmospheric pressure.
  • the chemotherapy nano drug Abraxane was dissolved in normal saline to prepare a 0.83 mg/mL solution
  • the chemotherapy drug GEM was dissolved in normal saline to prepare a 0.67 mg/mL solution
  • the chemotherapy nano drug Abraxane and the chemotherapy drug GEM was also dissolved in normal saline to prepare a solution of 0.83mg/mL Abraxane+0.67mg/mL GEM.
  • mice An orthotopic tumor model of pancreatic cancer Panc02 in mice was established, and the tumor-bearing mice were randomly divided into 8 groups (Saline group, HBO group, GEM group, HBO+GEM group, Abraxane group, HBO+Abraxane group, GEM+Abraxane group) one week later. group and HBO+GEM+Abraxane group) and recorded as the first day, the HBO group, HBO+GEM group, HBO+Abraxane group and HBO+GEM+Abraxane group were administered on the 1st, 2nd, 3rd, 4th, 7th and 10th day respectively The mice in the group were treated with hyperbaric oxygen.
  • the treatment method was to place the mice in a closed cabin, gradually introduce pure oxygen to increase the pressure in the cabin to 2.5 times the atmospheric pressure, and after maintaining it for 1.5 hours, slowly deflate the air to reduce the pressure in the cabin to atmospheric pressure.
  • each mouse in the HBO+GEM group was given 0.2mg/0.3mL GEM by tail vein injection, and each mouse in the HBO+Abraxane group was given 0.25mg/0.3mL Abraxane, and HBO+GEM was given to each mouse +Abraxane group 0.2mg/0.3mL GEM and 0.25mg/0.3mL Abraxane per mouse.
  • mice were sacrificed on the 11th day, and the tumor tissues of the mice were isolated. After a part of the tumor tissue was fixed and embedded in paraformaldehyde, the content of tumor-associated fibroblasts in the tumor tissue was detected by ⁇ -SMA staining.
  • Another part of the tumor tissue was chopped with scissors, digested with collagenase and DNase for 40 minutes, ground with a rubber stopper of a 50 mL syringe and filtered through a 70 ⁇ m cell mesh to obtain a single cell suspension.
  • Tumor-associated fibroblasts (CD45 - Ter-119 - Ep-CAM - CD31 - ) to all cells.
  • Figure 1 is the quantitative results of ⁇ -SMA immunofluorescence staining of sections. It can be seen from the figure that compared with chemotherapy drugs alone (including GEM group, Abraxane group and GEM+Abraxane group), after hyperbaric oxygen treatment (respectively corresponding to HBO+GEM group, HBO+Abraxane group and HBO+GEM+Abraxane group) group) the proportion of tumor-associated fibroblasts in tumor tissues was reduced by about 28%.
  • Figure 2 shows the ratio of CD45 - Ter-119 - Ep-CAM - CD31-tumor - associated fibroblasts to all cells in the tumor tissue of each group detected by flow cytometry after the treatment of Panc02 pancreatic cancer in situ. It can be seen from the figure that the ratio of tumor-associated fibroblasts decreased by 10% compared with the control group with HBO alone, and the results were significantly different.
  • the ratio of tumor-associated fibroblasts in the HBO+GEM group and HBO+Abraxane group decreased by 9% compared with the respective monotherapy groups, while the tumor-associated fibroblasts in the HBO+GEM+Abraxane group compared with the GEM+Abraxane group
  • the ratio of fibroblasts decreased by 12%, and the results were significantly different. This result indicated that hyperbaric oxygen treatment could significantly reduce the ratio of tumor-associated fibroblasts in tumor tissues.
  • the chemotherapy nano drug Abraxane was dissolved in normal saline to prepare a 0.83 mg/mL solution
  • the chemotherapy drug GEM was dissolved in normal saline to prepare a 0.67 mg/mL solution
  • the chemotherapy nano drug Abraxane and the chemotherapy drug GEM was also dissolved in normal saline to prepare a solution of 0.83mg/mL Abraxane+0.67mg/mL GEM.
  • mice An orthotopic tumor model of pancreatic cancer Panc02 in mice was established, and the tumor-bearing mice were randomly divided into 8 groups (Saline group, HBO group, GEM group, HBO+GEM group, Abraxane group, HBO+Abraxane group, GEM+Abraxane group) one week later. group and HBO+GEM+Abraxane group) and recorded as the first day, the HBO group, HBO+GEM group, HBO+Abraxane group and HBO+GEM+Abraxane group were administered on the 1st, 2nd, 3rd, 4th, 7th and 10th day respectively The mice in the group were treated with hyperbaric oxygen.
  • the treatment method was to place the mice in a closed cabin, gradually introduce pure oxygen to increase the pressure in the cabin to 2.5 times the atmospheric pressure, and after maintaining it for 1.5 hours, slowly deflate the air to reduce the pressure in the cabin to atmospheric pressure.
  • each mouse was given 0.2mg/0.3mL GEM by tail vein injection in the HBO+GEM group, 0.25mg/0.3mL Abraxane was given to each mouse in the HBO+Abraxane group, and HBO+GEM was given +Abraxane group 0.2mg/0.3mL GEM and 0.25mg/0.3mL Abraxane per mouse.
  • mice were sacrificed on the 11th day, and the tumor tissues of the mice were isolated. After the sections were fixed and embedded in paraformaldehyde, the contents of type I collagen and fibronectin in tumor tissues were detected by Collagen I and Fibronectin staining.
  • Figure 3 is the quantitative results of the slices stained with Collagen I immunofluorescence. It can be seen from the figure that compared with the Saline group and the chemotherapy nano-drug Abraxane group alone, the proportion of Collagen I in the tumor tissue was reduced by 42% after hyperbaric oxygen treatment. The proportion of Collagen I was reduced by 41% and 46% in HBO+GEM group and HBO+GEM+Abraxane group compared with the respective chemotherapy drug treatment groups. This result indicates that hyperbaric oxygen treatment can significantly reduce the proportion of Collagen I in the extracellular matrix of tumor tissue.
  • Fig. 4 is the quantification result of the section by Fibronectin immunofluorescence staining. It can be seen from the figure that after hyperbaric oxygen therapy in Saline group, GEM group, Abraxane group and GEM+Abraxane group, the proportion of Fibronectin decreased by 40%, 44%, 48% and 49%, respectively. It shows that hyperbaric oxygen treatment can significantly reduce the proportion of Fibronectin in the extracellular matrix of tumor tissue.
  • mice An orthotopic tumor model of pancreatic cancer Panc02 was established in mice. After one week, the tumor-bearing mice were randomly divided into two groups (Saline group, HBO group) and recorded as the first day, and were administered on the first, second, third, and fourth day, respectively.
  • Hyperbaric oxygen therapy for mice in the HBO group the treatment method is to place the mice in a closed cabin, gradually introduce pure oxygen to increase the pressure in the cabin to 2.5 times the atmospheric pressure, and after maintaining it for 1.5 hours, slowly deflate the pressure in the cabin to reduce the pressure. to atmospheric pressure.
  • mice in the Saline group were given 0.2 mL of normal saline by tail vein injection. After the treatment, the mice were sacrificed on the 5th day, and the tumor tissues of the mice were separated, fixed and embedded in paraformaldehyde, and the morphology and density of blood vessels in the tumor tissues were detected by CD31 staining.
  • Figure 5 is the statistical result of the blood vessel density in the CD31 immunofluorescence staining section. It can be seen from the figure that hyperbaric oxygen treatment can significantly reduce the blood vessel density in the tumor tissue.
  • Figure 6 is the statistical result of the degree of vascular curvature in the slices. It can be seen from the figure that the degree of vascular curvature is significantly reduced after hyperbaric oxygen treatment, which indicates that HBO treatment can promote the normalization of tumor vascular structure.
  • Abraxane was labeled with Cy5-NHS ester, and excess Cy5 molecules were removed by dialysis. Then the labeled Abraxane was dissolved in normal saline to prepare a 10 mg/mL solution.
  • the orthotopic tumor model of mouse breast cancer Panc02 was established.
  • the tumor-bearing mice were randomly divided into two groups (Abraxane group and HBO+Abraxane group)
  • the mice in the HBO+Abraxane group were given hyperbaric oxygen therapy every day.
  • 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 it for 1.5 hours, slowly release the air to The cabin pressure drops to atmospheric pressure. After the end of hyperbaric oxygen therapy on the fourth day, it was recorded as the 0th hour, and the mice in the Abraxane group and the Abraxane group were injected with Cy5 fluorescently labeled Abraxane immediately through the tail vein.
  • mice Use a small animal imaging system to image the mice at 0, 0.5, 1, 2, 4, 8, 12, and 24 hours to detect the distribution of Abraxane in the mice, and sacrifice the mice at 24 hours to separate the heart and liver For spleen, lung, kidney and tumor, the distribution of Abraxane in various tissues of mice was detected using a small animal imaging system.
  • Figure 7 shows the time-dependent distribution of chemotherapeutic nano-drugs in living mice detected by small animal imaging. It can be seen from the figure that at hour 0, the mice in the two groups had no fluorescent signal. After injection of fluorescently labeled Abraxane, fluorescent signals of different strengths appeared in the two groups of mice over time. The fluorescence signals of Abraxane in tumor sites of mice in the HBO+Abraxane group were stronger than those in the Abraxane group alone at different time points.
  • Figure 8 is the quantitative results of the distribution of Abraxane in the tumor tissue of living mice detected by small animal imaging over time. It can be seen from the figure that the enrichment of Abraxane in the tumor tissues of the mice in each group gradually increased with the prolongation of time, and the enrichment of Abraxane in the tumor site in the HBO+Abraxane treatment group was significantly higher than that of Abraxane alone. Group.
  • Figure 9 is the distribution map of isolated tissues after 24 hours. The results show that the fluorescence signal of Abraxane in the mouse tumor tissue of the HBO+Abraxane group is stronger than that of the Abraxane group alone, and the drug also has a higher enrichment in the lungs.
  • Figure 10 shows the quantification results of Abraxane in the isolated tumor tissue detected by small animal imaging. It can be seen that the accumulation of Abraxane in the tumor tissue of mice in the HBO+Abraxane group increased by 91.4% compared with that in the Abraxane group alone.
  • Figure 11 is the quantitative results of Abraxane distribution in isolated tissues detected by small animal imaging. It can be seen from the figure that, except that the drug accumulation in the HBO+Abraxane group in the tumor tissue was significantly increased compared with that in the Abraxane alone group, there was no significant difference in the accumulation in other organs. Enrichment.
  • Abraxane was labeled with Cy5-NHS ester, and excess Cy5 molecules were removed by dialysis. Then the labeled Abraxane was dissolved in normal saline to prepare a 10 mg/mL solution.
  • An orthotopic tumor model of pancreatic cancer Panc02 was established in mice.
  • the tumor-bearing mice were randomly divided into two groups (Abraxane group and HBO+Abraxane group)
  • the mice in the HBO+Abraxane group were given hyperbaric oxygen therapy every day.
  • 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 it for 1.5 hours, slowly release the air to The cabin pressure drops to atmospheric pressure.
  • Cy5-Abraxane was administered to mice in HBO+Abraxane group through tail vein injection, and Cy5-Abraxane was injected into Abraxane group mice at a dose of 2mg/0.2mL through tail vein. After the treatment, the mice were sacrificed on the 5th day, and the tumor tissues of the mice were separated, fixed and embedded in paraformaldehyde, and the distance of the drug out of the blood vessel was observed by CD31 staining.
  • Figure 12 is the statistical data of the fluorescence intensity of the drug in the immunofluorescent stained section, and the results also show that the hyperbaric oxygen treatment can significantly increase the accumulation of the drug in the tumor tissue.
  • Figure 13 is the quantitative result of the distance of the drug away from the blood vessel in the slice. It can be seen from the figure that the hyperbaric oxygen treatment can significantly increase the penetration distance of the drug in the tumor tissue.
  • Abraxane was labeled with Cy5-NHS ester, and excess Cy5 molecules were removed by dialysis. Then the labeled Abraxane was dissolved in normal saline to prepare a 10 mg/mL solution.
  • An orthotopic tumor model of pancreatic cancer Panc02 was established in mice.
  • the tumor-bearing mice were randomly divided into two groups (Abraxane group and HBO+Abraxane group)
  • the mice in the HBO+Abraxane group were given hyperbaric oxygen therapy every day.
  • 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 it for 1.5 hours, slowly release the air to The cabin pressure drops to atmospheric pressure.
  • Cy5-Abraxane was administered to mice in HBO+Abraxane group through tail vein injection, and Cy5-Abraxane was injected into Abraxane group mice at a dose of 2mg/0.2mL through tail vein.
  • the mice were sacrificed on the 5th day, and the tumor tissues of the mice were isolated. After cutting the tumor tissue with scissors, adding collagenase and DNase to digest for 40 minutes, using a 50mL syringe rubber stopper to grind and filtering through a 70 ⁇ m cell mesh to obtain a single cell suspension. The uptake of Cy5-Abraxane by tumor cells was detected by flow cytometry.
  • Figure 14 is the quantitative statistical results of the fluorescence intensity of the uptake of Cy5-Abraxane by tumor cells in each group after the treatment of Panc02 pancreatic cancer in situ tumors. It can be seen from the figure that the uptake of Cy5-Abraxane by tumor cells in the HBO+Abraxane group is single With 2.08 times of Abraxane group, this result indicates that hyperbaric oxygen treatment can improve the uptake of chemotherapeutic nanodrugs by tumor cells.
  • Anti-tumor activity test based on hyperbaric oxygen combined with chemotherapy drugs in mice bearing pancreatic cancer in situ.
  • the chemotherapy nano drug Abraxane was dissolved in normal saline to prepare a 0.83 mg/mL solution
  • the chemotherapy drug GEM was dissolved in normal saline to prepare a 0.67 mg/mL solution
  • the chemotherapy nano drug Abraxane and the chemotherapy drug GEM was also dissolved in normal saline to prepare a solution of 0.83mg/mL Abraxane+0.67mg/mL GEM.
  • An orthotopic tumor model of Panc02 pancreatic cancer was established in mice, and the tumor-bearing mice were randomly divided into 8 groups (Saline group, HBO group, GEM group, HBO+GEM group, Abraxane group, HBO+Abraxane group, GEM+Abraxane group) after one week. group and HBO+GEM+Abraxane group) and recorded as the first day, the HBO group, HBO+GEM group, HBO+Abraxane group and HBO+GEM+Abraxane group were administered on the 1st, 2nd, 3rd, 4th, 7th and 10th day respectively The mice in the group were treated with hyperbaric oxygen.
  • the treatment method was to place the mice in a closed cabin, gradually introduce pure oxygen to increase the pressure in the cabin to 2.5 times the atmospheric pressure, and after maintaining it for 1.5 hours, slowly deflate the air to reduce the pressure in the cabin to atmospheric pressure.
  • each mouse in the HBO+GEM group was given 0.2mg/0.3mL GEM by tail vein injection, and each mouse in the HBO+Abraxane group was given 0.25mg/0.3mL Abraxane, and HBO+GEM was given to each mouse +Abraxane group 0.2mg/0.3mL GEM and 0.25mg/0.3mL Abraxane per mouse.
  • mice were sacrificed on day 24, and the tumors were stripped and weighed. The body weight of the mice was weighed and recorded every two days during the treatment.
  • mice were killed, the whole blood was taken out to detect the blood cell components and content, and the blood was collected to separate the serum, and the blood biochemical indicators were analyzed, including four items of alanine aminotransferase, aspartate aminotransferase, total urea nitrogen and myocardial creatine kinase. index.
  • the complete intestines of the mice were taken out, and the number of intestinal metastatic nodules was recorded and photographed.
  • the five major organs of the mouse heart, liver, spleen, lung, and kidney were taken out, fixed with 4% paraformaldehyde, and then routinely embedded in paraffin, stained with HE, and observed under a microscope for tissue structure.
  • Figure 15 shows the statistical results of tumor weighing after the mice were treated. It can be seen from the figure that the tumor weight of the GEM, Abraxane and GEM+Abraxane chemotherapy drug treatment groups was significantly lower than that of the Saline group. At the same time, the tumor weight of the HBO combined with chemotherapy nano drug treatment group The tumor weight was significantly lower than that of the nano-chemotherapy group alone, but the tumor weight of the HBO+GEM group was not significantly different from that of the GEM alone group, which indicated that HBO selectively enhanced the anti-tumor effect of chemotherapeutic nano-drugs.
  • Figure 16 shows the statistical results of the number of intestinal metastatic nodules after treatment in each group of mice. It can be seen from the figure that the number of intestinal metastatic nodules in the combined treatment groups of HBO+GEM, HBO+Abraxane, and HBO+GEM+Abraxane was significantly less than that of chemotherapy alone In the drug treatment group, the number of intestinal metastatic nodules in the HBO group alone was also significantly reduced compared with the Saline group, which indicated that HBO treatment had a significant inhibitory effect on tumor metastasis.
  • Figure 17 is a diagram of the body weight changes of the above 8 groups during the administration period. It can be seen from the figure that compared with the Saline group, the body weight of the mice in the 7 experimental groups has no obvious trend of decline or rise, which shows that the use of HBO or chemotherapy alone Neither the drug treatment, nor the HBO combination treatment affected the health of the mice.
  • Figure 18 shows the blood cell detection results after the treatment in each group. It can be seen from the figure that compared with the Saline group, there was no significant difference in the white blood cell, red blood cell, platelet content and average hemoglobin content in the blood of the mice in the 7 experimental groups after the treatment, which indicated that the treatment with HBO or chemotherapy drugs alone, as well as the combined treatment All have good security.
  • Figure 19 shows the results of blood biochemical analysis after treatment in each group, in which alanine aminotransferase and aspartate aminotransferase are indicators of liver function, and the increase of these indicators represents that the drug has severe liver toxicity; myocardial creatine kinase is an indicator of heart function, and its An increase in the value means pathological changes in the heart; total urea nitrogen is an indicator of kidney function, and an increase in this indicator means that the kidney function is abnormal.
  • the figure shows that the four blood biochemical indicators of the 7 experimental groups have no significant changes compared with the control group. The above results show that the use of HBO or chemotherapeutic drugs alone, as well as the combined treatment, will not affect the major organs of the mice. Injury occurs, so the treatment has a good safety profile.
  • FIG. 20 is a structural diagram of a tissue section, and the scale bar is 250 ⁇ m. It can be seen from the figure that the structure of the heart, liver, spleen, lung and kidney in the Saline group and the 7 experimental groups is clear, without obvious pathological changes, without bleeding and inflammatory infiltration. The main organs of the mice were damaged, which has good safety.
  • the chemotherapy nano drug Abraxane was dissolved in normal saline to prepare a 0.83 mg/mL solution
  • the chemotherapy drug GEM was dissolved in normal saline to prepare a 0.67 mg/mL solution
  • the chemotherapy nano drug Abraxane and the chemotherapy drug GEM was also dissolved in normal saline to prepare a solution of 0.83mg/mL Abraxane+0.67mg/mL GEM.
  • An orthotopic tumor model of Panc02 pancreatic cancer was established in mice, and the tumor-bearing mice were randomly divided into 8 groups (Saline group, HBO group, GEM group, HBO+GEM group, Abraxane group, HBO+Abraxane group, GEM+Abraxane group) after one week. group and HBO+GEM+Abraxane group) and recorded as the first day, the HBO group, HBO+GEM group, HBO+Abraxane group and HBO+GEM+Abraxane group were administered on the 1st, 2nd, 3rd, 4th, 7th and 10th day respectively The mice in the group were treated with hyperbaric oxygen.
  • the treatment method was to place the mice in a closed cabin, gradually introduce pure oxygen to increase the pressure in the cabin to 2.5 times the atmospheric pressure, and after maintaining it for 1.5 hours, slowly deflate the air to reduce the pressure in the cabin to atmospheric pressure.
  • each mouse in the HBO+GEM group was given 0.2mg/0.3mL GEM by tail vein injection, and each mouse in the HBO+Abraxane group was given 0.25mg/0.3mL Abraxane, and HBO+GEM was given to each mouse +Abraxane group 0.2mg/0.3mL GEM and 0.25mg/0.3mL Abraxane per mouse.
  • the normal saline, GEM, Abraxane and GEM+Abraxane were injected through the tail vein to corresponding to the experimental group of mice. After the treatment, the survival status of the mice was observed and recorded every day.
  • Figure 21 is the Kaplan-Meier diagram of the survival period of mice in each group, the median of Saline group, HBO group, GEM group, HBO+GEM group, Abraxane group, HBO+Abraxane group, GEM+Abraxane group and HBO+GEM+Abraxane group
  • the survival period was 34, 39, 39, 43, 41, 43, 42 and 52 days in turn.
  • the survival of HBO+GEM+Abraxane was significantly prolonged by 10 days compared with GEM+Abraxane alone.
  • the analysis of the above results showed that HBO combined with clinical first-line treatment GEM+Abraxane can significantly prolong the survival of mice bearing Panc02 pancreatic cancer in situ tumor.

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Description

一种抑制肿瘤相关成纤维细胞及调控肿瘤基质正常化的方法 【技术领域】
本发明属于肿瘤治疗领域，更具体地，涉及一种抑制肿瘤相关成纤维细胞及调控肿瘤基质正常化的方法。
【背景技术】
肿瘤相关成纤维细胞是一种激活状态下的成纤维细胞，是肿瘤微环境中的重要组分之一，通过与肿瘤细胞相互作用来调控癌细胞增殖、迁移、侵袭和耐药等生物学过程。
细胞外基质是由细胞合成、分泌到细胞外间质中的大分子物质构成的复杂网络结构，是肿瘤微环境中的重要组成部分，主要包括胶原、纤维连接蛋白、透明质酸等，在肿瘤生长、侵袭和转移过程中扮演着重要角色。肿瘤细胞外基质的沉积不仅可以挤压肿瘤血管，减少肿瘤部位的血液供应，从而减少药物递送；同时，致密的胞外基质还作为物理屏障进一步阻碍药物的穿透，大大降低药物疗效。
【发明内容】
针对现有技术的缺陷，本发明提供了一种抑制肿瘤相关成纤维细胞及调控肿瘤基质正常化的方法，其目的在于通过高压氧治疗降低患者肿瘤组织中肿瘤相关成纤维细胞及胞外基质组分的比例，增强药物递送效率从而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，本发明提供了一种抑制肿瘤组织中肿瘤相关成纤维细胞比例的方法，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肿瘤相关成纤维细胞的比例；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基质正常化的方法，对肿瘤患者进行高压氧治疗，以调控肿瘤患者其肿瘤基质正常化；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，具有以下有益效果：
(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肿瘤相关成纤维细胞(α-SMA)的比例，比如某实施例中对Panc02胰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，小鼠肿瘤组织中肿瘤相关成纤维细胞(α-SMA)的比例降 低了28％。
(2)本发明将高压氧治疗与静脉注射化疗药物相结合，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肿瘤相关成纤维细胞(α-SMA)的比例。优选实施例中，与单用化疗药物相比，高压氧联合化疗药物GEM、Abraxane和GEM+Abraxane在不增加毒副作用的同时，肿瘤相关成纤维细胞(α-SMA)的占比都降低了29％。
(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降低肿瘤细胞外基质沉积，比如某实施例中对Panc02胰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，小鼠肿瘤组织中Collagen I和Fibronectin的比例分别降低了42％和40％。
(4)本发明将高压氧治疗与静脉注射化疗药物相结合，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正常化肿瘤细胞外基质，降低肿瘤血管整体密度及弯曲程度，增加纳米药物在肿瘤部位的富集与穿透，同时提高肿瘤细胞对药物的摄取。优选实施例中，与单用化疗药物相比，高压氧联合化疗药物GEM、Abraxane和GEM+Abraxane在不增加毒副作用的同时，肿瘤组织中Collagen I的比例分别降低了41％、42％和46％，Fibronectin的比例分别降低了44％、48％和49％。因而，纳米药物Abraxane在治疗部位的富集量、穿透深度和肿瘤细胞对化疗纳米药物的摄取量分别明显提高了1.91倍、4.78倍和2.08倍。
(5)本发明将高压氧治疗与静脉注射化疗药物相结合，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肿瘤相关成纤维细胞的比例，同时调控肿瘤细胞外基质，降低血管整体密度和弯曲程度，增加化疗纳米药物在肿瘤部位的富集与穿透，同时提高肿瘤细胞对药物的摄取，最终增强肿瘤化疗疗效，并且具有良好的安全性。在Panc02原位胰腺癌模型的实施例中，HBO联合胰腺癌临床一线治疗方案GEM+Abraxane组与其他组相比，小鼠中位生存期至少延长了10天。
(6)本发明提出的高压氧及联合化疗药物用于肿瘤治疗的方法都已应用于临床，简单易于操作，安全可靠，临床转化将更加容易。
【附图说明】
图1为本发明实施例5进行的肿瘤相关成纤维细胞比例实验中肿瘤相关成纤维细胞(α-SMA)的比例；
图2为本发明实施例5进行的肿瘤相关成纤维细胞比例实验中肿瘤相关成纤维细胞 (CD45 -Ter-119 -Ep-CAM -CD31 -)的比例；
图3为本发明实施例6进行的肿瘤胞外基质比例实验中Collagen I的比例；
图4为本发明实施例6进行的肿瘤胞外基质比例实验中Fibronectin的比例；
图5为本发明实施例7进行的肿瘤血管形态检测实验中血管密度统计结果；
图6为本发明实施例7进行的肿瘤血管形态检测实验中血管弯曲度统计结果；
图7为本发明实施例8各实验组小动物成像检测小鼠活体荧光成像图；
图8为本发明实施例8各实验组小动物成像检测活体肿瘤组织化疗纳米药物富集量-时间曲线；
图9为本发明实施例8各实验组小动物成像检测离体组织荧光成像图；
图10为本发明实施例8各实验组小动物成像检测化疗纳米药物在肿瘤组织富集定量图；
图11为本发明实施例8各实验组小动物成像检测各组织中化疗纳米药物分布定量图；
图12为本发明实施例9各实验组肿瘤药物蓄积的定量结果；
图13为本发明实施例9各实验组肿瘤血管药物穿透深度的定量结果；
图14为本发明实施例10各实验组对药物摄取的定量结果；
图15为本发明实施例11进行的抗肿瘤活性实验中小鼠的瘤重结果；
图16是本发明实施例11进行的抗肿瘤活性实验中小鼠的肠转移结节数；
图17为本发明实施例11进行的抗肿瘤活性实验中小鼠的体重-时间曲线；
图18为本发明实施例11各实验组血细胞指标的结果；
图19为本发明实施例11各实验组血生化指标的结果；
图20为本发明实施例11各实验组组织切片的显微观察图片；
图21为本发明实施例12各组小鼠生存期的结果。
【具体实施方式】
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
本发明提供的一种降低肿瘤组织中肿瘤相关成纤维细胞的比例和/或调控肿瘤基质正常化的方法，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肿瘤相关成纤维细胞的比例，并调控促进肿瘤患者其肿瘤组织中的肿瘤基质正常化；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，对肿瘤患者进行一次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肿瘤相关成纤维细胞的比例，并有效改善肿瘤组织中的肿瘤胞外基质沉积。
本发明一些实施例中提供的降低肿瘤组织中肿瘤相关成纤维细胞的比例和/或调控肿瘤基质正常化的方法，通过将高压氧治疗与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肿瘤相关成纤维细胞的比例，并调控促进肿瘤患者其肿瘤组织中的胞外基质正常化，具体包括如下步骤：
(1)对肿瘤患者施用化疗药物；
(2)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。
原则上本发明不限定高压氧治疗和静脉注射化疗药物治疗的先后顺序。
优选实施例中，先对肿瘤患者静脉注射化疗药物，再对肿瘤患者进行单次或多次高压氧治疗。实验发现对肿瘤患者进行高压氧治疗，可以改善肿瘤的乏氧环境，降低肿瘤组织中肿瘤相关成纤维细胞的占比，尤为重要地，能够显著调控肿瘤胞外基质并增加化疗纳米药物在肿瘤部位的富集和穿透，提高化疗纳米药物的抗肿瘤疗效。
根据美国水下及高气压医学学会(UHMS)的定义，高压氧(Hyperbaric oxygen，HBO)治疗是指在高于一个大气压下间歇呼吸100％氧气。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在高于常压(1ATA)的压力下摄入纯氧来提高血液和组织中的溶氧量。此外，高压氧治疗还可以减轻水肿，激活血管生成以及增加胶原合成。因此，高压氧被用作减压病(DCS)、动脉气体栓塞、失血性贫血和严重一氧化碳中毒等疾病的主要治疗手段。然而，本发明将高压氧与化疗药物联合，实验发现能够显著降低肿瘤组织中肿瘤相关成纤维细胞的占比，尤为重要地，能够显著调控肿瘤胞外基质正常化并增加化疗纳米药物在肿瘤部位的富集和穿透，提高化疗纳米药物的抗肿瘤疗效。
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，对肿瘤患者进行一次高压氧治疗具体为：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。化疗药物治疗方法为：通过静脉注射化疗药物。
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。
本发明对肿瘤患者施用化疗药物，施用方式为静脉注射。
高压氧治疗与化疗药物给药的先后次序可变，在一个优选实施方式中，为先静脉注射化疗药物，随后高压氧治疗。
本发明对患者施用化疗药物与高压氧治疗之间的间隔时间为0-12小时。可以在施用化疗药物之后立即进行高压氧治疗，也可以间隔一段时间之后再进行高压氧治疗。
本发明对肿瘤患者进行静脉注射化疗药物之前或之后，对肿瘤患者给予3-4次高压氧治疗。
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，重复治疗不少于1次。
本发明可根据实际应用情况调整化疗药物的剂量，也依据肿瘤化疗药物种类的不同设置合适的药物剂量。比如，一些优选实施例中，采用化疗药物Abraxane或GEM，每一至三周对肿瘤患者静脉注射化疗药物一次，其单次注射剂量为100-1000mg/m 2。
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Abraxane一次，其单次注射剂量为125mg/m 2。
另一些实施例中，每一至三周对肿瘤患者静脉注射化疗药物GEM一次，其单次注射剂量为1000mg/m 2。
本发明优选实施例中，每一次静脉注射化疗药物之后，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或每隔两天的高压氧治疗。在一个优选实施方式中，每天给予高压氧治疗1次。
化疗药物的注射剂量可依据个体差异而定，比如静脉注射每周或每2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，根据确定的化疗药物的用量和用法，在每次注射化疗药物之后，进行每周1-3次的高压氧治疗。根据患者病情以及耐受程度设置相应的治疗方案。
本发明采用的化疗药物为可应用于临床的各种药物，包括Abraxane和GEM。本发明化疗药物的静脉注射剂量可参考不同的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。Abraxane在临床上的推荐剂量为125mg/m 2，每周给一次药。GEM在临床上的推荐剂量为1000mg/m 2，每周给一次药。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)对肿瘤患者静脉注射化疗纳米药物；
(2)静脉注射化疗药物后，间隔0-12小时，然后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；按照一定的频率，比如每天或每三天一次高压氧治疗，重复高压氧治疗2-4次；
(3)重复步骤(1)和步骤(2)以完成一次治疗，重复治疗不少于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，包括非小细胞肺癌、乳腺癌、胰腺癌、卵巢癌、膀胱癌、子宫颈癌、肝癌、胆道癌、鼻咽癌、睾丸肿瘤、淋巴瘤、间皮瘤和头颈部癌等，高压氧联合治疗能够显著提高化疗纳米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。
与单用化疗药物相比，本发明高压氧联合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，降低肿瘤组织中肿瘤相关成纤维细胞的占比，显著调控肿瘤胞外基质正常化并增加化疗纳米药物在肿瘤部位的富集和穿透，提高肿瘤细胞对化疗纳米药物的摄取，增强化疗纳米药物的抗肿瘤疗效。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2至实施例12中采用的化疗药物为石药集团的Abraxane和齐鲁制药的GEM。
实施例1
本实施例提供一种高压氧降低肿瘤组织中肿瘤相关成纤维细胞的比例和/或促进肿瘤胞外基质正常化的方法，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4天。
实施例2
本实施例提供一种高压氧联合化疗药物用于肿瘤治疗的方法，该方法包括化疗药物注射和高压氧治疗两部分。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4天；第4天在高压氧治疗结束后立即通过静脉注射化疗纳米药物治疗，具体方法为：静脉注射125mg/m 2化疗纳米药物Abraxane一次。
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，该方法包括化疗药物注射和高压氧治疗两部分。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，第四天在静脉注射125mg/m 2化疗纳米药物Abraxane和1000mg/m 2化疗药物GEM及二者联合静脉注射后立即进行高压氧治疗。随后每间隔两天静脉注射一次化疗药物，注射后立即给予一次高压氧治疗，共三次。
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，该方法包括化疗药物注射和高压氧治疗两部分。化疗药物治疗方法为：通过静脉注射125mg/m 2化疗纳米药物Abraxane、静脉注射1000mg/m 2化疗药物GEM及两者联合静脉注射。高压氧治疗方法为：在静脉注射化疗纳米药物后，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5
基于高压氧联合化疗纳米药物对荷乳腺癌原位瘤小鼠肿瘤相关成纤维细胞比例的测试。
实验药物的配制：将化疗纳米药物Abraxane溶解在生理盐水中配制成0.83mg/mL的溶液，将化疗药物GEM溶解在生理盐水中配制成0.67mg/mL的溶液，将化疗纳米药物Abraxane和化疗药物GEM同时溶解在生理盐水中配制成0.83mg/mL Abraxane+0.67mg/mL GEM的溶液。
建立小鼠胰腺癌Panc02原位瘤模型，一周后将荷瘤小鼠随机分为8个组(Saline组、HBO组、GEM组、HBO+GEM组、Abraxane组、HBO+Abraxane组、GEM+Abraxane组和HBO+GEM+Abraxane组)并记为第1天，分别在第1、2、3、4、7、10天给予HBO组、HBO+GEM组、HBO+Abraxane组和HBO+GEM+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4、7、10天在高压氧治疗之前尾静脉注射给予HBO+GEM组每只小鼠0.2mg/0.3mL GEM，HBO+Abraxane组每只小鼠0.25mg/0.3mL Abraxane，给予HBO+GEM+Abraxane组每只小鼠0.2mg/0.3mL GEM和0.25mg/0.3mL Abraxane。并分别将生理盐水、GEM、Abraxane和GEM+Abraxane组以0.3mL/只、0.2mg/0.3mL、0.25mg/0.3mL和0.2mg/0.3mL+0.25mg/0.3mL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。一部分肿瘤组织经多聚 甲醛固定包埋切片后，通过α-SMA染色检测肿瘤组织中肿瘤相关成纤维细胞的含量。另一部分肿瘤组织用剪刀剪碎后，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FITC-CD45、APC-Ter-119、PE-Ep-CAM和PC7-CD31流式抗体与细胞悬液共孵育后流式细胞仪分析肿瘤相关成纤维细胞(CD45 -Ter-119 -Ep-CAM -CD31 -)占所有细胞的比率。
图1是α-SMA免疫荧光染色的切片定量结果。从图中可以看出，与单用化疗药物(包括GEM组、Abraxane组和GEM+Abraxane组)相比，高压氧处理后(分别对应HBO+GEM组、HBO+Abraxane组和HBO+GEM+Abraxane组)肿瘤组织中肿瘤相关成纤维细胞的比例都降低了28％左右。
图2为流式检测Panc02胰腺癌原位瘤治疗结束后各组肿瘤组织中CD45 -Ter-119 -Ep-CAM -CD31 -的肿瘤相关成纤维细胞占所有细胞的比率。从图中可以看出，单用HBO与对照组相比，肿瘤相关成纤维细胞的比率降低了10％，结果具有显著性差异。同时，HBO+GEM组与HBO+Abraxane组相比于各自的单药治疗组，肿瘤相关成纤维细胞的比率都下降了9％，而HBO+GEM+Abraxane组相对于GEM+Abraxane组，肿瘤相关成纤维细胞的比率下降了12％，结果都具有显著性差异。这个结果表明高压氧处理能明显降低肿瘤组织中肿瘤相关成纤维细胞的比率。
实施例6
基于高压氧联合化疗纳米药物对荷乳腺癌原位瘤小鼠肿瘤细胞外基质比例的测试。
实验药物的配制：将化疗纳米药物Abraxane溶解在生理盐水中配制成0.83mg/mL的溶液，将化疗药物GEM溶解在生理盐水中配制成0.67mg/mL的溶液，将化疗纳米药物Abraxane和化疗药物GEM同时溶解在生理盐水中配制成0.83mg/mL Abraxane+0.67mg/mL GEM的溶液。
建立小鼠胰腺癌Panc02原位瘤模型，一周后将荷瘤小鼠随机分为8个组(Saline组、HBO组、GEM组、HBO+GEM组、Abraxane组、HBO+Abraxane组、GEM+Abraxane组和HBO+GEM+Abraxane组)并记为第1天，分别在第1、2、3、4、7、10天给予HBO组、HBO+GEM组、HBO+Abraxane组和HBO+GEM+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4、7、10天在高压氧治疗之前尾静脉注射给予HBO+GEM组每只小鼠0.2mg/0.3mL GEM，HBO+Abraxane组每只小鼠0.25mg/0.3mL  Abraxane，给予HBO+GEM+Abraxane组每只小鼠0.2mg/0.3mL GEM和0.25mg/0.3mL Abraxane。并分别将生理盐水、GEM、Abraxane和GEM+Abraxane组以0.3mL/只、0.2mg/0.3mL、0.25mg/0.3mL和0.2mg/0.3mL+0.25mg/0.3mL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。经多聚甲醛固定包埋切片后，通过Collagen I、Fibronectin染色检测肿瘤组织中I型胶原及纤维连接蛋白的含量。
图3是Collagen I免疫荧光染色的切片定量结果。从图中可以看出，与Saline组和单用化疗纳米药物Abraxane组相比，高压氧处理后肿瘤组织中Collagen I的比例都降低了42％。HBO+GEM组和HBO+GEM+Abraxane组相比于各自的化疗药物治疗组，Collagen I的比例分别降低了41％和46％。这个结果表明高压氧处理能明显降低肿瘤组织胞外基质中Collagen I的比例。
图4是Fibronectin免疫荧光染色的切片定量结果。从图中可以看出，Saline组、GEM组、Abraxane组和GEM+Abraxane组在高压氧治疗后，Fibronectin比例分别降低40％、44％、48％和49％。说明高压氧处理能明显降低肿瘤组织胞外基质中Fibronectin的比例。
实施例7
基于高压氧治疗对荷胰腺癌原位瘤小鼠肿瘤血管形态影响的测试。
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2个组(Saline组、HBO组)并记为第1天，分别在第1、2、3、4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、3、4天通过尾静脉注射给予Saline组小鼠0.2mL生理盐水。治疗结束后，在第5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5是对CD31免疫荧光染色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6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结构正常化。
实施例8
基于高压氧联合化疗纳米药物对荷胰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10mg/mL的溶液。
建立小鼠乳腺癌Panc02原位瘤模型，一周后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、3、4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4天高压氧治疗结束后记为第0小时，立即通过尾静脉注射给予Abraxane组和Abraxane组小鼠Cy5荧光标记的Abraxane。使用小动物成像系统在第0、0.5、1、2、4、8、12、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7为小动物成像检测到化疗纳米药物在活体小鼠体内分布随时间的变化情况。从图中可以看出，在第0小时，两个组小鼠均无荧光信号。在注射荧光标记的Abraxane之后，两组小鼠体内随时间延长均出现不同强弱的荧光信号。HBO+Abraxane组小鼠肿瘤部位Abraxane在不同时间点的荧光信号均强于单用Abraxane组。
图8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在各组小鼠体内肿瘤组织中的富集逐渐增多，且HBO+Abraxane治疗组中肿瘤部位Abraxane的富集量均显著高于单用Abraxane组。
图9为24小时后离体组织分布图，结果显示HBO+Abraxane组小鼠肿瘤组织中Abraxane的荧光信号强于单用Abraxane组，且药物在肺部也有较高的富集量。
图10为小动物成像检测的离体肿瘤组织中Abraxane定量结果，可以看出，HBO+Abraxane组小鼠肿瘤组织中Abraxane的蓄积量相对于单用Abraxane组增加了91.4％。
图11为小动物成像检测的离体组织中Abraxane分布定量结果。从图中可以看出，除了肿瘤组织中HBO+Abraxane组药物蓄积量相对于单用Abraxane组显著增加外，其余各器官富集量无显著性差异，结果表明HBO提高Abraxane在小鼠肿瘤部位的富集。
实施例9
基于高压氧联合化疗纳米药物对荷胰腺癌原位瘤小鼠肿瘤中药物蓄积及穿透的测试。
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10mg/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2个组(Abraxane组和HBO+Abraxane组)并记为第1天，分别在第1、2、3、4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4天在高压氧之后通过尾静脉注射给予HBO+Abraxane组小鼠2mg/0.2mL Cy5-Abraxane，并将Cy5-Abraxane以2mg/0.2mL的剂量通过尾静脉注射到Abraxane组小鼠体内。治疗结束后，在第5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染色观察药物出血管的距离。
图12是对免疫荧光染色切片中药物荧光强度的统计数据，结果同样表明，高压氧处理会显著增加肿瘤组织中药物的蓄积。
图13是对切片中药物远离血管距离的定量结果，从图中可以看出，高压氧处理能明显增加肿瘤组织中药物的穿透距离。
实施例10
基于高压氧联合化疗纳米药物对荷胰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10mg/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2个组(Abraxane组和HBO+Abraxane组)并记为第1天，分别在第1、2、3、4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4天在高压氧之后通过尾静脉注射给予HBO+Abraxane组小鼠2mg/0.2mL Cy5-Abraxane，并将Cy5-Abraxane以2mg/0.2mL的剂量通过尾静脉注射到Abraxane组小鼠体内。治疗结束后，在第5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Cy5-Abraxane的摄取量。
图14是Panc02胰腺癌原位瘤治疗后各组肿瘤细胞对Cy5-Abraxane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Cy5-Abraxane的摄取量是单用Abraxane组的2.08倍，这个结果表明高压氧处理可以提高肿瘤细胞对化疗纳米药物的摄取。
实施例11
基于高压氧联合化疗药物对荷胰腺癌原位瘤小鼠抗肿瘤活性的测试。
实验药物的配制：将化疗纳米药物Abraxane溶解在生理盐水中配制成0.83mg/mL的溶液，将化疗药物GEM溶解在生理盐水中配制成0.67mg/mL的溶液，将化疗纳米药物 Abraxane和化疗药物GEM同时溶解在生理盐水中配制成0.83mg/mL Abraxane+0.67mg/mL GEM的溶液。
建立小鼠Panc02胰腺癌原位瘤模型，一周后将荷瘤小鼠随机分为8个组(Saline组、HBO组、GEM组、HBO+GEM组、Abraxane组、HBO+Abraxane组、GEM+Abraxane组和HBO+GEM+Abraxane组)并记为第1天，分别在第1、2、3、4、7、10天给予HBO组、HBO+GEM组、HBO+Abraxane组和HBO+GEM+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4、7、10天在高压氧治疗之前尾静脉注射给予HBO+GEM组每只小鼠0.2mg/0.3mL GEM，HBO+Abraxane组每只小鼠0.25mg/0.3mL Abraxane，给予HBO+GEM+Abraxane组每只小鼠0.2mg/0.3mL GEM和0.25mg/0.3mL Abraxane。并分别将生理盐水、GEM、Abraxane和GEM+Abraxane组以0.3mL/只、0.2mg/0.3mL、0.25mg/0.3mL和0.2mg/0.3mL GEM+0.25mg/0.3mL Abraxane剂量通过尾静脉注射到对应实验组小鼠。治疗结束后，在第24天处死小鼠，剥出肿瘤并称重。治疗过程中每两天称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肠，记录肠转移节结数并拍照。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切片、HE染色，在显微镜下观察组织结构。
图15为小鼠治疗结束后取出肿瘤称重的统计结果，由图可知，GEM、Abraxane和GEM+Abraxane化疗药物治疗组的肿瘤重量明显低于Saline组，同时，HBO联合化疗纳米药物治疗组的瘤重显著低于单用纳米化疗药物组，但是HBO+GEM组的瘤重相对于单独GEM组没有显著性差异，这表明HBO选择性地增强化疗纳米药物的抗肿瘤效果。
图16为各组小鼠治疗结束后肠转移结节数统计结果，由图可知，HBO+GEM、HBO+Abraxane、HBO+GEM+Abraxane联合治疗组肠转移结节数均显著少于单用化疗药物治疗组，且单独HBO组相对于Saline组的肠转移结节数也明显减少，这表明HBO治疗对于肿瘤转移具有明显的抑制效果。
图17为以上8个组在给药期间的体重变化图，图中可以看出，相比于Saline组，7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或化疗药物治疗，以及HBO联合治疗均不会影响小鼠的健康状况。
图18为各组治疗结束后血细胞检测结果。由图可知，与Saline组相比，7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或化疗药物治疗，以及联合治疗均具有良好的安全性。
图19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功能出现异常。图中显示，7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，因此治疗具有良好的安全性。
图20为组织切片的结构图，比例尺为250μm。由图可知，Saline组与7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12
基于高压氧联合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：将化疗纳米药物Abraxane溶解在生理盐水中配制成0.83mg/mL的溶液，将化疗药物GEM溶解在生理盐水中配制成0.67mg/mL的溶液，将化疗纳米药物Abraxane和化疗药物GEM同时溶解在生理盐水中配制成0.83mg/mL Abraxane+0.67mg/mL GEM的溶液。
建立小鼠Panc02胰腺癌原位瘤模型，一周后将荷瘤小鼠随机分为8个组(Saline组、HBO组、GEM组、HBO+GEM组、Abraxane组、HBO+Abraxane组、GEM+Abraxane组和HBO+GEM+Abraxane组)并记为第1天，分别在第1、2、3、4、7、10天给予HBO组、HBO+GEM组、HBO+Abraxane组和HBO+GEM+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4、7、10天在高压氧治疗之前尾静脉注射给予HBO+GEM组每只小鼠0.2mg/0.3mL GEM，HBO+Abraxane组每只小鼠0.25mg/0.3mL Abraxane，给予HBO+GEM+Abraxane组每只小鼠0.2mg/0.3mL GEM和0.25mg/0.3mL Abraxane。并分别将生理盐水、GEM、Abraxane和GEM+Abraxane以0.3mL/只、0.2mg/0.3mL、0.25mg/0.3mL和0.2mg/0.3mL GEM+0.25mg/0.3mL Abraxane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21为各组小鼠生存期Kaplan-Meier图，Saline组、HBO组、GEM组、HBO+GEM组、Abraxane组、HBO+Abraxane组、GEM+Abraxane组和HBO+GEM+Abraxane组的中位生存期依次为34、39、39、43、41、43、42和52天。HBO+GEM+Abraxane的生存期相较于单用GEM+Abraxane显著延长了10天。以上结果分析表明，HBO联合临床一线治疗方案GEM+Abraxane可以显著延长荷Panc02胰腺癌原位瘤小鼠生存期。
结合以上动物实验、血管形态、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，降低肿瘤组织中肿瘤相关成纤维细胞的占比，显著调控肿瘤胞外基质正常化，降低血管整体密度和弯曲程度，并增加化疗纳米药物在肿瘤部位的富集和穿透，提高肿瘤细胞对化疗纳米药物的摄取，增强化疗纳米药物的抗肿瘤疗效。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肿瘤相关成纤维细胞比例的方法，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肿瘤相关成纤维细胞的比例；
    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. 一种调控肿瘤基质正常化的方法，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基质正常化；
    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，对肿瘤患者进行一次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，将高压氧治疗与化疗药物治疗相结合，具体包括如下步骤：
    (1)对肿瘤患者施用化疗药物；
    (2)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。
  5. 如权利要求4所述的方法，其特征在于，步骤(1)对患者通过静脉注射施用化疗药物；对肿瘤患者静脉注射化疗药物后，再对肿瘤患者进行单次或多次高压氧治疗。
  6. 如权利要求4所述的方法，其特征在于，步骤(1)对患者通过静脉注射施用化疗药物，静脉注射化疗药物之前或之后，对肿瘤患者给予1-4次高压氧治疗。
  7. 如权利要求4所述的方法，其特征在于，步骤(2)高压氧治疗与步骤(1)化疗药物治疗之间的间隔时间为0-12小时。
  8. 如权利要求4所述的方法，其特征在于，步骤(1)每一至三周对肿瘤患者静脉注射化疗药物一次。
  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，重复治疗不少于一次。
  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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