A kind of somatostatin receptor agonist polypeptide and application thereof
Technical field
The present invention relates to somatostatin receptor agonist polypeptide 1 and application thereof, be specifically related to that there is promotion somatostatin receptor activity, the polypeptide for the treatment of hepatocarcinoma.
Background technology
Hepatocarcinoma is one of modal malignant tumor of China, and since the nineties in 20th century, China's mortality of liver cancer has been raised to the 2nd of malignant tumor, there are about 130,000 people every year and dies from hepatocarcinoma.Primary hepatocarcinoma PD is fast, if without treatment, Natural Survival phase average out to 4.3 months, even if excision prognosis is also undesirable, within 5 years, survival rate is 37.2%, and 5 years survival rates of small liver cancer Post operation are also only 53.5%.The main cause affecting liver cancer patient surgical result is recurrence and transfer.Recurrence of PHC rate may be up to more than 60%, even if small liver cancer Postoperative recurrent rate is also up to 40%.Current Biological target therapy tumor is very promising, and targeted therapy is for blocking certain or multiple signal path in tumor generating process, reaches the purpose for the treatment of tumor.
Somatostatin is except having suppression intestinal hormone secretion, also has the effect suppressing the tumor cell proliferations such as digestive tract tumor such as gastric cancer, cancer of pancreas, hepatocarcinoma, thus somatostatin becomes the medicine of potential treatment tumor, especially for hepatocarcinoma, the effects such as it has suppression tumor growth, inducing cell apoptosis.Existing bibliographical information, is used for the treatment of advanced liver cancer by the long-acting somatostatin anologues of synthetic and obtains certain effect at present.Pharmaceutical research confirms, somatostatin suppresses the effect of tumor growth with somatostatin receptor (SSTR) the specific bond competence exertion in tumor tissues.All there is the expression of SSTR in the many entity tumors including hepatocarcinoma, and in vitroreceptorautoradiography and radioligand binding are it have also been found that liver cancer tissue exists SSTR.Therefore, somatostatin receptor agonist, it is possible to promote the activity of SSTR, it is suppressed that the growth of tumor, is prevention and the novel targets for the treatment of liver cancer recurrence.But, not yet have the medicine of the treatment hepatocarcinoma of the somatostatin receptor agonist polypeptide of exploitation maturation.
Somatostatin receptor agonist polypeptide 1 in this patent has turned out in hepatocarcinoma effective, has the prospect of exploitation in other tumor models.
Summary of the invention
Goal of the invention
The present invention provides brand-new sequence, this sequence somatostatin receptor agonist, and prevention and treatment liver cancer recurrence are had good curative effect.
Technical scheme
Somatostatin receptor agonist polypeptide, it is characterised in that its sequence is VALPQEPASAGLYFFVV.
A kind of pharmaceutical composition, it is characterised in that it comprises polypeptide as claimed in claim 1 and more than one pharmacy
Upper acceptable excipient, filler, binding agent, lubricant, disintegrating agent or stabilizer.
Described pharmaceutical composition, it is characterised in that described compositions is injection.
Described polypeptide is characterized in that effective dose is 10mg/kg.
The application in preparation treatment liver-cancer medicine of the described somatostatin receptor agonist polypeptide.
Beneficial effect
Utilizing solid-phase synthesis chemosynthesis somatostatin receptor agonist polypeptide, this polypeptide has brand-new sequence, and this polypeptide can promote the activity of somatostatin receptor, prevention and treatment liver cancer recurrence.
Detailed description of the invention
The present invention relates to polypeptide by gill biochemical (Shanghai) synthesis.
Embodiment 1
The effect to HepG2 cell proliferation of the somatostatin receptor agonist polypeptide.
Mtt assay detection somatostatin receptor agonist polypeptide is adopted to suppress the activity of HepG2 cell growth.
HepG2 cell 37 DEG C, the incubator of 5%CO2 is cultured to more than 90% degree of converging time collect with trypsinization, with culture fluid re-suspended cell and count under the microscope, cell concentration is adjusted to 2 × 104/ml, by in cell suspension inoculation to 96 orifice plates, 100 μ l/ holes, and in 37 DEG C, overnight incubation in 5%CO2 incubator.Somatostatin receptor agonist polypeptide culture fluid is diluted to each predetermined concentration.RhEndostatin culture fluid is diluted to final concentration.After cell is completely adherent, each diluent is separately added in 96 orifice plates (100 μ l/ hole).Using add somatostatin receptor agonist polypeptide diluent as administration group, to add rhEndostatin, paclitaxel as positive controls, to be not added with the culture fluid of any medicine as negative control group.At 37 DEG C, 5%CO2 incubator hatches 48h.In 96 orifice plates, add the MTT of 5mg/ml, every hole 20 μ l, continue to cultivate 4h.Sopping up culture medium, every hole adds 150 μ lDMSO and dissolves, and shaking table mixes for 10 minutes gently.Being 570nm by microplate reader measuring wavelength, reference wavelength is 630nm place mensuration light absorption value, and calculates growth inhibition ratio (proliferationinhibition, PI), and formula is as follows:
PI (%)=1-administration group/feminine gender group
The result that test obtains represents with mean ± SD, and carries out statistics T inspection, and * P < 0.05 is significant difference, and * * P < 0.01 is pole significant difference.
Table 1 somatostatin receptor agonist polypeptide is to HepG2 Cells Cell Proliferation inhibitory action
Result: in Table 1, compared with negative control, somatostatin receptor agonist polypeptide can significantly inhibit the propagation of HepG2 cell in vitro, and presents obvious dose-dependence.
Embodiment 2
The effect to HepG2 cell migration of the somatostatin receptor agonist polypeptide.
Being diluted with 1:3 by 10mg/mlMatrigel (BD company, the USA) cell culture fluid of serum-free, coat on Transwell cell (Greiner company, USA) film, room temperature is air-dry.By cultivating, the HepG2 arriving exponential phase is cells trypsinised, collects, resuspended with serum-free cell culture medium, in counted under microscope, cell concentration adjusts 1 × 105/ml.Prepare each group of test liquid, be grouped as follows: blank group: for the serum-free cell culture medium of not drug containing;RhEndostatin group: the rhEndostatin liquid storage of 5mg/ml is diluted to predetermined concentration with the serum-free cell culture medium of not drug containing;Somatostatin receptor agonist polypeptide group: somatostatin receptor agonist polypeptide is diluted to each predetermined concentration with the serum-free cell culture medium of not drug containing.Seed cells in Transwell cell, every hole 100 μ l, and each group of test liquid is added in cell.24 orifice plates add the 0.6ml cell culture fluid containing 5% hyclone and 1% endothelial cell growth factor (ECGF) (ECGS) and stimulates cell migration, in 5%CO2, hatch 24 hours for 37 DEG C.Discarding Kong Zhongpei liquid, fix 30 minutes with dehydrated alcohol room temperature, 0.1% crystal violet room temperature dyes 10 minutes, and clear water rinses, and dabs off the non-migrating cell in upper strata with cotton swab, and basis of microscopic observation also randomly chooses four visuals field and takes pictures counting.According to formula computation migration suppression ratio (migrationinhibition, MI):
MI (%)=(1-Ntest/Ncontrol) × 100%
Wherein Ntest is the cell migration number of test group, and Ncontrol is the cell migration number of blank group.The result that test obtains represents with mean ± SD, and carries out statistics T inspection, and * P < 0.05 is significant difference, and * * P < 0.01 is pole significant difference.
Table 2 polypeptide is to HepG2 cellular migration inhibition effect
Result: under the effect of polypeptide, the HepG2 cell number of migration substantially reduces.Compared with blank group, many Toplink suppress the migration of the HUVEC of 5% hyclone and 1%ECGS induction.Under 0.5 μ g/ml and two dosage of 1 μ g/ml, the inhibitory action of polypeptide on cell migration has pole significant difference compared with blank, when the dosage of polypeptide is 0.5 μ g/ml, reaches maximum to the suppression ratio of HepG2 cell migration.
Embodiment 3
The inhibition test that HepG2 cell nude mouse xenograft tumor is grown by polypeptide
Take the logarithm the HepG2 cell strain of trophophase, aseptically prepare into 5 × 107/ml cell suspension, be inoculated in axillary fossa on the right side of nude mice with 0.1ml subcutaneous.With vernier caliper measurement transplanted tumor in nude mice diameter, by animal random packet after tumor growth to 100-200mm3.Use the method measuring tumor footpath, dynamically observe the antitumous effect of tested polypeptide.The pendulous frequency of diameter of tumor is survey 1 time for every 2 days.Administering mode all adopts tail vein injection.Negative control group injection normal saline, every day 1 time;Paclitaxel group 10mg/kg, Per-Hop behavior 1 time;RhEndostatin group 2.5mg/kg, is administered 1 every day;High, normal, basic group of polypeptide, respectively with 20mg/kg, 10mg/kg, 5mg/kg, is administered 1 every day.After off-test, sacrifice, operation strips tumor block and weighs.
The inhibitory action that HepG2 cell nude mouse xenograft tumor is grown by table 3 polypeptide
Therefore, HepG2 cell transplanted tumor in nude mice growth inhibition test result is shown by polypeptide, compared with negative control group, the growth of HepG2 cell transplantation tumor is had the inhibitory action of pole significance by polypeptide 20mg/kg group, and the growth of HepG2 cell transplantation tumor is had the inhibitory action of significance by polypeptide 10mg/kg group.Compared with positive controls paclitaxel, the body weight of laboratory animal is had not significant impact by polypeptide, has no obvious toxicity.
SEQUENCELISTING
<110>Suzhou Pu Luoda bio tech ltd
<120>a kind of somatostatin receptor agonist polypeptide and application thereof
<130>
<160>1
<170>PatentInversion3.3
<210>1
<211>17
<212>PRT
<213>artificial sequence
<400>1
ValAlaLeuProGlnGluProAlaSerAlaGlyLeuTyrPhePheVal
151015
Val