CN106265760A - Clostridium ghonii domestication strain application in preparing radiotherapeutic sensitizer - Google Patents
Clostridium ghonii domestication strain application in preparing radiotherapeutic sensitizer Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
-
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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Abstract
The present invention relates to Clostridium ghonii domestication strain application in preparing radiotherapeutic sensitizer, the domestication strain of described Clostridium ghonii is preserved in Australian National quantitative study institute, and strain number is V12/001485.Present invention firstly discovers that Clostridium ghonii domestication strain has the Apoptosis to tumor.Clostridium ghonii domestication strain can distribution of specific being maintained in tumor tissues, and there is suppression tumor cell proliferation and tumour cell cycle is blocked increase the tumor cell effect to radiation sensitivity in G2/M phase and inducing apoptosis of tumour cell etc..
Description
Technical field
The present invention relates to Clostridium ghonii domestication strain application in preparing radiotherapeutic sensitizer, tame particularly to Clostridium ghonii
Strain (Derivatives of Clostridium ghonii, DCG) increases nasopharyngeal carcinoma (nasopharyngeal in preparation
Carcinoma, NPC) to the application in X-ray sensitive drug, belong to biomedicine technical field.
Background technology
Radiotherapy is one of three kinds of traditional means of oncotherapy, its in oncotherapy in occupation of critical role.In recent years with
Radiation therapy technology and equipment record, achieve and to a certain degree improve, but most of tumor all exists certain Radioresistence
Property, i.e. use the exposure dose of maximum possible or change illuminating method, in radiation is wild, tumor recurrence still exists.Research at present is recognized
For, the reason of radiation opposing is many, most important of which is that owing to generally having 10%～50% in entity tumor weary
Oxygen tumor cell, has resistance effect to ray, limits the effect of tumor radiotherapy.At present the most clinically for radiotherapy sensitizer with
Chemicals is main, such as docetaxel, 5-fluorouracil, cisplatin and amycin etc., though but when chemicals carries with chemotherapy combined radiotherapy
High tumor cytotoxicity effect, increases Normocellular toxic and side effects as well as inorganizable Specific Distribution.
Therefore, in the urgent need to finding one to disclosure satisfy that performance Apoptosis in solid tumor, avoid normal tissue thin simultaneously
The hypersitization medicine of cellular damage.
At present, the research of Clostridium ghonii domestication strain focuses primarily upon that the body of hydrolytic enzyme and the mediation utilizing it secrete is anti-to swell
Tumor immunoreation suppression kills tumor, so far there are no the report used as radiotherapy hypersitization medicine by antibacterial.
As Chinese patent literature CN104473973A (application number 201410803072.X) discloses a strain domestication Ge Shi shuttle
The application in preparation treatment non-small cell lung cancer drug of the bacterium MW-DCG-HNCv-18 bacterial strain.This invention also discloses domestication Ge Shi
Clostridium MW-DCG-HNCv-18 bacterial strain and docetaxel are medicine associated with active ingredient.It finds MW-DCG-HNCv-18 first
Bacterial strain has specific inhibitory effect to nonsmall-cell lung cancer, is significantly better than nonsmall-cell lung cancer inhibition existing known
Other similar bacterial strains, and when finding that it is combined with docetaxel injection by screening, to nonsmall-cell lung cancer inhibition more
Add prominent, provide new approach for treatment nonsmall-cell lung cancer.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, it is provided that Clostridium ghonii domestication strain application in preparing radiotherapeutic sensitizer.
Technical solution of the present invention is as follows:
One strain Clostridium ghonii domestication strain application in preparing radiotherapeutic sensitizer, the domestication strain of described Clostridium ghonii is preserved in Australia
Big Leah national measurement academy, strain number is V12/001485.
Above-mentioned application, described radiotherapeutic sensitizer specificity is applied to nasopharyngeal carcinoma.
Above-mentioned application, described radiotherapeutic sensitizer includes and pharmaceutically can connecing with Clostridium ghonii domestication strain bacterial strain for effective ingredient
The adjuvant being subject to.
According to currently preferred, described radiotherapeutic sensitizer dosage form is injectable powder；Described Clostridium ghonii domestication strain bacterial strain is
Spore lyophilized powder, pharmaceutically acceptable adjuvant is solvent.It is further preferred that described solvent is dense selected from ringer's solution, quality
Degree is normal saline, the phosphate buffer of 0.9%.
Described solvent needs to meet the requirement of non-toxic, injectable.By sides such as vein, tumor intratumor injection and abdominal cavities
Formula is administered.
It is further preferred that the preparation method of described dagger-axe Salmonella domestication strain spore lyophilized powder is as follows:
Clostridium ghonii is tamed strain spore liquid precooling 1～1.5h under the conditions of-80～-70 DEG C, then condenser temperature-
50～-40 DEG C, lyophilization 7～9h under the conditions of vacuum 15～25Pa.
Beneficial effect
1, present invention firstly discovers that Clostridium ghonii domestication strain has the Apoptosis to tumor.Clostridium ghonii domestication strain
Can distribution of specific being maintained in tumor tissues, and have suppression tumor cell proliferation and tumour cell cycle is blocked in
It is sensitive to radiotherapy that G2/M phase (cell cycle phase most sensitive to radiotherapy) and inducing apoptosis of tumour cell etc. increase tumor cell
The effect of property.
2, Clostridium ghonii is tamed strain and is applied in the radiotherapeutic sensitizer of nasopharyngeal carcinoma by the present invention first, is relatively applied to other classes
Type cancerous cell has more significantly effect of enhanced sensitivity.
Accompanying drawing explanation
Fig. 1, the Clostridium ghonii domestication strain metabolite influence curve figure to CEN-2Z cell proliferation；
In figure, left figure is the change with concentration of the CEN-2Z cell proliferation inhibition rate；Right figure is CEN-2Z cell inhibitory effect
Rate is over time；
Fig. 2, Clostridium ghonii domestication strain metabolite is to CEN-2Z Cell cycle influences figure；
In figure, left figure is matched group CEN-2Z cell cycle distribution；Right figure is experimental group CEN-2Z cell cycle distribution；
Fig. 3, tumor growth curve figure；
Fig. 4, tissue homogenate RCM-Agar Anaerobic culturel result photo；
Fig. 5, tumor tissue section's Gram stain (40 ×) result photo.
Detailed description of the invention
Below in conjunction with embodiment, technical scheme is further elaborated, but institute of the present invention protection domain is not limited to
This.
Bacterium source
Clostridium ghonii domestication strain derives from Australian National quantitative study institute, and bacterial strain preserving number is V12/001485；
Docetaxel is purchased from Qilu Pharmaceutical Co., Ltd., lot number: 4040071TA, date of manufacture: on 04 30th, 2014；
Toy radiotherapy instrument PXI produces, model: X-RAD225；
Cell cycle and the green skies of apoptosis detection kit, article No.: C1052；
The triumphant base of Annexin V-FITC/PI apoptosis kit is biological, article No.: GA105-KGA108.
Embodiment 1
The preparation method of dagger-axe Salmonella domestication strain spore lyophilized powder is as follows:
The Clostridium ghonii domestication strain spore liquid obtained is added 50g/L aseptic mannitol cryoprotective agent, detects before pre-freeze
Total living spores amount (5 × 10 in lyophilizing liquid filled container7cfu).Respectively spore liquid is placed in-20 DEG C ,-40 DEG C ,-80 DEG C of refrigerators enter
Row pre-freeze, the time is respectively 0.5h, 1.0h, 2.0h and 4.0h.Under the conditions of condenser temperature-45 DEG C, vacuum 20Pa, freezing dry
Dry 8h obtains Clostridium ghonii domestication strain spore lyophilized powder, counts Number of spores.
Result
As shown in Table 1, Clostridium ghonii domestication strain spore liquid pre-freeze pre-freeze 0.5h under the conditions of-20 DEG C ,-40 DEG C and-80 DEG C
Spore number declines less compared with before pre-freeze, but pre-freeze effect is poor, in mucus shape, is unfavorable for follow-up vacuum lyophilization, its
In be best with-80 DEG C of pre-freeze effects.Compared with Clostridium ghonii domestication strain-20 DEG C ,-40 DEG C pre-freeze 1h of spore liquid pre-freeze, though-80
DEG C pre-freeze 1h Number of spores reduces the most on a small quantity, but the order of magnitude is still 107, and spore liquid pre-freeze is complete, pre-freeze physical state is wanted
It is substantially better than the above two.Clostridium ghonii domestication strain spore liquid pre-freeze under the conditions of-20 DEG C ,-40 DEG C and-80 DEG C pre-freeze 2.0h and
Though it is effective that 3.0h Number of spores reduces lyophilizing less, pre-, but in view of economy and the factor of saving of time, should not select this two
Person.
Table 1 pre-freezing temperature and with the impact on Number of spores of the pre-freeze time
To sum up thinking, Clostridium ghonii domestication strain spore liquid vacuum lyophilization process is-80 DEG C of pre-freeze 1h, cold-trap temperature
Spend-45 DEG C, lyophilization 8h under the conditions of vacuum 20Pa.
Embodiment 2
Research method:
(1) impact on CEN-2Z cell proliferation
By dagger-axe Salmonella domestication strain metabolite lyophilized powder (0mg/ml, 3mg/ml, 5mg/ml, 10mg/ of different final concentrations
Ml, 13mg/ml, 17mg/ml and 20mg/ml) respectively with CEN-2Z co-culture of cells 24h after, carry out MTT experiment and detect it to carefully
The dose-dependent relationship of born of the same parents' propagation.Each concentration sets 6 multiple holes and arranges zeroing hole.
Final concentration of 10mg/ml dagger-axe Salmonella domestication strain metabolite lyophilized powder is co-cultured 0h respectively with CEN-2Z cell,
6h, 12h, 18h, 24h, 48h carry out MTT experiment and detect the time-dependent sexual relationship of its cell proliferation.Often group sets 6 multiple holes,
Each set of time zeroing hole.
Result
Result of study shows, along with action time and the increase of concentration, cell proliferation inhibition rate is gradually increased, and increases cell
Grow inhibitory action and show significant time-dependent sexual relationship mediating recipe amount dependency relationships (as described in Figure 1).
(2) impact on CEN-2Z cell cycle
By final concentration of 10mg/ml Clostridium ghonii domestication strain metabolite lyophilized powder and RCM (inoculum) respectively with
After CEN-2Z co-cultures 24h, carry out Flow cytometry experimental group and cellular control unit DNA with cell cycle detection kit
The change of content is to determine the phase of the cycles residing for cell.
Result
Compared with the control, 10mg/ml Clostridium ghonii domestication strain metabolite can make G2/M phase cell proportion significantly increase
(0.29VS12.62), it is seen that it can be by CEN-2Z cell block in phase sensitive period-G2/M (Fig. 2) of radiotherapy.
(3) impact apoptotic on CEN-2Z
Final concentration of 10mg/ml Clostridium ghonii domestication strain metabolite lyophilized powder and CEN-2Z are co-cultured respectively 12h and
After 24h, carry out the apoptotic situation of flow cytometer detection with Annexin V-FITC/PI apoptosis kit.
Result
Compared with matched group, it is visible getting up early apoptosis after Clostridium ghonii domestication strain metabolite and CEN-2Z effect 12h.
After effect 24h, there is more late apoptic phenomenon (table 2) in CEN-2Z cell.Result shows, Clostridium ghonii domestication strain metabolite
CEN-2Z cell had apoptosis-induced effect.
The domestication strain impact apoptotic on CEN-2Z of table 2 Clostridium ghonii
| Matched group | 12h | 24h | |
| Cell survival (%) | 98.43 | 94.85 | 90.16 |
| Viable apoptotic cell (%) | 0.87 | 4.28 | 3.25 |
| Later stage apoptosis (%) | 0.47 | 0.51 | 5.54 |
In sum, Clostridium ghonii domestication strain both can significantly inhibit CEN-2Z cell proliferation and induction CEN-2Z cell withers
Die, tumor cell can be increased to radiation sensitivity by cell cycle arrest in the G2/M phase again.
Embodiment 3
Research method:
1. mice with tumor model is set up
Use cell subcutaneous injection method that nasopharyngeal carcinoma CEN-2Z cell is slowly injected into female Balb/c-nu/nu nude mouse (5
Week old, 20g) the subcutaneous place of thigh lateral, right back, treats that gross tumor volume length is to 300mm3Time, carry out in vivo test.
2. animal packet and process
Matched group: n=10 only (n represents the size of animal often organized), tail vein injection PBS 0.2ml；
Clostridium ghonii domestication strain spore group: n=10, the 1st day, the 5th day, the 8th day and the 11st day tail vein injection Ge Shi
Clostridium domestication strain spore, dosage is 3.0 × 105cfu/(kg·body weight)。
Individually X-ray group: n=10 is only, the 4th day, the 7th day, the 10th day, the 13rd day radiotherapy, 1 times/day, dosage
400cGy/ time.
Only, within the 1st day, tail vein injection Clostridium ghonii tames strain bud to Clostridium ghonii domestication strain spore associating X-ray group: n=10
Spore, dosage is 3.0 × 105Cfu/ (kg body weight), radiotherapy in the 4th day, dosage 400cGy.5th day tail vein injection dagger-axe
Family name clostridium domestication strain spore, dosage is 3.0 × 105Cfu/ (kg body weight), radiotherapy in the 7th day, dosage 400cGy.8th
It tail vein injection Clostridium ghonii domestication strain spore, dosage is 3.0 × 105Cfu/ (kg body weight), the 10th day puts
Treat, dosage 400cGy.11st day tail vein injection Clostridium ghonii domestication strain spore, dosage is 3.0 × 105cfu/(kg·body
Weight), radiotherapy in the 13rd day, dosage 400cGy.
Docetaxel associating X-ray group: n=10, the 4th day, the 7th day, the 10th day and the 13rd day lumbar injection
Docetaxel, dosage is 2.5mg/ (kg body weight), radiotherapy 1 time, dosage 400cGy/ time after 6h.
3. Testing index
With vernier caliper measurement transplanted tumor in nude mice major diameter (a) and transverse diameter (b), 2～3 times weekly.
4. evaluation criterion
Gross tumor volume (V=1/2 × a × b is calculated according to the tumor major diameter measured and transverse diameter data every time2), draw curve,
Dynamically observe the change of tumor.
Tumour inhibiting rate (%)=(matched group mean tumor volume-experimental group mean tumor volume)/matched group mean tumor volume ×
100%；
Tumor regrowth delay (tumor growth delay, TGD)=treatment group tumors volume doubling time-matched group swells
Tumor volume doubling time；
Enhancer (Enhancement Factor, EF)=(TBacterium combined-TAntibacterial)/(TRadiotherapy-TComparison)。
5. result
Compared with X-ray group, Clostridium ghonii domestication strain spore associating X-ray group tumour inhibiting rate (60.56%) is significantly higher than list
Solely combination radiotherapy group (36.88%) and independent Clostridium ghonii domestication strain spore group (13.61%), its tumour inhibiting rate is higher than simultaneously
Docetaxel combines X-ray group, but difference is not notable (as shown in table 3).Tumor growth curve shows simultaneously, and Clostridium ghonii is tamed and dociled
After changing strain spore combined radiotherapy, gross tumor volume whole observation process tames strain and independent combination radiotherapy group less than Clostridium ghonii, again smaller than
Docetaxel combines X-ray group gross tumor volume, and the most obvious (as shown in Figure 3) with the later stage.
When transplanted tumor gross tumor volume length to 500mm3Time, observe 5 groups of transplanted tumor tumor doubling (300mm3To 500mm3)
Time, and calculate growth of xenografted and delay.Result of study shows, compared with matched group, and Clostridium ghonii domestication strain spore group (dosage
3.0×105Cfu/ (kg body weight)) tumor doubling time (8.33vs9.17), TGD (0vs0.84) and tumor suppression
Rate (0%vs13.61%) is all without significant difference.But when Clostridium ghonii domestication strain spore is combined with X-ray, its gross tumor volume times
Increasing time (23.00vs15.17, p=0.012), TGD (14.67vs6.84) and tumour inhibiting rate (60.56%vs36.88%) are the most aobvious
Work is better than independent X-ray group.
Compared with matched group, Clostridium ghonii domestication strain spore associating X-ray group growth of xenografted delays 14.67d, beyond
The growth of xenografted that Clostridium ghonii domestication strain spore group and independent combination radiotherapy group cause respectively delays sum (7.68d) (such as table 3 institute
Show).
Clostridium ghonii domestication strain spore associating X-ray group EF=(23.00-9.17)/(15.17-8.33)=2.02, i.e. dagger-axe
The family name clostridium domestication strain spore associating growth of xenografted that causes of X-ray delays to be 2.02 times (as shown in table 3) of independent radiotherapy.
Compared with Docetaxel associating X-ray group, Clostridium ghonii domestication strain spore associating X-ray group tumor doubling
The enhanced sensitivities such as time (21.83vs23.00, p=0.728), TGD (14.67vs13.50) and tumour inhibiting rate (60.56%vs48.19%)
Index is superior to the Docetaxel sensitization to radiotherapy, but difference with insignificance (as shown in table 3).
As above analyze visible, be used alone Clostridium ghonii domestication strain spore and show certain antitumor action, but itself and
Chemotherapy combined radiotherapy shows the effect dramatically increasing radiation sensitivity.
Table 3 Clostridium ghonii domestication strain enhanced sensitivity Indexs measure
Note: compared with X-ray group,※p<0.05。
Embodiment 4
Research method:
1. mice with tumor model is set up
Use cell subcutaneous injection method that nasopharyngeal carcinoma CEN-2Z cell is slowly injected into female Balb/c-nu/nu nude mouse (5
Week old, 20g) the subcutaneous place of thigh lateral, right back, treats that gross tumor volume length is to 300mm3Time, carry out in vivo test.
2. animal packet and process
Matched group: n=8 only (n represents the size of animal often organized), tail vein injection PBS 0.2ml；
Individually X-ray group: n=8 is only, the 4th day, the 7th day, the 10th day, the 13rd day radiotherapy, 1 times/day, dosage
400cGy/ time.
Clostridium ghonii domestication strain spore-1 group: n=8, the 1st day, the 5th day, the 8th day and the 11st day tail vein injection Ge Shi
Clostridium domestication strain spore, dosage is 1.0 × 105cfu/(kg·body weight)。
Clostridium ghonii domestication strain spore-2 group: n=8, the 1st day, the 5th day, the 8th day and the 11st day tail vein injection Ge Shi
Clostridium domestication strain spore, dosage is 2.0 × 105cfu/(kg·body weight)。
Clostridium ghonii domestication strain spore-3 group: n=8, the 1st day, the 5th day, the 8th day and the 11st day tail vein injection Ge Shi
Clostridium domestication strain spore, dosage is 3.0 × 105cfu/(kg·body weight)。
Clostridium ghonii domestication strain spore-4 group: n=8, the 1st day, the 5th day, the 8th day and the 11st day tail vein injection Ge Shi
Clostridium domestication strain spore, dosage is 4.0 × 105cfu/(kg·body weight)。
Clostridium ghonii domestication strain spore-5 group: n=8, the 1st day, the 5th day, the 8th day and the 11st day tail vein injection Ge Shi
Clostridium domestication strain spore, dosage is 5.0 × 105cfu/(kg·body weight)。
Clostridium ghonii domestication strain spore associating X-ray-1 group: n=8, the 1st day, the 5th day, the 8th day, the 11st day tail vein
Injection Clostridium ghonii domestication strain spore, dosage is 1.0 × 105cfu/(kg·body weight).4th day, the 7th day, the 10th
My god, the 13rd day radiotherapy, 1 times/day, dosage 400cGy/ time.
Clostridium ghonii domestication strain spore associating X-ray-2 group: n=8, the 1st day, the 5th day, the 8th day, the 11st day tail vein
Injection Clostridium ghonii domestication strain spore, dosage is 2.0 × 105cfu/(kg·body weight).4th day, the 7th day, the 10th
My god, the 13rd day radiotherapy, 1 times/day, dosage 400cGy/ time.
Clostridium ghonii domestication strain spore associating X-ray-3 group: n=8, the 1st day, the 5th day, the 8th day, the 11st day tail vein
Injection Clostridium ghonii domestication strain spore, dosage is 3.0 × 105cfu/(kg·body weight).4th day, the 7th day, the 10th
My god, the 13rd day radiotherapy, 1 times/day, dosage 400cGy/ time.
Clostridium ghonii domestication strain spore associating X-ray-4 group: n=8, the 1st day, the 5th day, the 8th day, the 11st day tail vein
Injection Clostridium ghonii domestication strain spore, dosage is 4.0 × 105cfu/(kg·body weight).4th day, the 7th day, the 10th
My god, the 13rd day radiotherapy, 1 times/day, dosage 400cGy/ time.
Clostridium ghonii domestication strain spore associating X-ray-5 group: n=8, the 1st day, the 5th day, the 8th day, the 11st day tail vein
Injection Clostridium ghonii domestication strain spore, dosage is 5.0 × 105cfu/(kg·body weight).4th day, the 7th day, the 10th
My god, the 13rd day radiotherapy, 1 times/day, dosage 400cGy/ time.
3. Testing index
With vernier caliper measurement transplanted tumor in nude mice major diameter (a) and transverse diameter (b), 2～3 times weekly.
4. evaluation criterion
Tumour inhibiting rate (%)=(matched group mean tumor volume-experimental group mean tumor volume)/matched group mean tumor volume ×
100%.
Tumor regrowth delay (tumor growth delay, TGD)=treatment group tumors volume doubling time-matched group swells
Tumor volume doubling time
Enhancer (Enhancement Factor, EF)=(TBacterium combined-TAntibacterial)/(TRadiotherapy-TComparison)
5. result
Compared with matched group, independent Clostridium ghonii domestication strain spore respectively organize tumour inhibiting rate, tumor doubling time, TGD
Have no between notable difference, and independent Clostridium ghonii each group of strain spore of domestication and also have no notable difference.Visible, independent 1.0 × 105
～5.0 × 105The Clostridium ghonii domestication strain spore dosage antitumor action of cfu/ (kg body weight) is inconspicuous.But work as
1.0×105～5.0 × 105When the Clostridium ghonii of cfu/ (kg body weight) dosage tames strain spore combined radiotherapy respectively
All showing notable antitumor action, tumour inhibiting rate, tumor doubling time, TGD are all significantly better than independent X-ray group, and
Tumour inhibiting rate, TGD have exceeded each dosage Clostridium ghonii domestication strain spore group and independent combination radiotherapy group sum, show the super of superposition potentiation
Quick effect (as shown in table 4).
1.0×105～5.0 × 105Cfu/ (kg body weight) Clostridium ghonii domestication strain spore combined radiotherapy causes
Tumor regrowth delay be all higher than 1.55 times (as shown in table 4) of independent X-ray, each dosage group all shows notable enhanced sensitivity and makees
With.
With tumour inhibiting rate, tumor doubling time, TGD, EF and economy as index comprehensive evaluation, research thinks 1.0
×105～5.0 × 105Cfu/ (kg body weight) Clostridium ghonii domestication strain spore is the preferred model of combined radiotherapy enhanced sensitivity
Enclose, wherein with 3.0 × 105Cfu/ (kg body weight) dosage Clostridium ghonii domestication strain spore combined radiotherapy is Ge Shi shuttle
Bacterium domestication strain increases the nasopharyngeal carcinoma optimal dosage to radiation sensitivity.
Table 4 various dose Clostridium ghonii domestication strain enhanced sensitivity Indexs measure
Note: compared with X-ray group,※p<0.05。
Embodiment 5
Research method:
1. mice with tumor model is set up
Use cell subcutaneous injection method that nasopharyngeal carcinoma CEN-2Z cell is slowly injected into female Balb/c-nu/nu nude mouse (5
Week old, 20g) the subcutaneous place of thigh lateral, right back, treats that gross tumor volume length is to 300mm3Time, carry out Clostridium ghonii domestication strain distribution in vivo
Test.
2. tissue culture and the Gram stain of tissue paraffin section de
After tail vein injection DCG spore 96h, de-neck puts to death mice with tumor, is put in 75% ethanol solution by the mice with tumor of execution and kills
Bacterium 1min.Take mice with tumor heart, liver, spleen, lungs, kidney (2) and tumor tissues, and above-mentioned internal organs are divided into two
Part, a copy of it adds aseptic PBS with 10ml/g solid-to-liquid ratio, is homogenate by tissue grinder rapidly, operating process rigorous aseptic.
In taking respectively in above-mentioned tissue homogenate 300 μ l even spread RCM-Agar culture medium, 37 DEG C of Anaerobic culturel 96h, record each group
Knit internal organs bacterial growth situation, and bacterium colony is carried out Gram dyeing observation.
After another part is fixed with neutral formalin buffer, carry out 4 μm paraffin sections and make, and section is carried out Gram
Stain and 40 × microexamination, detect antibacterial distribution in above-mentioned main organs tissue and tumor.
Result
After mice main organs and tumor tissues homogenate, 37 DEG C of Anaerobic culturel find, the domestication strain of tail vein injection Clostridium ghonii
Only occurring circle after spore 96hs in the RCM-Agar culture medium that tumor tissues homogenate liquid is corresponding, white, surface is slightly sticky, wet
Profit, opaque colony (Fig. 4), its colonial morphology is identical with Clostridium ghonii domestication strain bacterium colony, and after Gram stain detects really
It is set to Clostridium ghonii domestication strain.
Mice main organs and tumor tissues paraffin section carry out Gram stain and 40X microexamination, are found only at swollen
Tumor tissue exist a large amount of in shaft-like, G+Clostridium ghonii domestication strain (Fig. 5), and other main organs tissues do not find this antibacterial
Exist.This result of study matches with tissue homogenate methods and results.
By above two detection method result it can be seen that Clostridium ghonii domestication strain as sensitizer can distribution of specific in
In tumor tissues, and radiotherapy hypersitization medicine is chemicals or Chinese medicine at present, can be distributed and align by whole body after intravenously administrable
Often histiocyte produces harm, inorganization Specific Distribution.Visible, Clostridium ghonii domestication strain shows the most excellent as sensitizer
Gesture.
Embodiment 6
Research method:
1. mice with tumor model is set up
Use cell subcutaneous injection method respectively by malignant melanoma A375, nasopharyngeal carcinoma CEN-2Z and human colon cancer cell
HCT116 cell is slowly injected into the subcutaneous place of thigh lateral, female Balb/c-nu/nu nude mouse (5 week old, 20g) right back, treats tumor
Volume length is to 300mm3Time, carry out in vivo test.
2. animal packet and process
CEN-2Z matched group: n=6 only (n represents the size of animal often organized), tail vein injection PBS 0.2ml；
CEN-2Z independent spore group: n=6 only, tame and docile by the 1st day, the 5th day, the 8th day and the 11st day tail vein injection Clostridium ghonii
Changing strain spore, dosage is 3.0 × 105cfu/(kg·body weight)(0.2ml)。
CEN-2Z independent X-ray group: n=6, the 4th day, the 7th day, the 10th day, the 13rd day radiotherapy, 1 times/day, agent
Measure 400cGy/ time.
CEN-2Z spore associating X-ray group: n=6, the 1st day, the 5th day, the 8th day, the 11st day tail vein injection Ge Shi shuttle
Bacterium domestication strain spore, dosage is 3.0 × 105cfu/(kg·body weight)(0.2ml).4th day, the 7th day, the 10th day,
13 days radiotherapies, 1 times/day, dosage 400cGy/ time.
3. Testing index
With vernier caliper measurement transplanted tumor in nude mice major diameter (a) and transverse diameter (b), 2～3 times weekly.
4. evaluation criterion
Gross tumor volume (V=1/2 × a × b is calculated according to the tumor major diameter measured and transverse diameter data every time2)。
Tumour inhibiting rate (%)=(matched group mean tumor volume-experimental group mean tumor volume)/matched group mean tumor volume ×
100%.
Tumor regrowth delay (tumor growth delay, TGD)=treatment group tumors volume doubling time-matched group swells
Tumor volume doubling time
Enhancer (Enhancement Factor, EF)=(TBacterium combined-TAntibacterial)/(TRadiotherapy-TComparison)
It is sensitive to radiotherapy that Clostridium ghonii domestication strain spore affects melanoma A375 cell and human colon carcinoma HCT116 cell
Property animal packet with process, Testing index and evaluation criterion be all with the research of nasopharyngeal carcinoma CEN-2Z.
5. result
The table 5 Clostridium ghonii domestication strain enhanced sensitivity Indexs measure to different tumor models
Result of study shows, the Clostridium ghonii domestication strain spore associating X-ray tumour inhibiting rate table to three different tumor models
Reveal: CEN-2Z spore associating X-ray group HCT116 spore associating X-ray group > A375 spore associating X-ray group
(62.11vs54.55vs46.58) (as shown in table 5).Meanwhile, CEN-2Z spore associating X-ray group tumor doubling time shows
Work is longer than A375 spore associating X-ray group, but combines X-ray group tumor doubling time with HCT116 spore without significant difference
(as shown in table 5).It is optimal that TGD also combines X-ray group with CEN-2Z spore, higher than other two groups of Clostridium ghoniis domestication strain spore
Associating X-ray group, shows as CEN-2Z spore associating X-ray group > HCT116 spore associating X-ray group > A375 spore associating X-
Ray group (14.77vs11.50vs8.50) (as shown in table 5).Three differences are swollen by Clostridium ghonii domestication strain spore associating X-ray
Tumor model slack time is 2.04 times of independent radiotherapy, 1.3 times and 1.5 times respectively.The domestication strain of the most visible Clostridium ghonii
Show specificity and increase the rhinopharyngocele cancer model effect to radiation sensitivity.
Claims (6)
1. a strain Clostridium ghonii domestication strain application in preparing radiotherapeutic sensitizer, it is big that the domestication strain of described Clostridium ghonii is preserved in Australia
Leah national measurement academy, strain number is V12/001485.
Apply the most as claimed in claim 1, it is characterised in that described radiotherapeutic sensitizer specificity is applied to nasopharyngeal carcinoma.
Apply the most as claimed in claim 1, it is characterised in that described radiotherapeutic sensitizer includes taming strain bacterial strain with Clostridium ghonii
For effective ingredient and pharmaceutically acceptable adjuvant.
Apply the most as claimed in claim 1, it is characterised in that described radiotherapeutic sensitizer dosage form is injectable powder；Described Ge Shi shuttle
Bacterium domestication strain bacterial strain is spore lyophilized powder, and pharmaceutically acceptable adjuvant is solvent.
Apply the most as claimed in claim 4, it is characterised in that described solvent is 0.9% selected from ringer's solution, mass concentration
Normal saline, phosphate buffer.
Apply the most as claimed in claim 4, it is characterised in that the preparation method of described dagger-axe Salmonella domestication strain spore lyophilized powder is such as
Under:
Clostridium ghonii is tamed strain spore liquid precooling 1～1.5h under the conditions of-80～-70 DEG C, then at condenser temperature-50
～-40 DEG C, lyophilization 7～9h under the conditions of vacuum 15～25Pa.
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| CN106868024A (en) * | 2017-04-01 | 2017-06-20 | 山东新创生物科技有限公司 | A kind of Clostridium ghonii specific PCR detection primer and method |
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| CN113995835A (en) * | 2021-10-09 | 2022-02-01 | 山东新创生物科技有限公司 | Application of clostridium goeri spore combined PD-1 antibody |
| CN115569193A (en) * | 2021-10-09 | 2023-01-06 | 山东新创生物科技有限公司 | Application of clostridium govier spore and palbociclizumab as well as medicine for treating colon cancer and preparation method of clostridium govier spore freeze-dried powder |
| WO2023056962A1 (en) * | 2021-10-09 | 2023-04-13 | 山东新创生物科技有限公司 | Use of spore of clostridium ghonii in combination with pembrolizumab |
| CN113855710B (en) * | 2021-10-09 | 2024-02-20 | 施慧达药业集团(吉林)有限公司 | Application of clostridium gossypii combined tumor angiogenesis inhibitor |
| CN115569193B (en) * | 2021-10-09 | 2024-04-02 | 施慧达药业集团(吉林)有限公司 | Application of clostridium gossypii spores combined with palbociclib monoclonal antibody and preparation method of drug for treating colon cancer and clostridium gossypii spore freeze-dried powder |
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