CN101167746A - Anti-tumor biologically active substance and preparation technology thereof - Google Patents

Abstract

The invention discloses an antineoplastic biological active substance, which can culture heart tissue to antineoplastic cardiocytes culture fluid. The concrete steps are that cell suspension is made, alternate freezing and thawing is processed and centrifugal is filtered. The invention proves that the disintegration liquid of the heart cell is anticancer reliably without toxic and side effects of treatment, which is divided into the liquid of medicament and vacuum freeze dried. The anticancer intensity and the toxic and side effects are both superior compared with traditional biologics, and the invention can be applied widely in clinical therapeutics of malignant tumour.

Description

A kind of anti-tumor biologically active substance and preparation technology thereof

Technical field

The present invention relates to biomedicine field, be specifically related to heart tissue is cultivated the myocardial cell culture fluid that becomes to have anti-tumor activity, with and preparation method thereof.

Background technology

Tumor be human tissue cell under inside and outside various harmful factors long terms, producer sudden change is expressed disorderlyly, regulates out of controlly, produces hyperplasia and breaks up formed neoplasm or vegetation unusually, often occurs with the lump form clinically.This neoplasm is not that body is required, not according to normal rule growth or unrestrained growth, lost the normal tissue cell function, and can destroy the original organ organizational structure, and then threat to life. malignant tumor has the i.e. 1. autonomys growth of three common traits, 2. infiltrative growth and metastasis form, and 3. the characteristic of cancerous cell can pass to its daughter cell.

The medicine of existing treatment tumor generally is divided into seven big classifications, is respectively the common medicine of alkylating agent, anti-metabolism, antitumor antibiotics, plant alkaloid, miscellany common medicine, hormones and the antitumor drug of biological engineering generation.

The development of biological preparation in recent years is rapid especially, and also flourish to the research of the active substance in the biological preparation, these active substances generally comprise 1. interferon: be mainly used in hair cell leukocyte, Kaposi sarcoma, low potential malignancy lymphoma; 2. interleukin IL-2 and LAK cell are united use, and cancerous cell is produced non-specific lethal effect, and the IL-11 increased platelets counts is used for the clinical treatment that chemotherapeutics uses the blood platelet reduction that causes; 3. monoclonal antibody such as IDEC-C2B8 Mabthera are used for the treatment of B cell lymphoma; 4. somatomedin GM-CSF, G-CSF; Promote granulocyte, macrophage differentiation and maturation, be used for the medication of antineoplastic auxiliary treatment and be used for the clinical treatment that chemotherapeutics uses the leucocytes reduction that causes; 5. tumor necrosis factor: topical application, intratumor injection can play certain local antitumor action.

The biological preparation activated species is various, does not still have effectively all kinds of malignant tumor of treatment of any medicine at present, and the common cause that its clinical practice is restricted is: 1. most of biological preparation all has toxic and side effects such as heating, weak, marrow function inhibition; 2. antitumor action is generally non-specific, its antitumor action a little less than, thereby the part biological preparation only has and strengthens or reconcile immunity and play the antitumor assosting effect; 3. most of biological preparation is owing to being subjected to reasons such as chemical technology extraction and synthetic production cost height, and its biological preparation costs an arm and a leg, thereby is applied to the clinical bigger restriction that is subjected to.

Summary of the invention

The objective of the invention is to overcome the shortcoming of above-mentioned prior art and a kind of nontoxic, broad-spectrum is provided, has antitumor myocardial cell lysate immunization and that be easy to obtain, another object of the present invention provides the preparation method of this antitumor myocardial cell lysate.

The object of the present invention is achieved like this:

Anti-tumor active substance of the present invention is a kind of myocardial cell lysate, and is concrete, and this myocardial cell lysate is to be obtained through following steps by heart tissue:

1) system cell suspension: heart tissue is made uniform cell suspension, place to be lower than-20 ℃ of environment freezing rapidly;

2) multigelation: take out after freezing 24 hours and place room temperature to melt, after treating to melt fully, place-20 ℃ of environment freezing once more, three times so repeatedly, obtain the homogenate of freezing-thawing and cracking;

3) centrifugal filtration:, behind the ultra-filtration filters of supernatant, collect molecular weight and be the myocardial cell lysate less than the filtrate of 10KD with interception 10KD with this homogenate under 3000 rev/mins condition centrifugal 30 minutes.

More specifically, described myocardial cell culture fluid is to be made by following steps:

1) system cell suspension: the cell suspension that heart tissue is made is in homogenate under 2000 rev/mins the condition after 30 minutes, places rapidly that to be lower than-20 ℃ of environment freezing;

2) multigelation: take out after freezing 24 hours and place room temperature to melt, after treating to melt fully, place-20 ℃ of environment freezing once more, three times so repeatedly, obtain the homogenate of freezing-thawing and cracking;

3) centrifugal filtration:, behind the ultra-filtration filters of supernatant, collect molecular weight and be the myocardial cell lysate less than the filtrate of 10KD with interception 10KD with this homogenate under 3000 rev/mins condition centrifugal 30 minutes.

The present invention is by reaching the experimentation in 3 years, and do not have the toxic and side effects of treatment.Its dosage form is divided into liquid preparation and two kinds of dosage forms of vacuum freeze-drying powder, be expected to be widely used in the clinical treatment of malignant tumor, during use with myocardial cell lysate liquid preparation through intravenous injection, perhaps the vacuum freeze-drying powder of myocardial cell lysate is dissolved in water for injection after intravenous administration, perhaps the vacuum freeze-drying powder of myocardial cell lysate is dissolved in 0.9% sodium chloride solution (being normal saline) thus after the intravenous administration approach realizes the antineoplaston purpose.Thereby this biological preparation can remedy the deficiency of traditional biological preparation, all is being better than the traditional biological preparation aspect its antitumor action intensity and the toxic and side effects.Its concrete experiment is as follows: antitumor activity research experiment in the myocardial cell cracking liquid, step is collected the filtrate of molecular weight less than 10KD, mouse inoculation S180 cell random packet for preparation small rat and adult rat cardiac muscle lysate.Negative control group is a normal saline; Positive controls is the cisplatin injection, experimental group is neonatal rat myocardial cell lysate (CMCLnr), adult rat myocardial cell lysate (CMCLar), CMCLnr, CMCLar organizes its inoculated tumour time of occurrence of comparing does not have significant difference, the tumor tumor weight average there was no significant difference that its inoculation grows, low molecule tumour-inhibitory substance group of two groups of different cardiac muscles and cisplatin group tumor time of occurrence are more late than normal saline group time of occurrence, thereby we draw, two groups of low molecule tumour-inhibitory substances of different cardiac muscles (are CMCLnr, CMCLar) and traditional anticancer chemotherapeutic agent cisplatin all have anticancer, tumor-inhibiting action, CMCLnr, CMCLar all has identical antitumor activity, all can suppress the growth of S180 tumor cell in the mice body, it presses down the tumor molecular weight less than 10KD.We observe mice body weight and two indexs of mice weight increase simultaneously, and cisplatin compares with normal saline group and CMCLnr, CMCLar respectively, and the difference of two indexs all has statistical significance; The equal not statistically significant of the difference of CMCLnr, CMCLar and normal saline group, and compare respectively between two groups of CMCLnr, the CMCLar, its difference is not statistically significant also.Experimental result shows that CMCLnr, CMCLar do not have obvious influence to the normal growth of mice, have anticancer, press down the good biological safety of tumor, and do not have that traditional anticancer chemotherapeutic agent has influence toxic and side effects such as body normal growth.(annotate: CMCLnr: neonatal rat myocardial cell lysate CMCLar adult rat myocardial cell lysate)

Description of drawings

Fig. 1: neonatal cardiac myocytes lysate and adult rat myocardial cell lysate homogenate picture;

Fig. 1 explanation: homogenate liquid in pipe in left side is the neonatal cardiac myocytes lysate, liquid is adult rat myocardial cell lysate in the homogenate of right side, the lysate that shows in the picture is that cardiac muscular tissue shreds and through after the refiner abundant homogenate, the 3rd time-20 ℃ take out the state that places room temperature to be in to melt after freezing 24 hours, as seen be suspended in the lysate frozen block in the pipe in the lysate in the both sides homogenate pipe.Neonatal cardiac myocytes lysate (left side pipe) lysate is faint yellow, it is darker slightly than lysate that Guan Zhongshang does not melt the frozen block color, be milk yellow, underage rat myocardial cell lysate (right side pipe) lysate is a pale red, it is darker slightly than lysate that Guan Zhongshang does not melt the frozen block color, is magneta colour.

Fig. 2: picture after neonatal cardiac myocytes lysate and the ultrafiltration of adult rat myocardial cell lysate.

Fig. 2 explanation: be the collected neonatal rat and the homogenate of the abundant freezing-thawing and cracking of adult rat myocardial cell among the figure, treatment conditions are all with 3000 rev/mins of centrifugal 30min, discard precipitation, get supernatant with 4000 rev/mins of the ultra-filtration centrifuge tubes of interception 10KD, 10min is centrifugal, collect the myocardial cell lysate ultrafiltrate of molecular weight less than 10KD, wherein the A pipe is neonatal cardiac myocytes lysate ultrafiltrate among the figure, appearance color is a weak yellow liquid, the B pipe is adult rat myocardial cell lysate ultrafiltrate among the figure, appearance color is a pink liquid, and red liquid is a myocardial cell culture fluid ultrafiltrate in all the other pipes.

The specific embodiment

The present invention is described in further detail with concrete experiment below in conjunction with accompanying drawing:

The effect of myocardial cell lysate is analyzed theoretically and is: Musculoskeletal is bulky, and blood flow is abundant, especially during strenuous exercise, so menses flow to the malignant tumor number that skeletal muscle sends out should be rather considerable, but the skeletal muscle metastatic tumor is rare.Heart is as systemic blood circulation major organs, and its blood supply is very abundant, and metastatic tumor is less, wherein mainly is that pericardium shifts, and cardiac muscle shifts rare.Cardiac muscle and skeletal muscle belong to the striped muscle system, all have malignant tumor and be difficult for transferring to the striped muscle system performance, foreign scholars such as the Djaldetti of Israel, domestic scholars such as Zhou Naikang, Luo Chenghua by experiment and prove in the skeletal muscle conditioned medium and to have low molecule antitumor activity material, it has inhibitory action widely to malignant cell, but not the propagation of tumor then is not subjected to obvious inhibition, even also be subjected to certain facilitation, think that this may be that skeletal muscle is resisted the neoplastic main mechanism that shifts.And cardiac muscle belongs to striped muscle with skeletal muscle, and myocardium metastatic tumor is also rare, infers that theoretically in the myocardium microenvironment be also to exist relevant tumour-inhibitory substance matter.Proved that by a series of related experiment the myocardial cell lysate has antitumor activity, and proved that there is low molecule antitumor activity material in the myocardial cell lysate.

Therefore, the present invention at first proposes a kind of myocardial cell lysate, and it is to be made by following steps:

1) system cell suspension: the cell suspension that heart tissue is made is in homogenate under 2000 rev/mins the condition after 30 minutes, places rapidly that to be lower than-20 ℃ of environment freezing;

2) multigelation: take out after freezing 24 hours and place room temperature to melt, after treating to melt fully, place-20 ℃ of environment freezing once more, three times so repeatedly, obtain the homogenate of freezing-thawing and cracking;

3) centrifugal filtration:, behind the ultra-filtration filters of supernatant, collect molecular weight and be the myocardial cell lysate less than the filtrate of 10KD with interception 10KD with this homogenate under 3000 rev/mins condition centrifugal 30 minutes.

Below by experimental results show that effect of the present invention:

1, experiment material and instrument:

1.1 laboratory animal

Wistar rat (1～3 day) is provided by the Luzhou Medical College animal center

1.2 experimental cell

Nasopharyngeal carcinoma cell (CNE2) is available from Zhongshan University's medical college

Mesangial cell (MCs) is provided by Luzhou Medical College premunition chamber

1.3 main experimental apparatus

Superclean bench (Suzhou cleaning equipment company)

Inverted phase contrast microscope (IMT-2 Japan OLYMPUS company)

5%CO2 constant temperature incubator (TC2323 U.S. Coulter company)

Low temperature autobalance centrifuge (LDZ5-2 Beijing Medical Centrifugal Machine Factory)

Freezer dryer (FD-1 eyela tokyo rikakikai co., ltd)

Electric heating constant temperature water bath (DSY-1-2 like in Beijing outstanding rosy clouds commerce trading center)

Enzyme-linked immunosorbent assay instrument (the EXL U.S.)

Electric drying oven with forced convection (PVG Rong Feng scientific instrument company limited)

Electro-heating standing-temperature cultivator (DG-407 type Chengdu electricity baking factory)

Cryogenic refrigerator (BCD-228B Qingdao Haier Group)

Cell counting count board, coverslip, microscope slide, wet box, color jar (Jiangsu Da Cang Medical Instruments factory)

6 well culture plates, 96 well culture plates (the magnificent company in Beijing)

2 main experiment reagents

2.1 cell culture reagent

DMEM (the magnificent company in Beijing)

Newborn calf serum (the magnificent company in Beijing)

0.25% trypsin Luzhou Medical Colledge Affiliated Hospital premunition chamber provides)

0.1% collagenase (the magnificent company in sigma product Beijing provides)

D-hanks liquid (Luzhou Medical Colledge Affiliated Hospital premunition chamber provides)

PBS (phosphate buffer, PH=7.0)

MTT (the magnificent company in Beijing)

DMSO (the magnificent company in Beijing)

2.2 immunohistochemistry reagent

10% poly-D-lysine (Chengdu Yun Hong technology ﹠ development Co.) 0.01mol/1, PH 7.2: take by weighing NaCl 8g, Na2HPO4 1.15g, KH2PO4 0.2g, adding distil water is transferred pH value to 7.2 to the 1000ml dissolving

Substrate solution DAB test kit (Bioisystech Co., Ltd of China fir Golden Bridge product comprises concentrated buffer in Beijing, and DAB solution concentrates hydrogenperoxide steam generator)

ABC test kit (Bioisystech Co., Ltd of China fir Golden Bridge product in Beijing comprises sealing and uses the normal serum working solution, biotin labeling goat anti-mouse igg, horseradish peroxidase labelling chain enzyme avidin working solution S-A/HRP)

Mouse anti rat striped muscle monoclonal actin antibody Mouse Anti-SarcomericActin 5C5; IgM (Wuhan doctor's moral reagent company limited)

3, detailed process step of the present invention

3.1 draw materials

(1) sterilization: small rat sterilization is taken out and is given birth to some of 1-3 days Wistar neonatal rats, and the cotton ball soaked in alcohol whole body with 75% is cleaned and sterilized, and is placed on the aseptic foam, with aseptic pin fixing limbs and head, with the alcohol disinfecting chest skin of abdomen of iodine tincture and 75%.

Some of adult Wistar rats are got in adult rat sterilization, about 200g/ only, dislocation method execution is fixed its extremity and head on plank with cord, cuts off chest abdominal part Mus hair with shears, with the alcohol disinfecting chest skin of abdomen of iodine tincture and 75%.

(2) cardiac muscle is drawn materials

Small rat is identical with adult rat cardiac muscle method of drawing material, changing shears earlier cuts off skin and peels off chest, change shears rib place on xiphoid-process and go into to cut, cut off rib, expose the thoracic cavity, the clip apex of the heart, normal saline is cleaned, and residual hemocyte in the flush away chambers of the heart is weighed on electronic balance, then it is cut into the piece of tissue of 1mm3 size, with normal saline in 20% (W/V) ratio mixing.

3.2 homogenate

With in adult rat myocardial cell suspension places different homogenate bottles respectively, treatment conditions are on refiner with 2000 rev/mins with the neonatal rat myocardial cell suspension, and small rat cardiac muscle and the adult rat cardiac muscle different homogenate of originating is made in homogenate 30 minutes.

3.3 multigelation

Small rat is all placed-20 ℃ of frozen 24h of refrigerator with the different homogenate in adult rat cardiac muscle source, place room temperature to melt afterwards, after treating to melt fully, it is frozen to put into-20 ℃ of refrigerators once more, three times repeatedly.

The multigelation ratio juris is freezing below-20 ℃ with cell, room temperature is melted, repeatedly several times, increase the swelling fragmentation that causes cell owing to the ice pellets crystalline solid that forms in the cell makes the salinity of residue cytosol, most tissues cell and intracellular granule can be melted brokenly.

3.4 ultrafiltration and lyophilization

Homogenate with collected neonatal rat and the abundant freezing-thawing and cracking of adult rat myocardial cell, treatment conditions are all with 3000 rev/mins of centrifugal 30min, discard precipitation, get supernatant with 4000 rev/mins of the ultra-filtration centrifuge tubes of interception 10KD, 10min is centrifugal, and the collection molecular weight is the myocardial cell lysate less than the filtrate of 10KD.

Further be, this myocardial cell lysate can be made freeze dried powder uses, concrete grammar is: the membrane filtration myocardial cell lysate of using 0.22um earlier, the degerming postlyophilization becomes dry powder, its freeze dried powder is the faint yellow of loose drying or yellow-white powder, and the freeze dried powder of its neonatal rat and adult rat myocardial cell lysate is all standby with-20 ℃ of cryopreservation.Concrete grammar is:

(1) pre-freeze: in packing the myocardial cell lysate into ground round bottom vial specific, soak, it is solidificated on bottle inwall uniformly, so that under vacuum, distil at low temperature heat-conduction liquid internal rotation.If do not freeze reality, then goods can overflow outside the bottle during evacuation.

(2) sublimation stage: also claim the primary drying stage, the goods drying baker of packing into starts vacuum pump, makes drying baker, water vessel obtain essential vacuum, sets up the pressure condition of vacuum freeze-drying; Heat up to hot plate, provide the ice crystal heat that distillation needs to goods.The vacuum of drying baker is advisable to be controlled at 10-30Pa, and the water vessel temperature should be less than-40 ℃; Shelf temperature is between-10 ℃～+ 10 ℃.

(3) desorption phase: also claim the redrying stage, although in primary drying, most water can be got rid of with the distillation of ice crystal, in order to obtain drying effect as well as possible, should carry out redrying.Freeze drying box vacuum remains on the 10-30Pa scope; The rising shelf temperature makes product temperature rise to the maximum temperature (＜25 ℃) that this product allows rapidly rapidly, finishes until drying, and whole dry run needs more than 10 hours approximately.

4, experiment 1: antitumor activity research in the myocardial cell cracking liquid

Preparation small rat and adult rat cardiac muscle lysate are collected the filtrate of molecular weight less than 10KD, mouse inoculation S180 cell random packet.Negative control group is a normal saline; Positive controls is the cisplatin injection, experimental group is neonatal rat myocardial cell lysate (CMCLnr), adult rat myocardial cell lysate (CMCLar), experimental result shows that CMCLnr, CMCLa all can suppress the growth of S180 tumor cell in the mice body, and it presses down the tumor molecular weight less than 10kd.CMCLnr, CMCLa relatively its tumor time of occurrence do not have significant difference, its tumor weight average there was no significant difference.

(see Table 1, table 2, table 3)

Table 1: the low molecule tumour-inhibitory substance of cardiac muscle is to the influence of S180 mice-transplanted tumor tumor time of occurrence

Group	Sample	Dosage (mg/kg)	The tumor time of occurrence (my god)
				Normal saline group cisplatin group CMCLnr CMCLar	10 10 10 10	25 2 60 60	5.90±0.88 7.70±0.82 7.30±0.82 7.30±0.67

Annotate: CMCLnr: neonatal rat myocardial cell lysate CMCLar adult rat myocardial cell lysate

As seen, low molecule tumour-inhibitory substance of two groups of different cardiac muscles and cisplatin group tumor time of occurrence are than normal saline group time of occurrence late (P＜0.05)

Table 2: the influence that the low molecule tumour-inhibitory substance of cardiac muscle is heavy to S180 mice-transplanted tumor tumor

Group	Dosage (mg/kg)	Tumor heavy (g)	Tumour inhibiting rate (%)	The P1 value	The P2 value
						Normal saline cisplatin CMCM CMCLnr CMCLar	- 2 60 60 60	1.4412±0.4257 0.2705±0.1102 0.4493±0.1168 0.4981±0.1059 0.4652±0.1158	- 81.23 68.82 65.44 67.72	- 0.000 0.000 0.000 0.000	0.000 - 0.024 0.002 0.012

Annotate: P ₁Be the comparison of each group with the normal saline group, P2 _ForThe comparison of each group and cisplatin group.

Table 3: respectively organize the mice body weight change before and after the experiment

Group (mg/kg)	Dosage	Body weight (g)		Weight increase (g)
					Before the administration	After the administration
		Normal saline cisplatin CMCLnr CMCLar F P value	- 2 60 60				20.40±0.86 20.35±0.75 20.45±1.01 20.35±1.03 0.064 0.992	30.55±0.80 26.70±1.00 31.25±1.03 30.95±0.86 42.463 0.000	10.20±0.95 6.35±0.71 10.90±0.46 10.85±0.53 92.733 0.000

Annotate: respectively organize mice body weight difference not statistically significant (P＞0.05) before the experiment.

Experiment finishes back mice body weight and two indexs of mice weight increase, and cisplatin compares with normal saline group and CMCLnr, CMCLar respectively, and the difference of two indexs all has statistical significance (P＜0.05); The equal not statistically significant of the difference of CMCLnr, CMCLar and normal saline group (P＞0.05), and compare respectively between two groups of CMCLnr, the CMCLar, its difference is not statistically significant (P＞0.05) also.

Experimental result: 1. neonate rat adult rat myocardial cell lysate all can suppress the growth of S180 tumor cell in the mice body, presses down the tumor molecular weight less than 10KD, and the normal growth of mice is not had obvious influence.

2. the antitumor activity no significant difference between neonatal cardiac myocytes lysate and the adult rat myocardial cell lysate all demonstrates effective antitumor activity.

Claims (2)

1. an anti-tumor biologically active substance is characterized in that, it is the myocardial cell lysate, and described myocardial cell lysate is obtained through following steps by heart tissue:

1) system cell suspension: heart tissue is made uniform cell suspension, place to be lower than-20 ℃ of environment freezing rapidly;

2) multigelation: take out after freezing 24 hours and place room temperature to melt, after treating to melt fully, place-20 ℃ of environment freezing once more, three times so repeatedly, obtain the homogenate of freezing-thawing and cracking;

3) centrifugal filtration:, behind the ultra-filtration filters of supernatant, collect molecular weight and be the myocardial cell lysate less than the filtrate of 10KD with interception 10KD with this homogenate under 3000 rev/mins condition centrifugal 30 minutes.

2. the preparation technology of an anti-tumor biologically active substance is characterized in that making the myocardial cell lysate by the cracking of heart tissue multigelation, and described myocardial cell lysate is obtained by following steps:

1) system cell suspension: the cell suspension that heart tissue is made is in homogenate under 2000 rev/mins the condition after 30 minutes, places rapidly that to be lower than-20 ℃ of environment freezing;

2) multigelation: take out after freezing 24 hours and place room temperature to melt, after treating to melt fully, place-20 ℃ of environment freezing once more, three times so repeatedly, obtain the homogenate of freezing-thawing and cracking;

3) centrifugal filtration:, behind the ultra-filtration filters of supernatant, collect molecular weight and be the myocardial cell lysate less than the filtrate of 10KD with interception 10KD with this homogenate under 3000 rev/mins condition centrifugal 30 minutes.