CN109675023A - A kind of method that BCG vaccine promotes epirubicin in treating bladder cancer - Google Patents
A kind of method that BCG vaccine promotes epirubicin in treating bladder cancer Download PDFInfo
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- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 title claims abstract description 89
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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
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A—HUMAN NECESSITIES
- 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
- A61K9/0034—Urogenital system, e.g. vagina, uterus, cervix, penis, scrotum, urethra, bladder; Personal lubricants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/10—Drugs for disorders of the urinary system of the bladder
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2503/00—Use of cells in diagnostics
- C12N2503/02—Drug screening
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Abstract
The invention discloses a kind of methods that BCG vaccine promotes epirubicin in treating bladder cancer, more particularly to the combination therapy field of the anti-bladder cancer in clinical medicine, including cellular level research BCG to the method for the synergistic effect of epirubicin and internal BCG to the method for the synergistic effect of epirubicin.By the present invention in that with cellular level research BCG to the method for the synergistic effect of epirubicin and internal BCG to the method for the synergistic effect of epirubicin, provide a kind of scheme of drug combination, to improve therapeutic efficiency, reduce toxic side effect when drug infusion, dabbling drug concentration of the present invention is not high, but the retention time is long in bladder, so that antitumous effect is better, and adverse reaction will not occur.
Description
Technical field
The present invention relates to the combination therapy technical fields of the anti-bladder cancer in clinical medicine, and more specifically, the present invention relates to
And a kind of BCG vaccine promotes the method for epirubicin in treating bladder cancer.
Background technique
Bladder cancer is the most common malignant tumour of China's urinary system, annual newly-increased 50,000 or more case.Wherein 90%
Bladder mucosa transitional epithelial cell is derived from above, and 5-10% is squamous carcinoma, and 2-3% is gland cancer.Treatment method mostly uses per urethra
Resection (TUR) thoroughly cuts off naked eyes visual tumors, and is aided with recurrence and progress that complementary perfusion therapy carrys out pre- preventing tumor.According to
It counts, in bladder cancer patients, the bladder cancer of 75-85% is shallow cancer, will be answered behind shallow cancer 1 year after surgery of 50-80%
Hair, the recurrent tumor of 10-30% and stroke degree increases.It is postoperative that the recurrence and progress of tumour can be effectively reduced by perfusion therapy,
It improves the quality of living.
It is Current therapeutic early stage shallow that treatment with chemotherapy drug (cytotoxic drug) or immune formulation, which carry out irrigation of bladder,
Tumor of bladder, the most popular method for preventing bladder cancer recurrence, but medicament categories are various, and method for filling is different, therapeutic effect
Also identical.It is generally acknowledged that dabbling drug concentration is higher, the retention time is longer in bladder, and antitumous effect is better, but adverse reaction
Also bigger, drug appropriate is selected, the reasonable dosage and method of being perfused seems most important.
The prior art related to the present invention:
Perfusion mainstay type include: BCG vaccine (BCG), interferon, epirubicin, pirarubicin, hydroxycamptothecin,
5 FU 5 fluorouracil etc..Intravesical BCG method are as follows: under room temperature, 120mg BCG freeze-dried powder is dissolved in bladder in 40-60ml physiological saline
Interior perfusion retains 2 hours.Generally start to be perfused within postoperative 2 weeks, once a week, be changed to after continuous 6 times monthly 1 time, continue 2 years with
On.
The 80mg of single irrigation of bladder for the first time in epirubicin postoperative 6 hours, later monthly 50mg are maintained in bladder
60min is maintained 8-10 months.
The mechanism of action difference to tumour is perfused in said medicine, and is all made of single drug mode and is perfused, and use in conjunction is
No to have better effect, there has been no the application of combined injection and reports at present.
And these methods have certain defect due to side effects of pharmaceutical drugs, can only be between drug dose and effect
Take balance.
Summary of the invention
In order to overcome the drawbacks described above of the prior art, the embodiment of the present invention provides a kind of BCG vaccine and Epi-ADM is promoted to control
The method for treating bladder cancer, by using cellular level research BCG to the method for the synergistic effect of epirubicin and BCG pairs internal
The method of the synergistic effect of epirubicin provides a kind of scheme of drug combination, to improve therapeutic efficiency, reduces drug infusion
When toxic side effect, dabbling drug concentration of the present invention is not high, but the retention time is long in bladder, so that antitumous effect is better,
And adverse reaction will not occur.
To achieve the above object, the invention provides the following technical scheme: a kind of BCG vaccine promotes epirubicin in treating bladder
The method of cancer, including cellular level research BCG to the method for the synergistic effect of epirubicin and internal BCG to epirubicin
The method of synergistic effect；
The present invention provides cellular level research BCG to the method for the synergistic effect of epirubicin, the specific steps are as follows:
Step 1: cell culture: in 37 DEG C, 5%CO2Under the conditions of using the MEM culture medium containing 10% fetal calf serum into
Row culture, liquid or passage were changed every 2-3 days；
Step 2: cytoactive detection: collecting logarithmic phase human bladder cancer UMUC3 using CCK8 cytoactive detection kit
Cell, adjustment cell concentration are 5 × 104/mL, are inoculated in 96 orifice plates by every 100 μ L of hole, are placed in incubator and stay overnight；
Step 3: relative medicine is added according to various concentration, after reaching preset time, discards old training after cell is adherent
It supports base and is washed 2 times with PBS；
Step 4: detecting cell activity using CCK8: the 100 μ L of MEM culture medium containing 10%CCK8 is added in every hole, in 37
Absorbance value (OD) is read in 450nm at after DEG C being incubated for 4 hours, is calculated further according to OD value, work of the detection drug to cell
Property influence；
The present invention also provides internal BCG to the method for the synergistic effect of epirubicin, the specific steps are as follows:
Step 1: selection: choosing SPF grades female sd inbred rats 106, weight 180g-200g is randomly divided into modeling group
(10), blank control group (24), EPI group (24), EPI+BCG group (24) and EPI+CpG group (24)；Prepare main
Reagent: citric acid, MNU reagent, chloraldurate, 4% paraformaldehyde fixer, rat interleukin-22 (IL-2) ELISA
Assay kit, epirubicin, BCG vaccine, CpG-ODN, CpG-ODN sequence；Prepare key instrument and equipment: speed-regulating type is mini
Centrifuge, HWS26 type electric-heated thermostatic water bath, electro-heating standing-temperature cultivator, microplate reader, inverted microscope, AR-1000 electronic balance
And slicer；
Step 2: starting to test:
1) establish rat original position bladder cancer models: SD rat is carried out 1 week adaptable fed by a., Rat Fast before being perfused
Prohibit water 12h, the as far as possible urine in empty bladder；B. perfusion tests MNU reagent used and is placed in 4 DEG C of refrigerators on the day before experiment
Overnight, it before use using the citrate buffer of pH6.0 as solvent, takes MNU reagent to be made into dense 20mg/ml, has been used in 40min
Finish；C. rat is with 10% chloraldurate intraperitoneal injection of anesthesia, fixation of lying on the back, 75% ethanol disinfection orificium urethrae externum 2 times；D. will
Alignment rat urethral orifice is slowly inserted into after the disposable epidural catheter of 1.0mm specification smears sterile paraffin oil, by ready MNU
Solution 0.1ml is poured into bladder through injection-tube, is exited after epidural catheter and is closed with pressing from both sides urethral orifice with small metal venous clamp, solution
It is detained 60min；E. rat carries out irrigation of bladder MNU, each 2mg, and 1 time every 2 weeks, totally 4 times, infusion time is the 1st, 3,5 and 7
In week, model foundation is completed within the 8th week, and rat is the object of this research at this time, is raised at 22-26 DEG C of temperature, humidity 20-70%'s
In SPF rank environment, the rat state of mind, feed drinking-water situation are observed daily, whether there is or not gross hematurias；
2) perfusion administration concentration control after modeling: except modeling group is 10 rats, every group of 24 rats of other groups, modeling
0.1ml is perfused in medication in 1 week afterwards every time, and infusion time is the 9th, 11,13 and 15 week, during perfusion administration herein: modeling group, nothing
Any processing；Physiological saline is perfused in control group；Epirubicin group is perfused EPI (2mg/ml)；Epirubicin+BCG vaccine group fills
It infuses EPI+BCG (2mg/ml+2mg/ml)；Epirubicin+CpG ODN group is perfused EPI+CpG ODN (2mg/ml+4 μM/ml)；
3) sample collection: a. serum collection collects whole blood sample in blood by eye socket blood collection method 1 week after each perfusion
4 DEG C are placed in clear separating pipe overnight, then 3000r/min is centrifuged 15 minutes, is taken supernatant, is carried out respective markers and be placed in -20
It DEG C saves, and is no more than 1 month, avoid multigelation, each each group collects 6 rats；
B. bladder body acquire, 1 week after each perfusion, using pull cervical vertebra method execution rat, every group each 6 groups；It cuts open the belly,
By its bladder complete resection, observes the general condition of inner bladder surface and cancer formation and take pictures, be placed in cryopreservation tube, carry out corresponding
Label is placed in -80 DEG C of preservations；The collection of specimens time is the 2nd, 4,6,8,10,12,14,16 week；
4) using IL2 content in the serum of the acquisition of ELISA method determination step six；
5) pathological change is observed using HE decoration method: each group bladder body being placed in 4% paraformaldehyde solution and fixes 15-
24 hours, the set time regard environment temperature and it is different；Then routine paraffin wax embeds, 5 μm of slices, and HE dyeing is observed under the microscope
Pathological change；
6) using the expression of p53 in Immunohistochemical Method detection histotomy, antibody working solution concentration is 1:100；
Step 3: statistical analysis: being analyzed using 19.0 statistical software of SPSS, calculate data with average ± mark
It is quasi- poorIt indicates, using the difference of the IL-2 concentration of one-way analysis of variance each group, P < 0.05 indicates that difference has significantly
Property；
Step 4: p53 ImmunohistochemistryResults Results judge: positive stained cells number < 10% is (-), positive stained cells number
10%~25% is (+), and positive stained cells number 25%~75% is (++).Positive stained cells number >=75% is (+++)；
Step 5: observation experiment result:
1) the bladder cancer morbidity situation for observing each group SD rat, compares the generation of control group Yu modeling group rat bladder cancer
Rate difference has conspicuousness, illustrate MNU induce bladder cancer rat have obvious effect, and after each treatment group's bladder cancer is formed respectively by
It is treated according to administration concentration, see Table 1 for details；
2) using the variation of IL-2 content in ELISA method detection rat blood serum, perfusion therapy, carries out IL-2 concentration ratio three times
Compared with；
3) it visually observes, observes by the naked eye the bladder feature of bladder cancer modeling group rat；
4) HE stained slice is analyzed, and puts to death rat, is taken bladder body to pass sequentially through hematoxylin-eosin (HE) dyeing, is set aobvious
Micro- microscopic observation pathological change；
5) it is expressed using p53 in Immunohistochemical Method detection histotomy, all stained slices are seen under an optical microscope
It examines, is saved after being taken pictures with Computer digital image analysis scanning, positive cell technology is calculated using image analysis software statistics,
The coloring of the p53 positive is located in neoplastic cell nuclei, and nucleus is dyed to sepia, and every slice takes 5 high power fields (400 at random
×), positive cell number is counted, the cell number of entire field of view is accounted for as percentage using positive cell number；It will be cut with a batch dyeing
Piece carries out tumour cell gray-scale statistical calculating with image analysis software.
In a preferred embodiment, the internal BCG is in the method for the synergistic effect of epirubicin, CpG-
ODN sequence includes 72 bases, and full sequence is modified by thiophosphoric acid.
In a preferred embodiment, the internal BCG is in the method for the synergistic effect of epirubicin, bladder cancer
Judgment criteria is as follows: a. no abnormality seen: mucosal epithelium is methodically arranged, queueing discipline, and cellular morphology, size, dyeing are consistent；B. light
Atypical hyperplasia: mucous membrane of urinary bladder is spent, nipple is in cone, and cell arrangement is consistent, no heteromorphism；C. severe atypical hyperplasia: thin
Born of the same parents are not of uniform size, disorganized, and pole is to disorder, and form of diverse, core is big and contaminates deeply, and karyoplasmic ratio increases, and nuclear fission increases；D. bladder
Cancer: cell size is different, disorganized, and pole is unevenly distributed to disappearance, form of diverse, core deformity, nuclear chromatin, and nuclear membrane increases
Thickness, kernel is loose, and caryoplasm ratio is big, it is seen that pathology nuclear fission picture is divided into I, II, III grade.
In a preferred embodiment, for the internal BCG in the method for the synergistic effect of epirubicin, observation is each
The bladder cancer morbidity situation of group SD rat: during experiment, blank control group does not occur death without an example tumor formation rat；8th week
When, modeling group is survived 9, and the incidence of bladder cancer is 77.8% (7/9)；Bladder cancer at remaining each group the 10th, 12,14,16 week
Incidence be for 66.7% (12/18), 72.2% (13/18), 61.1% (11/18), 55.6% (10/18) respectively.
In a preferred embodiment, the internal BCG is used in the method for the synergistic effect of epirubicin
The variation of IL-2 content in ELISA method detection rat blood serum: in chemotherapeutic EPI respectively with BCG, CpG ODN drug combination for the first time
After perfusion therapy tumor of bladder, the concentration of IL-2 is obviously higher than blank control group, model group and EPI group (P < 0.01)；This
When, EPI+BCG group IL-2 concentration highest, there were significant differences compared with EPI+CpG group (P < 0.05)；Second of perfusion therapy
Afterwards, EPI group, EPI+BCG group, the concentration of EPI+CpG group IL-2 have apparent raising, at this point, the concentration of three groups of IL-2 reaches
Peak in four perfusion therapies；Wherein EPI+BCG group IL-2 concentration highest, with EPI group, EPI+CpG group respectively compared with
There were significant differences (P < 0.05)；EPI group is not significantly different (P > 0.05) compared with EPI+CpG group；Third time perfusion is controlled
After treatment, EPI group, EPI+BCG group, the concentration of EPI+CpG group IL-2 are above modeling group and there were significant differences (P < 0.05)；EPI
Group is not significantly different (P > 0.05) compared with EPI+BCG group, EPI+CpG group；After last perfusion therapy, EPI+BCG
Group, the concentration of EPI+CpG group IL-2 are above its excess-three group, and there were significant differences (P < 0.05) with blank control group, modeling group；
At this point, the IL-2 concentration of modeling group reaches minimum；After the last administration, in the comparison of IL-2 concentration, model group and EPI+BCG
Group, EPI+CpG group compare, and difference has conspicuousness (P < 0.01)；EPI group is poor compared with EPI+BCG group, EPI+CpG group
It is different to have conspicuousness (P < 0.01)；EPI+BCG group, EPI+CpG group compare, no significant difference (P > 0.05).
In a preferred embodiment, the internal BCG passes through meat in the method for the synergistic effect of epirubicin
The intuitive observation of eye: the bladder volume of bladder cancer modeling group rat is obviously bigger than blank control group volume, and the bladder wall has different journeys
Degree thickens, and mucous membrane is rough fold, and diseased region is in bronzing, individual hyperemia occur, have cauliflower-shaped tumour protrusion, the bladder wall
Capillary increased significantly, and most mucosal protrusions are in nodositas and thicken, multiple in tumour, differ in size, and big can
Full of entire bladder；EPI group, EPI+BCG group, EPI+CpG group, after four administrations, bladder inner wall tumour protrusion has different journeys
The reduction of degree, individually with vesical calculus or ureteral calculi, gradually to normal bladder Form Development；Parallel comparison four times respectively
Tissue morphology after administration, mucous membrane of urinary bladder color are in by being deep to shallow variation, and mucous membrane of urinary bladder is in not only to slide into smooth variation, mucous membrane
Thickness is reduced.
In a preferred embodiment, the internal BCG uses HE in the method for the synergistic effect of epirubicin
Decoration method observes pathological change: the 10th week execution rat of control group, model group takes bladder body to pass sequentially through hematoxylin-eosin
(HE) it dyes, sets microscopically observation pathological change, blank control group coloration result no abnormality seen, bladder cancer modeling group migrates
Skin thickens, and is in visible peristalsis visible intestinal peristalsis, and cell level is more, and nucleus is larger, and nuclear chromatin is unevenly distributed, and is the Main Morphology of bladder canceration
Feature is determined as bladder cancer III level；
EPI group is from the 10th week to the 16th week, as there is no obvious for the morphological feature of number bladder cancer of medication treatment
Variation；
At the 10th week, cell arrangement was unevenly distributed EPI+BCG group, and cell heteromorphism is obvious, and cell quantity increases, carefully
Karyon dye levels are deep, are determined as I grades of bladder cancer；At the 12nd week, cell arrangement disorder, nuclear chromatin is unevenly distributed, form
Multiplicity, core is big and contaminates deeply, is determined as severe atypical hyperplasia；At the 14th week, squamous epithelization structure feature disappears, cell quantity
It significantly reduces, but cell quantity distribution is still uneven；At the 16th week, with the increase of administration number of times, cell is gradually decreased, carefully
Born of the same parents, which arrange, tends to rule, and level is more clear；
At the 10th week, cell quantity increased EPI+CpG group, and cell arrangement is inconsistent, and dye distribution is uneven, and cell is different
Shape is determined as II grades of bladder cancer；It at the 12nd week, cell arrangement disorder and is unevenly distributed, but cell quantity is reduced, and is sentenced
It is set to I grades of bladder cancer；At the 14th week, nuclei dyeing chromaticness is unevenly distributed, and cell quantity distribution is also uneven, but cell level
Tend to be obvious, is determined as severe atypical hyperplasia；By the 16th week, cell quantity was reduced, and the arrangement of cell level tends to rule, cell
Dye levels tend to consistent, but cell is still unevenly distributed and still has heteromorphism cell.
In a preferred embodiment, the internal BCG is in the method for the synergistic effect of epirubicin, using exempting from
Epidemic disease group method detects p53 expression in histotomy, and the relationship of bladder cancer p53 expression and each medication grouping: p53 positive cell is expressed
Percentage as a result, control group compared with modeling group, P < 0.05；EPI-2 group and EPI+CpG-2 group, EPI-2 group and EPI+
BCG_2 group compares, equal P < 0.05；EPI+CpG-1 group with EPI+BCG-1 group, EPI+CpG-4 compared with EPI+BCG-4, equal P <
0.05；Modeling group is compared with other groupings (removing EPI+BCG-1 group), equal P > 0.05；EPI+CpG-2 group and EPI+BCG-2 group and
EPI+CpG-3 group is compared with EPI+BCG-3 group, equal P > 0.05；
In a preferred embodiment, the internal BCG is in the method for the synergistic effect of epirubicin, using exempting from
Epidemic disease group method detects p53 expression in histotomy, is analyzed by gray value, identifies the percentage of p53 strong positive cell, as a result shows
Show, control group is compared with modeling group, P < 0.01, significant difference；Three groups of administration groups are with administration number of times p53 strong positive cell
Percentage is on a declining curve, and EPI+BCG group is compared with EPI+CpG group, and the percentage of p53 strong positive cell is lower, explanation
BCG inhibits the effect of tumour to be better than CpG, but CpG also can effectively inhibit the deterioration of tumour with the number of medication.
Technical effect and advantage of the invention:
By the present invention in that with cellular level research BCG to the method for the synergistic effect of epirubicin and BCG pairs internal
The method of the synergistic effect of epirubicin provides a kind of scheme of drug combination, to improve therapeutic efficiency, reduces drug infusion
When toxic side effect, dabbling drug concentration of the present invention is not high, but the retention time is long in bladder, so that antitumous effect is better,
And adverse reaction will not occur.
Detailed description of the invention
Fig. 1 is the variation diagram of each group bladder body form of the invention.
Fig. 2 is pathological section situation (× 200) figure of blank control group and modeling group of the invention.
Fig. 3 is pathological section situation (× 200) figure after independent EPI group four times administrations of the invention.
Fig. 4 is pathological section situation (× 200) figure after EPI+BCG tetra- times administrations of the invention.
Fig. 5 is pathological section situation (× 200) figure after EPI+CpG tetra- times administrations of the invention.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Embodiment 1:
The present invention provides a kind of methods that BCG vaccine promotes epirubicin in treating bladder cancer, including cellular level research
BCG is to the method for the synergistic effect of epirubicin and internal BCG to the method for the synergistic effect of epirubicin；
The present invention provides cellular level research BCG to the method for the synergistic effect of epirubicin, the specific steps are as follows:
Step 1: cell culture: in 37 DEG C, 5%CO2Under the conditions of using the MEM culture medium containing 10% fetal calf serum into
Row culture, liquid or passage were changed every 2-3 days；
Step 2: cytoactive detection: collecting logarithmic phase human bladder cancer UMUC3 using CCK8 cytoactive detection kit
Cell, adjustment cell concentration are 5 × 104/mL, are inoculated in 96 orifice plates by every 100 μ L of hole, are placed in incubator and stay overnight；
Step 3: relative medicine is added according to various concentration, after reaching preset time, discards old training after cell is adherent
It supports base and is washed 2 times with PBS；
Step 4: detecting cell activity using CCK8: the 100 μ L of MEM culture medium containing 10%CCK8 is added in every hole, in 37
Absorbance value (OD) is read in 450nm at after DEG C being incubated for 4 hours, is calculated further according to OD value, work of the detection drug to cell
Property influence.
The present invention also provides internal BCG to the method for the synergistic effect of epirubicin, the specific steps are as follows:
Step 1: selection: choosing SPF grades female sd inbred rats 106, weight 180g-200g is randomly divided into modeling group
(10), blank control group (24), EPI group (24), EPI+BCG group (24) and EPI+CpG group (24)；Prepare main
Reagent: citric acid, MNU reagent, chloraldurate, 4% paraformaldehyde fixer, rat interleukin-22 (IL-2) ELISA
Assay kit, epirubicin, BCG vaccine, CpG-ODN, CpG-ODN sequence, CpG-ODN sequence include 72 bases, and all
Sequence is modified by thiophosphoric acid；Prepare key instrument and equipment: the mini centrifuge of speed-regulating type, HWS26 type electric-heated thermostatic water bath,
Electro-heating standing-temperature cultivator, microplate reader, inverted microscope, AR-1000 electronic balance and slicer；
Step 2: starting to test:
1) establish rat original position bladder cancer models: SD rat is carried out 1 week adaptable fed by a., Rat Fast before being perfused
Prohibit water 12h, the as far as possible urine in empty bladder；B. perfusion tests MNU reagent used and is placed in 4 DEG C of refrigerators on the day before experiment
Overnight, it before use using the citrate buffer of pH6.0 as solvent, takes MNU reagent to be made into dense 20mg/ml, has been used in 40min
Finish；C. rat is with 10% chloraldurate intraperitoneal injection of anesthesia, fixation of lying on the back, 75% ethanol disinfection orificium urethrae externum 2 times；D. will
Alignment rat urethral orifice is slowly inserted into after the disposable epidural catheter of 1.0mm specification smears sterile paraffin oil, by ready MNU
Solution 0.1ml is poured into bladder through injection-tube, is exited after epidural catheter and is closed with pressing from both sides urethral orifice with small metal venous clamp, solution
It is detained 60min；E. rat carries out irrigation of bladder MNU, each 2mg, and 1 time every 2 weeks, totally 4 times, infusion time is the 1st, 3,5 and 7
In week, model foundation is completed within the 8th week, and rat is the object of this research at this time, is raised at 22-26 DEG C of temperature, humidity 20-70%'s
In SPF rank environment, the rat state of mind, feed drinking-water situation are observed daily, whether there is or not gross hematurias；
2) perfusion administration concentration control after modeling: except modeling group is 10 rats, every group of 24 rats of other groups, modeling
0.1ml is perfused in medication in 1 week afterwards every time, and infusion time is the 9th, 11,13 and 15 week, during perfusion administration herein: modeling group, nothing
Any processing；Physiological saline is perfused in control group；Epirubicin group is perfused EPI (2mg/ml)；Epirubicin+BCG vaccine group fills
It infuses EPI+BCG (2mg/ml+2mg/ml)；Epirubicin+CpG ODN group is perfused EPI+CpG ODN (2mg/ml+4 μM/ml)；
3) sample collection: a. serum collection collects whole blood sample in blood by eye socket blood collection method 1 week after each perfusion
4 DEG C are placed in clear separating pipe overnight, then 3000r/min is centrifuged 15 minutes, is taken supernatant, is carried out respective markers and be placed in -20
It DEG C saves, and is no more than 1 month, avoid multigelation, each each group collects 6 rats；
B. bladder body acquire, 1 week after each perfusion, using pull cervical vertebra method execution rat, every group each 6 groups；It cuts open the belly,
By its bladder complete resection, observes the general condition of inner bladder surface and cancer formation and take pictures, be placed in cryopreservation tube, carry out corresponding
Label is placed in -80 DEG C of preservations；The collection of specimens time is the 2nd, 4,6,8,10,12,14,16 week；
4) using IL2 content in the serum of the acquisition of ELISA method determination step six；
5) pathological change is observed using HE decoration method: each group bladder body being placed in 4% paraformaldehyde solution and fixes 15-
24 hours, the set time regard environment temperature and it is different；Then routine paraffin wax embeds, 5 μm of slices, and HE dyeing is observed under the microscope
Pathological change；
6) using the expression of p53 in Immunohistochemical Method detection histotomy, antibody working solution concentration is 1:100；
Step 3: statistical analysis: being analyzed using 19.0 statistical software of SPSS, calculate data with average ± mark
It is quasi- poorIt indicates, using the difference of the IL-2 concentration of one-way analysis of variance each group, P < 0.05 indicates that difference has significantly
Property；
Step 4: p53 ImmunohistochemistryResults Results judge: positive stained cells number < 10% is (-), positive stained cells number
10%~25% is (+), and positive stained cells number 25%~75% is (++).Positive stained cells number >=75% is (+++)；
Bladder cancer judgment criteria is as follows: a. no abnormality seen: mucosal epithelium is methodically arranged, queueing discipline, cellular morphology, big
Small, dyeing is unanimously；B. slight atypical hyperplasia: mucous membrane of urinary bladder, nipple is in cone, and cell arrangement is consistent, no heteromorphism；C. it weighs
Spend atypical hyperplasia: cell size is different, disorganized, and pole is to disorder, and form of diverse, core is big and contaminates deeply, and karyoplasmic ratio increases, core
Division increases；D. bladder cancer: cell size is different, disorganized, and pole is to disappearance, form of diverse, core deformity, nuclear chromatin distribution
Unevenly, thickening of nuclear membrane, kernel is loose, and caryoplasm ratio is big, it is seen that pathology nuclear fission picture is divided into I, II, III grade；
Step 5: observation experiment result:
1) the bladder cancer morbidity situation for observing each group SD rat, during experiment, blank control group without an example tumor formation rat,
Do not occur death；At the 8th week, modeling group is survived 9, and the incidence of bladder cancer is 77.8% (7/9)；Remaining each group the 10th, 12,
14,16 weeks when bladder cancer incidence be respectively for 66.7% (12/18), 72.2% (13/18), 61.1% (11/18),
55.6% (10/18), the incidence difference for comparing control group and modeling group rat bladder cancer have conspicuousness, illustrate that MNU induces wing
Guang cancer rat has obvious effect, and treats respectively according to administration concentration after each treatment group's bladder cancer is formed, and see Table 1 for details；
1 drug concentration of table
Each stage disease incidence of 2 each group of table
2) using the variation of IL-2 content in ELISA method detection rat blood serum, chemotherapeutic EPI respectively with BCG, CpG
ODN drug combination is for the first time after perfusion therapy tumor of bladder, the concentration of IL-2 obviously higher than blank control group, model group and
EPI group (P < 0.01)；At this point, EPI+BCG group IL-2 concentration highest, there were significant differences compared with EPI+CpG group (P <
0.05)；After second of perfusion therapy, EPI group, EPI+BCG group, the concentration of EPI+CpG group IL-2 have apparent raising, this
When, the concentration of three groups of IL-2 reaches the peak in four perfusion therapies；Wherein EPI+BCG group IL-2 concentration highest, with EPI
Group, EPI+CpG group compare respectively, and there were significant differences (P < 0.05)；EPI group is not significantly different compared with EPI+CpG group
(P > 0.05)；After third time perfusion therapy, EPI group, EPI+BCG group, the concentration of EPI+CpG group IL-2 be above modeling group and
There were significant differences (P < 0.05)；EPI group is not significantly different (P > 0.05) compared with EPI+BCG group, EPI+CpG group；?
After last perfusion therapy, EPI+BCG group, the concentration of EPI+CpG group IL-2 are above its excess-three group, with blank control group, modeling
There were significant differences (P < 0.05) for group；At this point, the IL-2 concentration of modeling group reaches minimum, see Table 3 for details, table 4；
All each treatment stage peripheral bloods of grouping of table 3 ask the level of IL-2
The variation of 4 each group treatment cycle IL-2 concentration of table
After the last administration, in the comparison of IL-2 concentration, model group is compared with EPI+BCG group, EPI+CpG group, difference
There is conspicuousness (P < 0.01)；For EPI group compared with EPI+BCG group, EPI+CpG group, difference has conspicuousness (P < 0.01)；EPI+
BCG group, EPI+CpG group compare, and no significant difference (P > 0.05), see Table 5 for details；
After the treatment of 5 last dose of table, the comparison of IL-2 concentration between each group
3) it visually observes, observes by the naked eye the bladder feature of bladder cancer modeling group rat, bladder cancer modeling group rat
Bladder volume is obviously bigger than blank control group volume, and the bladder wall has and thickens in various degree, and mucous membrane is rough fold, lesion
Position is in bronzing, and individual hyperemia occur, have cauliflower-shaped tumour protrusion, the bladder wall capillary increased significantly, most mucous membranes
Protuberance is in nodositas and thickens, multiple in tumour, differs in size, and big is full of entire bladder；EPI group, EPI+BCG group,
EPI+CpG group, after four administrations, bladder inner wall tumour protrusion has different degrees of reduction, individually with vesical calculus or defeated
Ureteral calculi, gradually to normal bladder Form Development；Tissue morphology after four administrations of parallel comparison respectively, mucous membrane of urinary bladder color
In by being deep to shallow variation, mucous membrane of urinary bladder is reduced in smooth variation, mucosal thickness is not only slided into；
4) HE stained slice is analyzed, and the 10th week execution rat of control group, model group takes bladder body to pass sequentially through bush
Essence-Yihong (HE) dyeing, sets microscopically observation pathological change, blank control group coloration result no abnormality seen, bladder cancer modeling
Group transitional epithelium thickens, and is in visible peristalsis visible intestinal peristalsis, and cell level is more, and nucleus is larger, and nuclear chromatin is unevenly distributed, and is bladder canceration
Main morphological features are determined as bladder cancer III level；
EPI group is from the 10th week to the 16th week, as there is no obvious for the morphological feature of number bladder cancer of medication treatment
Variation；
At the 10th week, cell arrangement was unevenly distributed EPI+BCG group, and cell heteromorphism is obvious, and cell quantity increases, carefully
Karyon dye levels are deep, are determined as I grades of bladder cancer；At the 12nd week, cell arrangement disorder, nuclear chromatin is unevenly distributed, form
Multiplicity, core is big and contaminates deeply, is determined as severe atypical hyperplasia；At the 14th week, squamous epithelization structure feature disappears, cell quantity
It significantly reduces, but cell quantity distribution is still uneven；At the 16th week, with the increase of administration number of times, cell is gradually decreased, carefully
Born of the same parents, which arrange, tends to rule, and level is more clear；
At the 10th week, cell quantity increased EPI+CpG group, and cell arrangement is inconsistent, and dye distribution is uneven, and cell is different
Shape is determined as II grades of bladder cancer；It at the 12nd week, cell arrangement disorder and is unevenly distributed, but cell quantity is reduced, and is sentenced
It is set to I grades of bladder cancer；At the 14th week, nuclei dyeing chromaticness is unevenly distributed, and cell quantity distribution is also uneven, but cell level
Tend to be obvious, is determined as severe atypical hyperplasia；By the 16th week, cell quantity was reduced, and the arrangement of cell level tends to rule, cell
Dye levels tend to consistent, but cell is still unevenly distributed and still has heteromorphism cell；
5) it is expressed using p53 in Immunohistochemical Method detection histotomy, all stained slices are seen under an optical microscope
It examines, is saved after being taken pictures with Computer digital image analysis scanning, positive cell technology is calculated using image analysis software statistics,
The coloring of the p53 positive is located in neoplastic cell nuclei, and nucleus is dyed to sepia, and every slice takes 5 high power fields (400 at random
×), positive cell number is counted, the cell number of entire field of view is accounted for as percentage using positive cell number；It will be cut with a batch dyeing
Piece carries out tumour cell gray-scale statistical calculating, the relationship of bladder cancer p53 expression and each medication grouping: p53 with image analysis software
Positive cell express percentage as a result, control group compared with modeling group, P < 0.05；EPI-2 group and EPI+CpG-2 group, EPI-
2 groups compared with EPI+BCG_2 group, equal P < 0.05；EPI+CpG-1 group and EPI+BCG-1 group, EPI+CpG-4 and EPI+BCG-4
Compare, equal P < 0.05；Modeling group is compared with other groupings (removing EPI+BCG-1 group), equal P > 0.05；EPI+CpG-2 group and EPI
+ BCG-2 group and EPI+CpG-3 group are compared with EPI+BCG-3 group, and equal P > 0.05, see Table 6 for details.
6 p53 of table positive cell accounting in each group
It is analyzed by gray value, identifies the percentage of p53 strong positive cell, the results show that control group is compared with modeling group,
P < 0.01, significant difference；Three groups of administration groups are on a declining curve with the percentage of administration number of times p53 strong positive cell, EPI+
For BCG group compared with EPI+CpG group, the percentage of p53 strong positive cell is lower, illustrates that BCG inhibits the effect of tumour to be better than
CpG, but CpG also can effectively inhibit the deterioration of tumour with the number of medication, and see Table 7 for details；
The high positive expression accounting of 7 p53 of table
By above-mentioned two method, the present invention provides a kind of schemes of drug combination to reduce medicine to improve therapeutic efficiency
Toxic side effect when object is perfused.
Embodiment 2:
In embodiment 1, cell activity is detected using CCK8, and calculated according to OD value, detects work of the drug to cell
Property influence, specific experiment is as follows:
Independent epirubicin acts on human bladder cancer UMUC3 cell 48,72 hours with various concentration, with the increase of concentration
It is reduced with the activity of the extension of time, human bladder cancer's UMUC3 cell；48 hours IC50 are 0.3321 μM, and IC50 is within 72 hours
0.1473μM；
Independent BCG vaccine acts on human bladder cancer UMUC3 cell 24,48 hours with various concentration, with concentration increase and
The activity of the extension of time, human bladder cancer's UMUC3 cell reduces；
The BCG vaccine for combining various concentration with (0.3 μM) of epirubicin acts on human bladder cancer cell 24,48 hours, with
The increase of BCG vaccine concentration and the extension of time, human bladder cancer's UMUC3 cell activity reduce；
Independent (0.3 μM) BCG vaccine, epirubicin joint BCG vaccine act on human bladder cancer cell UMU3 with various concentration
Cell 48 hours, the cell survival rate of joint group was significantly lower than independent BCG vaccine group, and has statistical difference.
The several points that should finally illustrate are: the foregoing is only a preferred embodiment of the present invention, is not limited to this
Invention, all within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in this hair
Within bright protection scope.
Claims (9)
1. a kind of method that BCG vaccine promotes epirubicin in treating bladder cancer, it is characterised in that: study BCG pairs including cellular level
The method of the method for the synergistic effect of epirubicin and internal BCG to the synergistic effect of epirubicin；
Wherein, method of the cellular level research BCG to the synergistic effect of epirubicin, the specific steps are as follows:
Step 1: cell culture: in 37 DEG C, 5%CO2Under the conditions of trained using the MEM culture medium containing 10% fetal calf serum
It supports, liquid or passage was changed every 2-3 days；
Step 2: cytoactive detection: it is thin to collect logarithmic phase human bladder cancer UMUC3 using CCK8 cytoactive detection kit
Born of the same parents, adjustment cell concentration are 5 × 104/mL, are inoculated in 96 orifice plates by every 100 μ L of hole, are placed in incubator and stay overnight；
Step 3: relative medicine is added according to various concentration, after reaching preset time, discards old culture medium after cell is adherent
And it is washed 2 times with PBS；
Step 4: detecting cell activity using CCK8: the 100 μ L of MEM culture medium containing 10%CCK8 is added in every hole, incubates in 37 DEG C
Absorbance value (OD) is read at 450nm after educating 4 hours, is calculated further according to OD value, active shadow of the detection drug to cell
It rings；
In addition, method of the internal BCG to the synergistic effect of epirubicin, the specific steps are as follows:
Step 1: selection: choosing SPF grades female sd inbred rats 106, weight 180g-200g is randomly divided into modeling group (10
Only), blank control group (24), EPI group (24), EPI+BCG group (24) and EPI+CpG group (24)；Prepare main examination
Agent: citric acid, MNU reagent, chloraldurate, 4% paraformaldehyde fixer, (IL-2) ELISA of rat interleukin-22 are surveyed
Determine kit, epirubicin, BCG vaccine, CpG-ODN, CpG-ODN sequence；Prepare key instrument and equipment: speed-regulating type it is mini from
Scheming, HWS26 type electric-heated thermostatic water bath, electro-heating standing-temperature cultivator, microplate reader, inverted microscope, AR-1000 electronic balance and
Slicer；
Step 2: starting to test:
1) establish rat original position bladder cancer models: SD rat is carried out 1 week adaptable fed by a., and Rat Fast prohibits water before being perfused
12h, the as far as possible urine in empty bladder；B. perfusion tests MNU reagent used and is placed in 4 DEG C of refrigerator mistakes on the day before experiment
Night takes MNU reagent to be made into dense 20mg/ml, has used in 40min before use using the citrate buffer of pH6.0 as solvent
Finish；C. rat is with 10% chloraldurate intraperitoneal injection of anesthesia, fixation of lying on the back, 75% ethanol disinfection orificium urethrae externum 2 times；D. will
Alignment rat urethral orifice is slowly inserted into after the disposable epidural catheter of 1.0mm specification smears sterile paraffin oil, by ready MNU
Solution 0.1ml is poured into bladder through injection-tube, is exited after epidural catheter and is closed with pressing from both sides urethral orifice with small metal venous clamp, solution
It is detained 60min；E. rat carries out irrigation of bladder MNU, each 2mg, and 1 time every 2 weeks, totally 4 times, infusion time is the 1st, 3,5 and 7
In week, model foundation is completed within the 8th week, and rat is the object of this research at this time, is raised at 22-26 DEG C of temperature, humidity 20-70%'s
In SPF rank environment, the rat state of mind, feed drinking-water situation are observed daily, whether there is or not gross hematurias；
2) perfusion administration concentration control after modeling: except modeling group is 10 rats, every group of 24 rats of other groups, 1 after modeling
0.1ml is perfused in all medications every time, and infusion time is the 9th, 11,13 and 15 week, during perfusion administration herein: modeling group, without any
Processing；Physiological saline is perfused in control group；Epirubicin group is perfused EPI (2mg/ml)；EPI is perfused in epirubicin+BCG vaccine group
+BCG(2mg/ml+2mg/ml)；Epirubicin+CpG ODN group is perfused EPI+CpG ODN (2mg/ml+4 μM/ml)；
3) sample collection: a. serum collection collects whole blood sample in serum point by eye socket blood collection method 1 week after each perfusion
From placing 4 DEG C in pipe overnight, then 3000r/min is centrifuged 15 minutes, is taken supernatant, is carried out respective markers and be placed in -20 DEG C of guarantors
It deposits, and is no more than 1 month, avoid multigelation, each each group collects 6 rats；
B. bladder body acquire, 1 week after each perfusion, using pull cervical vertebra method execution rat, every group each 6 groups；It cuts open the belly, by it
Bladder complete resection observes the general condition of inner bladder surface and cancer formation and takes pictures, is placed in cryopreservation tube, carries out respective markers
It is placed in -80 DEG C of preservations；The collection of specimens time is the 2nd, 4,6,8,10,12,14,16 week；
4) using IL2 content in the serum of the acquisition of ELISA method determination step six；
5) pathological change is observed using HE decoration method: each group bladder body is placed in 4% paraformaldehyde solution to fix 15-24 small
When, the set time regard environment temperature and it is different；Then routine paraffin wax embeds, 5 μm of slices, and pathology is observed in HE dyeing under the microscope
Variation；
6) using the expression of p53 in Immunohistochemical Method detection histotomy, antibody working solution concentration is 1:100；
Step 3: statistical analysis: being analyzed using SPSS19.0 statistical software, calculate data with average ± standard deviationIt indicates, using the difference of the IL-2 concentration of one-way analysis of variance each group, P < 0.05 indicates that difference has conspicuousness；
Step 4: p53 ImmunohistochemistryResults Results judge: positive stained cells number < 10% is (-), positive stained cells number 10%
~25% is (+), and positive stained cells number 25%~75% is (++).Positive stained cells number >=75% is (+++)；
Step 5: observation experiment result:
1) the bladder cancer morbidity situation for observing each group SD rat, compares control group and the incidence of modeling group rat bladder cancer is poor
It is different to have conspicuousness, illustrate MNU induce bladder cancer rat have obvious effect, and after each treatment group's bladder cancer is formed respectively according to
Concentration treatment, see Table 1 for details；
2) using the variation of IL-2 content in ELISA method detection rat blood serum, perfusion therapy, carries out IL-2 concentration and compares three times；
3) it visually observes, observes by the naked eye the bladder feature of bladder cancer modeling group rat；
4) HE stained slice is analyzed, and puts to death rat, is taken bladder body to pass sequentially through hematoxylin-eosin (HE) dyeing, is set microscope
Lower observation pathological change；
5) it is expressed using p53 in Immunohistochemical Method detection histotomy, all stained slices are observed under an optical microscope, are used
Computer digital image analysis scanning saves after taking pictures, and positive cell technology is calculated using image analysis software statistics, and p53 is positive
Coloring is located in neoplastic cell nuclei, and nucleus is dyed to sepia, and every slice takes 5 high power fields (400 ×) at random, counts
Positive cell number accounts for the cell number of entire field of view as percentage using positive cell number；Image will be used with a collection of stained slice
It analyzes software and carries out the calculating of tumour cell gray-scale statistical.
2. the method that a kind of BCG vaccine according to claim 1 promotes epirubicin in treating bladder cancer, it is characterised in that: institute
Internal BCG is stated in the method for the synergistic effect of epirubicin, CpG-ODN sequence includes 72 bases, and full sequence is by sulphur
It is modified for phosphoric acid.
3. the method that a kind of BCG vaccine according to claim 1 promotes epirubicin in treating bladder cancer, it is characterised in that: institute
Internal BCG is stated in the method for the synergistic effect of epirubicin, bladder cancer judgment criteria is as follows: a. no abnormality seen: mucosal epithelium
It is methodically arranged, queueing discipline, cellular morphology, size, dyeing are consistent；B. slight atypical hyperplasia: mucous membrane of urinary bladder, nipple are in circular cone
Shape, cell arrangement is consistent, no heteromorphism；C. severe atypical hyperplasia: cell size is different, disorganized, and pole is to disorder, form
Multiplicity, core is big and contaminates deeply, and karyoplasmic ratio increases, and nuclear fission increases；D. bladder cancer: cell size is different, disorganized, and pole is to disappearing
It loses, form of diverse, core deformity, nuclear chromatin is unevenly distributed, thickening of nuclear membrane, and kernel is loose, and caryoplasm ratio is big, it is seen that pathology core
Mitotic figure is divided into I, II, III grade.
4. the method that a kind of BCG vaccine according to claim 1 promotes epirubicin in treating bladder cancer, it is characterised in that: institute
Internal BCG is stated in the method for the synergistic effect of epirubicin, observes the bladder cancer morbidity situation of each group SD rat: experiment periods
Between, blank control group does not occur death without an example tumor formation rat；At the 8th week, modeling group is survived 9, and the incidence of bladder cancer is
77.8% (7/9)；At remaining each group the 10th, 12,14,16 week the incidence of bladder cancer be respectively for 66.7% (12/18),
72.2% (13/18), 61.1% (11/18), 55.6% (10/18).
5. the method that a kind of BCG vaccine according to claim 1 promotes epirubicin in treating bladder cancer, it is characterised in that: institute
Internal BCG is stated in the method for the synergistic effect of epirubicin, uses the change of IL-2 content in ELISA method detection rat blood serum
Change: in chemotherapeutic EPI respectively and after BCG, CpG ODN drug combination for the first time perfusion therapy tumor of bladder, the concentration of IL-2 is bright
It is aobvious to be higher than blank control group, model group and EPI group (P < 0.01)；At this point, EPI+BCG group IL-2 concentration highest, with EPI+
CpG group compares, and there were significant differences (P < 0.05)；After second of perfusion therapy, EPI group, EPI+BCG group, EPI+CpG group IL-2
Concentration have apparent raising, at this point, the concentration of three groups of IL-2 reaches the peak in four perfusion therapies；Wherein EPI+
BCG group IL-2 concentration highest, with EPI group, EPI+CpG group respectively compared with there were significant differences (P < 0.05)；EPI group and EPI+
CpG group, which compares, is not significantly different (P > 0.05)；After third time perfusion therapy, EPI group, EPI+BCG group, EPI+CpG group
The concentration of IL-2 is above modeling group and there were significant differences (P < 0.05)；EPI group compared with EPI+BCG group, EPI+CpG group,
It is not significantly different (P > 0.05)；After last perfusion therapy, EPI+BCG group, the concentration of EPI+CpG group IL-2 are above it
Excess-three group, there were significant differences (P < 0.05) with blank control group, modeling group；At this point, the IL-2 concentration of modeling group reaches minimum
Value；After the last administration, in the comparison of IL-2 concentration, compared with EPI+BCG group, EPI+CpG group, difference has significantly model group
Property (P < 0.01)；For EPI group compared with EPI+BCG group, EPI+CpG group, difference has conspicuousness (P < 0.01)；EPI+BCG group,
EPI+CpG group compares, no significant difference (P > 0.05).
6. the method that a kind of BCG vaccine according to claim 1 promotes epirubicin in treating bladder cancer, it is characterised in that: institute
Internal BCG is stated in the method for the synergistic effect of epirubicin, passes through the intuitive observation of naked eyes: the bladder of bladder cancer modeling group rat
Volume is obviously bigger than blank control group volume, and the bladder wall has and thickens in various degree, and mucous membrane is rough fold, and diseased region is in
Bronzing, individual hyperemia occur, have cauliflower-shaped tumour protrusion, the bladder wall capillary increased significantly, most mucosal protrusions
In nodositas and thicken, it is multiple in tumour, it differs in size, big is full of entire bladder；EPI group, EPI+BCG group, EPI+
CpG group, after four administrations, bladder inner wall tumour protrusion has different degrees of reduction, individually with vesical calculus or ureter
Calculus, gradually to normal bladder Form Development；Tissue morphology after four administrations of parallel comparison respectively, mucous membrane of urinary bladder color be in by
It is deep to shallow variation, mucous membrane of urinary bladder is reduced in smooth variation, mucosal thickness is not only slided into.
7. the method that a kind of BCG vaccine according to claim 1 promotes epirubicin in treating bladder cancer, it is characterised in that: institute
Internal BCG is stated in the method for the synergistic effect of epirubicin, observes pathological change: control group, model group using HE decoration method
10th week execution rat takes bladder body to pass sequentially through hematoxylin-eosin (HE) dyeing, sets microscopically observation pathological change,
Blank control group coloration result no abnormality seen, bladder cancer modeling group transitional epithelium thicken, and are in visible peristalsis visible intestinal peristalsis, cell level is more, cell
Core is larger, and nuclear chromatin is unevenly distributed, and is the main morphological features of bladder canceration, is determined as bladder cancer III level；
EPI group is from the 10th week to the 16th week, as there is no significantly becoming for the morphological feature of number bladder cancer of medication treatment
Change；
At the 10th week, cell arrangement was unevenly distributed EPI+BCG group, and cell heteromorphism is obvious, and cell quantity increases, nucleus
Dye levels are deep, are determined as I grades of bladder cancer；At the 12nd week, cell arrangement disorder, nuclear chromatin is unevenly distributed, form of diverse,
Core is big and contaminates deeply, is determined as severe atypical hyperplasia；At the 14th week, squamous epithelization structure feature disappears, and cell quantity is obvious
It reduces, but cell quantity distribution is still uneven；At the 16th week, with the increase of administration number of times, cell is gradually decreased, cell row
Column tend to rule, and level is more clear；
At the 10th week, cell quantity increased EPI+CpG group, and cell arrangement is inconsistent, and dye distribution is uneven, cell abnormity
Property, it is determined as II grades of bladder cancer；It at the 12nd week, cell arrangement disorder and is unevenly distributed, but cell quantity is reduced, determines
It is I grades of bladder cancer；At the 14th week, nuclei dyeing chromaticness is unevenly distributed, and cell quantity distribution is also uneven, but cell level becomes
In obvious, it is determined as severe atypical hyperplasia；By the 16th week, cell quantity was reduced, and the arrangement of cell level tends to rule, cell dye
Color degree tends to consistent, but cell is still unevenly distributed and still has heteromorphism cell.
8. the method that a kind of BCG vaccine according to claim 1 promotes epirubicin in treating bladder cancer, it is characterised in that: institute
Internal BCG is stated in the method for the synergistic effect of epirubicin, is expressed using p53 in Immunohistochemical Method detection histotomy, wing
Guang cancer p53 expresses the relationship that is grouped with each medication: p53 positive cell expression percentage as a result, control group compared with modeling group,
P < 0.05；EPI-2 group with EPI+CpG-2 group, EPI-2 group compared with EPI+BCG_2 group, equal P < 0.05；EPI+CpG-1 group with
EPI+BCG-1 group, EPI+CpG-4 are compared with EPI+BCG-4, equal P < 0.05；Modeling group and other groupings (remove EPI+BCG-1
Group) compare, equal P > 0.05；EPI+CpG-2 group with EPI+BCG-2 group and EPI+CpG-3 group compared with EPI+BCG-3 group, equal P
> 0.05.
9. the method that a kind of BCG vaccine according to claim 1 promotes epirubicin in treating bladder cancer, it is characterised in that: institute
Internal BCG is stated in the method for the synergistic effect of epirubicin, is expressed, is led to using p53 in Immunohistochemical Method detection histotomy
Gray value analysis is crossed, the percentage of p53 strong positive cell is identified, the results show that control group, compared with modeling group, P < 0.01 is poor
It is different significant；Three groups of administration groups are on a declining curve with the percentage of administration number of times p53 strong positive cell, EPI+BCG group and EPI
+ CpG group compares, and the percentage of p53 strong positive cell is lower, illustrates that BCG inhibits the effect of tumour better than CpG, but CpG with
The number of medication also can effectively inhibit the deterioration of tumour.
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