In-vivo evaluation method for curative effect of NK cells on human ovarian cancer ascites tumor and application

Abstract

The invention discloses an in-vivo evaluation method for the curative effect of NK cells on human ovarian cancer ascites tumor, which comprises the following steps: inoculating human ovarian cancer cells into the abdominal cavity of a female healthy mouse, and establishing a nude mouse model of the human ovarian cancer ascites tumor; nude mouse models are grouped, and intraperitoneal injection administration is carried out on each group of mice; and carrying out fluorescence tracing on the dosed mouse by adopting a fluorescence imaging method, observing the health and death conditions of the mouse at the same time, and judging the treatment effect of the NK cells on the human ovarian cancer ascites tumor. An ovarian cancer mouse model is established through intraperitoneal injection of an SK-OV-3 cell strain, the treatment effect of NK cells on human ovarian cancer ascites tumor in an in-vivo peritoneal complex environment is evaluated, the result is closer to the actual situation, and the detection result is accurate. The compound is applied to preparation of medicines for treating human ovarian cancer ascites tumor, and can significantly prolong the lifetime, reduce the death rate, reduce the tumor load and improve the life quality.

Description

Technical Field

[0001] This invention relates to the field of biochemical detection, specifically to an in vivo evaluation method and application of NK cell therapy for the treatment of human ovarian cancer ascites. Background Technology

[0002] Ovarian cancer is one of the three major malignant tumors of the female reproductive system. With the advancement of early diagnosis and treatment technologies for cervical cancer and endometrial cancer, ovarian cancer has become a type of malignant tumor that seriously threatens women's lives. Ascites is a common symptom in advanced ovarian cancer, often accompanied by symptoms such as increased abdominal girth, abnormal weight changes, and increased intra-abdominal tumor burden. Carboplatin injection is a commonly used chemotherapy drug for treating ovarian cancer, but it also brings some side effects, such as bone marrow suppression leading to decreased blood cell count, gastrointestinal reactions, ototoxicity, neurotoxicity, and allergic reactions, making it difficult to meet clinical treatment needs. Therefore, developing a treatment method that can effectively prolong the survival of patients with ovarian cancer and ascites, reduce tumor burden, and improve quality of life has become an urgent technical problem to be solved in this field.

[0003] Chinese invention patent application CN118291577A discloses a method for evaluating the in vitro killing effect of NK cells on ovarian cancer organoids. This method involves providing a solid matrix gel; adding ovarian cancer organoids and allowing them to stand for 12 hours; adding NK cells and co-culturing for 12 hours; and detecting the cytotoxicity of NK cells on ovarian cancer organoids. This method can be used to evaluate NK cells from various sources and predict their killing effect, but it cannot provide an evaluation of the effect in the complex peritoneal environment in vivo. Chinese invention patent application CN105219731A discloses an ovarian cancer stem cell vaccine and its preparation method. The CD117+CD44+ ovarian cancer stem cell vaccine, in animal experiments, can increase serum IFN-γ levels, downregulate TGF-β expression, enhance NK cell activity, significantly reduce the number of CD117+CD44+ ovarian cancer stem cells, and inhibit tumor growth. However, it does not directly use NK cells and cannot objectively determine the impact of NK cells on human ovarian cancer ascites tumors. Summary of the Invention

[0004] To develop a treatment method that can effectively prolong the survival of patients with ovarian cancer ascites tumors, reduce tumor burden, and improve quality of life, this invention establishes an ovarian cancer mouse model by intraperitoneal injection of SK-OV-3 cell line, evaluates its killing and inhibitory effect on ovarian cancer cells, and prolongs survival time. NK cells (natural killer cells) account for approximately 5%-10% of human peripheral blood single cells and are the main bearers of the body's innate immunity. Because NK cells can produce a killing effect on virus-infected cells and tumor cells without prior stimulation, they are important effector cells in tumor immunotherapy. The first aspect of this invention provides an in vivo evaluation method for the efficacy of NK cells against human ovarian cancer ascites tumors, including the following steps:

[0005] Human ovarian cancer cells were intraperitoneally inoculated into healthy female mice to establish a nude mouse model of human ovarian cancer ascites tumor;

[0006] The nude mouse model was divided into groups, and the mice in each group were injected with the drug intraperitoneally.

[0007] Fluorescence imaging was used to trace the fluorescence of mice after drug administration, and the health and mortality of the mice were observed to determine the therapeutic effect of NK cells on human ovarian cancer ascites tumors.

[0008] In one embodiment, the human ovarian cancer cells are a cell suspension, and the inoculation concentration of the cell suspension is 1×10⁻⁶. 7 -1×10 8 The inoculation amount of human ovarian cancer cells is 0.2-0.4 mL / ovarian.

[0009] In one embodiment, the human ovarian cancer cells are a cell suspension, and the inoculation concentration of the cell suspension is 1×10⁻⁶. 7 The inoculation amount of human ovarian cancer cells is 0.2 mL / ovarian.

[0010] In one embodiment, the human ovarian cancer cells are the SK-OV-3 cell line.

[0011] As one implementation method, the nude mouse model was divided into four groups: the model group, the carboplatin injection group, the NK cell group, and the NK cell group + carboplatin injection group.

[0012] As one implementation method, the dosage of the carboplatin injection group, converted to mouse body weight, is 50-60 mg / kg.

[0013] In one implementation, the dosage of the carboplatin injection group, converted to mouse body weight, is 52 mg / kg.

[0014] In one implementation, the drug concentration of the NK cell group is 1×10⁻⁶. 9 -1×10 10per mL.

[0015] In one implementation, the drug concentration of the NK cell group is 1×10⁻⁶. 9 per mL.

[0016] As one implementation method, the mice in each group were administered the drug via intraperitoneal injection 1-2 times.

[0017] As one implementation method, the time interval between the two intraperitoneal injections of the drugs in each group of mice was 20-25 days.

[0018] As one implementation method, the time interval between the two intraperitoneal injections of the drugs in each group of mice was 21 days.

[0019] In one implementation, the health status of the mouse includes changes in the mouse's abdominal circumference and body weight.

[0020] As one implementation method, the mice were evaluated before administration and at 4h, 24h, 72h, 1 week, and 2 weeks after administration.

[0021] The second aspect of the present invention provides an application of an in vivo evaluation method for the efficacy of NK cells against human ovarian cancer ascites tumors, which is used in the preparation of drugs for treating human ovarian cancer ascites tumors.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The in vivo evaluation method of NK cell efficacy against human ovarian cancer ascites tumor described in this invention establishes an ovarian cancer mouse model by intraperitoneal injection of SK-OV-3 cell line, and evaluates the therapeutic effect of NK cell on human ovarian cancer ascites tumor in the complex intraperitoneal environment. The results are closer to the actual situation and the detection results are accurate.

[0024] (2) The in vivo evaluation method of NK cell efficacy against human ovarian cancer ascites tumor described in this invention extends the survival period of model animals and reduces mortality by controlling the dosage of NK cell group.

[0025] (3) The in vivo evaluation method of the efficacy of NK cells against human ovarian cancer ascites tumor described in this invention involves two administrations to the NK cell group, which can reduce tumor burden, inhibit tumor cell proliferation, and reduce intra-abdominal tumor fluorescence intensity.

[0026] (4) The in vivo evaluation method of NK cell efficacy against human ovarian cancer ascites tumor described in this invention can determine the effect of tumor on the body's nutritional status by assessing the changes in abdominal circumference and body weight of mouse models, and indirectly assess the basic health status of mouse models.

[0027] (5) The in vivo evaluation method of NK cell efficacy against human ovarian cancer ascites lesions described in this invention is applied to the preparation of drugs for the treatment of human ovarian cancer ascites lesions, providing a theoretical basis for clinical treatment and drug research. Attached Figure Description

[0028] Figure 1 Survival curves for mice with ovarian cancer.

[0029] Figure 2 These are fluorescence images of nude mouse models of human ovarian cancer ascites tumors after the first dose. Figure 2 In the middle, from left to right, the animals were treated for 4 hours, 24 hours, 72 hours, one week, and two weeks. From top to bottom, the animals were the model group, the carboplatin injection group, the NK cell group, the NK cell group + carboplatin injection group, 6 animals (carboplatin was injected first, followed by NK cells), and 6 animals (carboplatin was injected).

[0030] Figure 3 Fluorescence imaging images of nude mouse models of human ovarian cancer ascites tumors after the second dose. Figure 2 In the middle, from left to right, the groups are 4h, 24h, 72h, one week, and two weeks after drug administration. From top to bottom, the groups are the model group, the carboplatin injection group, the NK cell group, and the NK cell group + carboplatin injection group, with 6 animals (carboplatin was injected first, followed by NK cells). Detailed Implementation

[0031] Example

[0032] An in vivo evaluation method for the efficacy of NK cells against human ovarian cancer ascites tumors includes the following steps:

[0033] Human ovarian cancer cells were intraperitoneally inoculated into healthy female mice to establish a nude mouse model of human ovarian cancer ascites tumor;

[0034] The nude mouse model was divided into groups, and the mice in each group were injected with the drug intraperitoneally.

[0035] Fluorescence imaging was used to trace the fluorescence of mice after drug administration, and the health and survival of the mice were observed to determine the therapeutic effect of NK cells on human ovarian cancer ascites tumors.

[0036] The human ovarian cancer cells mentioned are a suspension of human ovarian cancer SK-OV-3 cells, and the inoculation concentration of the cell suspension is 1×10⁻⁶. 7 The cells were purchased from the Chinese Academy of Sciences, and the inoculation amount of human ovarian cancer cells was 0.2 mL per ovary.

[0037] The method for establishing a nude mouse model of human ovarian cancer ascites tumor is as follows: Sixty qualified SPF-grade Balb / c-nu nude mice, female, weighing 12-15g, were selected. Ovarian cancer cells (SK-OV-3) in the logarithmic growth phase were extracted at a concentration of 1×10⁻⁶. 70.2 mL of the solution was injected intraperitoneally into 55 female Balb / c-nu nude mice. The injection volume was 0.2 mL per mouse. After injection, the abdominal circumference of the nude mice increased significantly, ascites was visible in the abdomen, and the fluorescence intensity of the abdomen was high in fluorescence imaging, indicating that the model was successfully established.

[0038] The SPF-grade Balb / c-nu nude mice were purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd., with animal quality certificate number 44829700038282 and laboratory animal production license number SCXK (Guangdong) 2022-0063. They were housed in Area D of the Barrier Environment Animal Laboratory at Hunan Prima Drug Research Center, with laboratory animal use license number SYXK (Hunan) 2020-0015.

[0039] The nude mouse model was divided into four groups: model group (n=10), carboplatin injection group (n=10), NK cell group (n=11), and NK cell group + carboplatin injection group (n=11).

[0040] Five uninoculated mice were used as a normal control group.

[0041] The dosage of carboplatin injection in the group, converted to mouse body weight, was 52 mg / kg. The concentration of carboplatin injection was 2.6 mg / mL, the dosing frequency was once, and the dosing volume was 20 mL / kg.

[0042] The concentration of the NK cells was 1×10⁻⁶. 9 The dosage is 0.1 mL / mL, administered twice daily, with a dosage volume of 0.1 mL / kg.

[0043] The two administration intervals for the NK cell group were 21 days.

[0044] The drug concentration of the NK cell group + carboplatin injection group was 1×10⁻⁶. 9 The dosage is 0.1 mL / kg, administered twice daily, with a volume of 0.1 mL / kg, and injected simultaneously.

[0045] The annotated drug for the model group and the normal control group was 0.9wt% sodium chloride injection, and the administration volume was 20mL / kg.

[0046] The health status of the mice includes changes in their abdominal circumference and body weight.

[0047] The abdominal circumference of the mice was measured using vernier calipers before and after drug administration.

[0048] The drug administration parameters for the survival status of the mice in each group are shown in Table 1.

[0049] The in vivo imaging detection in mice was performed using an in vivo imaging instrument before drug administration and at 4h, 24h, 72h, 1 week, and 2 weeks after drug administration to detect fluorescence imaging tracers in mice and calculate the changes in fluorescence intensity of ovarian cancer cells in vivo.

[0050] Note: Significant figures in this experiment were rounded to the nearest whole number, and statistical analysis was performed according to SOPs. SPSS 23.0 was used for statistical analysis. Quantitative data were expressed as mean ± standard deviation (X̄ ± S), and the homogeneity of variance was tested using Leven's test. If there was no statistical significance (P > 0.05), one-way ANOVA was used. If the ANOVA was statistically significant (P ≤ 0.05), the LSD test (parametric method) was used for comparative analysis. If the variances were unequal (P ≤ 0.05), the Kruskal-Wallis test was used. If the Kruskal-Wallis test was statistically significant (P ≤ 0.05), Dunnett's test (nonparametric method) was used for comparative analysis. The statistical results were compared with α = 0.05, where P ≤ 0.05 indicated statistical significance, and P ≤ 0.01 indicated a highly significant difference.

[0051] Performance testing

[0052] 1. Evaluation of the quality of life of mice with ovarian cancer ascites tumors using NK cells;

[0053] The test results are shown in Table 1 and Figure 1 .

[0054] Table 1

[0055] Group Dosage (mg / kg) Dosage frequency Survival time (days) Number of animals (individuals) Number of dead animals (individuals) mortality rate(%) Model group 25.70 ± 10.26 10 9 90 Carboplatin injection group 1.95 2 19.10 ± 6.62 10 10 100 NK cell group 10 2 31.55 ± 9.03## 11 8 72.7 NK cell group + carboplatin injection group 10 2 19.09 ± 6.85 11 11 100 Carboplatin injection + NK cell group (6 animals) 40 2 12.00 ± 6.78 6 6 100

[0056] Note: Compared with the carboplatin injection group. ## P ≤ 0.01.

[0057] The NK cell group + carboplatin injection group consisted of simultaneous injection of NK cells and carboplatin injection.

[0058] The carboplatin injection + NK cell group (6 animals) was administered carboplatin first, followed by NK cell injection 48 hours later.

[0059] As shown in Table 2, Figure 1As shown, 9 mice died in the model group, 10 mice died in the carboplatin injection group, 8 mice died in the NK cell group, 11 mice died in the NK cell + carboplatin injection group, and 6 mice died in the carboplatin + NK cell group (6 mice). Compared with the model group, the survival time of mice in the NK cell group was prolonged, but the difference was not statistically significant. Compared with the carboplatin injection group, the survival time of mice in the NK cell group was prolonged (P ≤ 0.01). The mortality rates of mice in the model group, carboplatin injection group, NK cell group, NK cell + carboplatin injection group, and carboplatin + NK cell group (6 mice) were 90%, 100%, 72.7%, 100%, and 100%, respectively.

[0060] 2. Evaluation of changes in abdominal circumference in mice with ovarian cancer ascites tumors using NK cells; the results of a single administration are shown in Table 2, and the results of a two-administration administration are shown in Table 3.

[0061] Table 2

[0062] Group normal control group Model group Carboplatin injection group NK cell group NK cell group + carboplatin injection group Six animals (after injection of carboplatin) Six animals (after NK injection) D0cm 5.72±0.08 <![CDATA[6.18±0.24 ++ ]]> <![CDATA[5.50±0.21 ∗∗ ]]> <![CDATA[5.84±0.18 ∗∗ ]]> <![CDATA[5.76±0.2 1∗∗ ]]> <![CDATA[5.78±0.42 ∗∗ ]]> <![CDATA[5.82±0.28 ∗∗ ]]> D1cm 5.78±0.11 <![CDATA[6.22±0.23 ++ ]]> <![CDATA[5.57±0.22 ∗∗ ]]> 5.88±0.13∗∗ <![CDATA[5.79±0.18 ∗∗ ]]> <![CDATA[5.82±0.28 ∗∗ ]]> 5.95±0.49 D4cm 5.90±0.10 <![CDATA[6.37±0.25 ++ ]]> <![CDATA[5.72±0.3 ∗∗ ]]> <![CDATA[5.96±0.18 ∗∗ ]]> <![CDATA[5.95±0.12 ∗∗ ]]> - 6.05±0.49 D7cm 6.62±0.45 <![CDATA[6.53±0.33 ++ ]]> <![CDATA[5.46±0.53 ∗∗ ]]> 6.15±0.46 <![CDATA[5.53±0.64 ∗∗ ]]> - <![CDATA[5.75±0.35 ∗ ]]> D10cm 6.16±0.34 6.25±0.21 <![CDATA[5.57±0.41 ∗∗ ]]> 6.10±0.11 <![CDATA[5.58±0.40 ∗∗ ]]> - - D13cm 6.36±0.30 6.28±0.24 <![CDATA[5.84±0.26 ∗∗ ]]> 6.19±0.17 <![CDATA[5.43±0.62 ∗∗ ]]> - -

[0063] Compared with the normal control group, ++ P≤0.01; compared with the model group, ∗ P≤0.05, ∗∗ P≤0.01.

[0064] The NK cell group + carboplatin injection group consisted of simultaneous injection of NK cells and carboplatin injection.

[0065] Six animals (after carboplatin injection): Results of the test performed after carboplatin injection.

[0066] Results of a test involving 6 animals (after NK cell injection): NK cell injection 48 hours after carboplatin injection.

[0067] Table 3

[0068] Group D1 (cm) D4 (cm) D7 (cm) D10 (cm) D13 (cm) normal control group 6.70±0.22 6.47±0.25 6.40±0.30 6.27±0.31 6.20±0.26 Model group <![CDATA[6.22±0.15 + ]]> <![CDATA[5.70±0.18 + ]]> 5.35±0.21++ <![CDATA[5.20±0.18 ++ ]]> <![CDATA[4.90±0.10 ++ ]]> Carboplatin injection group 6.00±0.41 6.00±0.00 - - - NK cell group 6.38±0.32 5.76±0.13 5.43±0.31 5.23±0.31 5.03±0.35 NK cell group + carboplatin injection group <![CDATA[5.70±0.53 ∗ ]]> 5.90±0.00 - -

[0069] Compared with the normal control group, + P≤0.05; compared with the model group, ∗ P≤0.05.

[0070] As shown in Table 2, compared with the normal control group, the abdominal circumference of mice in the model group was significantly increased from D0 to D7 after the first administration (P ≤ 0.01); compared with the model group, the abdominal circumference of mice in the carboplatin injection group and the NK cell group + carboplatin injection group was significantly decreased from D0 to D13 after the first administration (P ≤ 0.01); the abdominal circumference of mice in the NK cell group was significantly decreased from D0 to D4 after the first administration (P ≤ 0.01); the abdominal circumference of 6 animals (injected with carboplatin) was significantly decreased from D0 to D1 after the first administration (P ≤ 0.01), and the abdominal circumference of 6 animals (injected with NK) was significantly decreased from D0 to D7 after the first administration (P ≤ 0.01 or P ≤ 0.01).

[0071] As shown in Table 3, compared with the normal control group, the abdominal circumference of mice in the model group was significantly reduced on days 1 to 13 after the second administration (P ≤ 0.01); compared with the model group, the abdominal circumference of mice in the NK cell group + carboplatin injection group was significantly reduced on day 1 after the second administration (P ≤ 0.05).

[0072] 3. Evaluation of NK cell-induced weight changes in mice with ovarian cancer ascites tumors; the results of a single administration are shown in Table 4, and the results of a two-administration administration are shown in Table 5.

[0073] Table 4

[0074] Group D0(g) D1(g) D4(g) D7(g) D10(g) D13(g) normal control group 16.02±0.74 16.20±0.81 16.88±1.27 17.24±1.54 16.92±1.41 16.28±1.36 Model group <![CDATA[14.60±0.84 + ]]> <![CDATA[14.70±0.80 + ]]> <![CDATA[15.33±0.68 + ]]> 15.94±0.73 16.15±0.90 15.24±1.04 Carboplatin injection group 14.29±1.55 14.38±1.57 13.41±1.65∗∗ <![CDATA[12.61±1.95 ∗∗ ]]> <![CDATA[12.28±1.64 ∗∗ ]]> <![CDATA[12.84±1.29 ∗∗ ]]> NK cell group 14.40±1.32 14.47±1.35 15.15±1.53 15.86±1.80 15.93±1.79 15.76±1.88 NK cell group + carboplatin injection group 15.08±1.07 15.14±1.07 <![CDATA[14.17±0.82 ∗ ]]> <![CDATA[13.23±1.68 ∗∗ ]]> <![CDATA[12.91±1.66 ∗∗ ]]> <![CDATA[12.41±2.01 ∗∗ ]]> Six animals (after injection of carboplatin) <![CDATA[16.22±0.89 ∗ ]]> 14.20±1.34 - - - - Six animals (after NK injection) 14.20±1.34 15.50±0.14 15.50±0.0 15.00±0.14 - -

[0075] Compared with the normal control group, + P≤0.05; compared with the model group, ∗ P≤0.05 ，∗∗ P≤0.01.

[0076] Table 5

[0077] Group D1(g) D4(g) D7(g) D10(g) D13(g) normal control group 15.5±2.66 16.53±1.62 16.20±1.73 16.13±1.79 15.90±1.73 Model group 15.78±2.55 13.88±1.11 <![CDATA[12.20±1.10 ++ ]]> <![CDATA[10.65±0.81 ++ ]]> <![CDATA[9.37±0.76 ++ ]]> Carboplatin injection group <![CDATA[12.43±0.66 ∗ ]]> <![CDATA[10.90±0.00 ∗ ]]> - - - NK cell group 15.38±2.69 13.82±0.60 12.70±0.79 11.30±0.82 10.13±0.65 NK cell group + carboplatin injection group <![CDATA[11.87±2.39 ∗ ]]> <![CDATA[11.40±0.00 ∗ ]]> - -

[0078] Compared with the normal control group, ++ P≤0.01; compared with the model group, ∗ P≤0.05.

[0079] As shown in Table 4, compared with the normal control group, the body weight of mice in the model group was significantly reduced from D0 to D4 after the first administration (P ≤ 0.05); compared with the model group, the body weight of mice in the carboplatin injection group and the NK cell group + carboplatin injection group was significantly reduced from D4 to D13 after the first administration (P ≤ 0.05 or P ≤ 0.01), and the body weight of 6 animals (injected with carboplatin) was significantly increased from D0 after the first administration (P ≤ 0.05).

[0080] As shown in Table 5, compared with the normal control group, the body weight of mice in the model group was significantly reduced from D7 to D13 after the second administration (P ≤ 0.01); compared with the model group, the body weight of mice in the carboplatin injection group and the NK cell group + carboplatin injection group was significantly reduced from D1 to D4 after the second administration (P ≤ 0.05).

[0081] 4. Evaluation of NK cell fluorescence intensity changes in mice with ovarian cancer ascites tumors; results after a single dose are shown in Table 6. Figure 2 The test results after two doses are shown in Table 7. Figure 3 .

[0082] Table 6

[0083] Group Tumor fluorescence intensity in groups Tumor fluorescence intensity 4 hours after drug administration Tumor fluorescence intensity 24 hours after drug administration Tumor fluorescence intensity 72 hours after drug administration Tumor fluorescence intensity one week after administration Tumor fluorescence intensity two weeks after administration Model group 171573000.0±17189100 153680000.0±12906700.0 134450000.0±1212626.0 135095000.0±14900249.7 101074400.0±11261903.1 4306162.5±3080301.2 Carboplatin injection group 172175000.0±11562300 <![CDATA[6798320.0±5806700.0 ∗ ]]> 14489000.0±12014797.8 <![CDATA[25188000.0±16559453.7 ∗ ]]> 8425000.0±8786501.7 2095833.3±30368150.7 NK cell group 171800900.9±20483700 <![CDATA[5887123.6±9957640.0 ∗ ]]> <![CDATA[3984545.5±3972974.5 ∗∗ ]]> 6216727.3±6482715.0 <![CDATA[1864138.0±3948110.0 ∗ ]]> 46620500.0±65289046.3 NK cell group + carboplatin injection group 17253363.6±620661700 <![CDATA[6823781.8±6749390.0 ∗ ]]> 7120909.1±4678432.3 6169636.4±6605458.5 4281500.0±6552091.4 3599042.9±5642242.3 Six animals were injected with carboplatin first, followed by NK cells 48 hours later. 85580000.0±71716295.2 96460000.0±9889791.7 (4 hours after carboplatin injection) 30822000.0±372002470.9 (24 hours after NK injection) 49833333.3±237023346.6 (72 hours after NK injection) 31700000.0±44830569.9 (One week after NK injection) - Six animals (injected with carboplatin) 85580000.0±71716295.2 134760000.0±85585238.2 (Carboplatin injection) 139440000.0±85901472.6 (Carboplatin injection) - - -

[0084] Compared with the model group, ∗ P≤0.05, ∗∗ P≤0.01.

[0085] Table 7

[0086] Group Tumor fluorescence intensity 4 hours after drug administration Tumor fluorescence intensity 24 hours after drug administration Tumor fluorescence intensity 72 hours after drug administration Tumor fluorescence intensity after 1 week of drug administration Tumor fluorescence intensity 2 weeks after administration Model group 6750000.0±5768340.3 117600000.0±8989357.6 10380000.0±10291967.7 17433187.5±22062387.3 66833333.3±66746410.6 Carboplatin injection group 10269750.0±8655976.0 8135000.0±8076015.9 7104500.0±9986005.1 - - NK cell group 11944700.0±24029507.8 <![CDATA[3068267.0±3323205.2 ∗ ]]> 3138947.5±3845506.0 10995250.0±8292705.0 4906266.7±4304254.5 NK cell group + carboplatin injection group 721750.0±583230.9 <![CDATA[1444667.1±121207.5 ∗ ]]> 95900.0±0.0 - - Six animals were injected with carboplatin first, followed by NK cells 48 hours later. 20600000.0±291327993.9 - - - -

[0087] Compared with the model group, ∗ P≤0.05, ∗∗ P≤0.01.

[0088] As shown in Table 6, compared with the model group, the fluorescence intensity of mice in the carboplatin injection group, NK cell group, and NK cell + carboplatin injection group was significantly reduced 4 hours after administration (P ≤ 0.05); the fluorescence intensity of mice in the NK cell group was significantly reduced 24 hours and 1 week after administration (P ≤ 0.05 or P ≤ 0.01); and the fluorescence intensity of mice in the carboplatin injection group was increased 72 hours after administration (P ≤ 0.05).

[0089] As shown in Table 7, compared with the model group, the fluorescence intensity of mice in the NK cell group and the NK cell + carboplatin injection group was significantly reduced 24 hours after administration (P ≤ 0.05).

[0090] Conclusion: The results of this experiment demonstrate that the NK cell group and the NK cell + carboplatin injection group can significantly reduce the fluorescence intensity of mice 4 hours after administration; the NK cell group can significantly reduce the fluorescence intensity of mice 24 hours and 1 week after administration; the NK cell group and the NK cell + carboplatin injection group can significantly reduce the fluorescence intensity of mice 24 hours after administration, indicating that administration of NK cells alone can significantly prolong the survival time of mice.

Claims

1. An in vivo evaluation method for the efficacy of NK cells against human ovarian cancer ascites tumors, characterized in that, Includes the following steps: Human ovarian cancer cells were intraperitoneally inoculated into healthy female mice to establish a nude mouse model of human ovarian cancer ascites tumor; The nude mouse model was divided into groups, and the mice in each group were injected with the drug intraperitoneally. Fluorescence imaging was used to trace the fluorescence of mice after drug administration, and the health and mortality of the mice were observed to determine the therapeutic effect of NK cells on human ovarian cancer ascites tumors.

2. The in vivo evaluation method for the efficacy of NK cells against human ovarian cancer ascites tumors according to claim 1, characterized in that, The human ovarian cancer cells are a cell suspension, and the inoculation concentration of the cell suspension is 1×10⁻⁶. 7 -1×10 8 The inoculation amount of human ovarian cancer cells is 0.2-0.4 mL / ovarian.

3. The in vivo evaluation method for the efficacy of NK cells against human ovarian cancer ascites tumors according to claim 1, characterized in that, The nude mouse model was divided into four groups: the model group, the carboplatin injection group, the NK cell group, and the NK cell group + carboplatin injection group.

4. The in vivo evaluation method for the efficacy of NK cells against human ovarian cancer ascites tumor according to claim 3, characterized in that, The dosage of carboplatin injection in the group was converted to mouse body weight and was 50-60 mg / kg.

5. The in vivo evaluation method for the efficacy of NK cells against human ovarian cancer ascites tumor according to claim 3, characterized in that, The drug concentration administered to the NK cell group was 1×10⁻⁶. 9 -1×10 10 per mL.

6. The in vivo evaluation method for the efficacy of NK cells against human ovarian cancer ascites tumor according to claim 1, characterized in that, The mice in each group were administered the drug via intraperitoneal injection 1-2 times.

7. The in vivo evaluation method for the efficacy of NK cells against human ovarian cancer ascites tumors according to claim 6, characterized in that, The time interval between the two intraperitoneal injections in each group of mice was 20-25 days.

8. The in vivo evaluation method for the efficacy of NK cells against human ovarian cancer ascites tumor according to claim 1, characterized in that, The health status of the mice includes changes in their abdominal circumference and body weight.

9. The in vivo evaluation method for the efficacy of NK cells against human ovarian cancer ascites tumor according to claim 1, characterized in that, The mice were evaluated before administration and at 4 h, 24 h, 72 h, 1 week, and 2 weeks after administration.

10. The application of an in vivo evaluation method for the efficacy of NK cells against human ovarian cancer ascites tumor according to any one of claims 1-9, characterized in that, It is used in the preparation of drugs for treating ascites cysts in human ovarian cancer.

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