Study On Effection Of Expression Of The Tumor Cells' Polysaccharide Molecules After S180 Tumor-bearing Mice Were Sequential Injected With Space Streptococcus

1,Background:B Streptococcus preparation in the clinical application shows that there is significantly anti-tumor effect and enhance immunity.Early in 1967,at the research of Okamoto who is a Japanese scholars to prove:Streptococcus anti-tumor activity is mainly produced by bacteria-induced S hemolysin,and show a variety of hemolytic streptococcus transplanted animals have anti-tumor effects,Including Ai sascites carcinoma,S180,S37,Bron-pearce carcinoma,Krebs-2 carcinoma,Yoshida ascites sarcoma,fibroma,lymphatic leukemia(SN36,L4946),ascites hepatoma(AH-130, AH-66).Contact with cancer cells,cancer cells can directly undermine the RNA transcription process,so that the expansion of cancer cells and the collapse of vacuolization necrosis.In addition can also activate cells and humoral immunity to tumor necrosis factor,interferon,IL-such as levels of cytokines significantly in -creased,thus surrounded by infiltration and attachment of tumor cells at up to degeneration and necrosis,reduction in cancer cells to control tumor growth Therefore,hemolytic streptococcus preparation through direct anti-tumor cells and activate the immune system of non-specific anti-tumor cells,both anti-cancer drugs can also Immunomodulator.According to the principle of development and production of OK-432β-hemolytic streptococcus anti-tumor biological preparation. The drug in Japan,has been widely used in adjuvant therapy for tumor is a strong immunity induced by bacterial agents,low toxicity and efficient hemolytic strep -tococcus strain SIPI722 after treatment by penicillin,such as freezedried powder made from.Our country has the same preparations(trade names:Sapylin) is also widely used in clinical tumor.During the decade,Chinese manned-space-technology is developing rapidly, manned spacecraft space technology conditions microbial mutagenesis(Space Strepto -coccus) breeding biology are cultivating new strains become a reality.The principle is that through the outer special physical and chemical environment,that is, microgravity,cosmic radiation,high-energy particles,alternat -ing magnetic field and ultra-clean environment,etc.,caused by strain variation of DNA molecules and reorganization,changes in strain genetic traits,in order to obtain vigorous,fast growing,high rate of metabolites to obtain high quality and stability of genetic strains of high yield and efficiency.D33 # will hemolytic streptococcus has repeatedly carried by the "Shenzhou" series spacecraft and technological experiment satellites,to make it into outer-space,implementation of the mutation,and ultimately to obtain high yield and efficiency,has independent intellectual property rights,innovation and strain of hemolytic streptococcus T33#.Injection polyactin A(mannan-peptide) of the strains cultivated in the process at our country has been widely used in clinical anti-tumor treatment,a significant effect.The drug for 33 # hemolytic streptococcus strains derived Deep Sounds of the immune activity of a-mannan peptide substances have inhibitory effect on S180 sarcoma,or mouse Ehrlich ascites carcinoma DNA synthesis,an increase of mouse abdominal reticuloendothelial system phagocytic -ability and lymphocyte transformation function,promote metabolism of thymus lymphocytes,CTX antagonist inhibittory effect on the immune mechanism. Immunoenhancer used.With enhanced immune function and activation of phagocytic cells,increased the role of peripheral leukocytes;can improve bone marrow hematopoietic function and stress the capacity of the body.As we all know,cell biology and blood polysaccharide complexes exist. Saccharide chain is the third chain of life after second protein,nucleic acid.As a class of complex biological substances,widely exist in the human body,more and more studies have shown that glycoconjugates are important in vivo information elements, in cell differentiation,signal transduction,molecular recognition and many other life activeities plays a very important role.Glycoconjugates of the oligonu cleotide chain are between the molecules and cells to identify the signs.Through this recognition,many of the biological response has been initiated.Under normal circumstances,a variety of saccharide chain expression level is relatively stable.When malignant transformation of cells,saccharide chain,the nature and quantity will be a series of changes happened,and reflect the lectin on the bind -ing capacity.Agglutinin(Lectin) refers to a variety of plants,invertebrates and higher animals glycoprotein purified protein or a combination of sugar,which can agglutinate red blood cells(including blood group substances),named lectin.Lectins are a class of natural immunogenicity,to identify saccharide chain structure of proteins,with sugar to show the combination of specificity,similar to antigen-antibody binding specificity.Lectins such natural features,can be used as a probe detected tumor cell surface,cells and serum polysaccharide levels.Sugar in the current clinical treatment of them has an important role.Agglutinin and saccharide according to this specific binding can be divided into 7 major categories:(1)D-man nose(D---glucose)group;(2)N---acetylglucosamine group;(3)acetylgalactosa mine group;(4)D---galactose group;(5)L---rock bath sugar group;(6) sialicacid group; (7)group of complex sugars.Various types of lectin -polysaccharide molecules and tumor-related morbidity and clinics are more closely related.When the tumor appears, glycoconjugates and lectin binding changes will happen,but the cell membrane of tumor cells accompanied by changes in glycosylation on the cell membrane will produce corresponding changes can be detected lectin.Substantial study found that lectins can be used as markers of tumor tissue-derived,tumor spe -cific diagnostic sign of malignant tumor markers and differentiaton of different tumor markers.2,Purpose:At different types of lectins have selected five representative lectin,respectively, are concanavalin agglutinin(ConA),wheat germ agglutinin(WGA),Sambucus lectin (SNA),RCA Su-(RCAâ… ),phyto hemagglutinin(PHA).Use of lectin and saccharide-specific binding characteristics of injection of streptococcal preparation on S180 tumor cells in tumor-bearing mice a high mannose N-type,terminal GlcNAc, the end of Neu5 Aca2-6Gal,terminal Galβ1-4GlcNAc and Gal-NAC polysaccharide molecules expression of changing circumstances;observed by sequential growth of tumor after injection;explore the inhibitory rate of Streptococcus preparation and saccharide chain expression levels.3,Method:3.1,Set up the animal models:Randomly divided into 4 groups,①②③④,10 each group.1) The first group of miceâ‘ â‘¡systemic immunization,Methods:intra -peritoneal injection of streptococcal preparation(both dose 0.1mg).③④group and intra-abdominal injection of normal saline;2)After about a week,4 groups of mice were inoculated subcutaneously with S180 sarcoma cells,about two weeks to grow about 1cm in diameter of the tumor;3) Remove the groupâ‘ (intraperitoneal injection of Streptococcus one of agents 1 Group 2 Group),as well as group (intraperitoneal injection of saline one of Group 2 Group 1),direct intratumoral injection at the same dose of streptococcus preparation,and the remaining 2â‘¡â‘£group injected with normal saline.Following the above treatment a week later,mice were sacrificed to remove the tumor tissue,isolated from a single cell suspension with PBS buffer solution to adjust into the same concentration of 100 mg / ml,to ultra so -nic crushing tumor cells,select the supernatant.-70℃storage after collection,to be seized.3.2,With horseradish peroxidase labeled lectin preparation:Through improved sodium code coupling the two.Check the HRP lectin binding rate and titer.3.3,Specimen collection and treatment:S180 sarcoma cell line Specimen ultra -sound broken after repeated freezing and thawing,at ultrasonic crusher broken instrument.By ultrasound indicated that the sarcoma cells and PBS mixture,and adjust to put the concentration of each sample was 100 mg/ml(through microscopy, cell count of about 7×10⁸/ml) Power:800W,total time:4 minutes/Samples,broken time:2 minutes(5 seconds to open,Guan 5 seconds).According to the formula: relative broken cell rate =(1-ultrasonic precipitation after cell wet weight/ultrasono gr aphy before cell sediment wet weight)%to calculate the broken rate of 98.7%.Take the supematant,centrifuge 20min(12000r/s),Supematant preserved at -20℃refrigerator.3.4,Lectin recognition of polysaccharide molecules Detected:Samples in accordance with the above will be the most appropriate concentration of coated ELISA plate 96,each hole 100μl,4℃overnight,PBS wash buffer 1,the respective serum seized add 100μl,4℃reaction of 4h,PBS wash 3 times,add horseradish peroxidase labeled glycoprotein,each hole 100μl,4℃reaction 4h,PBS wash 3 times, TMB₂H₂O₂ Color 10min,the suspension after the reaction,using enzyme-linked immunosorbent Detector Detect wavelength 450nm OD value.Calculation of inhibition rate:[model group tumor weight-tumor weight of experimental grou -p]/model group,tumor weight.3.5,Experimental control:The broken mouse red blood cells to replace the super -natant sample for positive control;with PBS to do in lieu of negative control serum; other steps remain unchanged.3.6,Statistical data SPSS13.0 deal with statistical software.4,Result4.1,Calculation IR%and the united administration treatment group,systemic administration treatment group and local administration treatment group,respectively, the inhibitory rate:35.1%,8.8%,19.9%。The results suggest that:United administration treatment group than the systemic treatment group and the local administration Group more significant anti-tumor effect.4.2,The marked rate of ConA-HRP/WAG-HRP/SNA-HRP/PHA-HRP/RCA1-HRP were 0.73,0.78,0.75,0.70,0.71.ELISA assay to titers were 1:1000.4.3,A variety of tumor cells in mice to identify the relevant ConA/WAG/SNA/ PHA/RCA-1 polysaccharide molecular content OD values:United administration treatment group polysaccharide molecular samples to iden -tify ConA/WAG/SNA/PHA/RCA-1 content OD values were:1.306±0.020,1.313±0.033,1.259±0.031,1.255±0.021,1.251±0.028Systemic administration treatment group:1.856±0.025,1.737±0.033,1.444±0.028,1.669±0.035,1.566±0.035Local administration treatment group:1.362±0.024,1.384±0.033,1.386±0.025,1.346±0.033,1.417±0.031Negative control group:1.952±0.024,1.764±0.032,1.534±0.025,1.802±0.034,1.689±0.030These results for the importation of SPSS13.0 factorial analysis(intra-abdominal injection and intratumoral injection) main effects were statistically significant differences in A Streptococcus by intraperitoneal injection of P=0.000;B streptoc -occus intratumoral injection P=0.000,can consider intra-abdominal injection and intratumoral injection of streptococcus factorial decreased ConA/WAG/SNA/ PHA/RCA-1 related polysaccharides expression;five factors related to the two polysaccharide interactions and intra-abdominal injection in both tumor(general administration) when the space streptococcus,ConA/WAG/SNA/PHA/RCA-1 polysaccharide molecules related to decreased expression of the most significant.5,Conclusion:5.1,The first partial delivery after systemic administration of the sequential treatment of S180 sarcoma by the growth of two-factor factorial analysis of interaction has the effect of intraperitoneal injection of mice immunized with intratumoral injection of synergies have that S180 tumor-bearing mice vaccinated Sequential streptococcus preparation space than only intratumoral injection of anti-tumor effect.At the same time,space and proved that both anti-tumor streptococcal preparation role,but also has the role of immune regulation.Cancer treatment for clinical reference.Space streptococcus has prove that there is significantly anti-tumor effect and enhance immunity.5.2,The first partial delivery after systemic administration of the sequential treatment of the polysaccharide molecule expression levels by factorial analysis of exchange effects also exist,S180 tumor-bearing mice by sequential injection of streptococcal preparation space after the sarcoma cells polysaccharide molecules ConA/WAG/SNA/PHA/RCA-1 related content were significantly decreased,shows that the overall expression level of polysaccharide molecules down and the extent of tumor glycosylation reduced.