Lung micronucleus detection method

Abstract

The invention discloses a lung micronucleus detection method. The method comprises the following steps: S1, sample treatment: carrying out enzymolysis treatment on lung tissue fragments by adopting collagenase V digestive juice, and resuspending by adopting a neutral formalin solution to obtain a cell suspension; s2, dyeing and counting: dyeing the cell suspension by using an SYBR Gold dyeing solution; counting the micronucleus number of at least 1000 complete cells, and calculating the micronucleus rate; the micronucleus rate is calculated according to the following formula: p = (x / n) * 100%; wherein x is the micronucleus number obtained by dyeing counting, and n is the number of complete cells. The lung micronucleus detection method has excellent specificity and compatibility, and the repeatability of the test result is good.

Description

Technical Field

[0001] This invention relates to the field of non-clinical evaluation technology of drugs, specifically to a method for detecting lung micronuclei in the lungs for the detection of genotoxicity of inhaled formulations, and is particularly applicable to the lung micronucleus test under repeated administration conditions in rats. Background Technology

[0002] Existing technologies, based on the International Council for Harmonisation of Technical Requirements for Genetic Toxicity (ICH) S2 (R1) guidelines, and traditional micronucleus assays primarily target bone marrow and peripheral blood or liver, have the following key drawbacks:

[0003] (1) Insufficient specificity: The metabolic characteristics of lung cells (such as high expression of CYP1A1 / CYP2B6 in the CYP enzyme system) are significantly different from those of bone marrow / liver. Some precursor carcinogens activated by lung metabolism (such as benzo[a]pyrene) only produce genotoxicity in the lungs. Traditional methods are prone to false negatives (false negative rate of about 40%).

[0004] (2) Lack of standardization: There is no unified protocol in the existing lung micronucleus test. There is no consensus on sample processing (such as the selection of enzyme digestion reagents and incubation time) and the dosage of positive control drugs (such as cyclophosphamide), resulting in poor reproducibility of results.

[0005] (3) Poor compatibility: It cannot be integrated into repeated-dose toxicity studies, requires separate animal housing, and does not comply with the 3R principle of animal experiments (reduce / replace / optimize). Summary of the Invention

[0006] The technical problem this invention aims to solve is to overcome the deficiency in existing technologies regarding the lack of methods for detecting pulmonary genotoxicity, and to provide a method for detecting lung micronuclei and its applications. This lung micronuclei detection method exhibits excellent specificity and compatibility, and the test results show good repeatability.

[0007] The present invention solves the above-mentioned technical problems through the following technical solution:

[0008] This invention provides a method for detecting micronuclei in the lungs, comprising the following steps:

[0009] S1. Sample processing: Lung tissue fragments were enzymatically digested using collagenase V digestion solution and then resuspended in neutral formalin solution to obtain a cell suspension.

[0010] S2. Staining and Counting: The cell suspension was stained with SYBR Gold staining solution; the number of micronuclei in at least 1000 intact cells was counted, and the micronucleus rate was calculated;

[0011] The micronucleus rate is calculated using the following formula:

[0012] P = (x / n) * 100%; where x is the number of micronuclei obtained by staining and counting, and n is the number of intact cells.

[0013] In some embodiments, step S2 includes the step of mixing diluted SYBR Gold staining solution with the cell suspension at a volume ratio of (0.5-1.5):1; the volume ratio is preferably 1:1.

[0014] In some preferred embodiments, in step S2, the SYBR Gold staining solution is diluted by a factor of 5,000-15,000, for example, by a factor of 10,000.

[0015] In some embodiments, in step S1, the collagenase V digestion solution includes collagenase V and phosphate buffer, wherein the phosphate buffer is preferably HBSS buffer, more preferably HBSS buffer pre-cooled at 2-8°C.

[0016] In some embodiments, in step S1, the concentration of the collagenase V digestion solution used is 0.01%-0.1% (m / m), preferably 0.05%.

[0017] In some embodiments, in step S1, the temperature of the enzymatic hydrolysis treatment is 35-40°C, preferably 37°C.

[0018] In some implementations, the enzymatic hydrolysis time in step S1 is 1-24 hours, preferably 1 hour.

[0019] In some implementations, step S1 includes filtration and centrifugation steps after the enzymatic hydrolysis and before resuspending.

[0020] The filtration process preferably uses a 100-300 mesh cell sieve, and more preferably a 200 mesh sieve.

[0021] The centrifugal speed is preferably 450-650 rpm, more preferably 500 rpm.

[0022] The centrifugation time is preferably 1-10 min, more preferably 5 min.

[0023] In some embodiments, step S2, after resuspension, further includes washing; the washing is preferably with phosphate buffer until the cell suspension is white. The phosphate buffer is preferably HBSS buffer, more preferably HBSS buffer pre-cooled at 2-8°C.

[0024] In some implementations, in step S1, the lung tissue fragments are obtained by the following method:

[0025] Take lung tissue, rinse with phosphate buffer, and cut into small pieces.

[0026] The fragments are preferably 0.5-1.5 mm in size. 3 Size, preferably 1mm 3 size.

[0027] The phosphate buffer is preferably an HBSS buffer, and more preferably a 2-8℃ pre-cooled HBSS buffer.

[0028] In some implementations, in step S1, the lung tissue fragments are lung lobe tissue fragments.

[0029] In some implementations, in step S1, the neutral formalin solution is a formalin buffer solution with a volume percentage of 4%-10%.

[0030] Preferably, the formalin buffer solution has a volume percentage of 10%.

[0031] In some implementations, in step S1, the neutral formalin solution is a neutral formalin phosphate buffer solution.

[0032] In some embodiments, in step S2, the counting is performed under a fluorescence microscope at 900-1200x magnification, preferably 1000x.

[0033] In some implementations, step S2 is followed by the following steps:

[0034] S3. Data Processing and Statistics: After testing the normality of the micronucleus rate, one-way ANOVA combined with Dunnett's t-test was used to compare the differences between the experimental group and the control group; the dose-response relationship was analyzed by linear regression, with p < 0.05 considered statistically significant; when p < 0.05, the experimental group was considered to have significant cytotoxicity relative to the control group, and when p >= 0.05, the experimental group was considered to have no significant cytotoxicity relative to the control group.

[0035] In this invention, the lung tissue is administered intratracheally for three consecutive days before processing.

[0036] This invention belongs to the field of non-clinical genotoxicity evaluation technology of drugs, specifically involving the lung-specific genotoxicity detection of inhaled preparations. Its core application is in the preclinical safety evaluation of inhaled drugs for the treatment of lung diseases such as asthma, chronic obstructive pulmonary disease, and Covid-19, filling the technological gap of "the lungs being the direct target organ of inhaled drugs but lacking a dedicated genotoxicity detection method".

[0037] In this invention, the collagenase is extracted and prepared from Clostridium histolyticum. Its chemical name is collagen hydrolase, which can specifically hydrolyze the three-dimensional helical structure of natural collagen under physiological pH and temperature conditions without damaging other proteins and tissues. Currently, commercially available collagenases are mainly classified into types I, II, III, IV, and V.

[0038] Collagenase I, primarily derived from animal fibroblasts, exhibits substrate-specificity and can efficiently degrade all type I collagen, while showing almost no degradation effect on other proteins. For this reason, collagenase I is commonly used for the separation of epithelial, lung, adipose, and adrenal gland cells. It effectively digests connective tissues, facilitating cell separation, and is particularly suitable for the separation of mammalian cells.

[0039] Collagenase II, derived from mammalian embryonic tissue, exhibits different substrate specificity compared to type I. It can specifically degrade type II collagen, but has only a weak ability to degrade type I collagen. In practical applications, collagenase II is more suitable for the isolation of cells from liver, bone, thyroid, heart, and salivary gland tissues.

[0040] Collagenase V contains at least seven protease components with molecular weights ranging from 68 to 130 kDa. This complex composition endows it with unique functions, enabling it to be used for the isolation of pancreatic islet tissue and successfully separating connective tissue into single cells.

[0041] The positive and progressive effects of this invention are as follows:

[0042] (1) High specificity: Compounds designed for high local exposure to the lungs or lung metabolic characteristics have excellent detection rates of lung-specific genotoxicity;

[0043] (2) High sensitivity: The detection rate of micronuclei was 2.3 times higher after 3 consecutive days of administration than after a single administration (cyclophosphamide 16mg / kg group, micronucleus rate of single administration was 0.69±0.15%, and 1.58±0.21% after continuous administration); the cell viability of cells incubated with collagenase for 1 hour was 14.2% and 27.4% higher than that of cells incubated for 6 hours and 24 hours, respectively, ensuring the accuracy of counting;

[0044] (3) High degree of standardization: Key parameters such as reagent type, dosage, and time are clearly defined, and the coefficient of variation of repeated verification results in different laboratories is <10% (the coefficient of variation of existing non-standardized schemes is about 25%).

[0045] (4) Good compatibility: It can be carried out simultaneously with repeated-dose toxicity tests (such as weight monitoring and lung pathological examination), and multiple indicators can be detected in the same batch of animals. The number of animals used is reduced by 50%, which is in line with the 3R principle (reducing the number of animals).

[0046] (5) Convenient operation: The enzymatic digestion-purification process only takes 3-4 hours (existing methods take 6-8 hours), and SYBR Gold staining does not require fixative treatment. The steps are simplified by 30%, making it suitable for high-throughput detection. Attached Figure Description

[0047] Figure 1 The digestive effects of different types of collagenases.

[0048] Figure 2 The results show the effects of different digestion times (400×). a: Fluorescence staining results after 1 h of digestion; b: Fluorescence staining results after 24 h of digestion.

[0049] Figure 3 To refine the activity rate for different enzymatic hydrolysis processes.

[0050] Figure 4 A representative image (400×) of positive micronucleus results in the lungs after SYBR Gold staining. Figure 4 The arrow in the field of view has 3 micronuclei.

[0051] Figure 5 A representative image (400×) of positive micronucleus results in the lungs after SYBR Gold staining. Figure 5 Each arrow in the field of view has a micronucleus. Detailed Implementation

[0052] The present invention will be further illustrated by way of embodiments below, but the present invention is not limited to the scope of the embodiments described herein.

[0053] Laboratory animals and grouping

[0054] Animal type: SPF grade SD rats (6-9 weeks old, weighing 200±20g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., license number SYXK(Su)2023-0053).

[0055] Grouping: Negative control group (physiological saline), positive control group (cyclophosphamide), 5 animals / sex / group per group. Husbandry environment: Temperature: 20~26 ℃, Humidity: 40%~70%, Ventilation: ≥15 air changes per hour, using 100% fresh air (no air circulation), Lighting: Automatic lighting, alternating between light and dark every 12 hours, lights off around 19:00, lights on again around 07:00 the next day.

[0056] Feed: via Co 60 The maintenance feed for SPF rats and mice sterilized by irradiation was provided by Keao Cooperation (Tianjin) Feed Co., Ltd.

[0057] Drinking water: Animals have free access to homemade deionized water.

[0058] Bedding material: Ordinary grade corn cob bedding material is provided by Nantong Weiye Biotechnology Co., Ltd.

[0059] Dosing regimen

[0060] Positive control group: Cyclophosphamide (purchased from Sigma-Aldrich) was administered intratracheally at doses of 4 mg / kg, 8 mg / kg, and 16 mg / kg, once daily for 3 consecutive days; Negative control group: Normal saline (purchased from Jiangxi Kelun Pharmaceutical Co., Ltd.), with the same frequency as the high-dose positive control group.

[0061] Sample collection and processing

[0062] Euthanasia: Intraperitoneal injection of a mixture containing 40 mg / mL ketamine (purchased from Jiangsu Zhongmu Beikang Pharmaceutical Co., Ltd.) + 5 mg / mL xylazine (purchased from Dunhua Shengda Animal Pharmaceutical Co., Ltd.), followed by exsanguination after anesthesia.

[0063] Lung processing: After euthanasia, lung lobe tissue was collected, rinsed with HBSS buffer (pre-cooled at 2-8℃, purchased from Gibco), and minced to 1 mm³; Enzymatic digestion: 10 mL of 0.05% collagenase I, II, or V digestion solution (purchased from Sigma-Aldrich, diluted with HBSS buffer (pre-cooled at 2-8℃)) was added, and the cells were incubated at 37℃ with shaking for 1 h / 6 h / 24 h; Cell purification: The cells were filtered through a 200-mesh sieve, centrifuged at 500 rpm for 5 min, resuspended in 10% neutral formalin (Jiangsu Suyuan Pathology Diagnostic Center Co., Ltd., specification 20 L), and washed 3 times with HBSS buffer (pre-cooled at 2-8℃) until the cell clumps turned white, thus obtaining a cell suspension.

[0064] Staining and counting

[0065] Mix 20 μL cell suspension with 20 μL SYBR Gold staining solution (purchased from Thermo Fisher) (diluted using QuickBlock™ immunostaining primary antibody dilution buffer (Beyotime, 100 mL / bottle, dilution factor 10000)); count: under a 1000x fluorescence microscope (Olympus), count the number of micronuclei in 1000 intact cells and calculate the micronucleus rate (% MNPUL).

[0066] The micronucleus rate is calculated using the following formula:

[0067] p = (x / n) * 100%; where x is the number of micronuclei obtained from staining and counting, and n is the number of intact cells.

[0068] Experimental data processing and statistics

[0069] Statistical analysis was performed using SPSS software.

[0070] After testing the normality of % MNPUL, one-way ANOVA combined with Dunnett's t-test was used to compare the differences between the experimental group and the control group.

[0071] The dose-response relationship was analyzed by linear regression, with p < 0.05 considered statistically significant. When p < 0.05, the experimental group was considered to have significant cytotoxicity compared to the control group, and when p >= 0.05, the experimental group was considered to have no significant cytotoxicity compared to the control group.

[0072] For the first time, a micronucleus model of the lungs of SD rats was established by intratracheal administration for 3 consecutive days. Cyclophosphamide (4-16 mg / kg) was used as a positive control to specifically address the problem of genotoxicity detection of inhaled formulations in the lungs.

[0073] Achieve compatibility with general toxicity tests: It can be integrated into repeated-dose toxicity studies (general toxicity observation and micronucleus detection are carried out simultaneously on the same batch of animals), reducing the number of animals used by 29% (the calculation method is shown in Table 1), which complies with the 3R principle.

[0074] Table 1

[0075]

[0076] Example 1

[0077] Specific procedures for the repeated-dose lung micronucleus test in SD rats

[0078] Animals: SPF-grade SD rats, 7 weeks old, weighing 200±20g, 5 males and 5 females / sex / group, purchased from Beijing Vital River (License No. SYXK(Su)2023-0053);

[0079] Instruments: Fluorescence microscope, constant temperature shaking incubator, centrifuge;

[0080] Grouping and administration: The animals were divided into a negative control group (physiological saline), a positive low-dose group (cyclophosphamide 4 mg / kg), a positive medium-dose group (8 mg / kg), and a positive high-dose group (16 mg / kg), with 5 males and 5 females in each group; the drugs were administered via intratracheal injection once a day for 3 consecutive days.

[0081] Sample processing: On the 3rd day of drug administration, animals were anesthetized by intraperitoneal injection of ketamine 40 mg / kg + xylazine 5 mg / kg, euthanized by exsanguination, and lung tissue was collected. After rinsing with HBSS pre-cooled at 2-8℃, the tissue was cut into 1 mm³ and 10 mL of 0.05% collagenase V digestion solution (stock concentration of 1 g / mL, final concentration of 0.5 mg / mL, obtained by dilution with HBSS pre-cooled at 2-8℃) was added. The samples were then incubated at 37℃ with shaking for 1 h (1 animal per group at each time point).

[0082] Cell purification: Filter through a 200-mesh sieve, centrifuge at 500 rpm for 5 min, resuspend in 10% formalin, and wash three times with pre-cooled HBSS buffer at 2-8℃ until the cell clusters turn white to obtain a cell suspension; take 20 μL of cell suspension and mix with 20 μL of SYBR Gold (diluted 10,000 times using QuickBlock™ immunostaining primary antibody dilution buffer), and smear the mixture;

[0083] Counting and statistics: Count the micronuclei of 1000 intact cells under a 1000x microscope (count 2 replicates per sample), calculate %MNPUL, and perform ANOVA + Dunnett t test using SPSS 2.0;

[0084] The micronucleus rate is calculated using the following formula:

[0085] p = (x / n) * 100%; where x is the number of micronuclei obtained by staining and counting, and n is the number of intact cells (specifically 1000 in this embodiment).

[0086] The micronucleus results are shown in Table 2.

[0087] Depend on Figure 2 It can be seen that, under the same incubation time of 1 h, the micronucleus rate of each dose group of cyclophosphamide was significantly higher than that of the negative control group (p<0.01), and it was dose-dependent, proving that the model is effective.

[0088] Among them, the positive results of lung micronuclei after SYBR Gold staining when the cyclophosphamide dose was 8 mg / kg are shown in the figure. Figure 4 and Figure 5 . Figure 4 and Figure 5 These are representative images showing the results from different viewpoints, among which... Figure 4 The arrow in the field of view has 3 micronuclei. Figure 5 Each arrow in the field of view has a micronucleus.

[0089] Example 2-3

[0090] The only difference between this embodiment and Example 1 is the change in the incubation time during the enzymatic hydrolysis process. Specifically, 0.05% collagenase V was selected, and the incubation times were set to 6 hours and 24 hours respectively (one animal per group at each time point). The results are shown in Table 2 and... Figure 3 As shown in Table 2, the cell viability (86.4%-87.1%) and micronucleus rate (1.58%-1.59%) of the 1-hour incubation group were superior to those of the 6-hour and 24-hour groups, indicating that the 1-hour digestion time yielded better results. Note: Figure 3 In the mean time interval, compared with the 1-hour result, *P<0.05, **P<0.01.

[0091] Examples 4-5

[0092] The only difference between this embodiment and Embodiment 1 is the change in digestion time. Specifically, digestion was performed at 37°C on a shaker for 1 hour and 24 hours, respectively. See the results for details. Figure 2 a: Fluorescent staining results after 1 hour of digestion; b: Fluorescent staining results after 24 hours of digestion. Figure 2 It can be seen that intact cells are visible after 1 hour, while cells are clearly clumped together after 24 hours, and the digestion time of 1 hour is more effective.

[0093] Comparative Examples 1-2

[0094] Collagenase I, collagenase II, and collagenase V were used for digestion treatment, respectively.

[0095] The digestive effects of different types of collagenases are shown in the figure. Figure 1 Since collagenase I and collagenase II had poor digestion effects (no cell precipitation) and did not achieve the expected results, only collagenase V showed obvious cell precipitation. Collagenase V can be used for digestion treatment.

[0096] Table 2

[0097]

[0098] **: p≤0.01 compared with the negative control group of the same sex.

[0099] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and the scope of protection of the present invention is defined by the appended claims. Those skilled in the art can make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, but all such changes and modifications fall within the scope of protection of the present invention.

Claims

1. A method for detecting lung micronuclei, characterized in that, It includes the following steps: S1. Sample processing: Lung tissue fragments were enzymatically digested using collagenase V digestion solution and then resuspended in neutral formalin solution to obtain a cell suspension. S2. Staining and Counting: The cell suspension was stained with SYBR Gold staining solution; the number of micronuclei in at least 1000 intact cells was counted, and the micronucleus rate was calculated; The micronucleus rate is calculated using the following formula: p = (x / n) * 100%; where x is the number of micronuclei obtained by staining and counting, and n is the number of intact cells.

2. The lung micronucleus detection method as described in claim 1, characterized in that, In step S2, the staining includes mixing diluted SYBR Gold staining solution with the cell suspension at a volume ratio of (0.5-1.5):1; the volume ratio is preferably 1:

1. The preferred dilution ratio of the SYBR Gold staining solution is 5000-15000 times, for example, 10000 times.

3. The lung micronucleus detection method as described in claim 1 or 2, characterized in that, In step S1, the collagenase V digestion solution includes collagenase V and phosphate buffer, wherein the phosphate buffer is preferably HBSS buffer, and more preferably HBSS buffer pre-cooled at 2-8℃. And / or, in step S1, the concentration of the collagenase V digestion solution used is 0.01%-0.1% (m / m), preferably 0.05%; And / or, in step S1, the temperature of the enzymatic hydrolysis treatment is 35-40°C, preferably 37°C; And / or, in step S1, the enzymatic hydrolysis treatment time is 1-24 hours, preferably 1 hour.

4. The method for detecting lung micronuclei as described in any one of claims 1-3, characterized in that, In step S1, the enzymatic hydrolysis and resuspension process further includes filtration and centrifugation steps. The filtration is preferably carried out using a 100-300 mesh cell sieve, and more preferably 200 mesh; The centrifugation speed is preferably 450-650 rpm, more preferably 500 rpm; The centrifugation time is preferably 1-10 min, more preferably 5 min.

5. The method for detecting lung micronuclei as described in any one of claims 1-4, characterized in that, In step S2, the resuspension process further includes a washing step; the washing is preferably with phosphate buffer until the cell suspension is white; the phosphate buffer is preferably HBSS buffer, more preferably HBSS buffer pre-cooled at 2-8℃.

6. The method for detecting lung micronuclei as described in any one of claims 1-5, characterized in that, In step S1, the lung tissue fragments are obtained by the following method: Take lung tissue, rinse with phosphate buffer, and then cut it into small pieces; The fragments are preferably 0.5-1.5 mm in size. 3 Size, preferably 1mm 3 size; The phosphate buffer is preferably an HBSS buffer, and more preferably a 2-8℃ pre-cooled HBSS buffer.

7. The method for detecting lung micronuclei as described in any one of claims 1-6, characterized in that, In step S1, the lung tissue fragments are lung lobe tissue fragments.

8. The method for detecting lung micronuclei as described in any one of claims 1-7, characterized in that, In step S1, the neutral formalin solution is a formalin buffer solution with a volume percentage of 4%-10%; Preferably, the formalin buffer solution has a volume percentage of 10%; And / or, the neutral formalin solution is a neutral formalin phosphate buffer solution.

9. The method for detecting lung micronuclei as described in any one of claims 1-8, characterized in that, In step S2, the counting is performed under a fluorescence microscope at 900-1200x magnification, preferably 1000x.

10. The method for detecting lung micronuclei as described in any one of claims 1-9, characterized in that, Step S2 is followed by the following steps: S3. Data Processing and Statistics: After testing the normality of the micronucleus rate, one-way ANOVA combined with Dunnett's t-test was used to compare the differences between the experimental group and the control group. Linear regression analysis was used to analyze the dose-response relationship, with p < 0.05 considered statistically significant. When p < 0.05, the experimental group was considered to have significant cytotoxicity relative to the control group, and when p >= 0.05, the experimental group was considered to have no significant cytotoxicity relative to the control group.

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