Method for monitoring and evaluating human hematopoietic stem cell transplantation imaging by gold cluster fluorescent probe

Abstract

The invention discloses application of a gold cluster fluorescent probe to in-vivo evaluation of transplanted human hematopoietic stem cells. The animal model of hematopoietic stem cell injury caused by radiation injury is constructed to simulate the severe damage state of the hematopoietic system and the immune function in the human body, and then the hematopoietic system and the immune function are recovered by transplanting the human hematopoietic stem cells. And the in-vivo evaluation of the transplanted human hematopoietic stem cells is realized by using an infrared two-region targeting gold cluster probe with high sensitivity and specificity. According to the method, the in-vivo effectiveness of the human hematopoietic stem cells transplanted in vivo is evaluated by detecting the infrared two-region fluorescence intensity value of the targeting gold cluster probe. By accurately evaluating the number of transplanted hematopoietic stem cells, the transplantation effect can be better monitored and evaluated, so that an accurate and personalized treatment scheme is provided for a patient.

Description

Technical Field

[0001] The present invention relates to the fields of biomedical imaging and nanotechnology, and specifically provides a fluorescent probe based on gold clusters for targeted labeling and in vivo imaging of human hematopoietic stem cells transplanted into the body, which has important application value in biomedical research and cell therapy evaluation. Background Art

[0002] In recent years, with the rapid development of cell therapy and regenerative medicine, the transplantation of human hematopoietic stem cells (HSCs) has shown broad application prospects in the treatment of various blood diseases, immunodeficiency states and some malignant tumors. Although traditional cell tracking technologies, such as isotope labeling, fluorescent labeling and biological imaging, can provide information on the distribution and survival status of cells to a certain extent, these methods are generally limited by insufficient imaging depth, high signal noise and low temporal resolution, and are difficult to meet the needs of real-time and accurate monitoring of transplanted cells in deep tissues. Therefore, accurately monitoring and evaluating the dynamic characteristics of human hematopoietic stem cells after transplantation, such as survival status, migration path and functional changes, in vivo has great research value and significance for optimizing treatment plans and improving clinical efficacy.

[0003] In recent years, NIR-II (1000-3000nm) fluorescence imaging technology has attracted widespread attention due to its high tissue penetration and low fluorescence background noise. Using NIR-II imaging technology, deep tissue penetration and high-resolution imaging can be achieved, and problems such as fluorescence quenching and background interference in traditional imaging methods can be effectively reduced. In addition, gold clusters, as a new type of nanomaterial, have good optical properties, stability and biocompatibility, and their surfaces can be functionalized by chemical modification to achieve targeted recognition of specific cells. Therefore, the targeting of infrared-II gold cluster fluorescent probes will provide an effective solution for the in vivo dynamic characteristics of human hematopoietic stem cell transplantation, thereby providing important technical support for in-depth research on the biological characteristics and clinical applications of transplanted cells.

[0004] Those skilled in the art are eager to develop an in vivo imaging technology for human hematopoietic stem cell transplantation based on a gold cluster fluorescent probe to address deficiencies in the prior art and open up new research directions for human hematopoietic stem cell transplantation monitoring and efficacy evaluation. The targeted gold cluster fluorescent probe in the present invention can specifically bind to hematopoietic stem cells in vivo and emit fluorescence, and can specifically detect the location and number of hematopoietic stem cells in combination with an infrared two-zone imaging system. It can be used for continuous in vivo observation of transplanted human hematopoietic stem cells and dynamic monitoring of the survival, migration, and distribution of transplanted cells. Summary of the invention

[0005] In order to solve the problems of shallow imaging depth, high signal noise and low temporal resolution in the prior art, the present invention aims to provide an in vivo monitoring and evaluation method for human hematopoietic stem cell transplantation to support preclinical research and potential clinical applications.

[0006] To achieve the above object, the present invention provides a gold cluster fluorescent probe for in vivo evaluation of transplanted human hematopoietic stem cells, wherein the probe has targeting properties.

[0007] As a limitation of the above method, the gold clusters have specific ligands or modification groups to improve the affinity and stability of specific binding with human hematopoietic stem cells.

[0008] As a further limitation of the above method, the probe emits near-infrared second-zone fluorescence to improve the tissue penetration depth and signal-to-noise ratio of biological imaging, and is suitable for real-time monitoring in vivo.

[0009] As a further limitation of the above embodiment, the low-temperature storage condition of the gold cluster fluorescent probe is 0-4°C.

[0010] As a further limitation of the above method, the above-mentioned required antibodies are one or more of CD34, CD133 (Prominin-1), CD90 (Thy-1), CD38, CD45RA, CD49f (Integrin α6), CD117 (c-Kit) and CXCR4.

[0011] Through the above technical scheme, the targeted gold cluster fluorescent probe provided by the present invention can be used for in vivo transplantation of human hematopoietic stem cells detection, by irradiating myeloablative mice to simulate patient hematopoietic stem cell damage and transplantation treatment, so as to dynamically monitor the survival, migration and distribution of transplanted cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments:

[0013] Figure 1 This is a study on the targeting of gold cluster fluorescent probes to hematopoietic stem cells at different time points in normal mice.

[0014] Figure 2 This study is a study of the targeting of gold cluster fluorescent probes to hematopoietic stem cells at different time points in mice after myeloablation caused by radiation damage.

[0015] Figure 3 After myeloablative mice were transplanted with human hematopoietic stem cells, the targeting of hematopoietic stem cells in the left leg of mice was studied at different time points.

[0016] Figure 4After myeloablative mice underwent human hematopoietic stem cell transplantation, the targeting of hematopoietic stem cells in the right leg of mice was studied at different time points.

[0017] Figure 5 Quantitative analysis of infrared zone II fluorescence signals in the left and right legs of mice at different time points after human hematopoietic stem cell transplantation. DETAILED DESCRIPTION

[0018] Those skilled in the art may refer to the contents described herein and adjust the process parameters appropriately to achieve corresponding improvements. It is particularly emphasized that all similar substitutions and modifications are obvious to those skilled in the art and should be deemed to be included within the scope of the present invention. The method and application of the present invention have been elaborated in detail through preferred embodiments, and relevant personnel are obviously able to make necessary modifications, appropriate adjustments or combinations to the method and application without departing from the core content and scope of the present invention to implement and apply the technology of the present invention.

[0019] In order to more clearly and explicitly explain the purpose, technical solution and advantages of the present invention, the present invention will be explained in depth below in combination with specific implementation cases and drawings of the specification. The experimental methods used in the implementation cases, unless otherwise specified, all follow conventional methods; the materials, reagents, methods and instruments used, unless otherwise specified, all belong to the conventional scope in the field, and can be obtained through commercial channels in the field.

[0020] The sources of raw materials and instrument models used in the examples are described below.

[0021] EDC and NHS coupling activators were purchased from Sigma. Thy1 / CD90 antibody, CD34 antibody and other antibodies were purchased from Abcam. Ultrafiltration tubes were purchased from Millipore. The magnetic stirrer used was the C-MAG HS4 digital model produced by IKA, Germany. The electronic analytical balance used was the BSA224S model produced by Sartorius, Germany. The desktop high-speed centrifuge was the TG16-WS model produced by Hunan Xiangyi Co., Ltd. Figure 1 , Figure 2 , Figure 3 and Figure 4The near-infrared zone II optical imaging optical path system used is achieved by an imaging system independently developed and constructed. The core equipment of the system is a near-infrared zone II camera model NIRvana:640ST, manufactured by Princeton Instruments, USA. At the same time, the system is also equipped with a laser model FLC300-0808-8W-F200, which is produced by Frankfurt Laser Company in Germany. With these high-precision equipment and carefully planned optical path design, researchers were able to successfully carry out high-quality near-infrared zone II optical imaging experiments. C57 male mice (6-8 weeks old) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. The hematopoietic stem cells used were derived from human umbilical cord blood and purchased from allcells.

[0022] The reagents and process equipment used in the present invention are commonly used in the prior art and are not critical to the present invention, so they are briefly described.

[0023] Embodiment 1

[0024] Preparation of gold cluster fluorescent probe in the near-infrared region II:

[0025] First, 12 mg EDC and 12 mg NHS were added to 2 mL of a 2 mg / mL gold cluster solution. Then, the mixture was stirred at a constant speed of 500 rpm at room temperature for 20 min. Subsequently, in order to effectively remove excess chemical reagents, two ultrafiltration operations were performed. Each ultrafiltration used a 10K ultrafiltration tube and centrifuged at 3000 rpm for 15 min. After the ultrafiltration step, 40 μL of the thy1 / CD34 antibody was added to the above solution, which can specifically bind to stem cell surface markers. After adding the antibody, the mixture was placed at 4 ° C and stirred at 500 rpm for 2 h to ensure that the antibody was fully bound to the gold clusters. After that, in order to remove the unbound antibody, it was necessary to perform two ultrafiltration operations again using a 10K ultrafiltration tube. Through the above steps, a near-infrared second-zone fluorescent probe with human stem cell targeting was successfully prepared.

[0026] Embodiment 2

[0027] Establishment of radiation injury mouse model:

[0028] Using a standard gamma-ray radiation source, 6- to 8-week-old C57BL / 6 male mice were irradiated with an irradiation dose of 8Gy, aiming to induce a significant decrease in the number of hematopoietic stem cells in mice. The irradiation operation was strictly carried out in a standard shielded irradiation device to ensure that the surrounding environment had adequate radiation protection measures. The irradiation dose was set at 8Gy, and the irradiation rate was controlled in the range of 0.7 to 1.0Gy / min, aiming to reduce the potential effects of irradiation on the physiological functions of mice. After the irradiation, the mice were quickly and safely transferred back to the cage, and their vital signs and behavioral changes were continuously and carefully monitored. 24 hours after irradiation, the mice were subjected to infrared zone II in vivo imaging experiments.

[0029] Embodiment 3

[0030] Mouse in vivo imaging assay:

[0031] The gold cluster fluorescent probe was dissolved in deionized water, and a C57 male mouse with hair treatment was anesthetized with 5% chloral hydrate, and the mouse was fixed on the stand of the infrared zone II camera. 200 μL (10 mg / mL) of the fluorescent probe was injected into the mouse through the tail vein. The self-built infrared zone II camera was used to excite at 808 nm with a power of 25 mW / cm 2 With an exposure time of 500ms, mice were imaged in vivo at different time points to observe the fluorescence intensity of their legs. Figure 1 As shown in the figure, after the fluorescent probe was injected into the tail vein, significant fluorescent signals were observed in the mouse legs, and these signals were located in the bone marrow area of ​​the legs. According to the time points specified in the experimental plan, we performed fluorescent imaging on the femur and tibia of the mouse legs. Even after 12 hours, the legs still maintained strong fluorescent signals. Figure 2 As shown in the figure, the fluorescence intensity in the femur and tibia area of ​​mice irradiated with a standard gamma-ray radiation source dropped to the lowest level. This result reveals that high-dose radiation almost completely destroyed the stem cells in the mice, making it difficult for the fluorescent imaging agent to effectively bind to the very few remaining stem cells, which led to the near disappearance of the fluorescent signal. This phenomenon clearly shows that the irradiation treatment caused significant damage to the stem cells in the mice, causing the number and activity of stem cells to decrease. Figure 3-4 As shown in the figure, mice received human hematopoietic stem cell transplantation after being subjected to radiation damage. In order to evaluate the effect of this hematopoietic stem cell transplantation, we used a gold cluster fluorescent probe for continuous monitoring. Through fluorescence imaging technology, it can be clearly observed that the transplanted mice showed significant fluorescence signals in their leg areas. This phenomenon shows that the transplantation of human hematopoietic stem cells effectively restored the number of hematopoietic stem cells in the mice. For mice that have lost their immune and hematopoietic functions due to radiation damage, the transplantation of hematopoietic stem cells has played a significant therapeutic role. Figure 5 As shown in the figure, we processed the fluorescence signal of the leg and selected the femur and tibia as the target area for quantitative analysis of fluorescence intensity. By calculating the average fluorescence intensity of these areas, we can reflect their fluorescence expression level.

[0032] The present invention obtains the change in the number of hematopoietic stem cells in irradiated mice based on the fluorescence recovery of the mouse bone marrow after transplantation of human hematopoietic stem cells, and establishes an in vivo tracing and evaluation scheme for the reinfusion of human hematopoietic stem cells into irradiated mice. The results show that the gold cluster fluorescent probe has excellent performance in hematopoietic stem cell imaging and tracking, and can accurately mark and locate transplanted human hematopoietic stem cells in mice, so that they produce stable fluorescent signals. The fluorescent probe has good hematopoietic stem cell targeting, good stability and safety, and has good application prospects and clinical transformation potential in hematopoietic stem cell detection and hematopoietic stem cell transplantation surgery monitoring.

[0033] Parts not elaborated in the specification of the present invention belong to the technical scope generally mastered by those skilled in the art. Although the present invention has been disclosed through the above preferred embodiments, this is not intended to limit the scope of the present invention. Any person proficient in the technology in this field can make various adjustments and improvements to the present invention without violating the core idea and protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the content defined in the claims.

Claims

1. A targeted gold cluster fluorescent probe for in vivo imaging monitoring of transplanted human hematopoietic stem cells and its evaluation method.

2. The use according to claim 1, characterized in that: The fluorescent probe has infrared second-zone fluorescence characteristics and hematopoietic stem cell targeting properties.

3. The use according to claim 1, characterized in that: The gold cluster has a specific ligand or modification group and has affinity and stability for specifically binding to human hematopoietic stem cells.

4. According to claim 1, it is characterized in that: The surface antibody of the targeted gold cluster fluorescent probe is one or more of CD34, CD133 (Prominin-1), CD90 (Thy-1), CD38, CD45RA, CD49f (Integrinα6), CD117 (c-Kit) and CXCR4.

5. The targeted gold cluster fluorescent probe according to claim 1, characterized in that: It can specifically bind to hematopoietic stem cells in the body and emit fluorescence. Combined with the infrared two-zone imaging system, it can specifically detect the location and number of hematopoietic stem cells.

6. Use of the gold cluster fluorescent probe according to any one of claims 1 to 5 in in vivo transplanted cell detection, characterized in that: The probe is used for in vivo evaluation of transplanted human hematopoietic stem cells and for dynamically monitoring the survival, migration and distribution of transplanted cells.

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