CN101738462A - Method for imaging live nucleus and cytoplasm and application thereof in monitoring live nucleus and cytoplasm signal pathway - Google Patents

Abstract

The invention discloses a method for nucleoplasmic imaging of living cells, comprising the following steps: (a) irradiating living cells with near-infrared femtosecond lasers focused through a microscope; (b) the effect of near-infrared femtosecond lasers focused on living cells through a microscope Generate two-photon fluorescence and second harmonic light; (c) Simultaneously image two-photon fluorescence and second harmonic light microscopy, two-photon fluorescence microscopy imaging target molecules marked by fluorescence, second harmonic light microscopy Image the nucleus. The invention also discloses the application of the above-mentioned live cell nucleoplasm imaging method in the monitoring of live cell nucleoplasm signal transduction pathway. The invention eliminates phototoxicity and photobleaching, can observe living cells for a long time without affecting the activity of cells, and maintains the functional environment of cells to the greatest extent. The invention can be applied to the research on the cell molecular level of the mechanism of tumor generation, and the research on the pharmacodynamic mechanism of traditional Chinese medicine.

Description

Live cell nucleoplasm imaging method and its application in the monitoring of nucleoplasm signal transduction pathway in live cells

technical field

The invention relates to the fields of cell molecular biology and nonlinear optical microscopic imaging, in particular to a living cell nucleoplasmic imaging method and its application in the monitoring of living cell nucleoplasmic signal transduction pathways.

Background technique

Multicellular organisms are a busy and orderly cell society. The maintenance of this kind of society not only depends on the material metabolism and energy metabolism of cells, but also depends on cell communication and signal transmission, so as to coordinate their behavior in different ways, such as Cell growth, division, death, differentiation and their various physiological functions.

The information transduction between organism cells can be realized through the direct contact of adjacent cells, but more importantly and more commonly, cells secrete various chemical substances to regulate the metabolism and function of themselves and other cells. Cells receive external signals, transduce extracellular signals into intracellular signals through a set of specific mechanisms, and finally regulate the expression of specific genes (in the nucleus) to cause cellular responses. This is the main line of the cell signaling system. The reaction series is called the cell signaling pathway (Signaling Pathway). Therefore, in the human body, the signal transduction pathway is usually composed of specific cells that secrete and release signal substances, information substances (including intercellular and intracellular proteins, molecules, ions, etc.), and target cells (including specific receptors, etc.).

Signal transduction usually includes the following steps: specific cells release information substances → information substances reach target cells through diffusion or blood circulation → specifically bind to receptors of target cells → receptors convert signals and start intracellular messenger systems → target cells produce biological effect.

Among them, transcription factors play a very important role in the transmission of information in cells. The gene transcription process is controlled by these regulatory proteins. Their structures contain specific regions, namely DNA binding regions, which can bind to target gene regulatory regions (i.e. Binding to specific DNA sequences on the promoter and enhancer). According to the difference of their DNA binding regions, transcription factors are divided into different families, such as nuclear factor NF-κB (Nuclear factor kappa B) (translated by Zhang Ning and Xu Yongjian Checking factor KB: a new therapeutic approach? German Medicine 1999, 5 :261), JAK/STAT, DREB, etc. Therefore, effective monitoring of the transnuclear-to-cytoplasmic (between the cytoplasm and the nucleus) translocation process of these transcription factors will help to enhance the understanding of intracellular signaling pathways, which will have important practical significance for us to further understand the mechanism of tumor generation and the mechanism of action of drugs.

Fluorescent labeling technology combines fluorescent markers with specific target molecules, and tracks the behavior of target molecules by studying the behavior of fluorescent markers. Fluorescent markers include all substances with fluorescent emitting groups, such as small molecule fluorescent dyes (Fluorescein, Rhodamine, Texas Red, etc.), bioluminescent macromolecules (GFP and its derivatives, etc.), quantum dots (Quantum Dots) fluorescent probes, etc. Needle etc. The binding methods of fluorescent markers and target molecules include: direct specific binding (DNA and ethidium bromide EB), indirect binding through antigen antibody mediation (immunofluorescence calibration technology), bioluminescent molecules and specific binding through genetic recombination The marking technology combined with the target molecule (such as the method for marking NF-κB with GFP used in the present invention), etc.

In the current study of single-photon confocal microscopy imaging of transcription factors (such as nuclear factor NF-κB) regulatory pathways in living cells, in order to obtain information on the dynamic distribution of fluorescently labeled NF-κB before and after activation in the nucleoplasm, it is first necessary to clarify The location of the nucleus, which is usually achieved by the following methods: (1) Fluorescently labeled proteins accumulate in the cytoplasm in large quantities, while the concentration in the nucleus is extremely low, and the location of the nucleus is roughly judged by the intensity of the fluorescence distribution; (2) Using other fluorescent The dye is used to calibrate the cell nucleus, and the concentration change of the target protein in the cell nucleus is determined by image superposition; (3) Switch to bright field to image the cell nucleus, etc. However, they bring corresponding methodological deficiencies: the premise of the implementation of method (1) is to screen for cell lines with high expression of fluorescent proteins, and this abnormally high expression will have a potential impact on the normal physiological activities of cells on the one hand, and on the other hand On the one hand, it also brings difficulties to the selection and maintenance of cell lines. Method (2) adds a nuclear fluorescent staining procedure in the sample preparation process, the entry of exogenous fluorescent dyes may have a certain impact on the physiological state of the cells, and if used in the case of low expression cell lines ( It is difficult to collect experimental data under the smallest possible external interference to the cells, so that it is impossible to realize the dynamic research under the state of causing the smallest possible external interference to the cells; method (3) frequent optical path changes bring cumbersome operations .

With the emergence of femtosecond laser technology in the 1990s, the rapid development of nonlinear two-photon microscopy imaging technology in recent years provides a more powerful tool for biomedical research at the molecular level in living cells.

Compared with single-photon confocal microscopy, two-photon microscopy has the following advantages:

(1) Since it relies on the simultaneous action of two photons in the focal plane, the possibility of non-focus planes is extremely small, with clear automatic three-dimensional (3D) tomography capabilities, while greatly reducing photobleaching outside the focal plane effects and phototoxicity, enabling long-term in vivo imaging of samples.

(2) At the same time, due to the use of longer near-infrared wavelengths, the absorption and scattering effects of biological tissues on light are reduced, and the imaging depth is greatly increased.

The first imaging mode in nonlinear two-photon optical imaging is two-photon fluorescence (Two-Photon Fluorescence, TPF) imaging mode. In recent years, due to the realization of the outstanding advantages of two-photon fluorescence imaging (TPF) over single-photon fluorescence imaging, and the maturity of corresponding technologies such as actual fluorescent probe technology, two-photon fluorescence microscopy imaging in living biological tissues and cells has been obtained. a wide range of applications.

There is another important mode in nonlinear two-photon optical imaging - Second-Harmonic Generation (SHG). The SHG phenomenon was first discovered by Franken et al. in 1961: when two photons with the same fundamental frequency interact with a sample with an asymmetric structure, the scattering produces a second harmonic optical signal (half wavelength, double energy).

Like TPF, SHG is a nonlinear optical phenomenon, because its production efficiency (p) has a nonlinear square dependence on the intensity of excitation light (I) (p=αI ² ), so it also has the ability to form high-resolution The basis of nonlinear optical imaging, thus possessing all the advantages of two-photon excited fluorescence imaging. But the two-photon fluorescence signal and the second harmonic signal have different characteristics from each other. Since SHG is a coherent scattering process, which is different from the absorption and re-emission processes involved in two-photon fluorescence generation, SHG also has its special advantage compared with fluorescence imaging: since there is no energy level transition involved in SHG, there is no energy transition in SHG. Loss, theoretically completely eliminates phototoxicity and photobleaching, and can act on living cells for a long time without damaging cell functions. At the same time, there are a large number of endogenous SHG components in biological tissues, so there is no need to add exogenous calibration substances, which minimizes possible damage and interference to biological tissues.

It should be noted that, in a large number of applications of second harmonics in biological tissues (mainly collagen fibers), what is most directly related to the present invention is that Sheppard et al obtained SHG imaging in dispersed DNA components (as shown in Figure 1 ) (herring sperm cells, purchased from Sigma) (R. Gauderson, P.B. Lukins, C.J.R. Sheppard, Simultaneous multichannel nonlinear imaging: combined two-photon excited fluorescence and second-harmonic generation microscopy, Micron 32: 685-689 (2001)).

On the imaging device, different laser light sources are used for two-photon fluorescence microscopy than for single-photon confocal microscopy. Due to the need to match the energy of absorbing two photons at the same time with the equivalent energy of absorbing one photon when a single photon is absorbed, long-wavelength (low-energy) near-infrared (Near-Infrared) light is generally used instead of a shorter wavelength ( High energy) blue-violet or ultraviolet light excitation. At the same time, the femtosecond pulsed light source generated by the femtosecond (10 ^-15 s) pulsed laser is a more commonly used two-photon excitation light source in recent years.

In a two-photon imaging system, two-photon fluorescence imaging and second harmonic imaging can be realized simultaneously.

US Patent US2005/0259A1 discloses a two-photon fluorescence and second harmonic microscopy imaging system. The system can realize excitation by the same excitation light source, and simultaneously record two-photon fluorescence and second harmonic signals (the system simultaneously collects forward-propagating second-harmonic signals and back-propagating fluorescence signals). The basic components of the system are Ti:Sappire (TS) laser (excitation wavelength 760-880nm), a Berek polarization compensator, scanning box, and a focusing objective lens (0.45-1.3NA). For the collection system, TPF is conjugated to collect the fluorescence emitted from the sample, which is divided into suitable wavelength channels by a spectroscope and an emission filter and collected by a photomultiplier tube. The second harmonic, which dominates the forward propagation, passes through the objective (0.95NA) plano-convex mirror, and the second harmonic signal is separated from excitation light and fluorescence by using bandpass and blue light filters. The photomultiplier tube is connected to the computer to obtain data.

U.S. Patent US6208886B1 discloses another schematic diagram of an optical system that can simultaneously detect two-photon fluorescence signals and second harmonic signals (excited by the same excitation light source). In this system, two-photon fluorescence signals and second harmonic signals are both back The way of collection and scanning is realized as the way of moving the platform. The mobile stage realizes the 3D mobile scanning of the sample. After the excitation light source passes through the reflector to the beam splitter, it enters the objective lens to focus on the sample, and the backpropagating fluorescence and second harmonic signals of the sample pass through the beam splitter, are focused by the lens, and are collected by the photomultiplier tube through the band-pass filter. Figure 2 shows the nonlinear spectrum obtained from chicken muscle fibers using the optical system under the excitation of 100 fs pulse and 625 nm excitation wavelength, and the SHG signal and two-photon fluorescence signal are obtained simultaneously.

Contents of the invention

The purpose of the present invention is to provide a method for imaging nuclei and cytoplasm of living cells without additional staining of nuclei.

Another object of the present invention is to provide the application of the above-mentioned nucleoplasmic imaging method in living cells in the monitoring of nucleoplasmic signal transduction pathways in living cells.

A method for living cell nucleoplasm imaging: comprising the steps of:

(a) Irradiating live cells with a near-infrared femtosecond laser focused by a microscope;

(b) Living cells produce two-photon fluorescence and second harmonic light under the action of a near-infrared femtosecond laser focused by a microscope;

(c) Simultaneously imaging two-photon fluorescence and second harmonic light microscopy, two-photon fluorescence microscopy imaging of target molecules labeled with fluorescence, and second harmonic light microscopy imaging of cell nuclei.

Further, the wavelength of the near-infrared femtosecond laser is 800-850 nm.

The application of the above-mentioned nucleoplasmic imaging method in living cells in the monitoring of nucleoplasmic signal transduction pathways in living cells, two-photon fluorescence microscopic imaging traces the distribution and migration of fluorescently labeled target molecules, and second harmonic light microscopic imaging traces the nucleus, thereby Monitoring nucleoplasmic signaling pathways in living cells.

Compared with the prior art, the present invention has the following beneficial effects:

(1) Use two-photon fluorescence microscopy (TPF) to track the activity of target molecules, and second harmonic microscopy (SHG) to locate the position of the nucleus (DNA). The combination of the two can accurately monitor the impact of stimulation on the target Molecular induction and regulation processes. Due to the characteristics of multiphoton—especially SHG imaging using endogenous cell nucleus (DNA), which theoretically eliminates phototoxicity and photobleaching—living cells can be observed for a long time without affecting the activity of cells , The functional environment of the cell is maintained to the greatest extent, so as to truly realize the dynamic, clear, and in vivo observation of the process of the target molecule being transported between the cytoplasm and the nucleus under slight external interference.

(2) Simultaneous imaging of TPF and SHG, especially the use of endogenous nuclear SHG imaging technology to explore the research of target molecular regulatory pathways, not only has a broad role in the study of tumor generation mechanisms at the cellular and molecular level, but also in traditional Chinese medicine It also has attractive application prospects in the study of the pharmacodynamic mechanism.

Description of drawings

Figure 1 is a SHG image of DNA in the nucleus of herring sperm cells.

Figure 2 is the second harmonic signal (SHG) and two-photon fluorescence signal (TPF) obtained simultaneously from chicken fibers using a two-photon optical imaging system.

Figure 3 is the SHG imaging of HeLa cancer cells (cells after the cell membrane is damaged after pouring out the culture medium PBS) under the excitation of 830nm near-infrared femtosecond laser (imaging spectral range is 405-425nm).

Figure 4 is the transmitted light imaging of HeLa cancer cells (cells after the culture solution PBS was poured out to cause cell membrane damage).

Fig. 5 is the spectrum (376-550nm) of the region where the HeLa cancer cells (the cell membrane is damaged after pouring out the culture solution PBS) are excited by the 830nm near-infrared femtosecond laser.

Fig. 6 is the technical route of two-photon fluorescence and second harmonic nonlinear optical microscopic imaging for studying the translocation of transcription factors between nucleus and cytoplasm in the present invention.

Fig. 7 is a schematic diagram of the combined optical system of two-photon fluorescence and second harmonic microscopic imaging to be used for monitoring the translocation of fluorescence-labeled transcription factors in the nucleoplasm of the present invention.

Figure 8 Schematic diagram of NF-κB transport between cytoplasm and nucleus.

Fig. 9 Schematic diagram of JAK/STAT signaling pathway.

Fig. 10 Schematic diagram of steroid receptor signaling channel.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

Figure 1 is a SHG image of DNA in the nucleus of herring sperm cells. DNA from herring sperm cells purchased from Sigma was dispersed and fixed in distilled water. The image size is 43x43um. The excitation wavelength is 800nm, and the laser power irradiated on the sample is <15mW. This figure illustrates that the DNA component of the nucleus is the endogenous (no additional staining required) SHG signal source material.

Figure 2 uses the optical imaging system (US Patent No. 6208886B1) to realize the detection of two-photon fluorescence signals and second harmonic signals from the nonlinear spectrum (100fs pulse, excitation at 625nm excitation wavelength) obtained from chicken muscle fibers, and two Subharmonic signal (SHG) and two-photon fluorescence signal (TPF). It should be noted that the legend results show that the same two-photon optical imaging system can simultaneously detect two-photon fluorescence signals and second harmonic signals, but the second harmonic signals come from fibrous tissue, not cell nuclei or DNA components.

Figure 3 is the SHG imaging of HeLa cancer cells (cells after the cell membrane is damaged after pouring out the culture medium PBS) under the excitation of 830nm near-infrared femtosecond laser (imaging spectral range is 405-425nm). Comparing the transmitted light imaging of cells shown in Figure 4 and the spectral characteristics shown in Figure 5, we can see that the image obtained in Figure 2 is a cell nucleus with a clear outline, and it is determined that the signal source of the image is the SHG signal.

Fig. 4 is a transmitted light imaging of HeLa cancer cells (cells after the culture medium PBS was poured out to cause cell membrane damage).

Fig. 5 is the spectrum of HeLa cancer cells (the cells after the cell membrane is damaged after pouring out the culture medium PBS) under the excitation of 830nm near-infrared femtosecond laser. According to the spectral characteristics of SHG, the narrow peak (415nm) is the characteristic of SHG signal. This legend shows that the complete nucleus formed by DNA can also be used as the source material of endogenous SHG signal, which further confirms that in the present invention, it is hoped to use the endogenous SHG signal of the nucleus to locate the nucleus position, display the outline of the nucleus, and trace the target molecule in the nucleoplasm Feasibility of inter-transfer.

Fig. 6 is the technical route of two-photon fluorescence and second harmonic nonlinear optical microscopic imaging for studying the translocation of transcription factors between nucleus and cytoplasm in the present invention. In the present invention, the transport of transcription factors (such as nuclear factor NF-κB) from the cytoplasm to the nucleus can be clearly observed, so on the one hand, the movement of transcription factors can be traced in living cells; at the same time, there is a clear outline of the nucleus to show transcription The movement position of the factor. Therefore, the second harmonic signal will be combined with the two-photon fluorescence signal to track and observe the whole process of the transcription factor regulation pathway from the cytoplasm to the nucleus in living cells in real time. Two-photon fluorescence signals track the activity of fluorescently labeled transcription factors (such as nuclear factor NF-κB-GFP fusion protein), and the SHG signal simultaneously images the position and outline of the nucleus, so the combined two-photon imaging can monitor the activity of transcription factors in real time , and then use the activity channels of transcription factors to explore various mechanisms related to it.

Fig. 7 is a schematic diagram of a combined two-photon fluorescence and second harmonic microscopic imaging optical system for the transport of specific proteins, ions, molecules, etc. in living cells between the cytoplasm and the nucleus of the present invention. The optical system can simultaneously realize two-photon fluorescent TPF to monitor specific proteins, ions, and molecules, and second harmonic microscopic imaging SHG to display the outline of the cell nucleus. Both two-photon fluorescence and second harmonic are back collection methods. The optical system consists of a femtosecond pulsed laser source and a two-photon microscopic imaging system (Leica TCS SP2). The laser is a Ti:Sapphire self-mode-locked femtosecond laser (wavelength adjustable range 700-1100nm, pulse width 110fs, pulse repetition frequency 76MHz, Coherent Mira 900-F); the excitation light is reflected by the beam splitter DBS and enters the scanning objective (focusing Objective lens, water immersion UplanApo/IR, 60×, NA=1.25, Olympus), focus on the sample through the objective lens, the backpropagating two-photon fluorescence and the second harmonic light generated by the sample are collected through the same objective lens, and filtered by the filter (IR Bantong, KP650, Zeiss) blocks the excitation light, focuses it through a lens, and splits the light through a prism spectrometer (the detectable spectral range is 400-850nm). The monochromatic light passing through the slit is collected by the PMT, and the two-photon excited fluorescence and the second harmonic light are respectively imaged, so as to realize high-throughput observation of the activity process of the calibration target protein between the cytoplasm and the nucleus. X, Y scanning is an optical scanning device, which realizes the moving scanning of the light beam in the X and Y directions, and the scanning in the Z direction is realized by moving the gantry.

Figure 8 is a schematic diagram of the transport of NF-κB between cytoplasm and nucleus. Nuclear factor κB (multiple refers to the heterodimer formed by p65 and p50), usually forms a trimer (NF-κB/IκB) with its inhibitory protein (NF-κB inhibitor, IκB) to inactivate (that is, have no biological activity ) state exists in the cytoplasm. Under stimulation, IκB is phosphorylated by IκB kinase (IKK) and then degraded by protease, nuclear factor κB is gradually released from the trimer, and rapidly translocates into the nucleus (nuclear translocation: exists in the cytoplasm After the protein is activated, it enters the nucleus through the nuclear membrane to exert biological activity), and specifically binds to the κB site of the promoter region on the target gene, thereby starting and regulating the corresponding gene (such as various cytokines such as TNF-α , adhesion factors and very early proteins, etc.) transcription. The NF-κB calibrated by gene recombination technology expresses the fluorescent protein GFP, so the activity of the target molecule NF-κB is tracked by two-photon fluorescence imaging, and the position and outline of the same nucleus are imaged simultaneously in the same optical imaging system, so it can be precisely monitored Regulatory pathway of the target molecule NF-κB in the nucleoplasm.

Fig. 9 is a schematic diagram of JAK/STAT signaling pathway. The JAK (Janus kinase)-STAT (signal transducer and activator of transcription) signaling pathway is a signaling pathway closely related to cell growth, proliferation and differentiation. The basic process of its signal transmission can be summarized as follows: cytokines and their corresponding receptors body binding; receptors and JAKs aggregate, and adjacent JAKs are activated by mutual phosphorylation; the JH1 domain of JAKs catalyzes the phosphorylation of tyrosine residues on the corresponding parts of STATs, and at the same time, the SH2 functional region of STATs interacts with the phosphate in receptors STATs are activated through the action of tyrosine residues; STATs enter the nucleus and interact with other transcription factors to regulate gene transcription. Utilize corresponding fluorescent technology calibration technology (gene recombination technology, immunofluorescence technology, quantum dot labeling technology) to carry out fluorescent labeling on STATs, obtain STATs target molecules with fluorescent characteristics, on this basis, combine the imaging method provided by the present invention to track target molecules The activity of STATs can accurately reveal the regulatory process of STATs crossing the nuclear membrane to initiate the expression of corresponding genes in the state of living cells. The transmembrane transport of STATs molecules is monitored through the nucleoplasmic boundary defined by TPF/SHG in the same optical system.

Fig. 10 Schematic diagram of steroid receptor signaling channel. In the figure, H refers to the ligand (steroid hormone), HSP90 refers to the heat shock protein (Heat Shock Protein), and HRE refers to the hormone response element (Hormone Response Element). Steroid hormones include glucocorticoids, mineralocorticoids, androgens, estrogens, and progestins. Steroid hormones have a small molecular weight, are insoluble in water and are soluble in fat solvents, and are easy to penetrate the lipid layer of the cell membrane and enter the cytoplasm by simple diffusion. The steroid hormone entering the cell first activates with the specific receptor in the cytoplasm to form a steroid-receptor complex. The cytoplasmic steroid receptor is a protein with a molecular weight between 50,000 and 150,000. It is highly specific and can recognize steroids. Due to the slight difference in structure, the cytoplasmic receptors of any target cell can only bind to the corresponding steroid hormone, and have only slight affinity or no binding to other steroid hormones. After the steroid hormone binds to the specific receptor protein, the conformation of the receptor protein changes, from the original inactive form to the active form, and then it can be transferred to the nucleus. The activated receptors all combine with the hormone response element (HRE) in the target gene in the form of dimer, thereby exerting its transcriptional regulation function. In this signaling pathway, steroid receptors are important protein molecules for signal transmission. In order to describe the process of nuclear translocation after the activation of the steroid receptor, the two-photon fluorescence imaging technology provided by the present invention combined with the second harmonic imaging technology is used to accurately monitor the nucleoplasmic distribution of the steroid receptor. The protein can be effectively fluorescently labeled, and the labeling method can also be pre-transcriptional gene recombination labeling or post-transcriptional immunofluorescence labeling. By performing TPF/SHG imaging on specific cells cultured in vitro in the same optical system, the position information of the cytoplasm and nucleus can be directly obtained without additional steps, and the distribution of fluorescently labeled steroid receptor molecules in the nucleus and cytoplasm can be obtained accordingly , thus inferring the transfer process of the labeled molecule.

Example 1

The first embodiment of the present invention is to use the method of the present invention to observe the translocation process of NF-κB marked by gene recombination technology GFP between nucleoplasm and tumor necrosis factor (Tumor necrosis factor) in human lung adenocarcinoma cells (ASTC-a-1). NecrosisFactor, TNF) as an activator of NF-kB signaling pathway.

Culture of human lung adenocarcinoma cells (ASTC-a-1): cell culture medium is RPMI 1640, add 15% fetal bovine serum, 2mmol/L glutamine (Glutamine), 25mmol/L HEPES, 100IU/ml penicillin and 100mg /mL streptomycin. For cell transfection, 30%-50% confluent human lung adenocarcinoma cells were co-cultured with a suspension of Lipofectin reagent and a sufficient amount of recombinant plasmid vector (gifted by Professor Schmid, University of Vienna) for 6 hours, and then supplemented with an appropriate amount of fresh culture medium for expansion. Increase culture. The process of transfection is the process of marking specific molecules in the cells. This patent adopts the method of gene recombination to carry out fluorescent labeling on the molecules, and recombines the green fluorescent protein (GFP) gene with the gene of the target molecule, and the combined gene (Exogenous gene) is transfected into the host cell, and the expression product of the foreign gene in the host cell is the fusion protein of NF-kB and GFP. This labeling method has basically no effect on the function of the target molecule itself and the physiological state of the host cell. There will be an effect, and the transfer process of target molecules can be traced by monitoring the change of fluorescence distribution.

The above-mentioned transfected cells (5ml petri dish) were cultured for 48 hours, and placed directly on the stage of a two-photon laser confocal microscope (with a cell workstation system to maintain the culture environment), using a high-magnification water mirror lens (UplanApo/IR, 60 ×, NA=1.25, Olympus) were directly immersed in the culture solution for TPF and SHG observations, and the wavelength of the near-infrared femtosecond laser was 830nm.

Firstly, the distribution of fluorescence intensity between nucleoplasm and cytoplasm in cultured cells in a resting state was recorded in real time, and then tumor necrosis factor (Tumor Necrosis Factor, TNF) with a final concentration of 5 ng/ml was added to the culture medium immediately as NF- Activator of the kB signaling pathway, the stimulating signal is transmitted to the NF-kB/I-kB complex through membrane surface receptors, resulting in the phosphorylation and degradation of I-kB, releasing NF-kB to expose its nuclear transfer site and translocate To the nucleus and start the transcription of related genes and translate them into proteins, so as to realize the response of cells to external stimuli (see Figure 6). The change in this process is reflected by the position change of the GFP fluorescent molecule linked to NF-kB. When the observed fluorescence intensity of the cytoplasm decreases while the fluorescence intensity of the nucleus increases, it reflects that the signal channel is activated and plays a role. corresponding feedback regulation.

Example 2

The second embodiment of the present invention is to use the present invention to observe the translocation process of STAT marked by the gene recombination technology GFP between nucleoplasm in human lymphocytes - Interferon (Interferon) is used as the activator of the signaling pathway STAT.

Lymphocytes were used as host cell lines for validation of the JAK/STAT signaling pathway. Whole blood comes from healthy blood donors at the blood bank, and lymphocytes are obtained from heparin-anticoagulated blood separated by lymphocyte separation fluid. RPMI1640 was used as the lymphocyte culture medium, and 10% fetal bovine serum, HEPES, antibiotics, interleukin-2 (IL-2), and phytohemagglutinin (PHA) were added. Construction of the transfection vector: The genes of the STATs family (STAT 1-6) were cloned into the carboxyl terminus of the plasmid vector pEGFP-C1 (Catalog#6084-1, Clontech). Lipofectin reagent and a sufficient amount of recombinant plasmid vectors were co-cultured with the lymphocytes cultured in vitro for 6 hours. The process of transfection is the process of labeling specific molecules in the cell, recombining the green fluorescent protein (GFP) gene with the gene of the target molecule, and transfecting the combined gene (exogenous gene) into the host cell. The expression product of the gene in the host cell is the fusion protein of STAT and GFP, and the transfer process of the target molecule can be traced by monitoring the change of the fluorescence distribution.

The above-mentioned transfected cells (5ml petri dish) were cultured for 48 hours, and placed directly on the stage of a two-photon laser confocal microscope (with a cell workstation system to maintain the culture environment), using a high-magnification water mirror lens (UplanApo/IR, 60 ×, NA=1.25, Olympus) were directly immersed in the culture solution for TPF and SHG observations, and the wavelength of the near-infrared femtosecond laser was 800 nm.

In this embodiment, interferon (Interferon) is used as a signal pathway stimulator. Interferon is a reaction product induced by host cells when the virus enters the body. After it is released from the cell, it can prompt other cells to resist the infection of the virus. After adding a small amount of interferon (final concentration 5ng/ml) to the transfected in vitro cultured lymphocytes, the interferon will bind to the interferon receptor on the cell surface, and the receptor subunit will dimerize after binding to the interferon It can activate the autophosphorylation of JAKs and affect the JAKs coupled with it. In this way, JAKs become a substrate in the process of mutual transfer of phosphate groups, and their catalytic function is activated by the phosphorylation of tyrosine residues on the activation loop of the JH1 domain, which in turn activates the inactive form of STATs in the cytoplasm. body to phosphorylate it. Activated STAT forms a dimer, and is also bound to GFP fluorescent marker, with a large molecular weight, and must pass through the nuclear pore complex (NPC) to enter the nucleus. The prerequisite for mutual recognition and interaction between STAT and Improtin is the activation of STAT. Therefore, the interferon signal passes through a series of transmissions, and finally the activated STAT enters the nucleus, thereby initiating the expression of related genes. The result of expression causes the proliferation of various lymphocytes to fight against bacteria, viruses, and foreign substances invading the body, or to attack tumor cells.

Example 3

Culture of human skeletal muscle cells: cell culture medium is DMEM, add 10% fetal bovine serum, 5uM growth factor (GrowthFactor), 2mM glutamine (Glutamine), 25mmol/L HEPES, 100IU/ml penicillin and 100mg/mL streptomycin white. For cell transfection, 30% to 50% confluent human skeletal muscle cells were co-cultured with a suspension of Lipofectin reagent and a sufficient amount of recombinant plasmid vector for 6 hours, and then an appropriate amount of fresh culture medium was supplemented for expansion culture. Construction of the transfection plasmid: the androgen receptor gene was inserted into the amino terminal of the commercialized EGFP plasmid vector, the green fluorescent protein (GFP) gene was recombined with the androgen receptor gene, and the combined gene (plasmid vector) was transfected Transfection into skeletal muscle cells, the expression product of this foreign gene in skeletal muscle cells is the fusion protein of androgen receptor and GFP. When using androgen to stimulate the above-mentioned skeletal muscle cells cultured in vitro, the transfer process of the target molecule can be traced by monitoring the change of fluorescence distribution. The proliferation level of skeletal muscle cells cultured in vitro for this signaling pathway is significantly enhanced compared with the normal culture state.

The above-mentioned transfected cells (5ml petri dish) were cultured for 48 hours, and placed directly on the stage of a two-photon laser confocal microscope (with a cell workstation system to maintain the culture environment), using a high-magnification water mirror lens (UplanApo/IR, 60 ×, NA=1.25, Olympus) were directly immersed in the culture solution for TPF and SHG observations, and the wavelength of the near-infrared femtosecond laser was 850 nm.

After the cultured cells are put into the test system, first look for cells expressing the plasmid vector (that is, search for cells with fluorescent characteristics through a fluorescence microscope), and use the TPF/SHG combination method proposed in this patent to record the fluorescence distribution between the nucleoplasm in real time. Then add androgen with a final concentration of 10, 20, and 50 ng/ml in the culture medium as an activator of the steroid receptor signaling pathway. The activator directly passes through the plasma membrane into the cytoplasm, and is specifically labeled with fluorescent Binding of the androgen receptor of the protein, causing receptor allosterism, resulting in the detachment of the heat shock protein dimer bound to the androgen receptor and exposing its DNA binding region, so that the receptor changes from an inactive form to one that can interact with DNA The active form of the action, the activated receptor protein is combined with the hormone response element (HRE) in the target gene in the form of a dimer. HRE is a short specific DNA sequence, mostly located near the promoter, generally in the Within 400bp upstream of the transcription initiation site, it indicates the key site of DNA binding to the receptor, that is, the recognition site of DNA and the receptor. The binding of HRE to the androgen receptor will lead to the expression of a series of downstream genes. One of the results is Induces the growth and proliferation of skeletal muscle cells.

From the previous discussion, it is clear that fluorescent probe labeling based on other biological methods, such as immunofluorescence, rather than purely based on gene recombination, and other fluorescent probes, such as in recent years The "Quantum dots" label used; the labeled object is not limited to other proteins, molecules, and ions of nuclear factor NF-κB; it also includes the monitoring of proteins, molecules, and ions caused by other stimulating ingredients (such as traditional Chinese medicine) , when two-photon fluorescence is used to track the marker, and second harmonic imaging is used to image the nucleus to observe the transport process of the marker between the cytoplasm and the nucleus, it is also covered by the scope of the present invention.

Claims (3)

1. a living cells caryoplasm imaging method is characterized in that comprising the steps:

(a) use the near infrared femtosecond laser irradiation living cells that focuses on through microscope;

(b) living cells produces two-photon fluorescence and second harmonic light under the effect of the near infrared femtosecond laser that microscope focuses on;

(c) simultaneously to two-photon fluorescence and second harmonic light micro-imaging, the two-photon fluorescence micro-imaging is by the target molecule of fluorescence calibration, second harmonic light micro-imaging nucleus.

2. living cells caryoplasm imaging method according to claim 1 is characterized in that: the wavelength of described near infrared femtosecond laser is 800～850nm.

3. the described living cells caryoplasm of claim 1 imaging method is in the application of living cells caryoplasm signal transduction pathway monitoring.

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