CN103749457A - Application of dequalinium chloride as mouse birth control medicine - Google Patents
Application of dequalinium chloride as mouse birth control medicine Download PDFInfo
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- CN103749457A CN103749457A CN201310739915.XA CN201310739915A CN103749457A CN 103749457 A CN103749457 A CN 103749457A CN 201310739915 A CN201310739915 A CN 201310739915A CN 103749457 A CN103749457 A CN 103749457A
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- ctbs
- dequalinium chloride
- muroid
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Abstract
The invention relates to an application of dequalinium chloride as a mouse birth control medicine. Researches find that CTBS (di-N-acetylchitobiase) is a unique sperm-egg binding receptor on the sperms of rodents, the inhibitor dequalinium chloride can effectively inhibit in vitro fertilization of a mouse, the semi-inhibition constant is less than 100nM, and the toxic effect on a human body is tiny; the researches also find that the CTBS is expressed on the sperms of rodents, the CTBS antibody can inhibit in vitro fertilization of the mouse, and the protein is not expressed on the sperms of the human body. According to the application provided by the invention, dequalinium chloride is used as a mouse birth control medicine and can effectively control the sperm-egg binding of rodents to achieve the aims of controlling the population quantity of mice and realizing mouse damage control without needing pesticides; moreover, after eating the medicine, the natural enemy of the mice does not cause harm.
Description
Technical field
The present invention relates to the birth control medicine of a kind of rodent, particularly a kind of Dequalinium Chloride is as the application of the birth control medicine of muroid.
Background technology
Dequalinium Chloride (Dequalinium chloride, CAS#522-51-0), as a kind of pkc inhibitor, as cancer therapy drug, use, its IC50 is 7-18 μ M, it is also a kind of selectivity apamin responsive type potassium-channel blocker simultaneously, and its IC50 is 1.1 μ M
[1,2,3,4].Recently, find that Dequalinium Chloride also can suppress the activity of microbe-derived chitin hydrolase in nanomole level
[5].Dequalinium Chloride is very micro-to the toxicity of human body, does not affect the mankind's reproduction, and eye and skin are had to spread effect.To the lethal dose of mouse, be: oral 150-1000mg/kg body weight, hypodermic injection 70mg/kg body weight, intravenous injection 1.9mg/kg body weight; To the oral lethal dose of rabbit and dog, be 500mg/kg body weight (MSDS).
And two-N-acetylchitobiose enzyme, Di-N-acetylchitobiase (being called for short CTBS) is a kind of of chitin hydrolase, in mammal, only the liver in rodent and mankind's body has expression
[6,7,8].
The reproduction isolation of species is a kind of general natural phenomenas, and on the carbohydrate on ovum oolemma and sperm, the interaction of protein is one of mechanism wherein.Have report to show, lewis oligosaccharide X (being Lewis X) can be incorporated into mouse sperm, and can suppress specifically the in vitro fertilization of mouse, the fertility isolation of pointing out the albumen of identifying Lewis X on its sperm may participate in mouse
[9], but be only a kind of conjecture.
The plague of rats refers to the harm that muroid causes productions such as agriculturals.Muroid belongs to Mammalia (Mam-malia) Rodentia (Rodentia) animal, has kind more than 1600.Often, the pregnancy period is short in muroid breeding, and farrowing rate is high, and sexual maturity is fast, and quantity can sharply increase in a short time; Its adaptability is very strong, except Antarctica, on the ground all over the world, underground, tree, can survive in water, no matter huge disaster, so that desert area has its trace, is often led to productions such as agriculturals in Plain, high mountain, forest, grassland.
Therefore be badly in need of providing a kind of medicine that can control muroid fertility, especially suppress the medicine of the smart ovum combination of muroid, and this medicine can suppress the plague of rats in the wild and its natural enemy is edible harmless, without adopting agricultural chemicals to suppress the plague of rats.
List of references of the present invention is as follows:
[1]ManettaA，et?al.Gynecol?Oncol，1993，50(1)，38-44。
[2]Castle?NA，et?al.Eur?J?Pharmacol，1993，236(2)，201-207。
[3]Dunn?PM，et?al.Eur?J?Pharmacol，1994，252(2)，189-194。
[4]Rotenberg?SA，et?al.J?Biol?Chem，1998，273(4)，2390-2395。
[5]Pantoom?S，et?al，J.Bi01.Chem.2011，286：24312-24323。
[6]Aronson?NN?Jr，et?al，Arch?ofBiochem?Biophy,1989，272(2)，290-300。
[7]Liu?B，et?al，Glycobiology,1999，9(6)，589-593。
[8]Park?C，et?al，J?Biol?Chem2005，280，37204-37216。
[9]Kerr?CL，et?al，Biol?Reprod，2004，71(3)，770-777。
[10]Rao?FV,et?al，?Chem?Bio，2005，12，973-980。
[11]Shur?BD，et?al，J?Biol?Chem1988,263，17706-17714。
Summary of the invention
The object of the present invention is to provide a kind of application that can control the medicine of muroid fertility, a kind of application of medicine Dequalinium Chloride of the smart ovum combination that can suppress muroid is especially provided, this medicine can be used for controlling the population quantity of muroid, thereby reach without adopting agricultural chemicals can control the effect of the plague of rats, and edible this medicine of the natural enemy of muroid is harmless.
To achieve these goals, the present invention adopts following technical scheme:
Dequalinium Chloride, as the application of the birth control medicine of muroid, for controlling the population quantity of muroid, thereby reaches the object of controlling the plague of rats.
Further, the birth control of described muroid is for adopting inhibitor Dequalinium Chloride to suppress the smart ovum combination of muroid.
Further, the smart ovum of described muroid in conjunction with on sperm specific expressed receptor protein be two-N-acetylchitobiose enzyme, i.e. Di-N-acetylchitobiase, referred to as CTBS; Find after deliberation, described Dequalinium Chloride be described muroid smart ovum in conjunction with in specific expressed receptor protein CTBS inhibitor on sperm, CTBS be muroid smart ovum in conjunction with in requisite albumen, therefore by Dequalinium Chloride as the special birth control medicinal application of muroid.
We find that CTBS is distinctive smart ovum bind receptor on rodent sperm after deliberation, and its inhibitor Dequalinium Chloride can suppress the in vitro fertilization of mouse effectively, very micro-to the toxic action of human body.
We find that CTBS only expresses on the sperm of rodent after deliberation, and CTBS antibody can suppress the inseminatio externalis of mouse, and on people's sperm, do not express this albumen.
The affinity that inhibitor Dequalinium Chloride of the present invention is combined with CTBS is strong, and the activity of smart ovum combination that suppresses muroid is high, and what experiment showed that it suppresses smart ovum combination partly suppresses constant < 100nM; Further, it partly suppresses constant is 50nM, therefore, can use by the birth control medicine using Dequalinium Chloride as muroid, and Dequalinium Chloride can suppress the in vitro fertilization of mouse, so Dequalinium Chloride can be used for controlling the population quantity of muroid, thereby reach the object of controlling the plague of rats.
The present invention adopts Dequalinium Chloride to use as the birth control medicine of muroid, can effectively control the smart ovum combination of rodent, reach the population quantity that controls muroid, realize and can control plague of rats object without agricultural chemicals, and edible this medicine of its natural enemy but can not produce harm.
Accompanying drawing explanation
Fig. 1 is the inhibition figure of Dequalinium Chloride to mouse in vitro fertilization.
Fig. 2 A is the evaluation thinking figure of Lewis X acceptor.
Fig. 2 B is the mouse sperm albumen figure that utilizes Lewis X probe to find.
Fig. 2 C is the mass spectral analysis collection of illustrative plates of albumen.
Fig. 3 is the picture that the antibody suppression Lewis X of CTBS is attached to mouse sperm.
Fig. 4 is that the binding site of Lewis X on mouse sperm and CTBS are at the positioning result picture of mouse sperm.
Fig. 5 A is that interactional schematic diagram occurs people source CTBS and the Mouse Eggs oolemma of restructuring.
Fig. 5 B is that interactional light field and fluorescence photo occur people source CTBS and the Mouse Eggs oolemma of restructuring.
Fig. 5 C is RFP or the analysis of RFP-CTBS protein immunoblot that the latex particle of CTBS antibody is enriched to.
Fig. 6 is the inductive effect figure of CTBS Antibody on Mouse sperm acrosome reaction.
Fig. 7 is the figure in vitro fertilization of the CTBS antibody suppression mouse of variable concentrations.
Fig. 8 is the protein blot analysis chart of mouse, rat and people's sperm lysate.
Embodiment
By particular specific embodiment explanation embodiments of the present invention, person skilled in the art scholar can understand other advantages of the present invention and effect easily by the disclosed content of this specification below.
Embodiment 1: Dequalinium Chloride is as the application of the birth control medicine of muroid
The mouse eggs >50 for each dosage experiments of CTBS variable concentrations, repeat 3 times, each dosage amounts to ovum >150, its experimental result as shown in Figure 1, it partly suppresses constant at 50nM, shows that Dequalinium Chloride can well suppress the in vitro fertilization of mouse.
Embodiment 2:
We find to express on the sperm of CTBS rodent through experiment, and CTBS antibody can suppress the inseminatio externalis of mouse, and on people's sperm, do not express this albumen.Its experimental technique is as follows:
(1) discovery of CTBS on mouse sperm:
Utilize lewis oligosaccharide X (be Lewis X, be abbreviated as LeX) probe to identify the CTBS on mouse sperm, and prove that CTBS can be incorporated into the ovum oolemma of mouse.
(1) first Lewis X acceptor is identified, its identity process as shown in Figure 2 A, comprises the identity process of the agglutinoid albumen that can identify Lewis X and the Identification of Fusion Protein flow process that the endogenic Lewis of containing X modifies.Its principle is that bovine serum albumin(BSA) (BSA) finishing Lewis X is made to Neoglycoproteins LeX-BSA, and this Neoglycoproteins is further modified to upper UV-crosslinked reagent [6-(biotin acylamino)-2-(p-azido benzoyl is amino) hexanoyl is amino] ethyl-1,3 '-bis-propane thioic acid sulfosuccinimide esters (Sulfo-NHS-2-(6-[biotinamido]-2-(p-azido benzamido)-hexanoamido) ethy1-1,3 '-dithiopropionate, is called for short Sulfo-SBED) as LewisX probe.This probe forms Lewis X probe-receptor complex through UV-crosslinked after mouse sperm, four rib lotus agglutinins (Lotus tetragonolobus agglutinin the is called for short LTA) resin that utilization can be identified Lewis X carrys out the Lewis X probe-receptor complex in separation and purification spermoblast lysate.Owing to may containing in mouse sperm, the Chi that endogenous Lewis X modifies is white, is provided with the Identification of Fusion Protein flow process that the endogenic Lewis of containing X modifies and distinguishes in contrast this class Chi white (control group).In general, the differential protein that two flow processs identify is likely the receptor protein of Lewis X.
(2) utilize the albumen on the mouse sperm that Lewis X probe finds:
Utilize Lewis X probe to find mouse sperm albumen, as shown in Figure 2 B.Differential band in Fig. 2 B 1,2,3,4 is taken off, for the Mass Spectrometric Identification of albumen, mass spectral analysis figure as shown in Figure 2 C, by analysis, likely as shown in table 1 with the albumen identity of carbohydrate effect.
Table 1
Note: the first hurdle in table 1 is nucleocytoplasmic ratio, the second hurdle is peptide sequence, third column is protein identity.
(3) antibody capable of CTBS inhibition Lewis X-fluorescence probe is attached to mouse sperm:
Lewis X-fluorescence probe LeX-BSA-Alexa430 adds fluorophor Alexa430 by the Neoglycoproteins LeX-BSA of LewisX and is prepared from.As shown in Figure 3, this probe can be incorporated on the mature sperm that mouse epididymis afterbody takes out (positive control).Antibody is the rabbit polyclonal antibody of being prepared by the CTBS recombinant protein in people source, purchased from Wuhan Sanying Bio-Technology Co., Ltd..
Find after deliberation to only have the antibody capable inhibition LeX-BSA-Alexa430 of CTBS to be attached to mouse sperm, as shown in Figure 3.And a kind of weak chitinase inhibitors oxpentifylline (Pentoxifylline is called for short PTX) also can suppress this combination
[10], got rid of nonspecific combination that the space steric effect of antibody itself causes and suppressed.
(4) binding site of Lewis X on mouse sperm is consistent in the location of mouse sperm with CTBS:
Lewis X-fluorescence probe LeX-BSA-Alexa430 adds fluorophor Alexa430 by the Neoglycoproteins LeX-BSA of LewisX and is prepared from.This probe can be incorporated on the mature sperm of mouse epididymis afterbody taking-up, as shown in Fig. 4 illustration, consistent with previous report in conjunction with situation
[9].Antibody is the rabbit polyclonal antibody of being prepared by the CTBS recombinant protein in people source, purchased from Wuhan Sanying Bio-Technology Co., Ltd..By immunofluorescence, locate, we find that the binding site of LeX-BSA-Alexa430 on mouse sperm is consistent with the CTBS protein localization of mouse sperm, as shown in Figure 4.
(5) interaction that the people source CTBS (being rCTBS) of restructuring and Mouse Eggs oolemma (ZP) occur:
A. experimental considerations as shown in Figure 5A, is fixed to red fluorescent protein (RFP) antibody on latex particle, and the restructuring RFP-CTBS protein enrichment that is expressed in 293 cells, to latex particle, and is added in the medium of Mouse Eggs.If CTBS can identify the carbohydrate on Mouse Eggs oolemma, on mouse oolemma by the latex particle of sclerozone red fluorescence.
B. Fig. 5 B is light field (left side) and red fluorescence (right side) photo.Found that the latex particle that is only enriched to RFP cannot be attached on the ovum oolemma of mouse (as shown in Fig. 5 B lastrow); And with the latex particle of RFP-CTBS, can be incorporated on the ovum oolemma of mouse (as shown in Fig. 5 B middle row), CTBS antibody can hinder this in conjunction with (as shown in Fig. 5 B next line).The fluorescence photo on right side has confirmed above-mentioned observation.
C. Fig. 5 C is western blot analysis result, illustrates result and shows, the latex particle that is combined with RFP antibody can enrichment RFP or RFP-CTBS.Wherein white arrow refers to RFP, and black arrow refers to RFP-CTBS.
The above results has proved that the people source CTBS of restructuring can interact with Mouse Eggs oolemma.
(2) CTBS has participated in the fertilization process of mouse.
(1) inductive effect of CTBS Antibody on Mouse sperm acrosome reaction:
Adopt the acrosome reaction of the CTBS antibody induction mouse sperm of variable concentrations between 0-30ug/ml, as shown in Figure 6, result proof CTBS antibody can inducing mouse sperm acrosome reaction, the feature that on this mouse sperm of reporting with prior art, smart ovum shows in conjunction with relevant receptor protein is consistent
[11].
(2) the CTBS antibody suppression mouse of variable concentrations is in vitro fertilization:
Adopt CTBS antibody suppression mouse in vitro fertilization of variable concentrations, as shown in Figure 7, result proof CTBS antibody capable well suppresses the in vitro fertilization of mouse, further show CTBS on mouse sperm really with the smart ovum of mouse in conjunction with relevant.
(3) the protein blot analysis of mouse, rat and people's sperm lysate.
Sperm lysate to mouse, rat and people carries out protein blot analysis, and the applied sample amount of sperm lysate is mouse 50ug, rat 60ug, people 60ug.Antibody is the rabbit polyclonal antibody of being prepared by the CTBS recombinant protein in people source, purchased from Wuhan Sanying Bio-Technology Co., Ltd.; The applied sample amount of human cell line Hela cell pyrolysis liquid is 10ug; Use Alpha-tubulin (α-tubulin) as the reference of albumen applied sample amount.Result as shown in Figure 8, shows that the CTBS in CTBS antibody on human source has very high sensitivity, has further proved that CTBS does not express on people's sperm, and only on the sperm of rodent, has had expression.
To sum up all results described in embodiment 1 and embodiment 2 show, CTBS is receptor protein specific expressed on rodent sperm, and it has participated in the physiology course of smart ovum combination, and CTBS antibody capable suppresses the in vitro fertilization of mouse.Dequalinium Chloride can well suppress the in vitro fertilization of mouse as the birth control medicine of muroid, and experiment shows that it partly suppresses constant < 100nM.
Above-described embodiment is illustrative principle of the present invention and effect thereof only, but not for limiting the present invention.Any person skilled in the art scholar all can, under spirit of the present invention and category, modify or change above-described embodiment.Therefore, such as in affiliated technical field, have and conventionally know that the knowledgeable, not departing from all equivalence modifications that complete under disclosed spirit and technological thought or changing, must be contained by claim of the present invention.
Claims (4)
1. Dequalinium Chloride is as the application of the birth control medicine of muroid.
2. Dequalinium Chloride as claimed in claim 1, as the application of the birth control medicine of muroid, is characterized in that, the birth control of described muroid is for adopting inhibitor Dequalinium Chloride to suppress the smart ovum combination of muroid.
3. Dequalinium Chloride as claimed in claim 2, as the application of the birth control medicine of muroid, is characterized in that, in the smart ovum combination of described muroid, on sperm, specific expressed receptor protein is CTBS; Described Dequalinium Chloride be described muroid smart ovum in conjunction with in the inhibitor of specific expressed receptor protein CTBS on sperm.
4. Dequalinium Chloride as claimed in claim 3, as the application of the birth control medicine of muroid, is characterized in that, what described inhibitor Dequalinium Chloride suppressed the combination of muroid essence ovum partly suppresses constant < 100nM.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070202177A1 (en) * | 2006-02-28 | 2007-08-30 | Becton, Dickinson And Company | Antimicrobial Compositions and Methods for Locking Catheters |
| CN101919807A (en) * | 2010-07-16 | 2010-12-22 | 钟术光 | Suppository composition |
| CN103387537A (en) * | 2013-07-24 | 2013-11-13 | 珠海经济特区生物化学制药厂 | Preparation method of compound requalinium chloride |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070202177A1 (en) * | 2006-02-28 | 2007-08-30 | Becton, Dickinson And Company | Antimicrobial Compositions and Methods for Locking Catheters |
| CN101919807A (en) * | 2010-07-16 | 2010-12-22 | 钟术光 | Suppository composition |
| CN103387537A (en) * | 2013-07-24 | 2013-11-13 | 珠海经济特区生物化学制药厂 | Preparation method of compound requalinium chloride |
Non-Patent Citations (2)
| Title |
|---|
| 史美平等: "地喹氯铵合成工艺的研究", 《河北化工》 * |
| 肖红俊等: "急性咽炎、扁桃体炎的细菌学特征及地喹氯铵口含片(泰乐奇)的疗效观察", 《临床耳鼻咽喉科杂志》 * |
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