CN107537027A - TNFSF15 albumen is preparing the purposes in treating melanoma medicine - Google Patents

Abstract

The present invention relates to TNFSF15 albumen to prepare the purposes in treating melanoma medicine, present invention discover that TNFSF15 can not only suppress endothelial cell growth, induces its apoptosis, can also suppress the growth and migration of B16 mouse melanoma cell line, inducing cell apoptosis.Present invention discover that during using TNFSF15 combined chemotherapy drug therapy melanomas, TNFSF15 not only can suppress the generation of tumour by suppressing tumor vascular generation, and can be by inducing melanoma cells apoptosis, lethal effect of the enhancing chemotherapeutics for melanoma cells.Finally, it has been found that TNFSF15 can strengthen the effect of plus cisplatin in treatment melanoma in the use in conjunction of the chemotherapeutic drugs Cisplatin with being usually used in treating melanoma.

Description

TNFSF15 albumen is preparing the purposes in treating melanoma medicine

Technical field

The present invention relates to field of medicaments, more particularly to TNFSF15 albumen is preparing the purposes in treating melanoma medicine, And prepare the purposes in treatment melanoma medicine with Cisplatin.

Background technology

Malignant mela noma (malignant melanoma, MM) be due to inherent cause, environmental factor joint effect and A kind of caused tumour occurred in skin or other organ melanocytes.This sick clinical manifestation is tenderness, itch, gone out Blood, ulcer etc., the elderly's symptom are attached most importance to compared with young man.The growth rate of this malignant tumour is fast more many than other kinds of, In past 30 years, the incidence of U.S.'s malignant mela noma increases one times.World Health Organization, every year in full generation There are 132,000 such malignant tumours to be diagnosed in the range of boundary.If malignant mela noma can be diagnosed in getting up early, The case that by surgery operating removing tumor, can have 80% is treated by this mode.But come for existing treatment means It is plain stubborn difficult to treat to say metastasis melanin tumor, and the prognosis of patient also can be very poor, six months of this some patients and 5 The average viability in year is below 5%.Therefore new therapeutic strategy is urgently developed.

Chemotherapy occupies an important position in malignant mela noma treatment.For course of disease middle and advanced stage or with high risk factor Early lesion should give regional or systemic chemotherapy.Chemotherapy can be used for perioperatively, before and after radiotherapy, or with putting Treatment is carried out simultaneously, so as to improve curative effect.At present chemotherapeutics mainly have Dacarbazine (dacarbazine, dacarbazine, DTIC), BCNU (sarmustine, BCNU), vincristine (vincristine, VCR), alkeran (melphalan, MEL), actinomycin D and cis-platinum (cisplatin, DDP) etc..Cis-platinum (DDP) is the master in current clinically a variety of chemotherapy regimens Want one of medicine, can by with cancer cell DNA (DNA) occur cross link, the apoptosis of induced tumor cell and rise Antitumaous effect.Melanoma cells are better than single medicine for chemotherapeutics relative insensitivity, Combination chemotherapy reactivity.Institute With searching and medicine associated with DDP, lethal effects of the DDP for melanoma cells can be strengthened, and reduce what DDP was brought Toxic side effect, the treatment for melanoma are significant.

The content of the invention

It is an object of the invention to provide TNFSF15 albumen to prepare the purposes in treating melanoma medicine.

It is a further object of the present invention to provide TNFSF15 albumen and Cisplatin in treatment melanoma medicine is prepared Purposes.

The technical solution adopted by the present invention is：

The invention provides TNFSF15 albumen to prepare the purposes in treating melanoma medicine.

Further, in the purposes, by the TNFSF15 albumen of therapeutically effective amount and pharmaceutically acceptable carrier and/or Excipient is mixed and made into composition.

Present invention also offers TNFSF15 albumen and Cisplatin to prepare the purposes in treating melanoma medicine.

Further, in the purposes, by TNFSF15 albumen, cis-platinum and the pharmaceutically acceptable carrier of therapeutically effective amount And/or excipient is mixed and made into composition.

Preferably, in such use, described TNFSF15 albumen is prepared by the purification process comprised the following steps 's：

(1) induced expression

A) will expression bacterial strain in the flat lining outs of LB containing ammonia benzyl resistance, cultivate 12h at 37 DEG C；

B) oese picking monoclonal, 37 DEG C in the LB culture mediums that 5mL contains ammonia benzyl resistance, 220rpm is in amplification 12h.

C) will amplification gained bacterium solution 1:100 are added in the LB culture mediums containing ammonia benzyl resistance, 37 DEG C, under the conditions of 220rpm Make bacterial growth to exponential phase, OD values 0.6~0.8；

D) bacterium solution is placed in 4 DEG C of standing 30min；

E) final concentration of 50 μM of IPTG is added after standing, 16 DEG C, 220rpm induces 24h.

(2) cellular lysate

A) 4 DEG C, 8000rpm centrifugations 10min collects thalline；

B) add 25mL lysates per 200mL bacterium solutions, with No. 630-0561 probe, ultrasonic 1s, suspend 1s, 30% power ice Upper ultrasonic 1min, totally 2 times；

C) 5mL 10%Triton X-100 are added, same time interval and power ultrasonic 1min on ice, totally 2 times；

D) bacterium solution is placed in and stands 30min on ice；

E) 4 DEG C of bacterium solution after standing, 5000rpm centrifugation 30min separation obtain cracking supernatant；

(3) upper prop elutes

A) 10 times of column volume ddH₂O rinses nickel post, 5 times of column volume equilibrium liquid balance nickel posts；

B) supernatant and Ni-Agarose will be cracked using volume ratio as 2:14 DEG C of 100rpm of ratio are sufficiently mixed 2h；

C) the solution upper prop that will be mixed, 4 DEG C of collection effluxes；

D) after efflux all flows out, 4 DEG C of washing foreign proteins of cleaning fluid of 8 times of column volumes are added；

E) with 4 DEG C of elutions of eluent of 4 times of column volumes, the eluent of 2 volumes and rear 2 volume before collecting respectively.

(4) desalination dispenses

A) using a kind of progress desalination in following two modes：

Mode one：The albumen being collected into is loaded into bag filter, albumen and dialyzate are with 1:100 volume ratio is in 4 DEG C of progress Dialysis, the mixing speed of magneton is 200rpm, and dialyse 3h every time, is dialysed 2 times；

Mode two：By the albumen being collected into and saline solution is changed with 1:Salt plug is changed in 1 ratio mixing, addition, 4 DEG C, 5000rpm from Heart 15min.The solution for changing salt plug upper strata is collected, repeats aforesaid operations 10 times.

B) gained albumen is degerming using 0.22 μM of membrane filtration, packing, -80 DEG C of preservations.

Further, the expression bacterial strain in described step (1) is：Express express target protein is VEGI-192 hypotypes, contains 192 Individual amino acid, the vector plasmid used is pET-21b (+), and expression competence is BL21 (DE3), and gained expression bacterial strain is ammonia benzyl Resistance.

Further, the lysate in described step (2) is NaCl 20mM, Tris-Cl 10mM, and lauryl creatine is sour Sodium 50mM, pH7.9.

Further, equilibrium liquid is 50mM NaH in described step (3)₂PO₄, 300mM NaCl, pH 8.0；Cleaning fluid is 50mM NaH₂PO₄, 300mM NaCl, 20mM imidazoles, pH 8.0；Eluent is 50mM NaH₂PO₄, 300mM NaCl, 250mM Imidazoles, pH 8.0.Preferably, the equilibrium liquid, cleaning fluid, eluent are using being preceding pre-chilled to 4 DEG C, all operating process Carried out in 4 DEG C of refrigerators.

Further, dialyzate and to change saline solution be PBS in described step (4), pH 8.0, being pre-chilled to 4 DEG C makes With.

Purification schemes of the present invention reduce albumen and existed by way of low temperature induction, and with very low IPTG induced concentrations Expression in inclusion body, lifts the content of soluble protein, and with more gentle cracking way of purification, improves the stabilization of albumen Property and activity.And easy to operate, fabrication cycle is short, consumption resource is few, convenient amplification production.

Beneficial effect possessed by the present invention：

Present invention discover that TNFSF15 can suppress the growth and migration of B16 mouse melanoma cell line, inducing cell withers Die.

Present invention discover that during using TNFSF15 combined chemotherapy drug therapy melanomas, TNFSF15 can not only pass through suppression Tumor vascular generation is made to suppress the generation of tumour, and chemotherapeutic can be strengthened by inducing melanoma cells apoptosis Lethal effect of the thing for melanoma cells.Finally, it has been found that TNFSF15 is in the chemotherapy with being usually used in treating melanoma During the use in conjunction of drugs Cisplatin, the effect of plus cisplatin in treatment melanoma can be strengthened.

Brief description of the drawings

Fig. 1 left figures are the results of B16 cells scratch experiment under the influence of various concentrations TNFSF15, and right figure is to this reality Test the statistical result of middle cell migration distance.

Fig. 2 is that various concentrations TNFSF15 stimulates the lower protein expression situations of B16 cells Cleaved-Caspase 3.

Fig. 3 left figures are that the TNFSF15 albumen that concentration is 5 μ g/mL detects for the growth inhibition of B16 cells, and right figure is DDP With DDP+5 μ g/mL TNFSF15 for B16 cell growth inhibition curves.

Fig. 4 left figures are that DDP groups/TNFSF15 groups/DDP+TNFSF15 groups/Buffer group C57BL/6J melanoma lotus knurls are small Mouse gross tumor volume statistical result.Right figure is to start injection medicine to put to death mouse taking-up melanoma after 7 days, is detected obtained by its weight Statistical result.

Embodiment

In order to understand the present invention, with reference to specific embodiment, the invention will be further described, but does not limit the present invention Protection domain.

Embodiment 1：TNFSF15 suppresses the transfer ability of B16 mouse melanoma cell line

A) B16 cells are at 37 DEG C, 5%CO₂Under conditions of culture grow into logarithmic phase, digest, count, be taped against in 6 orifice plates, Per hole 5X 10⁵Individual cell, adherent 12h, make the degrees of fusion of cell close to 100%；

B) rule with yellow pipette tips perpendicular to orifice plate along ruler, per hole every mono- line of 0.5cm, per three, hole, use PBS Rinse twice, rinse out non-attached cell；

C) 2mL serum free mediums are added per hole, is taken pictures under white light with 10X object lens 10X eyepieces, it is wide to record cut under 0h Degree；

D) TNFSF15 that final concentration is respectively 0,10,25,50 μ g/mL, at 37 DEG C, 5%CO are added₂Under conditions of cultivate 24h；

E) taken pictures under white light with 10X object lens 10X eyepieces, record scratch width under 24h.

As a result as shown in figure 1, can see TNFSF15 concentration since 10 μ g/mL by Fig. 1, it is possible to obvious to suppress Transfer ability of the B16 cells on orifice plate, the migration distance of cell reduce 16.08%.Raising protein concentration, TNFSF15 pairs It is even more to be obviously improved in the suppression of the transfer ability of B16 cells, the inhibition of metastasis rate under 25 μ g/mL and 50 μ g/mL concentration is divided Wei 75.11% and 78.37%.Prove that TNFSF15 can significantly inhibit the transfer ability of B16 mouse melanoma cell line.

Embodiment 2TNFSF15 inducing mouse melanoma cells B16 apoptosis

A) B16 cells are at 37 DEG C, 5%CO₂Under conditions of culture grow into logarithmic phase, digest, count, be taped against 12 orifice plates In, per hole 2.5X 10⁴Individual cell, adherent 12h.

B) TNFSF15 that final concentration is respectively 0,1,5,10 μ g/mL, at 37 DEG C, 5%CO are added₂Under conditions of cultivate 48h。

C) total protein of cell is extracted：Remove cell culture medium, quickly wash cell 3 times with the PBS of precooling；Add pre- per hole The cold μ L of protein lysis buffer 100 (50mM Tris-HCl.pH 7.4,1%NP-40,150mM NaCl, 1mM EDTA, 1mM PMSF), with cell scraper Assisted Cleavage cell；Collect in liquid mixture centrifuge tube, be incubated 30 minutes on ice；4 DEG C, 20000g Centrifuge 30min；Supernatant is taken, protein concentration is determined with BCA methods, Cleaved- in cell is detected using Western Blotting methods Caspase 3 protein level.

D) Western Blotting methods are：1) each μ g albumen of sample 10 is taken, polyacrylamide gel electrophoresis is carried out and (divides From gum concentration 12%)；Voltage 60V, flattens band, rise voltage to 120V, electrophoresis 1.5h for 0.5 hour；2) after electrophoresis, dismounting is solidifying Adhesive dispenser, and gel is taken out, it is placed in transferring film buffer solution and soaks 1 minute；3) pvdf membrane after methanol activation is taken, after immersion Gel, pvdf membrane, support filter paper, the fitting such as sponge compress, be fitted into membrane-transferring device；4) 100V constant pressures, 4 DEG C of transferring films 2 hours； 5) after transferring film terminates, pvdf membrane is taken out, with TBST buffer solutions vibration washing 3 times, every time 5 minutes；6) use and contain 5% skimmed milk power TBST solution closing, normal temperature, 1 hour；7) antibody (1 of Caspase 3 is added:2000), 4 DEG C, reaction overnight；8) suck anti- Body, cleaned 6 times, every time 5 minutes with TBST；9) the anti-rabbit secondary antibody (1 of HRP marks is added:5000), 37 DEG C, 1 hour；10) Antibody is sucked, is cleaned 6 times, every time 5 minutes with TBST；11) fluorescence developing Substrate cocktail is added in day energy gel imager 30s is incubated on to pvdf membrane, chooses the suitable time for exposure, preserves exposure results.(β-Actin are used as internal reference, operating process phase Together)

As a result it is as shown in Figure 2, it can be seen that after TNFSF15 is added, the albumen of B16 cells Cleaved-Caspase 3 Expression significantly rise.Caspase 3 is that an important apoptosis performs albumen, during the digestion of many critical proteins it There is more or less participation, Cleaved-Caspase 3 is Caspase 3 activated state obtained after shearing.Institute B16 cells Cleaved-Caspase 3 expression can be raised with TNFSF15, it was demonstrated that TNFSF15 can induce B16 cells Apoptosis.

Embodiment 3TNFSF15 can suppress B16 cell growths, and improve suppression energy of the cis-platinum for B16 cell growths Power

A) B16 cells are at 37 DEG C, 5%CO₂Under conditions of culture grow into logarithmic phase, digest, be taped against in 96 orifice plates, per hole 10³Individual cell, adherent 12h.

B) DDP groups/DDP+TNFSF15 groups are separately added into the DDP and final concentration of 5 μ g/mL of various concentrations TNFSF15 eggs In vain.The specific concentration of DDP such as following table, each 4 multiple holes of concentration gradient.

37 DEG C, 5%CO₂Under conditions of stimulate 72h.

C) culture medium in orifice plate is discarded, PBS 2 times, the nothing containing final concentration of 0.5 μ g/mL MTT is added per hole Blood serum medium.37 DEG C of 5%CO₂It is incubated 6h.

D) culture medium containing MTT, PBS 2 times, 150 μ L of the addition DMSO per hole are discarded.On 37 DEG C of shaking table 200rpm dissolves 10min.

E) 96 orifice plates detect its absorbance under 570nm on ELIASA, make cells survival curve, calculate IC50.

As a result as shown in figure 3, left figure be concentration be 5 μ g/mL TNFSF15 albumen for B16 cells growth inhibition examine Survey, the TNFSF15 of the concentration is 12.01% for the inhibition level of B16 cell growths.Right figure is DDP and DDP+5 μ g/mL TNFSF15 is 22.88 μM for B16 cell growth inhibition curves, DDP groups IC50, and DDP+5 μ g/mL TNFSF15 groups IC50 is 6.763μM.The growth of B16 cells can be suppressed by demonstrating TNFSF15, and can significantly be carried after having used TNFSF15 albumen Growth inhibition effect of the high cis-platinum for B16 cells.

Embodiment 4TNFSF15 and cis-platinum use in conjunction, killing ability of the enhancing cis-platinum for murine melanoma

A) B16 cells are at 37 DEG C, 5%CO₂Under conditions of culture grow into logarithmic phase, digest, be diluted to serum-free In DMEM culture mediums, concentration is 5X 10⁶/mL。

B) 8 weeks C57BL/6J female mices are divided into 4 groups (DDP groups/TNFSF15 groups/DDP+TNFSF15 groups/Buffer groups), every group 7.

C) every μ L 5X 10 of mouse subcutaneous injection 100⁵B16 cells.

D) the 10th day after inoculating cell, tumour is more than 100mm³, start to carry out within every 2 days once abdominal cavity injection administration, administration Dosage is：

DDP groups：Cis-platinum 5mg/kg

TNFSF15 groups：TNFSF15 5mg/kg.

DDP+TNFSF15 groups：Cis-platinum is dissolved in TNFSF15 solution, cis-platinum 5mg/kg, TNFSF15 5mg/kg.

Buffer groups：According to the Buffer liquid of three groups of same calculation injection certain volumes with more than.

E) with wide, gross tumor volume calculation is the length of every 2 days detection tumours：Long X is wide²/2。

F) the 7th day after being administered, mouse tumor is taken out, detects gross tumor volume and quality.

As a result as shown in figure 4, either from gross tumor volume still from the quality of tumour, TNFSF15 can may be used To suppress the growth of murine melanoma to a certain extent, and cis-platinum can be dramatically increased for mouse melanoma Inhibition.

Claims (5)

1.TNFSF15 albumen is preparing the purposes in treating melanoma medicine.

2. purposes according to claim 1, it is characterised in that：In the purposes, by the TNFSF15 albumen of therapeutically effective amount Composition is mixed and made into pharmaceutically acceptable carrier and/or excipient.

3.TNFSF15 albumen and Cisplatin are preparing the purposes in treating melanoma medicine.

4. purposes according to claim 3, it is characterised in that：By TNFSF15 albumen, cis-platinum and the pharmacy of therapeutically effective amount Upper acceptable carrier and/or excipient are mixed and made into composition.

5. according to the purposes described in claim any one of 1-4, it is characterised in that：Described TNFSF15 albumen is by including such as What the purification process of lower step was prepared：

(1) induced expression

A) will expression bacterial strain in the flat lining outs of LB containing ammonia benzyl resistance, cultivate 12h at 37 DEG C；

B) oese picking monoclonal, 37 DEG C in the LB culture mediums that 5mL contains ammonia benzyl resistance, 220rpm is in amplification 12h.

C) will amplification gained bacterium solution 1:100 are added in the LB culture mediums containing ammonia benzyl resistance, 37 DEG C, make under the conditions of 220rpm thin Bacteria growing is to exponential phase, OD values 0.6~0.8；

D) bacterium solution is placed in 4 DEG C of standing 30min；

E) final concentration of 50 μM of IPTG is added after standing, 16 DEG C, 220rpm induces 24h.

(2) cellular lysate

A) 4 DEG C, 8000rpm centrifugations 10min collects thalline；

B) 25mL lysates are added per 200mL bacterium solutions, with No. 630-0561 probe, ultrasonic 1s suspends 1s, and 30% power surpasses on ice Sound 1min, totally 2 times；

C) 5mL 10%Triton X-100 are added, same time interval and power ultrasonic 1min on ice, totally 2 times；

D) bacterium solution is placed in and stands 30min on ice；

E) 4 DEG C of bacterium solution after standing, 5000rpm centrifugation 30min separation obtain cracking supernatant；

(3) upper prop elutes

A) 10 times of column volume ddH₂O rinses nickel post, 5 times of column volume equilibrium liquid balance nickel posts；

B) supernatant and Ni-Agarose will be cracked using volume ratio as 2:14 DEG C of 100rpm of ratio are sufficiently mixed 2h；

C) the solution upper prop that will be mixed, 4 DEG C of collection effluxes；

D) after efflux all flows out, 4 DEG C of washing foreign proteins of cleaning fluid of 8 times of column volumes are added；

E) with 4 DEG C of elutions of eluent of 4 times of column volumes, the eluent of 2 volumes and rear 2 volume before collecting respectively.

(4) desalination dispenses

A) using a kind of progress desalination in following two modes：

Mode one：The albumen being collected into is loaded into bag filter, albumen and dialyzate are with 1:100 volume ratio is dialysed at 4 DEG C, The mixing speed of magneton is 200rpm, and dialyse 3h every time, is dialysed 2 times；

Mode two：By the albumen being collected into and saline solution is changed with 1:Salt plug is changed in 1 ratio mixing, addition, and 4 DEG C, 5000rpm is centrifuged 15min.The solution for changing salt plug upper strata is collected, repeats aforesaid operations 10 times.

B) gained albumen is degerming using 0.22 μM of membrane filtration, packing, -80 DEG C of preservations.