阅读说明：本技术 Rassf2调控内耳干细胞增殖分化方法及在毛细胞再生中的应用 (Rassf2 method for regulating proliferation and differentiation of inner ear stem cells and application of Rassf2 method in hair cell regeneration ) 是由 柴人杰 周姗 张莎莎 程诚 高下 于 2021-02-20 设计创作，主要内容包括：本发明涉及内耳干细胞领域,具体的是Rassf2调控内耳干细胞增殖分化方法及在毛细胞再生中的应用。调控方法,包括以下步骤：一、在野生型小鼠中检测Shh或Hippo和Rassf2在耳蜗中的表达情况；二、在离体实验中研究Shh或Hippo和Rassf2对流式分选出的内耳干细胞增殖分化的作用；三、在活体毛细胞新霉素损伤模型上,研究Shh或Hippo和Rassf2的作用。本发明使用Lgr5-EGFP-CreERT2工具鼠可使得内耳干细胞(Lgr5阳性细胞)的分离准确且大量,可达到较好的研究前提：分选出的小鼠Lgr5阳性内耳干细胞。此外,在离体实验中研究Shh、Hippo和Rassf2对分选出的内耳干细胞进行成球和分化实验后,可以深入探究Shh、Hippo和Rassf2在调控内耳干细胞增殖分化及毛细胞再生中的具体作用机制。(The invention relates to the field of inner ear stem cells, in particular to a method for regulating proliferation and differentiation of inner ear stem cells by Rassf2 and application of the inner ear stem cells in hair cell regeneration. The regulation and control method comprises the following steps: detecting the expression of Shh or Hippo and Rassf2 in cochlea in wild mice; secondly, researching the action of Shh or Hippo and Rassf2 on the proliferation and differentiation of the flow-sorted inner ear stem cells in an in vitro experiment; thirdly, the effects of Shh or Hippo and Rassf2 were studied on a live hair cell neomycin injury model. The invention uses Lgr5-EGFP-CreERT2 tool mice to separate a large number of inner ear stem cells (Lgr5 positive cells) accurately, and can achieve better research premise: selected mouse Lgr5 positive inner ear stem cells. In addition, after the experiments of balling and differentiating the selected inner ear stem cells are subjected to the Shh, Hippo and Rassf2 in an in vitro experiment, the specific action mechanisms of the Shh, Hippo and Rassf2 in regulating and controlling the proliferation and differentiation of the inner ear stem cells and the regeneration of hair cells can be deeply researched.)

A method for regulating proliferation and differentiation of inner ear stem cells by Rassf2, which is characterized by comprising the following steps:

detecting the expression of Shh or Hippo and Rassf2 in cochlea in wild mice;

secondly, researching the action of Shh or Hippo and Rassf2 on the proliferation and differentiation of the flow-sorted inner ear stem cells in an in vitro experiment;

thirdly, the effects of Shh or Hippo and Rassf2 were studied on a live hair cell neomycin injury model.

2. The Rassf2 method for regulating proliferation and differentiation of inner ear stem cells according to claim 1, wherein the detection method in the first step is: the expression of Shh or Hippo and Rassf2 was qualitatively and quantitatively determined at the gene and protein level using RT-PCR, Westernblotting, and immunological chemistry techniques.

3. The Rassf2 method for regulating proliferation and differentiation of inner ear stem cells according to claim 1, wherein the research method in the second step is as follows: the method comprises the steps of dissecting an Lgr5-EGFP-CreERT2 mouse cochlea, preparing a single cell suspension, screening Lgr5 positive inner ear stem cells by a flow cytometer, applying small molecules in the screened inner ear stem cells or constructing knock-down or over-expression viruses of Shh or Hippo and Rassf2, and regulating the activity of Shh or Hippo and Rassf2, so that the proliferation and differentiation of the inner ear stem cells are regulated, and the regeneration of hair cells is promoted.

4. The Rassf2 method for regulating proliferation and differentiation of inner ear stem cells according to claim 1, wherein the research method of step three is: the activity of Shh or Hippo and Rassf2 is regulated in cochlear tissue cells cultured in vitro through the screened small molecules or the construction of Shh and Rassf2 knockdown or over-expression viruses, so that the regeneration of hair cells in the damaged cochlear tissue is promoted.

Use of Rassf2 in hair cell regeneration, comprising Rassf2 as claimed in any of claims 1 to 4, and Rassf2 in hair cell regeneration.

Technical Field

The invention relates to the field of inner ear stem cells, in particular to a method for regulating proliferation and differentiation of inner ear stem cells by Rassf2 and application of the inner ear stem cells in hair cell regeneration.

Background

Auditory perception is one of the most important perceptions of humans and also the basis for verbal communication. Auditory perception serves as a basis for language recognition and understanding, and plays an important role in human social activities. Due to rapid development of social economy and industrialization, increasing aging of population, abuse of ototoxic drugs, noise and other environmental pollution factors, the number of people suffering from various degrees of otopathy is increasing year by year. Statistics by WHO2019 show that about 5% of the world population, i.e., 4.66 million people, suffer from disabled hearing loss, of which 3400 million are children.

Hearing loss and deafness have become global problems that severely threaten human health and affect people's quality of life, with sensorineural deafness accounting for about 63% of deaf patients. Sensorineural deafness is caused by abnormalities of the cochlear vestibulocochlear nerve or the central auditory system, and is mainly characterized by hearing vulnerability and outer hair cell damage in the high-frequency region of the cochlea. At present, methods for treating sensorineural deafness mainly comprise artificial cochlea, small molecule drugs, stem cell therapy, gene therapy and the like, and with continuous and deep research on hearing impairment mechanisms, more and more researches show that stem cell transplantation therapy has potential feasibility for sensing the sensorineural deafness. Stem cells are a population of cells with self-renewal, high proliferation and multipotentiality that can differentiate into tissue-specific cells depending on the surrounding environment. Damaged or degenerative recipient tissues can be repaired or replaced by implanting stem cells into the recipient tissues or activating endogenous stem cells, and on the other hand, stem cells can also be applied to gene therapy as therapeutic gene carrier cells. How to regulate stem cells to regenerate hair cells has great significance for treating sensorineural deafness. At present, the medical means is mainly to assist a patient to recover certain auditory function through the cochlear implant, but the treatment is only temporary and permanent, in addition, the cochlear implant effect varies from person to person, but only part of people can reach or approach the hearing level of normal people. Therefore, how to repair and regenerate inner ear hair cells after damage and loss is the focus of recent research in the hearing field.

The Hedgehog pathway is a highly conserved signaling pathway that plays a critical role in the early stages of inner ear development, including the proliferation of progenitor cells and their subsequent cell fate determination and differentiation. Most vertebrate species have three Hedgehog genes (Sonic Hedgehog (Shh), Indian Hedgehog (Ihh) and Desert Hedgehog (Dhh). the morphogen Shh is secreted by the spinal cord and the floor and plays an important role in dorsal/ventral mapping of the neural canal and paraxial structures.

Rassf2(Ras association domain family member 2) is one of the Ras family members. The Rassf family (Ras-associated region family), consisting of ten Rassf 1-Rassf 10 members, all of which contain a distinctive Ras-associated region (Ra domain), RASSF2 can bind directly to K-Ras in a GTP-dependent manner via the Ras effector domain. Thus, RASSF2 shows the basic features of the Ras effector. RASSF2 interaction with Ras appears to be directed against K-Ras, since only weak interaction with H-Ras is detected. Overexpression of RASSF2 inhibited lung tumor cell growth, rather than promoted transformation. Activated K-Ras enhances Ras sf 2-mediated growth inhibition, and appears to be involved in apoptosis and cell cycle arrest. Analysis of the expression of RASSF2 protein in a range of human lung tumor cell lines shows that the protein is often down-regulated. Besides the RA structural domain common to RASSF family members, the RASSF2 protein structure also comprises a SARAH structural domain, and the SARAH structural domain enables RASSF2 to regulate the activity and stability of MST1 and MST2 in a Hippo pathway. MST1 and MST2 have functions of inhibiting cell overgrowth and promoting apoptosis. Ras proteins regulate a wide range of biological processes by interacting with a variety of effector proteins. Although the activated forms of Ras are commonly associated with tumorigenesis, they may also cause growth antagonism. These include senescence, cell cycle arrest, differentiation and apoptosis. The underlying mechanisms by which these growth activities are inhibited are poorly understood. Thus, by inhibiting the expression of Rassf2, it is possible to relieve cell cycle arrest and promote differentiation and proliferation. The invention can promote hair cell regeneration by regulating Rassf2 and hippo pathway coordination, thereby providing a molecular mechanism basis for treating sensorineural deafness.

The defects existing in the prior art are as follows:

1: the inner ear stem cells are difficult to obtain, and the sorting efficiency is low: since mouse cochlear stem cells are mainly present in the supporting cells under hair cells, but not all supporting cells are stem cells, only a portion can have the potential to regenerate or differentiate into hair cells. Therefore, the accurate and efficient separation and obtaining of the inner ear stem cells is a current problem. At present, the simple regulation of wnt or notch channels has low hair cell differentiation rate, and regulatory genes bHLH, Atoh, EGF and the like for inner ear hair cell regeneration have been studied in a large quantity, but other molecular mechanisms are incomplete.

2: in the current research field, the research foundation for the relevant effects of Shh or Hippo and Rassf2 in the hearing field is very small, and therefore corresponding special reagents and drugs are lacking.

3: currently, the common Cochlear Implant (CI) is the main neural repair treatment for hearing recovery of deaf people at present, and it acts by directly stimulating the spiral ganglion neurons. However, the complete recovery of sensorineural hearing loss (SNHL) is still quite limited due to the loss or damage of either or both of inner ear sensory cells or helical ganglion neurons (SGN), which is the reason for limiting the recovery of most hearing-impaired patients, and therefore the effect depends entirely on the number and quality of remaining hair cells, and is palliative.

Disclosure of Invention

In order to solve the defects in the background art, the invention aims to provide a method for regulating and controlling proliferation and differentiation of inner ear stem cells by Rassf2 and application of the method in hair cell regeneration, and solves the problems that the inner ear stem cells are difficult to obtain and low in sorting efficiency.

The purpose of the invention can be realized by the following technical scheme:

the Rassf2 method for regulating proliferation and differentiation of inner ear stem cells comprises the following steps:

detecting the expression of Shh or Hippo and Rassf2 in cochlea in wild mice;

secondly, researching the action of Shh or Hippo and Rassf2 on the proliferation and differentiation of the flow-sorted inner ear stem cells in an in vitro experiment;

thirdly, the effects of Shh or Hippo and Rassf2 were studied on a live hair cell neomycin injury model.

Further, the detection method of the first step is as follows: the expression of Shh or Hippo and Rassf2 was qualitatively and quantitatively determined at the gene and protein level using RT-PCR, Westernblotting, and immunological chemistry techniques.

Further, the research method of the second step is as follows: the method comprises the steps of dissecting an Lgr5-EGFP-CreERT2 mouse cochlea, preparing a single cell suspension, screening Lgr5 positive inner ear stem cells by a flow cytometer, applying small molecules in the screened inner ear stem cells or constructing knock-down or over-expression viruses of Shh or Hippo and Rassf2, and regulating the activity of Shh or Hippo and Rassf2, so that the proliferation and differentiation of the inner ear stem cells are regulated, and the regeneration of hair cells is promoted.

Further, the research method of the third step is as follows: the activity of Shh or Hippo and Rassf2 is regulated in cochlear tissue cells cultured in vitro through the screened small molecules or the construction of Shh and Rassf2 knockdown or over-expression viruses, so that the regeneration of hair cells in the damaged cochlear tissue is promoted.

The application of Rassf2 in hair cell regeneration comprises the Rassf2, and the Rassf2 is applied in hair cell regeneration.

The invention has the beneficial effects that:

the invention uses Lgr5-EGFP-CreERT2 tool mice to separate a large number of inner ear stem cells (Lgr5 positive cells) accurately, and can achieve better research premise: selected mouse Lgr5 positive inner ear stem cells. In addition, after the experiments of balling and differentiating the selected inner ear stem cells are subjected to the Shh, Hippo and Rassf2 in an in vitro experiment, the specific action mechanisms of the Shh, Hippo and Rassf2 in regulating and controlling the proliferation and differentiation of the inner ear stem cells and the regeneration of hair cells can be deeply researched.

Drawings

The invention will be further described with reference to the accompanying drawings.

FIG. 1 is a schematic representation of the enhancement of Hair Cell (HC) differentiation of inner ear progenitor cells by Shh signaling in vitro in accordance with the present invention;

FIG. 2 is a schematic representation of the absence of neomycin treatment, without triggering of Shh signaling to support cell proliferation or formation of new HC, in accordance with the present invention;

FIG. 3 is a schematic representation of the inhibition of proliferation of Lgr5 positive cells by the knock-out Rassf2 of the present invention;

FIG. 4 is a schematic representation of the inhibition of differentiation of Lgr5 positive cells by the knock-out Rassf2 of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention researches the effect of Shh, Hippo and Rassf2 on the regeneration of inner ear hair cells: firstly, in a single cell suspension prepared by digesting a cochlea basement membrane of an Lgr5-EGFP-CreERT2 mouse, Lgr5 positive inner ear stem cells are sorted by a flow cytometer to be cultured, expression levels of Shh and Rassf2 in the cells are up-regulated or down-regulated by a cell transfection technology, and finally, in a mouse living hair cell neomycin injury model, the expression levels of Shh and Rassf2 in tissues are regulated and controlled by a virus infection mode, so that the function and the mechanism of Shh and Rassf2 in regulation and control of hair cell regeneration are researched.

The application of Shh and Rassf2 in regulating the proliferation and differentiation of inner ear stem cells in hair cell regeneration has been specifically studied and includes several aspects:

first, the expression of Shh or Hippo and Rassf2 in cochlea was detected in wild type mice

The experimental contents are as follows:

1. and (3) RT-PCR detection:

taking 15-20 wild mice of P3, taking out a cochlea basilar membrane, and extracting total RNA by using a TRIzol method; the extracted RNA is used as a template, oligo (dT) and reverse transcriptase and other components are added to perform reverse transcription reaction, and a specific primer is used for PCR reaction to amplify a target band. Identifying the expression of Shh or Hippo and Rassf2 in mouse cochlea by agarose gel electrophoresis;

2. westernblotting detection:

taking 15-20P 3 wild mice, taking out a cochlea basement membrane, adding a proper amount of protein lysate and a corresponding protease inhibitor, fully grinding on ice to ensure that the protein is fully released, centrifuging, removing tissue fragments which are not fully cracked, adding SDS (sodium dodecyl sulfate) and boiling to denature the protein; westernblotting experiments were performed using antibodies specific to Shh or Hippo and Rassf2 to identify Shh and Rassf2 expression in mouse cochlea;

3. immunological chemistry assay:

taking 2-3 wild mice of P3, taking out a complete cochlea basal membrane, and paving the intact cochlea basal membrane on a glass slide coated with Cell-Tak according to the original shape; adding 4% PFA solution, fixing for 1h at room temperature, washing with PBS solution containing 0.1% TritonX-100 for several times, and sealing for 1h at room temperature with sealing liquid; and (3) performing incubation by using specific antibodies of Shh or Hippo and Rassf2 and corresponding secondary antibodies, washing, adding an anti-fluorescence quenching mounting solution, and performing mounting. The expression of Shh or Hippo and Rassf2 proteins in mouse cochlea was identified by observation under a confocal laser microscope.

Secondly, research on the regulation mechanism of Shh or Hippo and Rassf2 on the proliferation and differentiation of flow-sorted inner ear stem cells in an ex vivo experiment

The experimental contents are as follows:

1. balling experiments (sphereaassay):

lgr5 positive inner ear stem cells were selected from Lgr5-EGFP-CreERT2 mouse cochlea and cultured, and then transfected with siRNA of Shh and Rassf2, so that the expression of Shh or Hippo and Rassf2 in the cells was inhibited. EdU is added into culture solution of cultured days 2, 3 and 4 to detect the proliferation condition of Lgr5 positive inner ear stem cells, and meanwhile, the influence of Shh or Hippo and Rassf2 knock-down on the cell balling capacity and the passaging capacity is observed through a cell balling experiment and a passage experiment. Lgr5 positive inner ear stem cells were selected from the cochlea of an Lgr5-EGFP-CreERT2 mouse and cultured, and then transfected with plasmids to overexpress Shh or Hippo and Rassf 2. Adding EdU into the culture solution of cultured days 2, 3 and 4 to detect the proliferation condition of Lgr5 positive inner ear stem cells, and simultaneously observing the influence of Shh or Hippo and Rassf2 on the cell balling capacity and the passaging capacity through a cell balling experiment and a passage experiment;

2. differentiation experiments (differential assays):

lgr5 positive inner ear stem cells were selected from the cochlea of an Lgr5-EGFP-CreERT2 mouse, cultured, and transfected with siRNAs against Shh or Hippo and Rassf2, so that the expression of Shh and Rassf2 in the cells was inhibited. And adding EdU into culture solution of cultured 2, 3 and 4 days to detect the proliferation condition of Lgr5 positive inner ear stem cells, and observing the change of hair cell number, cell ball number, EdU +/Myo7a + hair cell number after Shh and Rassf2 knockdown through a differentiation experiment. Lgr5 positive inner ear stem cells were selected from the cochlea of an Lgr5-EGFP-CreERT2 mouse and cultured, and then transfected with plasmids to overexpress Shh or Hippo and Rassf 2. The proliferation of Lgr5 positive inner ear stem cells was detected by adding EdU to the culture medium at days 2, 3 and 4 of culture, and the changes of hair cell number, cell ball number, EdU +/Myo7a + hair cell number after overexpression of Shh or Hippo and Rassf2 were observed through differentiation experiments. FIG. 1 shows that Shh signaling enhances Hair Cell (HC) differentiation of inner ear progenitor cells in vitro.

Thirdly, researching how Shh and Rassf2 regulate and control inner ear stem cells in a live hair cell neomycin damage model, thereby promoting hair cell regeneration

The experimental contents are as follows:

1. taking 2-3 wild mice of P3, dissecting under a dissecting mirror, taking out a complete cochlea basement membrane for culture, and simultaneously adding a proper amount of neomycin and specific damaged hair cells into a culture solution;

2. the culture solution is added with a virus vector for inducing or inhibiting the expression of Shh or Hippo and Rassf2, and cochlear cells can up-regulate or down-regulate the expression of Shh or Hippo and Rassf2 after cochlear tissues are infected. (Note: Here, experiments were carried out by selecting viral vectors that induce or inhibit the expression of Shh or Hippo and Rassf2 based on the results of the above two-part step 1 and step 2 experiments)

3. EdU was added to the culture medium on days 2, 3, and 4 of the culture to detect the proliferation of Lgr 5-positive inner ear stem cells, and the regeneration of hair cells was observed by Myo7a staining.

As shown in figure 2, activation of Shh signaling did not trigger the support of cell proliferation or new HC formation without neomycin treatment. Fig. 3 is a schematic diagram showing that the Rassf2 knockout inhibits proliferation of Lgr5 positive cells, and fig. 4 is a schematic diagram showing that Rassf2 knockout inhibits differentiation of Lgr5 positive cells.

In summary, the scheme of the present invention can be summarized as follows:

firstly, the method comprises the following steps: the expression of Shh or Hippo and Rassf2 was detected in wild type mouse cochlea: the expression of Shh and Rassf2 is detected qualitatively and quantitatively at the gene and protein level by using RT-PCR, Westernblotting and immunological chemistry technical methods;

II, secondly: effect of Shh or Hippo and Rassf2 in flow sorted Lgr5 positive inner ear stem cells: the method comprises the steps of dissecting an Lgr5-EGFP-CreERT2 mouse cochlea, preparing a single cell suspension, screening Lgr5 positive inner ear stem cells by a flow cytometer, applying small molecules in the screened inner ear stem cells or constructing knock-down or over-expression viruses of Shh or Hippo and Rassf2, and regulating the activity of Shh or Hippo and Rassf2, so that the proliferation and differentiation of the inner ear stem cells are regulated, and the regeneration of hair cells is promoted;

thirdly, the method comprises the following steps: the effects of Shh or Hippo and Rassf2 were studied on a live hair cell neomycin injury model: the activity of Shh or Hippo and Rassf2 is regulated in cochlear tissue cells cultured in vitro through some screened small molecules or Shh and Rassf2 knockdown or over-expression viruses, so that the regeneration of hair cells in damaged cochlear tissues is promoted.

Through the technical scheme, the regeneration of inner ear hair cells is promoted in vitro through small molecule and virus infection technology. Provides a new treatment idea and method for hearing impairment caused by hair cell injury in clinic.

The invention uses Lgr5-EGFP-CreERT2 tool mice to separate a large number of inner ear stem cells (Lgr5 positive cells) accurately, and can achieve better research premise: selected mouse Lgr5 positive inner ear stem cells. In addition, after the experiments of balling and differentiating the selected inner ear stem cells are divided by Shh, Hippo and Rassf2 in an in vitro experiment, the specific action mechanisms of Shh, Hippo and Rassf2 in regulating and controlling the proliferation/differentiation of the inner ear stem cells and the regeneration of hair cells can be deeply explored.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed.