CN105725963A - Evaluation method for tumour model effective intervention based on optical imaging technology - Google Patents
Evaluation method for tumour model effective intervention based on optical imaging technology Download PDFInfo
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Abstract
The invention belongs to the technical field of experiment animals and medical image processing and application, and specifically relates to an evaluation method for tumour model effective intervention based on an optical imaging technology. The method comprises: using bioluminescence and fluorescent techniques to mark tumour model cells, using optical detection instruments to directly detect activities of the marked cells of a tumour model and behaviors of marked genes, observing expression or interaction of specific cells, genes, and molecules, and meanwhile detecting a plurality of kinds of molecular events, tracking targets, and imaging and evaluating an intervening measure effect from molecular and cellular level. The method can observe growth and metastasis of tumour cells in vivo and reaction after intervention in real time. Further, the method can be used for simply, conveniently, and accurately screen out effective intervention measures or effective intervention compatibility measures for different histological pattern tumours.
Description
Technical field
The invention belongs to laboratory animal and Medical Image Processing and applied technical field, relate toRightThe evaluation methodology of tumor model effective prevention, is specifically related to the evaluation methodology to tumor model effective prevention of the optically-based imaging technique.
Background technology
Report display, in recent years, along with the continuous change of environmental pollution deterioration and people life style, cancer serious threat human health, its M & M rises to rapidly first place in the world.Research display, the immunotherapy of tumors method set up for method based on immunology principle, with immunological technique, because it can produce positive anticancer therapeutic, few side effects and body injury is little, gradually become the important means of clinical treatment Advanced cancers and placed high hopes by medical circle.Researcher is concerned about, how to improve body antineoplastic effect of immune response specifically, take precautions against the immunologic escape of tumor cell, improve the microenvironment etc. that tumor cell is depended on for existence, even reach the purpose of containment regressing tumors completely eventually through effectively transfer body entirety immune system, it it is the great direction of study hotspot and the tool prospect capturing tumor.Substantial amounts of experimentation data shows, acupuncture can effectively adjust immunity of organism, obtains definite anticancer therapeutic.Moxa-moxibustion is one of China's treatment method the earliest, rather prevailing in Ancient Times in China.The record of " pin is not, the institute of moxibustion is suitable " is just had as far back as " Ling Shu Miraculous Pivot or Divine Axis official energy "." Elementary Medicine " have " all sick medicines too late, pin unfavorable, it is necessary to moxibustion it " saying.Moxibustion therapy has the effect of dredge the meridian passage, damp eliminating cold expelling, dispersing swelling and dissipating binds, recuperating depleted YANG and rescuing the patient from collapse, ascending yang-energy, can rise the gas in blood, stagnant in ventilation, can lead to all warps and except all kinds of diseases and ailments.Treated with moxibustion tumor is with a long history, just on the books in "Nei Jing" one book, the method that " medical secrets of official " existing a thousand pieces of gold moxibustion treatment treats scrofula, and says that garlic moxibustion is suitable to " all scrofulas are on neck ".Additionally, control the record of " carcinoma of lip " (cheiloncus) in the Ming Dynasty's " Waike Zhengzong, Orthodox Manual of External Diseases " useful moxa-moxibustion；The record of useful Cera Flava moxibustion treatment " magic flower skin ulcer " (skin carcinoma) in Qing Dynasty's " surgery card controls pandect ".In the recent decade, Chinese scholars is to relevant Moxibustion treatment Carry out clinical and basic research, treated skin carcinoma such as Xia Shi etc. with moxibustion with electric warming needles, except treating with moxibustion with electric warming needles except local, give acupuncture needle acupuncture further accordance with medicine typing, obtain relatively good efficacy, total effective rate 92%；The clinical treatment of Tang Shi and Li Shi etc. also achieves similar good efficacy.Experimental data shows, acupuncture reaches, by the overall Immunomodulating effects of neuro-endocrine-immune system, the effect that acupuncture is for cancer；Wherein indicate the neural opioid peptides substance release of acupuncture stimulating maincenter and periphery, increase the lethality of natural killer cell, strengthen the Functional activity of reticuloendothelial system, the especially phagocytic activity of macrophage, and play antitumaous effect；Additionally, moxa-moxibustion coordinates other remedy measures, also improve clinical symptoms, improve late result, alleviate cancerous pain, alleviate chemotherapy, the untoward reaction etc. of radiotherapy.
There are some researches show, acupuncture and moxibustion treatment to the curative effect of tumor and Different Acupoint choose and the key element such as compatibility is closely related.Tumor belongs to the disease category such as " hysteria abdominal mass " " gathering " in motherland's medical science." gather " be seven emotions pent-up, taste are impaired, caused by diseases caused by exogenous pathogenic factor six climate exopathogens and deficiency of vital QI, and it is relevant to think that healthy energy is not only formed with tumor, also develops with its pathogenesis, prognosis all has substantial connection.So-called " being gathered of heresy, its gas must be empty ".Acupuncture antineoplastic main hypertensive TCM mechanism is in that strengthening the body resistance.Clinical acupuncture point selects above to take governor vessel and Yangming Channel cave and back shu points more, mostly is and has roborant acupuncture point, such as caves such as big vertebra, Guan Yuan, Shenques.Though acupuncture and moxibustion therapy tumor is based on strengthening the body resistance, but acupoint selection is still with determination for the treatment of based on pathogenesis obtained through differentiation of symptoms and signs for principle.So, on dialectical basis, it is necessary to take with different acupuncture points on the human bodys according to the difference of the different parts of tumor and symptom, good effect could be obtained.
Based on this, present inventor plans to build vertical a kind of specificationRightTumor model effective preventionEvaluateMethod, is specifically related to the evaluation methodology to tumor model effective prevention of the optically-based imaging technique.
Summary of the invention
The order ground of the present invention is in that to overcome the defect of prior art, it is provided that a kind of specificationRightTumor model effective preventionEvaluateMethod, is specifically related to the evaluation methodology to tumor model effective prevention of the optically-based imaging technique.
In the present invention, utilize optical image technology its quantitatively performance and functional imaging characteristic accurately, establish an evaluation methodology to tumor model effective prevention, by this appraisal procedure, for clinic, tumor model effective prevention and mode can be provided rich valuable reference.
In embodiments of the invention, the method for the effect of histological types tumor model is intervened in the assessment of optically-based imaging technique in acupuncture mode；The process of tumor is judged according to luminous intensity, easy growth and the transfer assessing tumor intuitively, for histological types tumor, there is the acupuncture compatibility acupuncture point of preferred means of intervention provide experimental animal model technology platform for filtering out rapidly and accurately, wherein, selecting the C57BL/6 mice with normal cell and immunity system or BALB/c mouse is experimentation animal, the malignant melanoma of skin subcutaneous tumor model of the B16 cell strain of bioluminescence or fluorescently-labeled Syngenic mice and the breast cancer model of 4T1 cell strain are carried in preparation, choose different acupoints respectively and compatibility is intervened, utilize the effect that optical in vivo imaging technology assessment various acupuncture means of intervention is anticancer, for clinicRightTumor model effective prevention and mode provide rich valuable reference.
In the present invention, based on following principle: luciferase gene is integrated into tumor cell chromosomal DNA with expressing luciferase, to the noctilcent principle of tumor cell, when ATP and oxygen exist, external source (abdominal cavity or intravenous injection) gives substrate luciferin (Luciferin), the oxidation reaction of luciferase catalytic fluorometry element produces luminescence, to just producing luminescence phenomenon in living cells, and the number linear correlation of the intensity of light and labeled cell；Each catalytic reaction of luciferase only produces a photon, utilizes the super-sensitive CCD camera under ultralow temperature and specially designed imaging camera bellows and the imaging software can accurate surveying recorded these photons；The present invention adopts bioluminescence and fluorescent technique marked tumor model cell, sensitive optical detecting instrument is utilized directly to detect the labeled cellular activity of tumor model and labeled gene behavior, the expression of observation specific cell, gene and molecule or Interaction, detection different kinds of molecules event simultaneously, follow the trail of target, from molecule and cellular level intervening measure effect carried out imaging and assess
In the present invention, bioluminescence adopts luciferase (Luciferase) genetic marker cell or DNA,
Fluorescent technique adopts fluorescent reporter group (GFP, RFP, Cyt and dyes etc.) to carry out labelling.
More specifically, the optically-based imaging technique of the present invention evaluation methodology to tumor model effective prevention, it is characterised in that it includes step:
1) tumor cell line of bioluminescence luciferase (Luciferase) gene is carried in preparation, including:
(1) structure carries Luciferase gene expression frame plasmid
Build the slow virus carrier pRRLsin-luc containing luc gene；
(2) slow virus packaged unit
With pRRLsin-luc, slow virus packaging plasmid Pcmvr8.74, slow virus envelope plasmid Pmd2.G cotransfection 293T cell, it is thus achieved that concentrate after viral supernatants ,-80 DEG C of preservations；
(3) slow virus infection and screening
The slow virus liquid of defrosting cryopreservation, adds 37 degree of incubated overnight in cell；Continue to cultivate；Select the stable cell clone infected；Continue to cultivate；
2) tumor model expressing Luc gene is set up；
3) different tumor models accept different dry preliminary experiment；
4) different intervention group tumor model imaging quantitative datas are analyzed by dynamically observing of situ tumor model difference intervention group, evaluate.
The present invention is with the tumor cell of luciferase gene (Luciferase) labelling histological types, after subcutaneous vaccination, by optical in vivo imaging technology, can the growth transfer in vivo of tumor cell described in Real Time Observation and the reaction after intervening.
Further, the evaluation methodology of tumor model effective prevention be can be used for simplicity, filters out the effective intervening measure of histological types tumor or effective intervention compatibility measure fast and accurately by the optically-based imaging technique of the present invention.
The experiment reagent adopted in the present invention and relevant plasmid all can be obtained by commercial channel.
It is an advantage of the current invention that: compared with traditional external imaging,
First, molecular imaging can reflect the room and time distribution of cell or gene expression, thus the related biological process understood in living animal body, specific gene function and interaction；
Second, owing to same research individuality can be carried out repeatedly following the tracks of imaging for a long time, both can improve the comparability of data, it is to avoid the individual variation impact on result of the test, not need again tupe animal, save expense；
3rd, can be used for drug development, overcome wherein owing to the medicine of entrance clinical research terminating causing the defect that substantial amounts of fund is wasted because of safety problem, obtain the data of more detailed molecule or gene level simultaneously, solve the insurmountable problem of conventional art.
Detailed description of the invention
Embodiment 1Set up the evaluation methodology to tumor model effective prevention of the optically-based imaging technique
Pass through following step:
1) tumor cell line of bioluminescence luciferase (Luciferase) gene is carried in preparation
B16 malignant melanoma and the 4T1 breast carcinoma cell strain of bioluminescence luciferase (Luciferase) gene is carried in foundation；
(1) structure carries Luciferase gene expression frame plasmid
Enzyme action pGL4.0(luc) obtain the luc gene segment of 1653bp, this purpose fragment is inserted slow virus carrier pRRLsin, builds the slow virus carrier pRRLsin-luc containing luc gene；
(2) slow virus packaged unit
Utilize jetPEI transfection reagent, pRRLsin-luc, slow virus packaging plasmid Pcmvr8.74, slow virus envelope plasmid Pmd2.G are according to the ratio three plasmid co-transfection 293T cell of 3:2:1, after 48 hours, by in the media transfer containing virus liquid to 15ml centrifuge tube, under low temperature 4 degrees celsius, 3000rpm, centrifugal 10min removes the cell debris of flaky precipitate, extracts viral supernatants；By viral supernatants with in 0.45um frit to concentration centrifuge tube, 2000rpm, 4 DEG C, 15min is centrifuged, and concentrated solution is collected in 500 μ l centrifuge tubes ,-80 DEG C of preservations；
(3) slow virus infection and screening
Infecting the previous day, digestion B16 or 4T1 cell also counts, and is laid on by cell in 24 orifice plates, 37 ° of C incubated overnight cells, the slow virus liquid of defrosting cryopreservation, and prepares in 1:2 viral dilution liquid 200 μ l addition cell；Add Polybrene to every hole and reach final concentration 6ug/ml, shake even orifice plate, 37 degree of incubated overnight, remove the culture medium containing slow virus, add 500 μ l complete mediums；3.5ug/mlpuromycin selects the cell clone of stable infection；With containing 10%FBSandpuromycin (2ug/ml) culture medium culturing B16/lucand4T1/luc cell；
2) the B16 maligna element Subcutaneous tumor expressing Luc gene and 4T1 breast cancer mouse tumor model are set up
The B16 malignant melanoma cell of stably express Luc gene and 4T1 breast cancer cell are expanded in a large number in culture dish, by the tumor cell of exponential phase through 0.25% trypsinization and PBS wash after 2 times, be 1 × 10 by PBS modulation density respectively7With 1 × 108The cell suspension of individual/ml, is placed in ice bath standby；The pentobarbital sodium 150ul of the C57BL/6 mice of about 20g or BALB/c mouse lumbar injection 1% is anaesthetized, in fluorescently-labeled B16 or the 4T1 tumor cell of right rear leg subcutaneous injection 100ul, set up B16 malignant melanoma subcutaneous model or 4T1 breast carcinoma subcutaneous tumor model；
3) C57BL/6 mice B16 and 4T1 breast carcinoma subcutaneous tumor model accepts the B16 malignant melanoma subcutaneous situ tumor model experiment of moxa-moxibustion experiment (1) labelling of Different Acupoint: 1. model group: except do identical with treatment group fixing except, be left intact；2. Guanyuan acupoint group: acupuncture point district, with reference to the animal acupuncture point standard setting method of side of Huaxing, is carried out shaving process, with moxa roll (diameter 4mm, length 12cm) direct contact moxibustion Guanyuan acupoint, ADT 1 time by Guanyuan acupoint acupoint selection；3. DAZHUI acupoint group: acupuncture point district, with reference to the animal acupuncture point standard setting method of side of Huaxing, is carried out shaving process by DAZHUI acupoint acupoint selection, with moxa roll (diameter 4mm, length 12cm) direct contact moxibustion Guanyuan acupoint, ADT 1 time；
(2) the 4T1 breast carcinoma subcutaneous tumor model experiment of labelling: 1. model group: except do identical with treatment group fixing except, be left intact；2. Guanyuan acupoint group: acupuncture point district, with reference to the animal acupuncture point standard setting method of side of Huaxing, is carried out shaving process by Guanyuan acupoint acupoint selection, with moxa roll (diameter 4mm, length 12cm) direct contact moxibustion Guanyuan acupoint, the next day, intervenes 1 time；
3. DAZHUI acupoint group: acupuncture point district, with reference to the animal acupuncture point standard setting method of side of Huaxing, is carried out shaving process by DAZHUI acupoint acupoint selection, with moxa roll (diameter 4mm, length 12cm) direct contact moxibustion Guanyuan acupoint, the next day, intervenes 1 time；
4) C57BL/6 mice and the BALB/c with fluorescently-labeled B16 and 4T1 breast carcinoma subcutaneous tumor model is raised by dynamically observing of situ tumor model different disposal group under SPF environment；From accepting each group of intervening measure, every 1 week by mouse anesthesia after put into living imaging system imaging, in Continuous Observation 2-3 week, obtain the consecutive image of every mice tumors grew situation；The specialized image utilizing living imaging instrument analyzes system, obtains the quantitative data of every each imaging time point of mice, and these data directly accurately show the size of tumor；
This method can be additionally used in the distant place micrometastasis stove of detection tumor, and quantitatively；According to according to the picture obtained and quantitative data, accurate evaluation implements the effect of each group of each intervening measure.
Claims (10)
1. the optically-based imaging technique evaluation methodology to tumor model effective prevention, it is characterized in that, adopt bioluminescence and fluorescent technique marked tumor model cell, optical detecting instrument is utilized directly to detect the labeled cellular activity of tumor model and labeled gene behavior, the expression of observation specific cell, gene and molecule or Interaction, detection different kinds of molecules event simultaneously, follows the trail of target, intervening measure effect is carried out imaging and assesses.
2., by the method described in claim 1, it is characterised in that wherein, bioluminescence adopts luciferase (Luciferase) genetic marker cell or DNA；Fluorescent technique adopts fluorescent reporter group GFP, RFP, Cyt and dyes to carry out labelling.
3. by the method described in claim 1, it is characterised in that it includes step:
1) tumor cell line of bioluminescence luciferase (Luciferase) gene is carried in preparation, including:
(1) structure carries Luciferase gene expression frame plasmid
Build the slow virus carrier pRRLsin-luc containing luc gene；
(2) slow virus packaged unit
With pRRLsin-luc, slow virus packaging plasmid Pcmvr8.74, slow virus envelope plasmid Pmd2.G cotransfection 293T cell, it is thus achieved that concentrate after viral supernatants ,-80 DEG C of preservations；
(3) slow virus infection and screening
The slow virus liquid of defrosting cryopreservation, adds 37 degree of incubated overnight in cell；Continue to cultivate；Select the stable cell clone infected；Continue to cultivate；
2) tumor model expressing Luc gene is set up；
3) different tumor models accept different dry preliminary experiment；
4) different intervention group tumor model imaging quantitative datas are analyzed by dynamically observing of situ tumor model difference intervention group, evaluate.
4. by the method described in claim 1, it is characterized in that, described bioluminescence adopts luciferase (Luciferase) genetic marker cell or DNA, and described fluorescent technique adopts fluorescent reporter group (GFP, RFP, Cyt and dyes etc.) to carry out labelling.
5. by the method described in claim 1 or 2 or 3, it is characterised in that described tumor model cell is the B16 malignant melanoma and the 4T1 breast carcinoma cell strain that carry bioluminescence luciferase (Luciferase) gene.
6. by the method described in claim 3, it is characterized in that, in step (1), by enzyme action pGL4.0(luc) obtain the luc gene segment of 1653bp, this purpose fragment is inserted slow virus carrier pRRLsin, builds the slow virus carrier pRRLsin-luc containing luc gene.
7. by the method described in claim 3, it is characterized in that, in step (2), slow virus packaged unit includes: utilize jetPEI transfection reagent, pRRLsin-luc, slow virus packaging plasmid Pcmvr8.74, slow virus envelope plasmid Pmd2.G are according to the ratio three plasmid co-transfection 293T cell of 3:2:1, after 48 hours, by in the media transfer containing virus liquid to 15ml centrifuge tube, under low temperature 4 degrees celsius, 3000rpm, centrifugal 10min removes the cell debris of flaky precipitate, extracts viral supernatants；By viral supernatants with in 0.45um frit to concentration centrifuge tube, 2000rpm, 4 DEG C, 15min is centrifuged, and concentrated solution is collected in 500 μ l centrifuge tubes ,-80 DEG C of preservations.
8. by the method described in claim 3, it is characterised in that step 2) in,
The B16 malignant melanoma cell of stably express Luc gene and 4T1 breast cancer cell are expanded in a large number in culture dish, by the tumor cell of exponential phase through 0.25% trypsinization and PBS wash after 2 times, be 1 × 10 by PBS modulation density respectively7With 1 × 108The cell suspension of individual/ml, is placed in ice bath standby；The pentobarbital sodium 150ul of the C57BL/6 mice of about 20g or BALB/c mouse lumbar injection 1% is anaesthetized, in fluorescently-labeled B16 or the 4T1 tumor cell of right rear leg subcutaneous injection 100ul, set up B16 malignant melanoma subcutaneous model or 4T1 breast carcinoma subcutaneous tumor model.
9., by the method described in claim 3, it is characterised in that in step 3), it is the moxa-moxibustion experiment that C57BL/6 mice B16 and 4T1 breast carcinoma subcutaneous tumor model accept Different Acupoint that different tumor models accept different dry preliminary experiment.
10. the evaluation methodology of tumor model effective prevention is screened the effective intervening measure of histological types tumor or the effective purposes intervened in compatibility measure being used for by the optically-based imaging technique of claim 1.
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