Application of intestinal flora marker in predicting intestinal injury caused by FLASH irradiation

Abstract

The invention discloses application of an intestinal flora marker in predicting intestinal injury caused by FLASH irradiation, and belongs to the field of diagnosis of intestinal injury caused by radiation. The invention provides application of an intestinal flora marker abundance detection reagent in preparation of a kit for diagnosing intestinal injury caused by FLASH irradiation. An intestinal flora marker comprises one or three of combined lactobacillus, liquid lactobacillus and duber's bacteria. The invention finds that the three intestinal bacteria are related to the intestinal injury caused by the ultrahigh dose rate irradiation for the first time, and when the three bacteria are combined as a detection marker to predict the intestinal injury caused by the ultrahigh dose rate irradiation, the sensitivity and the accuracy are higher, and the prediction mode is noninvasive. Therefore, a new approach is provided for diagnosis of intestinal injury caused by ultra-high dose rate irradiation, and the method has perspectiveness and high research value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of diagnosis of radiation-induced intestinal injury, and particularly relates to the application of intestinal flora markers in predicting intestinal injury caused by FLASH irradiation. BACKGROUND

[0002] At present, more than 50% of patients with pelvic and abdominal cancer will receive radiotherapy. Because the intestine is located in the abdominal and pelvic cavity, more than 50% of patients with pelvic and abdominal tumors have different degrees of radiation-induced intestinal injury after receiving local radiotherapy. Acute radiation-induced intestinal injury often occurs within 3 months after radiotherapy, and patients may have symptoms such as diarrhea, abdominal pain, and hematochezia; chronic radiation-induced intestinal injury usually occurs 3 months to several years after radiotherapy, and is manifested as intermittent diarrhea, hematochezia, intestinal stenosis and obstruction, etc. Radiation-induced intestinal injury seriously affects the quality of life of patients and the effect of radiotherapy, and also limits the use of radiotherapy. How to prevent or reduce radiation-induced intestinal injury and improve the quality of life of patients and the effect of radiotherapy is a difficult problem that clinicians have been trying to overcome.

[0003] Ultra-high dose rate irradiation (≥40 Gy / s), referred to as FLASH irradiation, has a "FLASH effect", that is, compared with conventional radiation, it can effectively reduce the radiation toxicity of normal tissues and achieve the same tumor control effect as conventional radiation. More and more preclinical studies have shown that FLASH irradiation has potential benefits. Montay et al. used X-ray 10 Gy whole brain irradiation in mice and found that the cognitive memory ability of mice in the FLASH irradiation group was preserved, while the hippocampal cell division of mice in the conventional irradiation group was greatly reduced, and reactive astrocyte proliferation occurred, causing irreversible memory changes. Zhang et al. found that FLASH proton irradiation could reduce skin contraction, epidermal thickness and collagen deposition in mice. Ruan et al. found that compared with the conventional irradiation group, the number of residual intestinal crypts in the FLASH irradiation group was more, and the composition of intestinal flora changed less, and FLASH irradiation could reduce acute normal tissue toxicity in the intestine. At present, FLASH therapy has also been applied to clinical studies of cancer patients, and the results show that most patients can obtain pain relief and have no obvious adverse reactions after using FLASH proton palliative treatment for patients with bone metastases in the extremities.

[0004] Intestinal flora is the second genome of the human body, as it carries about 100 times the genes of the human genome. Intestinal flora has a variety of physiological functions and plays a key role in host health and intestinal homeostasis. The interaction between intestinal flora and cancer radiotherapy is a two-way function, and cancer radiotherapy can destroy intestinal flora, leading to intestinal flora imbalance, which is usually manifested as a decrease in the abundance and diversity of intestinal flora, an increase in harmful bacteria and a decrease in beneficial bacteria, and these disruptions can affect the effectiveness of anticancer radiotherapy and the severity of treatment-induced gastrointestinal toxicity.

[0005] Compared with conventional radiation, ultra-high dose rate radiation can reduce the radiation toxicity of normal tissues and alleviate radiation damage, and intestinal flora plays an important role in intestinal radiation damage, so it is necessary to further study the influence of ultra-high dose rate radiation on intestinal flora, contribute to the early application of ultra-high dose rate radiation in clinic, and provide beneficial exploration for the final clinical treatment and prevention of radiation-induced intestinal injury. SUMMARY

[0006] In view of the deficiencies of the prior art, the purpose of the present application is to provide the application of intestinal flora markers in predicting intestinal injury caused by FLASH irradiation, which solves the problems in the prior art.

[0007] The purpose of the present application can be achieved by the following technical solutions:

[0008] The application of the intestinal flora marker abundance detection reagent in the preparation of a FLASH irradiation-induced intestinal injury diagnosis kit, the intestinal flora marker comprising: one or three combinations of Lactobacillus confervae, Lactobacillus liquid, Dubosiella.

[0009] Further, the detection reagent comprises: specific primers for detecting the abundance of the intestinal flora marker in feces.

[0010] Further, the specific primers are primers for amplifying the 16S rRNA of the intestinal flora marker.

[0011] Further, the primers for amplifying the 16S rRNA of the intestinal flora marker comprise: forward primers and reverse primers; the nucleotide sequences of the forward primers and the reverse primers are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

[0012] Further, the dose rate of the FLASH irradiation is greater than or equal to 40 Gy / s.

[0013] A kit comprising an intestinal flora marker abundance detection reagent, the intestinal flora marker comprising: one or three combinations of Lactobacillus confervae, Lactobacillus liquid, Dubosiella.

[0014] The beneficial effects of the present application are:

[0015] (1) The present application first discovers that Lactobacillus confervae, Lactobacillus liquid and Dubosiella are related to intestinal injury caused by ultra-high dose rate irradiation, and these three genera are significantly reduced in mice receiving ultra-high dose rate irradiation.

[0016] (2) The experimental results of the present application show that any one of Lactobacillus conjunctus, Lactobacillus liquidus and Dubosiella as a diagnostic marker for intestinal injury caused by ultra-high dose rate irradiation has high specificity and sensitivity, and when the three bacteria are combined for detection, the sensitivity and specificity reach 1.0, so the bacteria can be used as a detection marker for intestinal injury caused by ultra-high dose rate irradiation, and the detection method is safe, non-invasive and high in accuracy.

[0017] (3) The present application provides a detection means for diagnosing intestinal injury caused by ultra-high dose rate irradiation, and provides a bacterial source basis for intestinal injury and intestinal flora disorder caused by ultra-high dose rate irradiation, and provides a basis for intestinal bacteria transplantation in the later stage. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments or the prior art description will be briefly introduced as follows, and obviously, other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0019] Figure 1 HE diagram of intestinal injury changes of mice in different dose ultra-high dose rate irradiation groups (F10, F15, F20, F25) and control group mice (F0);

[0020] Figure 2 Statistical diagram of intestinal injury scores of mice in different dose ultra-high dose rate irradiation groups (F10, F15, F20, F25) and control group mice (F0);

[0021] Figure 3 Relative abundance result diagram of Lactobacillus conjunctus of mice in different dose ultra-high dose rate irradiation groups (F10, F15, F20, F25) and control group mice (F0);

[0022] Figure 4 Relative abundance result diagram of Lactobacillus liquidus of mice in different dose ultra-high dose rate irradiation groups (F10, F15, F20, F25) and control group mice (F0);

[0023] Figure 5 Relative abundance result diagram of Dubosiella of mice in different dose ultra-high dose rate irradiation groups (F10, F15, F20, F25) and control group mice (F0);

[0024] Figure 6 ROC curve diagram of Lactobacillus conjunctus, Lactobacillus liquidus and Dubosiella;

[0025] Figure 7 ROC curve diagram of three bacteria combined. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0027] Embodiment 1

[0028] In this embodiment, the collection of related samples is introduced.

[0029] 1. Construction of mouse intestinal injury model induced by ultra-high dose rate irradiation and grouping

[0030] 25 healthy male C57BL / 6J mice, SPF level, 6-8 weeks old, weighing 18-22 g, purchased from Chengdu Dashuo Experimental Animal Co., Ltd. (Production License No.: SCXK (Chuan) 2020-030), and raised in the barrier environment of the specific pathogen-free animal room of Suzhou University, with a temperature of 18-25℃ and a relative humidity of 40%-70%. After the mice were raised for one week to adapt to the environment, they were randomly divided into a control group (5, F0 group) and a FLASH whole abdominal irradiation group (25). The FLASH whole abdominal irradiation group was divided into four groups according to different irradiation doses, namely F10 group (irradiation dose of 10 Gy, n=5), F15 group (irradiation dose of 15 Gy, n=5), F20 group (irradiation dose of 20 Gy, n=5), and F25 group (irradiation dose of 25 Gy, n=5).

[0031] 2. Instruments and irradiation

[0032] The mice were anesthetized by intraperitoneal injection of isoflurane. The mice were fixed on a plastic plate with adhesive tape to prevent them from moving during radiation exposure. The whole abdominal FLASH irradiation was performed using the PARTER research platform of the Chengdu Terahertz Free Electron Laser Facility, with a dose rate of 100 Gy / s for single-dose 10 Gy, 15 Gy, 20 Gy, and 25 Gy irradiation of the mouse abdomen, a source-skin distance of 19 cm, and a field of 3 cm x 3 cm. The control group of mice received 0 Gy sham irradiation under the same conditions.

[0033] 3. Fecal collection

[0034] The mouse feces were collected 3.5 days after irradiation. Since one mouse in each of the F20 and F25 groups had substandard fecal quality, they were removed, so the total number of mouse feces collected was 23, which were: F0 group, 5; F10 group, 5; F15 group, 5; F20 group, 4; and F25 group, 4.

[0035] 4. Small intestinal tissue HE staining

[0036] The mice were euthanized 3.5 days after irradiation, and the small intestinal tissues were taken; after fixation, paraffin embedding, sectioning, hematoxylin-eosin staining (HE), the intestinal tissue damage score was performed, referring to the Chiu' scoring system

[0037] Example 2

[0038] In this example, the extraction of fecal DNA and 16S rRNA sequencing are introduced

[0039] The mouse fecal genomic DNA was extracted using a magnetic bead method soil DNA extraction kit (Omega, USA), and the DNA was quantified using Nanodrop and detected by 1.2% agarose gel electrophoresis to detect the quality of DNA extraction. According to the conserved region (V3 and V4) of microbial ribosomal RNA sequence, primers were designed, including F primer and R primer, the sequences are shown in SEQ ID NO. 1 and SEQ ID NO. 2 (F primer: 5, ACTCCTACGGGAGGCAGCA 3, R primer: 5, GGACTACHVGGGTWTCTAAT 3), and then the variable region gene fragment of rRNA gene was amplified by PCR. The amplification product was purified and quantified using Quant-iTPicoGreen dsDNA Assay Kit and Microplate reader (BioTek, FLx800). According to the fluorescence quantitative results and the sequencing abundance requirements, the samples were mixed in proportion. The sequencing library was constructed using TruSeq Nano DNA LT Library prepkit of Illumina Company, and paired-end DNA sequencing was performed using Illumina MiSeq / NovaSeq platform. The 16S rRNA gene of Greengenes database (Release 13.8, http: / / greengenes.secondgenome.com / ) was annotated using the classifysklearn algorithm of QIIME2 analysis software, and considering that the microbial sequences in the current database are not completely covered, some specific species cannot be identified (unclassified).

[0040] Example 3

[0041] In this example, biomarkers are screened, and diagnosis and prediction are made for intestinal damage caused by FLASH irradiation.

[0042] The intestinal tissues of FLASH irradiation group and control group mice were subjected to HE staining and intestinal damage scoring, and the results are shown in Figure 1Compared with the control group, the intestinal tissues of the FLASH irradiation group mice showed varying degrees of intestinal injury, and the degree of intestinal injury increased with the increase of the irradiation dose. The F10 group mainly showed expansion of the intestinal epithelial sub-interstitial space and separation of the mucosal layer and the lamina propria; the F15 group mainly showed large areas of intestinal mucosal epithelium lifting and partial villus tip shedding; and the F20 and F25 groups mainly showed intestinal villus shedding, lamina propria degeneration, and hemorrhage and ulcer formation. The intestinal injury score histogram is shown in Figure 2 .

[0043] The different dose ultra-high dose rate irradiation groups and the control group were compared and analyzed respectively, and the intestinal flora with differences (absolute value of logFC > 1.5, P value < 0.05) was screened out. Then the screened intestinal flora was intersected, and the intestinal flora existing in each comparison between the ultra-high dose rate irradiation group and the control group was screened out, that is, the intestinal differential flora common to the ultra-high dose rate irradiation group was screened out;

[0044] Through relative abundance comparison analysis, three kinds of intestinal microorganisms with significantly reduced relative abundance in the ultra-high dose rate irradiation group were screened out, which were Lactobacillus conjunctus, Lactobacillus liquid and Dubosiella. The relative abundance results of the three markers are shown in Figures 3 to 5 , and the three bacteria are intestinal injury related bacteria caused by ultra-high dose rate irradiation, which are first discovered.

[0045] The specificity and sensitivity calculation and ROC curve drawing were completed by using IBM SPSS Statistics (v27) statistical software. The ROC curve was constructed by 1-specificity and sensitivity, and the area under the ROC curve was AUC. In order to calculate the specificity and sensitivity of a certain index, the Youden coefficient (Youden coefficient = sensitivity + specificity - 1) was calculated first, and the specificity and sensitivity corresponding to the maximum Youden coefficient were the specificity and sensitivity of a certain index.

[0046] The relative abundance value of a single microbial marker was directly subjected to receiver operating characteristic test (ROC) analysis, and the results are shown in Figure 6 . It can be seen that when Lactobacillus conjunctus is used alone for detection, the AUC value is 0.989, the sensitivity is 1.0, and the specificity is 0.944; when Lactobacillus liquid is used alone for detection, the AUC value is 0.889, the sensitivity is 0.8, and the specificity is 1.0; when Dubosiella is used alone for detection, the AUC value is 0.8, the sensitivity is 0.8, and the specificity is 1.0. The ROC analysis of the combination of the three bacteria is shown in Figure 7 . It can be seen that the AUC value is 1.0, and the sensitivity and specificity points are also 1.0.

[0047] The AUC, P value, sensitivity and specificity of single bacteria and three bacteria combined to predict intestinal injury caused by ultra-high dose rate irradiation are shown in Table 1:

[0048] Table 1 ROC diagnostic curve results

[0049]

[0050]

[0051] From the above results, it can be seen that the prediction (diagnosis) ability of Lactobacillus conjunctus, Lactobacillus liquidus and Dubosiella for intestinal injury caused by ultra-high dose rate irradiation is very high, and the prediction effect is the best when the three bacteria are combined, the AUC of the prediction result of the three bacteria combination is 1.0, and the sensitivity and specificity are both 1.0.

[0052] In summary, the present application first found that Lactobacillus conjunctus, Lactobacillus liquidus and Dubosiella are related to intestinal injury caused by ultra-high dose rate irradiation, and the abundance is significantly lower than that of the healthy control group, which can be used as a biological marker for detecting intestinal injury caused by ultra-high dose rate irradiation, and by determining whether the relative abundance of one or more of these markers is reduced in the intestinal flora of the subject, the occurrence of intestinal injury caused by ultra-high dose rate irradiation in the subject can be effectively monitored. When the three genera of bacteria are used as predictors of intestinal injury caused by ultra-high dose rate irradiation, the accuracy is high and the prediction method is non-invasive, and the present application provides a new way for the diagnosis and treatment of intestinal injury caused by ultra-high dose rate irradiation.

[0053] In the description of the present specification, the description of the terms "one embodiment", "example", "specific example" and the like means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0054] The basic principles, main features and advantages of the present application are shown and described above. It should be understood by those skilled in the art that the present application is not limited by the above embodiments, and the above embodiments and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application.

Claims

1. Use of a test agent for the determination of the abundance of a gut microbiota marker in the manufacture of a diagnostic kit for intestinal damage caused by FLASH irradiation, characterized in that, The intestinal flora marker comprises one or three combinations of Lactobacillus conjunctus, Lactobacillus liquidus and Dubosiella.

2. Use according to claim 1, characterized in that, The detection reagent comprises specific primers for detecting the abundance of the intestinal flora marker in feces.

3. Use according to claim 2, characterized in that, The specific primers are primers for amplifying 16S rRNA of the intestinal flora marker.

4. Use according to claim 3, characterized in that, The primers for amplifying 16S rRNA of the intestinal flora marker comprise a forward primer and a reverse primer, and the nucleotide sequences of the forward primer and the reverse primer are shown in SEQ ID NO. 1 and SEQ ID NO. 2 respectively.

5. The use according to claim 1, characterized in that, The dose rate of the FLASH irradiation is greater than or equal to 40 Gy / s.

6. A kit characterized in that, The detection reagent comprises specific primers for detecting the abundance of the intestinal flora marker in feces.

7. The kit of claim 6, wherein The specific primers are primers for amplifying 16S rRNA of the intestinal flora marker.

8. The kit of claim 7, wherein The primers for amplifying 16S rRNA of the intestinal flora marker comprise a forward primer and a reverse primer, and the nucleotide sequences of the forward primer and the reverse primer are shown in SEQ ID NO. 1 and SEQ ID NO. 2 respectively.

9. The kit of claim 8, wherein The dose rate of the FLASH irradiation is greater than or equal to 40 Gy / s.

10. The kit of claim 6, wherein ​

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