CN105920683B

CN105920683B - A kind of titanium that can prevent bacterium infection used for gastrointestinal disease is followed closely

Info

Publication number: CN105920683B
Application number: CN201610257300.7A
Authority: CN
Inventors: 李蒙
Original assignee: Hebei Weiquan Medical Technology Co Ltd
Current assignee: Hebei Weiquan Medical Technology Co., Ltd.
Priority date: 2016-04-24
Filing date: 2016-04-24
Publication date: 2019-02-01
Anticipated expiration: 2036-04-24
Also published as: CN105920683A

Abstract

The present invention relates to will sterilize in vivo and the titanium of infection-proof bacterium follow closely, by by titanium follow closely immerse polyethyleneimine aqueous solution, titanium nail surface formed polymer base coat；It is subsequently placed in the methanol solution of 4,4 '-Diphenyl disulfide alcohol to react and be reacted under horseradish peroxidase and hydrogen peroxide effect with polypeptide again, obtain the titanium nail of surface bond antibacterial polypeptide.Titanium nail, preparation is simple, low in cost, has the effect of preventing bacterium infection, has high application value.

Description

A kind of titanium that can prevent bacterium infection used for gastrointestinal disease is followed closely

Technical field

It is the present invention relates to medical instruments field, in particular to a kind of to prevent bacterium infection for what gastrointestinal disease used Titanium nail and preparation method thereof.

Background technique

For most people, upper abdomen pain and other gastrointestinal diseases are common chronic problems.It is examined by doctor In the individual looked into and diagnosed, many people have the disease of such as peptic ulcer or other ulcer.Certainly there are also gastrointestinal diseases It is as caused by external mechanical wounding, for example traffic accident, blunt are stabbed.The treatment of gastrointestinal disease is carried out using operation at present Very big ratio is accounted for, wherein the excision of stomach is common type of surgery.

And it inevitably needs to suture in performing the operation.Simple suture is usually used in the seam of skin, subcutaneous tissue, muscle, aponeurosis (aponeuroses) It closes；Everting suture keeps edge of wound inner face smooth, is usually used in the suture of blood vessel, peritonaeum, relaxation skin；Inverting suture is kept outside edge of wound Face is smooth, is usually used in the suture of enteron aisle, bladder.1908, Hungary doctor Hultl and Fisher invented stitching unstrument.1967 Year U.S. surgery company (Auto Suture) founder's Leon Hirsh invention titanium follows closely stapler, and realizes titanium nail stapler It commercially produces, there is landmark meaning in mechanical stitch technograph.The product being commercialized at present has: end end kiss Clutch, anastomosis ring applicator, linear anastomat, line style Endo-GIA, arc incision anastomat, hemorrhoid stapler, purse-string suture device, Skin fascia stitching unstrument.1892, Murphy doctor, which invents, coincide button (Murphy button).1976, Hardy doctor Anastomosis ring is invented.

The identical method of the gastrointestinal tract of clinical use has manual suture, the stitching of metal anastomotic device, anastomosis ring to fasten these three sides Method.Manual suture is with a long history, is a kind of suturing skill with suture repairing wound.The technical operation is cumbersome, when suture Between depending on surgical experience, generally require 30 minutes, in addition, the puncture repeatedly of suture to gastrointestinal tissue damage compared with Greatly, a possibility that postoperative intestinal inflammatory generation and granuloma etc. occur is increased.With the appearance of Titanium nail stapler, it is born A kind of novel Sutureless anastomosis with surgical instrument.The stapler operate it is more convenient, suture mouth It more can standardization control.

But instrument of the titanium nail as implantation human body, at enteron aisle or gastric operation bacterium colony can be formed on the surface that titanium is followed closely Microenvironment causes to be easy inflammation in wound, causes lesion, influences the recovery effects of operation.

Porous, degradable composite coating carried with antibiotics drug system is disclosed in CN 103705294A is mainly used in orthopaedics Implant solves and fixes bring infection risk in current implantation material, higher in particular for open fracture infection rate The case where.By a kind of novel local administration mode, antibiotic sustained release system, antibiotic sustained release system are prepared in implant surface It can control its rate of release, make drug in local cluster, concentration increases, and is conducive to directly play drug effect；Drug is among carrier, It is discharged with vector degradation, accretion rate slows down, and the therapeutic effect time is lasting, and carrier itself is biodegradation material, can be Eubolism is participated in vivo, is had no toxic and side effect to human body, is finally excreted.

And for the titanium nail in gastrointestinal disease, it develops effective coating and is studied in the prior art there is no corresponding, because This, develops a kind of preferable coating of effect, has extremely strong application prospect.

Summary of the invention

One of technical solution of the invention provides a kind of preparation method of titanium nail surface covering, and 10g titanium is followed closely and is immersed The aqueous solution of polyethyleneimine, the concentration of polyethyleneimine is 5-10mg/ml in the aqueous solution, containing soaking in 0.14mol/LNaCl Bubble 75 minutes or so can form polymer base coat on the surface that titanium is followed closely；Being subsequently placed in 100mL mass percent concentration is 0.01% 4,4 '-Diphenyl disulfide alcohol methanol solution in react 5 hours, it is dry that titanium after reaction follows closely the vacuum after methanol sufficiently embathes It is dry, it is any with SEQ ID NO:1-16 shown in polypeptide respectively horseradish peroxidase and hydrogen peroxide effect under react, obtain Titanium to surface bond antibacterial polypeptide is followed closely.When titanium nail is contacted with bacterial cell, titanium follows closely the disulfide bonds on surface, reduction release Polypeptide plays bactericidal effect.

The present invention additionally provides a kind of preparation method of antibacterial polypeptide, the screening step of the polypeptide includes:

One, prediction screening: downloading the genome sequence of ascidian, finds small open reading wherein with potential encoding function These small open reading frame are translated corresponding amino acid sequence respectively, therefrom screen antibacterial peptide by frame；

Two, antibacterial peptide is prepared: according to the antibacterial peptide sequence for predicting screening in step 1, wherein using solid-phase synthesis synthesis Part of polypeptide, and make C-terminal amidation, N-terminal does not contain methionine, prepares antibacterial peptide, and carry out Mass Spectrometric Identification；

Three, antibiotic property detects: it is 2mg/ml that antibacterial peptide, which is dissolved into concentration with distilled water, is carried out using agar diffusion method Antibacterial activity detection；

Four, hemolytic detects: utilizing the haemocylolysis of sheeps blood erythrocyte detection antibacterial peptide；

Five, broad-spectrum antibacterial polypeptide: the polypeptide with broad-spectrum antiseptic function finally screened is prepared.

Further, in the step 3 bacterial strain used in agar diffusion method be Escherichia coli, Staphylococcus aureus, Tetrads, Candida albicans, proteus, Pseudomonas aeruginosa, Streptococcus mutans, one or more of groups of helicobacter pylori It closes.

Further, the polypeptide prepared in the step 5, sequence are as follows: shown in SEQ ID NO:1-16 is any.

The beneficial effects of the present invention are: the titanium nail with antibacterial peptide of this method preparation, having prevents titanium nail in vivo The risk that will lead to inflammation generation after over time, with great application prospect and important social value value.

Specific embodiment

The preparation of 1 antibacterial polypeptide of embodiment

One, the bioinformatics screening of antibacterial peptide

Ascidian DNA is extracted, No. 3 DNA sequence sequences are obtained by genome sequencing, according to antibacterial peptide molecular weight Small feature, screening from genome sequence first with small open reading frame analysis software sORFfinder has potential coding The small open reading frame (sORFs) of function.These sORFs are translated into corresponding amino acid sequence respectively, it is pre- using antibacterial peptide It surveys software CAM P and screens antibacterial peptide from sORFs.More than 300 item of sequence that possibility is more than 70% is predicted altogether.

Two, the antibacterial activity identification of polypeptide

Selecting 17 possibilities is more than 95% sequence, and the raw work in commission Shanghai is using solid-phase synthesis synthesis, and by C-terminal Amidation purifies after synthesis through HPLC, and purity reaches 99.86%, and identifies (table 1) through mass spectral analysis.

1 synthetic peptide sequence table of table

Three, antibiotic property detects:

It is 2mg/ml that polypeptide, which is dissolved into concentration with distilled water, carries out antibacterial activity detection using agar diffusion method.Fine jade Bacterial strain used in lipolysaccharide diffusion method be Escherichia coli, Staphylococcus aureus, tetrads, Candida albicans, proteus, The combination of Pseudomonas aeruginosa, Streptococcus mutans, helicobacter pylori, serratia marcesens.After bacterial strain recovery, it is inoculated in common LB culture medium After 37 DEG C are incubated overnight, (Candida albicans are inoculated in PD culture medium and are incubated overnight for 30 DEG C) takes 50 microlitres of bacterium solution coatings respectively In LB solid medium.After standing band bacterium solution absorption, it is separately added into 10 microlitres of polypeptide solution that concentration is 2mg/ml.37 DEG C of cultures After 48 hours, the presence or absence of observation inhibition zone and size.It observes and whether is changed in inhibition zone one week simultaneously, judge that the antibacterial time is long It is short.It the results are shown in Table 2.

2 polypeptide Analysis of Antimicrobial Activity result of table

+: there is antibacterial activity；- without antibacterial activity

As can be seen from Table II, KJ-1-16 all has preferable antibacterial characteristics, and KJ-17 antibacterial characteristics are poor.

Four, the minimum inhibitory concentration analysis of the Staphylococcus aureus of KJ-1~16

Using the minimum inhibitory concentration of blood agar plate detection KJ-1~16 pair Staphylococcus aureus.By 50 microlitres of bacterium solution Half an hour is stood after being coated on blood agar plate, respectively by 10 microlitre 0,20,30,50,100,200,500,1000 micrograms/ml Culture dish is added in KJ-1-16 solution, marks.After 37 DEG C are cultivated 48 hours, the presence or absence of observation inhibition zone.The result shows that KJ- 1,2,4,5,7,9,10,11,13,15,16 minimum inhibitory concentrations are 20 micrograms/ml, and KJ-3,6,8,12,14 are minimum antibacterial dense Degree is 30 micrograms/ml.

Five, the hemolytic detection of antibacterial peptide

Utilize the haemocylolysis of sheeps blood erythrocyte detection antibacterial peptide；The solution of antibacterial peptide KJ-1~16 for being 2mg/ml by concentration 10 microlitres are added dropwise in blood dish surface, are compareed with distilled water.It is placed in 37 DEG C 24 hours or more, then observes having for haemolysis circle Nothing.As a result all tested antibacterial peptides do not occur haemolysis circle.PRELIMINARY RESULTS shows the antibacterial peptide, and there is no hemolytic toxicities.

The preparation of the peptide modified titanium nail of embodiment 2

10g titanium is followed closely to the aqueous solution for immersing polyethyleneimine, the concentration of polyethyleneimine is 5-10mg/ in the aqueous solution Ml can form polymer base coat on the surface that titanium is followed closely containing impregnating 75 minutes or so in 0.14mol/LNaCl；It is subsequently placed in 100mL It is reacted 5 hours in the methanol solution for 4, the 4 '-Diphenyl disulfide alcohol that mass percent concentration is 0.01%, the titanium after reaction follows closely warp Methanol is dried in vacuo after sufficiently embathing, with SEQ ID NO:1-16 any one polypeptide respectively in horseradish peroxidase and peroxidating Hydrogen effect is lower to react 8h, obtains the titanium nail of surface bond antibacterial polypeptide.

It modifies titanium and follows closely compliance test result

The titanium nail that KJ-1~16 are modified respectively is respectively placed in and is coated on the bacterium solution of 50 microlitres of Staphylococcus aureus Blood agar plate center.37 DEG C culture 3 days after, observe inhibition zone, by measurement, discovery antibacterial circle diameter be respectively 3.5cm, 3.9cm、3.8cm、3.4cm、4.1cm、3.3cm、3.9cm、3.8cm、3.7cm、4.2cm、4.5cm、3.4cm、3.6cm、 3.8cm,4.1cm,4.3cm.With extremely strong fungistatic effect.

The above are specific embodiments of the present invention, its role is to be further described in detail to the contents of the present invention, It is more readily understood reader, but is not constituted to protection of the presently claimed invention.

Sequence table

110 > Li Meng of <

A kind of titanium that can prevent bacterium infection used for gastrointestinal disease of 120 > of < is followed closely

〈210〉1

〈211〉18

〈212〉PRT

〈400〉KJ-1

VDCWPHDVKLRLPHHQGK

〈210〉2

〈211〉18

〈212〉PRT

〈400〉KJ-2

VIQIEFFQLYNWWTHYKP

〈210〉3

〈211〉18

〈212〉PRT

〈400〉KJ-3

YHQAYYRKHCKQSNRGPK

〈210〉4

〈211〉17

〈212〉PRT

〈400〉KJ-4

TCVQGRLCQYDTIQGVV

〈210〉5

〈211〉17

〈212〉PRT

〈400〉KJ-5

WALQDSQEWHGSLFAKR

〈210〉6

〈211〉17

〈212〉PRT

〈400〉KJ-6

TQLSRIGWPTSGGGEGS

〈210〉7

〈211〉18

〈212〉PRT

〈400〉KJ-7

FPSPIFHHRSPIIDDCGG

〈210〉8

〈211〉18

〈212〉PRT

〈400〉KJ-8

PHCKYSWKWAPAASWTPR

〈210〉9

〈211〉16

〈212〉PRT

〈400〉KJ-9

HGNQLQSGWWSPLWSS

〈210〉10

〈211〉16

〈212〉PRT

〈400〉KJ-10

PHKNGQCNVYRYPWIW

〈210〉11

〈211〉18

〈212〉PRT

〈400〉KJ-11

NKHQQDWGRQMTSQDHVN

〈210〉12

〈211〉18

〈212〉PRT

〈400〉KJ-12

KYWSWWGASRVQWEESEN

〈210〉13

〈211〉18

〈212〉PRT

〈400〉KJ-13

WASNQFDPGPSVRDSGIT

〈210〉14

〈211〉18

〈212〉PRT

〈400〉KJ-14

CWTEHGSMRFPIGQNQRC

〈210〉15

〈211〉18

〈212〉PRT

〈400〉KJ-15

HWFMQDSYVSKLMYRWPM

〈210〉16

〈211〉18

〈212〉PRT

〈400〉KJ-16

WVPCKWICNDPDEVWLRS

〈210〉17

〈211〉18

〈212〉PRT

〈400〉KJ-17

VHFWDVAYLHMNDMKWDN

Claims

1. a kind of polypeptide, sequence is shown in SEQ ID NO:1-16 is any.

2. purposes of the polypeptide described in claim 1 in the titanium nail for being used to prepare bacterial-infection resisting.