CN103340905B - Anti-tumor bioactive substance and preparation method thereof - Google Patents
Anti-tumor bioactive substance and preparation method thereof Download PDFInfo
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- CN103340905B CN103340905B CN201310308948.9A CN201310308948A CN103340905B CN 103340905 B CN103340905 B CN 103340905B CN 201310308948 A CN201310308948 A CN 201310308948A CN 103340905 B CN103340905 B CN 103340905B
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Abstract
The invention discloses a small molecule bioactive substance, namely, a cardiac muscle tissue lysate with the anti-tumor activity. The lysate is prepared by homogenating cardiac muscle tissues of non-bearing cancer healthy animals of different ages, species and resources and carrying out multigelation, pyrolysis and ultrafiltration, and the lysate is prepared into two dosage forms, namely, a liquid preparation and a vacuum freeze-dried powder preparation. The cardiac muscle tissue lysate is definite in anti-tumor effect, has the function of radiosensitization, and is free of therapeutic toxic and side effects; and moreover the cardiac muscle tissue lysate has prospects in anti-tumor capability, application range and toxic and side effect when being compared with the conventional biological agents, and has the hope to be widely applied to clinical treatment on malignant tumors.
Description
Technical field
The present invention relates to biomedicine field, be specifically related to healthy animal cardiac muscular tissue to make cardiac muscular tissue's pyrolysis product with antitumor activity and Radiosensitizing and preparation method thereof.
Background technology
Tumor is the common frdquently encountered disease affecting human health, harm people's life.At present, the medicine for the treatment of tumor generally has alkylating agent, anti-metabolism, antitumor antibiotics, plant alkaloid, miscellany, hormones and monoclonal antibody, micromolecule targeted drug etc.
The development of biological preparation is in recent years rapid especially, and also flourish to the active matter Quality Research in biological preparation, these active substances generally comprise:
1, interferon: be mainly used in hairy cell leukemia, Kaposi sarcoma, low potential malignancy lymphoma;
2, interleukin: IL-2 and LAK cell conbined usage, produces non-specific lethal effect to cancerous cell, IL-11 increased platelets counts, for the clinical treatment of the platelet reduction that chemotherapeutics causes;
3, monoclonal antibody such as IDEC-C2B8 Mabthera is used for the treatment of B cell lymphoma;
4, somatomedin GM-CSF, G-CSF: promote that granulocyte, macrophage differentiation are ripe, for the medication of antineoplastic auxiliary treatment and the clinical treatment using the leukocyte reduction caused for chemotherapeutics;
5, tumor necrosis factor: topical application, intratumor injection can play certain local anti-tumor effect.
From the above, biological agent activity substance classes is various, and there is no any medicine at present and effectively treat all kinds of malignant tumor, the common cause that its clinical practice is restricted is:
1, most of biological preparation all has the toxic and side effects such as heating, weak, Inhibitory effect of marrow function;
2, antitumor action is generally non-specific, and its antitumor action is more weak, and part biological preparation only has enhancing or reconciles immunity thus play antitumor assosting effect;
The factors such as 3, most of biological preparation is owing to being subject to complicated process of preparation, and production cost is high impact, its biological preparation is expensive, thus is applied to and is clinically subject to larger restriction.
The patent No. be CN200710050378.2 patent discloses a kind of anti-tumor biologically active substance and preparation technology thereof, in above-mentioned patent, described anti-tumor biologically active substance is induction of myocardial cell lysate, this induction of myocardial cell lysate needs first heart tissue to be made cell suspension in preparation, complex operation, production cost is higher, Production Time is longer, because cell resistance power is weak, requirement for environmental conditions is harsh to external world, in addition cell manipulation is microtechnique, naked eyes cannot well regulate and control whole operation, there are many cells excessively can digest due to enzyme in manufacturing process unavoidably, the reasons such as mechanical damage are dead, therefore stock utilization is low, effect of drugs is unstable.
Summary of the invention
The object of the invention is to overcome the problem such as the large and antitumous effect of anti-tumor biologically active substance complicated process of preparation in prior art, side effect is poor, there is provided a kind of nontoxic, wide spectrum, stable curative effect definite and be easy to the anti-tumor biologically active substance that obtains and cardiac muscular tissue's lysate, this material tumor-inhibiting action definitely and have radiosensitizing effect, has very large using value simultaneously.
Another object of the present invention is to provide a kind of preparation method of above-mentioned anti-tumor biologically active substance.
In order to realize foregoing invention object, the technical solution used in the present invention is as follows:
There is provided a kind of anti-tumor biologically active substance, it is cardiac muscular tissue's lysate.
Described cardiac muscular tissue lysate is divided into liquid preparation and vacuum freeze-drying powder two kinds of dosage forms; During use, if the liquid preparation of cardiac muscular tissue's pyrolysis product then can directly through intravenous injection; If vacuum freeze-drying powder, be then dissolvable in water after water for injection or normal saline through intravenous administration.
The preparation method of above-mentioned anti-tumor biologically active substance, comprises the following steps:
A, cardiac muscular tissue is cut into 1mm
3piece of tissue, add normal saline mixing, obtained cardiac muscular tissue suspension;
B, by the homogenate 30 minutes under the condition of 2000 revs/min of cardiac muscular tissue's suspension, obtain ventricular homogenate liquid;
C, that homogenate obtained for step b is placed in-70 DEG C of environment is frozen, takes out to be placed in room temperature and to melt after 24 hours, after melting completely, is again placed in-70 DEG C of environment frozen, three times so repeatedly, the ventricular homogenate liquid of obtained freezing-thawing and cracking;
D, ventricular homogenate liquid obtained for step c is crossed 200 mesh sieves, get filtrate under the condition of 4000 revs/min centrifugal 30 minutes, by the ultra-filtration centrifuge tube of supernatant interception 10KD under the condition of 4000 revs/min centrifugal 10 minutes again, collect the filtrate that molecular weight is less than 10KD, after using 0.22um membrane filtration again, get filtrate, the liquid preparation of obtained cardiac muscular tissue lysate, i.e. cardiac muscular tissue's lysate.
Cardiac muscular tissue's lysate obtained for steps d is placed in-20 DEG C to be refrigerated to and to freeze reality completely, then carries out vacuum lyophilization process, the vacuum freeze-drying powder of Ji get cardiac muscular tissue lysate.
Described cardiac muscular tissue is the cardiac muscular tissue of the non-tumor animal from all ages and classes, different genera source.
The application of above-mentioned anti-tumor biologically active substance in neoplasm growth and radiation sensitization.
In sum, anti-tumor biologically active substance cardiac muscular tissue of the present invention lysate is without the need to preparing cell suspension, operating process does not need rigorous aseptic, have no side effect, repdocutbility is good, is prepared into power high, prepares material requested wide material sources and is easy to obtain, preparation method is simple, is more conducive to large-scale production; And can effectively grow by Tumor suppression, tumor-inhibiting action is definite, and effect is more stable, has radiosensitizing effect, has range of application, larger use value and application prospect widely simultaneously.
Accompanying drawing explanation
Fig. 1 is under 2Gy radiation situation, the cell clone growing state of irradiation group merely, topotecan+irradiation group and cardiac muscular tissue's lysate+irradiation group.
Fig. 2 is CNE-2 cell survival curve after simple irradiation group, topotecan+irradiation group and cardiac muscular tissue's lysate in experimental example of the present invention+irradiation group is irradiated.
Fig. 3 in invention experimental example through irradiate after blank group, topotecan+irradiation group, cardiac muscular tissue's lysate+irradiation group, topotecan group, cardiac muscular tissue's lysate group cell residing for cell cycle ratio comparatively.
Fig. 4 be in invention experimental example through irradiate after, blank group, topotecan+irradiation group, cardiac muscular tissue's lysate+irradiation group, topotecan group, cardiac muscular tissue's lysate group apoptosis rate compare.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in detail, but they are not to further restriction of the present invention.
Embodiment
Anti-tumor biologically active substance cardiac muscular tissue of the present invention lysate is obtained by following steps:
1) cardiac muscular tissue's suspension is prepared: get the non-tumor animal of health, after iodine tincture, alcohol disinfecting, animal thoracic cavity is cut open with cutting, the clip animal apex of the heart, removes the tissues such as heart surface peplos, obtains comparatively simple cardiac muscular tissue, with normal saline cyclic washing cardiac muscular tissue, wash away hemocyte residual in the chambers of the heart, weigh on electronic balance, be then cut into 1mm
3the piece of tissue of size, adds normal saline mixing in 20% (W/V) ratio; Above-mentioned cardiac muscular tissue derives from the non-tumor animal of all ages and classes, different genera.
2) homogenate: be placed in homogenate bottle by above-mentioned cardiac muscular tissue suspension, the speed homogenate with 2000 revs/min on refiner makes ventricular homogenate liquid in 30 minutes.
3) multigelation: the homogenate obtained is placed in-70 DEG C of refrigerators and takes out after frozen 24 hours, after room temperature is melted completely, again put into-70 DEG C of refrigerators frozen, three times repeatedly, obtain the ventricular homogenate liquid of freezing-thawing and cracking;
4) centrifugal ultrafiltration: by the ventricular homogenate liquid of above-mentioned freezing-thawing and cracking by 200 object filtered through gauze to remove fragment of tissue, get the centrifugal 30min under the condition of 4 DEG C, 4000 revs/min of the homogenate after filtration, Aspirate supernatant (not containing thin precipitation) is in centrifuge tube, with ultra-filtration centrifuge tube centrifugal 10min under the condition of 4 DEG C, 4000 revs/min of interception 10KD, collect the filtrate that molecular weight is less than 10KD, again with getting the liquid preparation that namely filtrate obtain peach cardiac muscular tissue pyrolysis product after the membrane filtration of 0.22um, i.e. cardiac muscular tissue's lysate.This liquid preparation in 4 DEG C of short-term preservations or can save backup in-20 DEG C for a long time.
5) the vacuum freeze-drying powder of cardiac muscular tissue's lysate is prepared: above-mentioned cardiac muscular tissue lysate is placed in-20 DEG C of refrigerators and carries out pre-freeze until lysate freezes reality completely and keeps a period of time, lysate good for pre-freeze is taken out, put into freezer dryer, carry out vacuum lyophilization process, the Powdered thing of rose pink loose drying can be collected, this is the vacuum freeze-drying powder of cardiac muscular tissue's pyrolysis product, this vacuum freeze-drying powder can at-20 DEG C in conventional seals bottle low temperature save backup for a long time.
Experimental example
Below for cardiac muscular tissue's lysate, with specific experiment, more detailed describing is done to effect of the present invention by reference to the accompanying drawings.
Test 1 cardiac muscular tissue's lysate to the external inhibiting tumor assay of CNE-2
1.1, different genera cardiac muscular tissue lysate is to the external inhibiting tumor assay of CNE-2
Adopt method described in above-described embodiment to obtain cardiac muscular tissue's lysate of fish, the frog, chicken, Mus, pig, aborted fetus, cardiac muscular tissue's lysate of originating with the above-mentioned different genera of mtt assay observation analysis is on the impact of nasopharyngeal carcinoma (CNE-2) cell strain growth in vitro; Wherein:
Experimental result display (see table 1): fish, the frog, chicken, Mus, pig, aborted fetus cardiac muscular tissue lysate to compare with negative control group CNE-2 survival rate and demonstrate obvious antitumor activity; Fish, the frog, chicken, Mus, pig, human myocardial tissues's lysate to CNE-2 survival rate mutually more then without statistical difference, illustrate that a species specificity is not had in the effect of cardiac muscular tissue's lysate.
Table 1 different genera cardiac muscular tissue lysate is on the impact of CNE-2 survival rate
Note: ★ represents that difference has statistical significance compared with negative group, inspection level α=0.05; * represent that difference has statistical significance compared with DDP group, inspection level α=0.05.
1.2, all ages and classes rat heart muscle Tissue lysates is to the external inhibiting tumor assay of CNE-2
The cardiac muscular tissue's lysate obtained with the cardiac muscular tissue of mtt assay observation analysis tire Mus, newborn rat, adult rats is on the impact of CNE-2 growth in vitro.Experimental result display (see table 2): cardiac muscular tissue's lysate of tire Mus, newborn rat, adult rats to compare with negative control group the impact of CNE-2 survival rate obvious antitumor activity; Tire Mus, newborn rat, adult rats cardiac muscular tissue lysate to CNE-2 survival rate mutually more then without statistical difference; This experiment reconfirms that cardiac muscular tissue's lysate acts on the neoplasm growth (tumor suppression) of CNE-2 tumor cell, and illustrates that its tumor killing effect has nothing to do with animal age of drawing materials.
Table 2 all ages and classes rat heart muscle Tissue lysates is on the impact of CNE-2 survival rate
Note: ★ represents that difference has statistical significance compared with negative group, inspection level α=0.05; * represent that difference has statistical significance compared with DDP group, inspection level α=0.05.
Test 2 cardiac muscular tissue's lysates to the radiosensitizing effect of nasopharyngeal carcinoma CNE-2 cell strain
2.1, cardiac muscular tissue's lysate and topotecan are on the impact of CNE-2 cell strain radiosensitivity
Experiment is divided into blank group, simple irradiation (RT) group, simple medicine group, medicine+irradiation group, and its Chinese medicine+irradiation component is two subgroups: cardiac muscular tissue's lysate+irradiation group (CMTL+RT), topotecan+irradiation group (TPT+RT).By the number of cell clones in count plates, draw cloning efficiency after each process of cell under this growth conditions and CNE-2 cell colony formation rate; Result shows: Cell colonies assay reduces along with the increase of radiological dose (as shown in table 3), under same exposure dose, the CNE-2 cell colony formation rate irradiation group more merely of cardiac muscular tissue's lysate+irradiation group obviously reduces (as shown in Figure 1), cardiac muscular tissue's lysate+irradiation group compares the CNE-2 cell D0 value of simple irradiation group, Dq value, N value all reduce, SF2 also reduces (as shown in table 4 and Fig. 2), and above result shows that cardiac muscular tissue's lysate has radiosensitizing effect to nasopharyngeal carcinoma cell.
CNE-2 Cell colonies assay (%) after each experimental group of table 3 corrects
The enhanced sensitivity calculated after the matching of table 4 one-hit multitarget compares result
2.2, cardiac muscular tissue's lysate and topotecan are on the impact of apoptosis rate and cell cycle
Experiment be divided into blank group, topotecan medicine (TPT) group, cardiac muscular tissue's lysate (CMTL) medicine group, irradiation group (RT), topotecan enhanced sensitivity group (TPT+RT) and cardiac muscular tissue's lysate enhanced sensitivity group (CMTL+RT), flow cytomery respectively organizes apoptosis rate and cell cycle stage; Result shows: CMTL+RT group compares with RT group, and apoptosis rate obviously increases, and S phase ratio increases, and G2/M compares decline (as shown in table 5, Fig. 3 and Fig. 4).
Table 5 respectively group natural death of cerebral cells and cell cycle compares
This experiment 2 confirms the radiosensitizing effect of cardiac muscular tissue's lysate to CNE-2 cell strain, and enhance-ment ratio is 1.153, and its nude mice is relevant with cell death inducing and S phase cell cycle arrest.
In sum, above-mentioned experiment 1 and experiment 2 confirm that cardiac muscular tissue's lysate that the present invention obtains is to the tumor-inhibiting action of nasopharyngeal carcinoma cell (CNE-2), demonstrates this cardiac muscular tissue's lysate simultaneously and has radiosensitizing effect, and without the toxic and side effects for the treatment of; In addition, cardiac muscular tissue of the present invention lysate raw material sources is extensive, cardiac muscle (fish, the frog, chicken, Mus, pig, the aborted fetus etc.) tissue of different genera animal can be taken from, to animal age not tool strictly limit (as this experiment confirm adult rats, tire Mus, newborn rat source cardiac muscular tissue's lysate all have an anti-tumor biological), and in anti-tumor capacity, range of application and toxic and side effects, compare traditional biological preparation may have more prospect.
Although describe in detail the specific embodiment of the present invention in conjunction with specific embodiments, it is not the restriction to this patent protection domain.In claims limited range, the various amendment that those skilled in the art can make without creative work or adjustment are still by the protection of this patent.
Claims (3)
1. a preparation method for anti-tumor biologically active substance, is characterized in that, comprises the following steps:
A, cardiac muscular tissue is cut into 1mm
3piece of tissue, add normal saline mixing, obtained cardiac muscular tissue suspension;
B, by the homogenate 30 minutes under the condition of 2000 revs/min of cardiac muscular tissue's suspension, obtain ventricular homogenate liquid;
C, that homogenate obtained for step b is placed in-70 DEG C of environment is frozen, takes out to be placed in room temperature and to melt after 24 hours, after melting completely, is again placed in-70 DEG C of environment frozen, three times so repeatedly, the ventricular homogenate liquid of obtained freezing-thawing and cracking;
D, ventricular homogenate liquid obtained for step c is crossed 200 mesh sieves, get filtrate under the condition of 4000 revs/min centrifugal 30 minutes, by the ultra-filtration centrifuge tube of supernatant interception 10KD under the condition of 4000 revs/min centrifugal 10 minutes again, collect the filtrate that molecular weight is less than 10KD, use 0.22um membrane filtration again, get filtrate, the liquid preparation of obtained cardiac muscular tissue lysate, i.e. cardiac muscular tissue's lysate.
2. the preparation method of anti-tumor biologically active substance according to claim 1, it is characterized in that: cardiac muscular tissue's lysate that steps d is obtained is placed in-20 DEG C and freezes reality, carry out vacuum lyophilization process again, the vacuum freeze-drying powder of Ji get cardiac muscular tissue lysate.
3. the preparation method of anti-tumor biologically active substance according to claim 1, is characterized in that: described cardiac muscular tissue is the cardiac muscular tissue of the non-tumor animal from all ages and classes, different genera source.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1624113A (en) * | 2004-11-17 | 2005-06-08 | 中国医学科学院北京协和医院 | Biological differentiation revellent, its preparation process and application differenting inducing in from stem cells to cardiacy muscular tissue cells |
| CN101167746A (en) * | 2007-11-02 | 2008-04-30 | 泸州医学院附属医院 | Anti-tumor biologically active substance and preparation technology thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1624113A (en) * | 2004-11-17 | 2005-06-08 | 中国医学科学院北京协和医院 | Biological differentiation revellent, its preparation process and application differenting inducing in from stem cells to cardiacy muscular tissue cells |
| CN101167746A (en) * | 2007-11-02 | 2008-04-30 | 泸州医学院附属医院 | Anti-tumor biologically active substance and preparation technology thereof |
Non-Patent Citations (2)
| Title |
|---|
| 乳鼠心肌裂解液对S180小鼠移植瘤抑瘤作用的实验研究;蒋晓睿等;《四川医学》;20100815;第31卷(第8期);第1064页第1.2节，第1065页第3节 * |
| 心肌细胞裂解液对鼻咽癌CNE-2细胞株的放射增敏作用及其机制的研究;刘华文等;《临床肿瘤学杂志》;20120815;第17卷(第8期);第687页1.3节，688页第2.1节至689页2.4节和表1-3 * |
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