CN1562375A - Combination of medication produced from attenuated salmonella bacteria possessing effect of radiation protection and anti tumour - Google Patents
Combination of medication produced from attenuated salmonella bacteria possessing effect of radiation protection and anti tumour Download PDFInfo
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- CN1562375A CN1562375A CNA2004100187705A CN200410018770A CN1562375A CN 1562375 A CN1562375 A CN 1562375A CN A2004100187705 A CNA2004100187705 A CN A2004100187705A CN 200410018770 A CN200410018770 A CN 200410018770A CN 1562375 A CN1562375 A CN 1562375A
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Abstract
A composite attenuated salmonella medicine with radiation-protecting and antineoplastic actions is prepared through cloning the gene fragment generated by the antiradiation and antineoplastic blood vessel into the eucaryotic expression carrier carrying green fluorescin and transferring the carrier into the attenuated salmonella by electric transfer. Said salmonella can apply the gene fragment to the target in tissue.
Description
Technical field
The pharmaceutical composition that hemopoietic recovers and prolong life cycle after the present invention relates to a kind of antineoplastic vascular new life and promoting radiotherapy, especially relate to bacteria carrier and clone's the cDNA carrier and the expression in animal body thereof of attenuation, the detection of various indexs and the observation of life cycle behind newborn treatment of antineoplastic vascular and the normal mouse radiation exposure after the mouse inoculation tumor.
Background technology
Damage in various degree all appears in blood system after the tumour patient radiotherapy, and bone marrow depression also appears in some patients that contact radioactive substance for a long time or be subjected to radiation damage.PF4 is a blood-platelet specific albumen, CXC chemotactic factor family member.Can promote the recovery of hemopoietic behind the radiation injury, hematopoietic cell there is reversible inhibitory action, temporarily suppress its propagation, make hematopoietic cell stop at the S phase, can improve myeloid element to radiating toleration, can also stimulate the propagation of early stage hematopoietic stem cell, thereby promote the recovery of irradiation back myeloid element effectively.PF417-70 is the genetic fragment of PF41-70, has the PF41-70 and the PF417-70 of report adenovirus coding to suppress growth of tumor by angiogenesis inhibiting.And proof suppresses tumor and present operation and chemicotherapy by anti-angiogenic rebirth, and to compare toxicity low.Its characteristics of attenuated bacteria strain are that by oral route can pass lymphoid tissue that the mucous membrane of small intestine epithelial cell invades the intestinal wall particularly aggregated lymphatic follicles and the solitary lymph follicle of ileum lower end, and prolong lymphatic vessel to mesenteric lymph node.In these lymphoid tissues, by macrophage phagocytic, and breeding therein; Enter blood through thoracic duct on the other hand, the pathogenic bacteria in the blood is very fast to be engulfed macrophage in system such as liver, spleen, bone marrow and the lymph node by the whole body monokaryon.Because the picked-up of the macrophage in bone marrow pathogenic bacteria is more, life period is longer, so bone marrow cultivation positive rate can be up to 90%.The influence of the microenvironment in tumor tissues, the Salmonella of attenuation can optionally be collected in the tumor tissues, and its specificity is compared 1000: 1 with normal structure.Become a kind of carrier of valuable Antioncogene treatment.Attenuated strain is owing to can not synthesize P-hydroxybenzoic acid (PAB) and 2, and 3-dihydroxy benzenes (DHB) is so can not become auxotrophic attenuated strain in the mammalian cell internal breeding.
Summary of the invention
Problem to be solved by this invention provides a kind of attenuation salmonella pharmaceutical composition with radiological protection and antitumor action, by the bacterial strain of attenuation and clone's cDNA carrier expression in animal body.
In order to solve the problems of the technologies described above; the technical solution used in the present invention is: a kind of attenuation salmonella pharmaceutical composition with radiological protection and antitumor action; carry the nucleotide sequence of SEQ ID NO.1/or the carrier for expression of eukaryon of SEQ ID NO.3 nucleotide sequence fragment; by integrating in vivo after oral; express corresponding polypeptide in vivo by recipient cell; reach Antiradiation injury and antitumor action; drug regimen be gene fragment clone by will having radioprotective and antineoplastic vascular new life in the carrier for expression of eukaryon that carries green fluorescent protein, and the pulsating carrier of clone gene is transferred in the Salmonella of attenuation by electric transform mode.
Used Salmonella is an attenuated strain.
Described carrier for expression of eukaryon is PIRES2-EGFP, through the attenuation salmonella mediation, shifts to integrate arriving each tissue of whole body.
Can express polypeptide factor in vivo after oral, prolong the life span of radiation injury animal, promote the recovery of hemopoietic with radioprotection.
Can express polypeptide factor in vivo after oral, significantly suppress the growth of malignant tumor with neoplasm growth.
The invention has the beneficial effects as follows: the salmonella strain by attenuation is that bacteria carrier is brought into play therapeutical effect with the gene segment targeting ground that transforms in tissue.Method is simple, effective, has no side effect, and damages little suitable prolonged application.
Description of drawings
Fig. 1 is a Construction of eukaryotic.
Fig. 2 is the expression of green fluorescent protein in the mouse tumor tissue.
Fig. 3 is the expression of green fluorescent protein in myeloid tissue.
Fig. 4 is the expression of green fluorescent protein in peripheral blood.
Fig. 5 is the expression of green fluorescent protein in liver.
Fig. 6 is the expression of green fluorescent protein in spleen.
Fig. 7 is the expression of green fluorescent protein in kidney.
Fig. 8 is the expression of green fluorescent protein in small intestinal.
Fig. 9 is the observation of gross tumor volume.
Figure 10 is the survival curve of irradiation back mice.
Figure 11 is the survival rate of mice behind the inoculated tumour.
After Figure 12 irradiation is the comparison of animal peripheral hemogram.
Figure 13 shines the comparison of back animal bone marrow total cellular score.
Figure 14 shines the comparison of back animal bone marrow high value-added potential colony forming unit number.
Figure 15 shines the comparison of back animal bone marrow grain list colony forming unit number.
Survival curve behind Figure 16 mice irradiation 7Gy.
The specific embodiment
The present invention is described further below in conjunction with embodiment, but that the present invention is not limited to is following
Embodiment.
Embodiment 1: the preparation of the cDNA that is used to clone
As shown in Figure 1, by gene runner design primer sequence, by Shanghai biotech firm synthetic primer, the primer both sides have EcoRI and BamHI restriction enzyme site respectively, have the PF417-70 (SEQ ID NO.3) of people MCP-1 signal peptide (27 aminoacid) and the gene segment of PF41-70 (SEQ ID NO.1) from carrier PCMV-PF417-70 and PCMV-PF41-70 amplification.The segment of amplification is approximately 240bp, 291bp (comprising signal peptide).PCR product after EcoRI and BamHI enzyme action carrier and glue reclaim, method (the Sambrook of molecular cloning main reference Sambrook etc., T.etal.Molecular Cloning:A Laboratory Manual, 1989, Cold Spring HarborLaboratory, New York), the PIRES2-EGFP plasmid is purchased the company in invitrogen, attenuated strain is purchased in Canadian antibacterial storehouse, the fragment that two ends are had EcoRI and BamHI joint cDNA respectively is inserted in the plasmid of the PIRES2-EGFP of EcoRI and BamHI double digestion, uses the junctional complex transformed into escherichia coli, screening positive clone, carry out enzyme action, electrophoresis and order-checking identify that correct clone is used for expressing.
Embodiment 2: the preparation of carrying the attenuated strain of clone gene fragment carrier
Attenuated strain is cultured to OD600=0.4 in the LB culture medium, preparation competence attenuated strain will carry the segmental carrier of clone gene by the method for electroporation and be transformed into attenuated strain.Screening positive clone.
Embodiment 3: the detection of transfection in the body
As Fig. 2 to Fig. 8, the expression by green fluorescent protein in the detection bodies inner tissue comes the distribution in vivo of side light transgenic.Liver, spleen, kidney, small intestinal, bone marrow, peripheral blood and the tumor tissues fluorescence microscope of getting irradiation back and inoculated tumour mice respectively detect the expression of fluorescence, under Laser Scanning Confocal Microscope, all can see the expression of green fluorescent protein, express stronger in the tumor tissues.And do not see the expression of green fluorescent protein in the control group mice section.Show that Salmonella can import allogenic gene in the mice body and carries out the organ specifically expressing.
Embodiment 4: the observation of gross tumor volume
As shown in Figure 9, with 1 (10
5) the pernicious melanocyte of B16 is inoculated in the right side of mice oxter, grows to naked eyes when visible when tumor, conversion had the attenuated strain of genetic fragment be cultured in containing the LB culture medium of 50ug/ml kanamycin, OD600=0.4, collect thalline, after PBS cleans, 100g/LNaCHO
3Solution suspends and adjusts bacterial population is 1 * 10
9Individual/ml.Laboratory animal is divided into four groups every group 6, be respectively blank group, green fluorescent protein group, empty bacterium group (SL3261), SL3261/PF417-70 group, each is organized Mus and raised the corresponding antibacterial 0.1ml of clothes every three days with stomach tube, and the blank group is raised clothes equivalent 100g/L NaCHO
3, measure the size of tumor every three days.Mouse tumor volume through treatment obviously dwindles (P＜0.05)
Embodiment 5: observe life cycle
As shown in figure 10, the survival condition of mice irradiation back its state of record and mice.The attenuated strain that carries transformed clone genetic fragment carrier is cultured to OD600=0.4,10%NaHCO
3Bacterial population is adjusted to 1 * 10
9/ ml gets 100ul by the oral feed of esophageal probe.Healthy male Balb/c mice is divided into five groups at random: experimental group PF41-70, experimental group PF417-70, empty carrier group, attenuated strain group and PBS group, every group all in pre-irradiation every three days feed, gave 7.0cGy radiation exposure in second day behind the feed for the second time.Shine and gave feed in back second day, later on once every three days feed.Feed is 7 times altogether.Compare obvious prolongation the life cycle of the mice of process PF41-70 and PF417-70 treatment with matched group.The mice of PF417-70 treatment survives at duration of test always, and the mice of PF41-70 treatment has 2/5ths death, does not have the mice of treatment all dead.
As shown in figure 11, tumor inoculation is after compare obvious prolongation 50% life cycle of the mice of SL3261/PF417-70 treatment with matched group.
Embodiment 6: the comparison of animal peripheral hemogram
As shown in figure 12, irradiation back we detect every group of mice at the 7th day peripheral blood leucocyte, platelet and erythrocytic counting.It is not obvious that each organizes platelet and the erythrocyte difference of mice.Through PF41-70 and PF417-70 treatment, though leukocyte count be lower than normal value, compare obviously with matched group and increase (P＜0.05).
Embodiment 7: the comparison of animal bone marrow total cellular score
As shown in figure 13, irradiation back in the time of the 7th day every group get three mices, the cervical vertebra dislocation method is put to death mice, takes out femur and tibia, goes out the quantity of mononuclearcell (MNC) in the medullary cell counting bone marrow.BMNC number through PF41-70 and PF417-70 treatment group increases (P＜0.05) than matched group.
Embodiment 8: irradiation back bone marrow colony is cultivated
Shown in Figure 14,15, irradiation back fortnight is got bilateral tibial and the femur mononuclearcell 5 * 10 of mice
4Colony is cultivated PF41-70 and PF417-70 treatment group, and the quantity of high proliferation potential colony forming unit (HPP-CFC), the single colony forming unit of grain (GM-CFU) have been compared obvious difference (P＜0.05) with matched group.
Embodiment 9: chemosynthesis artificial blood function and protecting peptide is to the radioprotection of mice
The recombinant vector that carries for the function series antibacterial of the Radiological Defense hemopoietic of verifying above-mentioned pharmaceutical composition is expressed due to the hemopoietic function protection peptide in vivo.The present invention adopts chemical synthesis process, has synthesized this polypeptide.And carried out animal vivo test with highly purified polypeptide.Specifically, behind the female C57 mice irradiation 7Gy, every day, the subcutaneous injection polypeptide was 1 time, injected altogether 4 times, each every injected in mice 5 μ g.Observe the existence situation of mice then, the result as shown in figure 16, its survival rate of mice of accepting polypeptide injection has improved about 27%.
SEQUENCE LISTING (sequence table)
＜110〉Inst. of Hematology, Chinese Academy of Medical Sciences
＜120〉has the attenuation salmonella pharmaceutical composition of radiological protection and antitumor action
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Claims (5)
1; a kind of attenuation salmonella pharmaceutical composition with radiological protection and antitumor action; it is characterized in that carrying the nucleotide sequence of SEQ ID NO.1/or the carrier for expression of eukaryon of SEQ ID NO.3 nucleotide sequence fragment; by integrating in vivo after oral; express corresponding polypeptide in vivo by recipient cell; reach Antiradiation injury and antitumor action; drug regimen be gene fragment clone by will having radioprotective and antineoplastic vascular new life in the carrier for expression of eukaryon that carries green fluorescent protein, and the pulsating carrier of clone gene is transferred in the Salmonella of attenuation by electric transform mode.
2,, it is characterized in that used Salmonella is an attenuated strain according to the described Pharmaceutical composition of claim 1.
3, according to the described Pharmaceutical composition of claim 1, it is characterized in that described carrier for expression of eukaryon is PIRES2-EGFP, through the attenuation salmonella mediation, shift to integrate arriving each tissue of whole body.
4, according to the described Pharmaceutical composition of claim 1, it is characterized in that can expressing polypeptide factor in vivo after oral with radioprotection, prolong the life span of radiation injury animal, promote the recovery of hemopoietic.
5, according to the described Pharmaceutical composition of claim 1, it is characterized in that can expressing polypeptide factor in vivo after oral with neoplasm growth, significantly suppress the growth of malignant tumor.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100187705A CN100435850C (en) | 2004-03-22 | 2004-03-22 | Combination of medication produced from attenuated salmonella bacteria possessing effect of radiation protection and anti tumour |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100187705A CN100435850C (en) | 2004-03-22 | 2004-03-22 | Combination of medication produced from attenuated salmonella bacteria possessing effect of radiation protection and anti tumour |
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| CN1562375A true CN1562375A (en) | 2005-01-12 |
| CN100435850C CN100435850C (en) | 2008-11-26 |
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| CNB2004100187705A Expired - Fee Related CN100435850C (en) | 2004-03-22 | 2004-03-22 | Combination of medication produced from attenuated salmonella bacteria possessing effect of radiation protection and anti tumour |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114736860A (en) * | 2022-03-18 | 2022-07-12 | 江苏靶标生物医药研究所有限公司 | Monocyte or macrophage loaded with attenuated salmonella as well as preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IL134936A0 (en) * | 1997-09-10 | 2001-05-20 | Vion Pharmaceuticals Inc | Genetically modified tumor-targeted bacteria with reduced virulence |
| BR0014491A (en) * | 1999-10-04 | 2004-03-09 | Vion Pharmaceuticals Inc | Tumor-marked attenuated bacteria, pharmaceutical composition, use of attenuated bacteria, and fusion protein |
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- 2004-03-22 CN CNB2004100187705A patent/CN100435850C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114736860A (en) * | 2022-03-18 | 2022-07-12 | 江苏靶标生物医药研究所有限公司 | Monocyte or macrophage loaded with attenuated salmonella as well as preparation method and application thereof |
| CN114736860B (en) * | 2022-03-18 | 2024-05-17 | 江苏靶标生物医药研究所有限公司 | Monocyte or macrophage loaded with attenuated salmonella and preparation method and application thereof |
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| CN100435850C (en) | 2008-11-26 |
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