CN1520886A - Application of mycoplasma-resistant pharmaceutical and P37 protein blocking agent in preparing antineoplastic medicine - Google Patents
Application of mycoplasma-resistant pharmaceutical and P37 protein blocking agent in preparing antineoplastic medicine Download PDFInfo
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Abstract
The present invention relates to the application of mycoplasma resisting medicine or P37 protein blocker in preparing medicine for treating or preventing tumor and tumor metastasis. The mycoplasma resisting medicine or P37 protein blocker of the present invention may be administrated together with routine antitumor medicine. Therefore, the present invention also relates to medicine composition or kit containing mycoplasma resisting medicine or P37 protein blocker and routine antitumor medicine as active components.
Description
Technical field
The present invention relates to anti-mycoplasma drug or the agent of the P37 protein blocking application in the medicine of preparation treatment or prophylaxis of tumours or neoplasm metastasis.Anti-mycoplasma drug of the present invention or the agent of P37 protein blocking can be with conventional antitumor drug administrations.Therefore, the invention still further relates to and contain anti-mycoplasma drug or the agent of P37 protein blocking and conventional antitumor drug pharmaceutical composition or medicine box as active component.
Background technology
Mycoplasma extensively is present in nature, parasitizes the mankind and other mammals, reptile, Fish, arthropod and plant.Mycoplasma is usually expressed as organ and tissue specificity.Because the general immunity function was suppressed after AIDS caused immunologic hypofunction and organ transplantation, mycoplasma obviously reduces at this class patient tissue specificity.Cell culture medium is the autoeciously common of mycoplasma under the non-natural environment.From from the bibliographical information of country variant as can be seen, mycoplasma contamination from 10% to 87% does not wait in the laboratory cultures cell, and the common mycoplasma bacterial strain that pollutes cultured cell is mycoplasma hyorhinis, Mycoplasma orale, mycoplasma arginini and mycoplasma laidlawii.
Mycoplasma hyorhinis is that a kind of animal is pathogenic former, and it can cause arthritis, and polyserositis reaches and cause chronic experiment arthritis in its natural reservoir (of bird flu viruses) porcupine body, and also is the source of infection common in the cell culture, the parasitic and propagation at cell surface.
Nineteen ninety-five, Japanese scientist finds that from fresh gastric cancer specimen mycoplasma hyorhinis not only exists in cancerous cell line, and also exists in stomach organization, and 11 parts have mycoplasma infection in 23 parts of gastric cancer specimen, and wherein 4 parts is mycoplasma hyorhinis.See Jpn.J.Cancer Res.86,791-794, nineteen ninety-five JIUYUE.
The gastric cancer specific monoclonal antibody PD4 of the applicant's preparation, prove the monoclonal antibody of anti-mycoplasma hyorhinis recently, utilize this monoclonal antibody to gastric cancer, intestinal cancer, the esophageal carcinoma, pulmonary carcinoma is carried out the detection of SABC and is found that positive rate all has more than 50%, and in corresponding normal structure, positive rate is starkly lower than cancerous tissue.
Because may there be antigenic cross-reaction in SABC, above-mentioned positive findings can not be as the positive evidence that has mycoplasma hyorhinis in the tumor tissues.
In addition, though the infection rate of prior art prompting mycoplasma hyorhinis in some tumor tissues is higher.But this high infection rate can be interpreted as mycoplasma hyorhinis and these tumors follow relation or mycoplasma merely have a liking for the tumor phenomenon, perhaps mycoplasma hyorhinis directly causes the generation of tumor.But the admissible evidence that can directly not cause tumor in the prior art about mycoplasma hyorhinis.
Summary of the invention
The present inventor first from tumor tissues separation and Culture obtained mycoplasma, obtained to exist in the tumor tissues positive evidence of mycoplasma.Isolating these mycoplasma mycoplasma hyorhinis and mycoplasma fermentanses all have mycoplasma hyorhinis all positive cultivations in the specimen, and the part tumor tissues has mycoplasma fermentans simultaneously.The inventor discovers that further the P37 albumen of mycoplasma hyorhinis can strengthen the infiltration activity of tumor cell by directly promoting the release of TNF-α.
Therefore, the present invention relates to the method for a kind of prevention or treatment tumor or neoplasm metastasis, comprise the anti-mycoplasma drug or the agent of P37 protein blocking that need the patient treatment of this treatment effective dose.
The invention still further relates to anti-mycoplasma drug or the agent of P37 protein blocking and be used for preventing or treat the application of the medicine of tumor or neoplasm metastasis in preparation.
The invention still further relates to a kind of method for the treatment of tumor or neoplasm metastasis, comprise the conventional antitumor drug and anti-mycoplasma drug or the agent of P37 protein blocking that need the patient treatment of this treatment effective dose, described anti-mycoplasma drug or the agent of P37 protein blocking before giving conventional antitumor drug, simultaneously or give afterwards.
The invention still further relates to anti-mycoplasma drug or the agent of P37 protein blocking and be used for the treatment of application in the medicine of tumor or neoplasm metastasis in preparation with conventional antitumor drug.
The invention still further relates to a kind of pharmaceutical composition for the treatment of tumor or neoplasm metastasis, it contains anti-mycoplasma drug or the agent of P37 protein blocking and a kind of conventional antitumor drug and pharmaceutical carrier.
The invention still further relates to a kind of medicine box, wherein contain container that anti-mycoplasma drug or P37 blocker are housed, a kind of container of conventional antitumor drug and optional drug use description are housed.
The invention still further relates to anti-mycoplasma drug or the agent of P37 protein blocking and be used for preventing or treating the application of the medicine of TNF-α excessive secretion relevant disease in preparation.
Detailed Description Of The Invention
The present invention collects fresh gastric cancer specimen under strict aseptic condition, carry out the separation and Culture of mycoplasma, identifies with PCR and SABC method simultaneously, to confirm to exist really in the stomach organization infection of mycoplasma.The present invention first successfully from tumor tissues separation and Culture gone out mycoplasma.And separation and Culture positive findings and immunohistochemical staining, the pcr amplification result rate of fitting like a glove are 87.5% (7/8); The PCR result of 61 routine specimen and SABC testing result, the identical rate between the two reaches 72.1% (44/61).Three kinds of method testing results have good concordance, have confirmed to exist in the gastric cancer infection of mycoplasma from different perspectives, and infection type is mainly two kinds of mycoplasma hyorhinis and mycoplasma fermentanses.Except that the fermentation of isolating from patient's AIDS Karpasi sarcoma being arranged and penetrating the report of mycoplasma, not seeing as yet so far has the report of isolating mycoplasma from common tumor in the world.The present invention separates from cancerous tissue first and obtains mycoplasma, for the infection that has mycoplasma in the tumor tissues provides positive evidence.
In addition, prior art does not report that separation and Culture goes out mycoplasma hyorhinis from tissue.Mycoplasma has special affinity highly to host, organ and tissue, does not generally parasitize animal from the isolating mycoplasma of human body.Result of the present invention shows that but mycoplasma hyorhinis also can colonize in human body except the nasal cavity parasitism of natural reservoir (of bird flu viruses)-pig, in the particularly human stomach organization.
In order to seek mycoplasma hyorhinis and tumorigenic relation, the present invention utilizes the fibroblast 32D of mycoplasma hyorhinis infecting mouse, finds that the ability that forms the clone at infection 10 all backs 32D cells on soft agar obviously increases.The present invention is that N87 handles with the major antigen albumen P37 of mycoplasma hyorhinis to human peripheral blood mononuclear cell and stomach cancer cell also.Found that P37 albumen can promote above-mentioned different cell release tumor necrosis factor α (TNF-α), and the antibody of P37 can in and the short TNF-α release action of P37.TNF-α is most important endogenous carcinogenic promoting agent in the inductive cytokine of infected by microbes institute of generally acknowledging at present, and is relevant with transfer with the generation of tumor.The influence of P37 to the infiltration behavior of stomach cancer cell N87 that the present invention has also used boyd cell infiltration experimental observation find that P37 can obviously promote the infiltration (being similar to tumor cell transfer in vivo) of tumor cell, and P37 antibody has neutralization to this.Thereby proved the positive evidence that causes tumor and neoplasm metastasis of mycoplasma hyorhinis and major antigen albumen P37 thereof, and proved that mycoplasma hyorhinis and P37 albumen are by raising the mechanism of TNF-α induced tumor and neoplasm metastasis.
A large amount of researchs about tumor are arranged in the prior art, find that wherein a large amount of infected by microbes and tumor have such-and-such the contact.But the venereal infection of finding tumor is because of being not an easy thing, in fact up to now have only the minority infection by microorganisms to be believed to cause tumor, (see H.KUPER etc., Journal of Internal Medicine2000:248:171-183 as hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), human papillomavirus (HPV), human herpes virus 8 (HHV8), helicobacter pylori, Liver fluke etc.Though the relation of in the prior art many bibliographical informations have been arranged mycoplasma and tumor, those skilled in the art can not draw one of immediate cause that mycoplasma causes tumor such conclusion according to these reports.People even to whether really having mycoplasma in the human tumor and existing which kind of mycoplasma (if any) to have very big query.Mycoplasma has the infected by microbes of certain relation as other a large amount of under a cloud and tumors, does not cause enough concerns of people.Therefore, before the present invention, those skilled in the art can not expect utilizing anti-mycoplasma drug, particularly anti-mycoplasma hyorhinis medicine or the agent of P37 protein blocking to treat or prophylaxis of tumours or neoplasm metastasis.Because the present invention has been separated to mycoplasma hyorhinis from body tumor tissue for the first time, and proved the proteic oncogenic function of mycoplasma hyorhinis and P37 definitely, drawn the technical scheme of utilizing anti-mycoplasma drug, particularly anti-mycoplasma hyorhinis medicine or the agent treatment of P37 protein blocking or prophylaxis of tumours or neoplasm metastasis naturally.
Anti-mycoplasma drug described in the present invention can be known mycoplasma medicine or the mycoplasma inhibitor of killing.Known anti-mycoplasma drug for example is: Tetracyclines such as tetracycline, oxytetracycline, chlortetracycline etc.; Macrolide such as erythromycin, josamycin etc.; Quinolones such as ciprofloxacin, norfloxacin etc.Described anti-mycoplasma drug also can be the polyclone or the monoclonal antibody of anti-mycoplasma.
P37 protein blocking of the present invention agent can be proteic polyclone of anti-P37 or monoclonal antibody, also can be any other materials that can block the P37 protein function.
Conventional antitumor drug of the present invention is meant that except that anti-mycoplasma drug of the present invention and the agent of P37 protein blocking other suppress the medicine of tumor growths or transfer.Conventional antitumor drug for example can be the chloroethene amine, as bendamustine, bofumustine, carmustine, tauromustine etc.; Vinca is as vinblastine, vincristine, vindesine, vinorelbine, vinglycinate etc.; Nuclear toxicity class is as mitindomide, mitoxantrone, mitoquidone etc.; Antifol such as methotrexate etc.; And other antineoplastic agents such as acivicin, altretamine, carboplatin, busulfan, chlormethine etc.
In a technical scheme of the present invention, prevent or treat tumor or neoplasm metastasis by the anti-mycoplasma drug or the agent of P37 protein blocking of patient treatment effective dose that needs treatment.
Therefore anti-mycoplasma drug or the agent of P37 protein blocking can be used for preparing the medicine that is used to prevent or treat tumor or neoplasm metastasis.
In another technical scheme of the present invention, treat tumor or neoplasm metastasis by uniting the conventional antitumor drug of patient treatment effective dose that needs treatment and anti-mycoplasma drug or the agent of P37 protein blocking, described anti-mycoplasma drug or the agent of P37 protein blocking can be before giving conventional antitumor drug, simultaneously or give afterwards.
Therefore, anti-mycoplasma drug or the agent of P37 protein blocking can be used for preparing the medicine that is used for the treatment of tumor or neoplasm metastasis with conventional antitumor drug.
Said medicine can be a kind of pharmaceutical composition for the treatment of tumor or neoplasm metastasis, and it contains anti-mycoplasma drug or the agent of P37 protein blocking and a kind of conventional antitumor drug and pharmaceutical carrier.
Said medicine can also be a kind of form of medicine box, wherein contains container that anti-mycoplasma drug or P37 blocker are housed, a kind of container of conventional antitumor drug and optional drug use description are housed.
The invention still further relates to anti-mycoplasma drug or the agent of P37 protein blocking and be used for preventing or treating the application of the medicine of TNF-α excessive secretion relevant disease in preparation.
But various medicine of the present invention or blocker be the interior injection administration of oral administration, non-gastrointestinal approach, Sublingual, transdermal route or tumor usually.The amount of the active component of administration depends on characteristic, seriousness and the patient's of the disease for the treatment of body weight when the method according to this invention treatment tumor or neoplasm metastasis.Concrete dosage can be determined according to a conventional method by the attending doctor.
The treatment tumor of administration or the pharmaceutical composition of neoplasm metastasis in the present invention is used for oral, Sublingual, subcutaneous, intramuscular, intravenous, transdermal, internal rectum or tumor, active component can be applicable to human body with dosage unit form.For example be cryodesiccated form or with the blended form of conventional medicine carrier.The appropriate dosage unit form comprises, oral form is as dispersible tablets, capsule, powder, granule and solution or suspension; And the form of administration in Sublingual and cheek; The form of administration in subcutaneous, intramuscular, intravenous or the tumor; The form of topical or rectally.
Tabletting forms the solid composite of tablet form by main active is mixed also with drug excipient.Excipient for example is gelatin, starch, lactose, magnesium stearate, Talcum, Radix Acaciae senegalis etc.Tablet can be with sucrose or other material coatings.Tablet can also be made slow releasing tablet, with the active component that continues, postpones and discharge scheduled volume continuously.
By packing in hard or the soft capsule, obtain the preparation of capsule form with active component and mixing diluents and with mixture.
Powder or granule that available water is disperseed can contain active component, dispersant or wetting agent, suspending agent and flavoring agent etc.
The suppository that is used to make administration can make with cocoa butter or Polyethylene Glycol.
Aqueous solution, saline solution, oil solution, suspension or the emulsion that parenteral uses can make by active component is dissolved or is dispersed in the suitable injection carrier.
To set forth the present invention in more detail by the following example and accompanying drawing, but scope of the present invention is not limited to these embodiment.
Description of drawings
Figure 1A is the result with the positive culture DNA of mycoplasma hyorhinis Auele Specific Primer pcr amplification.The wherein positive contrast in the 1st road; The negative contrast in the 2nd road; 3rd, 4,6,8,9,10 be respectively culture 3,4,6,8,9 and No. 10, its amplification is positive.
Figure 1B is the result with DNA in the positive culture of mycoplasma fermentans Auele Specific Primer pcr amplification.The wherein positive contrast in the 1st road; The negative contrast in the 2nd road; 5th, 6,7 roads are respectively culture the 5th, 6, No. 7, and its amplification is positive.
Fig. 2 is the agarose gel electrophoresis analysis result of RT-PCR amplified production.Wherein: 1.GST albumen stimulating group amplified production, 2.GST-P37 albumen stimulating group amplified production, 3.GST-P37 albumen stimulating group tumor necrosis factor amplified fragments HindIII enzyme action result.
Fig. 3 is for to show P37 albumen dosage dependent stimulation peripheral blood lymphocytes release tumor necrosis factor: the GST-P37 of variable concentrations and GST albumen stimulate the growth inhibited of monocytic supernatant to the L929 cell.
Fig. 4 represent in the PD4 monoclonal antibody and GST-P37 albumen to the stimulation of peripheral blood lymphocytes.
Fig. 5 represents to detect P37 albumen and the bonded result of cell with the ELISA method.
Fig. 6 represents that immunofluorescence detects P37 and the direct bonded result of AGS.
Fig. 7 Northern blot analyzes the expression that P37 albumen stimulates TNF in the ags cell: 1.PBS, 2.GST-P37+PD4, and 3.GST, 4.GST-P37,5.GST-P37+mIgG, 28s, 18s are the RNA standard control.
The Transwell Chamber method that shows Fig. 8 detects the result of P37 albumen to the influence of ags cell wetting capacity.Fig. 8 A is the rod figure that soaks into cell number under the various situations; Fig. 8 B is the microphotograph of cell in the microporous membrane.
The specific embodiment
The separation and Culture of mycoplasma hyorhinis and evaluation in the stomach organization
One, material
1. gastric cancer specimen: collect from Beijing Tumour Hospital's excision gastric cancer specimen in year March June to 2002 calendar year 2001 totally 61 examples, all learn diagnosis before the art in a organized way.All stomach organizations all in the half an hour of exsomatizing the sterile working draw materials, put into the PPLO culture medium that contains glucose, transport laboratory as early as possible under 4 ℃.The part stomach organization is carried out separation and Culture immediately, the packing of residue cancerous tissue ,-70 ℃ are frozen, are used for PCR and detect.Other collects the paraffin section of above-mentioned gastric cancer, is used for SABC and detects.
2. mycoplasma culture medium:
1) fluid medium: take by weighing PPLO powder (production of DIFCO company) 21g, be dissolved in deionized water 700ml, autoclaving 15min is packed as the 70ml/ bottle, and this is the PPLO basal medium.Get basal medium 70ml, aseptic interpolation deactivation rabbit anteserum 20ml, 25% fresh yeast immersion l0ml (pH value 8.0), 50% glucose 2ml, 0.4% phenol red 0.6ml, 1N NaOH0.4ml, 50mg/ml thaliium acetate 1.0ml and 200,000 U/ml penicillin 0.5ml, whole last pH value transfers to 7.8 behind the above composition mixing.
2) solid medium: contain the 0.8g agar powder in the 100ml fluid medium, divide to be filled in the aseptic plastics plate, natural cooling solidifies, and 4 ℃ of preservations are standby.
3. test kit: CLON-GEN mycoplasma sleeve type PCR test kit is available from Wuxi City clone genetic technique institute, test kit provides the overcoat universal primer and the corresponding interior cover Auele Specific Primer of 8 mycoplasma species that can increase, and can detect mycoplasma hyorhinis (Mhy), mycoplasma fermentans (Mf), mycoplasma pneumoniae (Mp) respectively, penetrates mycoplasma (Mpe), mycoplasma genitalium (Mg), separate urine urea substance (Uu), Mycoplasma pirum (Mpi) and mycoplasma hominis (Mh).T/A clone test kit is available from Promega company.
4. SABC antibody: Mus source property resists multiple mycoplasma monoclonal antibody, and inspection center provides by microorganism of military medical sciences academy; The Envision working solution is available from GIBCO company.
Two, method
1. the separation and Culture of mycoplasma: stomach organization was drawn materials the sterile working in the half an hour of exsomatizing, get the about 0.5g of cancerous tissue, clean surperficial clot of removal and downright bad composition with physiological saline solution, cut fragment, be transferred in the test tube that contains 2.0ml PPLO fluid medium into the about 1-2mm of diameter.Draw culture 0.2ml and move in another test tube that contains 1.8ml PPLO culture fluid, carry out 10 times of serial dilutions to 10
-3Use the plug stoppered test tube, put in 37 ℃ of incubators and cultivate.One week back is got the 0.2ml culture and is continued cultivation (being blind passage) after with 10 times of dilutions of fluid medium in first test tube.After this from the culture tube in a last week, got weekly and cultivate continuous 8 weeks after the 0.2ml culture dilutes 10 times.With the PPLO fluid medium as negative control, the next day observe the color of liquid in all culture tubes and turbidity changes, as color flavescence is arranged slightly, promptly it is carried out continuous transferred species; If culture its colour changed into yellow and do not have muddy the appearance, after going down to posterity for 1-2 time, get 1ul after 40000 times of dilutions at its growth vigor animated period (this moment, culture fluid just transferred orange colour to by aubergine), absorption 100ul diluent evenly is coated with to plant and (contains antibiotic and do not contain two kinds in antibiotic) to solid medium, observes colonial morphology after 3-7 days.
2. the criterion of cultivation results: liquid color is turned to yellowly and limpid transparent in the culture tube by aubergine, and then prompting is cultivated positive.Transferred species as is observed " fried egg " sample colonial morphology to solid medium, and is not containing no bacterial clump growth on the antibiotic culture medium, then can except the possibility of L-form, confirm as the mycoplasma bacterium colony.As if the culture fluid its colour changed into yellow but with obvious muddiness and precipitate generation then is germ contamination.60 days colors of culture fluid do not have significant change and then are judged to the cultivation feminine gender.
3. DNA extraction in the positive culture: get positive culture 0.5ml, the centrifugal 5min of 12000rpm abandons supernatant; After adding the washing of 1ml PBS mixing in the precipitate, the centrifugal 5min of 12000rpm abandons supernatant; Add 20 μ l deionized waters in the precipitation, boil 3min, the centrifugal 5min of 12000rpm, this supernatant promptly can be used as template DNA.
4. the DNA extraction in the stomach organization: get-70 ℃ of frozen about 1mm of fresh stomach organization
3, put into aseptic 1.5ml E.P. pipe, add lysate (10mMTris-CL PH7.6, SDS 0.5%, 10mM EDTA PH8.0,100mM NaCl) 300 μ l, E.C. 3.4.21.64 (20mg/ml) 10 μ l, 55 ℃ of water-baths are spent the night.Treat to use phenol respectively, phenol, chloroform, chloroform extracting after tissue digestion fully; Draw supernatant and add 30 μ l 3M sodium acetates (pH5.2) and 600 μ l dehydrated alcohol, mixing is put-70 ℃ of refrigerator 1h; 4 ℃ of centrifugal 20min of 12000rpm abandon supernatant; Add the cold ethanol desalinization of soil by flooding or leaching of 600 μ l75% in the precipitation; 4 ℃ of centrifugal 20min of 12000rpm abandon supernatant, and vacuum is drained.Add 20 μ l TE buffer, 4 ℃ of preservations are standby.
5. sleeve type PCR amplification: press the test kit operation instruction, 300 μ l reactant liquors 1 and reactant liquor 2 are added respectively in the E.P. pipe that contains the Taq enzyme, mixing is packed as 15 μ l/ and props up.Comprise the positive control template DNA in the test kit, with fluid medium or the negative contrast of lysate.At first carry out overcoat PCR: get DNA 1-2 μ l, add and contain in 15 μ l reactant liquors, 1 pipe of mycoplasma universal primer, add deionization H
2O to 20 μ l carries out pcr amplification, and reaction condition is: 93 ℃ of pre-degeneration 2min → 93 ℃ of 30s → 55 ℃ 30s → 72 ℃ of 60s, and after 35 circulations, 72 ℃ are extended 5min; Get PCR product 1 μ l one time, add deionization H
2O to 10 μ l gets 5 μ l adding and contains in the 15 μ l reactant liquors 2 of mycoplasma special primer behind the mixing, increase, and reaction condition is the same.
6.PCR the order-checking of product: behind the conventional T/A clone of PCR product, choose the recombiant plasmid order-checking.
7. SABC detects the mycoplasma in the stomach organization: the gastric cancer paraffin section is through dewaxing, entry, 3% hydrogen peroxide blocking-up endogenous peroxydase, 1%BSA antigen sealing 20min, with 1%BSA with 1: 1000 dilution anti-mycoplasma monoclonal antibody, normal mouse IgG (dilution in 1: 1000) makes negative control, and 4 ℃ are spent the night.Add under the two anti-Envision room temperatures and react 20min, the DAB colour developing.The result judges: occur pale brown color in the stomach cancer cell endochylema and and dye granule and be judged as the positive, no brown yellow granule dye negative.
Three, result
1. the separation and Culture of stomach organization mycoplasma: 61 routine fresh gastric cancer specimen are collected in this research altogether, and 9 examples by germ contamination, are effectively cultivated 52 examples in incubation.2-8 has 8 routine fluid mediums to change yellow into by aubergine respectively after week, and liquid is limpid transparent, and prompting has the mycoplasma growth.The culture transferred species in solid medium, can be seen typical case's " fried egg " sample bacterium colony in 3-7 days, do not see the bacterial clump growth on the solid medium of antibiotic-free, get rid of the probability of L type antibacterial.So the separation and Culture positive rate is 15.4% (8/52).Positive findings average time to occur be 33 days from beginning to cultivate.
2. the evaluation and the clone purification of mycoplasma type in the positive culture: for the mycoplasma type in the clear and definite positive culture, we increase with mycoplasma special primer not of the same race DNA to positive culture.The result shows that the 5 routine mycoplasma hyorhinis DNA cloning positives are arranged in 8 routine positive cultures, the 2 routine mycoplasma fermentans DNA cloning positives, other have 1 this two mycoplasma species DNA cloning of example all positive (Figure 1A, B).
3. the concordance of separation and Culture positive findings and PCR, SABC testing result relatively: in the male gastric cancer specimen of 8 routine mycoplasma separation and Culture, be male 7 examples that have with SABC and PCR detection, there have 1 routine PCR to detect to be positive, and SABC detects negative.Separation and Culture positive findings and SABC, pcr amplification result's the rate of fitting like a glove is 87.5% (7/8), with the identical rate of SABC testing result be 87.5% (7/8), with pcr amplification result's identical rate be 100% (8/8).
Three kinds of method testing results have good concordance, have confirmed to exist in the gastric cancer infection of mycoplasma from different perspectives, and infection type is mainly two kinds of mycoplasma hyorhinis and mycoplasma fermentanses.
Mycoplasma hyorhinis albumen P37 induces human peripheral blood mononuclear cell's release tumor necrosis factor
One, material
Gastric carcinoma cells MGC-803 cell strain, expression plasmid pGEX-4T-1, e. coli bl21-DE3 can obtain from commercial source.Monoclonal anti PD4 is that the applicant is a MGC803 cellular immunization BALB/c mouse with stomach cancer cell, obtains through cell screening.After studies confirm that, the antigen of this monoclonal antibody correspondence is mycoplasma hyorhinis antigen.The L929 cell strain is so kind as to give by medical courses in general institute virus.Rite-directed mutagenesis test kit, lymphocyte separation medium and M-MLV reverse transcriptase are available from Promega company.Diaminobenzidine (DAB), isopropylthio-(IPTG), polymyxin B, cycloheximide D and MTT are available from Sigma company.T4 dna ligase, Vent archaeal dna polymerase and various restricted enzyme are NEB company product.TNF-α is R﹠amp; D company product.RPMI1640 is a GIBCO company product.RNA extracts test kit available from Invitrogen company.
Two, method
1.GST-P37 Expression of Fusion Protein, purification and evaluation
Because coding of tryptophan is TGA in the mycoplasma, in the complete encoding sequence of P37, contain 7 TGA coding, in order to obtain the successful expression of total length p37 in escherichia coli, we utilize the sudden change test kit that 7 tryptophans TGA that encodes is all sported TGG from the mycoplasma hyorhinis genome that extracts behind the amplification P37 full length gene.With sudden change and the P37 genetic fragment through checking order and identifying, the prokaryotic expression carrier pGEX-4T-1 that recombinates behind the transformed into escherichia coli BL21-DE3, is 0.2mM at the IPTG final concentration, and abduction delivering spends the night under 30 ℃ of conditions.Collect and the cracking thalline, get supernatant after centrifugal, with Glutathione-Sepharose-4B adsorb repeatedly, eluting, the eluent that contains fusion rotein GST-P37 through sucrose concentrate and the PBS dialysis after, row SDS-PAGE identifies its purity and content, and confirms through Western blot.
Raise the experiment of peripheral blood lymphocytes expressing tumor necrosin 2.RT-PCR detect the GST-P37 fusion rotein
Get healthy people's venous blood 50mL to the sterilization centrifuge tube that contains heparin, add PBS gently mixing at 1: 1, be sub-packed in the 50mL centrifuge tube by 10mL, behind 1: 1 adding lymphocyte separation medium, the centrifugal 20min of 1500 * g.Slowly draw PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC) layer, after twice of the PBS rinsing, be suspended in again in serum-free 1640 culture medium.Behind the counting, with 10
7Individual PBMC/mL inoculated and cultured bottle, in 37 ℃, 5%CO
2Cultivate 2h in the incubator.After the PBS rinsing 4 times, add final concentration 0.1 μ g/mL GST-P37 fusion rotein and GST albumen effect 16h respectively, extract total RNA by operation instruction, and carry out the synthetic cDNA of reverse transcription as primer with Oligo (dt) with TRIZOL reagent.Get 4 μ L cDNA and do the reaction of template performing PCR, the tumor necrosis factor forward primer is that 5 '-CAGGCAGTCAGATCATCTTCTCA-3 ' (contains exon 2 and exon 3 partial sequence, cross over the 400bp intron), downstream primer is 5 '-CAAACATAAATAGAGGGAGCTGGC-3 ' (from exon 4) [GenBankaccession number NM-000594, Z15026].GAPDH is as internal reference
[17], forward primer is 5 '-ACCACAGTCCATGCCATCAC-3 ', downstream primer is 5 '-TCCACCACCCTGTTGCT GTA-3 '.The two reaction condition is: 94 ℃ of 5min, 94 ℃ of 1min, 52 ℃ of 1min, 72 ℃ of 1min, 30 circulations, 72 ℃ of 10min.
3.GST-P37 fusion rotein stimulates the experiment of peripheral blood lymphocytes release tumor necrosis factor
Separate PBMC, step is the same.Isolating PBMC is with 10
6After individual cell/mL inoculates 24 orifice plates, in 37 ℃, 5%CO
2Cultivate 2h in the incubator.After the PBS rinsing 4 times, add the GST-P37 fusion rotein and the GST albumen effect 16h of variable concentrations respectively, stimulate with serum-free 1640 simultaneously to compare, collect culture supernatant, behind the centrifugal 2min of 10000 * g, supernatant-70 is ℃ frozen standby.The L929 cell is with 10
4Individual cell/100 μ L are inoculated in 96 orifice plates, behind the 24h, add above-mentioned frozen supernatant 50 μ L/ holes and the cycloheximide D of final concentration 1 μ g/mL respectively, and row MTT detects behind the 24h
[18]Be calculated as follows inhibitory rate of cell growth:
4. monoclonal antibody PD4 stimulates the neutralization of peripheral blood lymphocytes to P37 albumen.Separate PBMC, step is the same.Isolating PBMC is with 10
6After individual cell/mL inoculates 24 orifice plates, add respectively after mixture behind GST-P37 fusion rotein, 0.1 μ g/mL PD4 monoclonal antibody albumen and the two 37 ℃ of incubation of 0.1 μ g/mL stimulates 16h, collect supernatant, the samely do the MTT experiment.The inhibitory rate of cell growth computational methods are the same.
Three, result:
1.P37 albumen can raise transcribing of peripheral blood lymphocytes expressing tumor necrosin
With 10
7Individual cell/mL concentration inoculation 5mL PBMC, 37 ℃, 5%CO
2Cultivate 2h in the incubator, after the PBS rinsing, the GST-P37 and the GST albumen that add 0.1 μ g/mL respectively stimulate 16h, extract RNA then and carry out reverse transcription, and with special primer by pcr amplification tumor necrosis factor (TNF α)) the cDNA fragment, the result shows, GST-P37 fusion rotein stimulating group is through amplifying the purpose band (Fig. 2 the 2nd swimming lane) about 930bp behind the RT-PCR, this band is through the HindIII enzyme action, the clip size consistent (Fig. 2 the 3rd swimming lane) of its result and expection, GST albumen stimulating group fails to amplify corresponding target band (Fig. 2 the 1st swimming lane), and internal reference GAPDH all obtains amplification (Fig. 2 the 1st, 2 swimming lane) at two groups.Illustrate that the GST-P37 fusion rotein can stimulate transcribing of tumor necrosis factor.
2.P37 albumen can stimulate the peripheral blood lymphocytes release tumor necrosis factor
The L929 cell is responsive to the cell growth inhibited reaction that TNF α causes, therefore, the MTT experiment that utilizes this cell to carry out is commonly used with the detection to TNF α.The experimental result of present embodiment shows, increase along with the GST-P37 protein concentration, the peripheral blood lymphocytes supernatant also progressively improves the L929 cell growth inhibition, when concentration reaches 0.1 μ g/ml, this stimulated peripheral mononuclear cells supernatant reaches 47.8 ± 0.3% to the growth inhibited of L929 cell, and the GST albumen of same concentrations stimulation supernatant suppression ratio is 12.5 ± 0.4%.The two significant difference (P＜0.01, t check).Illustrate that the GST-P37 fusion rotein can dose dependent ground stimulates the release (see figure 3) of tumor necrosis factor.
Monoclonal antibody PD4 can in and P37 albumen to the stimulation of peripheral blood lymphocytes
We are with PD4 and GST-P37 and peripheral blood lymphocytes coprocessing, the result shows, monoclonal antibody PD4 is when 0.1 μ g/mL concentration, to stimulate caused peripheral blood lymphocytes to discharge the TNF alpha active by 0.1 μ g/mLGST-P37 the obvious suppression effect is arranged, and PD4 itself there is not obvious influence to the activity of mononuclear cell release tumor necrosis factor.The activity that GST-P37 fusion rotein induced tumor necrosin is described is mediated by P37, point out simultaneously position (or around it) that P37 and PD4 combine may with P37 with combining of peripheral blood lymphocytes relevant (Fig. 4).
The combination of P37 albumen and stomach cancer cell AGS
One, ELISA detects the combination of P37 albumen and stomach cancer cell AGS
With stomach cancer cell AGS with every hole 2 * 10
4Cell inoculation is in 96 orifice plates.After the overnight incubation, discard the culture medium that contains serum, change serum-free medium.Add the doubling dilution liquid (initial final concentration is 64 μ g/ml) that contains GST albumen and GST-P37 albumen (its preparation method is seen embodiment 2) respectively, in 37 ℃, 5%CO
2Incubation is 3 hours in the incubator.Use PBS rinsing cell 2 times then, fix 10 minutes with the ratio room temperature of 0.125% (v/v), spend the night with 4 ℃ of sealings of 3%BSA/PBS with glutaraldehyde.After the reuse PBS rinsing 2 times, add anti-GST one and resist room temperature reaction 2 hours.After Tween20-PBS rinsing 5 times, the sheep anti mouse two that adds the HPR-labelling is anti-, room temperature reaction 2 hours.After the Tween20/PBS rinsing 5 times,, measure OD492 with the OPD colour developing.
The result as shown in Figure 5.The result shows that P37 can combine with the AGS-1 cell.
Two, immunofluorescence method detects the combination of P37 albumen and stomach cancer cell AGS
Stomach cancer cell AGS is inoculated in 6 orifice plates that are equipped with coverslip, allows its creep plate 24 hours.Add GST and GST-P37 albumen that final concentration is 10 μ g/ml then, 37 ℃, incubation 1 hour.With the PBS rinsing cell that contains 1%BSA 2 times, each 10 minutes.The sheep anti mouse two that adds the TRITC labelling is anti-, 37 ℃, reacts 1 hour.After using 1%BSA/PBS rinsing 2 times then, observed result under the fluorescence microscope.
The result as shown in Figure 6.The result shows that P37 can directly combine with ags cell.
Northern blot analyzes the rise that P37 albumen stimulates TNF in the ags cell:
The mixture (respectively being 10 μ g/ml) that in 80% ags cell that converges that is incubated in 5 bottles, adds PBS, GST albumen, GST-P37 albumen (final concentration 10 μ g/ml), GST-P37 and 2 hours mixture of PD4 precincubation (respectively being 10 μ g/ml) and GST-P37 and mIgG precincubation respectively.37 ℃, stimulated 24 hours under the 5%CO2 incubator condition.Extract total RNA respectively, utilize 28s, 18s RNA runs RNA degeneration glue with after sample is adjusted sample on spectrophotometer and the race glue, changes film, utilizes the TNF-α oligonucleotide probe hybridization of α-32P-dCTP labelling, detects the expression of TNF-α.The result shows: P37 albumen can raise TNF and express (see figure 7).
Mycoplasma hyorhinis albumen P37 promotes the infiltration of tumor cell
80% ags cell that converges is after digestion, with 10
6Cell/ml is suspended in the F-12K culture medium of serum-free.In each cell suspension, add PBS, GST, GST-P37, GST-P37+PD4 (pretreatment) mixture, GST-P37+TNF-α multi-resistance (pretreatment) mixture and GST-P37+mIgG (pretreatment) mixture respectively.Then the above-mentioned suspension of each 500 μ l is added respectively on the Boyden chamber in the chamber, following chamber adds the supernatant of the hungry NIH3T3 of cultivation cell as chemotactic factor.After 24 hours, the microporous membrane between the chamber is up and down taken out, with methanol fixing after, carry out Gimsa dyeing.Every film is chosen 9 visuals field arbitrarily, and microscopically * 200 are observed and carried out the cell numeration.3 parallel holes, calculating mean value are established in every group of experiment.
The results are shown in Figure 8A and 8B.Compare with the GST matched group with PBS, the GST-P37 protein groups has significantly improved the infiltration cell number.This infiltration cell number rising effect that is stimulated by P37 albumen can most ofly be neutralized by the proteic monoclonal antibody of anti-mycoplasma hyorhinis, also can be neutralized by TNF-α multi-resistance.The result shows: P37 albumen can obviously strengthen the wetting capacity of ags cell by rise TNF.
Claims (12)
1. anti-mycoplasma drug or the agent of P37 protein blocking are used for preventing or treat the application of the medicine of tumor or neoplasm metastasis in preparation.
2. the application of claim 1, wherein said anti-mycoplasma drug are tetracycline antibiotics, macrolide antibiotics or quinolone antibiotic.
3. the application of claim 1, the agent of wherein said P37 protein blocking are proteic monoclonal of anti-P37 or polyclonal antibody.
4. anti-mycoplasma drug or the agent of P37 protein blocking are used for the treatment of application in the medicine of tumor or neoplasm metastasis with conventional antitumor drug in preparation.
5. the application of claim 4, wherein said anti-mycoplasma drug are tetracycline antibiotics, macrolide antibiotics or quinolone antibiotic.
6. the application of claim 4, the agent of wherein said P37 protein blocking are proteic monoclonal of anti-P37 or polyclonal antibody.
7. pharmaceutical composition for the treatment of tumor or neoplasm metastasis, it contains anti-mycoplasma drug or the agent of P37 protein blocking and a kind of conventional antitumor drug and pharmaceutical carrier.
8. the compositions of claim 7, wherein said anti-mycoplasma drug is tetracycline antibiotics, macrolide antibiotics or quinolone antibiotic.
9. the application of claim 7, the agent of wherein said P37 protein blocking are proteic monoclonal of anti-P37 or polyclonal antibody.
10. medicine box wherein contains container that anti-mycoplasma drug or P37 blocker are housed, a kind of container of conventional antitumor drug and optional drug use description is housed.
11. the medicine box of claim 10, wherein said anti-mycoplasma drug are tetracycline antibiotics, macrolide antibiotics or quinolone antibiotic.
12. the medicine box of claim 10, the agent of wherein said P37 protein blocking are proteic monoclonal of anti-P37 or polyclonal antibody.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104138598A (en) * | 2014-08-15 | 2014-11-12 | 北京市肿瘤防治研究所 | Method and preparation for preventing mycoplasma hyorhinis from infecting cells |
| WO2019028646A1 (en) * | 2017-08-08 | 2019-02-14 | Sun Yat-Sen University | Methods and compositions for treatment of multi-drug resistant tumors |
-
2003
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104138598A (en) * | 2014-08-15 | 2014-11-12 | 北京市肿瘤防治研究所 | Method and preparation for preventing mycoplasma hyorhinis from infecting cells |
| WO2016023514A1 (en) * | 2014-08-15 | 2016-02-18 | 北京市肿瘤防治研究所 | Method and formulation of preventing cell infection by mycoplasma hyorhinis |
| WO2019028646A1 (en) * | 2017-08-08 | 2019-02-14 | Sun Yat-Sen University | Methods and compositions for treatment of multi-drug resistant tumors |
| CN111315446A (en) * | 2017-08-08 | 2020-06-19 | 中山大学 | Methods and compositions for treating multidrug resistant tumors |
| JP2020530035A (en) * | 2017-08-08 | 2020-10-15 | スン・ヤット−セン・ユニバーシティＳｕｎ Ｙａｔ−Ｓｅｎ Ｕｎｉｖｅｒｓｉｔｙ | Methods and Compositions for Treating Multidrug-Resistant Tumors |
| AU2017426847B2 (en) * | 2017-08-08 | 2021-11-18 | Sun Yat-Sen University | Methods and compositions for treatment of multi-drug resistant tumors |
| US11576930B2 (en) * | 2017-08-08 | 2023-02-14 | Sun Yat-Sen University | Methods and compositions for treatment of multi-drug resistant tumors |
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