CN101229362A - Use of thymulin on preparing protective medicine of antineoplastic agent, tumour physiatry and chemotherapeutic medicine - Google Patents
Use of thymulin on preparing protective medicine of antineoplastic agent, tumour physiatry and chemotherapeutic medicine Download PDFInfo
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Abstract
The invention discloses applications of serum thymus factor (FTS) in preparation of anti-tumor medicines, protective medicine or immunity intensified medicines for physical treatment and chemical treatment of tumor. Experimental results reveal that all groups of tumor-bearing mice and W256 rat fed with medicines are significantly different from groups fed with physiological saline and the tumor inhibition rates of all the groups fed with the medicines are more than 30 percent, thus confirming the tumor inhibition effect of the FTS. The FTS can also enhance the immunity activity and strengthen the immunity function of living organisms in a plurality of aspects; the FTS has no toxicity and less side effects; the effective wide dose scope and wide safety range. The FTS has obvious protective function to immunity indexes of living organisms, which is discovered in the study of the protective medicines for the physical and chemical treatment medicines of tumor. Therefore, the FTS can provide a new target and development for the clinical treatment of tumor.
Description
Technical field
The present invention relates to the new purposes of a kind of biochemical in pharmaceutical engineering, more particularly serum thymic factor is in the protection medicine of preparation antitumor drug, tumor physics and chemotherapeutic agent or the purposes of enhance immunity prescription face.
Background technology
Cancer is to cause one of human main causes of death.According to World Health Organization (WHO) statistics, the patient that cancer is died from the whole world every year has 5,000,000 approximately, and recent years because the deterioration of our living environment, the sickness rate of cancer rises, and presents the trend that the patient becomes younger.As seen the prevention of cancer and treatment are very urgent.Drug therapy is one of main treatment means of cancer.Since the nearly century, the Drug therapy of cancer has obtained important achievement, has developed tens kinds of antitumor drug, has prolonged patient's life effectively or has improved patient's life quality.The medication effect highly significant of some tumor wherein is as Drug therapy acute leukemia of children etc.Adopt rational administering mode or combine, can improve the therapeutic effect of medicine usually with other treatment means.But the drug research of tumor and exploitation still face great challenge, more single as present tumour medicine kind, majority is a cell toxicity medicament, can kill the tumor cell of various growth cycles, but its side effect also clearly, reason is that these medicines in the kill tumor cell, have also limited the performance of these curative effect of medication.In addition, to most solid tumors such as pulmonary carcinoma, hepatocarcinoma and cancer of pancreas etc., the therapeutic effect of chemicals is often not obvious, and up to the present, most of chemotherapeutics are " targeting " treatment tumor with a definite target in view, often when killing and wounding cancerous cell, normal histiocyte is exerted an influence and damages, so when producing therapeutical effect, often with in various degree untoward reaction.The drug resistance problem that also has antitumor drug simultaneously.The solution of above-mentioned these problems will depend on the further investigation of oncomolecularbiology on the one hand, also will therefrom develop the new type antineoplastic medicine of high-efficiency low-toxicity by further research to find more to have in a large number the medicine of anti-tumor activity on the other hand.
Modernization along with science and technology and people's life, environmental pollution brings increasing adverse effect for people's health, especially in the Clinics and Practices process of disease, as in tumor radiotherapy, chemotherapy process, some drugs has immeasurable radiation effects to the mankind.Also some has the Job body of encroaching on people all at all times of radioactivity, is damaging human beings'health.Therefore, the research and development antiradiation drug has been subjected to people's very big attention.
Radiation and chemotherapy is the main mode of treatment tumor, but all has significant side effects.As cyclophosphamide (CY) is a kind of commonly used and effective chemotherapeutics that is used for the treatment of malignant tumor and autoimmune disease clinically, but its toxic and side effects is bigger, particularly suppress bone marrow hematogenesis, cause erythrocyte in the peripheral blood, leukocyte and thrombocytopenia, and significantly gastrointestinal reaction makes the decline of patient's appetite, hypoimmunity.Because these toxic and side effects all are restricted the consumption of CY and the course of treatment.In order to alleviate the toxic and side effects of chemotherapeutics, some drug for increasing white cells of Chang Fuyong clinically, as leucogen, creatinine etc., but effect is undesirable.
Thymus is the central immune organ that body weight for humans is wanted, and is the major organs that the T cell maturation is grown, and is necessary for keeping and developing normal immunne response and earn a bare living.It can secrete multiple thymosin, and (facteur thymic serique FTS) is exactly the excretory nonapeptide of its epithelial cell to serum thymic factor.FTS is made up of nine aminoacid Glu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn.
FTS at first by JF Bach etc. from the Sanguis sus domestica purification, since delivering on the Nature, caused extensive concern in 1977.Contain equimolar zinc ion in the natural FTS molecule, then lose if its biologic activity is removed in its complexation.Compare with natural FTS, the FTS of synthetic has identical biologic activity.East China University of Science's journal the 31st volume the 6th phase 727-730 page or leaf disclose utilize chemical method synthesize serum thymic factor (FTS) and to the synthetic serum thymic factor content of having carried out determination of activity.
It is reported, autoimmune disease such as systemic lupus erythematosus (sle), rheumatoid arthritis, multiple sclerosis etc., the FTS secretion of its thymus reduces; FTS content among the acquired immune deficiency syndrome (AIDS) patients serum also reduces.What relation FTS and these sick causes of disease have still indeterminate, still give FTS treatment back patient's the immunologic hypofunction and the state of immunologic derangement and all can correct.As if FTS can both the generation effect to the panimmunity cell.In thymus, FTS plays an important role to the growth of birds and mammiferous T cell precursors, there are some researches prove that FTS can regulate birds CD4
+: CD8
+Ratio.FTS also can increase the vigor of natural killer cell.In the chicken model, FTS can increase the expression of IL-2R.FTS also is suggested influences people IFN-γ expression of receptor.But, up to the present, yet there are no patent and the research report of FTS both at home and abroad as the application of antitumor and enhance immunity prescription face.
Summary of the invention
The object of the present invention is to provide serum thymic factor in the protection medicine of preparation antitumor drug, tumor naturopathy and chemotherapeutic agent or the purposes aspect the enhance immunity medicine.
The serum thymic factor that pharmacological experiment adopts among the present invention can be naturally extracted from animal blood, also can be by the chemical synthesis process synthetic.
Used material and test method in the pharmacological experiment test of the present invention:
One, material
1, experimental animal:
Experimental animal among the present invention is experiment kunming mice, body weight 20-22g; The Wistar rat, body weight 100-120g is the SPF level, and male female half and half.The animal quality certification number: the capital is moving is betrothed to (2000) No. 017, is provided by Nat'l Pharmaceutical ﹠ Biological Products Control Institute's Experimental Animal Center.Laboratory animal room is the filtrated air cascade filtration, and man-day's light modulation 12h throws light on automatically, constant temperature and humidity, temperature 22-24 ℃, relative humidity 40%-50%.The mice with tumor kind is provided by institute of Materia Medica,Chinese Academy of Medical Sciences.People's hepatocarcinoma Heps tumor is available from the medicine institute that coordinates in the Chinese Academy of Medical Sciences.Nude mice is provided by Nat'l Pharmaceutical ﹠ Biological Products Control Institute's animal breeding field, cleaning level, and the quality certification number is: SCXK capital (2005-2004).
2, main agents
Serum thymic factor among the present invention (FTS) is provided by China Research Centre of Medicine Biological Products Standardization, it is the serum thymic factor (FTS) of synthetic, synthetic method is seen East China University of Science's journal the 31st volume the 6th phase 727-730 page or leaf, is white lyophilized powder, with preceding pure water dilution; Cyclophosphamide is provided by SHANXI POWERDONE PHARMACEUTICAL.,LTD, lot number 14023686; 5-fluorouracil is provided by Shanghai Xudong Hipu Medicine Co., Ltd, lot number H31020593; India ink is sincerely believed in meticulous preparation factory by Beijing to be provided; The RPMI-1640 culture fluid is provided by the prosperous grand technology company limited of Beijing astronomical phenomena.
Two, method
1, dosage setting
According to the beneficial effect curve result high, normal, basic three dosage groups are set
Mice: high dose 0.25mgkg
-1, middle dosage 0.125mgkg
-1, low dosage 0.0625mgkg
-1The positive control 5-fluorouracil dosage 25mgkg of EAC ascites tumor
-1, H22 solid tumor positive control drug cyclophosphamide dosage 20mgkg
-1The administration volume is 10mlkg
-1The blank normal saline.
Rat: high dose 2.2mgkg
-1, middle dosage 1.1mgkg
-1, low dosage 0.55mgkg
-1Rat w256 solid tumor positive control drug is cyclophosphamide 89.2mg.kg
-1, the administration volume is 2mlkg
-1The blank normal saline.
Nude mice: high dose 0.125mgkg
-1Middle dosage 0.0625mgkg
-1Low dosage 0.032mgkg
-1Positive controls: cyclophosphamide 10mgkg
-1, only being administered once, the administration volume is 0.1ml10g
-1The blank normal saline.
2, animal model preparation
Ascites tumor: get the 7th day the EAC tumor in abdominal cavity that is inoculated in mice and be made into tumor cell suspension and be inoculated in allogeneic Mus abdominal cavity, every Mus inoculation contains the cell suspension 0.4ml of 106 oncocytes.
Solid tumor:<1〉mice: getting the 7th day the H22 tumor in abdominal cavity that is inoculated in mice, to be made into cell suspension inoculation subcutaneous in allogeneic Mus forelimb axillary fossa, and every Mus inoculation contains the cell suspension 0.2ml of 106 oncocytes;<2〉get and be inoculated in the 7th day Walker-256 tumor of rat abdominal cavity and be made into tumor cell suspension and be inoculated in allogeneic rat right side leg muscle, every Mus inoculation contains the cell suspension 0.2ml of 106 oncocytes.
People's hepatocarcinoma Heps tumor: available from Chinese Academy of Medical Sciences consonance medicine institute, getting the tumor oxter, to be inoculated in nude mice forelimb axillary fossa subcutaneous, and the inoculation back is observed and survived the back administration and experimentize.Administration is 17 days altogether, and dissection in second day claims that tumor is heavy, measures line of apsides volume calculated after the last administration.
3, immunologic function test
With lymphocyte transformation test (mtt assay): experiment in vitro finishes mouse orbit blood-letting next day in administration and causes death the aseptic spleen of getting.Make 3 * 10 with the RPMI1640 culture fluid
6-8 * 10
6The lymphocyte suspension of/mL is standby.Cultivate lymphocyte with 96 well culture plates, every hole contains standby lymphocyte liquid 100 μ L, every kind of concentration 3 multiple holes.Except that the blank hole, add sample by test requirements document respectively, sample+zinc each 100 μ L and ConA10ug/50 μ L.Lymphocyte is at 37 ℃ of CO
2Hatch 72h in the incubator, stop hatching preceding 4h, every hole adds MTT20ul, 5mg/mL.Cell culture adds dimethyl sulfoxide 200 μ L after finishing, and treats that purple crystal all dissolves the back and surveys the OD value at the 570nm place with enzyme-linked immunosorbent assay instrument.
The test of mice carbon clearance: get last administration mouse tail vein injection next day and dilute good indian ink juice 0.1ml/10g, the vena ophthalmica clump blood of adopting 5min and 10min respectively adds to 0.1%Na
2CO
32ml, reuse 721 type spectrophotometers survey the OD value at the 600nm place.
4, test rating
Solid tumor:
Increase in life span and tumour inhibiting rate all need clear and definite greater than 30% antitumor action;
Stimulation index SI=experimental group OD value/matched group OD value;
Clean up index K=(logOD
1-logOD
2)/(t
2-t
1)
OD wherein
1And OD
2Be respectively t at a time
1With t
2Measured optical density value;
The computing formula of gross tumor volume is: V=(L * W
2)/2
The minor axis of the major diameter W----tumor of L----tumor wherein
Serum thymic factor of the present invention is as the protection medicine of preparation antitumor drug, tumor physics and chemotherapeutic agent or the purposes of enhance immunity prescription face.The biological response modifier series antineoplastic medicament itself has the antiproliferative effect except that the minority kind, reaches the anticancer growth by enhancing body's immunological function mostly and kills cancerous cell.The visible FTS of animal vivo test result is that significantly high, middle dosage all reaches good tumor killing effect to the EAC mice to the tumor inhibition effect of described four kinds of mice with tumor among the present invention, and significant difference has been analysed in the matched group credit that takes statistics.With FTS during as anti-human liver cancer Heps tumor medicine, high, medium and low dosage is all obvious to people's hepatocarcinoma Heps tumor mice tumor-inhibiting action among the present invention, and significant difference has been analysed in the matched group credit that takes statistics.Therefore the FTS antitumor action is certain.FTS can also many-sided immunocompetence that improves body, the enhancing human body immunity function.And FTS itself is not a cell toxicant class antineoplastic agent; acute toxicity test shows; FTS is nontoxic; and the side effect that produces is very little; and its useful effect dosage range is very wide; safety range is bigger; in research, find as the protection medicine of tumor physics and chemotherapeutic agent; FTS has significant protective effect to the immune indexes of raying mice; total white blood cells and phagocytic activity all are improved; thymus index also increases, and under higher dose (7.0Gy), FTS has also improved survival rate and the mean survival time of mice.Therefore FTS can provide a new targeting and developing direction for treating tumor clinically.
Description of drawings
Fig. 1 FTS is to the influence of T cell propagation effect stimulation index
The concentration of Fig. 2 FTS is to the influence of phagocytic index
Fig. 3 FTS is to the influence (6.0Gy) of raying mice survival rate
Fig. 4 FTS is to the influence (7.0Gy) of raying mice survival rate
Fig. 5 FTS is to the influence (6.0Gy) of raying mice time-to-live
Fig. 6 FTS is to the influence (7.0Gy) of raying mice time-to-live
Fig. 7 FTS is to the influence (1 is blank, and 2 is FTS5 μ g/, and 3 is FTS50 μ g/) of tumor-bearing mice chemotherapy survival rate
Fig. 8 FTS is to the influence (1 is blank, and 2 is FTS5 μ g/, and 3 is FTS50 μ g/) of tumor-bearing mice chemotherapy time-to-live
The specific embodiment
Below in conjunction with specific embodiments the present invention is made a more detailed description, should be pointed out that all embodiment that list at this only are illustrative, and do not mean that the scope of the invention is limited.
Embodiment one
Present embodiment relates to EAC ascites tumor group result
The subcutaneous injection administration, the 0th day inoculation animal checked the cultivation situation on the 1st day, as pollution-free, then animal being weighed also, random packet begins administration.The tumor model mice is divided into 5 groups at random, is respectively high dose group, middle dosage group, low dose group, positive controls and blank group, and dosage is respectively high dose 0.25mgkg
-1, middle dosage 0.125mgkg
-1, low dosage 0.0625mgkg
-1, be administered once every day except that the positive drug group, continuous 14 days; Positive drug group 5-fluorouracil, the next day once, totally three times.The result is as shown in table 1:
The table 1FTS be used for anti-EAC ascites tumor medicine the result (x ± s, n=10)
Group | Life span (my god) | Increase in life span |
Dosage low dosage positive control blank in the high dose | 18.3±3.6 △△ 17.9±3.1 △△ 16.9±3.3 △△ 24.3±4.1 △△△ 13.3±2.5 | 37.6% 34.6% 27.1% 82.7% |
△ △P<0.01,
△ △ △P<0.001 and blank group contrast; Increase in life span and blank group contrast.
The result of table 1 shows and observes survival condition after the last administration: high dose group, two groups of increase in life span of middle dosage group are all greater than 30%, and certain dose-effect relationship is arranged, and curative effect determines that administration group and matched group all have significant difference (P<0.01).Illustrate that FTS effect when the purposes of the anti-EAC ascites tumor medicine of conduct preparation is remarkable.
Embodiment two
Present embodiment relates to H22 solid tumor group result
The subcutaneous injection administration, the 0th day inoculation animal checked the cultivation situation on the 1st day, as pollution-free, then animal being weighed also, random packet begins administration.The tumor model mice is divided into 5 groups at random, is respectively high dose group, middle dosage group, low dose group, positive controls and blank group, and dosage is respectively high dose 0.25mgkg
-1, middle dosage 0.125mgkg
-1, low dosage 0.0625mgkg
-1, be administered once every day except that the positive drug group, continuous 14 days; The disposable administration of cyclophosphamide.The results are shown in Table 2.
The table 2FTS be used for anti-H22 solid tumor medicine the result (x ± s, n=10)
Group | The weight of solid tumor (g) | Tumour inhibiting rate |
Dosage low dosage positive control blank in the high dose | 2.5±0.9 △△ 2.7±0.7 △△ 3.1±1.4 0.6±0.5 △△△ 4.2±1.0 | 40.7% 31.2% 26.8% 84.8% |
△ △P<0.01,
△ △ △P<0.001 and blank group contrast; Tumour inhibiting rate contrasts with blank group.
The presentation of results of table 2: the administration high dose group, middle dosage group tumour inhibiting rate is greater than 30% and a certain amount of effect relationship is arranged, and curative effect is clear and definite.High dose group, middle dosage group and matched group have significant difference (P<0.01).The increase of high dose group and middle dosage group object tumor weight is lacked than the increase of low dose group and blank group weight.Illustrate that FTS effect when the purposes of the anti-H22 solid tumor medicine of conduct preparation is remarkable.
Embodiment three
Present embodiment relates to Walker-256 tumor group result
The subcutaneous injection administration, the 0th day inoculation animal checked the cultivation situation on the 1st day, as pollution-free, then animal being weighed also, random packet begins administration.The tumor model rat is divided into 5 groups at random, is respectively high dose group, middle dosage group, low dose group, positive controls and blank group, and dosage is respectively high dose 2.2mgkg
-1, middle dosage 1.1mgkg
-1, low dosage 0.55mgkg
-1, be administered once every day except that the positive drug group, continuous 14 days; The disposable administration of cyclophosphamide.The results are shown in Table 3.
Dissection taking-up next day solid tumor is weighed after the administration of administration group last, calculates tumour inhibiting rate.Administration group tumour inhibiting rate is all greater than 30% and a certain amount of effect relationship is arranged, and curative effect is clear and definite.Administration group and matched group all have significant difference (P<0.001).The results are shown in Table 3.
The table 3FTS be used for anti-Walker-256 tumor effect (x ± s, n=10)
Group | The weight of tumor (g) | Tumour inhibiting rate (%) |
Dosage low dosage positive control blank in the high dose | 6.2±2.7 △△△ 9.2±2.5 △△△ 11.2±4.1 △△△ 2.6±1.7 △△△ 17.8±3.8 | 65.2% 48.4% 37.1% 85.4% |
△ △ △P<0.001 and blank group contrast; Tumour inhibiting rate contrasts with blank group.
The presentation of results of table 3: administration group tumour inhibiting rate is all greater than 30% and a certain amount of effect relationship is arranged, and curative effect is clear and definite, and administration group and matched group all have significant difference (P<0.001); The weight relationships of the tumor that high dose group, middle dosage group and low dose group record is: high dose group<middle dosage group<low dose group<blank group.Illustrate that FTS effect when the purposes of the anti-Walker-256 tumor medicine of conduct preparation is remarkable.
Embodiment four
Present embodiment relates to people's hepatocarcinoma Heps tumor group result
The subcutaneous injection administration, the 0th day inoculation animal checked the cultivation situation on the 1st day, as pollution-free, then animal being weighed also, random packet begins administration.The tumor model rat is divided into 5 groups at random, is respectively high dose group, middle dosage group, low dose group, positive controls and blank group, and dosage is respectively high dose 0.125mgkg
-1, middle dosage 0.0625mgkg
-1, low dosage 0.032mgkg
-1, be administered once every day except that the positive drug group, and administration is 17 days altogether; The disposable administration of cyclophosphamide, dosage are 10mgkg
-1The administration volume is 10mL/kg.The results are shown in Table 4.
The table 4FTS be used for anti-human liver cancer Heps tumor effect (x ± s, n=10)
Group | The weight of tumor (g) | Tumour inhibiting rate | Tumor size (mm 3) |
Dosage low dosage positive control blank in the high dose | 0.72±0.23 *** 0.90±0.19 ** 0.99±0.24 ** 0.57±0.12 *** 1.51±0.52 | 52.28% 40.36% 34.19% 62.43% | 287.52±192.56 *** 519.61±301.00 ** 660.83±448.81 * 99.73±68.60 *** 1224.08±701.42 |
*P<0.05,
*P<0.01,
* *P<0.001 and blank group contrast
The presentation of results of table 4: dissection taking-up next day solid tumor is weighed after the administration of administration group last, calculates tumour inhibiting rate.Result of the test shows, administration group tumour inhibiting rate is all greater than 30% and a certain amount of effect relationship is arranged, and curative effect is clear and definite.Administration group and matched group all have significant difference (P<0.001).The weight magnitude relationship of the tumor that high dose group, middle dosage group and low dose group record is: high dose group<middle dosage group<low dose group<blank group.The tumor size magnitude relationship that high dose group, middle dosage group and low dose group record is: high dose group<middle dosage group<low dose group<blank group.
Embodiment five
Present embodiment relates to the influence of FTS to kunming mice spleen lymphocyte proliferation effect
Experiment in vitro finishes mouse orbit blood-letting next day in administration and causes death the aseptic spleen of getting.Make 3 * 10 with the RPMI1640 culture fluid
6-8 * 10
6The lymphocyte suspension of/mL is standby.Cultivate lymphocyte with 96 well culture plates, every hole contains standby lymphocyte liquid 100 μ L, every kind of concentration 3 multiple holes.Except that the blank hole, add sample by test requirements document respectively, sample+zinc each 100 μ L and ConA10ug/50 μ L.Lymphocyte is at 37 ℃ of CO
2Hatch 72h in the incubator, stop hatching preceding 4h, every hole adds MTT20ul, 5mg/mL.Cell culture adds dimethyl sulfoxide 200 μ L after finishing, and treats that purple crystal all dissolves the back and surveys the OD value at the 570nm place with enzyme-linked immunosorbent assay instrument.
Table 5FTS to the influence of In vitro culture spleen lymphocyte proliferation effect (x ± s, n=10)
△P<0.05,
△ △P<0.01,
△ △ △P<0.001 compares with blank group; The influence that the ConA stimulating group is arranged is greater than no ConA stimulating group.
By the result of Fig. 1 and table 5 as seen: have or when not having ConA and stimulating, each is organized 0.002~0.008mg/ml concentration and relies on ground and strengthen the T cell propagation effect, and when concentration during greater than 0.016mg/ml facilitation weaken.The FTS+Zn group is all strong than FTS group effect simultaneously, and ConA stimulates back T cell propagation effect obviously to strengthen, and the multiplication effect that shows at the 0.016mg/ml place is the strongest, and both sides are on a declining curve.
Embodiment six
Present embodiment relates to the carbon clearance result of the test
The ability of carbon granule in the clearance in mice blood flow is just represented in this test with phagocytic index.After the standard of dose,optimum was injected in mice, in the 30min, the Cl of control group mice will be starkly lower than by the activated clearance in mice rate of positive drug.Wherein the dosage of high dose group is 2.2mgkg
-1, middle dosage group dosage be 1.1mgkg
-1, low dose group dosage be 0.55mgkg
-1, the results are shown in Figure 2.
The result of Fig. 2 shows: high dose group 2.2mgkg
-1, middle dosage group 1.1mgkg
-1, low dose group 0.55mgkg
-1Three groups of phagocytic index are all apparently higher than the blank group, and wherein high dose group and cyclophosphamide group result are approaching, and phagocytic index is all greater than 5, and the phagocytic index of middle dosage group and low dose group is all greater than 4, and the phagocytic index of blank group is then less than 2.This a series of presentation of results FTS can strengthen the immunity of living organism significantly.
Embodiment seven
Present embodiment relates to the result of FTS acute toxicity test
Get 15 hours kunming mice of fasting, 20/group, male and female half and half, body weight are 22-24g, and the each tail vein injection volume of mice is the 10mL/kg body weight, observes untoward reaction and the death state of animal in 7 days continuously.Reach the body weight of observing when finishing before the record administration.Behind the intravenous administration, observed continuously 4 hours, the mice no abnormality seen does not see that Deviant Behavioies such as central nervous excitation or inhibition appear in mice.Administration mice next day is in high spirits, and behavior is normal, activity freely, the respiratory frequency no change, optoacoustic etc. is stimulated responsive, moist by hair; Food consumption, drinking-water and feces etc. are all normal, do not find any toxic reaction or death, and redness, congestion and necrosis are not found in the tail vein injection part.See table 6 for details.
Table 6FTS The acute toxicity tests
Group | Administration number of times/day | Dosage (mg/kg) | Administration concentration (mg/ml) | Number of mice (only) | Dead (%) | Body weight (g) before the administration | Body weight after the administration (g) |
1 | 1 | 5.0 | 0.5 | 20 | 0 | 22.3±2.1 | 28.3±2.4 |
2 | 1 | 10.0 | 1.0 | 20 | 0 | 23.4±3.1 | 29.7±2.1 |
3 | 1 | 50.0 | 5.0 | 20 | 0 | 23.6±2.5 | 28.6±3.2 |
4 | 1 | 100.0 | 10.0 | 20 | 0 | 23.1±2.8 | 29.8±2.0 |
The result of table 6 shows: administration survival mice average weight on the 7th increases 4-6g, and administration 5.0mg/kg group, 10.0mg/kg group, 50.0mg/kg group, 100.0mg/kg group mice are not all found any toxic reaction or death in the test.It is 4mg/ time/day that the clinical people of FTS intends consumption, and in maximum test dose 280mg/ people/sky, when being equivalent to 70 times of clinical consumption, it is unusual to there is no mice, does not see that Deviant Behavioies such as central nervous excitation or inhibition appear in mice, mice is in high spirits, behavior is normal, activity freely, the respiratory frequency no change, responsive to stimulations such as optoacoustics, moist by hair; Food consumption, drinking-water and feces etc. are all normal, do not find any toxic reaction or death, and redness, congestion and necrosis are not found in the tail vein injection part.Therefore, think that FTS is nontoxic antineoplastic polypeptide.
Embodiment eight
Present embodiment relates to the result of FTS in the purposes of the protection prescription face of conduct preparation tumor physics and chemotherapeutic agent.
Material therefor and instrument
Gentian Violet (the kindness company difficult to understand in Beijing), india ink (Beijing sincerely believe in meticulous preparation factory), cyclophosphamide (Shanxi Tai Sheng pharmaceutical Co. Ltd), serum thymic factor (FTS) is provided by China Research Centre of Medicine Biological Products Standardization.Cobalt source (Beijing Radiomedicine Inst. of institute of section of army), and microscope (CH20BIMF 200, Olympus), spectrophotometer (UV-2550, SHIMADZU).
Used method:
1, numeration of leukocyte
The leukocyte diluent: get glacial acetic acid 1.5mL and 1% Gentian Violet 1mL, mixing, adding distil water is to 100mL.
(1) in small test tube, adds 0.38mL leukocyte diluent;
(2) get blood from the Mus tail,, clean the blood that suction pipe attaches outward with the suction pipe 20 μ L that accurately suck blood.
(3) rapidly blood is put into above-mentioned small test tube gently, and wash suction pipe 2-3 time, shake up with supernatant.
(4) the leukocyte suspension is splashed in the counting chamber, left standstill 2-3 minute, treat to count after leukocyte sinks.
(5) under low power lens, leukocyte is rounded, starch bright, nuclear be atropurpureus, refractive power is arranged slightly, characteristics can be distinguished mutually with impurity whereby.All leukocyte counts in 4 big grids that counting chamber is four jiaos multiply by 50 with sum, promptly get every 1mm
3Leukocyte count in the blood.
Calculate principle: each big grid area is 1mm
3, the counting chamber degree of depth is 0.1mm, so 20 times of hemodilution are every 1mm
3Leukocyte count is leukocyte count * 10 * 20 ÷ 4 of 4 big grids in the blood.
2, carbon clearance method
Operating procedure: body weight 18-22g white mice, male and female half and half are divided into experimental group and matched group at random, give and medicine and solvent respectively.Every caudal vein injection india ink 0.05mL/10g body weight during experiment is in 1 (t
1) and 5 (t
2) minute after, get blood 20 μ L from the eye socket vein respectively, be added to 2ml, 0.1%Na
2CO
3Shake up in the solution, use spectrophotometer in the 680nm colorimetric, photometry density.
3, result
The immune indexes of FTS radiation resistance detects
Kunming mouse 17 ± 1g, 10/group, male and female half and half.650 rad Co60 irradiation, exposure rate 236.1 human relations/minute, irradiation time is 2 minutes 39.4 seconds.
Negative control group subcutaneous injection every day 0.1mL normal saline, the FTS:0.5 μ g/ of administration group subcutaneous injection every day various dose are only.Positive control is a nilestriol, disposable filling stomach: get 2 of nilestriol (2mg), pulverize, add 4mL water, add 0.5% sodium carboxymethyl cellulose (CMC), stir evenly, only irritate stomach 0.1mL/, promptly 0.05mg/ only.One week back detection blood leucocyte sum, carbon clearance exponential sum phagocytic index, thymus index and spleen index.The significance of difference that compares administration group and negative control group with the T check.The result is as shown in table 7:
The index of table 7FTS radiation resistance detects
Group | Weight increase | The leukocyte number is (individual/mm 3) | Clean up index K | Phagocytic index α | Thymus index (mg/10g) | Spleen index (mg/10g) |
Not radiation | 7.16 (p<0.05) | 5878 (p<0.01) | 0.041 | 5.48 | 32.82 (p<0.01) | 84.69 (p<0.01) |
Negative control | 3.93 | 985 | 0.041 | 7.41 | 20.45 | 17.76 |
Positive control | 3.47 | 2693 (p<0.01) | 0.033 | 6.00 | 28.32 (p<0.05) | 8.96 (p<0.01) |
0.5μg | 4.16 | 1445 (p<0.01) | 0.053 | 7.54 | 24.71 | 13.32 |
5μg | 4.69 | 1794 (p<0.01) | 0.060 (p<0.05) | 8.15 (p<0.05) | 25.60 | 11.56 (p<0.05) |
50μg | 2.56 (p<0.05) | 1761 (p<0.01) | 0.063 (p<0.05) | 8.23 (p<0.05) | 26.47 (p<0.05) | 10.17 (p<0.05) |
The result of table 7 shows that the total white blood cells of administration group obviously increases, and has statistical significance, and the exponential sum phagocytic index of cleaning up of administration group also obviously improves, and wherein 5 μ g group and 50 μ g group have significance.The thymus index of administration group also increases, and wherein 50 μ g group has significance.FTS truly has protective effect to the raying mice on immune indexes, and 5 μ g/ dosage only is better.
(1) FTS is to the influence of raying mice survival rate
Negative control subcutaneous injection every day 0.1mL normal saline, the FTS:0.5 μ g/ of administration group subcutaneous injection every day various dose, 5 μ g/, 50 μ g/, 200 μ g/ only.The back drug withdrawal of two weeks continued to observe, till 30 days.Positive controls is a nilestriol, and disposable filling stomach is got 2 of nilestriol (2mg), pulverizes, and adds 4mL water, adds 0.5% sodium carboxymethyl cellulose (CMC), stirs evenly, and only irritates stomach 0.1mL/, and promptly 0.05mg/ only.Result such as Fig. 3, Fig. 4, Fig. 5, shown in Figure 6.The result shows that after the Co60 irradiation, subcutaneous injection FTS has obviously reduced mortality of mice.No matter be than low radiation dose 600 rads or higher dose 700 rads, FTS has protective effect to mice.
(2) FTS is to the effect of ascites tumor mice
Get the kunming mice of 20 ± 1g, female, 10/group, the secondary experimental animal room is raised.Hepatocarcinoma H22 cell goes down to posterity to protect through mouse peritoneal and plants, and gets its ascites tumor, dilution in 1: 1.5, abdominal cavity inoculation 0.4mL.Subcutaneous injection administration every day, in continuous two weeks, blank injecting normal saline, FTS administration 50 μ g/ are only.It is heavy to investigate ascites on the 15th day, spleen index and thymus index.Check the significance of difference that compares between administration group and the blank group with t.The result is as shown in table 8.
Table 8FTS is to the influence of ascites tumor mouse thymus exponential sum spleen index
Group | Weightening finish (g) | Ascites weight (g) | Spleen index (mg/10g) | Thymus index (mg/10g) |
Blank | -2.075 | 16.075 | 56.981 | 13.518 |
FTS | -2.021 | 16.235 | 55.219 | 19.632(p<0.05) |
The result of table 8 shows: FTS group thymus index obviously improves, and has the significance meaning.But ascites weight is obviously difference not, and the expression ascites cells is not suppressed.As seen FTS has the certain protection effect to the ascites tumor mice, by spleen index and thymus index initial reaction immunocompetence improve.
(3) FTS is to the result of tumor-bearing mice chemotherapy
Get the kunming mice of 20 ± 1g, female, 10/group, the secondary experimental animal room is raised.Get the switching of hepatocarcinoma H22 ascites tumor and be solid tumor, two weeks back collection solid tumor cell, cell dispersion, back subcutaneous vaccination.Intraperitoneal injection of cyclophosphamide every day (100mg/kg), the synthetic polypeptide FTS of subcutaneous injection variable concentrations in continuous two weeks, continues the injection polypeptide afterwards simultaneously.Motility rate and mean survival time recorded in postscript in 20 days.Check the significance of difference that compares between administration group and the blank group with experiment of card side and t.Result such as Fig. 7, shown in Figure 8.
The result of Fig. 7, Fig. 8 shows to the 20th day survival rate certain difference is arranged, but because the effect of cyclophosphamide, mice does not all have tangible tumor growth.The result has mainly embodied the side effect that medicine reduces cyclophosphamide.And the effect of 5 μ gFTS is more obvious than the effect of 50 μ gFTS.
Claims (1)
1. serum thymic factor is in the protection medicine of preparation antitumor drug, tumor naturopathy and chemotherapeutic agent or the purposes aspect the enhance immunity medicine.
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CN104906575A (en) * | 2014-03-12 | 2015-09-16 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Application of LSECtin as melanoma immunotherapy target |
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CN104906575A (en) * | 2014-03-12 | 2015-09-16 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Application of LSECtin as melanoma immunotherapy target |
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