TWI630272B - Method for inhibiting heat shock protein 27 promoting differentiation of placenta-derived multifunctional cells into nerve cells and use thereof - Google Patents

Abstract

The invention provides a method for promoting differentiation of a placenta-derived multifunctional cell into a nerve cell, which comprises the following steps: (1) preparing a placenta-derived multifunctional cell and inhibiting the expression of heat shock protein 27 in a placenta-derived multifunctional cell; (2) The placenta-derived multi-functional cells inhibited by heat shock protein 27 were induced to differentiate with 3-isobutyl-1-methylxanthine to obtain nerve cells. The present invention achieves an effect of promoting the differentiation of placenta-derived multifunctional cells into neurons by 3-isobutyl-1-methylxanthine by inhibiting the expression of heat shock protein 27.

Description

Method for inhibiting heat shock protein 27 and promoting differentiation of placenta-derived multifunctional cells into nerve cells and use thereof

The present invention relates to a method for promoting differentiation of a placenta-derived multifunctional cell into a neural cell, particularly by inhibiting heat shock protein 27. The invention further relates to the use of a ribonucleic acid which inhibits the expression of heat shock protein 27, in particular to a medicament for preparing a multi-function cell for promoting placenta-derived cells to induce differentiation into nerve cells by 3-isobutyl-1-methylxanthine. .

Regenerative medicine is a treatment method in which autologous cells are used to repair damage or functional failure of tissues and organs, and they are unable to repair themselves by themselves. Because they are less likely to produce autoimmune system-related rejection, patients can be exempted from disease. The pain of having to take anti-rejection drugs for life is limited by receiving transplanted organs. How to use stem cells to cultivate functional cells is also a challenge for regenerative medicine in clinical treatment. If the proliferation and differentiation of cells cannot be effectively controlled, it may cause them to evolve into cancer cells and harm the human body. Therefore, it is a very important issue to fully understand the message transmission process during cell differentiation. If we can find out the decision-making factors of stem cell differentiation, and infer the mechanism of its signal transmission, we can induce or block it, and artificially regulate the differentiation direction of stem cells and regulate the fate of cells.

Regarding the application of neural stem cells, it has been progressed to implant neural stem cells into animals, and in addition to successful survival, they can differentiate into neurons and glial cells, which shows that it is feasible to treat them with stem cells. However, it is not easy to treat with neural stem cells. In addition to whether the tissue is repulsive, it is necessary to consider the invasive process of acquiring neural stem cells, involving legal and moral perceptions, etc., which are faced in clinical application. Difficulties.

Placenta-Derived Multipotent Cells (PDMCs) have superior differentiation ability into adipocytes, osteoblasts, hepatocytes, and nerve cells, and the source is not ethical, and is the preferred adult mesenchymal The source of stem cells. However, there are still many problems to be solved about how to regulate the placenta-derived multi-functional cells to become nerve cells.

In view of this, how to develop a method that achieves a source of unethical problems and can be quickly divided into nerve cells, the existing technology needs to be improved.

In order to overcome the shortcomings of the prior art, it is an object of the present invention to provide a method for inhibiting the expression of heat shock protein 27 (HSP27, also known as heat shock protein beta-1, HSPB1) to achieve a multi-function of placenta source. The effect of cell differentiation into nerve cells.

In order to achieve the above object, the present invention provides a method for promoting differentiation of placenta-derived multifunctional cells (PDMCs) into neural cells, comprising the steps of: (1) preparing a placenta-derived multifunctional cell and in a placenta-derived multifunctional cell; Inhibition of heat shock protein 27 expression; and, (2) induction of placenta-derived multifunctional cells inhibited by heat shock protein 27 by 3-isobutyl-1-methylxanthine (IBMX) Differentiate and obtain nerve cells.

The "nerve cell" of the present invention, also known as neuron, is one of the structural and functional units of the nervous system.

Preferably, in the step (1), the inhibition of heat shock protein 27 expression comprises expression of a gene which inhibits heat shock protein 27 or inhibition of protein expression of heat shock protein 27.

Preferably, the gene expression inhibiting heat shock protein 27 is via RNA interference (RNAi).

Preferably, the ribonucleic acid interference comprises using small interfering RNA (siRNA), small molecule ribonucleic acid (microRNA), small hairpin ribonucleic acid (shRNA), double-stranded ribonucleic acid (dsRNA) or analog.

Preferably, the nerve cells are glutamatergic neurons.

The present invention further provides a use of a ribonucleic acid which inhibits the expression of heat shock protein 27, which is used for the preparation of a medicament for promoting differentiation of placenta-derived multifunctional cells into neural cells by 3-isobutyl-1-methylxanthine.

Preferably, the pharmaceutical product comprises a pharmaceutically acceptable carrier.

More preferably, the carrier comprises a liposome and other carriers similar or suitable for use in the present invention.

An advantage of the present invention is that by inhibiting the expression of heat shock protein 27, an effect of promoting differentiation of placenta-derived multifunctional cells into neuronal cells by 3-isobutyl-1-methylxanthine is achieved; wherein heat shock protein 27 is inhibited; The main pathway for the differentiation of nerve cells.

1A is an immunofluorescence staining diagram of cells of the present invention with or without overexpression of heat shock protein 27; fluorescence of cells on the first, second, and third days after induction of differentiation into two groups by two groups And phase contrast microscopy; the red arrow points to the location of the nerve cells, and the yellow arrow points to the location of the cells that overexpress the heat shock protein 27.

Figure 1B is a bar graph of the number of neurons in the present invention with or without overexpression of heat shock protein 27; observed by phase contrast microscopy in two groups (no overexpression of heat shock protein 27, no overexpression of heat shock protein 27) The results of counting and quantifying the cells with neuronal cell morphology in six observation fields were randomly selected; * indicates p<0.05.

1C is an immunofluorescence staining pattern of overexpression of heat shock protein 27 of the present invention; the photos of the group are all in the same field of view, but heat shock protein 27 (green) and pan-neuronal cell marker protein (Pan-) are observed at different excitation wavelengths. Neuronal Marker, PNM, red) and nucleus (4',6-diamidino-2-phenylindole) 哚, 4', 6-diamidino-2-phenylindole, DAPI, blue), the rightmost photo is the result of superimposing the three photos on the left (Merge); the red arrow points to the pancreatic nerve cells The location of the cell of the marker protein, indicated by the yellow arrow, is the location of the cell that overexpresses heat shock protein 27.

1D is an immunofluorescence staining diagram of the present invention without overexpression of heat shock protein 27; the photos of the group are all in the same field of view, but no excessive expression of heat shock protein 27 (green), pan-neuronal cell marker protein observed at different excitation wavelengths (PNM, red) and the nucleus (DAPI, blue), the rightmost photo is the result of stacking the three photos on the left (Merge).

2A is a bar graph showing the presence or absence of inhibition of heat shock protein 27 gene expression in the present invention; short hairpin RNA luciferase (shLuc) is used as a control group, and small hairpin RNA inhibits heat shock protein. 27 (shHSP27) was used as an experimental group for inhibiting the expression of heat shock protein 27.

Fig. 2B is an electrophoresis pattern showing the presence or absence of inhibition of the protein expression of heat shock protein 27 in the present invention.

Fig. 2C is a photograph of a cell which inhibits the expression of heat shock protein 27 according to the present invention; the presence of PDMCs which inhibit heat shock protein 27 expression (shHSP27) and does not inhibit heat shock protein 27 expression (shLuc), the left side is Before induction of differentiation (untreat), the right side was induced by differentiation with IBMX 0.4 mM.

Figure 2D is a diagram showing the immunocytochemical staining (ICC) of the present invention for inhibiting the expression of heat shock protein 27; observed for PDMCs which inhibit heat shock protein 27 expression (shHSP27) and no heat shock protein 27 expression (shLuc) Inducing differentiation of neural cells; the target of immunocytochemical staining is neuron specific enolase (NSE), in which the neural network formed by nerve cells that inhibit heat shock protein 27 and simultaneously differentiated by IBMX is on the right side. The three images (a, b, c) of the partial enlargement are indicated by red arrows.

2E is an immunofluorescence staining pattern for inhibiting the expression of heat shock protein 27 according to the present invention; immunofluorescence staining is performed on neuronal important marker proteins, including Tau protein and second microtubule protein (microtubule). -associated protein 2, MAP2), Tuj 1 protein (neuron- Specific class III beta-tubulin), and medium-sized neurofilament medium (NFM); fluorescein isothiocyanate (FITC) as a secondary antibody with green fluorescence and DAPI with blue for nuclei Where, and the result of superimposing FITC and DAPI (Merge).

Figure 2F is a bar graph showing the presence or absence of inhibition of heat shock protein 27 expression in the present invention; quantification of Tau, MAP2, Tuj 1 and NFM, respectively; * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001.

Fig. 3A is a graph showing the measurement of calcium ion concentration in the heat shock protein 27 PDMCs of the present invention; the arrow indicates the time point at which glutamate is added.

FIG. 3B is an immunofluorescence staining diagram showing the presence or absence of inhibition of heat shock protein 27 in the differentiation of PDMCs into glutaminic neurons; from left to right, respectively, an important biomarker of glutamate neurons: first type capsule Vesicle glutamate transporter 1 (VGLUT1), synaposomal-associated protein 25 (SNAP25), N-methyl-D-aspartate receptor (N-methyl -D-aspartate receptor, NMDAR); FITC as a biomarker for secondary antibodies with green fluorescence, DAPI with blue for the nucleus, and FITC and DAPI superimposed; red arrow (arrow) for dendrites The red triangle (arrowhead) represents the synapse.

Fig. 3C is an immunofluorescence staining diagram showing the presence or absence of inhibition of heat shock protein 27 in the differentiation of PDMCs into glutamateurgic neurons; the left column is an important biomarker of other types of neurons from top to bottom: dopaminergic nerve Choline acetyltransferase (ChAT), alanine butyric acid neuron in tyrosine hydroxylase (TH), cholinergic neuron (dopaminergic neuron) GABAergic neuron) glutamic acid decarboxylase (GAD67); FITC as a biomarker for secondary antibodies with green fluorescence, DAPI with blue for the nucleus, and FITC and DAPI superimposed result. The middle column is a phase difference photo. The right column PC12 cells (a conventional neural cell strain) were used as positive control groups as various biomarkers.

The technical means adopted by the present invention for achieving the object are further explained below in conjunction with the drawings and the preferred embodiments of the present invention.

Preparation Example 1

Refer to the Taiwan invention patent I299752 for separation of placenta-derived multi-function cells (PDMCs): after obtaining a full-term placenta in vitro, wash the tissue block with phosphate-buffered saline (PBS) and use 0.25 at 37 °C. The slurry was obtained by digesting with ethylene diaminetetraacetic acid (EDTA) for 10 minutes. Subsequently, the homogeneous slurry was centrifuged and centrifuged in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal calf serum, 100 U/mL penicillin and 100 μg/mL streptomycin to obtain PDMCs. And cultured at 37 ° C, 5% CO ₂ .

Preparation Example 2

The HSP27 gene (human placenta cDNA; purchased from Sigma-Aldrich, St. Louis, MO, USA) was transfected into pEGFP-C3 (purchased from Clontech Laboratories, Mountain View, CA, USA) to obtain pEGFP-C3/HSP27, and The integrity of the sequence was confirmed by DNA sequencing.

Preparation Example 3

pLKO.1-puro plasmid-based shRNA was obtained from the National RNAi Core Facility (National RNAi Core Facility, Academia Sinica, Taipei, Taiwan), including shLuc (Clone ID: TRCN0000072249) and shHSP27 (Clone ID: TRCN0000072249). Purified shLuc and shRNA plasmids are shHSP27 of liposomes (Lipofectamine ^® available from Invitrogen / Life Technologies, Carlsbad, CA , USA), expression plasmid (pMD2.G) and a packaging vector (psPAX2) together transfected into cells H293T To generate a lentivirus containing shRNA, and then infect each lentivirus into PDMCs.

Example 1 Overexpression of heat shock protein 27 inhibits neuronal differentiation of PDMCs

Preparation Example 1 taken from the logarithmic growth phase to PDMCs treated to 0.4mM IBMX 6 hours followed using FuGENE ^® HD reagent (available from Roche Diagnostics GmbH, Mannheim, Germany) prepared in Example pEGFP-C3 2 / HSP27 and pEGFP- C3 was transfected into PDMCs for 2 days, in which pEGFP-C3/HSP27 was preceded by a heat shock protein (GFP) gene. If transfected with PDMCs, it will emit green fluorescence, which may indicate excessive expression of heat shock protein 27. However, pEGFP-C3 transfected cells showed only green fluorescence, which was used as the control group of the experiment; in addition, PNM was used as a marker for pan-neuronal cells.

Referring to Figure 1A, the group with overexpression of heat shock protein 27 (pEGFP-C3/HSP27) has a relatively small number of neurons compared to the group without overexpression of heat shock protein 27 (pEGFP-C3). Figure 1B shows the results of dividing the number of nerve cells in the two groups with or without overexpression of heat shock protein 27 by the total number of cells. The number of nerve cells in the group with excessive expression of heat shock protein 27 is the first day, the second day. Days or days 3 were less than those without overexpression of heat shock protein 27.

Referring to Figure 1C, in the group with overexpression of heat shock protein 27 (green fluorescence on the left side in Figure 1C), the cell has no neuronal differentiation; in addition, there is excessive expression of heat shock protein 27 The cell will not be infected with the pan-neuronal cell marker protein (red); conversely, if the cell is stained with the pan-neuronal cell marker protein (red), the cell will not have a green fluorescent response.

Please refer to Figure 1D, but in the group without excessive expression of heat shock protein 27, it will be found that both green and red fluorescence appear in the same cell. And the cells stained with green fluorescence will show the appearance of nerve differentiation. It can be confirmed that in PDMCs, if the expression level of heat shock protein 27 is too high, the neural differentiation ability of PDMCs will be inhibited.

Example 2 Inhibition of heat shock protein 27 promotes neuronal differentiation of PDMCs

The PDMCs infected with shLuc of Preparation Example 3 and the PDMCs infected with shHSP27 were used, and RNAi of heat shock protein 27 was shRNA-mediated to silence the heat shock protein 27 gene. Referring to Figures 2A and 2B, the gene expression of heat shock protein 27 was reduced to 10% (Fig. 2A) after 18 days of infection, and the amount of protein expression was also significantly reduced (Fig. 2B).

The PDMCs with shLuc and PDMCs with shHSP27 were infected for 18 days (or the PDMCs with shHSP27 showed a gene expression of heat shock protein 27 of 10%), and induced with 0.4 mM IBMX for 6 hours. Referring to Figure 2C, the number of neurons differentiated by PDXs inhibiting heat shock protein 27 expression group (shHSP27) stimulated by IBMX was stimulated by IBMX compared to PDMCs without inhibition of heat shock protein 27 expression group (shLuc). The number of differentiated nerve cells is significantly increased. Referring to FIG. 2D, there are three graphs that inhibit the expression of heat shock protein 27 and further form a neural network by nerve cells in the IBMX-stimulated differentiation group, such as a partial enlargement of the immunocytochemical staining map (Fig. 2D right a, b) , c) where the red arrow points.

The nerve cells inhibiting the expression of heat shock protein 27 were subjected to immunofluorescence staining, and immunofluorescence staining of important neuronal marker proteins, including Tau protein, MAP2 protein, Tuj 1 protein and NFM protein, respectively. Referring to Fig. 2E, it was found that the expression level of the important marker proteins of each neuron in the heat shock protein 27 group (shHSP27) was higher than that of the non-inhibitory heat shock protein 27 group (shLuc). Light intensity, and the cells stained have the appearance of nerve cells. Further, these fluorescence intensities were quantified, as shown in Fig. 2F, and the results showed that the expression of the important marker proteins of each neuron in the heat shock protein 27 group (shHSP27) was inhibited from the heat shock protein 27 group (shLuc). ) is about five times better. This proves that in cells, if the expression of heat shock protein 27 is inhibited, it will promote the differentiation of nerve cells.

Example 3 Inhibition of heat shock protein 27 promotes differentiation of PDMCs into glutamatergic neuron

In the same manner as in Example 2, PDMCs having shLuc of Preparation Example 3 and PDMCs having shHSP27 were taken. The green fluorescent calcium indicator fluo-4 AM was used as a method for detecting the behavior of calcium ions in and out of cells. 20 μM glutamic acid was added to the IBMX-induced heat shock protein 27 expression group (shHSP27) and the IBMX-induced non-inhibitory heat shock protein 27 expression group (shLuc), and the intracellular calcium concentration was observed. .

Referring to Figure 3A, in a functional analysis, it was found that there was an inhibition of heat shock protein 27 expression (shHSP27) group added to glutamate compared with no inhibition heat shock protein 27 expression group (shLuc), calcium ion influx The ability of the cells is significantly enhanced, demonstrating that this differentiated cell has a functional NMDA receptor. Referring to FIG. 3B, the immunosuppressive protein 27 expression group (shHSP27) was subjected to immunofluorescence staining, and it was found that the heat-shock protein 27 regulates the differentiation of the neural cells with important biomarkers of glutamate neurons. Contains VGLUT1, SNAP25 and NMDAR. These marker proteins demonstrate that this differentiated cell functions as a functional NMDA receptor with glutamate neurons.

In addition, we also perform immunofluorescence staining of important biomarkers of other types of neurons, including important biomarkers of other types of neurons such as tyrosine hydrolase (TH) and cholinergic neurons in dopaminergic neurons. Choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD67) in alanine butyric neurons. Referring to FIG. 3C, none of the above marker proteins can be expressed in nerve cells which inhibit the expression of heat shock protein 27.

Therefore, the results of the above functional studies and immunofluorescence staining of important biomarkers are sufficient to demonstrate that the reduction of heat shock protein 27 will contribute to the differentiation of nerve cells.

It is apparent to those skilled in the art that various modifications and variations can be made without departing from the scope and spirit of the invention. Although the present invention has been described in terms of specific preferred embodiments, it should be understood that the invention should not be In fact, in fact Various modifications to the above-described modes of the invention are also apparent to those skilled in the art.

Claims (4)

A method for promoting differentiation of a placenta-derived multifunctional cell into a neural cell, comprising the steps of: (1) preparing a placenta-derived multifunctional cell and inhibiting the expression of heat shock protein 27 in a placenta-derived multifunctional cell; and, (2) 3-isobutyl-1-methylxanthine induces differentiation of placenta-derived multifunctional cells inhibited by heat shock protein 27 to obtain nerve cells; wherein the nerve cells are glutamyl nerve cells. The method according to claim 1, wherein in the step (1), the inhibition of heat shock protein 27 exhibits a gene expression comprising inhibition of heat shock protein 27 or inhibition of protein expression of heat shock protein 27. The method of claim 2, wherein the gene expression that inhibits heat shock protein 27 is via ribonucleic acid interference. The method of claim 3, wherein the ribonucleic acid interference comprises the use of small interfering ribonucleic acid, small molecule ribonucleic acid, small hairpin ribonucleic acid, double ribonucleic acid or the like.