Method for detecting distribution of human stem cell-derived nanovesicles in tissue and use

Abstract

Provided are a method for detecting distribution of human stem cell-derived nanovesicles (SC-NVs) in tissue and a use, relating to the technical field of biological detection. The method comprises: extracting nucleic acids from tissue to be detected, carrying out reverse transcription on the obtained nucleic acids to obtain cDNA, detecting a specific sequence sRNA of the human SC-NVs in the cDNA by means of a PCR method, detecting the distribution of nanovesicles (NVs) on the basis of comparison of a copy number of the sRNA and an exogenous standard curve, and finally realizing the in vivo quantitative distribution research of NVs. The method is an efficient, sensitive and specific method for detecting the distribution of human SC-NVs in tissue, biological distribution of SC-NVs per milligram of tissue can be quantitatively measured, and the method has the potential for drug loading research of the human SC-NVs.

Description

A method for detecting the distribution of human stem cell nanovesicles in tissues and its application Technical Field

[0001] The present invention belongs to the field of biological detection technology, and specifically relates to a method and application for non-labeling, high-sensitivity quantitative detection of the distribution of human stem cell nanovesicles in tissues. Background Art

[0002] Stem cell-derived nanovesicles (SC-NVs) are a class of nanoscale vesicles derived from stem cells, either autocrine or engineered, with diameters ranging from 50 to 200 nm. They contain biomolecules such as proteins, lipids, and small RNAs (sRNAs). These contents share many of the biological characteristics of the parental cells from which they originate, exhibiting diverse biological functions such as anti-apoptosis, immunomodulation, aging reversal, and angiogenesis promotion, and hold broad application prospects in regenerative medicine. SC-NVs can be combined with other chemical drugs to achieve synergistic effects. For example, combining traditional Chinese medicine (TCM) monomers with nanovesicles can improve their solubility and enable sustained release, long-term circulation, and targeted delivery. The pharmacological properties of SC-NVs have been extensively studied, and they hold promising translational potential. The in vivo stability, cellular uptake, intracellular fate, and drug circulation of SC-NVs are crucial for pharmacokinetic studies and clinical translation. Several tracing methods have been developed and applied to the in vivo behavior and mechanism exploration of nanovesicles (NVs). Most tracing methods use fluorescent dyes, fluorescent proteins, radioactive elements, etc. to directly label NVs or indirectly label parent cells, and use fluorescence imaging, nuclear magnetic resonance, tomography, and other detection methods to visually track the labeled NVs. However, membrane markers easily form nonspecific images in vivo, the half-life of radioisotope labels is inconsistent with that of NVs, affecting the detection results, and all exogenous markers will affect the biodistribution of NVs. Therefore, the current SC-NVs tracing method cannot meet the pharmacokinetic approval requirements for SC-NVs clinical drugs, which limits the clinical translation of SC-NVs. There is an urgent need to develop a reliable, non-labeled, and highly sensitive method for quantitatively detecting the distribution of stem cell nanovesicles in tissues for SC-NVs tracing.

[0003] Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method and application for non-labeling, highly sensitive quantitative detection of the distribution of human stem cell nanovesicles in tissues. The present invention uses the identified species-specific stem cell nanovesicle-related sRNA to exclude the background of similar species tissues, develop species-specific primers, and optimize the PCR system, thereby obtaining an efficient, sensitive and specific determination method that can quantitatively measure the biological distribution of SC-NVs per milligram of tissue.

[0005] The object of the present invention is to achieve the following goals:

[0006] The present invention provides a method for detecting the distribution of human stem cell nanovesicles in tissues, which mainly comprises the following steps:

[0007] (1) Extracting nucleic acid from the tissue to be tested;

[0008] (2) reverse transcribing the nucleic acid obtained in step (1) to obtain cDNA;

[0009] (3) Detecting the specific sequence sRNA of human stem cell nanovesicles in the cDNA obtained in step (2) by PCR, and detecting the distribution of nanovesicles based on the comparison of the copy number of sRNA with the exogenous standard curve.

[0010] Based on the above technical solution, further, the human stem cells include umbilical cord mesenchymal stem cells, neural stem cells and other human stem cells containing specific sRNA sequences of human stem cell nanovesicles.

[0011] Based on the above technical solution, further, the method for obtaining the nanovesicles includes mechanical force extrusion method and gradient centrifugation method.

[0012] Based on the above technical solution, further, the tissues in step (1) include liver, heart, spleen, lung, kidney, and brain tissues.

[0013] Based on the above technical solution, further, the source of the tissue in step (1) includes rats and mice.

[0014] Based on the above technical solution, further, the process of extracting nucleic acid in step (1) uses a QIAGEN kit.

[0015] Based on the above technical solution, further, a QIAGEN kit is used in the reverse transcription process in step (2).

[0016] Based on the above technical solution, further, the reverse transcription program in step (2) is: 40°C for 15 min, 95°C for 5 min.

[0017] Based on the above technical solution, further, the PCR method described in step (3) includes qPCR and ddPCR

[0018] Based on the above technical solution, further, the nucleotide sequence of the sRNA described in step (3) is shown as SEQ ID NO: 1.

[0019] Based on the above technical solution, further, the nucleotide sequence of the stem-loop RT primer in the qPCR in step (3) is shown as SEQ ID NO: 2; the nucleotide sequence of the forward primer is shown as SEQ ID NO: 3; the nucleotide sequence of the reverse primer is shown as SEQ ID NO: 4; and the probe is: FAM-CAGTTCTGATAACCC-MGB.

[0020] The present invention also provides application of the above method in the study of pharmacokinetics of drug-loaded human stem cell nanovesicles.

[0021] The present invention has the following beneficial effects compared to the prior art:

[0022] 1. Current tracing of nanovesicles is based on exogenous markers, and these detection methods have limited sensitivity and accuracy. The present invention has developed and implemented a new ddPCR-based molecular platform to detect the biodistribution of stem cell nanovesicles in vivo, which can achieve vesicle biodistribution detection without exogenous labeling of nanovesicles and maternal cells.

[0023] 2. This method detects species-specific stem cell nanovesicle-associated sRNA to exclude background from tissues of similar species, develops species-specific primers, and optimizes ddPCR to obtain an efficient, sensitive, and specific assay that can quantitatively measure the biodistribution of SC-NVs per milligram of tissue.

[0024] 3. In the presence of hydrophilic and hydrophobic Chinese medicine monomers, this method always operates stably, suggesting that this method has the potential to be used in the study of drug delivery of human stem cell nanovesicles. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0026] Figure 1 shows the validation of umbilical cord mesenchymal stem cells, including: (A) osteogenic differentiation of umbilical cord mesenchymal stem cells; (B) adipogenic differentiation of umbilical cord mesenchymal stem cells; (C) chondrogenic differentiation of umbilical cord mesenchymal stem cells; (D) identification of umbilical cord mesenchymal stem cells using the surface marker CD34; (E) identification of umbilical cord mesenchymal stem cells using the surface marker CD45; (F) identification of umbilical cord mesenchymal stem cells using the surface marker HLA / DR; (G) identification of umbilical cord mesenchymal stem cells using the surface marker CD73; (H) identification of umbilical cord mesenchymal stem cells using the surface marker CD105; and (I) identification of umbilical cord mesenchymal stem cells using the surface marker CD90.

[0027] Figure 2 shows the characterization data of the prepared umbilical cord mesenchymal stem cell nanovesicles, including: (A) NTA analysis of umbilical cord mesenchymal stem cell nanovesicles; (B) electron microscopy image of umbilical cord mesenchymal stem cell nanovesicles; and (C) surface markers of umbilical cord mesenchymal stem cell nanovesicles.

[0028] Figure 3 shows the validation of neural stem cells, including (A) neural stem cell morphology; (B) identification of neural stem cell surface marker Sox2; (C) identification of neural stem cell surface marker Nestin; (D) tri-lineage differentiation of neural stem cells;

[0029] Figure 4 shows the characterization data of the prepared neural stem cell nanovesicles, including: (A) NTA analysis of neural stem cell nanovesicles; (B) electron microscopy image of neural stem cell nanovesicles; and (C) surface markers of neural stem cell nanovesicles.

[0030] Figure 5 shows the key data for the construction and application of the tracing method, including: (A) neural stem cell nanovesicle sequencing results; (B) the designed primer probe can effectively distinguish human and nude mouse sRNA through qPCR; (C) species-specific sequence secondary structure; (D-F) optimization of the primer probe reaction conditions in ddPCR; (G) schematic diagram of the tracing method; (H) umbilical cord mesenchymal stem cell nanovesicle tracing results using the tracing method.

[0031] FIG6 shows the in vitro tracing of neural stem cell nanovesicles using the tracing method: (A) schematic diagram of the tracing method; (B) tracing results.

[0032] FIG7 shows the application of the tracing method in the context of the hydrophobic Chinese medicine monomer curcumin: (A) schematic diagram of the tracing method; (B) tracing results.

[0033] FIG8 shows the application of the tracing method in the context of the hydrophilic Chinese medicine monomer paclitaxel: (A) schematic diagram of the tracing method; (B) tracing results. DETAILED DESCRIPTION

[0034] The present invention is described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments obtained without creative work all fall within the scope of protection of the present invention.

[0035] Example 1:

[0036] The preparation process of human umbilical cord mesenchymal stem cell-derived nanovesicles (UMSC-NVs) is as follows:

[0037] 1. Cell culture:

[0038] The human UMSCs used in this experiment were purchased commercially. During the cell culture process, the cells were passaged when the degree of confluence reached 80%-90%. First, discard the old cell culture medium in the culture flask and rinse the cells once with PBS buffer. Add trypsin to digest the cells until a large area falls off, and then add complete culture medium at a ratio of 1:2 to neutralize. Blow the cells apart and transfer them to a centrifuge tube, centrifuge at 1000rpm for 5 minutes. Discard the supernatant and resuspend the cells in fresh culture medium. Inoculate them into culture flasks according to the appropriate ratio. Label the cell type, cell generation, passage date, etc. on the flask. Finally, culture them in a 37°C, 5% CO2 incubator.

[0039] The validation results of human UMSC cells are shown in Figure 1. The cells adhered to the surface of the cell wall, expressed >95% of the CD73, CD90, and CD105 surface antigens, and lacked the hematopoietic markers HLA / DR, CD34, and CD45. MSCs possessed the ability to differentiate into bone, adipose, and cartilage tissue cells. Experimental studies have demonstrated that the UMSCs used in this study met the certification standards of the International Society for Cellular Therapy.

[0040] 2. Isolation and purification of nanovesicles:

[0041] UMSC cells were cultured to the logarithmic growth phase, digested, centrifuged at 300 g for 5 min, and the supernatant was discarded. 6Cells / mL were dispersed in PBS solution to prepare a single-cell suspension. Using a small extruder, the cell suspension was sequentially passed through 10, 5, and 1 μm polycarbonate membrane filters, extruded five times at each diameter, and the resulting cell suspension was centrifuged at 4°C and 2000g for 15 minutes to remove cell debris. The supernatant was centrifuged at 4°C and 20,000g for 15 minutes. The precipitate was repeatedly pipetted and sonicated with sterile PBS to ensure uniform dispersion. After protein quantification, a portion was taken and frozen in a -80°C refrigerator as needed for subsequent experiments. The characterization results of the nanovesicles are shown in Figure 2. The nanovesicles are uniform in size, relatively stable, and evenly dispersed, with a diameter between 100-200 nm.

[0042] Example 2:

[0043] The preparation of human neural stem cell-derived nanovesicles (NSC-NVs) is as follows:

[0044] 1. Cell passaging:

[0045] NSC cell lines were purchased commercially. NSCs were collected during the logarithmic growth phase, when they had grown into uniform, translucent spheres. The cells were centrifuged at 300g for 5 minutes, and the supernatant discarded. The cells were digested with an appropriate amount of Accutase for 6-10 minutes, pipetted 50-100 times, and the digestion solution was neutralized with two volumes of culture medium. The cells were centrifuged again, and the supernatant discarded. The cells were resuspended in culture medium and plated onto T75 plates according to the size of the bottom surface. The flasks were labeled with cell type, passage number, and plating date. The cells were then cultured in a 37°C, 5% CO2 incubator.

[0046] The validation results of NSC cells are shown in Figure 3. Neural stem cells grew as spherical aggregates in suspension culture. Specific antigen expression is a key indicator in the identification of neural stem cells. Nestin and Sox2 are representative markers of neural stem cells. Under specific culture conditions, neural stem cells can undergo trilineage differentiation into neurons, oligodendrocytes, and astrocytes.

[0047] 2. Isolation and purification of nanovesicles:

[0048] NSCs were cultured to the logarithmic growth phase, digested, centrifuged at 300 g for 5 min, and the supernatant was discarded. 6Cells / mL were blown away in PBS solution to prepare a single cell suspension. A small extruder was used to pass the cell suspension through 10, 5 and 1 μm polycarbonate membrane filters in sequence, and each diameter was extruded continuously for 5 times. The resulting cell suspension was centrifuged at 4°C and 2000g for 15 minutes to remove cell debris, and the supernatant was centrifuged at 4°C and 20000g for 15 minutes. The precipitate was repeatedly blown and sonicated with sterile PBS to ensure uniform dispersion. After taking a portion for protein quantification, it was divided and frozen in a -80°C refrigerator as needed for subsequent experiments. The results are shown in Figure 4. The nanovesicles are uniform in size, relatively stable, evenly dispersed, and have a diameter between 100-200nm.

[0049] Example 3:

[0050] 1. sRNA extraction (using QIAGEN kit throughout the process):

[0051] UMSC-NVs (50 μg) prepared in Example 1 were injected into the tail vein of nude mice (n=3 per group). One hour later, the mice were sacrificed, and 25 mg of each of the liver, heart, spleen, lung, kidney, and brain tissues were removed. Each 25 mg of tissue was directly placed in 700 μL of QIAzol lysis reagent and homogenized in a 3 mL grinding tube until the sample was uniform. The homogenate tube was placed on the bench at room temperature for 5 minutes. After adding 140 μL of chloroform, the homogenate tube was incubated at room temperature for 3 minutes. Centrifuged at 12,000 g for 15 minutes at 4°C, the clear supernatant was collected, 525 μL of anhydrous ethanol was added, and the mixture was added to a miRNeasy spin column. The column was centrifuged at 8000 g for 1 minute, and the liquid was discarded. 700 μL of RWT was added to the spin column, and the column was centrifuged at 8000 g for 1 minute, and the liquid was discarded. 500 μL of RPE was added to the spin column, and the column was centrifuged at 8000 g for 1 minute, and the liquid was discarded. Finally, add 500 μL of RPE to the spin column and centrifuge at 8000 g for 2 minutes to discard the liquid. Replace the spin column base with a new one and centrifuge at 23000 g for 2 minutes to dry out the ethanol. Replace the spin column base with a new 1.5 mL EP tube to collect sRNA. Add 40 μL of enzyme-free water and centrifuge at 8000 g for 1 minute. Then, check the nucleic acid quality and concentration using a spectrophotometer.

[0052] 2. Reverse transcription and cDNA synthesis (using QIAGEN kit throughout the process):

[0053] Measure RNA concentration using a spectrophotometer. Use a maximum of 1 μg of template in the reaction. Prepare the reverse transcription system on ice, referring to Table 1. Perform reverse transcription on a PCR instrument using the following protocol: 40°C for 15 minutes, then 95°C for 5 minutes. Dilute the reverse-transcribed cDNA at a 1:5 ratio, label it, and store it at -20°C until use. Excess RNA should be labeled and stored at -80°C until use.

[0054] Table 1 Reverse transcription system

[0055] 3. Real-time quantitative PCR (using bio-rad kit throughout the process):

[0056] The obtained cDNA samples were prepared into PCR reaction solutions according to Table 2. The vesicle templates and nude mouse tissue templates were amplified using the primers designed in Table 3. The amplification program was as follows: Initial heat activation: 95°C for 2 min, 2-step cycling: 95°C for 5 s, 56°C for 30 s, 40 cycles.

[0057] Table 2 PCR reaction solution

[0058] Table 3 Primer sequences

[0059] 4. The distribution of nanovesicles can be determined by comparing the sRNA copy number with an exogenous standard curve.

[0060] The test results, shown in Figures 5G-H, showed that UMSC-NVs were distributed in all organs, with RNA copy numbers of 236, 253, 25, 6269, 57, and 12 copies / mg in the liver, heart, spleen, lung, kidney, and brain, respectively. The lung was most abundant, followed by the liver, heart, and kidney. Importantly, the presence of NVs in brain tissue was also observed, suggesting that NVs can enter the brain through the capillary network, indicating that NVs have the potential to assist drugs in crossing the blood-brain barrier.

[0061] Example 4:

[0062] 1. sRNA extraction:

[0063] The NSC-NVs (50 μg) prepared in Example 2 were injected into the tail vein of three nude mice. The mice were sacrificed 1 hour later, and 25 mg of liver, heart, spleen, lung, kidney, and brain tissues were collected from the animals. Each 25 mg of tissue was directly placed in 700 μl of QIAzol lysis reagent and homogenized in a 3 mL grinding tube until the sample was uniform. The homogenate tube was placed on the bench at room temperature for 5 minutes. After adding 140 μl of chloroform, the homogenate tube was placed in the room temperature tube for 3 minutes. Centrifuge at 12000g for 15 minutes at 4°C. The clear supernatant after separation was added with 525 μl of anhydrous ethanol, mixed, and added to a miRNeasy spin column. The mixture was centrifuged at 8000g for 1 minute, and the liquid was discarded. 700 μl of RWT was added to the spin column, centrifuged at 8000g for 1 minute, and the liquid was discarded. 500 μl of RPE was added to the spin column, and the mixture was centrifuged at 8000g for 1 minute, and the liquid was discarded. Finally, add 500 μL of RPE to the spin column and centrifuge at 8000 g for 2 minutes to discard the liquid. Replace the spin column base with a new one and centrifuge at full speed for 2 minutes to dry out the ethanol. Replace the spin column base with a new 1.5 mL EP tube to collect sRNA. Add 40 μL of enzyme-free water and centrifuge at 8000 g for 1 minute. Then, check the nucleic acid quality and concentration using a spectrophotometer.

[0064] 2. Reverse transcription and cDNA synthesis:

[0065] Measure RNA concentration using a spectrophotometer. Use a maximum of 1 μg of template in the reaction. Prepare the reverse transcription system on ice, referring to Table 1. Perform reverse transcription on a PCR instrument using the following protocol: 40°C for 15 minutes, then 95°C for 5 minutes. Dilute the reverse-transcribed cDNA at a 1:5 ratio, label it, and store it at -20°C until use. Excess RNA should be labeled and stored at -80°C until use.

[0066] 3. Real-time quantitative PCR:

[0067] The obtained cDNA samples were prepared into PCR reaction solutions according to Table 2, and the vesicle template and nude mouse tissue template were amplified using the primers designed in Table 3. The amplification procedures are as follows:

[0068] Initial heat activation: 95℃ 2min, 2-step cycling: 95℃, 5s, 56℃, 30s, 40Cycle.

[0069] 4. The distribution of nanovesicles can be determined by comparing the sRNA copy number with an exogenous standard curve.

[0070] The results are shown in Figure 6. This method can be successfully applied to the in vivo tracing of NSC-NVs. One hour after injection, NSC-NVs were distributed in all organs in the body. The RNA copy numbers in the liver, heart, spleen, lung, kidney, and brain were 239, 213, 25, 5242, 71, and 15 copies / mg, respectively, which were basically consistent with those of UMSC-NVs.

[0071] Example 5:

[0072] This example uses the tracer method to study the distribution of nanovesicles in the context of traditional Chinese medicine monomers.

[0073] 1. 50 μg of UMSC-NVs were dissolved in 200 μL of 37.5 μg / μL curcumin solution and injected into the tail vein of nude mice. One hour later, liver, heart, spleen, lung, kidney, and brain tissues were harvested. RNA was extracted, reverse transcribed, and then analyzed by PCR. The sequence copy number per mg of tissue was calculated based on the PCR reads.

[0074] The results are shown in Figure 7, which are essentially consistent with those obtained with UMSC-NVs alone. One hour after UMSC-NVs injection, RNA was distributed throughout all organs in the body, with RNA copy numbers of 189, 167, 16, 4542, 58, and 9 copies / mg in the liver, heart, spleen, lung, kidney, and brain, respectively. This demonstrates that the system can stably detect hydrophobic TCM monomers.

[0075] 2. Dissolve 50 μg of UMSC-NVs in 200 μL of 15 μg / μL paclitaxel solution and inject into the tail vein of nude mice. One hour later, liver, heart, spleen, lung, kidney, and brain tissues were harvested. RNA was extracted, reverse transcribed, and analyzed by PCR. The sequence copy number per mg of tissue was calculated based on the PCR reads.

[0076] The results are shown in Figure 8, which are essentially consistent with those obtained with UMSC-NVs alone. One hour after UMSC-NVs injection, RNA was distributed throughout all organs in the body, with RNA copy numbers of 177, 197, 28, 5542, 64, and 13 copies / mg in the liver, heart, spleen, lung, kidney, and brain, respectively. This demonstrates that the system can operate stably in the presence of hydrophilic TCM monomers.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

[0078] Sequence Listing

[0079] Sequence Listing

Claims

1. A method for detecting the distribution of human stem cell nanovesicles in tissues, characterized in that: The main steps include: (1) extracting nucleic acid from the tissue to be tested; (2) reverse transcribing the nucleic acid obtained in step (1) to obtain cDNA; (3) Detecting the specific sequence sRNA of human stem cell nanovesicles in the cDNA obtained in step (2) by PCR, and detecting the distribution of nanovesicles by comparing the copy number of sRNA with the exogenous standard curve.

2. The method according to claim 1, characterized in that The human stem cells include umbilical cord mesenchymal stem cells, neural stem cells and other human stem cells containing specific sRNA sequences of human stem cell nanovesicles; the methods for obtaining the nanovesicles include mechanical force squeezing method and gradient centrifugation method.

3. The method according to claim 1, characterized in that The tissues described in step (1) include liver, heart, spleen, lung, kidney, and brain tissues; and the sources of the tissues include rats and mice.

4. The method according to claim 1, characterized in that: The process of extracting nucleic acid in step (1) uses a QIAGEN kit.

5. The method according to claim 1, characterized in that The QIAGEN kit was used in the reverse transcription process in step (2).

6. The method according to claim 5, characterized in that Reverse transcription procedure in step (2): 40°C for 15 min, 95°C for 5 min.

7. [Corrected 07.10.2023 according to Rule 91] The method according to claim 1, characterized in that The PCR method described in step (3) includes qPCR and ddPCR.

8. The method according to claim 1, characterized in that The nucleotide sequence of the sRNA described in step (3) is shown in SEQ ID NO:

1.

9. The method according to claim 7, characterized in that: The nucleotide sequence of the stem-loop RT primer in the qPCR in step (3) is shown in SEQ ID NO: 2; the nucleotide sequence of the forward primer is shown in SEQ ID NO: 3; the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 4; and the probe is: FAM-CAGTTCTGATAACCC-MGB.

10. Use of the method according to any one of claims 1 to 9 in the pharmacokinetic study of drug-loaded human stem cell nanovesicles.

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