CN115976194A - Nervous system disease diagnosis method based on exosome micro RNA - Google Patents
Nervous system disease diagnosis method based on exosome micro RNA Download PDFInfo
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Abstract
The invention discloses a nervous system disease diagnosis method based on exosome micro RNA, which belongs to the technical field of disease diagnosis, and the scheme comprises the steps of extracting exosome miRNA in plasma of a patient, dividing the exosome miRNA into equal parts, culturing the equal parts in a first culture solution and a plurality of culture solutions, marking the exosome miRNA in the culture solutions by using a methyl green-piro red dyeing principle, establishing a stack diagram model according to color change ranges of the exosome miRNA in the culture solutions, comparing the stack diagram model with a comparison model in the first culture solution, inhibiting normal functions of the exosome miRNA by regulating immune cells, promoting invasion and development of malignant cells by using the malignant cell exosome miRNA, rapidly propagating the malignant cell exosome miRNA in the culture solutions, comparing the malignant cell miRNA with an initial exosome miRNA range to judge whether a large number of malignant cells exist in the plasma of the patient, finally detecting miR-125b in the miRNA by a real-time fluorescent RTFQ-PCR method, and judging whether the patient has Alzheimer disease or not by using the standard.
Description
Technical Field
The invention relates to the technical field of disease diagnosis, in particular to a method for diagnosing nervous system diseases based on exosome microRNA.
Background
The exosome is a membrane vesicle with the size of 30-150 nm secreted by cells, is released after the microvesicle is fused with a cell membrane, usually shows a cup-shaped or dish-shaped form, the outer membrane of the exosome is similar to the cell membrane, and contains a phospholipid bilayer, the exosome contains bioactive substances such as proteins, lipids, mRNA and the like, and also contains a plurality of non-coding RNAs including miRNA, long-chain non-coding RNA and circular RNA, the exosome is widely present in body fluid and various cells, and plays a direct or indirect role along with the circulation of the body, and different exosomes have different functions such as cell proliferation induction, immunosuppression, cell secretion promotion, signal transmission between cells and the like according to the source of the cells.
In the age-old population age, the number of the old people is more and more, the old people are more likely to suffer from some diseases, such as Alzheimer's disease, and the implementation of the diagnosis mode of the old people has certain limitation due to poor physical quality of the old people, so that the diagnosis time is easily prolonged in the detection process, the precious time for treating the disease is delayed, and the spread of the disease is aggravated.
Therefore, a method for diagnosing nervous system diseases based on exosome microRNAs is proposed to solve the above problems.
Disclosure of Invention
1. Technical problem to be solved
Aiming at the problems in the prior art, the invention aims to provide a method for diagnosing nervous system diseases based on exosome microRNA, which can realize the establishment of a stack map model according to the color change ranges of a plurality of culture solutions of the exosome microRNA, compare the stack map model with a comparison model in a first culture solution, compare the stack map model with the miRNA range of an initial exosome to judge whether a large number of malignant cells exist in the plasma of a patient, finally detect miR-125b in miRNA through a real-time fluorescence RTFQ-PCR method, and judge whether the patient has Alzheimer's disease according to the standard.
2. Technical scheme
In order to solve the above problems, the present invention adopts the following technical solutions.
A method for diagnosing nervous system diseases based on exosome microRNA comprises the following steps:
s1: extracting exosome miRNA in plasma of a patient by using an ultracentrifugation method, dividing the exosome miRNA into equal parts, and putting the equal parts into a comparison culture solution and a plurality of comparison culture solutions for simultaneous culture;
s2: carrying out color marking on exosome miRNA in a first culture solution; establishing a comparison model for the areas displayed outside;
s3: after culturing for a certain period of time, sequentially carrying out color marking on a plurality of contrast culture solutions, and sequentially recording the marking time;
s4: establishing a comparison model aiming at color areas in a plurality of comparison culture solutions, and comparing the comparison model with the comparison model established according to the first culture solution;
s5: and detecting the first culture solution exosome miRNA by a real-time fluorescent RTFQ-PCR method.
Further, the temperature of the S1 culture solution was set at 4 ℃ and the time for the culture was controlled within 48 hours.
Further, the ultracentrifugation in S1 utilizes the separation of exosomes with different characteristics, further removing other impurities by combining with other filtration steps or using sucrose density gradients.
Furthermore, in the S1 and the S3, the culture dishes of the plurality of control culture solutions need to be labeled sequentially when the culture is carried out, and the labels are recorded sequentially from small to large when the color labels of the plurality of control culture solutions are recorded.
Furthermore, the color marking of the exosome miRNA in S2 and S3 can adopt a methyl green-pirox dyeing principle.
Furthermore, when a control model is established for a plurality of control culture solutions in S4, a stacking graph method may be used for establishment.
Further, when the comparison model and the comparison model are compared in S4, the continuously changing marker region of the comparison model is always increased and is all larger than the comparison marker region, and it is determined that the possibility of the patient suffering from the disease exists in the approximate probability.
Further, in the S5, the miR-125b in the first culture solution is detected by a real-time fluorescence RTFQ-PCR method.
3. Advantageous effects
Compared with the prior art, the invention has the advantages that:
(1) According to the scheme, a stacked graph model is established according to the color change range in a plurality of culture solutions and is compared with a comparison model in a first culture solution, the initial exosome miRNA range is compared, whether a large number of malignant cells exist in blood plasma of a patient is judged, miR-125b in miRNA is finally detected through a real-time fluorescence RTFQ-PCR method, and whether the patient suffers from Alzheimer's disease is judged according to the standard.
Drawings
FIG. 1 is a schematic diagram of the method for diagnosing a nervous system disease by exosome microRNA of the present invention.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention; it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and all other embodiments obtained by those skilled in the art without any inventive work are within the scope of the present invention.
In the description of the present invention, it should be noted that the terms "upper", "lower", "inner", "outer", "top/bottom", and the like indicate orientations or positional relationships based on those shown in the drawings, and are only for convenience of description and simplification of description, but do not indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus should not be construed as limiting the present invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
In the description of the present invention, it should be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "disposed," "sleeved/connected," "connected," and the like are to be construed broadly, e.g., "connected," which may be a fixed connection, a detachable connection, or an integral connection; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in a specific case to those of ordinary skill in the art.
Example 1:
referring to fig. 1, a method for diagnosing a nervous system disease based on exosome microrna includes the following steps:
s1: extracting exosome miRNA in plasma of a patient by using an ultracentrifugation method, dividing the exosome miRNA into equal parts, and putting the equal parts into a comparison culture solution and a plurality of comparison culture solutions for simultaneous culture;
s2: carrying out color marking on exosome miRNA in the first culture solution; establishing a comparison model for the area displayed outside;
s3: after culturing for a certain period of time, sequentially carrying out color marking on a plurality of contrast culture solutions, and sequentially recording the marking time;
s4: establishing comparison models aiming at color areas in a plurality of comparison culture solutions, and comparing the comparison models with the comparison model established according to the first culture solution;
s5: and detecting the first culture solution exosome miRNA by a real-time fluorescent RTFQ-PCR method.
According to the scheme, exosome miRNA in plasma of a patient is extracted and then divided into equal parts to be cultured in a first culture solution and a plurality of culture solutions, exosome miRNA in the culture solutions is marked by utilizing a methyl green-piro red staining principle, the culture solutions are marked in sequence according to marks in a plurality of culture dishes when being marked, the time interval between adjacent culture solution marks is the same, a stack graph model is established according to the color change range of the culture solutions and compared with a comparison model in the first culture solution, malignant cell miRNA is utilized to control immune cells, so that the normal functions of the immune cells are inhibited, invasion and progress of malignant cells are promoted, malignant cell exosome miRNA in the culture solutions is propagated rapidly, the propagation speed is far higher than that of the normal cells, whether malignant cells exist in the plasma of the patient is judged by comparing with the initial exosome miRNA range, and finally whether miR-125b in the miRNA is detected by a real-time fluorescence RTFQ-PCR method, and whether miR-125b existing in the plasma of the patient is far higher than the normal level of the patient, and whether the patient suffers from Alzheimer's disease is judged by a human body.
The temperature of the S1 culture solution was set at 4 ℃ and the time of the culture was controlled within 48 hours.
According to the scheme, the temperature of the culture solution in the S1 is set to be 4 ℃, so that the exosome miRNA extracted from the culture solution can be normally and stably stored for 48h, the detection and comparison time needs to be controlled within 48h, and the exosome miRNA is prevented from being influenced by other factors to cause the accidental performance of the contrast test when the contrast test is carried out, so that the diagnosis result is deviated.
The ultracentrifugation method in S1 utilizes the different characteristics of exosomes for separation, further removing other impurities by combination with other filtration steps or using sucrose density gradients.
The scheme is characterized in that an ultracentrifugation method is adopted for extracting miRNA of the exosome, cells, cell fragments and other organelles are separated out through different sedimentation coefficients of the exosome and other organelles, and therefore the pure exosome is obtained.
And (3) sequentially marking the culture dishes of the plurality of control culture solutions in the S1 and the S3 when the culture solutions are cultured, and sequentially recording the color marks of the plurality of control culture solutions according to the sequence from small to large.
This scheme is through when carrying out the culture mark to a plurality of contrast culture solution, through carry out the back to a plurality of culture dish in proper order, can record according to the order of mark number from small to big when recording the culture solution in a plurality of culture dish to the time of recording keeps unanimous between two adjacent, thereby make when more convenient when the model is established in the record, also control its unique variable, also be exactly that the spaced time is in a definite value, thereby eliminate the influence of other factors to the detection and comparison to the at utmost.
In S2 and S3, the color marking of the exosome miRNA can adopt a methyl green-pirox red dyeing principle.
According to the scheme, the exosome miRNA is subjected to color marking by utilizing a methyl green-pirored dyeing principle, green is presented by combining methyl green with DNA with high polymerization degree easily, and the pirored is presented red by combining RNA with low polymerization degree, namely the RNA is dyed into red due to high affinity to the pirored, and the DNA is dyed into green due to high affinity to the methyl green, so that the breeding condition of the exosome miRNA can be recorded by observing the range size of a green area.
In S4, when the contrast models are established for a plurality of contrast culture solutions, a stacking graph method can be adopted for establishment.
According to the scheme, the model is established by adopting a stacked graph method, so that the change range presented by the comparison model can be clearly seen through the change area of the graph and the recording time, the comparison can be conveniently and better carried out, and meanwhile, the size of the change range can be clearly reflected.
And S4, when the comparison model is compared with the comparison model, the continuously changed marking area of the comparison model is always increased and is all larger than the comparison marking area, and then the possibility of illness of the patient is judged.
The scheme utilizes malignant cell exosome miRNA to inhibit normal functions of immune cells and promote invasion and development of malignant cells by regulating the immune cells, so that malignant cell exosome miRNA in a plurality of culture solutions can be rapidly propagated, the propagation speed is far higher than that of normal cells, and malignant cells can be judged to exist in plasma of a patient by comparing the propagation speed with the initial exosome miRNA range,
and in S5, detecting miR-125b in the first culture solution by a real-time fluorescent RTFQ-PCR method.
The scheme detects miR-125b by a real-time fluorescence RTFQ-PCR method, a reporter group R is marked at the 5' end of a probe to emit a fluorescence signal, a fluorescence quenching group Q is marked at the 3' end to absorb the fluorescence signal, the probe is actually an oligonucleotide chain, an oligonucleotide chain (DNA/RNA) is added in the PCR reaction process, when the probe is complete, the fluorescence signal emitted by the reporter group is absorbed by the quenching group and is not detected, when the PCR is amplified to the position of the probe, the exonuclease activity of TaqDNA polymerase cuts off the reporter group at the 5' end of the probe to ensure that the reporter group is far away from the probe, and the generated fluorescence signal cannot be absorbed by the quenching group and is detected by an instrument. Every DNA chain is amplified, a fluorescent molecule is generated, the change of the amount of each cycle of amplified products in the whole PCR reaction is monitored in real time through the accumulation amount of fluorescent signals, finally, the unknown template is quantitatively analyzed through a standard curve, the fluorescent threshold value is a value set artificially on the fluorescent amplification curve, the fluorescent signals of the first 15 cycles of the PCR reaction are used as fluorescent background signals, and the default set value of the general fluorescent threshold value is 10 times of the standard deviation of the fluorescent signals of 3-15 cycles, so that the monitoring of miR-125b is completed, and the method is beneficial to judging whether a patient suffers from Alzheimer's disease.
The foregoing is only a preferred embodiment of the present invention; the scope of the invention is not limited thereto. Any person skilled in the art should be able to cover the technical scope of the present invention by equivalent or modified solutions and modifications within the technical scope of the present invention.
Claims (8)
1. A method for diagnosing nervous system diseases based on exosome microRNA is characterized in that: the method comprises the following steps:
s1: extracting exosome miRNA in plasma of a patient by using an ultracentrifugation method, dividing the exosome miRNA into equal parts, and putting the equal parts into a comparison culture solution and a plurality of comparison culture solutions for simultaneous culture;
s2: carrying out color marking on exosome miRNA in the first culture solution; establishing a comparison model for the areas displayed outside;
s3: after culturing for a certain period of time, sequentially carrying out color marking on a plurality of contrast culture solutions, and sequentially recording the marking time;
s4: establishing a comparison model aiming at color areas in a plurality of comparison culture solutions, and comparing the comparison model with the comparison model established according to the first culture solution;
s5: and detecting the first culture solution exosome miRNA by a real-time fluorescent RTFQ-PCR method.
2. The method of claim 1 for diagnosing a neurological disease based on exosome micrornas, wherein: the temperature of the culture solution of the S1 is set to be 4 ℃, and the culture time is controlled within 48 h.
3. The method of claim 1 for diagnosing a neurological disease based on exosome micrornas, wherein: the ultracentrifugation in S1 utilizes the different characteristics of exosomes for separation, further removing other impurities by combination with other filtration steps or using sucrose density gradients.
4. The method of claim 1 for diagnosing a neurological disease based on exosome micrornas, wherein: and in the S1 and the S3, the culture dishes of the plurality of control culture solutions need to be labeled in sequence when the culture is carried out, and the labels are recorded in sequence from small to large when the color labels of the plurality of control culture solutions are recorded.
5. The method of claim 1 for diagnosing a neurological disease based on exosome micrornas, wherein: in S2 and S3, the color marking of the exosome miRNA can adopt a methyl green-pirox red dyeing principle.
6. The method of claim 1 for diagnosing a neurological disease based on exosome micrornas, wherein: when the control model is established for a plurality of control culture solutions in the S4, the control model can be established by adopting a stacking diagram method.
7. The method of claim 1 for diagnosing a neurological disease based on exosome micrornas, wherein: and S4, when the comparison model and the comparison model are compared, the continuously changed marking area of the comparison model is always increased and is totally larger than the comparison marking area, and then the possibility of illness of the patient is judged.
8. The method of claim 1 for diagnosing a neurological disease based on exosome micrornas, wherein: and in the S5, the miR-125b in the first culture solution is detected by a real-time fluorescent RTFQ-PCR method.
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| CN105861712A (en) * | 2011-04-18 | 2016-08-17 | 迪阿米尔有限责任公司 | Methods of using mirna from bodily fluids for early detection and monitoring of mild cognitive impairment (mci) and alzheimer's disease (ad) |
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