CN1903374A - Application of colon bacillus for preparing medicine for diagnosis and treatment of tumor - Google Patents
Application of colon bacillus for preparing medicine for diagnosis and treatment of tumor Download PDFInfo
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Abstract
An application of the colibacillus in preparing the medicines for diagnosing and treating tumor is disclosed. Said colibacillus can be located in the tumor tissue to suppress it.
Description
Technical field
The present invention relates to the medical application of antibacterial, relate in particular to the purposes of escherichia coli in preparation diagnosing tumor or medicine.
Background technology
Tumor is the disease of serious threat human health and life, and traditional oncotherapy pattern such as excision are put, chemotherapy etc., have tumor-targeting poor, patient's normal structure is damaged shortcomings such as big, easily recurrence, side reaction are big.Press for a kind of new technology or method at present, differentiation tumor tissues and normal structure that can be correct reach the special identification and the purpose of killing tumor cell.Along with to the deep understanding of antibacterial and the progress of modern molecular biology, it is found that, a lot of antibacterials such as clostruidium, bifidus bacillus, vibrio cholera, Li Site bacterium, salmonella and escherichia coli etc. have the special tumor tissues that is gathered in, and can be in tumor tissues the characteristic of growth and breeding, some antibacterial also has effect (the Pawelek J.M that suppresses tumor growth, Low K.B, Bermudes D.Bacteria as tumor-targeting vectors.LancetOncol, 2003,4:548-556.).Utilize this characteristic, started a major transformation of in neoplasm targeted therapy, using about antibacterial.The targeted therapy that antibacterial is applied to tumor has incomparable advantage:
1. in tumor tissues, antibacterial mainly is gathered in outside the tumor cell, and is comparatively responsive to antibiotic, can remove quickly, safer.
2. the genome of antibacterial is very big, can express genes of interest fast with oncotherapy effect, make it at more special, the stronger antitumor action of specific part performance, simultaneously the antibacterial of tumor locus can growth and breeding, make that effector can be at the tumor locus continuous expression, thus bigger, the more persistent tumor killing effect of performance.
3. no matter some antibacterial is at allogeneic tumor model or xenograft tumor graft model, and tumor-localizing effect is preferably all arranged, and it is big in amount that tumor tissues gathers, and promptly less antibacterial is positioned tumor tissues just can be at this growth and breeding.
4. a lot of antibacterials are through behind the genetic modification, all do not change its tumor-targeting as the attenuated strain of some antibacterial, the antibacterial of carrying some oncotherapy gene etc., and do not change it to antibiotic sensitivity yet, provide more wide prospect for antibacterial being applied to oncotherapy.
5. antibacterial is used for oncotherapy multiple notes bacterium mode, as intravenous injection, intraperitoneal injection, intratumor injection etc., provides more selection for antibacterial from now on is applied to oncotherapy.
6. antibacterial can not only be positioned primary tumor site, also can be positioned the metastasis of tumor, so antibacterial can not only also can be used for the early discovery and the diagnosis of tumor as the carrier of oncotherapy.
At present, be used for oncotherapy and mainly contain two big bacterioids, a class is bacillus and the bifidus bacillus of anaerobism Pseudomonas, and another kind of is the Salmonella of facultative anaerobe.Bacillus is to find early, and study also more antibacterial, the close tumprigenicity and the tumor size of bacillus have certain relation, promptly have only tumor long after a certain size, bacillus just can be positioned in the tumor, and the sign that do not reduce of the tumor of most patient.The toxicity of bacillus and shortage clinical effectiveness have influenced its application at anti-tumor aspect.Up to the nineties in last century, utilize the close tumprigenicity of bacillus, it has been obtained good effect as the Graft Versus Tumor Vectors in Gene Therapy, but its toxic action remains its major obstacle that is applied to clinical treatment (Liu SC, Minton NP, Giaccia AJ, et al.Anticancer efficacy of systemically deliveredanaerobic bacteria as gene therapy vectors targeting tumor hypoxia/necrosis.Gene Ther, 2002,9:291-296.).
Bifidobacterium Grain-positive anaerobe is the nontoxic and safe antibacterial of a class.Researcheres are found, when injecting tumor-bearing mice body in intravenous mode bifidus bacillus, can find to have in the tumor tissues antibacterial to assemble, and it is in tumor tissues and ratio＞1000 of measuring in normal structure: 1, but find no the phenomenon that tumor reduces or disappears, and it can only assemble in diameter 1.5cm or bigger tumor tissues, the antibacterial of same dosage then can't assemble in little tumor, the prompting bifidus bacillus is used for oncotherapy and also depends on the tumor size, and certain limitation is arranged.But its carrier as oncotherapy is still attracting the attention of numerous researcheres and is obtaining good experimental result (Geng-Feng Fu, Xi Li, Ya-Yi Hou, et al.Bifidobacteriumlongum as an oral delivery system of endostatin for gene therapy on solid liver cancer.CancerGene Therapy, 2005,12:133-140.).
The Salmonella gram negative bacilli, be a newfound class facultative anaerobe with tumor-targeting in nearly ten years, it has the tumor-localizing ability equally, but because its strong toxic action, old friends try to explore to seek its attenuated strain or low virulent strain, make it be used for oncotherapy better.VNP20009 is the Salmonella that a class has knocked out msbB and purI gene, and its toxicity greatly weakens, and has kept its tumor-targeting.Zhao Ming etc. has developed a kind of auxotroph Salmonella, this kind low virulent strain has kept tumor-targeting, when the intratumor injection Salmonella, the tumor tissues of mice disappears fully, shown application prospect (the Zhao Ming of attenuation Salmonella at anti-tumor aspect, Yang Meng, Li Xiao-Ming, et al.Tumor-targeting bacterial therapy with amino acid auxotrophs of GFP-expressing Salmonellatyphimurium, PNAS, 2005,102:755-760.).The attenuation Salmonella is used for the existing many reports of neoplasm targeted therapy as the carrier of some effector such as tumor necrosis factor, angiostatin, suicide gene etc., and obtained gratifying achievement (Ivan King in zoopery, David Bermudes, Stanley Lin, et al.Tumor-targeted salmonella expressing cytosine deaminase as an anticancer agent.Human GeneTherapy, 2002,22:3261-3266; ), but attenuation Salmonella be not totally nontoxic, it is applied to clinical certain restriction that also has.
From as can be seen above, bacillus, bifidus bacillus and Salmonella are used for oncotherapy respectively the deficiency of itself, so pressing at present and seeking a class is the nontoxic tumor size restriction that is not subjected to again, the antibacterial that has good tumor-targeting simultaneously again is used for oncotherapy.
The Colibacter gram negative bacilli is to parasitize the intravital normal flora of human and animal.Present engineered colibacillus engineering mainly contains DH5 α, BL21, JM101, JM103～JM109, K802, K803 etc., their genome is stable, genetic background is clear, the more important thing is that it is nontoxic, be widely used in engineered various aspects, as cloning vehicle or some effector gene expression carrier etc.
Escherichia coli also as other antibacterial, have tumor-targeting, when the intravenous injection antibacterial is in the tumor-bearing mice body, annotating second day of bacterium, can find germy existence in tumor tissues, researcher is observed this phenomenon, does not announce relevant colibacillary detailed data.(Yong?A?Yu，ShahrokhShabahang，Tatyana?M?Timiryasova，et?al.Visulization?of?tumors?and?metastases?in?live?animalswith?bacteria?and?vaccinia?virus?encoding?light-emitting?proteins.Nature?Biotechnology2004；22：313-21)。Also still have nothing to do at present in utilizing colibacillary tumor-targeting performance antitumor action, and whether escherichia coli have the report that suppresses the tumor growth effect.
Summary of the invention
In order to solve the problem of bacteria that lacking of present existence is suitable for diagnosing tumor or treatment, the invention discloses the purposes of escherichia coli at preparation diagnosing tumor or medicine.
The present invention found through experiments escherichia coli can be collected in the animal tumor tissue specifically, finds that also the multiple injection escherichia coli can suppress tumor growth.
Each kind that escherichia coli can belong to for escherichia coli among the present invention and pass through genetically engineered colibacillus engineering on this basis such as DH5 α, BL21, JM101, JM103～JM109, K802, K803 etc.
Tumor can be various solid tumors among the present invention, as Mus melanin tumor B16-F10, people's hepatocarcinoma BEL7405 and SMMC7721, human prostata cancer PC-3, human breast carcinoma MCF-7, human fibrosarcoma cell HT1080, Mus sarcoma of bladder cell MB-49 and Mus glioma C6.
Diagnosing tumor is gathered in tumor tissues by escherichia coli and realizes that described tumor tissues can be primary tumors, also can be the neoplasm metastasis kitchen range in the purposes of the present invention.Oncotherapy in the purposes of the present invention is to be gathered in tumor tissues by escherichia coli, and suppresses the growth realization of tumor tissues.
Being used for the colibacillary dosage of oncotherapy in the purposes of the present invention is 5 * 10
9Cfu/ time/kg body weight, weekly, continuous three times, being used for the colibacillary dosage of diagnosing tumor is 5 * 10
8The cfu/kg body weight.Diagnosing tumor or medicine route of administration are intravenously administrable in the purposes of the present invention.
Purposes of the present invention is by following experimental verification:
One, the foundation of animal tumor model and metastatic tumor model
At first carry out tumor cell culture, wherein tumor cell comprises various solid tumor cells such as B16-F10, MCF-7, PC-3, MB-49, HT1080, C6 glioma, BEL7405 and SMMC7721.Carry out cell counting after the cultivation, be prepared into finite concentration cell suspension.
Carry out the foundation of tumor model and metastatic tumor model then, tumor cell suspension inoculation animal with preparation, wherein the animal of tumor model can be BALB/C mice and nude mice etc., vaccination ways is the right rear leg subcutaneous injection, the metastasis models animal can be nude mice etc., and vaccination ways is a tail vein injection.
Two, structure of photobacteria and detection
At first prepare photobacteria: prepare the competence escherichia coli according to a conventional method, with plasmid pXen-1 (luxABCDE) (FDrancis KP, Joh D, Bellinger-kawahara C, et al.Monitoring bioluminescentstrphylococcus aureus infections in living mice using a novel luxABCDE construct.InfectImmun; 2000,68 (6): 3594～3600.) be transformed into escherichia coli, use luminous situation and the efficient that the IVIS imaging system is observed the clone behind the coated plate.The monoclonal bacterium that luminous efficiency is higher is cultivated in the culture medium that has resistance, collects antibacterial, and makes certain density bacterial suspension, annotates bacterium in normal and tumor animal, can adopt tail vein injection etc.
Three, antibacterial distribution characteristics and tumor-localizing characteristic in animal body
The characteristic distributions of antibacterial in normal mouse.The intravenous injection antibacterial is in normal mouse, and visible antibacterial is distributed in lung earlier, gradually assembles to liver then, finally is positioned liver.Animal postmortem result shows that also antibacterial enters that the short time mainly is gathered in lungs and liver in the animal body, and along with time lengthening, antibacterial only is distributed in liver.
The characteristic distributions of antibacterial in tumor-bearing mice.The intravenous injection antibacterial is in tumor-bearing mice, annotate at the beginning of the bacterium, antibacterial at the intravital distribution of tumor-bearing mice and distributional class in normal mouse seemingly, but at second day that annotates bacterium, the tumor locus of tumor-bearing mice promptly had the existence of light.Dissect mice results suggest antibacterial and all be distributed in tumor locus.For bigger tumor, antibacterial mainly is distributed in the periphery of tumor tissues, illustrate that escherichia coli mainly are gathered in the relatively abundanter tumor region of oxygen part, and less antibacterial can be located in tumor also.
Four, antibacterial is to the effect of tumor growth
By once annotating bacterium and repeatedly annotating bacterium, observe the effect of escherichia coli itself to tumor growth in tumor animal.The result shows that the growth of animal tumor volume changes once annotating between bacterium group and matched group and do not have significant difference, but repeatedly annotate the bacterium group with once annotate bacterium organize between or and matched group between then difference is obvious.Illustrate that repeatedly annotating bacterium can suppress growth of tumor.
Five, the application of antibacterial in metastatic tumor is found
Make up animal metastatic tumor model as stated above, animal can be adopted nude mice etc., and tumor can be melanoma, hepatocarcinoma and breast carcinoma etc., and by the lumbar injection substrate, living imaging judges whether the metastatic tumor model successfully constructs.Tail vein injection escherichia coli in the animal model that successfully constructs observe escherichia coli and have the ability that is positioned metastatic tumor then.
Escherichia coli are applied to the diagnosis and the treatment of tumor, and except that the advantage with antibacterial itself, also have its exclusive characteristics: at first, the Colibacter extracellular bacteria is removed by antibiotic than bacterium in the born of the same parents is easier; Secondly, the non-pathogenic escherichia coli are nontoxic antibacterials, must not carry out attenuation to antibacterial earlier or weak malicious transformation can be used; Once more, more comprehensive to colibacillary genome understanding, it is easier to transform; At last, escherichia coli are facultative anaerobes, can assemble in the necrotic zone of tumor, also can assemble at the abundant position of oxygen, are not subjected in tumor size and the tumor whether anoxybiotic restriction aspect the selection of tumor.The present invention has proved escherichia coli by experiment first in the animal body of tumor growth is arranged, and to tumor tissues, comprises that metastatic tumor has the specific localization characteristics, and can suppress growth of tumor.Can utilize escherichia coli to the targeting of tumor and to the inhibitory action of tumor growth, add acceptable accessories, to keep colibacillary activity and stability, be used for preparing the medicine that is used for diagnosing tumor or treatment, have boundless market prospect.
Description of drawings
Fig. 1 is the characteristic distributions of photobacteria in normal mouse.Wherein A figure is tail vein injection E.coliDH5 α (1 * 10
9The cfu/100ul/ Mus) in the imaging situation of normal mouse after 10～20 minutes, visible photobacteria is distributed in two lungs of mice, and B figure is tail vein injection E.coli DH5 α (1 * 10
9The cfu/100ul/ Mus) the imaging situation after 3 days, visible photobacteria is distributed in the liver of mice, and the distribution situation of other colibacillus engineerings in normal mouse or nude mice is similar therewith.
Fig. 2 is the characteristic distributions of photobacteria in tumor-bearing mice.Wherein A figure is tail vein injection E.coliDH5 α (1 * 10
8The cfu/100ul/ Mus) in lotus the imaging situation of melanoma mice in the time of 1 day arranged, B figure is tail vein injection E.coli DH5 α (1 * 10
8The cfu/100ul/ Mus) in lotus the imaging situation of melanoma mice in the time of 2 days arranged, shown in the circle is the position of mouse tumor inoculation, be that tumor inoculation is subcutaneous in the mice right rear leg, luminous zone is that photobacteria is gathered in tumor tissues, and other colibacillus engineerings have the distribution situation in other solid tumor mices similar therewith melanoma mice or lotus.
Fig. 3 is tail vein injection E.coli DH5 α (1 * 10
8The cfu/100ul/ Mus) anatomy of melanoma mice mice after 13 days is arranged in lotus.Wherein A figure is the exposure status of Different Organs: the 1-liver, and the 2-lungs, the 3-heart, the 4-kidney, the 5-spleen, 6-stomach and intestinal, time of exposure: 300s, all organs are not seen the existence that photobacteria is arranged.B figure is the exposure status of whole tumor, and as seen time of exposure: 30s has the existence of a large amount of photobacterias.C figure is the tumor exposure status of cutting in half, time of exposure: 30s, and from C figure as can be seen: photobacteria mainly is present in the marginal area of tumor, then exists seldom in the intermediary necrotic zone of tumor.Other colibacillus engineerings have the situation in melanoma or other solid tumor mices similar therewith lotus.
Fig. 4 has the inhibitory action of melanoma mouse tumor growth to lotus for bacillus coli DH 5 alpha.Bacillus coli DH 5 alpha has the inhibitory action of other solid tumor mouse tumor growths similar therewith to lotus.
Fig. 5 has the inhibitory action of melanoma mouse tumor growth to lotus for e. coli bl21.E. coli bl21 has the inhibitory action of other solid tumor mouse tumor growths similar therewith to lotus.
Fig. 6 has the inhibitory action of melanoma mouse tumor growth to lotus for e. coli jm109.E. coli jm109 has the inhibitory action of other solid tumor mouse tumor growths similar therewith to lotus.
Fig. 7 is positioned nude mice melanoma lung metastasis for E.coli DH5 α.Wherein A figure takes from the lungs of annotating the melanoma metastasis model nude mice of bacterium after four days, has been covered with big and small melanotic tumor metastasis on the visible lung; B figure is the lungs with left figure, and behind the exposure 120s, visible photobacteria is positioned the neoplasm metastasis kitchen range of lungs.
The specific embodiment
The foundation of embodiment one animal tumor model and metastatic tumor model
One, experiment material:
1, cell, antibacterial, plasmid and culture medium: tumor cell B16-F10-luc-G5, MCF-7-luc-F5, BEL7405-luc and SMMC7421-luc purchase the company in Xenogen, and PC-3, C6, MB-49 and HT1080 purchase the company in ATCC.Bacillus coli DH 5 alpha, BL21, JM109 etc. are the international standard strain, and be commercial, and this laboratory is preserved.Cell culture medium: RPMI-1640, DMEM, hyclone are Sigma company product.Bacteria culture media: each composition peptone, yeast extract are purchased the company in OXOID among the LB, and sodium chloride is homemade analytical pure.PXen-1 (luxABCDE) plasmid is purchased the company in Xenogen.
2, laboratory animal: BALB/C mice and nu/nuBALB/C nude mice, in 5～6 ages in week, body weight 20 ± 5g provides by Military Medical Science Institute's animal center.
3, instrument and equipment: IVIS imaging system (Xenogen company); UV-1601 type ultraviolet spectrophotometer (Bio Rad company); Steri-cycle CO
2Incubator (Thermo ELECTRON company); Low temperature supercentrifuge (Sigma company); The OLYMPUS microscope; PCR instrument (Biometra company).
Two, methods and results
1, the cultivation of tumor cell and preparation
The culture medium constituent and the ratio of tumor cell are:
B16-F10, MCF-7, PC-3, MB-49 and HT1080 cell: DMEM 90%, FBS 10%.C6 glioma, BEL7405 and SMMC7421 cell: RPMI-164090%, FBS 10%.Tumor cell is placed 37 ℃ of 5%CO
2Cultivate in the incubator, change liquid or go down to posterity according to cell growth state.
Conventional method digestion, collecting cell, the centrifugal 10min of 1200rpm abandons supernatant, and washes cell three times with normal saline, at last with the blood counting chamber counting cells.Cell is resuspended in the normal saline the most at last, and making its final concentration is 5 * 10
7Cell/ml or 5 * 10
6Cell/ml.
2, the foundation of tumor model and metastatic tumor model
The tumor model of BALB/C mice and nude mice all adopts right rear leg subcutaneous vaccination tumor cell (5 * 10
6Cell/100 μ l/ Mus), the metastasis models of nude mice all adopts the tail vein to inject the mode (5 * 10 of tumor cell
5Cell/100 μ l/ Mus).
3, result: the tumor model of BALB/C mice and nude mice can form palp tumor behind 7～10 days of inoculated tumour cell; The metastatic tumor model of nude mice forms inject 7～14 days of cell of tumor at vein after, and the metastasis site lung shifts and accounts for more than 80%, and other also can have metastasis as liver, lymph node, fat, bone etc.
Structure and the detection of embodiment two photobacterias
One, experiment material
With embodiment one.
Two, methods and results
1, the preparation of photobacteria
Bacteria culture media LB prepares routinely, bacillus coli DH 5 alpha, and BL21, competent cells such as JM109 prepare routinely.
According to a conventional method with pXen-1 (luxABCDE) difference transformed into escherichia coli DH5 α, BL21, JM109, the luminous situation and the efficient that adopt the observation of IVIS imaging system to clone behind the coated plate.
2, the processing of antibacterial before the experiment
1. in the previous day of annotating bacterium to mice, the higher monoclonal bacterium colony of picking luminous efficiency in the LB that has resistance, overnight incubation in 37 ℃ of shaking tables, next day is by 1: 100 this bacterium of switching, continue to shake to the logarithmic growth middle and late stage be OD600=0.6～1.0.
2. press 1OD=8 * 10
8Cfu/ml calculates the bacterium amount, and is centrifugal then, removes LB, and thoroughly gives a baby a bath on the third day after its birth time with physiological saline solution, finally is diluted to 1 * 10
9Cfu/ml.
3. from the bacterium liquid for preparing, get the tail vein injection that 100 μ l are used for animal.
3, annotate bacterium in tumor-bearing mice
When mouse inoculation tumor after 7～10 days, annotate bacterium in the mode of tail vein injection to mice, annotating the microbial inoculum amount is 1 * 10
8Cfu/100 μ l/ Mus.
The result: by the photobacteria of above method preparation, luminous efficiency is higher, be injected in the animal body after, can under the live body situation, observe antibacterial distribution in animal body, thereby follow the trail of the location (seeing accompanying drawing 1～2) of antibacterial.
Embodiment three antibacterials distribution characteristics and tumor-localizing characteristic in animal body
One, experiment material
With embodiment one.
Two, methods and results
1, the characteristics that in normal mouse, distribute of antibacterial
Vein gives 2 * 10
9Cfu/100 μ l/ Mus or 1 * 10
9Cfu/100 μ l/ Mus is in the intravital 10～20min of normal mouse, and visible antibacterial is distributed in lung, and antibacterial gradually assembles to liver then, and is positioned liver, sees accompanying drawing 1, and wherein A figure is tail vein injection E.coli DH5 α (1 * 10
9The cfu/100ul/ Mus) in the imaging situation of normal mouse after 10～20 minutes, visible photobacteria is distributed in two lungs of mice; B figure is tail vein injection E.coli DH5 α (1 * 10
9The cfu/100ul/ Mus) the imaging situation after 3 days, visible photobacteria is distributed in the liver of mice; The distribution situation of other colibacillus engineerings in normal mouse or nude mice is similar therewith.Most of mice can not tolerate greater than 1 * 10
9The antibacterial of cfu/100 μ l/ Mus dosage, most mices are in the death in second day of annotating bacterium, and the postmortem result shows: antibacterial mainly is gathered in lungs and the liver of mice, and dead mice postmortem result then shows after dead or a couple of days: antibacterial only is distributed in liver.Intravenous injection 1 * 10
8The mice of cfu/100 μ l/ Mus antibacterial then can all survive, and bacterial distribution finally also only is distributed in liver.
2, the characteristics that in tumor-bearing mice, distribute of antibacterial
Intravenous injection 1 * 10
8Cfu/100 μ l/ Mus antibacterial is in tumor-bearing mice, annotate at the beginning of the bacterium, antibacterial at the intravital distribution of tumor-bearing mice and distributional class in normal mouse seemingly, but annotating second day of bacterium, the tumor locus of tumor-bearing mice promptly has the existence of light, see accompanying drawing 2, wherein A figure is the E.coli DH5 α (1 * 10 of tail vein injection
8The cfu/100ul/ Mus) in lotus the imaging situation of melanoma mice in the time of 1 day arranged, B figure is the E.coli DH5 α (1 * 10 of tail vein injection
8The cfu/100ul/ Mus) in lotus the imaging situation of melanoma mice in the time of 2 days arranged, shown in the circle is the position of mouse tumor inoculation, be that tumor inoculation is subcutaneous in the mice right rear leg, luminous zone is that photobacteria is gathered in tumor locus, and other colibacillus engineerings have the distribution situation in other solid tumor mices similar therewith melanoma mice or lotus.Dissect mice, get normal structure and tumor tissues respectively, find to have only tumor locus that stronger light is arranged, normal structure such as liver, lung, the heart, kidney and gastrointestinal etc. are then unglazed, and the prompting antibacterial all is distributed in tumor tissues.And we find that also for bigger tumor, antibacterial mainly is distributed in the periphery of tumor tissues, it is less that necrotic zone in tumor center, antibacterial then distribute, and illustrates that escherichia coli mainly are gathered in the relatively abundanter tumor region of oxygen part, see accompanying drawing 3, wherein A figure is the exposure status of Different Organs: 1-liver, 2-lungs, the 3-heart, the 4-kidney, 5-spleen, 6-stomach and intestinal, time of exposure: 300s, all organs are not seen the existence that photobacteria is arranged; B figure is the exposure status of whole tumor, and as seen time of exposure: 30s has the existence of a large amount of photobacterias; C figure is the tumor exposure status of cutting in half, time of exposure: 30s, and from C figure as can be seen: photobacteria mainly is present in the marginal area of tumor, then exists seldom in the intermediary necrotic zone of tumor.Other colibacillus engineerings have the situation in melanoma or other solid tumor mices similar therewith lotus.This research is also found, tail vein injection 1 * 10
7The tumor-bearing mice of cfu/100 μ l/ Mus antibacterial after dissecting in second day that annotates bacterium, and tumor locus just has the existence of light, and is then unglazed in normal structure, points out less antibacterial also can locate in tumor.
Embodiment four antibacterials are to the effect of tumor growth
One, experiment material
With embodiment one.
Two, methods and results
Get three groups of mices: be respectively matched group (normal saline group), once annotate the bacterium group and repeatedly annotate the bacterium group, observe the effect of escherichia coli itself to tumor growth.
At inoculated tumour the 7th day is divided into three groups at random with 15 mices, 5 every group.Once annotate bacterium group and repeatedly annotate the bacterium group and inject 1 * 10 in the mode of tail vein injection
8Cfu/100 μ l/ Mus bacterial suspension, matched group are injected the normal saline of equal volume, repeatedly annotate bacterium group mice 7 days, 14 days after annotating bacterium for the first time, re-inject the bacterium liquid of equal volume and concentration.The index of observation is that the growth of mouse tumor volume changes, and the computing formula of gross tumor volume is: V=L * W
2* 0.5 (wherein L is the major axis of tumor, and W is the minor axis of tumor).Statistical method adopts one factor analysis of variance, carries out comparing in twos of sample average, and what method adopted is the Student-Newman-Keuls check, i.e. q check.
The result shows: the growth of mouse tumor volume changes once annotating between bacterium group and matched group does not have significant difference (P＞0.05), but repeatedly annotate bacterium group with once annotate between the bacterium group or and matched group between difference obvious (P＜0.05) then, it is slow to show as the mouse tumor volume growth of repeatedly annotating the bacterium group, the mouse tumor that has has the phenomenon of dwindling, prompting: repeatedly annotate bacterium and can suppress growth of tumor, see accompanying drawing 4～6, show bacillus coli DH 5 alpha respectively, the inhibitory action that BL21 and JM109 have the melanoma mouse tumor to grow to lotus, these three kinds of engineering bacterias have the effect of other solid tumor mouse tumors similar therewith to lotus.
The application of embodiment five antibacterials in metastatic tumor is found
One, experiment material
With embodiment one.
Two, methods and results
Make up metastatic tumor models such as nude mice melanoma, hepatocarcinoma and breast carcinoma as stated above, after 1～2 week, press 0.1ml/10g lumbar injection substrate, behind the living imaging, can judge whether the metastatic tumor model successfully constructs.
After the model construction success, as stated above, the tail vein is annotated bacterium, observes escherichia coli and whether has the ability that is positioned metastatic tumor.
The result: tail vein injection melanoma, tumor cells such as hepatocarcinoma and breast carcinoma are in nude mouse, and the visible lungs metastatic tumor of living imaging successfully constructs after 10 days, and the tail vein injects 1 * 10
8The escherichia coli of cfu/100 μ l/ Mus are after 3～4 days, dissect the visible lungs of mice and be covered with the metastasis that varies in size, imaging shows, there is photobacteria to exist in the lungs metastasis, illustrate that escherichia coli can be positioned the metastasis of tumor, see accompanying drawing 7, and do not having other internal organs of neoplasm metastasis kitchen range, then do not see the existence that photobacteria is arranged; The location situation of other colibacillus engineerings in metastatic tumor is similar therewith.
Claims (8)
1. the purposes of escherichia coli in preparation diagnosing tumor or medicine.
2. purposes according to claim 1, wherein escherichia coli are each kind of escherichia coli genus or pass through genetically engineered colibacillus engineering on this basis.
3. purposes according to claim 1 and 2, wherein escherichia coli are DH5 α, BL21, JM101, JM103～JM109, K802 or K803.
4. purposes according to claim 1, wherein tumor is a solid tumor.
5. purposes according to claim 4, wherein tumor is Mus melanin tumor B16-F10, people's hepatocarcinoma BEL7405 and SMMC7721, human prostata cancer PC-3, human breast carcinoma MCF-7, human fibrosarcoma cell HT1080, Mus sarcoma of bladder cell MB-49 or Mus glioma C6.
6. according to the described purposes of claim 1, wherein the colibacillary dosage of diagnosing tumor is 5 * 108cfu/k body weight.
7. according to the described purposes of claim 1, wherein the colibacillary dosage of oncotherapy is 5 * 10
9Cfu/ time/kg body weight, weekly, continuous three times.
8. according to the described purposes of claim 1, wherein the route of administration of diagnosing tumor or medicine is an intravenously administrable.
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| CN103301163A (en) * | 2012-06-21 | 2013-09-18 | 桂林医学院 | Application of escherichia coli mixed entamoeba histolytica in preparation of medicine for treating liver cancer solid tumor |
| CN112513071A (en) * | 2018-04-30 | 2021-03-16 | 阿米库斯治疗学公司 | Gene therapy constructs and methods of use |
| CN113398275A (en) * | 2020-03-17 | 2021-09-17 | 中国科学院福建物质结构研究所 | Bacterial vector for photodynamic therapy and preparation method and application thereof |
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2005
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103301163A (en) * | 2012-06-21 | 2013-09-18 | 桂林医学院 | Application of escherichia coli mixed entamoeba histolytica in preparation of medicine for treating liver cancer solid tumor |
| CN112513071A (en) * | 2018-04-30 | 2021-03-16 | 阿米库斯治疗学公司 | Gene therapy constructs and methods of use |
| CN113398275A (en) * | 2020-03-17 | 2021-09-17 | 中国科学院福建物质结构研究所 | Bacterial vector for photodynamic therapy and preparation method and application thereof |
| CN113398275B (en) * | 2020-03-17 | 2023-07-25 | 中国科学院福建物质结构研究所 | Bacterial vector for photodynamic therapy and preparation method and application thereof |
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