CN111020012A - Method for gene screening and bioinformatics analysis - Google Patents
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Abstract
The invention belongs to the technical field of gene screening, and particularly relates to a method for gene screening and bioinformatics analysis, which comprises the following steps: the method comprises the following steps: preparing an experimental model; step two: RNA extraction and gene chip analysis are carried out; step three: performing a molecular biology experiment; step four: concluding the final result, designing an effective experimental model according to different changes of pathophysiology at the near end and the far end after the nerve injury, starting from the overall angle of regeneration after the peripheral nerve injury by utilizing a gene chip technology, and according to the most important change characteristics in the injury and regeneration processes: axon guiding, axon regeneration, myelination, extracellular matrix and signal transduction are used for selecting genes with obvious expression difference, comparative analysis is carried out on the biological process of the genes at different time points by applying the conventional biotechnology software to obtain reliable data, and the reliable data is verified by applying a common experimental method of molecular biology.
Description
Technical Field
The invention relates to the technical field of gene screening, in particular to a method for gene screening and bioinformatics analysis.
Background
With the progress of social modernization, various traumatic events increase year by year. In many traumatic events, the resulting injuries to the limbs, especially combined peripheral nerve injuries, are increasing due to labor manipulation.
Although more and more people suffer from peripheral nerve injury, the patients suffer from pain and are accompanied with neurological sequelae due to the lack of a special effective treatment method at present. The limb dysfunction caused by peripheral nerve injury not only has great influence on the life quality and the working capacity of patients, but also adds a heavy burden to the society.
Peripheral nerve injury refers to a clinical condition in which certain traumatic factors cause nerve or nerve axon rupture, resulting in dysfunction of nerve conduction, resulting in sensory, motor, and sympathetic dysfunction of the trunk and limbs. The peripheral nerve injury is caused by many reasons, and can be classified into mechanical injury and non-mechanical injury according to the injury mechanism, wherein the former includes traction, extrusion, cutting, tearing and the like, and the latter includes ischemic, electric shock, radioactive injury and the like. Acute and chronic injuries are classified according to the onset time. Peripheral nerves also respond differently to injury, to varying degrees.
The damage causes are many, the damage forms are various, and the damage modes of the nerves are different. In the aspect of nerve regeneration, the mechanism is complex, and the influence of external factors is large, so that the slow nerve regeneration speed is poor, the effect is poor, the recovery of the nerve function of a patient with peripheral nerve injury is poor, the prognosis is poor, and the labor capacity and the life quality of the patient are seriously influenced.
The repair of nerve regeneration after injury has also been a focus of common concern for traumatology and neurosurgery. With the development of advanced science and technology in society, some progress has been made on the research on nerve regeneration, such as the discovery of far-end Wallerian degeneration after nerve injury, the migration, proliferation and differentiation functions of Schwann cells, and the intervention of a series of exogenous substances such as neurotrophic factors, and the like, which effectively reveal some essential phenomena of nerve regeneration, but deeper understanding is not yet fully obtained. Since the speed of nerve regeneration in human is very slow, the intervention of tissue engineering materials opens up a new therapeutic situation, but at present, autologous nerve transplantation is still the best therapeutic method. Therefore, although scholars at home and abroad make a great deal of theoretical and clinical researches on the regeneration and repair of peripheral nerve injury, the scholars focus on a certain field and have not obtained the overall view of regeneration after nerve injury. The reason for this is that peripheral nerve injury and regeneration factors have diversity and mechanism complexity, and the study lacks systematicness, and relatively complete understanding of the mechanism of change of injured nerves is lacking.
With the continuous deepening of research fields and the continuous innovation of treatment methods, the regeneration and repair of peripheral nerve after damage are greatly progressed. The previous research on the peripheral nerve regeneration process is started from morphological changes, and the research in the field of protein and enzymology is gradually increased along with the development of molecular biology. Many studies have been made to dissect peripheral nerves from the genetic field, but the findings obtained are limited, and the dynamic changes of all genes and the changes of biological processes reflected by different genes during peripheral nerve injury and regeneration are still lack of intensive systematic studies. In organic organisms, genes are in a complex dynamic network, and the functions of the genes depend on the abundance degree of expression of the genes, the internal environment and external stimulation conditions and are influenced by the regulation of various factors. Therefore, there is an urgent need to fully understand the development rule of nerve regeneration from the deeper gene level.
Early neural studies were mostly morphologically initiated and were not fully comparable in function. The molecular biology and cell biology techniques developed in the later stage can research the relevant factors for repairing and regenerating after nerve injury from the aspects of proteomics, cell functions and the like and the exogenous action of neurotrophic factors. These methods are all remedial measures for the injured nerve, but have no decisive effect and significance for fundamentally changing the injured characteristic.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made in view of the above and/or the problems occurring in the existing gene screening and bioinformatic analysis methods.
Therefore, the present invention aims to provide a method for gene screening and bioinformatics analysis, which can start from the overall point of view of regeneration after peripheral nerve injury by using gene chip technology, according to the most important change characteristics in the injury and regeneration processes: axon guiding, axon regeneration, myelination, extracellular matrix and signal transduction are used for selecting genes with obvious expression difference, comparative analysis is carried out on the biological process of the genes at different time points by applying the conventional biotechnology software to obtain reliable data, and the reliable data is verified by applying a common experimental method of molecular biology.
To solve the above technical problem, according to an aspect of the present invention, the present invention provides the following technical solutions:
a method for gene screening and bioinformatics analysis, which comprises the following steps:
the method comprises the following steps: preparing an experimental model;
step two: RNA extraction and gene chip analysis are carried out;
step three: performing a molecular biology experiment;
step four: a summary of the final results was performed.
As a preferable embodiment of the method for gene screening and bioinformatics analysis according to the present invention, wherein: the second step comprises the following specific steps of RNA extraction and gene chip analysis:
the method comprises the following steps: taking materials;
step two: preparing a gene chip;
step three: gene screening and bioinformatics analysis.
As a preferable embodiment of the method for gene screening and bioinformatics analysis according to the present invention, wherein: the molecular biology experiment in the third step comprises timed quantitative PCR analysis, western blot analysis and immunofluorescence staining.
As a preferable embodiment of the method for gene screening and bioinformatics analysis according to the present invention, wherein: the conclusion of the fourth step is to summarize the number of the differential genes, perform classification analysis according to different times intervals to obtain the general trend and the classification trend, and combine the general trend and the classification trend with the result of the meristem experiment to obtain the final conclusion.
Compared with the prior art: the method designs an effective experimental model according to different changes of pathophysiology at the near end and the far end after nerve injury, starts from the overall angle of regeneration after peripheral nerve injury by utilizing a gene chip technology, and is characterized by comprising the following steps of: axon guiding, axon regeneration, myelination, extracellular matrix and signal transduction are used for selecting genes with obvious expression difference, comparative analysis is carried out on the biological process of the genes at different time points by applying the conventional biotechnology software to obtain reliable data, and the reliable data is verified by applying a common experimental method of molecular biology.
The research aims to obtain biological information of the most important characteristics of the near-end nerve segment and the far-end nerve segment in the process of injury and regeneration after the sciatic nerve of the rat, and extract the change trends of cell proliferation, cell migration and apoptosis gene characteristics at different time points. Therefore, the gene genus and the cell factor which are the most key for regulating and controlling the regeneration after nerve injury can be obtained, the expression of the gene genus and the cell factor can be promoted or blocked by applying an intervention technology, the efficient regulation and control effect on nerve regeneration is realized, and a better method direction is provided for clinical application and treatment.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the present invention will be described in detail with reference to the accompanying drawings and detailed embodiments, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise. Wherein:
FIG. 1 is a schematic diagram of the flow structure of a method for gene screening and bioinformatics analysis according to the present invention.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described herein, and it will be apparent to those of ordinary skill in the art that the present invention may be practiced without departing from the spirit and scope of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Next, the present invention will be described in detail with reference to the drawings, wherein for convenience of illustration, the cross-sectional view of the device structure is not enlarged partially according to the general scale, and the drawings are only examples, which should not limit the scope of the present invention. In addition, the three-dimensional dimensions of length, width and depth should be included in the actual fabrication.
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail with reference to the accompanying drawings.
The invention provides a method for gene screening and bioinformatics analysis,
according to the experimental method, adult male SD rats are bred, materials are obtained at a proper experimental time point, sciatic nerve segments required by the experiment are obtained, the sciatic nerve segments are further divided into different groups, gene chip analysis, real-time fluorescence quantitative RT-PCR analysis, WesternBlot western blot analysis and histochemical immunofluorescence staining analysis are respectively carried out, and axon guidance, axon regeneration, myelination, extracellular matrix and signal transduction, which are biological characteristics vital to nerve injury regeneration, are obtained from the gene level and the molecular biology level.
The method comprises the following steps:
the method comprises the following steps: experimental model preparation, healthy adult SPF SD male rats weighing 220 + -20 g and 180 animals are anesthetized by intraperitoneal injection with compound anesthetic (30mg/kg), left hip oblique incisions are made under aseptic conditions, muscles are separated in a blunt manner, sciatic nerves are exposed in left gluteus gaps of the rats, the sciatic nerves are transected at a position 0.5cm away from the lower edge of piriformis, the proximal ends are folded to prevent reconnection of the nerve-broken ends, and wounds are sutured. Animals are kept under suitable temperature and humidity conditions, and are exposed to light for 12h every day, and the animals are fed with water freely in normal diet. The control group was a normal group without any treatment;
animal molding technology: animal disease models are used primarily for experimental physiology, experimental pathology, and experimental therapeutics (including new drug screening) studies. The development of human diseases is very complex, people themselves are taken as experimental objects to deeply discuss disease occurrence mechanisms and promote the slow development of medicine, and the clinically accumulated experience has limitations in time and space and limits in practicalities and methods. By means of indirect research of the animal model, factors which are impossible or difficult to eliminate under natural conditions can be changed consciously, so that the experimental result of the model can be observed more accurately and compared with human diseases for research, the occurrence and development rules of the human diseases can be known more conveniently and more effectively, and prevention and treatment measures can be researched.
Step two: RNA extraction and gene chip analysis are carried out, and the gene chip technology comprises the following steps: gene chip (also called DNA chip, biological chip) technology refers to that a large number of probe molecules (usually the density of the lattice per square centimeter is higher than 400) are fixed on a support and then hybridized with labeled sample molecules, and the number and sequence information of the sample molecules are obtained by detecting the hybridization signal intensity of each probe molecule. In general, a two-dimensional DNA probe array is formed by fixing tens of thousands or even millions of DNA fragments (gene probes) of a specific sequence on a support such as a silicon wafer or a glass slide of 2cm2 by a microfabrication technique, and is very similar to an electronic chip of a computer, and is called a gene chip. The gene chip is mainly used for gene detection. The gene chip technology can simultaneously fix a large number of probes on a support, so that a large number of sequences of a sample can be detected and analyzed at one time, and the defects of complex operation, low automation degree, small number of operation sequences, low detection efficiency and the like of the traditional nucleic acid blotting hybridization (southern blotting, northern blotting and the like) technology are overcome. Moreover, by designing different probe arrays and using a specific analysis method, the technology has various application values, such as gene expression profiling, mutation detection, polymorphism analysis, genome library mapping, hybridization sequencing and the like;
step three: the molecular biology experiment is carried out, and the molecular biology technology can be applied to the research of genetic diseases, the detection of pathogens, the research of etiology, pathogenesis, diagnosis, treatment and the like of tumors, and the level of gene molecules is improved. For example, reverse transcription PCR, the transcription of one RNA strand into complementary DNA (cdna), called "reverse transcription", is performed by an RNA-dependent DNA polymerase (reverse transcriptase). Subsequently, the other strand of DNA is completed by a deoxynucleotide primer and a DNA-dependent DNA polymerase, multiplied with each cycle, i.e. usual PCR. The original RNA template is degraded by RNase H, leaving complementary DNA. Exponential amplification by RT-PCR is a very sensitive technique that can detect RNA at very low copy numbers. RT-PCR is widely used in the diagnosis of genetic diseases and can be used to quantitatively monitor the content of certain RNAs. For example, Western blotting is a method in which a sample of the obtained protein is electrophoresed through SDS-polyacrylamide gel to separate proteins having different molecular weights, and then the proteins separated on the gel are transferred to a solid support by transfer electrophoresis. The solid support includes NC (nitrocellulose) membrane, nylon membrane, PVDF (polyvinylidene fluoride) membrane, and the like. The mode of transferring the film is wet-transferring and semi-dry-transferring. Hybridizing a non-labeled antibody (primary antibody) resisting the target protein with the membrane, specifically binding the antibody with the target protein, then binding the antibody with a horseradish peroxide-labeled secondary antibody, finally reacting with an ECL reagent, and carrying out a series of reactions such as X-ray film exposure and development to achieve the purpose of detecting the target protein;
step four: a summary of the final results was performed.
Wherein, the RNA extraction and gene chip analysis in the second step comprises the following specific steps:
the method comprises the following steps: taking materials, taking 5 rats at each time point of 0d, 4d, 7d, 14d, 21d and 28d after operation, taking the sciatic nerve at the proximal end and the distal end of the transection by 0.5cm respectively, analyzing by using a gene chip, and repeating the biology three times;
step two: preparing a gene chip, extracting RNA and precipitating, measuring an RNA sequence, respectively synthesizing first-strand and second-strand cDNA, purifying, marking synthesized cRNA by biotin, purifying and transcribing in vitro to obtain a product, measuring the concentration of the cRNA by a spectrophotometer, detecting by gel electrophoresis, fragmenting the cRNA, hybridizing a molecular probe, eluting and dyeing to finally obtain gene data;
step three: gene screening and bioinformatics analysis, wherein an Affymetrix gene difference analysis scanning system (each time point and different gene ratios are set) is used for analyzing gene difference, and the significance of expression trend of different genes under long and short time sequences is respectively analyzed; selecting the gene with the chip result p <0.05 and foldchange >2 as a differential expression gene, performing function enrichment analysis on the classification of the gene according to the EntrezGeneID number of the gene recorded by the data by using the analysis function of GeneOntology (GO) of a DAVID database, finding out the genes related to cell proliferation, migration and apoptosis, and classifying to obtain the biological process in which the differential genes participate in regulation and control.
The molecular biology experiment in the third step comprises timed quantitative PCR analysis, western blot analysis and immunofluorescence staining, specifically, the timed quantitative PCR analysis is to design a primer sequence in advance, 5 rats are taken at each time point of 0d, 4d, 7d, 14d, 21d and 28d after operation, the preparation of total RNA of the nerve segment line obtained by material collection, reverse transcription reaction amplification and 96wellsReal-timePCR detection are utilized, and single-factor ANOVA (namely one-wayANOVA) processing is carried out by adopting STATA7.0 software statistics to obtain data;
performing western blot analysis, namely taking 5 rats at each time point of 0d, 4d, 7d, 14d, 21d and 28d after operation, extracting the tissue protein by using the obtained nerve fragments, determining the total protein concentration by using a BCA method, performing SDS-PAGE gel electrophoresis, cutting gel, transferring the membrane and the antibodies, and developing, fixing and scanning to obtain an image;
and (3) performing immunofluorescence staining, namely, respectively taking 5 rats at each time point of 0d, 4d, 7d, 14d, 21d and 28d after operation, performing perfusion material taking and gradient sucrose treatment, performing frozen section, sealing, incubating antibody and tissue immunofluorescence double-label staining, and then shooting by using a fluorescence microscope to obtain a picture, wherein the picture is processed by using software.
The result in the fourth step is specifically summarized as summarizing the number of the different genes, carrying out classification analysis according to different times intervals to obtain the general trend and the classification trend, and combining with the result of the meristem experiment to obtain the final conclusion.
In the method, a rat sciatic nerve dissociation model is established by adopting a nerve reverse-folding method after the sciatic nerve is dissociated. In the prior method, the extraction anatomical position of the sciatic nerve of the rat is low, and the operation difficulty is high. In the past, nerve dissection often needs to remove a part of the nerve of the tissue, so that a sufficient amount of nerve tissue fragments cannot be obtained, and the experimental result is inaccurate. And the broken end is not protected after the disconnection, so that the nerve often grows by mistake, and the nerve can be bridged again to cause the molding failure, and the obtained data is not accurate enough.
In the aspect of gene analysis method, after different gene ratios are set by the identified feasible Affymetrix gene difference analysis scanning system, the expression trend analysis is respectively carried out on the genes with significant difference under long and short time sequences according to the variable obtained at each time point. The specific method comprises the following steps: setting the range as p <0.05, foldchange >2, selecting the number sequence of genes with different expressions as the chip result according to the screening range, substituting the Entrez Gene ID number of the Gene recorded by the data into a DAVID database, and performing enrichment analysis on the characteristics which are set by people and regenerated after nerve injury, namely axon guidance, axon regeneration, myelination, extracellular matrix and signal transduction by utilizing the Gene Ontology (GO) analysis function in the database to find the Gene related to the characteristics. The obtained genes are arranged, and the high-expression genes (key genes) which are regenerated after peripheral nerve injury is regulated through the characteristics are found through expression trend division. Furthermore, the regeneration and repair process of peripheral nerves after injury can be promoted by taking measures such as key gene knockout, modification, blocking, intervention and the like.
Examples
At the beginning of the experiment, i.e., day 0, 10 healthy adult SPF-grade SD male rats weighing about 200g were anesthetized by intraperitoneal injection with pentobarbital sodium anesthetic (30mg/kg), left hip oblique incisions were made under aseptic conditions, muscles were separated bluntly, sciatic nerves were exposed in the left hip gap of the rat, and sciatic nerves were transected at a distance of 0.5cm from the lower edge of piriformis. 5 rats are taken as a control group for 0 day, proximal and distal nerve tissues are cut off by 0.5cm respectively to obtain nerve fragments required by the experiment, and then the rats are killed according to animal ethical operation specifications. The procedure was repeated for 5 additional rats, after exposure of the sciatic nerve and transection, the proximal end was invaginated, and washed with saline to protect, prevent reconnection of the severed nerve ends, and then the wound was sutured. And then repeating the operations on the 1 st day of the experiment, taking the near-end nerve tissues and the far-end nerve tissues, and finely trimming and removing the adhesion parts to form nerve segments required by the experiment, so as to obtain the experimental group material on the 1 st day.
After different gene ratios are set by a gene difference analysis scanning system, according to variables obtained at each time point, expression trend analysis is respectively carried out on genes with significant differences under long and short time sequences. Through preliminary screening, the expression characteristics of the genes in the general trend can be obtained. For example, the gene expression number of proximal nerve segments after sciatic nerve dissociation increases with time, increases rapidly in the early stage, reaches a peak at day 7, and then gradually decreases to a relatively stable level, and the up-regulated gene has a preponderance over the down-regulated gene in the whole aspect. The gene expression number of the distal end nerve segment after sciatic nerve amputation obviously increases along with time, reaches a small peak at day 7 and then gradually decreases. The genes with up-regulated expression in the early stage of the experiment have a more obvious quantitative advantage than the genes with down-regulated expression in the early stage of the experiment, which is the same as the data trend of the proximal end, and the down-regulated genes have more up-regulated genes in the overall quantity aspect as the experiment time is prolonged. In previous experimental studies, it was found that cytokines such as Il1, Il6 and Il10 and chemical molecules such as Cxcl1, Cxcl2, Cxcl5, Ccr1, Ccr5, Ccl2, Ccl7 and Ccl20 have significant changes in early stages after nerve disruption, and these changes are closely related to inflammatory reactions. Meanwhile, the antibody expression genes such as Cd8a, Cd8b, Tnfrf11, Itgal and the like and macrophage activation genes such as Lbp, Fcgr3a, Cx3cr1 and the like which are closely related to immune response are found to be obviously expressed after nerve interruption. Furthermore, a high-expression gene can be screened from the characteristic and intervened to improve the nerve repair process.
While the invention has been described above with reference to an embodiment, various modifications may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In particular, the various features of the disclosed embodiments of the invention may be used in any combination, provided that no structural conflict exists, and the combinations are not exhaustively described in this specification merely for the sake of brevity and resource conservation. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.
Claims (4)
1. A method for gene screening and bioinformatics analysis, comprising: the method comprises the following steps:
the method comprises the following steps: preparing an experimental model;
step two: RNA extraction and gene chip analysis are carried out;
step three: performing a molecular biology experiment;
step four: a summary of the final results was performed.
2. The method of claim 1, wherein the method comprises: the second step comprises the following specific steps of RNA extraction and gene chip analysis:
the method comprises the following steps: taking materials;
step two: preparing a gene chip;
step three: gene screening and bioinformatics analysis.
3. The method of claim 1, wherein the method comprises: the molecular biology experiment in the third step comprises timed quantitative PCR analysis, western blot analysis and immunofluorescence staining.
4. The method of claim 1, wherein the method comprises: the conclusion of the fourth step is to summarize the number of the differential genes, perform classification analysis according to different times intervals to obtain the general trend and the classification trend, and combine the general trend and the classification trend with the result of the meristem experiment to obtain the final conclusion.
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