CN104017051B - A kind of Cyclin D protein inhibitor polypeptide and application thereof - Google Patents

Abstract

The present invention relates to drug world, be specifically related to that there is suppression Cyclin D protein expression, the polypeptide for the treatment of lymphoma mantle cell.Its sequence be ETIRRAYPDAANLLNDRVL be brand-new sequence, this polypeptide can vitro inhibition lymphoma mantle cell cell Mino cell proliferation, migration, treat lymphoma mantle cell；In body lotus tumor model experiment, successfully add the survival rate of mice, there is potential new drug development value.

Description

A kind of Cyclin D protein inhibitor polypeptide and application thereof

Technical field

The present invention relates to Cyclin D protein inhibitor polypeptide 1 and application thereof, be specifically related to that there is suppression Cyclin D protein expression, the polypeptide for the treatment of lymphoma mantle cell.

Background technology

Lymphoma mantle cell (MCL) is a kind of with t (11；14)(q13；And cyclin D1 is excessive q32) Being expressed as the B cell non-Hodgkin lymphoma of feature, belong to the lymphoma that moderate is pernicious, long-term survival rate is very Low.It is common in old people, in the majority with male, the median age 60 years old.MCL easily recurs, and is long-dated survival The lymphoma hypotype that rate is minimum, median survival interval is 3～5 years.Clinical manifestation is aggressive, and disease progression is relatively Hurry up, have extensive lymph node involvement more, tie outer pathological changes common, liver, spleen, peripheral blood and bone marrow have infiltration more, often Accompany multiple gastrointestinal tract involve and nervous system infiltration, when the patient of 80%～90% makes a definite diagnosis be Ann Arbor III～ IV phase.Treatment at present is based on chemotherapy or merges stem cell transplantation, although improves MCL to some extent and suffers from The CR of person leads, but still can not improve the long-term survival rate of patient.Biological target therapy tumor is very at present Promising, targeted therapy is for blocking certain or multiple signal path in tumor generating process, reaches treatment The purpose of tumor.

Rest that Cyclin albumen controls cell as " checkpoint " guarder, the cycle growing and dividing. Wherein, Cyclin D is the important target spot of growth cycle controlling cell.Cyclin D determines when cell starts Generate DNA, form new cell for division and prepare.Cyclin D overexpression in the most eurypalynous cancer, The too fast growth of cell is stimulated to form tumor.Research finds, blocks cyclin D1 albumen and can order about breast cancer cell Enter aging state, irreversibly terminate their growth cycle.Cyclin is suppressed in T-ALL leukemia mouse D can cause cancerous cell self-destruction (a programmed death process being referred to as apoptosis).Therefore, suppression Cyclin D egg White expression, suppression lymphoma mantle cell development, is the novel targets for the treatment of lymphoma mantle cell.But, not yet have The medicine of the treatment lymphoma mantle cell of the Cyclin D protein inhibitor polypeptide that exploitation is ripe

Cyclin D protein inhibitor polypeptide 1 in this patent is proved in lymphoma mantle cell effectively, tool There is the prospect of exploitation in other tumor models.

Summary of the invention

Goal of the invention

The present invention provides brand-new sequence, and this sequence suppression Cyclin D protein expression, to treatment jacket cell lymph Tumor has good curative effect.

Technical scheme

Cyclin D protein inhibitor polypeptide 1, it is characterised in that its sequence is ETIRRAYPDAANLLNDRVL.

A kind of pharmaceutical composition, it is characterised in that its comprise polypeptide as claimed in claim 1 and more than one Pharmaceutically acceptable excipient, filler, binding agent, lubricant, disintegrating agent or stabilizer.

Described pharmaceutical composition, it is characterised in that described compositions is injection.

Described Cyclin D protein inhibitor polypeptide 1, it is characterised in that effective dose is 10mg/kg.

The Cyclin D protein inhibitor polypeptide 1 application in treatment lymphoma mantle cell medicine.

Beneficial effect

Cyclin D protein inhibitor polypeptide 1, this polypeptide has brand-new sequence, and this polypeptide can vitro inhibition set Cell lymphoma Mino cell proliferation, migrates, and treats lymphoma mantle cell；Body lotus tumor model experiment becomes The suppression tumor of merit increases, and adds the survival rate of mice, has potential new drug development value.

Accompanying drawing explanation

Detailed description of the invention

The present invention relates to polypeptide ETIRRAYPDAANLLNDRVL, by the synthesis of gill biochemistry (Shanghai) Co., Ltd..

Embodiment 1

The effect that Cyclin D protein inhibitor polypeptide 1 people lymphoma mantle cell cell Mino migrates.

Use scratch experiment.First with marker pen at 24 orifice plates behind, compare with ruler, uniform must draw horizontal stroke Line, per every about 0.5～1cm together, crosses via.Every hole is through 3 lines；The Mino of logarithmic growth will be become Cell, adds in 24 well culture plates with 1.0 × 105, cultivates 24h.Within second day, compare ruler with 10 μ l rifle heads, It is perpendicular to horizontal line cut behind, with the cross point of cut and horizontal line behind for Orientation observation site；Experimental port, Positive drug control hole is separately added into Experimental agents Cyclin D protein inhibitor polypeptide 1 and the positive of variable concentrations Control drug vincristine；Blank group adds the solvent of same volume, and every hole sets five multiple holes；Put into 37 DEG C, 5%CO₂Incubator, cultivates.By 0,6,12,24,36 hours, take pictures；Measure 0,6,12,24, 36h scratch width.With different time points, record the change of scratch width at fixed position, three, every hole, i.e. For cell migration distance.According to formula computation migration rate (migration rate, MR): mobility (MR)= (testing scratch width-experiment scratch width of the 0th hour of n-th hour) × 100%/experiment the 0th hour Scratch width.As a result,

Group	MR
		Blank group	100%
Positive controls 32 μ g/ml	77.43
		Experimental group 32 μ g/ml	76.35

Result shows, when dosage is 32 μ g/ml, has pole significant difference (p < 0.01), with positive group effect Quite.Illustrate that Cyclin D protein inhibitor polypeptide 1 can suppress people lymphoma mantle cell cell Mino to migrate.

Embodiment 2

The effect of Cyclin D protein inhibitor polypeptide 1 people lymphoma mantle cell cell Mino propagation.

Use the activity of mtt assay detection polypeptide suppression Mino cell growth.Mino cell is at 37 DEG C, 5%CO₂ Incubator in cultivate to more than 90% degree of converging time with trypsinization collect, use culture fluid re-suspended cell And count under the microscope, cell concentration is adjusted to 2 × 10⁴Individual/ml, by cell suspension inoculation to 96 orifice plates In, 100 μ l/ holes, and in 37 DEG C, 5%CO₂Overnight incubation in incubator.Polypeptide culture fluid is diluted to each Predetermined concentration.RhEndostatin culture fluid is diluted to final concentration.After cell is the most adherent, by each diluent respectively Add in (100 μ l/ hole) in 96 orifice plates.Using add AP25 diluent as administration group, with Addition rhEndostatin, paclitaxel are as positive controls, to be not added with the culture fluid of any medicine as negative control group. At 37 DEG C, 5%CO₂Incubator hatches 48h.The MTT of 5mg/ml, every hole 20 μ l is added in 96 orifice plates, Continue to cultivate 4h.Sopping up culture medium, every hole adds 150 μ l DMSO and dissolves, and shaking table mixes for 10 minutes gently. Being 570nm by microplate reader measuring wavelength, reference wavelength is to measure light absorption value 630nm at, and calculates growth Suppression ratio (proliferation inhibition, PI), formula is as follows:

PI (%)=1-administration group/feminine gender group

The result that test obtains represents with mean ± SD, and carries out adding up T inspection, and * P < 0.05 is significance Difference, * * P < 0.01 is pole significant difference.

Table 2. AP25 is to people's gastric cancer cell line MGC-803 inhibited proliferation

Result: be shown in Table 2, compared with negative control, it is thin that polypeptide can significantly inhibit polypeptide suppression Mino in vitro The propagation of born of the same parents, and present obvious dose-dependence.

Embodiment 3

Cyclin D protein inhibitor polypeptide 1 is raw to people's lymphoma mantle cell cell Mino nude mouse xenograft tumor Long inhibition test

Take the logarithm people's lymphoma mantle cell cell Mino cell strain of trophophase, be aseptically prepared as 5×10⁷/ ml cell suspension, is inoculated in axillary fossa on the right side of nude mice with 0.1ml subcutaneous.Move with vernier caliper measurement nude mice Plant tumor diameter, treat that tumor growth is to 100-200mm³After by animal random packet.Use the method measuring tumor footpath, Dynamically observe the antitumous effect of tested polypeptide.The pendulous frequency of diameter of tumor is to survey 1 time for every 2 days.To prescription Formula all uses 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 once daily；High, normal, basic group of polypeptide is respectively With 20mg/kg, 10mg/kg, 5mg/kg, it is administered once daily.After off-test, sacrifice, operation Strip tumor mass to weigh.

The inhibitory action that people's lymphoma mantle cell cell Mino nude mouse xenograft tumor is grown by table 3 polypeptide

Result: be shown in Table 1, paclitaxel 10mg/kg group is to people's lymphoma mantle cell cell Mino transplanted tumor in nude mice Tumour inhibiting rate be 73.11%；RhEndostatin 2.5mg/kg group is to people's lymphoma mantle cell cell Mino transplanted tumor in nude mice Tumour inhibiting rate be 38.47%；People lymphoma mantle cell cell Mino nude mice is moved by polypeptide high, medium and low dosage group The tumour inhibiting rate planting tumor reaches 78.19%, 76.21%, 69.73% respectively.But paclitaxel toxicity is relatively big, the weight of animals Declining, in experimentation, animal has death.And nude mice body weight is not had significance to affect by polypeptide.

Therefore, people's lymphoma mantle cell cell Mino transplanted tumor in nude mice growth inhibition test result is shown by polypeptide, Compared with negative control group, polypeptide 20mg/kg, 10mg/kg and 5mg/kg group is thin to people's lymphoma mantle cell The growth of born of the same parents' Mino transplanted tumor is respectively provided with the inhibitory action of pole significance.Compared with positive controls paclitaxel, The body weight of laboratory animal is had not significant impact by polypeptide, has no obvious toxicity, and survival rate improves.

SEQUENCE LISTING

<110> Pu Luoda bio tech ltd, Suzhou

<120> A kind of Cyclin D protein inhibitor polypeptide and application thereof

<130>

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 19

<212> PRT

<213> Artificial sequence

<400> 1

Glu Thr Ile Arg Arg Ala Tyr Pro Asp Ala Ala Asn Leu Leu Asn Asp

1 5 10 15

Arg Val Leu

Claims (5)

1.Cyclin D protein inhibitor polypeptide 1, it is characterised in that its sequence is ETIRRAYPDAANLLNDRVL.

2. a pharmaceutical composition, it is characterised in that it comprises polypeptide as claimed in claim 1 and more than one pharmaceutically acceptable filleies, binding agent, lubricant, disintegrating agent or stabilizer.

3. pharmaceutical composition as claimed in claim 2, it is characterised in that described compositions is injection.

4. Cyclin D protein inhibitor polypeptide 1 as claimed in claim 1, it is characterised in that effective dose is 10mg/kg.

5. the Cyclin D protein inhibitor polypeptide 1 as claimed in claim 1 application in preparation treatment lymphoma mantle cell medicine.