Bladder perfusion synergistic preparation and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a bladder perfusion synergistic preparation and application thereof. The gemcitabine gel comprises the following components: gemcitabine and hyaluronic acid with the molecular weight of 200-1000KD, and the mass ratio of gemcitabine to hyaluronic acid is (4-6): 1. Compared with the prior art, the invention has the beneficial effects that hyaluronic acid and gemcitabine are combined for use, the growth of tumors is inhibited by down-regulating the expression of Ki67, the migration, invasion and multiplication capacity of bladder cancer cells can be inhibited, the apoptosis of the bladder cancer cells can be promoted, the immunosuppression induced by GEM can be reversed, and the progress of the bladder cancer can be inhibited; the combined application of hyaluronic acid and gemcitabine is suitable for clinical chemotherapy sensitization and has a definite conversion prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a bladder perfusion synergistic preparation and its application. Background Art

[0002] The tumor microenvironment (Tumor Microenvironment, TME) refers to the cells, extracellular matrix, cytokines, etc. around the tumor, and it has plasticity. Macrophages are one of the most important immune cells in the TME and are also key factors mediating the chemotherapy-induced immunosuppressive tumor microenvironment. Different polarization forms of macrophages have different effects on the occurrence and development of tumors.

[0003] Hyaluronic acid (Hyaluronic Acid, HA), as one of the components of the extracellular matrix, has a profound impact on the TME. HA was first isolated from the vitreous humor of bovine eyes by Karl Meyer and John Palmer. However, its structure was described by Laurent in 1970, and it is the main component of the extracellular matrix. In most cases, hyaluronic acid appears as a long-chain polymer, namely high molecular weight hyaluronic acid (High Molecular Weight Hyaluronic Acid, HMWHA), which mainly plays an immunosuppressive and anti-angiogenic role. While low molecular weight hyaluronic acid (Low Molecular Weight Hyaluronic Acid, LMWHA) plays a pro-inflammatory and pro-angiogenic role. HA can bind to receptors on the surface of macrophage membranes and affect the polarization of macrophages, which mainly depends on the molecular weight of HA. Existing studies have shown that: (1) HMWHA can inhibit the polarization of macrophages induced by IL-1β by inhibiting the activation of the GRP78-NF-κB signaling pathway. (2) When using mid-MWHA to treat osteoarthritis models in vitro, HA can neutralize the upregulation of IL-1β, TNF-α, and Gal-1 in osteoarthritis and inhibit the pro-inflammatory effect of macrophages, and instead polarize macrophages towards an anti-inflammatory phenotype. (3) Using mannose-modified LMWHA to prepare a new cancer vaccine to increase the targeting of M2 macrophages for tumor treatment. The results showed that when mouse macrophages RAW264.7 were co-cultured with tumor cells, HA could reprogram tumor-associated macrophages into a pro-inflammatory and anti-tumor M1 phenotype. In addition, a study based on patients with knee osteoarthritis showed that after injecting HA, the proportion of M1 macrophages decreased and the proportion of M2 macrophages increased. The above research results show that the effects of HA on macrophages are different, which mainly depends on its different molecular weights. And it is precisely this characteristic of different molecular weights having different effects that determines its treatment diversity. Currently, research on HA treatment has been used in fields such as skin, eyes, arthritis, heart, and tumors.

[0004] Bladder cancer is one of the most common malignant tumors in the urinary system. The main treatment for bladder cancer is surgical resection supplemented by chemotherapy, radiotherapy, and immunotherapy. In the treatment of bladder cancer, most clinical chemotherapy drugs have relatively low overall sensitivity, which may be related to chemotherapy-induced immunosuppression. Studies have found that some clinical chemotherapy drugs can cause macrophages to polarize more towards the M2 type, which may be the main reason for the changes in the tumor microenvironment (TME) and immunosuppression. The problem to be solved is how to reverse the immunosuppressive tumor microenvironment and enhance chemotherapy sensitivity in the treatment of bladder cancer. Summary of the Invention

[0005] To solve the problems existing in the prior art, the present invention provides a bladder perfusion synergistic preparation and its application.

[0006] The specific technical solution is as follows: A bladder perfusion synergistic preparation, the components of which include: gemcitabine and hyaluronic acid with a molecular weight of 200 - 1000 KD, and the mass ratio of gemcitabine to hyaluronic acid is 4 - 6:1.

[0007] Preferably, the molecular weight of hyaluronic acid is 1000 KD, and the mass ratio of gemcitabine to hyaluronic acid is 5:1.

[0008] The application of the above-mentioned bladder perfusion synergistic preparation in the preparation of a drug for reversing the immunosuppressive tumor microenvironment.

[0009] The use of the above-mentioned bladder perfusion synergistic preparation in enhancing the chemotherapy sensitivity of bladder cancer.

[0010] Mechanistic explanation: Through experiments, it was found that GEM treatment would reduce the ratio of lymphocytes to myeloid cells, and this phenomenon could be reversed after combining with 1000 KD HA. The combined use of drugs significantly enhanced the expression levels of CD86 and iNOS, reduced the expression level of CD206, while the expression level of F4 / 80 remained basically unchanged, which suggested that 1000 KD HA might reverse the macrophage-related immunosuppression caused by GEM by affecting the polarization of macrophages towards the M1 type. Therefore, it is speculated from the mechanism that this process mainly plays a role by directly binding of HA to Toll-like receptor TLR4 and activating the JAK2 / STAT3 signaling pathway.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: It is proposed to use hyaluronic acid in combination with gemcitabine, which can inhibit tumor growth by downregulating the expression of Ki67, can inhibit the migration, invasion and proliferation ability of bladder cancer cells, can promote the apoptosis of bladder cancer cells, can reverse the immunosuppression induced by GEM, and inhibit the progression of bladder cancer; the combined application of hyaluronic acid and gemcitabine activates the TLR4 / JAK2 / STAT3 pathway, can enhance the chemotherapy sensitivity of bladder cancer, is suitable for clinical chemotherapy sensitization, and has a clear transformation prospect. Brief Description of the Drawings

[0012] Figure 1 It is a diagram of the grouping and drug administration patterns in animal experiments.

[0013] Figure 2 It is a physical picture of a mouse tumor after 14 days of treatment.

[0014] Figure 3 It is a graph showing the change in the volume of a mouse tumor over time.

[0015] Figure 4 It is a graph showing the change in the weight of a mouse tumor over time.

[0016] Figure 5 It is a Western-blot result diagram.

[0017] Figure 6 It is a CCK8 result diagram of T24.

[0018] Figure 7 It is a CCK8 result diagram of MB49.

[0019] Figure 8 It is a Transwell result diagram of T24.

[0020] Figure 9 It is a Transwell result diagram of MB49.

[0021] Figure 10 It is a diagram showing the apoptosis in mouse tumor tissues by Tunel staining.

[0022] Figure 11 It is a diagram showing the expression of Ki67 in mouse tumors by IHC staining.

[0023] Figure 12 It is a statistical graph showing the expression score of Ki67 in mouse tumors by IHC staining.

[0024] Figure 13 It is a diagram showing the expression of cleaved-caspase3 in mouse tumors.

[0025] Figure 14 It is an IHC staining diagram of F4 / 80, CD86, iNOS, and CD206 in mouse tumor tissues.

[0026] Figure 15 It is a scatter plot of CD45+ cells in mouse tumor tissues.

[0027] Figure 16 It is a flow cytometry statistical graph of CD45+ cells in mouse tumor tissues.

[0028] Figure 17It is a scatter plot of CD45+CD11b+ cells in mouse tumor tissues.

[0029] Figure 18 It is a flow cytometry statistical graph of CD45+CD11b+ cells in mouse tumor tissues.

[0030] Figure 19 It is a scatter plot of CD45+CD11b+F4 / 80+CD86+ cells in mouse tumor tissues.

[0031] Figure 20 It is a flow cytometry statistical graph of CD45+CD11b+F4 / 80+CD86+ cells in mouse tumor tissues.

[0032] Figure 21 It is a scatter plot of CD45+CD11b+F4 / 80+CD206+ cells in mouse tumor tissues.

[0033] Figure 22 It is a flow cytometry statistical graph of CD45+CD11b+F4 / 80+CD206+ cells in mouse tumor tissues.

[0034] Figure 23 It is a schematic diagram showing that HA promotes M1-like polarization of macrophages and reverses the GEM-induced immunosuppressive pattern related to macrophages.

[0035] In the figure, Control represents the control group, HA represents the HA group, GEM represents the GEM group, and G+HA represents the GEM+HA group. Detailed implementation manners

[0036] For the convenience of understanding the present invention, the present invention will be described in more detail below with reference to the accompanying drawings and specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosed content of the present invention more thorough and comprehensive.

[0037] A bladder perfusion synergistic preparation mainly uses hyaluronic acid HA and gemcitabine GEM in combination. Below, a control group, a GEM group, an HA group, and a GEM+HA group are set up for specific description.

[0038] I. Cell culture The human bladder cancer cell line T24 was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The murine bladder cancer cell line MB49 was purchased from iCell Bioscience Inc. (Shanghai, China). The reagents required for this experiment included Dulbecco's Modified Eagle Medium (DMEM) complete culture medium containing 10% fetal bovine serum and 1% double antibody (100 U / mL penicillin-streptomycin) (subsequently referred to as DMEM complete culture medium for short), and trypsin. All cells were cultured in a constant temperature incubator at 37°C and 5% CO2 until the logarithmic growth phase.

[0039] General process: 1. Cell resuscitation (1) Take out the cryopreservation tube containing cells from the liquid nitrogen tank, immediately place it in a 37°C water bath for rapid thawing. After the cryopreservation solution has completely melted, immediately transfer the liquid in the cryopreservation tube to a 15 ml centrifuge tube in a laminar flow hood, and add 7 ml of DMEM complete culture medium containing serum and double antibody. Use a centrifuge to centrifuge at 1000 rpm for 5 min to obtain a white cell pellet.

[0040] (2) Carefully discard the supernatant, resuspend the cells with 6 ml of DMEM complete culture medium, mix well and transfer them to a 25 cm culture flask, and culture them at 37°C and 5% CO2 until the cells enter the logarithmic growth phase and are in good condition.

[0041] (3) Observe the cell attachment and cell status under the microscope the next day. Judge whether subculture is needed according to the cell status and the number of adherent cells. When the cell density reaches about 90%, cell subculture can be carried out. If subculture is not required, change the medium. After discarding the old culture medium, rinse the cells twice with sterile PBS and replace with fresh DMEM complete culture medium to continue observing the cell growth status.

[0042] 2. Cell subculture (1) Observe the growth status of the cells in the 25 cm culture flask under an optical microscope. When the cell confluence reaches about 90%, cell subculture can be carried out.

[0043] (2) First, sterilize the biosafety cabinet with ultraviolet light for at least 30 minutes, and pre-prepare the complete culture medium, 0.25% trypsin and cell PBS and other liquids and place them in a 37°C water bath for preheating.

[0044] (3) After the disinfection of the biosafety cabinet is completed, spray the reagents required for cell culture with 75% alcohol and transfer them to the laminar flow hood.

[0045] (4)Carefully discard the old culture medium in the bottle, and rinse the cells twice with pre-warmed sterile PBS. Then add an appropriate amount of trypsin-EDTA digestion solution and gently shake to completely cover the cells at the bottom of the bottle with the liquid, and digest for the corresponding time. The digestion time varies for different cells, generally ranging from a few minutes to more than ten minutes. You can observe the cell digestion status under a light microscope at regular intervals.

[0046] (5)After the corresponding time, observe the cell digestion status under a microscope. If most cells are observed to be round in shape and suspended in the culture medium under the microscope, immediately place the cells in a laminar flow hood, add an appropriate amount of complete culture medium to terminate the digestion of the trypsin-EDTA digestion solution, and then gently blow down the cells.

[0047] (6)Transfer the above suspension to a 15 ml centrifuge tube, centrifuge at 1000 rpm for 5 minutes at room temperature, then transfer it back to the laminar flow hood. After discarding the supernatant, add fresh complete culture medium and mix well to resuspend the cells, and then perform subculture operations according to the cell quantity.

[0048] 3. Cell Preservation and Cryopreservation (1)Before using the cells for subsequent experiments, perform preservation and cryopreservation treatments according to requirements. For the cells to be preserved, select maternal or primary cells with as early passage numbers as possible to ensure small cell variability and no contamination. Before cryopreserving the cells, first observe whether the cell growth status is good and in the logarithmic growth phase. If the cell status is suitable for cryopreservation, carefully discard the original old culture medium.

[0049] (2)Wash the cells twice with sterile PBS, then perform the above cell subculture operation to obtain cell pellets. After carefully discarding the supernatant, add 0.8 - 1.5 ml of cell cryopreservation solution (specifically determined according to the cell quantity), and aliquot the cell suspension into cell cryopreservation tubes.

[0050] (3)Seal the cell cryopreservation tubes containing the cell cryopreservation solution with sealing film. If using a programmed cryopreservation solution, place the cryopreservation tubes in a programmable freezing box, freeze overnight in an -80°C refrigerator, and transfer to a liquid nitrogen tank for long-term storage the next day; if using a non-programmed cryopreservation solution, there is no need to use a programmed freezing box, and directly place the cell cryopreservation tubes in an -80°C refrigerator and store overnight.

[0051] 4. Cell Counting (1)Take a cell counting plate and its cover glass, disinfect and set aside. Under an optical microscope, check whether there are foreign objects in the counting grid of the counting chamber of the counting plate, whether it is clear and the boundaries are distinct, then wash it with 75% alcohol and air dry it in a laminar flow hood for later use.

[0052] (2)Resuspend the digested cells and add 1 ml of complete culture medium to resuspend. Use a pipette to slowly add 10 μL of the suspension along the edge of the cover glass, a total of 20 μL on both sides, so that the cell suspension naturally infiltrates into the counting chamber.

[0053] (3) Using an optical microscope, count the cells in the cell grid lines and perform calculations according to the principle of “count the top but not the bottom, count the left but not the right, and count clustered cells as one cell”.

[0054] The main solution preparation used: (1) Cell culture system (taking 50 ml volume as an example): Add 5 ml of fetal bovine serum to DMEM or 1640 medium to a final concentration of 10%, supplemented with penicillin-streptomycin solution (100 U / ml) and 0.5 ml of double antibody (final concentration of 1%), and store at 4°C.

[0055] (2) Pancreatic enzyme-EDTA digestion solution: Weigh 2.5 g of powdered pancreatic enzyme using an electronic balance and add 0.2 g of EDTA to dissolve in 1000 ml of PBS; then filter and sterilize through a 0.22 μm disposable filter, divide into portions and store in a refrigerator at 4°C.

[0056] (3) Cell-grade phosphate buffered saline (PBS): Add 1 pack of PBS powder to 2000 ml of deionized water; stir to dissolve. When the liquid is clear, mix thoroughly and sterilize by high pressure. After cooling, store in a refrigerator at 4°C.

[0057] (4) 10×PBS: Weigh 160 g NaCl, 28.8 g Na2HPO4, 4.8 g KH2PO4, and 4 g KCl respectively, add the three compounds into a beaker, add deionized water to 1.2 L, and use a stirrer to fully dissolve. When the liquid is clear, adjust the volume to 2 L and transfer to a special container for storage at room temperature.

[0058] 2. Construction of mouse subcutaneous tumor model 1. Purchase 6-8 week old C57BL / 6J male mice and feed them for 3 days, and prepare their skin and shave their hair in advance.

[0059] 2. 5.5×10 5 MB49 cells, a mouse bladder cancer cell line, were trypsinized for 5-10 minutes and then resuspended in a volume of about 100 μL. 50 μL of matrix gel was added to increase the tumor formation rate and shorten the tumor formation time.

[0060] 3. Disinfect the injection area with iodine tincture, and then use an insulin needle to inject the cell suspension into the left armpit of a 6-week-old male C57BL / 6J mouse. At this time, a round or oval skin mound is clearly seen under the skin. Note that after the needle is inserted, it should be inserted parallel to the skin for 0.5 cm before injecting the cell suspension. When removing the needle, gently rotate the needle 180° before pulling it out to ensure that there will be no leakage after the needle is removed.

[0061] 4. Observe the changes at the inoculation site. When the inflammatory reaction at the inoculation site subsides and a hard mass can be felt at the inoculation site, drug treatment of the mice can be considered. After the start of drug treatment, measure the length L (mm) and width W (mm) of the tumor each time the drug is administered and record them. Then evaluate the tumor volume according to the formula: Tumor volume (mm 3 ) = L × W 2 / 2.

[0062] III. Intervention Strategies for Subcutaneous Tumor Models in Mice 1. Inject different drugs subcutaneously around the tumors of the mice once every two days. Divide all the mice into 4 groups, namely: control group, GEM group, HA group, and GEM+HA group. Administer 150 ul of the drug to each mouse once. The control group is PBS; the GEM group is Gem 8 mg / ml; the HA group is HA 1.6 mg / ml; the GEM+HA group is Gem 8 mg / ml + HA 1.6 mg / ml. The grouping and drug administration mode of the animal experiment are referred to Figure 1 for understanding.

[0063] 2. Raise the mice until the tumors are about 14 days old and within the scope of animal ethics, that is, the maximum diameter grows to nearly but not exceeding 2 cm and the tumor volume does not exceed 2000 mm 3 . Euthanize the mice and dissect the tumors: First, disinfect the peritumoral area of the mice, and then use tissue scissors and tissue forceps to dissect the mice. Find and carefully dissect the tumor by blunt dissection. The removed tumor is soaked in ice-bathed PBS or physiological saline to maintain the viability of tissue cells.

[0064] 3. After all the tumors are dissected, lay them flat on a specimen board with a scale for photographing and data collection. The results show that the tumor growth rate in the combined group is significantly slowed down. Combine Figures 2 - 4 for understanding. Then divide the tissue into three parts. The first part is prepared into a single-cell suspension by mechanical method, and the proportions and inter-group differences of CD45, F4 / 80, CD86, and CD206 positive and negative cells are detected by flow cytometry. The second part is fixed in formalin liquid for subsequent immunohistochemistry experiments. The third part is used for subsequent molecular experiments and stored in a -80°C refrigerator.

[0065] IV. Western-blot The main processes include: protein extraction, protein concentration determination by BCA method, protein denaturation, gel preparation system, electrophoresis, membrane transfer, blocking, primary antibody incubation, washing of primary antibody, secondary antibody incubation, washing of secondary antibody, and development to collect picture data. Specifically as follows: Protein extraction: After the target cells are routinely plated and intervened, carefully aspirate and discard the old medium, wash twice with 1×PBS, and add a certain volume of strong SDS sample buffer to lyse the cellular proteins according to the cell growth density in the well plate (taking a 24-well plate as an example, 80 μL is added to each well when the bottom surface is fully covered with cells). It is advisable to place the sample on ice or at 4°C during protein lysis. Use a cell scraper or a 200-μL pipette tip to stir and assist in lysis to make it more thorough. Then, continue to physically break the protein sample through ultrasonic waves to make it further thoroughly and evenly lysed. After that, centrifuge at 13,000 rpm for 5 min at 4°C using an ultra-low temperature centrifuge to remove the cell debris precipitate, harvest the supernatant with higher purity protein, and continue with the subsequent steps or store it temporarily at -80°C.

[0066] Protein concentration determination by BCA method: For the purified protein, we use a Thermo protein concentration detection kit to measure the protein concentration. The specific operation steps are as follows: S1. According to the total amount of the protein sample and the standard, prepare the BCA working solution by mixing reagent A and reagent B in a volume ratio of 1:50, and mix the above mixture thoroughly with a vortex mixer.

[0067] S2. Standard preparation: Prepare the standards according to the following table.

[0068] Table 1 BCA standard preparation table

[0069] S3. Add the standards in step S2 to a 96-well plate in sequence, 20 μL per well.

[0070] S4. Add 18 μL of double-distilled water to the 96-well plate, and then add 2 μL of the protein sample to the 96-well plate. At this time, the test sample is diluted 10 times.

[0071] S5. Add the prepared BCA working solution to each well, 200 μL per well; then incubate the 96-well plate at 37°C for 30 min; after incubation, shake the well plate thoroughly, and use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance of each protein sample at a wavelength of 562 nm.

[0072] S6. Draw a standard curve based on the obtained absorbance values and calculate the protein concentration.

[0073] Protein denaturation: Based on the sample concentration and the required loading amount (25 μg of protein loading mass), calculate the volume required for each protein. If the volume of the loaded protein is less than 20 μL, make it up with SDS. Subsequently, add 1 μL of a mixture composed of equal volumes of β-mercaptoethanol and 1% bromophenol blue to each protein sample. Then place the samples in a 95°C metal bath and heat for 5 min.

[0074] Gel preparation system: Prepare the separating gel mixture according to the gel preparation system table. After thoroughly shaking and mixing the mixture evenly, add it into the glass plate. After adding an appropriate amount of the mixture, add isopropanol to flatten the surface of the separating gel. Let it stand at room temperature for an appropriate time (until the separating gel is completely solidified), then pour out the isopropanol and blot the remaining liquid with absorbent paper. According to the gel preparation system table, thoroughly shake the mixture evenly and add it into the glass plate. Insert the comb into the stacking gel and let it stand at room temperature for 15 minutes.

[0075] Table 2 Gel preparation system table

[0076] Electrophoresis: Install the glass plate in the fixing clamp, add fresh 1× electrophoresis buffer to the fixing clamp and the electrophoresis tank. Gently and vertically pull out the comb from the SDS-PAGE gel plate. Then add the protein samples and Marker into the sample wells in turn. The electrophoresis conditions are 80 V constant voltage for 30 min, and then adjusted to 120 V for 60 min.

[0077] Transfer: After electrophoresis, take out the gel, activate the PVDF membrane with methanol, and make a "sandwich" model according to black cotton, filter paper, gel, PVDF membrane, filter paper, black cotton (avoid generating bubbles). Then place the transfer clamp in the transfer tank, add the pre-cooled transfer buffer to the transfer tank, and at the same time place a foam box with crushed ice outside the transfer tank (to prevent heat generation during the transfer process). Connect the power supply, adjust the current to 280 - 300 mA, and adjust the transfer time according to the molecular weight, which ranges from 40 - 120 min.

[0078] Blocking: After transfer, take out the PVDF membrane, mark the front and back sides and the position of the target protein by cutting a corner at the upper right and using a ballpoint pen. Then put it into the blocking solution (a mixture of 0.5 g skim milk powder and 10 mL PBST), and slowly rotate and block it on a shaker at room temperature for 1 hour to cover the non-specific binding sites of the PVDF membrane.

[0079] Primary antibody incubation: Prepare the primary antibody dilution solution (5% BSA): Add 25 mg bovine serum albumin + 0.5 mL sodium azide to PBS and make up the volume to 50 mL. Take 10 μL of the primary antibody stock solution and 10 mL of the primary antibody dilution solution (1:1000) to prepare the working solution, add it to the incubation box containing the PVDF membrane, and then incubate it on a shaker at 4°C overnight or at least 4 hours.

[0080] Wash the primary antibody: The primary antibody is recovered and stored in a 15 mL centrifuge tube at 4°C and can be reused multiple times. Take out the membrane and wash it 3 times with PBST for 10 minutes each time.

[0081] Secondary antibody incubation: The secondary antibody dilution is prepared with 5% skim milk powder and PBST. The ratio of the secondary antibody to the secondary antibody dilution is 1:10000. Place the PVDF membrane in the secondary antibody solution and incubate it slowly on a shaker at room temperature for 1.5 hours.

[0082] Wash the secondary antibody: After recovering the secondary antibody, store it in a 4°C refrigerator. Wash the PVDF membrane 3 times with PBST for 10 minutes each time.

[0083] Develop and collect picture data: Prepare the developing solution according to the instructions (Solution A: Solution B = 1:1), prepare it fresh as needed, evenly drip the ECL developing solution onto the PVDF membrane, and obtain the differential images of protein expression through a developer. Use β-actin as an internal reference to compare and analyze the differences in relative protein expression levels. For images with difficult-to-distinguish differences, use ImageJ software for gray value quantitative analysis and comparison.

[0084] In this experiment, different molecular weight HAs were used for testing, and the results are as Figure 5 shown. The results show that 1000KD HA significantly enhanced the activation level of PARP, and 1000KD HA can enhance GEM-induced apoptosis of bladder cancer cells. Unless otherwise specified, the molecular weight of HA used in other experiments of this application is 1000KD.

[0085] V. Detection of cell proliferation by CCK8 method 1. Seed human bladder cancer cells T24 and murine bladder cancer cells MB49 into 96-well plates at a density of 1000 cells per well. Note that the wells around the perimeter of the plate should not be seeded with cells, but filled with sterile PBS to prevent evaporation.

[0086] 2. After the cells are normally adherent and spread, give different interventions according to the experimental grouping and culture them normally.

[0087] 3. At the corresponding time point when the cells are normal, then add CCK8 solution at a ratio of 10 μL / 100 μL, incubate at 37°C in the dark for 2 - 4 hours, and measure the absorbance at a wavelength of 450 nm. The results are as Figures 6 - 7 shown, and this result proves that 1000KD HA can improve the anti-tumor ability of GEM.

[0088] VI. Detection of cell migration and invasion ability by Transwell method 1. Detection of cell migration ability by Transwell method (1)Take bladder cancer cells T24 and MB49 in the logarithmic growth phase and give different interventions according to the experimental protocol.

[0089] (2)After the cells are intervened according to the experimental protocol for sufficient time, use trypsin to digest and collect the cells, resuspend and mix the cells with serum-free medium, and perform cell counting. The cell counting method is as described above.

[0090] (3)Take a new 8-μm Transwell chamber and place it in a 24-well plate. Uniformly seed 50,000 cells in the upper chamber and add normal culture medium containing serum to the lower chamber for culture.

[0091] (4)After 24 hours, take out the chamber, gently rinse the chamber with PBS, fix the cells with 4% paraformaldehyde for 30 minutes, and then stain the cells with 0.1% crystal violet solution for 30 minutes.

[0092] (5)After staining, rinse the chamber with PBS and gently wipe the inner bottom surface of the chamber with a clean cotton swab.

[0093] (6)Use an inverted microscope to observe and take pictures.

[0094] 2. Detection of cell invasion ability by Transwell method (1)Take a new 8-μm Transwell chamber and place it in a 24-well plate. Mix the Matrigel and serum-free medium at a ratio of 1:8 and gently spread it on the inner bottom surface of the chamber, and incubate at 37 °C for 2 hours.

[0095] (2)Take bladder cancer cells T24 and MB49 in the logarithmic growth phase and give different interventions according to the experimental protocol.

[0096] (3)After the cells are intervened according to the experimental protocol for sufficient time, use trypsin to digest and collect the cells, resuspend and mix the cells with serum-free medium, and perform cell counting. The cell counting method is as described above.

[0097] (4)Uniformly seed 80,000 cells in the upper chamber and add normal culture medium containing serum to the lower chamber for culture.

[0098] (5)After 24 hours, take out the chamber, gently rinse the chamber with PBS, fix the cells with 4% paraformaldehyde for 30 minutes, and then stain the cells with 0.1% crystal violet solution for 30 minutes.

[0099] (6)After staining, rinse the chamber with PBS and gently wipe the inner bottom surface of the chamber with a clean cotton swab.

[0100] (7)Use an inverted microscope to observe and take pictures.

[0101] The results are asFigures 8 - 9 As shown in the figure, the results showed that the combined use of drugs significantly inhibited the migration and invasion abilities of bladder cancer cells T24 and MB49, and these results demonstrated that 1000KD HA could enhance the anti-tumor ability of GEM.

[0102] VII. Tunel Staining 1. Fixation and Permeabilization (1) Immerse the paraffin sections in xylene for dewaxing for 10 min * 2 times; soak the sections in absolute ethanol for 5 min * 2 times; then soak the sections in 90%, 80%, and 70% ethanol aqueous solutions for 3 min each.

[0103] (2) Immerse the dewaxed sections in PBS and rinse 3 times, 5 minutes each time.

[0104] (3) Blot dry the moisture around the section tissue, dilute the 100× proteinase K solution to 1× with PBS, and then add 100 μL of the 1× proteinase K working solution to each sample and react at 37 °C for 20 minutes.

[0105] (4) Rinse the sections with PBS 3 times, 5 minutes each time.

[0106] 2. Labeling Table 3 Formulation of Labeling Working Solution

[0107] Prepare the labeling working solution according to the above system for standby.

[0108] (1) Add 100 μL of TdT equilibration buffer to each tissue section, and incubate the sections in a 37 °C wet box for 10 - 30 min.

[0109] (2) After incubation, aspirate the buffer with absorbent paper (keep the tissue moist); add 50 μL of the labeling working solution to each sample, and react in a 37 °C wet box in the dark for 60 min; after the reaction, rinse 3 times with PBS.

[0110] (3) Stain the nucleus: After aspirating the residual liquid, add the DAPI working solution and stain in the dark at room temperature for 5 min; immerse the samples in PBS and rinse 4 - 5 times, 5 minutes each time.

[0111] (4) After aspirating the excess liquid, mount the sections with an anti-quenching mounting medium.

[0112] (5) Immediately observe the fluorescence quantity and intensity under a fluorescence microscope. If not detected immediately, the sections can be stored in the dark at 4 °C in a refrigerator.

[0113] The detection results are as Figure 10 shown. The results showed that the apoptosis level of tumors in the combined group increased significantly, indicating that 1000KD HA could improve the efficacy of GEM.

[0114] VIII. Immunohistochemical Staining (IHC) 1. Immunohistochemistry Procedure (1) Immerse the paraffin sections in xylene for dewaxing twice for 10 min each; soak the sections in absolute ethanol twice for 5 min each; then soak the sections in 90%, 80%, and 70% aqueous ethanol solutions for 3 min each.

[0115] (2) Antigen retrieval: Heat retrieve the antigens of the paraffin sections in a microwave oven using citric acid antigen retrieval buffer (pH 6.0). Take out the sections after 20 minutes (note to prevent the sections from drying). Wash the sections with PBS for 5 minutes three times after they cool down naturally.

[0116] (3) Serum blocking: Absorb the excess liquid at the edges of the sections with absorbent paper. Add 100 - 150 μL of 3% BSA blocking solution to each section and incubate at room temperature for 30 minutes.

[0117] (4) Primary antibody incubation: After blocking, add Ki67 antibody or cleaved - caspase antibody (dilution ratio 1:100) to the sections and place them in a wet box at 4°C overnight.

[0118] (5) Secondary antibody incubation: Wash the sections in the same way. After draining the liquid, add a secondary antibody of the same species as the primary antibody and incubate at room temperature for 1 h.

[0119] (6) DAB color development: Wash the sections in the same way. After draining the liquid, add DAB color development solution. Closely observe the color of the sections. Immediately stop the color development with tap water when it turns brown - yellow.

[0120] (6) Counterstaining of cell nuclei: Stain the sections with hematoxylin for 3 minutes. After rinsing with running water, blue them with differentiating solution and then rinse with running water again.

[0121] (7) Dehydration and mounting: Gradient dehydrate the sections in 50% ethanol, 75% ethanol, and absolute ethanol for 2 minutes each. After dehydration, mount the sections with neutral balsam.

[0122] (8) Observe the images under a microscope and perform image acquisition and analysis.

[0123] 2. Immunohistochemistry Scoring The immunohistochemical scoring criteria are based on the product of the staining intensity of tissue cells and the proportion score of the stained area as the scoring reference. Ki-67 is mainly expressed in the nucleus, and Cleaved-caspase is mainly expressed in the cytoplasm. The staining intensity evaluation criteria are divided into non-staining (0 points), light yellow (1 point), brown yellow (2 points), and tan (3 points) according to 0-3 respectively. The proportion scoring criteria for the positive area of tissue staining are 0-4 points: the staining range below 5% is 0 points, the staining range accounting for 5-25% is 1 point, the staining range accounting for 26%-50% is 2 points, the staining range accounting for 51%-75% is 3 points, and the staining range greater than 75% is 4 points. The final immunohistochemical score of the tissue is the product of the two scores, and the scoring grades are: a score of 0-8 is low expression, and a score of 8-12 is high expression. The immunohistochemical results are determined by two pathologists above the attending physician level.

[0124] 3. Result Explanation The expression of Ki67 in mouse tumors was shown by IHC staining as Figure 11 shown, and the scoring situation was as Figure 12 shown. The results showed that the expression of Ki67 in tumors of the combined group was significantly down-regulated, indicating that 1000KD HA could enhance the anti-tumor effect of GEM in vivo.

[0125] By performing IHC staining on mouse tumor sections, it was found that the activation level of caspase3 in the tumor tissues of the combined group of mice was significantly up-regulated, as Figure 13 shown, suggesting that the combined drug use might increase the apoptosis level of bladder cancer caused by GEM.

[0126] IHC staining of F4 / 80, CD86, iNOS, and CD206 was performed on the above-mentioned tumor tissues, and the results were as Figure 14 shown. The results showed that the combined drug use significantly enhanced the expression levels of CD86 and iNOS, decreased the expression level of CD206, while the expression level of F4 / 80 basically did not change, which suggested that 1000KD HA might reverse the macrophage-related immunosuppression caused by GEM by affecting macrophage polarization.

[0127] Flow Cytometry 1. Preparation of single-cell suspension by mechanical method (1) The dissected mouse tumors were rinsed clean with PBS, and fat, fibers, and necrotic areas were carefully removed, then placed in serum-free 1640 medium and kept on ice.

[0128] (2) The tumor tissues were minced as much as possible with tissue scissors, noting not to cut for too long and keeping the whole process on ice at low temperature. The remaining tissue pieces could be gently ground in a sieve.

[0129] (3) After allowing the supernatant in the centrifuge tube to stand for several seconds, transfer it to a 70-μm cell strainer using a pipette. At this time, single cells will pass through the strainer and flow into the 15-ml centrifuge tube below, while tumor tissue blocks cannot pass through. Perform the same operation for each sample, and pay attention to avoiding cross-contamination of cells between different groups or within the same group. The entire process should be carried out at low temperature on ice.

[0130] (4) After filtration, centrifuge all the cells at 4°C and 300 g for 5 minutes to obtain cell pellets.

[0131] (5) Carefully discard the supernatant, wash the cells with PBS, and centrifuge again at 4°C and 300 g for 5 minutes.

[0132] 2. Red blood cell lysis (1) According to the instructions, dilute the 10× red blood cell lysis buffer to a 1× working solution with sterile deionized water.

[0133] (2) Add 1 ml of the 1× red blood cell lysis buffer to each tube and resuspend the cells for red blood cell lysis. After allowing the lysis to proceed at room temperature for 3 - 5 minutes, add 10 volumes of PBS to terminate the lysis. Then, centrifuge at 300 g for 5 minutes and discard the supernatant. If only a pale yellow or white cell pellet can be seen at the bottom of the tube at this time, it indicates that the red blood cells have been lysed sufficiently and the next step can be carried out. If the upper part of the pellet is red, it means that the red blood cell lysis is incomplete, and the dose of the lysis buffer needs to be increased or the lysis time needs to be extended for re-lysis. Note that the number of re-lysis steps should be minimized to ensure minimal cell loss.

[0134] 3. Viable and dead cell staining (1) Resuspend each tube with 2 ml of autoclaved PBS, gently pipette to resuspend on ice, and then centrifuge at 300 g for 5 minutes to obtain cell pellets.

[0135] (2) Resuspend the cells with 2 ml of autoclaved PBS and adjust the cell density to 1 - 10×10 6 / mL. Then, add 1 μL of the viable and dead cell dye (APC-cy7) to each milliliter and incubate at 2 - 8°C for 30 minutes.

[0136] (3) After centrifuging at 4°C and 300 g for 5 minutes, discard the supernatant. Add 2 ml of FACS to each sample group to resuspend and wash the cells once, then continue centrifuging to discard the supernatant and add an appropriate amount of FACS for subsequent experiments.

[0137] 4. Fc receptor blocking (1) After resuspending with a certain volume of FACS, adjust the cell density to 2×10 7 / mL.

[0138] (2) Add 2 μL of Anti-Mouse CD16 / 32 Antibody to each sample and incubate at room temperature for 10 minutes.

[0139] 5. Extracellular Staining (1) After centrifuging at 300 g for 5 min at 4°C, discard the supernatant. Add an appropriate amount of FACS to resuspend each tube, and then take out blank tubes and single-staining tubes (divided into APC-Cy7, FITC, PE, Percp-Cy5.5, APC, PE-Cy7) for standby. The remaining samples are reserved for other staining.

[0140] (2) Sequentially add antibodies against extracellular markers (CD45, CD11b, F4 / 80, CD86) to the sample tubes to observe the proportions of different subsets of macrophages in different groups. Add 5 μL of all antibodies according to the instructions, and operate on ice throughout the process. Stain for half an hour at 4°C in the dark.

[0141] (3) After the antibody incubation is completed, centrifuge at 600 g for 5 min, discard the supernatant, and then add 500 μL of FACS to resuspend the cells in each ep tube and prepare for machine detection.

[0142] 6. Intracellular Staining (1) Add stimulants to the single-cell suspension after lysing red blood cells, let it act for 5 - 8 hours, and then perform the above operations.

[0143] (2) After extracellular staining, centrifuge at 300 g for 5 min at 4°C, discard the supernatant, and add 100 μL of fixative to fix the cells. Incubate at room temperature in the dark for 10 - 50 minutes.

[0144] (3) Add 2 mL of 1× permeabilization solution to the fixed samples, centrifuge at 300 g for 5 minutes at room temperature, discard the supernatant, and then repeat this step once.

[0145] (4) Resuspend the cell pellet in 100 μL of 1× permeabilization solution, and add the CD206 antibody. Incubate in the dark for 30 minutes.

[0146] (5) After the incubation is completed, centrifuge at 300 g for 5 min at 4°C, discard the supernatant, and add 2 ml of 1× permeabilization solution. Centrifuge at 300 g for 5 minutes at room temperature, discard the supernatant, and then repeat this step once.

[0147] (6) Use FAC to resuspend the cells and perform subsequent machine operations.

[0148] 7. Machine Operation (1) First, use the blank tube to adjust the voltage, and then use the single-staining tubes for live and dead cell dyes, CD45, CD11b, F4 / 80, CD86, CD206 respectively. Gate the cells and perform cell population analysis according to the machine data.

[0149] (2) Perform flow cytometry on each control group and experimental group, collect all data and analyze the differences between groups.

[0150] 8. Result Analysis The results are shown in Figures 15 - 22 , Figure 16 , 18 , 20, and 22 correspond to the statistical charts of Figure 15 , 17 , 19, and 21 in sequence. The results show that GEM treatment will reduce the proportions of lymphocytes and myelocytes, while the combination with 1000KD HA can reverse this phenomenon, Figures 15 - 18 . At the same time, the combination with 1000KD HA can reverse the down-regulation of the proportions of CD45 + CD11b + F4 / 80 + CD86 + cells and the up-regulation of the proportions of CD45 + CD11b + F4 / 80 + CD206 + cells, Figures 19 - 22 . The above results indicate that the combination with 1000KD HA can reverse the immunosuppression caused by GEM and improve the chemotherapy efficacy. In addition, gemcitabine and hyaluronic acid with a molecular weight of 200 - 1000KD, and the mass ratio of gemcitabine to hyaluronic acid is 4 - 6:1, both have a certain effect of improving the chemotherapy effect of GEM, further confirming the advantages of combination therapy.

[0151] In this application, in vivo and in vitro experiments prove that HA can induce macrophages to polarize towards the M1 phenotype and reverse the inhibition of GEM on the TME; and the combination can significantly improve the chemotherapy effect of GEM. Speculating from the mechanism, this process mainly exerts its effect by HA directly binding to Toll-like receptor 4 (TLR4) and activating the JAK2 / STAT3 signaling pathway. Refer to Figure 23 for understanding.

[0152] Finally, the relevant experimental equipment, reagent information, and the statistical analysis methods used in the experiments involved in the specific implementation manners are described.

[0153] Table 4 Information on Relevant Experimental Instruments and Equipment

[0154] Table 5 Information on Relevant Reagents

[0155] Statistical analysis: The data processing and statistical charts were processed using GraphPad Prism 6.0 and SPSS Statistics 23.0 software. The data obtained in this experiment were recorded as mean ± standard deviation, and independent-samples t-test and one-way ANOVA tests were used respectively. (ns represents no statistical significance, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, **** represents p < 0.0001).

[0156] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

Claims

1. A bladder perfusion co - preparation, characterized in that, The components include gemcitabine and hyaluronic acid with a molecular weight of 200-1000 KD, and the mass ratio of gemcitabine to hyaluronic acid is 4-6:

1.

2. The bladder perfusion synergistic preparation according to claim 1, characterized in that, The molecular weight of hyaluronic acid is 1000 KD, and the mass ratio of gemcitabine to hyaluronic acid is 5:

1.

3. Use of the intravesical perfusion synergistic preparation according to claim 1 or 2 in the preparation of a drug for reversing an immunosuppressive tumor microenvironment.

4. Use of the intravesical perfusion synergistic preparation according to claim 1 or 2 in enhancing the chemosensitivity of bladder cancer.

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