CN1217341A - Shark-cartrilage blood-vessel generation inhibitory factor and separating and purifying method therefor - Google Patents

Abstract

The present invention relates to a shark chondroangiogenesis inhibition factor and its separation and purification method. The silver dyeing of SDS-PAGE electrophoretic result of said shark chondroangiogenesis inhibition factor SCAIF-1 displays a strip, and its molecular weight is 18000. Said SCAIF-1 is obtained by using shark cartilage as raw material through the processes of guanidinium hydrochloride solution extraction, organic solvent stage precipitation, Resource Q ion-exchange chromatography, Sephacryl S-300 molecular sieve chromatography and preparation electrophoresis, and its inhibition effect research for inhibiting angioendothelial cell growth, neogenetic hemangiogenesis and mouse tumor growth shows that its purity is high, activity is strong and specificty is good.

Description

Shark-cartrilage blood-vessel generation inhibitory factor and separation purification method thereof

The invention belongs to medical technical field, is the method for a kind of shark-cartrilage blood-vessel generation inhibitory factor and separation and purification thereof.

Adult's vasculogenesis is only limited to wound healing and women's reproductive cycle process usually, proved that the every other vasculogenesis of adult all is ill, and can cause pathology widely, as growth of tumor, chaff urine characteristic of disease retina hyperplasia, endotheliocyte cancerate (as vascular tumor), atherosclerosis and sacroiliitis etc.

Harvard University professor Persian in 1971 the researcher Judah Folkman of children's hospital that pauses proposes " tumor growth is to rely on blood vessel ", and 20 years of researches results have fully proved this theory.In most of normal human the tumour that is in dormant state is arranged all, have only 1mm ³Size does not constitute a threat to human body; Have only when it to generate new vessel, obtain nutrition supply and route of metastasis, tumour could grow and shift, and threatens HUMAN HEALTH.

Growth of tumor exists the no blood vessel phase and the blood vessel phase is arranged, and in the no blood vessel stage of beginning, tumor tissues is with linear mode growth at a slow speed, and volume can not surpass 1mm ³, the propagation and the apoptosis of tumour cell are in a dynamic balance state in period in this.To the initial stage that the transition of blood vessel phase is arranged, tumor tissues can discharge a kind of vasculogenesis stimulating factor of solubility, induce capillary endothelial cell propagation and migration, and the formation of blood vessel endothelium bud and new vessel generation, cause tumor tissues to be grown with exponential manner.Suppress nutrition supply and blood road route of metastasis that vasculogenesis can cut off tumour, thereby suppress growth of tumor and transfer.

Abroad, particularly the Folkman laboratory is studied for many years to suppressing new vessel, found that some cytokines have the effect that suppresses the new vessel generation, synthesized some simultaneously and can suppress the organic compound of vasculogenesis, but most of owing to toxicity is restricted greatly.Afterwards, the scientific worker had forwarded research emphasis to naturally occurring biomacromolecule, find all may to have in natural animal-plant and the microorganism Angiostatin (angiogen-esis inhibitory factor, AIF).Folkman laboratory in 1994 finds that propagation and new vessel that Profibrinolysin fragment-angiostatin of 38KD can special inhibition vascular endothelial cell generate, and can significantly suppress the Lewis lung cancer metastasis growth (O ' Reilly M S, Holmgren L, Shing Y, et al.Angiostatin:A novelangiogenesis inhibitor that mediates the suppression of metastases by a Lewis lungcarcinoma.Cell, 1994; 79:315-328).They C-terminal fragment-endostatin of reporting the 20KD of collagen X VIII again in 1997 have same activity (O ' Reilly M S, Boehm T, Shing Y, et al.En-dostatin:An endogenous inhibitor of angiogenesis and tumor growth.Cell, 1997; 38:277-285).

Cartilage is the main vesselless tissue of animal body, contains more rich Angiostatin, and as far back as the mid-80, people just find that cartilage can resist the intrusion of tumour cell, seldom bearing tumor.Nineteen ninety Moses report is from the calf cartilage Angiostatin of calf tracheal cartilage purifying homogeneous, molecular weight is 27650 (Moses AM, Sudhalter J, Langer R.Identification of an inhibitor of neovascularization fromcartilage.Science, 1990248:1408-1410).Afterwards they also to obtain molecular weight from chondrocytes cultured be 35550 same merit albumen.Because ox cartilage content is less, processing difficulties does not see that always its product enters the report in clinical or market.

Shark belongs to chondrichthyes, and nineteen eighty-three Lee report shark suft bone contains abundant AIF.Doctor Lane is widely used in the treatment of tumour patient with shark suft bone crude extract and shark cartilage powder, has obtained curative effect preferably.Since the early 1990s, states such as the U.S., New Zealand, Canada, Australia, Japan approvals shark cartilage powder or crude extract come into the market to be used for the treatment of tumour patient with protective foods, have risen the anticancer upsurge of international shark suft bone.U.S. FDA enters the clinical experiment of II phase in approval shark cartilage powder in 1994, and Canada's approval in 1997 shark suft bone crude extract enters the clinical experiment of III phase.We began the antitumor action of shark and calf cartilage is studied the end of the eighties, had taken the lead in obtaining food, protective foods and the inner preparation authentication code of shark suft bone crude extract at home, and had taken the lead in carrying out clinical application research.Result of study in the past few years shows that the shark suft bone crude extract has certain curative effect to patients with solid tumor.But shark cartilage powder and shark suft bone crude extract still have its shortcoming.Cartilage contains abundant Angiostatin, wherein also contains the vasculogenesis stimulating factor simultaneously, and Cartilage powder or crude extract are not purified, fail to remove vasculogenesis stimulating factor wherein, can influence it and suppress angiogenic action.In addition, contain abundant calcium in the cartilage, take the hypercalcemia that shark cartilage powder or crude extract can cause tumour patient.Therefore, many in recent years laboratory attempts are separation and purification shark-cartrilage blood-vessel generation inhibitory factor (shark cartilage angiogenesis inhibitory fac-tor from shark suft bone, but up to the present, do not see the report of achieving success yet SCAIF).

The object of the present invention is to provide a kind of from the Angiostatin of shark suft bone acquisition and the method for separation and purification thereof.

The present invention has prepared a kind of new protein-shark cartilage angiogenesis inhibitory factor-(brief note is the SCAIF-I) from shark suft bone, can significantly suppress the growth of vasculogenesis and mouse tumor, identify and molecular weight determination through purity, its SDS-PAGE electrophoresis result silver dyes and shows a band, as shown in Figure 1.It is pure to illustrate that the SCAIF-I reaches electrophoresis.Recording its molecular weight is 18000.

The SCAIF-I is obtained by following separation purification method among the present invention, with shark suft bone through the extracting of Guanidinium hydrochloride (Gu.HCl) solution, use the organic solvent fractionation precipitation, carry out ion exchange chromatography with Resource Q again, obtain 3 elution peaks (as Fig. 3), collect the 3rd peak, carry out Sephacryl S-300 sieve chromatography after concentrating, obtain 3 tangible elution peaks (as Fig. 4) equally, regather the 3rd peak, carry out SDS-PAGE after concentrating and prepare electrophoresis, obtain 4 elution peaks (as Fig. 5), regather the 3rd peak, concentrated freeze-dried, promptly get white powder material SCAIF-I.

To the SCAIF-I that is obtained by the aforesaid method separation and purification, the present invention has carried out series of experiment research.

1.SCAIF-I is to the retarding effect research of vascular endothelial cell growth.

MTT method with reference to Mosmann is carried out (Mosmann T.Rapid colorimetric assay for cel-lular growth and survival:application to prolifereation and cytotoxicity assays.J Im-muno Met, 1983; 65:55-63), be research object with calf aortic blood endothelial cell, with the skin flbroblast normal control.The result is as shown in table 1.Wherein, μ g/ml represents the concentration of SCAIF-I, and BaEc is a calf aortic blood endothelial cell, and FC is an inoblast, and P is a significance.

The result represents that the SCAIF-I has the obvious suppression effect to the growth of calf aortic blood endothelial cell, and tangible dose-dependence is arranged; When dosage was 0.1 μ g/ml, inhibiting rate still reached 47.71%; And to normal skin flbroblast unrestraint effect, counter when high density have a promoter action, the growth that prompting SCAIF-I can more special inhibition vascular endothelial cell.

Table 1 SCAIF-I is to the retarding effect of vascular endothelial cell growth

Concentration (μ g/ml)	????BaEC	????P	????FC	????P
					50 10 2 0.5 0.1 physiological saline	?0.115±0.005 ?0.201±0.038 ?0.291±0.013 ?0.313±0.039 ?0.217±0.033 ?0.415±0.119	＜0.01 ＜0.01 ＜0.01 ＜0.01 ＜0.01 ??-	?2.500±0.001 ?2.500±0.001 ?2.500±0.001 ?1.716±0.076 ?1.714±0.038 ?1.721±0.125	＜0.01 ＜0.01 ＜0.01 ＞0.05 ＞0.05 ??-

2.SCAIF-I is to the retarding effect research of chick chorioallantoic membrane vasculogenesis.

Improved and (pair thought of a way Lu Yinglin, Zhang Chaoshan etc. with reference to paying the method that waits of thinking of a way.Institute of Military Medical Science Institute periodical, 1993; 17 (4): 294-296).To hatch the 9th day application of sample and change the 4th day application of sample into, be the reagent contrast with physiological saline, the positive contrast of ANG, and SCAIF-I concentration of treatment is 0.5mg/L, the application of sample amount is 10 μ l, photographic recording result (seeing shown in Figure 6).

The result shows that physiological saline group angiogenic growth is vigorous, and caliber is thick and branch is many; SCAIF-I treatment group is then opposite.Illustrate that the SCAIF-I can significantly suppress new vessel and generate.

3.SCAIF-I is to the retarding effect research of mouse tumor growth.

With reference to methods such as Wang Hengwen (Wang Hengwen, Cheng Li chief editor.The experimental oncology basis.Beijing: People's Health Publisher, 1992.296-297)。C57BL/6 mouse inoculation Lewis lung cancer is inoculated beginning administration in the 2nd day, and dosage is 1mg/kg.d, and every day 1 time, subcutaneous or abdominal injection claims after continuous 18 days that knurl is heavy, calculates tumour inhibiting rate.Result such as table 2 and shown in Figure 7.The SCAIF-I significantly suppresses the growth of C57BL/6 Mice Bearing Lewis Lung Cancer, and the experimental group tumour is obviously little than control group.Subcutaneous injection group inhibitory rate 96.25%, abdominal injection inhibitory rate 92.76% has all almost completely suppressed growth of tumor, has shown that good research and development are worth.

Table 2 SCAIF-I is to the retarding effect of C57BL/6 Mice Bearing Lewis Lung Cancer

Group	Knurl heavy (g) (x ± s)	Tumour inhibiting rate (%)	????P
				Physiological saline SCAIF-I/ip SCAIF-I/si	????3.73±0.81 ????0.27±0.23 ????0.14±0.14	?????- ????92.76 ????96.25	??????- ????＜0.01 ????＜0.01

Result of study explanation of the present invention, SCAIF-I are the Angiostatin that purity is higher, active by force, specificity is good, can generate highly significant by the inhibition new vessel and suppress growth of tumor, and be a kind of protein that gets a good eye DEVELOPMENT PROSPECT.

SCAIF has the advantage of general anti-angiogenic hyperplasia medicine, also has oneself outstanding advantage simultaneously:

1.SCAIF-I is as anti-angiogenic hyperplasia medicine, its effect target is the vascular endothelial cell of genetic material quite stable, the neurocyte that is only second to longest-lived in the life-span of blood in human body in endothelial cell, be difficult for producing drug tolerance, and existing cell toxicant based chemotherapy medicine mostly acts on tumour cell, its genetic material is extremely unstable, is easy to generate multidrug resistance.

2. the same with other anti-angiogenic hyperplasia medicines, the SCAIF-I just only acts on the vascular endothelial cell at growth and breeding, and does not act on existing " immobilized " vascular endothelial cell, not to human body normal blood vessels generation effect.Human body except women's physiological period, wound healing and fetus and growth of children process, does not have new vessel to generate under normal physiological condition.Existing vascular endothelial cell metabolism is slow, is in relative static conditions.Therefore, anti-angiogenic hyperplasia medicine does not have obvious toxic and side effects.

3.SCAIF-I directly obtains from animal tissues's purifying, the toxicity of chemotherapeutics as none.

4.SCAIF-may there be higher conservative property in I in organism, do not find tangible anaphylaxis in experimentation on animals.

5. shark all exists in each Large Marine Ecosystem in a large number, and its fecundity is very strong, as the abundant source of cartilage, has guaranteed the raw material supply of SCAIF-I.

Description of drawings:

Fig. 1 dyes the result for the silver of SDS-PAGE SCAIF-I, and wherein Fig. 1-1 and Fig. 1-2 are the SCAIF-I, and Fig. 1-3 is Marker.

Fig. 2 is the mass spectroscopy of SCAIF-I

Fig. 3 Resource Q ion exchange chromatography (1.6 * 20cm)

Fig. 4 Sephacryl S-300 sieve chromatography (1.6 * 30cm)

Fig. 5 SDS-PAGE prepares electric arteries and veins wash-out collection of illustrative plates

Fig. 6 SCAIF-I is to chick chorioallantoic membrane vasculogenesis retarding effect

Wherein Fig. 6-1 is a physiology saline control group, and Fig. 6-2 is a SCAIF-I treatment group, and Fig. 6-3 is the ANG positive controls

Fig. 7 SCAIF-I treatment group and control group mice tumour size are relatively

Embodiment:

Adopt China East Sea whale shark cartilage 300g,, the centrifugal 25-35 of 8000rpm minute,,, obtain 3 elution peak P with Resource Q ion exchange chromatography with the acetone fractional precipitation of 45%-65% through 1mol/l Gu HCl solution extracting 45-50 hour ₁₁, P ₁₂, P ₁₃(as shown in Figure 3), buffer A:20mM Tris-Cl wherein, pH8.5 Buffer B:1M NaCl, 20mM Tris-Cl, pH7.6 collects the 3rd peak; Carry out the SephacrylS-300 sieve chromatography after concentrating, obtain 3 tangible elution peak P equally ₂₁, P ₂₂, P ₂₃, as shown in Figure 2; Carry out SDS-PAGE after again the collection of the 3rd peak being concentrated and prepare electrophoresis elution, obtain 4 peak P ₃₁, P ₃₂, P ₃₃, P ₃₄, as shown in Figure 5; Regather the 3rd peak, obtaining 0.5mg white powder material after concentrated freeze-dried is the SCAIF-I.It is carried out purity identifies, method with reference to Sambrook etc. is carried out (Sambrook J, Friescn E F, Maniatis T.Moleoular Cloning-a laboratory manual.2nd ed, cold Spring Harbor laboratotryPress, 1989.), concentrated glue with 5%, 12% separation gel adopts slab-electrophoresis, and SDS-PAGE electrophoresis silver dyes and shows a band, as shown in Figure 1, it is pure to illustrate that the SCAIF-I reaches electrophoresis, and according to the mobility of standard protein, recording its molecular weight is 18128.In addition, the molecular weight that adopts mass spectroscopy further to record the SCAIF-I is 18415, as shown in Figure 2.

Claims (2)

1. a shark-cartrilage blood-vessel generation inhibitory factor is characterized in that the SDP-PAGE electrophoresis silver of this factor dyes demonstration one band, and its molecular weight is 18000.

2. the separation purification method of a shark-cartrilage blood-vessel generation inhibitory factor, it is characterized in that shark suft bone through the guanidine hydrochloride solution extracting, behind the organic solvent fractionation precipitation, with Resource Q ion exchange chromatography, obtain 3 elution peaks, collect the 3rd peak, carry out Sephacryl S-300 sieve chromatography after concentrating, obtain 3 elution peaks, collect the 3rd peak, carry out SDS-PAGE after concentrating and prepare electrophoresis, wash-out obtains 4 peaks, regather the 3rd peak, concentrate dry freeze, promptly get white powder material SCAIF-I.

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