CN103990106A - Application of cholecystokinin octapeptide in medicine preparation - Google Patents
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Abstract
The invention relates to novel application of cholecystokinin octapeptide (CCK-8) in the field of medicine preparation. The cholecystokinin octapeptide can obviously reduce behavior change caused by taking METH for a long term, is beneficial to preventing and treating neurological complications generated and developed due to long-term taking of METH, can also obviously reduce death of nerve cells caused by METH and prevent nerve injury caused by long-term taking of METH, and is also able to inhibit the generation of METH-caused microglial cell proinflammatory cytokine, and therefore, CCK-8 has the central anti-inflammatory function, and is of important significance in reduction of neurological complications caused by taking METH for a long term.
Description
Technical field
The present invention relates to the purposes of CCK-8 (CCK-8), the application of CCK-8 in pharmacy specifically.
Background technology
Cholecystokinin (cholecystokinin, CCK, PZ), claims again cholecystokinin, and the polypeptide of separating from pig small intestine extract, being made up of 33 aminoacid, was proved to be and purifies in nineteen twenty-eight, nineteen forty-three.CCK is extensively present in gastrointestinal tract and central nervous system, its by with target cell membrane on specific receptor CCKR(cholecystokinin receptor, cholecystokinin receptor) in conjunction with and performance Cell regulate effect.CCKR is the class glycoprotein on cell membrane, belongs to G albumen coupling superfamily.The CCKR tissue distribution difference of different types of structure, mediates different cytological effects.Sankaran in 1980 etc. have found CCK1R(cholecystokinin 1 receptor in pancreatic acini, cholecystokinin 1 receptor), diverse CCK2R(cholecystokinin 2 receptor of a kind of pharmacological property in brain, are found again the same year, cholecystokinin 2 receptors).CCK1R is mainly distributed in peripheral tissues, but also has part to distribute at central nervous tissue; CCK2R is mainly present in central nervous tissue, but also has part in periphery tissue distribution.
After CCK and its receptors bind, bring into play different physiological roles.CCK is to stimulate pancreatin secretion with synthetic in the effect of gastrointestinal major physiological, strengthens the secretion of pancreas bicarbonate and gastric emptying, stimulates gallbladder contraction and Oddi's sphincter lax, also can increase hepatic bile secretion, regulates many-sided functions such as small intestinal, Colon Movement; Bring into play the effect of neurotransmitter central nervous system, the physiological process such as pain sensation impression, appetite stimulator, memory are participated in, also there is the effect that regulates pituitary hormone to discharge, relevant with pathological manifestations such as terrified, anxiety, epilepsies, meanwhile, CCK also has the effect of control axis respiratory rhythm and adjusting cardiovascular anxiety.
With the CCK of trypsinization 33 peptides, can make 8 aminoacid parallel offs of its c-terminus out, be called CCK-8 (CCK-8), CCK-8 has whole activity of whole CCK molecule, is the minimum active fragment of CCK PZ molecule.Tyrosine the 7th of CCK molecule c-terminus is sulfation, the sulfate group on it to exist for its biological activity of performance institute essential.The aminoacid sequence of CCK-8 is H-Asp-Tyr (SO
3h)-Met-Gly-Trp-Met-Asp-Phe-NH
2, molecular formula is C
49h
62n
10o
16s
3, molecular weight is 1143.27.Knownly can be used as gallbladder contraction diagnostic reagent.
Summary of the invention
The object of the present invention is to provide the new purposes of CCK-8, i.e. application in pharmacy.
The object of the present invention is achieved like this:
The invention provides CCK-8 in preparation treatment or improve the application in the dystropy medicine that life-time service methamphetamine causes.
The invention provides CCK-8 and cause the application in nerve injury medicine in preparation treatment or prevention methamphetamine.
The invention provides the application of CCK-8 in the maincenter inflammation damnification medicine that preparation is treated or prevention methamphetamine causes.
The present invention has verified by following pharmacological evaluation the above-mentioned new purposes that CCK-8 has in pharmaceutical field.
Preparation CCK-8 injection: buy Sulfated CCK-8 from Sigma company of the U.S., 33.7 μ L, 25% ~ 28% ammonia are diluted to 10 mL with normal saline, obtain 0.05 mol/L ammonia (matching while using).5 mg CCK-8 and 0.05 mol/L ammonia 5 mL are fully vibrated and mixed, obtain 1 mg/mL CCK-8 solution, 50 μ L/ pipe subpackages, ice bath operation ,-80 DEG C of preservations, half a year effect duration.By normal saline dilution to 1 mg/L concentration, avoid multigelation with front.
1, CCK-8 suppresses the C57BL/6J mice behavior sensitization that methamphetamine (METH) causes
The preparation of experimental animal:
1. experimental animal: C57BL/6J mice (SPF level), body weight 18 ~ 22g, by Beijing, laboratory animal company limited of dimension tonneau China provides.
2. raise condition: the random sub-cage rearing of mice, 6/cage, 22 ± 1 DEG C of room temperatures, humidity 50% ± 10%, light application time 7:00-19:00, drinking water is not limit.
3. after 40mg/kg pentobarbital sodium intraperitoneal injection of anesthesia mice, be fixed on mouse brain stereotaxic instrument, carry out the operation of tricorn pipe laying.Postoperative adaptive response is raised (single cage raising) 1 week, then random packet, 12 every group.All mices are first pre-adaptation 3d before experiment starts, and spontaneous behaviour activity data in observed and recorded 30min, using as basic value and screen out the individual foundation that peels off.
1.1, set up behavior sensitization model
The process of establishing of behavior sensitization model is divided into 3 stages: behavior sensitization induction period, behavior sensitization During The Withdrawal Period and behavior sensitization detection period.
Get 72 of ready experimental animals, be divided at random 6 groups, 12 every group, the 1st group is matched group, and 2nd ~ 6 groups is experimental group, specific as follows:
The 1st group: normal saline group (Saline group);
The 2nd group: METH(1mg/kg) group;
The 3rd group: METH(2mg/kg) group;
The 4th group: g) group of CCK-8(0.1 μ;
The 5th group: g) group of CCK-8(0.01 μ;
The 6th group: g) group of CCK-8(0.001 μ.
Wherein, 1st ~ 3 groups of mices adopt the administration of lumbar injection (ip) mode, and the same time of every morning is surveyed body weight, presses 10mL/kg administration volume and calculate dosage in experiment; 4th ~ 6 groups of mices adopt the administration of micro sample adding appliance tricorn, and administration volume is 2 μ L/.
1.1.1 behavior sensitization induction period (d1-d7):
The 1st group of mice gives normal saline by 1 time/d lumbar injection, and d1, d3, d5 days give after normal saline, immediately mice put into spontaneous activity case, records the spontaneous behaviour activity data of mice in 30min.
The 2nd group of mice gives METH by 1mg/kg, 1 time/d lumbar injection, and d1, d3, d5 days give after METH, immediately mice put into spontaneous activity case, records the spontaneous behaviour activity data of mice in 30min.
The 3rd group of mice gives METH by 2mg/kg, 1 time/d lumbar injection, and d1, d3, d5 days give after METH, immediately mice put into spontaneous activity case, records the spontaneous behaviour activity data of mice in 30min.
4th, 5,6 groups of mices give CCK-8 by the amount intracerebroventricular injection of 0.1 μ g/2 μ L, 0.01 μ g/2 μ L, 0.001 μ g/2 μ L respectively, after d1, d3, d5 days give CCK-8, mice is put into spontaneous activity case by 15min, records the spontaneous behaviour activity data of mice in 30min.
1.1.2 behavior sensitization During The Withdrawal Period (d8-d13):
Under 1st ~ 6 groups of mice normal conditions, raise 6 days, do not carry out any processing.
1.1.3 behavior sensitization detection period (d14):
The 1st group of mice gives normal saline by 1 time/d lumbar injection, gives immediately mice to be put into spontaneous activity case after normal saline, records the spontaneous behaviour activity data of mice in 30min.
The 2nd group of mice gives METH by 1mg/kg, 1 time/d lumbar injection, gives immediately mice to be put into spontaneous activity case after METH, records the spontaneous behaviour activity data of mice in 30min.
The 3rd group of mice gives METH by 2mg/kg, 1 time/d lumbar injection, gives immediately mice to be put into spontaneous activity case after METH, records the spontaneous behaviour activity data of mice in 30min.
4th, 5,6 groups of mices, respectively by the amount intracerebroventricular injection CCK-8 of 0.1 μ g/2 μ L, 0.01 μ g/2 μ L, 0.001 μ g/2 μ L, are put into spontaneous activity case by mice after 15min after injection CCK-8, record the spontaneous behaviour activity data of mice in 30min.
The data that the spontaneous behaviour activity of mice is recorded comprise: total distance, moving speed, range of activity.
Data to the activity of 1st ~ 3 groups of mice spontaneous behaviour are carried out sorting-out in statistics, and result as shown in Figure 1.As seen from Figure 1, within d14 days, give after METH, the spontaneous activity distance of the 2nd group, the 3rd group mice obviously increases, can know with the 3rd group of data by comparing the 2nd group in addition, the operating range that increases animal of METH time, dose dependent, comprehensive above analysis, behavior sensitization model is successfully prepared.
Data to the 1st, 4,5 groups of mice spontaneous behaviour activities are carried out sorting-out in statistics, and result as shown in Figure 2.As seen from Figure 2, the administration of CCK-8 tricorn does not make significant difference to animal spontaneous activity, and CCK-8 itself can not form behavior sensitization by induced animal.
1.2, METH(1mg/kg group) in the behavior sensitization model process of establishing of induction, the impact on life-time service METH animal behavior variation of CCK-8 different times and METH concomitant dosing.
1.2.1, the administration of CCK-8 induction period suppresses the formation of the behavior sensitization of METH induction:
1.2.1.1, behavior sensitization induction period (d1-d7):
Get 48 of ready experimental animals, be divided at random 4 groups, 12 every group, the 1st group is matched group, and 2nd ~ 4 groups is experimental group, specific as follows:
The 1st group: METH(1mg/kg)+normal saline group;
The 2nd group: METH(1mg/kg)+CCK-8 (g) group of 0.1 μ;
The 3rd group: METH(1mg/kg)+CCK-8 (g) group of 0.01 μ;
The 4th group: METH(1mg/kg)+CCK-8 (g) group of 0.001 μ.
Each group above, METH adopts the administration of lumbar injection mode, and the same time of every morning is surveyed body weight, presses 10mL/kg and calculate administration volume and administration concentration in experiment; Normal saline and CCK-8 adopt the administration of micro sample adding appliance tricorn, and administration volume is 2 μ L/.
(the 1st group: normal saline 2 μ L of each group difference administration of same time of every day; The 2nd group: CCK-8 0.1 μ g; The 3rd group: CCK-8 0.01 μ g; The 4th group: CCK-8 0.001 μ g), give after normal saline (or giving CCK-8) 15 min, each group mice gives METH by 1mg/kg, 1 time/d lumbar injection, d1, d3, d5 days give after METH, immediately mice is put into spontaneous activity case, record the behavioral activity data of mice in 30min.
1.2.1.2, behavior sensitization During The Withdrawal Period (d8-d13):
Under 1st ~ 4 groups of mice normal conditions, raise 6 days, do not carry out any processing.
1.2.1.3, behavior sensitization detection period (d14):
Each group mice gives to give METH by 1mg/kg, 1 time/d lumbar injection, gives immediately mice to be put into spontaneous activity case after METH the spontaneous behaviour activity data of record 30 min mices.
Data to the activity of 1 ~ 4 group of mice spontaneous behaviour are carried out sorting-out in statistics, and result as shown in Figure 3.As seen from Figure 3, g) induction period and METH concomitant dosing of CCK-8(0.1 μ g, 0.01 μ g, 0.001 μ, can obviously suppress the expression of detection period animal behavior on same day sensitization.
Conclusion: CCK-8 concomitant dosing can reduce the sensitivity of animal to METH, improves the dystropy that life-time service METH causes.
The expression of the behavior sensitization that 1.2.2, CCK-8 detection period administration inhibition METH causes:
1.2.2.1, behavior sensitization induction period (d1-d7):
Get 48 of ready experimental animals, all mices same time of every day adopts lumbar injections to give METH (1mg/kg, 1 time/d), after d1, d3, d5 days administration, immediately mice is put into spontaneous activity case, record the spontaneous behaviour activity of mice in 30min.After administration in d7 days, mice is divided into 4 groups at random, 12 every group, the 1st group is matched group, and 2nd ~ 4 groups is experimental group.
1.2.2.2, behavior sensitization During The Withdrawal Period (d8-d13):
Under 1st ~ 4 groups of mice normal conditions, raise 6 days, do not carry out any processing.
1.2.2.3, behavior sensitization detection period (d14):
The 1st group: METH(1mg/kg)+normal saline group;
The 2nd group: METH(1mg/kg)+CCK-8 (g) group of 0.1 μ;
The 3rd group: METH(1mg/kg)+CCK-8 (g) group of 0.01 μ;
The 4th group: METH(1mg/kg)+CCK-8 (g) group of 0.001 μ.
Each group above, METH adopts the administration of lumbar injection mode, and the same time of every morning is surveyed body weight, presses 10mL/kg and calculate administration volume and administration concentration in experiment; Normal saline and CCK-8 adopt the administration of micro sample adding appliance tricorn, and administration volume is 2 μ L/.
(the 1st group: normal saline 2 μ L of each group difference administration; The 2nd group: CCK-8 0.1 μ g; The 3rd group: CCK-8 0.01 μ g; The 4th group: CCK-8 0.001 μ g), give after normal saline (or giving CCK-8) 15 min, each group mice gives METH by 1mg/kg, 1 time/d lumbar injection, gives immediately mice to be put into spontaneous activity case after METH mice spontaneous behaviour activity data in record 30 min.
Spontaneous behaviour activity data to 1 ~ 4 group of mice carries out sorting-out in statistics, and result as shown in Figure 4.As seen from Figure 4, CCK-8(0.1 μ g, g) detection period administration on the same day of 0.01 μ g, 0.001 μ, can obviously suppress the animal behavior sensitization that METH 1mg/kg causes.
Conclusion: CCK-8 improves the dystropy that life-time service METH causes, has defencive function.
The protective effect of the neural cell injury that 2, CCK-8 p-Methylamphetamine causes
PC-12 cell derived is in Rattus norvegicus Adrenal Pheochromocytoma, and the general features of tool neuroendocrine cell, for the in vitro study of nervous system disease.
The take the logarithm PC-12 cell of trophophase, makes single cell suspension after trypsinization, adjusting cell concentration is 10
5/ ml, with 10
4/ 100 μ L/ holes are inoculated in 96 orifice plates.
Experiment point matched group and METH (0.1,0.5,1.0,2.0,4.0mmol/L) group, CCK-8 (0.1,0.5,1.0 μ mol/L) group.CCK-8 and METH coprocessing groups of cells; Experiment is grouped into METH 2 mmol/L groups; METH 2 mmol/L+CCK-8 (0.1,0.5,1.0 μ mol/L); METH 1 mmol/L group; METH 1 mmol/L+ CCK-8 (0.1,0.5,1.0 μ mol/L) group.Establish 6 multiple holes for every group.Establish the blank well that only adds culture medium is zeroing hole simultaneously.Dosing after Growth of Cells to 80% fusion.After 24 h, add MTT 20 μ l/ holes, continue to cultivate 4 h, discard culture fluid in hole, add DMSO 150 μ l/ holes, be placed on shaking table and shake 10 min, after crystallization is fully dissolved, put microplate reader λ=490 place and survey each hole absorbance.Cell survival rate is pressed formula and is calculated: cell survival rate=D490(processed group-blank)/D490(matched group-blank) × 100%.
Result: as shown in Fig. 5 ~ 8.As seen from Figure 5, the survival rate of the reduction PC12 cell of METH (0.1,0.5,1.0,2.0,4.0 mmol/L) dose dependent, shows as the coup injury effect to neurocyte.As seen from Figure 6, CCK-8 (0.1,0.5,1.0 μ mol/L) individual processing cell does not affect PC12 cell survival rate, shows that CCK-8 itself does not have damaging action to neurocyte.Can be found out CCK-8 (0.1,0.5,1.0 μ mol/L) and METH(1mmol/L, 2 mmol/L by Fig. 7,8) coprocessing cell, can obviously alleviate the damage of METH to cell, there is defencive function.
Conclusion: CCK-8 itself is harmless to neurocyte, but can obviously alleviate the damage of METH to cell.
The inhibitory action of the proinflammatory cytokine (TNF-α, IL-1 β, IL-6 and MCP1) that 3, CCK-8 p-Methylamphetamine causes
Large quantity research shows that life-time service METH causes one of reason of nerve injury, is to activate microglia, and the proinflammatory factor level of its secretion is raise.N9 cell is mice microglia system, possesses the biological function of microglia.
The take the logarithm N9 cell of trophophase, makes single cell suspension after trypsinization, adjusting cell concentration is 10
5/ ml, is then seeded to 6 orifice plates, and every hole 2mL, treats Growth of Cells to 80% fusion, removes serum, places 6h, and cell is processed in dosing.
Test dose divides matched group, METH (0.1,0.5,1.0,2.0mmol/L) group and LPS(1 μ g/mL) group, processing time 3h.Experimental period is divided into METH 1mM and processes 1h, 3h, 6h, 12h group.Add afterwards CCK-8 and METH coprocessing cell, experiment is grouped into METH 2 mmol/L groups, METH 2 mmol/L+CCK-8 (0.01,0.1,1.0 μ mol/L) group, CCK-8 (1.0 μ mol/L) group; METH 1 mmol/L group, METH 1 mmol/L+CCK-8 (0.1,0.5,1.0 μ mol/L), CCK-8 (1.0 μ mol/L) group, processing time 3h.
Collect each porocyte, extract total RNA, reverse transcription, real-time fluorescence quantitative PCR.Result is using GAPDH as reference gene, the relative expression of △ △ Ct method analyzing gene.Regard matched group as 100%, all the other each groups all compare with it, using each group with the ratio of matched group as statistical value.Each experiment all in triplicate.
Result: as shown in Fig. 9 ~ 20.Can find out that from Fig. 9 ~ 12 1mM METH processes N9 cell 3h and can cause that proinflammatory inflammation factor TNF-α, IL-1 β, IL-6 and MCP1 express obviously rising.Can find out that from Figure 13 ~ 16 1mM METH processes N9 cell 1 ~ 12h and can cause that proinflammatory factor TNF-α, IL-1 β, IL-6 and MCP1 express obviously rising.Can find out that from Figure 17 ~ 20 CCK-8 and METH coprocessing cell can obviously suppress the short inflammatory Cytokines Expression rising that METH causes, and CCK-8 individual processing cell are to urging inflammatory Cytokines Expression without obvious effect.
Conclusion: CCK-8 itself without impact, can obviously suppress the short inflammatory Cytokines Expression rising that METH causes on microglia inflammatory Cytokines Expression.
From above result, can draw and the invention has the advantages that:
1, the behavior that life-time service METH causes that is significantly improved of CCK-8 (CCK-8) tool changes, and is conducive to generation and the development of neurological complication due to prevention and treatment METH life-time service.
2, CCK-8 can significantly reduce the death of the neurocyte that METH causes, the nerve injury that prevention life-time service METH causes.
3, CCK-8 can suppress the generation of the microglia proinflammatory cytokine that METH causes, shows CCK-8 performance maincenter antiinflammatory action, significant to improving the neurological complication that life-time service METH causes.
Brief description of the drawings
Fig. 1 is the formation (Mean ± SD, n=12) that METH 1mg/kg, 2mg/kg can induce C57BL/6J mice behavior sensitization.
*represent and matched group comparison,
p<0.05;
*represent and matched group comparison,
p<0.01;
* *represent and matched group comparison,
p<0.001.
Fig. 2 is that CCK-8 0.1,0.01,0.001 μ g can not induce C57BL/6J mice to form behavior sensitization (Mean ± SD, n=12).
Fig. 3 is CCK-8 0.1,0.01,0.001 μ g induction period administration, can suppress formation and the expression (Mean ± SD, n=12) of C57BL/6J mice behavior sensitization.
*represent and matched group comparison
p<0.05;
*represent and matched group comparison
p<0.01;
* *represent and matched group comparison
p<0.001.
Fig. 4 is CCK-8 0.1,0.01,0.001 μ g detection period administration, can suppress the expression (Mean ± SD, n=12) of C57BL/6J mice behavior sensitization.
*represent and matched group comparison
p<0.05;
* *represent and matched group comparison
p<0.001.
Fig. 5 is METH 0.1,0.5,1.0,2.0,4.0 mM dose-dependent inhibition PC12 cell survival rates (Mean ± SD, n=3).
* *represent and matched group comparison
p<0.001.
Fig. 6 is that CCK-8 0.1,0.5,1.0 μ M do not affect (Mean ± SD, n=3) to PC12 cell survival rate.
Fig. 7 is the reduction (Mean ± SD, n=3) that CCK-8 0.5,1.0 μ M can improve the PC12 cell survival rate that METH 1mM causes.
* *represent and matched group comparison
p<0.001,
###represent to compare with METH 1mM group
p<0.001.
Fig. 8 is the reduction (Mean ± SD, n=3) that CCK-8 0.5,1.0 μ M can improve the PC12 cell survival rate that METH 2mM causes.
* *represent and matched group comparison
p<0.001;
##represent to compare with METH 2 mM groups
p<0.01;
### represent to compare with METH 2 mM groups
p<0.001.
Fig. 9 is that METH various dose 2.0,1.0,0.5,0.1 mM processes the TNF-α secretion level variation that cell 3h causes,
*represent and matched group comparison
p<0.01;
* *represent and matched group comparison
p<0.001.LPS organizes positive control drug group, can obviously cause that cellular inflammation factor secretion increases.
Figure 10 is that METH various dose 2.0,1.0,0.5,0.1mM process the IL-1 β secretion level that cell 3h causes and change,
* *represent and matched group comparison
p<0.001.LPS organizes positive control drug group, can obviously cause that cellular inflammation factor secretion increases.
Figure 11 is that METH various dose 2.0,1.0,0.5,0.1 mM processes the IL-6 secretion level variation that cell 3h causes,
*represent and matched group comparison
p<0.05;
* *represent and matched group comparison
p<0.001.LPS organizes positive control drug group, can obviously cause that cellular inflammation factor secretion increases.
Figure 12 is that METH various dose 2.0,1.0,0.5,0.1 mM processes the MCP-1 secretion level variation that cell 3h causes,
* *represent and matched group comparison
p<0.001.LPS organizes positive control drug group, can obviously cause that cellular inflammation factor secretion increases.
Figure 13 is that the TNF-α secretion level that METH 1 mM processing 1h, 3h, 6h, 12h cause changes,
* *represent and matched group comparison
p<0.001.
Figure 14 is that the IL-1 β secretion level that METH 1 mM processing 1h, 3h, 6h, 12h cause changes,
*represent and matched group comparison
p<0.05;
*represent and matched group comparison
p<0.01;
* *represent and matched group comparison
p<0.001.
Figure 15 is that the IL-6 secretion level that METH 1 mM processing 1h, 3h, 6h, 12h cause changes,
* *represent and matched group comparison
p<0.001.
Figure 16 is that the MCP-1 secretion level that METH 1 mM processing 1h, 3h, 6h, 12h cause changes,
* *represent and matched group comparison
p<0.001.
Figure 17 is CCK-8(0.01,0.1,1.0 μ M) 1 mM METH is processed to the impact (Mean ± SD, n=3) that the TNF-α secretion level that causes of 3h changes,
*represent and matched group comparison
p<0.05;
#represent to compare with METH group
p<0.05.
Figure 18 is CCK-8(0.01,0.1,1.0 μ M) 1 mM METH is processed to the impact (Mean ± SD, n=3) that the IL-1 β secretion level that causes of 3h changes,
* *represent and matched group comparison
p<0.001;
#represent to compare with METH group
p<0.05;
###represent to compare with METH group
p<0.001.
Figure 19 is CCK-8(0.01,0.1,1.0 μ M) 1 mM METH is processed to the impact (Mean ± SD, n=3) that the IL-6 secretion level that causes of 3h changes,
* *represent and matched group comparison
p<0.001;
##represent to compare with METH group
p<0.01;
###represent and relatively P < 0.001 of METH group.
Figure 20 is CCK-8(0.01,0.1,1.0 μ M) 1 mM METH is processed to the impact (Mean ± SD, n=3) that the MCP-1 secretion level that causes of 3h changes,
*represent and matched group comparison
p<0.01;
##represent to compare with METH group
p<0.01;
###represent to compare with METH group
p<0.001.
Detailed description of the invention
To describe several embodiments of the present invention below, but content of the present invention is not limited to this completely.
Embodiment 1: preparation CCK-8 injection
Buy Sulfated CCK-8 from Sigma company of the U.S.; 33.7 μ L 25% ~ 28% ammonia are diluted to 10 mL with normal saline, obtain 0.05 mol/L ammonia (matching while using).
5 mg CCK-8 and 0.05 mol/L ammonia 5 mL are fully vibrated and mixed, obtain 1 mg/mL CCK-8 solution, 50 μ L/ pipe subpackages, ice bath operation ,-80 DEG C of preservations, half a year effect duration.By normal saline dilution to 1 mg/L concentration, avoid multigelation with front.
Cell administration has been calculated concentration by molar concentration meter, dilutes.Join in the unparalleled anti-cell culture medium of serum-free.
Embodiment 2: animal tricorn medication
1 mg/mL CCK-8 solution dilutes 20 times to 0.05 μ g/ μ L with sterile water for injection, then 10 times are diluted to 0.005,0.0005 μ g/ μ L, administration volume 2 μ L.
When injection, sleeve pipe nook closing member is extracted, syringe (external diameter 0.67 mm, internal diameter 0.3 mm) in inserting, connects PE pipe and microsyringe and injects.The injection cannula locator data of tricorn: 0.22 mm after bregma, other 0.94 mm of bregma; 2.20 mm under skull outer surface, (AP ,-0.22; ML, ± 0.94; DV ,-2.20), 0.22 mm after the bregma of interior syringe location, other 0.94 mm of bregma; 2.40 mm(AP under skull outer surface ,-0.22; ML, ± 0.94; DV ,-2.40).Intracerebroventricular injection volume 2 μ l, injection speed 1 μ l/min, let the acupuncture needle remain at a certain point after having injected 5 min.
CCK-8 belongs to polypeptide drug, and Peptides and proteins poor stability is easily hydrolyzed in gastrointestinal tract, and can exist absorbed problem, therefore use injection, using method is intravenously administrable more.
But because the biological half-life of this type of medicine is short, when clinical practice, usually need duplicate injection administration, bring misery and burden to patient.In order to reduce administration number of times, reduce the side reaction that accident rate and duplicate injection cause, the Injectable sustained release of polypeptide drug or controlled-release administrating system in recent years, CCK-8 can be prepared into slow releasing agent by existing conventional method, also can make particulate delivery system, can also make injectable gel, also can play slow releasing function.
According to the general Drug therapy cycle, 7 days be one-period the course for the treatment of.But concrete treatment time length, should abuse METH dosage according to cerebral injury in patients degree and patient and determine.
The contraindication that may exist and points for attention: CCK-8 has in periphery the cholecystokinetic function of receipts, may cause that digestive function waits side effect extremely.Therefore,, in using this medicine, should note monitoring gallbladder contraction function and digestive function.
Claims (3)
1. CCK-8 is in preparation treatment or improve the application in the dystropy medicine that life-time service methamphetamine causes.
2. CCK-8 causes the application in nerve injury medicine in preparation treatment or prevention methamphetamine.
3. the application of CCK-8 in the maincenter inflammation damnification medicine that preparation is treated or prevention methamphetamine causes.
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Citations (3)
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