CN100372571C - Chitosan-immune RNA composite formulation and preparation method thereof - Google Patents
Chitosan-immune RNA composite formulation and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a chitosan-immune RNA compound preparation which mainly contains compounds prepared from chitosan and immune RNA, wherein the compounds are nanometer granules or micron granules. The present invention overcomes the defect of the existing immune RNA of easy degradation in vivo and is a new stable immune RNA preparation prepared from chitosan. The chitosan-immune RNA compound preparation can effectively protect immune RNA from rapid degradation, prolong the acting time of the immune RNA and promote the immune RNA to enter lymphocyte. Therefore, the immune RNA can better perform functions, the activity of the immune RNA is enhanced, and curative effects are improved. The present invention also discloses a method for preparing the chitosan-immune RNA compound preparation.
Description
Technical field
The present invention relates to a kind of medical preparation, more particularly it is a kind of chitosan-immune RNA composite formulation, and the present invention has also announced the preparation method of this complex formulation simultaneously; This chitosan-immune ribonucleic acid preparation can be applicable to the treatment in fields such as viral disease and malignant tumor.
Background technology
Immune ribonucleic acid (Immune RNA, immune ribonucleic acid) is the active substance that a class surmounts kind boundary premunition function, the specific immune response ability that donor had can be passed to the body or the lymphocyte of receptor.A large amount of studies show that, if give animal immune with a kind of antigen, it is produced this antigenic immunoreation (comprising humoral immunization and cellular immunization), again from the lymphatic organ (lymph node and spleen) and the lymphocyte isolation of RNA (RNA) of sensitized animal, this kind RNA can pass to never contacted this antigenic receptor to the immunoreation ability of specific antigen with donor animal, make its can produce to this antigenic special body fluid and cell immune response (referring to Zheng Changxue. from experiment, clinical and view of theory immune ribonucleic acid [J]. Chinese biochemical drug, 1999,20 (5): 267-269), therefore claim that this RNA is an immune ribonucleic acid.As can be seen, immune ribonucleic acid is the RNA of a class from immune organ and energy premunition function.The immunological role mechanism of immune ribonucleic acid do not illustrate fully as yet (referring to Feng Laikun, intercalation respectful, Liu Jinghui, etc.Curative effect from the view of theory immune ribonucleic acid.China's biochemical drug, 1999,18 (2): 106-107), that most of experimental results are all supported to work in the immune ribonucleic acid is mRNA, and it may be the direct template of antibody or the direct template of some activate immunity response factor.
Immune ribonucleic acid is as a kind of immunotherapeutic agent, and clinical practice had for two more than ten years, but very easily is subjected to owing to immune ribonucleic acid enters that the RNase degraded loses activity in the human body behind the human body, so its immunocompetence and clinical efficacy are still disputable (referring to Zheng Changxue.Some problems about current immune ribonucleic acid production, quality control and application facet.China's biochemical drug, 1997,18 (6): 320-321)., as a kind of immunotherapeutic agent the therapeutical effect of chronic hepatitis is extremely paid attention to as anti-hepatitis B immune RNA, but the clinical application effect instability.We think that the immune ribonucleic acid of present clinical practice is the lyophilized powder that extracts lymphoid tissue (spleen, the lymph node) RNA of sensitized animal, real RNA for exposing, after directly subcutaneous (some or even intramuscular injection) injection enters human body, RNase in being organized is hydrolyzed into immunocompetence rapidly to be reduced, and causes the clinical efficacy instability.Therefore for improve immune ribonucleic acid enter in the body after immunocompetence, successful premunition information, making up the immune ribonucleic acid carrier systems that can effectively protect immune ribonucleic acid also can be organized better absorption is that ten minutes is very necessary.
Chitosan (chitosan) is called soluble chitin again, or chitosan, chitosan, chitin etc., and formal name used at school is β-1, and 4-is poly--D-glucosamine, molecular formula (C
6H
11NO
4)
n, trade name Flonac is by producing through deacetylation behind the refining chitin (chitin) of aquatic products processing garbages such as Eriocheir sinensis, shrimp extraction; contain β-(1; 4)-and 2-acetylaminohydroxyphenylarsonic acid D-glucose unit and β-(1,4)-2-amino-unitary polymer of D-glucose, the latter generally is no more than 65%.Can get the different chitosans that take off acetyl degree and mean molecule quantity according to different preparation methoies.The chitosan rich raw material sources has many unique chemical physical propertys, also can be prepared into multi-purpose product according to reactions such as its acidylate, Sulfation and oxidation, grafting and crosslinked, hydroxyethylation, methylolations.
Chitosan molecule is positively charged under acid condition, nucleic acid molecules is electronegative under alkali condition, chitosan can form surface positively charged microgranule (referring to Ishii T with nucleic acid molecules in acetic acid or saline solution, Okahata Y, Sato T.Mechanism of cell transfectionwith plasmid/chitosan complexes.Biochimica et Biophysica Acta (BBA)/Biomembranes200l, 1514 (1): 51-64), nucleic acid is wrapped in and is difficult in the nanoparticle by nuclease degradation, stability significantly improves (referring to Richardson SCW, KolbeHVJ, Duncan R.Potential of low molecular mass chitosan as a DNAdelivery system:biocompatibility.body distribution and ability tocomplex and protect DNA.International Journal of Pharmaceutics1999,178 (2): 231-243).Chitosan-nucleic acid nano complex also can serve as a controlled release preparation in the nucleic acid process that discharges parcel, chitosan-nucleic acid nano polymer has good prospect as the carrier of nucleic acid, but chitosan has not yet to see report as the carrier of RNA.
Summary of the invention
The objective of the invention is to overcome the defective of existing immune ribonucleic acid preparation, and a kind of chitosan-immune RNA composite formulation is provided, another object of the present invention provides the method for the above-mentioned chitosan-immune RNA composite formulation of a kind of preparation.Said preparation can protect immune ribonucleic acid to avoid degraded on the one hand, and the activity of simultaneously all right enhance immunity RNA improves curative effect.
The objective of the invention is to reach by following measure: a kind of chitosan-immune RNA composite formulation, it mainly comprises chitosan and the formed complex of immune ribonucleic acid, and described complex is nanoscale or micron particles.
In technique scheme, described compound particles diameter is between 100 nanometers～100 micron.
In technique scheme, described immune ribonucleic acid is the resistance of hepatitis B immune ribonucleic acid.
In technique scheme, described immune ribonucleic acid is antineoplastic immune RNA.
Prepare the method for above-mentioned chitosan-immune RNA composite formulation, which comprises at least following steps:
A. chitosan is dissolved in 1% acetum and obtains 0.5% chitosan solution, sodium hydroxide transfers pH to arrive
5.5, aseptic filtration;
B. immune ribonucleic acid is dissolved in 5% to 20% the metabisulfite solution, immune ribonucleic acid concentration is 10-20 μ g/ml;
C. immune ribonucleic acid solution is dropwise joined in equal-volume 0.5% chitosan solution, stir simultaneously with 500 rev/mins speed, treat that whole immune ribonucleic acids all drip after, stirred 1 hour, be the glutaraldehyde of 1% adding concentration 12.5% by volume, continue then to stir 1 hour;
D. with centrifugal 10 minutes of mixed solution 12000rpm to collect chitosan-immune RNA composite;
E. add distilled water washing chitosan-immune RNA composite, 12000rpm repeated 3 times to collect chitosan-immune RNA composite in centrifugal 10 minutes;
F. vacuum lyophilization obtains the freeze dried powder of chitosan-immune RNA composite.
The present invention be with chitosan as carrier, the preparation chitosan-immune RNA a kind of new formulation.
Chitosan is the polysaccharide that is formed by the different proportion polymerization by glucamine monomer and N-acetyl group glucamine monomer, owing on the glucamine monomer free amino is arranged, so have positive charge, its pKa value is 6.5.When pH 6 when following, the chitosan dissolving, its one-level is amino can be protonated.The chitosan that has positive charge can form chitosan-immune ribonucleic acid microgranule with the immune ribonucleic acid that has negative charge by electrostatic interaction.Formed chitosan-immune ribonucleic acid mean particle dia is generally between 100 nanometers～100 micron.Can protect immune ribonucleic acid to avoid the degraded of enzyme after chitosan and the immune ribonucleic acid combination, increase the stability of immune ribonucleic acid, improve the biologic activity and the clinical efficacy of immune ribonucleic acid.In addition, chitosan is a kind of natural biomaterial, and avirulence has no side effect, and is the environmental type nucleic acid carrier, and user is easy to accept.Chitosan itself has certain immunological enhancement simultaneously, can improve patient's non-specific immunity simultaneously.Preparation technology of the present invention is simple, and equipment requirements is not high, and the response time is short, mild condition, easy operating.And material is easy to get, and is with low cost.
Description of drawings
Fig. 1 is chitosan of the present invention-RNA complex RNA enzymatic degradation protection experimental result picture.
The specific embodiment
Protect result of experiment below in conjunction with accompanying drawing labor chitosan of the present invention-RNA complex RNA enzymatic degradation:
Consult Fig. 1 as can be known: after RNA and chitosan formed complex, chitosan-immune RNA composite was arrested in the well and is not degraded.Among Fig. 1: A is the immune ribonucleic acid that does not have protection, and B is RNA+RNase effect 15min, can see that RNA substantially has been degraded; C, D are the RNase effect 15min of chitosan-immune RNA composite+variable concentrations; E, F are the RNase effect 15min of the chitosan-immune RNA composite+variable concentrations of 1/2 concentration in C, the D hole; as can be seen because chitosan and RNA have formed complex; chitosan plays retardation to RNA, and RNA all is arrested in the well, and is protected; not by the degraded of RNase; the contrast of four holes, the concentration maximum of Rnase in the F hole, the RNA in chitosan-RNA complex partly is degraded.
Embodiment 1:
The preparation and the effect analysis of chitosan-hepatitis b immune RNA complex formulation
1, the preparation of chitosan-hepatitis b immune RNA new formulation
(1) chitosan is dissolved in 1% acetum and obtains 0.5% chitosan solution, stirred 24 hours, the ammonia of adding 1%, make dissolved chitosan precipitation, filter, the vacuum lyophilization precipitation is dissolved in 1% acetum again with gained chitosan after the drying and obtains 0.5% chitosan solution, transfer pH to 5.5, aseptic filtration with sodium hydroxide;
(2) the 0.5mg immune ribonucleic acid is dissolved in 5% the metabisulfite solution of 50ml, immune ribonucleic acid solution is dropwise joined in equal-volume 0.5% chitosan solution, stir simultaneously with 500 rev/mins speed, after treating that whole immune ribonucleic acids all drip, stirred 1 hour, the glutaraldehyde that adds 1ml concentration 12.5% continues to stir 1 hour then, with centrifugal 10 minutes of mixed solution 12000rpm to collect chitosan-immune RNA composite;
(3) add distilled water washing chitosan-immune RNA composite, 12000rpm repeated 3 times to collect chitosan-immune RNA composite in centrifugal 10 minutes;
(4) vacuum lyophilization obtains one of the freeze dried powder of chitosan-immune ribonucleic acid.
2. chitosan-RNA complex RNA enzymatic degradation protection experiment
Experimental procedure is as follows:
(1) preparation method by above-mentioned chitosan-hepatitis b immune RNA new formulation prepares chitosan-RNA complex, gets a freeze dried powder and is dissolved in 0.5ml ddH
2O.
(2) are the immune ribonucleic acids that do not have protection by following operation preparation A, B, C, D, six sample: A of E, F, B is RNA+RNase effect 15min, can see that RNA substantially has been degraded; C, D are the RNase effect 15min of chitosan-immune RNA composite+variable concentrations, and E, F are the RNase effect 15min of the chitosan-immune RNA composite+variable concentrations of 1/2 concentration in C, the D hole; Carry out agarose gel electrophoresis then.
3, mouse test
Get 20 of healthy mices, body weight 28～34g, female, four of the freeze dried powders of chitosan-immune ribonucleic acid of preparation are dissolved in the distilled water of 10ml, in oxter or the groin subcutaneous injection of mice, every injection 0.5ml, 1 time weekly, totally 7 times, duplicate injection is 2 times after 30 days.Also set up normal saline group (injecting normal saline 0.5ml), chitosan group (injecting 0.1% chitosan 0.5ml), immune ribonucleic acid group (injecting immune RNA solution 0.5ml simultaneously, contain immune ribonucleic acid 0.1mg) in contrast, every group of 20 white mice, the injection site is identical with the time.Mice is carried out in the last injection respectively after 10 days hepatitis B surface antibody HbsAb mensuration, the interior leukocyte adhesion inhibition of body (LAI) test, external leucocyte adherence inhibition assay and splenocyte identification HBsAg ELISA test.
(1) hepatitis B surface antibody HbsAb measures: from mouse tail blood sampling 0.5ml, treat separation of serum behind the blood coagulation, measure the intravital hepatitis B surface antibody HbsAb level of mice, adopt hepatitis B surface antibody HbsAb kit measurement HbsAb, concrete grammar is undertaken by operation instructions.
(2) leukocyte adhesion suppresses (LAI) test:
Take off cervical vertebra and put to death mice, put into 75% ethanol and soak 5min, the preparation splenocyte suspension, concrete grammar is as follows: take out the back right arm reclining, sterilization left dorsal abdomen intersection skin is with the aseptic taking-up mouse spleen of shears, flush away bloodstain in the incomplete culture fluid of the RPMI-1640 that does not contain serum, cut off fat and fascia, place the aseptic little plate that is lined with 200 order nylon wires; Glass syringe inner core crushing spleen with aseptic adds 1640 liquid washing and filtering, and the cell suspension under the collecting net is gone in the aseptic centrifuge tube, with incomplete culture fluid washing of the RPMI-1640 that does not contain serum 2 times and suspension again; Separate mononuclearcell: the above-mentioned splenocyte suspension that has prepared slowly places on the lymphocyte separation medium (proportion 1.080) that has prepared, the centrifugal 20min of 2000r/min, draw the nebulous low-density cell of the second layer, the incomplete culture fluid 1000r/min of RPMI-1640 that reuse does not contain serum washs 2 times; Regulate cell concentration to 5 * 10
6Individual/ml.
Get 96 orifice plates, set up test hole and control wells (5 parallel holes are established in each hole) respectively, press following application of sample: experimental port adds mouse boosting cell suspension 100 μ l, hepatitis B surface antigen 50 μ l, 10% calf serum RPMI-1640 liquid, 50 μ l; Control wells adds mouse boosting cell suspension 100 μ l, 10% calf serum RPMI-1640 liquid, 100 μ l.5%CO
2, 37 ℃ cultivate 2h, supernatant discarded gently, in each hole, add incomplete culture fluid of RPMI-1640 and the 20 μ lMTT solution that 180 μ l do not contain calf serum, cultivate 4h again, abandon supernatant, every hole adds 200 μ lDMSO again, and vibration evenly, room temperature is placed 15min, and microplate reader is surveyed the optical density value (A value) at wavelength 490nm.Calculate adhesion inhibition index (LAI value) by following formula:
(3) splenocyte specific bond HBsAg ELISA test
Get 96 orifice plates, press following application of sample: mouse boosting cell suspension 50 μ l, hepatitis B surface antigen 50 μ l, 10% calf serum RPMI-1640 liquid, 100 μ l, 5%CO
2, 37 ℃ cultivate 2h, centrifugal microwell plate (1000g, 15min), supernatant discarded adds the RPMI-1640 liquid 50 μ l that contain 10% calf serum gently; Establish positive control hole (positive serum 50 μ l) and negative control hole (negative serum 50 μ l) simultaneously, all establish 5 parallel holes; Add the antibody 50 μ l of horseradish peroxidase-labeled in each hole, pat mixing; Hatch 25min for 37 ℃, equilibrium at room temperature 5min; With after the cleaning mixture washing, abandon supernatant after centrifugal, repeats 5 times, wash afterwards and detained dried (keeping 30～60s soak time) at every turn; In each hole, add substrate buffer solution and each 50 μ l of substrate, pat mixing, secretly put 15min for 37 ℃; Every hole adds stop buffer 50 μ l, mixing; Measure the OD value (A value) in each hole under the dual wavelength 450/630nm condition, sample OD value/(2.1 * negative control hole OD value) 〉=1 are positive.
4, the effect analysis of chitosan-hepatitis b immune RNA new formulation
After immune RNA composite RNA enzymatic degradation protection test result showed that (referring to Fig. 1): RNA and chitosan form complex, chitosan-immune RNA composite was arrested in the well and is not degraded.As shown in Figure 1: A is the RNA blank that extracts, and B is RNA+RNase effect 15min, can see that RNA substantially has been degraded; C, D, E, F are because chitosan and RNA have formed complex, and chitosan plays retardation to RNA, and RNA all is arrested in the well; and be protected, not by the degraded of RNase, the contrast of four holes; the concentration maximum of Rnase in the F hole, the RNA in chitosan-RNA complex partly is degraded.
Hepatitis B surface antibody HbsAb measurement result shows that the positive rate (referring to table 3) of anti-Hbs antibody positive rate (referring to table 1), leukocyte adhesion inhibition index (referring to table 2) and the splenocyte identification HBsAg of the mice of use chitosan-immune RNA composite all is significantly higher than normal saline group, chitosan group and immune ribonucleic acid group.
Table 1 is anti-HBs antibody (HBsAb) production through chitosan-immune ribonucleic acid polymer treatment group and control group mice, is significantly higher than normal saline group, chitosan group and immune ribonucleic acid group by anti-HBs antibody (HBsAb) positive rate of the visible chitosan of table 1-immune RNA composite group mice.Show that chitosan-immune RNA composite can effectively protect immune ribonucleic acid to avoid degraded, and the remarkable activity of enhance immunity RNA.
The antibody production of table 1, four groups of mices
| Group | n | Anti-HBs antibody (HBsAb) positive rate (%) |
| Normal saline group chitosan group immune ribonucleic acid group chitosan-immune RNA composite group | 20 20 20 20 | 0 0 15 75 * |
*Compare P＜0.01. with the immune ribonucleic acid group
Table 2 is the leucocyte adherence inhibition assay result through chitosan-immune ribonucleic acid polymer treatment group and control group mice, is significantly higher than normal saline group, chitosan group and immune ribonucleic acid group by the leukocyte adhesion inhibition index of the visible chitosan of table 2-immune RNA composite group mice.Show that chitosan-immune RNA composite can effectively protect immune ribonucleic acid to avoid degraded, and the remarkable activity of enhance immunity RNA.
The leukocyte adhesion inhibition index of table 2, mice relatively
| Group | n | LAI |
| Normal saline group chitosan group immune ribonucleic acid group chitosan-immune RNA composite group | 20 20 20 20 | 0.21±0.24 4.99±2.33 12.95±6.13 35.79±12.33 * |
*Compare P＜0.01 with the immune ribonucleic acid group.
Table 3 is through chitosan-immune ribonucleic acid polymer treatment group and control group mice splenocyte specific bond HBsAg situation, is significantly higher than normal saline group, chitosan group and immune ribonucleic acid group by the positive rate of the visible chitosan of table 3-immune RNA composite group mouse boosting cell specific bond HBsAg.Show that chitosan-immune RNA composite can effectively protect immune ribonucleic acid to avoid degraded, and the remarkable activity of enhance immunity RNA.
The ELISA testing result of table 3, mouse boosting cell specific bond HBsAg
| Group | n | Positive rate (%) |
| Normal saline group chitosan group immune ribonucleic acid group chitosan-immune RNA composite group | 20 20 20 20 | 0 0 5 90 * |
*Compare P＜0.01 with the immune ribonucleic acid group.
Embodiment 2
The preparation and the effect analysis of chitosan-tumour immunity RNA new formulation
1, the preparation of chitosan-tumour immunity RNA new formulation
(1) chitosan is dissolved in 1% acetum and obtains 0.5% chitosan solution, stirred 24 hours, the ammonia of adding 1%, make dissolved chitosan precipitation, filter, the vacuum lyophilization precipitation is dissolved in 1% acetum again with gained chitosan after the drying and obtains 0.5% chitosan solution, transfer pH to 5.5, aseptic filtration with sodium hydroxide;
(2) the 0.5mg immune ribonucleic acid is dissolved in 5% the metabisulfite solution of 50ml, immune ribonucleic acid solution is dropwise joined in 0.5% chitosan solution, stir simultaneously with 500 rev/mins speed, after treating that whole immune ribonucleic acids all drip, stirred 1 hour, add 12.5% glutaraldehyde 1ml again, continue then to stir 1 hour, with centrifugal 10 minutes of mixed solution 12000rpm to collect chitosan-immune RNA composite;
(3) add distilled water washing chitosan-immune RNA composite, 12000rpm repeated 3 times to collect chitosan-immune RNA composite in centrifugal 10 minutes;
(4) vacuum lyophilization obtains the freeze dried powder of chitosan-immune ribonucleic acid.
2. mouse test
Get 40 of healthy mices, body weight 28～34g, female, eight of the freeze dried powders of chitosan-immune ribonucleic acid of preparation are dissolved in the distilled water of 20ml, in oxter or the groin subcutaneous injection of mice, every injection 0.5ml, 1 time weekly, totally 7 times, duplicate injection is 2 times after 30 days.Also set up normal saline group (injecting normal saline 0.5ml), chitosan group (injecting 0.02% chitosan 0.5ml), immune ribonucleic acid group (injecting immune RNA solution 0.5ml simultaneously, contain immune ribonucleic acid 0.5mg) in contrast, every group of 40 white mice, the injection site is identical with the time.Last injection after 10 days every group get that leukocyte adhesions suppress (LAI) test, cytotoxic t cell activities test in 20 anti-tumour antibodies that carry out mice respectively, the body.20 every groin subcutaneous vaccination 0.1ml H22 tumor cell (1 * 10 in addition
5Individual), treat to carry out the survival rate determination test after solid tumor occurs.
(1) anti-tumour antibody: from mouse tail blood sampling 0.5ml, treat separation of serum behind the blood coagulation, measure the intravital anti-tumour antibody of mice, concrete grammar: separate the H22 cellular antigens with the salt precipitation method, the bag quilt is as on 96 orifice plates, and every hole adds mice serum 50 μ l, and negative control is set.Hatched 30 minutes for 37 ℃, discard liquid in the hole, wash 5 times, add horseradish peroxidase-labeled goat anti-mouse igg two and resist, hatch half an hour for 37 ℃, discard liquid in the hole, wash 5 times, every hole adds substrate buffer solution 50 μ l, add substrate 50 μ l again, pat mixing, 37 ℃ were reacted 15 minutes.Under dual wavelength 450nm/630nm condition, read each hole OD value.Sample OD value 〉=(the average OD value of 0.12+ negative control) is antibody positive.
(2) leukocyte adhesion suppresses (LAI) test:
Take off cervical vertebra and put to death mice, put into 75% ethanol and soak 5min, the preparation splenocyte suspension, concrete grammar is as follows: take out the back right arm reclining, sterilization left dorsal abdomen intersection skin is with the aseptic taking-up mouse spleen of shears, flush away bloodstain in the incomplete culture fluid of the RPMI-1640 that does not contain serum, cut off fat and fascia, place the aseptic little plate that is lined with 200 order nylon wires; Glass syringe inner core crushing spleen with aseptic adds 1640 liquid washing and filtering, and the cell suspension under the collecting net is gone in the aseptic centrifuge tube, with incomplete culture fluid washing of the RPMI-1640 that does not contain serum 2 times and suspension again; Separate mononuclearcell: the above-mentioned splenocyte suspension that has prepared slowly places on the lymphocyte separation medium (proportion 1.080) that has prepared, the centrifugal 20min of 2000r/min, draw the nebulous low-density cell of the second layer, the incomplete culture fluid 1000r/min of RPMI-1640 that reuse does not contain serum washs 2 times; Regulate cell concentration to 5 * 10
6Individual/ml.
Get 96 orifice plates, set up test hole and control wells (5 parallel holes are established in each hole) respectively, press following application of sample: experimental port adds mouse boosting cell suspension 100 μ l, tumor-cell antigen 50 μ l, 10% calf serum RPMI-1640 liquid, 50 μ l: control wells adds mouse boosting cell suspension 100 μ l, 10% calf serum RPMI-1640 liquid, 100 μ l.5%CO2,37 ℃ of cultivation 2h, supernatant discarded gently, in each hole, add incomplete culture fluid of RPMI-1640 and the 20 μ lMTT solution that 180 μ l do not contain calf serum, cultivate 4h again, abandon supernatant, every hole adds 200 μ lDMSO again, and vibration evenly, room temperature is placed 15min, and microplate reader is surveyed the optical density value (A value) at wavelength 490nm.Calculate adhesion inhibition index (LAI value) by following formula:
(3) cytotoxic T cell (CTL) activity test is further purified above-mentioned isolating mice mononuclearcell through the nylon cotton column absorption method, isolates the T cell.In 96 orifice plates, add 2 * 10 of 50 μ l complete culture solutions preparation
6Individual/ml CTL suspension; Add 50 μ l2 * 10
6The H22 cell adds 40 μ l complete culture solutions and 10ul1%Triton X-100 and always discharges control wells in enzyme in detecting the hole, and the blank hole adds 50 μ l complete culture solutions: put 37 ℃ and cultivate 4h; 4 ℃, the centrifugal 5min of 1100r/min draws 50 μ l culture supernatant and changes in the test tube night, adds the freshly prepared BLT substrate of 950 μ l (20mmol/L N-α benzene oxygen hydroxyl-L-lysine thio phenyl methyl ester), 37 ℃ of water-bath 20min.Then test tube is put ice bath immediately, and add PMSF (Phenylmethanesulfonyl fluoride) the 10 μ l of 0.1mol/L immediately, add 1.0ml PBS and make final concentration reach 0.05mol/L, survey each hole OD value down in the 412nm wavelength, and be calculated as follows granule secretion of esterase percentage rate.
Granule secretion of esterase percentage rate (%)=100 * (E-B) * (T-B)
E is the inductive CTL cell conditioned medium of a target cell liquid average light absorption value in the formula, and B represents background or the average light absorption value of untreated CTL cell hole; T represents the total burst size of enzyme (i.e. the CTL cell conditioned medium of handling with Triton X-100).
(4) after survival rate is tested four groups of mices and injected chitosan-immune ribonucleic acid as previously mentioned, get 20 groin subcutaneous vaccination 0.1ml H22 tumor cells (1 * 10 for every group
5Individual), measure 30 days survival rates of every group.
3, the effect analysis of chitosan-tumour immunity RNA new formulation
Experimental result shows that anti-Hbs antibody positive rate (referring to table 4), leukocyte adhesion inhibition index (referring to table 5) and the cytotoxic t cell activity (referring to table 6) of the mice of use chitosan-immune RNA composite all are significantly higher than normal saline group, chitosan group and immune ribonucleic acid group.30 days survival rates behind the mouse inoculation H22 tumor cell of use chitosan-immune RNA composite also are significantly higher than its excess-three group matched group (referring to table 7)
Table 4 is the anti-tumour antibody production through chitosan-immune ribonucleic acid polymer treatment group and control group mice, is significantly higher than normal saline group, chitosan group and immune ribonucleic acid group by the anti-tumour antibody positive rate of the visible chitosan of table 4-immune RNA composite group mice.Show that chitosan-immune RNA composite can effectively protect immune ribonucleic acid to avoid degraded, and the remarkable activity of enhance immunity RNA.
The antibody production of table 4, four groups of mices
| Group | n | Anti-tumour antibody positive rate (%) |
| Normal saline group chitosan group immune ribonucleic acid group chitosan-immune RNA composite group | 20 20 20 20 | 0 0 10 80 * |
*Compare P＜0.01. with the immune ribonucleic acid group
Table 5 is the leucocyte adherence inhibition assay result through chitosan-immune ribonucleic acid polymer treatment group and control group mice, is significantly higher than normal saline group, chitosan group and immune ribonucleic acid group by the leukocyte adhesion inhibition index of the visible chitosan of table 5-immune RNA composite group mice.Show that chitosan-immune RNA composite can effectively protect immune ribonucleic acid to avoid degraded, and the remarkable activity of enhance immunity RNA.
The leukocyte adhesion inhibition index of table 5, mice relatively
| Group | n | LAl |
| Normal saline group chitosan group immune ribonucleic acid group chitosan-immune RNA composite group | 20 20 20 20 | 0.67±1.24 4.43±2.17 15.28±7.45 37.85±13.49 * |
*Compare P＜0.05 with the immune ribonucleic acid group.
Table 6 is through chitosan-immune ribonucleic acid polymer treatment group and matched group CTL activity, is significantly higher than normal saline group, chitosan group and immune ribonucleic acid group by the activity of the visible chitosan of table 6-immune RNA composite group mice CTL.Show that chitosan-immune RNA composite can effectively protect immune ribonucleic acid to avoid degraded, and the remarkable activity of enhance immunity RNA.
Table 6, the active testing result of CTL
| Group | n | The CTL activity |
| Normal saline group chitosan group immune ribonucleic acid group chitosan-immune RNA composite group | 20 20 20 20 | 12.5％ 24.8％ 36.1％ 83.4％ * |
*Compare P＜0.05 with the immune ribonucleic acid group.
Table 7 is 30 days survival rates behind chitosan-immune ribonucleic acid polymer treatment group and control group mice inoculation H22 tumor cell.Survival rate by the visible chitosan of table 7-immune RNA composite group mice is significantly higher than normal saline group, chitosan group and immune ribonucleic acid group.Show that chitosan-immune RNA composite can effectively protect immune ribonucleic acid to avoid degraded, and the remarkable activity of enhance immunity RNA.
30 days survival rates of table 7 mice relatively
| Group | n | 30 days survival rates | 30 days survival rates |
| Normal saline group chitosan group immune ribonucleic acid group chitosan-immune RNA composite group | 20 20 20 20 | 10％ 15％ 15％ 70％ * | 0 5％ 5％ 30％ |
*Compare P＜0.05 with the immune ribonucleic acid group.
Need to prove: to those of ordinary skill in the art, can also make some changes or distortion to the present invention under the prerequisite that does not change the principle of the invention, this belongs to protection scope of the present invention equally.
Claims (2)
1. chitosan-immune RNA composite formulation, it mainly comprises chitosan and the formed complex of immune ribonucleic acid, described compound particles diameter is between 100 nanometers～100 micron, and described immune ribonucleic acid is resistance of hepatitis B immune ribonucleic acid or antineoplastic immune RNA.
2. the preparation method of chitosan-immune RNA composite formulation as claimed in claim 1 is characterized in which comprises at least following steps:
A. chitosan is dissolved in 1% to 5% acetum and obtains 0.5% chitosan solution, sodium hydroxide is transferred pH to 5.5, aseptic filtration;
B. immune ribonucleic acid is dissolved in 5% to 20% the metabisulfite solution, immune ribonucleic acid concentration is 10-20 μ g/ml;
C. immune ribonucleic acid solution is dropwise joined in 0.5% chitosan solution, stir simultaneously with 500 rev/mins speed, treat that whole immune ribonucleic acids all drip after, stirred 1 hour, be the glutaraldehyde of 1% adding concentration 12.5% by volume, continue then to stir 1 hour;
D. with centrifugal 10 minutes of mixed solution 12000rpm to collect chitosan-immune RNA composite;
E. wash chitosan-immune RNA composite with distilled water, centrifugal collection chitosan-immune RNA composite repeats 3 times;
F. lyophilisation obtains the freeze dried powder of chitosan-immune RNA.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1175397A (en) * | 1996-09-03 | 1998-03-11 | 深圳市龙岗区新业有限公司 | Effervescent tablet for preparing gargle |
| JP2004166685A (en) * | 2002-11-19 | 2004-06-17 | Coletica | Method for identifying change produced in at least one biological parameter as result by using living cell under stress and living cell freed from stress |
| US20040228884A1 (en) * | 2003-05-15 | 2004-11-18 | Gupta Shyam K. | Ion-pair delivery system for cosmetic and pharmaceutical compositions |
| CN1548056A (en) * | 2003-05-08 | 2004-11-24 | 天津市金士力药物研究开发有限公司 | Application of chitin, chitosan and their derivatives in preparing antiviral |
| CN1786163A (en) * | 2004-12-07 | 2006-06-14 | 广州暨南生物医药研究开发基地有限公司 | Method of using bio material as siRNA trans dyeing carrier |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1175397A (en) * | 1996-09-03 | 1998-03-11 | 深圳市龙岗区新业有限公司 | Effervescent tablet for preparing gargle |
| JP2004166685A (en) * | 2002-11-19 | 2004-06-17 | Coletica | Method for identifying change produced in at least one biological parameter as result by using living cell under stress and living cell freed from stress |
| CN1548056A (en) * | 2003-05-08 | 2004-11-24 | 天津市金士力药物研究开发有限公司 | Application of chitin, chitosan and their derivatives in preparing antiviral |
| US20040228884A1 (en) * | 2003-05-15 | 2004-11-18 | Gupta Shyam K. | Ion-pair delivery system for cosmetic and pharmaceutical compositions |
| CN1786163A (en) * | 2004-12-07 | 2006-06-14 | 广州暨南生物医药研究开发基地有限公司 | Method of using bio material as siRNA trans dyeing carrier |
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