JP2000300509A - Fluorescent observation device for endoscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fluorescent observation device which enables positive fluorescent observation by using sufficient fluorescence light intensity with an inexpensive apparatus structure. SOLUTION: A vector containing a gene for developing a kind of protein that emits fluorescence with a sufficient light intensity is injected into a tissue to be observed by a treating device 7, and the tissue to be observed is irradiated with excitation light which has transmitted an excitation light transmitting filter 33 to observe a fluorescent component of an observation image through an observation light transmitting filter 41. This enables accurate fluorescent observation with fluorescence of sufficient light intensity with an inexpensive structure without employing a high-sensitive image-pickup device or the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an endoscope fluorescence observation apparatus for conducting fluorescence observation by introducing a gene that expresses a protein that emits fluorescence into a tissue to be observed.

[0002]

2. Description of the Related Art For example, in order to observe a lesion such as a tumor or a cancer cell, an agent which easily gathers on the lesion such as a tumor or a cancer cell and emits fluorescence when irradiated with excitation light is injected into a living body. The technique of performing fluorescence observation by using has been used. As a fluorescence observation apparatus for performing such fluorescence observation, for example, Japanese Patent Application No. Hei 9
In JP-A-109668, excitation light obtained through a filter that transmits the excitation light is supplied to an endoscope, and the excitation light is irradiated by the endoscope toward a target portion of a living body into which a fluorescent agent has been injected. There is shown means for obtaining an optical image due to the fluorescence of a drug through observation, and observing the subject image through a filter transmitting the fluorescence, thereby performing a diagnosis for specifying a lesion in an observation target site.

[0003]

However, the fluorescence-emitting drugs described in the prior art vary in fluorescence intensity depending on the type of tumor, cancer cells, and the like, and individual differences in living organisms.
In some cases, tumors or cancer cells could not be reliably identified due to weak fluorescence. Conventionally, even if the intensity of the excitation light is increased in order to improve this problem, an effect for sure observation has not been obtained. In addition, if a high-sensitivity imaging device is introduced to observe weak fluorescence, there is a problem that the device becomes expensive. The present invention has been made in view of the above circumstances,
It is an object of the present invention to provide an endoscope fluorescence observation device that can perform reliable fluorescence observation with a sufficient amount of fluorescence with an inexpensive device configuration.

[0004]

Means for Solving the Problems In order to achieve the above object, the present invention provides an introduction means for introducing a gene which expresses a protein which emits fluorescence into an observation target tissue, and an illumination light emitted from a light source for the observation target tissue. An endoscope for irradiating the observation target tissue to obtain an observation image of the observation target tissue, first transmission wavelength limiting means provided on an optical path of the illumination light and transmitting a wavelength for exciting the protein, and an optical path of the observation image And a second transmission wavelength limiting means for transmitting the wavelength of the fluorescence emitted from the protein.

[0005]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS (First Embodiment) An embodiment of the present invention will be described below with reference to the drawings. 1 to 5 relate to a first embodiment of the present invention, FIG. 1 is an explanatory diagram showing a configuration of an endoscope fluorescence observation apparatus, FIG. 2 is an explanatory diagram showing a configuration and operation of a pEGFP vector, FIG. Is GFP and E
FIG. 4 is an explanatory view showing an amino acid sequence related to each fluorescent function of GFP, FIG. 4 is an explanatory view showing characteristics of respective wavelength bands of excitation light and fluorescence of GFP and EGFP, and FIG. 5 is an excitation light transmission filter and an observation light transmission filter. FIG. 4 is an explanatory diagram showing characteristics of respective transmission wavelength bands.

As shown in FIG. 1, an endoscope fluorescence observation apparatus 1 according to the present embodiment includes an endoscope 2 which is inserted into a body cavity or the like to obtain an observation image of a tissue to be observed, , A light source device 3 that supplies illumination light to the imaging device, an imaging device 4 that captures an observation image obtained by the endoscope 2 to obtain an imaging signal, and a video signal that can monitor and display the imaging signal obtained by the imaging device 4. , A monitor device 6 for displaying a video signal obtained by the video processor 5, and pEGG, a microorganism containing a gene that expresses a protein that emits fluorescence.
It has a treatment tool 7 for injecting an aqueous solution containing an FP vector into a site to be observed.

The endoscope 2 is provided with an elongated insertion portion 11 to be inserted into a body cavity or the like, and is provided continuously at a base end side of the insertion portion 11.
An operation unit 12 for grasping and operating the endoscope 2, an eyepiece unit 13 which is provided continuously to a base end side of the operation unit 12 and emits an observation target image obtained by the endoscope 2, A light guide cable 14 extending from, for example, a side portion of the unit 12 and receiving illumination light from the light source device 3; and a light guide cable 14 provided at an end of the light guide cable 14,
Light guide connector 15 for detachably connecting to
And the light guide cable 14 and the operation unit 12
And a light guide 1 that passes through the insertion portion 11 and guides illumination light obtained from the light source device 3 through the light guide connector 15 to the distal end portion 11a of the insertion portion 11.
6, a light distribution optical system 17 provided at the distal end portion 11a and distributing the illumination light guided by the light guide 16 toward the observation target site; Optical system 18 for guiding the optical image of
An image guide 19 that is inserted through the insertion section 11 and the operation section 12 and guides an optical image guided by the objective optical system 18 to the eyepiece section 13;
An eyepiece optical system 20 for emitting an optical image guided by the image guide 19, a treatment instrument introduction port 21 provided in the operation unit 12 for introducing the treatment instrument 7, and the distal end 11a
The treatment tool outlet 22 for guiding the treatment tool 7 introduced from the treatment tool inlet 21 is inserted into the operation unit 12 and the insertion unit 11, and is introduced from the treatment tool inlet 21. The treatment instrument 7 has a treatment instrument insertion conduit 23 for guiding the treatment instrument 7 to the treatment instrument outlet 22.

The light source device 3 includes a light source lamp 31 that emits illumination light, a power supply circuit 32 that supplies power to the light source lamp 31, and a protein that is provided on the illumination light path and that is expressed in an observation target site by a pEGFP vector. An excitation light transmission filter 33 that transmits the wavelength to be excited, and a condensing optical system 34 that condenses the illumination light on the light incident end face of the light guide 16.
A motor 35 for moving the excitation light transmission filter 33 so that the excitation light transmission filter 33 can be inserted into and removed from the illumination light path, an operation panel 36 for inputting an operation instruction to the light source device 3, The control circuit 37 includes at least a control circuit 37 that drives and controls the motor 35 in accordance with the operation of the operation panel 36.

The imaging device 4 includes an observation light transmission filter 41 that transmits a wavelength component of fluorescence of the protein of the observation light emitted from the eyepiece 13 of the endoscope 2, and an imaging device that forms an image of the observation light. The imaging optical system 42 includes an imaging optical system 42 and a CCD 43 as an imaging unit that captures an observation image formed by the imaging optical system 42 and obtains an imaging signal.

The treatment instrument 7 includes a needle 51 provided at the distal end of the treatment instrument 7 and a pEGFP vector attached to the proximal end of the treatment instrument 7 via the needle 51 to the tissue to be observed. It has a syringe 52 for injection.

As shown in FIG. 2, the pEGFP vector is
EGFP (Enhanced Green) that fluoresces
EGF for expressing a protein called Fluorescent Protein) in host cells of the tissue to be observed
It contains a gene called the P gene. This pEGF
The P vector can stably introduce a gene into a host cell, and is supplied as a product from Clonetech, USA.

[0012] The EGFP is a fluorescent GFP (Green Fl) contained in a wild jellyfish.
uoresent Protein) 23
It is generated by genetic manipulation to increase the fluorescence intensity of a protein composed of 8 amino acids. FIGS. 3A and 3B are excerpts of the amino acid sequences related to the fluorescent functions of the GFP and the EGFP, respectively. EGFP is represented by (Green Fluorescenten).
By replacing Ser (serine) contained in (t Protein) and GFP with Thr (threonine), the intensity of fluorescence is increased.

As shown in FIG. 4, EGFP has a characteristic of emitting stronger fluorescence than natural GFP. This EGFP has a characteristic that it is excited by excitation light having a peak wavelength of 488 nm and emits fluorescence having a peak wavelength of 510 nm.

As shown in FIG. 5, the excitation light transmission filter 33 is configured to transmit light having a wavelength of about 450 to 500 nm, and a wavelength band of about 488 nm at which the excitation sensitivity of the EGFP reaches a peak. Is transmitted. The excitation light transmitting filter 33 attenuates wavelengths other than the wavelength band of the excitation light of the EGFP, thereby preventing the living cells from being excited by another wavelength. The observation light transmission filter 41 is configured to attenuate the light having a wavelength band of the excitation light and a wavelength less than that. Thereby, the reflected light and the scattered light from the observation target tissue are removed from the observation light, and only the fluorescence can be extracted.

Next, the operation of the present embodiment will be described. First, a tissue to be transfected is specified endoscopically, the treatment tool 7 is inserted into the treatment tool insertion duct 23, and an aqueous solution containing the pEGFP vector is injected into the observation target tissue by the syringe 52. Then, EGFP is added to the host cells of the observation target tissue.
The gene has been introduced and the host cell has
EGFP generation is started according to the nucleotide sequence of the GFP gene.

Then, at the time when EGFP occurs, the motor 35 is operated by the operation panel 36 to insert the excitation light transmission filter 33 into the illumination light path. Then, excitation light is irradiated from the endoscope 2 toward the observation target tissue, and the EGFP irradiated with the excitation light is oxidized to form a chromophore. The chromophore of the EGFP is excited, and the EGFP emits fluorescence. Can be At this time, since EGFP does not show intracellular localization, the whole cell emits EGFP fluorescence. The observation image due to the fluorescence is reflected by the objective optical system 18
, Via the image guide 19 and the eyepiece optical system 20,
The light is emitted from the endoscope 2. From the observation image emitted from the endoscope 2, the fluorescence component is extracted by the observation light transmission filter 41, and formed on the imaging surface of the CCD 43 by the imaging optical system 42. As a result, the subject image due to the fluorescent light is displayed on the monitor device 6.

At this time, the part emitting the fluorescent light is often a small area in the whole observation part by the endoscope 2, and it is not known where the fluorescent part is located in the whole observation part. Sometimes. In such a case,
By retracting the excitation light transmission filter 33 from the illumination light path and irradiating the observation target tissue with visible light including the transmission wavelength of the observation light transmission filter 41, the entire observation region can be observed.

The tissue to be observed is not limited to human or animal tissues or cells, but may be tissues or cells of other organisms.

As described above, according to the present embodiment, the EGFP gene is introduced into the tissue to be observed by endoscopic operation, and EGFP is expressed, so that a sufficient amount of fluorescence emitted from EGFP is generated. Reliable fluorescence observation can be performed. Also, without introducing a high-sensitivity imaging device or the like, the endoscope fluorescence observation device 1 having an inexpensive configuration can be used.
Reliable fluorescence observation can be performed. Therefore, according to the present embodiment, it is possible to obtain an effect that reliable fluorescence observation with a sufficient amount of fluorescence can be performed with an inexpensive device configuration. (Modification of First Embodiment) Next, a modification of the first embodiment will be described. In this modification, points different from the first embodiment will be described. In this modification,
Instead of the pEGFP vector used in the first embodiment, a vector described below is used. The vector used in this modified example is configured by incorporating the EGFP gene described in the first embodiment into a vector that expresses a modified low-density lipoprotein receptor. A vector for expressing the modified low-density lipoprotein receptor may be a mammalian, microbial, yeast, or mammalian modified low-density lipoprotein receptor linked to a regulatory element derived from a bacteriophage or viral gene. Alternatively, it is composed of a DNA fragment generated via synthetic DNA (deoxyribonucleic acid) or cDNA (double-stranded DNA) encoding a biologically equivalent substance.

Next, the operation of the present modification will be described. In addition,
In the present modification, points different from the first embodiment will be described. The vector of this modification induces expression of a modified low-density lipoprotein receptor and EGFP by transformation, transduction or infection into a suitable cell line. Then, for example, the mammalian modified low-density lipoprotein receptor and EGFP
Is expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Thereby, the tissue in which the modified low-density lipoprotein receptor is produced can be observed with fluorescence.

According to Japanese Patent Application Laid-Open No. 9-98787,
Suitable cloning and expression vectors for use in bacterial, fungal, yeast and mammalian host cells are available from Powell (Po Po).
uwl) et al. (Cloning Vectors: A Laboratory Manual, Elsby, NY, (1985)).

(Second Embodiment) Next, a second embodiment of the present invention will be described.
An embodiment will be described. In the present embodiment, points different from the first embodiment will be described. In the present embodiment, pEGFP of the first embodiment will be described.
Instead of using a vector, EGF was added to the antitumor antibody described below.
A fusion antibody into which the P gene has been introduced is used. According to Reference 1, "Biomolecular Science of Antibodies" (Aichi Medical University, Kazuhiro Yoshikawa et al., Neurosurgery, September 1998), it can be used as a means of transporting isotopes, cytotoxins, or therapeutic genes, and direct tumors Antitumor antibodies have been described that suppress the expression of growth factor receptor molecules when expressed in E. coli.
The possibility of clinical application of this anti-tumor antibody to missile therapy for tumors has been studied, and it has become more realistic with recombinant antibodies of tumor single-chain antibodies. It has been confirmed that this anti-tumor antibody is an antibody that accumulates in tumors.

Further, according to Document 1, it is possible to produce proteins having different functions as one molecule by gene manipulation technology, so that antibodies can have various functions. Therefore, in the present embodiment, in order to express EGFP in cells or living tissues, a fusion antibody in which an EGFP gene has been introduced into an anti-tumor antibody by gene recombination technology is generated, and this fusion antibody is used.

Next, the operation of the present embodiment will be described. In this embodiment, points different from the first embodiment will be described. For example, when observing a resected tumor endoscopically, after resection of the tumor,
The anti-tumor antibody into which the gene has been introduced is injected or sprayed.
If there is a tumor in the tissue into which the anti-tumor antibody has been injected or sprayed, the anti-tumor antibody acts as an antibody against the tumor,
Accumulates at the site of the tumor. Then, at the site where the anti-tumor antibody is accumulated, when the EGFP gene is integrated into the cell and a new cell is generated by cell division, EGFP is expressed at that site. Thereby, fluorescence is emitted from the tumor cells, so that the fluorescence of the tumor cells can be observed, and the progress of the tumor cells can be observed under an endoscope. The observation target site may be any site as long as it is a body cavity such as a stomach, an intestine, or a bladder of a living body that can be observed under an endoscope. The anti-tumor antibodies used include monoclonal antibodies. The anti-tumor antibody to be used is not limited to a monoclonal antibody, but may be any as long as it can be a vector that can express EGFP by introducing an EGFP gene into a tumor cell.

According to the embodiment described above, the first
The following effects can be obtained in addition to the effects described in the embodiment. According to the present embodiment, a fusion antibody obtained by introducing an EGFP gene into an anti-tumor antibody such as a monoclonal antibody that accumulates in a tumor is injected or scattered into a tissue to be observed, and the tumor or the like is observed by fluorescence observation. It makes it easier to find and follow-up cancer cells.

The present invention is not limited to the above-described embodiment, but can be variously modified without departing from the gist of the invention.

[Supplementary note] (Supplementary note 1) Introducing means for introducing a gene that expresses a protein that emits fluorescence into a tissue to be observed, and illuminating light emitted from a light source onto the tissue to be observed to observe the tissue to be observed. An endoscope for obtaining an image, first transmission wavelength limiting means provided on an optical path of the illumination light for transmitting a wavelength for exciting the protein, and fluorescence emitted from the protein provided on an optical path of the observation image An endoscope fluorescence observation apparatus, comprising: a second transmission wavelength limiting means that transmits the wavelength.

(Additional Item 2) The endoscopic fluorescence observation apparatus according to Additional Item 1, wherein the protein includes a protein obtained by rearranging the amino acid sequence of a protein that emits fluorescence contained in Oan jellyfish to enhance fluorescence.

(Additional Item 3) In the endoscope fluorescence observation apparatus according to Additional Item 1, the gene includes a gene obtained by rearranging a gene contained in Oan jellyfish by genetic manipulation.

(Additional Item 4) In the endoscope fluorescence observation device according to Additional Item 1, the protein fluoresces when oxidized by excitation light.

(Additional Item 5) In the endoscope fluorescence observation apparatus according to Additional Item 1, the introduction means includes a microorganism into which the gene has been incorporated.

(Supplementary note 6) The endoscopic fluorescence observation apparatus according to supplementary note 1, wherein the introducing means expresses the protein in the cells by introducing the gene into cells of the tissue to be observed. This is the vector to be used.

(Additional Item 7) In the endoscope fluorescence observation apparatus according to additional item 1, the gene is generated by genetic modification.

(Additional Item 8) In the endoscope fluorescence observation apparatus according to additional item 1, the introduction means includes a fusion antibody in which the antitumor antibody and the gene are bound.

(Additional Item 9) In the endoscope fluorescence observation device according to Additional Item 8, the anti-tumor antibody includes a monoclonal antibody.

(Supplementary note 10) The endoscope fluorescence observation apparatus according to Supplementary note 1, wherein the excitation wavelength is approximately 488 n.
m.

(Additional Item 11) The endoscope fluorescence observation apparatus according to additional item 1, wherein the wavelength of the fluorescence is approximately 510 nm.
including.

(Additional Item 12) The endoscope fluorescence observation apparatus according to additional item 1, wherein the first transmission wavelength limiting means comprises:
At least light having a wavelength of about 488 nm is transmitted.

(Additional Item 13) The endoscope fluorescence observation apparatus according to additional item 1, wherein the second transmission wavelength limiting means comprises:
At least light having a wavelength of about 510 nm is transmitted.

(Additional Item 14) The endoscope fluorescence observation apparatus according to Additional Item 1, wherein the second transmission wavelength limiting means comprises:
At least light of a wavelength transmitted by the first transmission wavelength limiting means is attenuated.

(Additional Item 15) The endoscope fluorescence observation apparatus according to Additional Item 1, wherein the second transmission wavelength limiting means comprises:
Attenuates light having a wavelength of approximately 500 nm or less.

(Additional Item 16) The endoscope fluorescence observation apparatus according to additional item 1, wherein the second transmission wavelength limiting means comprises:
Light having a wavelength shorter than the wavelength of the fluorescence is attenuated.

(Additional Item 17) In the endoscope fluorescence observation apparatus according to Additional Item 1, the introduction means includes a treatment tool for guiding the gene to the observation target tissue.

(Additional Item 18) The endoscope fluorescence observation apparatus according to additional item 1, further comprising a treatment tool for guiding the introduction unit to the observation target tissue.

(Additional Item 19) The endoscope fluorescence observation apparatus according to additional item 1, further comprising a unit for freely inserting the first transmission wavelength limiting unit into the optical path of the illumination light.

[0046]

As described above, according to the present invention,
With an inexpensive device configuration, it is possible to obtain an effect that reliable fluorescence observation with a sufficient amount of fluorescent light can be performed.

[Brief description of the drawings]

FIG. 1 to FIG. 5 relate to a first embodiment of the present invention, and FIG. 1 is an explanatory view showing a configuration of an endoscope fluorescence observation apparatus.

FIG. 2 is an explanatory diagram showing the configuration and operation of a pEGFP vector.

FIG. 3 is an explanatory diagram showing amino acid sequences related to the respective fluorescent functions of GFP and EGFP.

FIG. 4 is an explanatory diagram showing the characteristics of the respective excitation light and fluorescence wavelength bands of GFP and EGFP.

FIG. 5 is an explanatory diagram showing characteristics of respective transmission wavelength bands of an excitation light transmission filter and an observation light transmission filter.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 ... Endoscope fluorescence observation device 2 ... Endoscope 3 ... Light source device 4 ... Imaging device 7 ... Treatment tool 33 ... Excitation light transmission filter 41 ... Observation light transmission filter

Claims (1)

[Claims] 1. Introducing means for introducing a gene that expresses a protein that emits fluorescence into an observation target tissue, and an endoscope for irradiating illumination light emitted from a light source to the observation target tissue to obtain an observation image of the observation target tissue. First transmission wavelength limiting means provided on an optical path of the illumination light and transmitting a wavelength for exciting the protein; and a first transmission wavelength limiting means provided on an optical path of the observation image and transmitting a wavelength of fluorescence emitted from the protein. 2. An endoscope fluorescence observation apparatus, comprising: