CN1626245A - New application of chromaffin cell of adrenal medulla or opium peptide energy cell - Google Patents
New application of chromaffin cell of adrenal medulla or opium peptide energy cell Download PDFInfo
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- CN1626245A CN1626245A CN 200410068367 CN200410068367A CN1626245A CN 1626245 A CN1626245 A CN 1626245A CN 200410068367 CN200410068367 CN 200410068367 CN 200410068367 A CN200410068367 A CN 200410068367A CN 1626245 A CN1626245 A CN 1626245A
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention relates to a new application of chromaffin cell of adrenal medulla or opium peptide energy cell, particularly an application of curing and/or releasing withdrawal syndrome caused by addiction to drugs or drug abuse.
Description
Invention field
The present invention relates to the new purposes of adrenal medullary chromaffin cell or opium peptide cell, especially it is used for the treatment of and/or alleviates because of being addicted to drug or the purposes of the withdrawal syndrome that drug dependence produces.
Background technology
Drug dependence and addiction due to drug abuse and the drug dependence have become serious social concern day by day.Common method of treatment has: substitute and reach decreasing therapy (as methadone, the many coffee of dihydro dust etc.), subhibernation therapy, tcm therapy etc., but still do not have the ideal method of generally acknowledging so far.
Think that at present the addiction mechanism of opioid drug is relevant with the function of opiate receptor.Under the normal physiological situation, opiate receptor is subjected to the effect of the endogenous opiate sample polypeptide (EOP) of certain foundation level, when giving exogenous opioid compounds (EOC) as morphine, morphine has captured remaining opiate receptor, has strengthened the analgesic effect of endogenous opiate sample polypeptide.When continuous, when giving exogenous morphine to volume, cause that by feedback regulation the amount that the EOP neuron discharges EOP falls sharply, and needs more exogenous morphine just can keep effects such as analgesia.Therefore, in case the morphine class medicine of stopping using, body is lacking endogenous opioid peptide, when not having exogenous morphinization in opiate receptor again, will cause the abnormal symptom that a series of other neurotransmitter secretions increase, i.e. the withdrawal symptom that occurs clinically.
Normal body exists the cell that can secrete endogenous opiatepeptide, as adrenal medullary chromaffin cell (MCC) etc.MCC can secrete three class materials: monoamines, as norepinephrine (NE), epinephrine (E), dopamine etc.; The endogenous opiatepeptide class is as bright deltorphin delta, first deltorphin delta, dynorphin etc.; And multiple somatomedin, as nerve growth factor, epidermal growth factor etc.MCC can secrete respective substance when the stimulation of being correlated with, generation stress, effect such as analgesia.At the early eighties in last century, two seminar of the U.S. and Switzerland attempt being implanted into internal therapy pain under the spinal arachnoid mater with the adrenal medulla tissue and the pheochromocyte (chromaffin cell) of (people, Mus) of the same race, have obtained satisfied effect.But because human adrenal medulla tissue and pheochromocyte source are rare, thus seminar's trial of the nineties U.S. with the spinal arachnoid mater of xenogeneic bovine adrenal medullary substance pheochromocyte (hereinafter to be referred as BCC) implantation cancer patient under to treat cancer pain.In order to overcome immunological rejection, they adopt length is the polyacrylamide hollow fiber conduit parcel BCC of 5cm, diameter 1mm.Because doughnut tube wall fenestra only allows micromolecular material to pass through, so the secretions of BCC can be slowly and is diffused out fiber pipe equably, the performance analgesic effect, and the intravital macromole immunoglobulin of host can not penetrate the tube wall fenestra, cell can be survived in host the long period (about 1 year) constantly secrete the pain relieving material, thereby pain patients is produced therapeutical effect.But the volume of hollow fiber conduit is bigger, on the one hand, because dead space is big in its pipe, has therefore influenced the diffusion of nutrient and metabolite, makes the pipe inner cell be difficult for long-term surviving; On the other hand, the fiber pipe of large volume is implanted easily spinal nerves to be produced under the spinal arachnoid mater to stimulate and compressing, thereby produces many to the disadvantageous side effect of human body; In addition,, implant spinal arachnoid mater following time and must adopt operating method to implant, bring certain damage to patient because the volume of hollow fiber conduit is big.Simultaneously, the biocompatibility of the hollow fiber conduit of making of material such as chitan, polyacrylamide and sodium carboxymethyl cellulose uses such as () U.S. is relatively poor, the tissue reaction that causes host easily.On the other hand, adopt sodium alginate-polylysine--the three-decker film microcapsule (hereinafter to be referred as the APA microcapsule) that sodium alginate is made, its volume little (diameter is 200-1000 μ m), biocompatibility height are beneficial to the survival of long-time in animal body intact existence of microcapsule and capsule inner cell longer-term.Experiment proves, but the cyst membrane cut of APA microcapsule make immunoglobulin and immunologically competent cell can not pass this film and enter capsule in destruction zooblast wherein greater than 110,000 Kd (dalton's) molecule, thereby have immanoprotection action preferably.Experiment also demonstrates the APA microcapsule and has better biocompatibility, but long period (about 1 year half) intact existence in little, large animal body.In the research of islets of langerhans, hepatocyte, parathyroid gland and recombinant somatropin's secretory cell transplantation treatment disease model animal, proved that having the protection xenograft avoids the destructive effect of host immune system.But, up to now, do not find as yet both at home and abroad to adopt APA microencapsulated bovine adrenal medullary substance pheochromocyte to be used for the treatment of and/or to alleviate the report of withdrawal syndrome.
Summary of the invention
The objective of the invention is to provide a kind of biocompatibility height, long action time, toxic and side effects little, easy and simple to handle being applicable to treatment or alleviating the product of the withdrawal syndrome that produces because of addiction.
The inventor after deliberation, have now found that adrenal medullary chromaffin cell or the opium peptide cell that obtains by methods such as genetic engineerings, especially APA microencapsulated bovine adrenal medullary substance pheochromocyte withdrawal syndrome that addiction is caused has good curing or remission effect, the present invention is based on above-mentioned discovery and now finishes.
Therefore, first aspect present invention relates to adrenal medullary chromaffin cell or opium peptide cell and is used for the treatment of and/or alleviates purposes in the product of the withdrawal syndrome that produces because of drug dependence or abuse in preparation.
Further aspect of the present invention relates to treats and/or alleviates the method that produces withdrawal syndrome because of drug dependence or abuse, and it comprises to medicine addiction or misuser injects adrenal medullary chromaffin cell or opium peptide cell.
Further aspect of the present invention relates to and being used for the treatment of or the product of the disconnected syndrome of alleviation formula, and it comprises especially APA microsaccular adrenal gland medullary substance pheochromocyte of adrenal medullary chromaffin cell or opium peptide cell.
Further aspect of the present invention relates to microencapsulated bovine adrenal medullary substance pheochromocyte or the opium peptide cell medicine that is used for the treatment of or alleviates withdrawal syndrome, it is characterized in that this medicine follows these steps to make:
(1) is that the solution of sodium alginate of 10-20g/L is mixed with bovine adrenal medullary substance pheochromocyte or opium peptide cell and a kind of concentration, makes suspension, make to contain 0.1-1 * 10 in every liter of suspension
10Individual cell;
(2) utilize suspension that sprayer unit obtains step (1) to be scattered in the calcium chloride or calcium lactate solution that a kind of concentration is 80-120mmol/L with the micro-droplet status of 150-1000 μ m diameter, the ratio of two kinds of liquid is for guaranteeing that every liter of mixed liquor contains 0.1-1 * 10
8Individual cell was placed 5-20 minute, treated to remove supernatant after precipitation fully, obtained to contain the calcium alginate pearl precipitation of bovine adrenal medullary substance pheochromocyte;
(3) precipitate that step (2) is obtained joins in the polylysine solution that a kind of concentration is 0.3-0.7g/L, and the ratio of the two is for guaranteeing that every liter of liquid contains 0.2-2 * 10
8Individual cell, mix homogeneously was placed 5-20 minute, treated to remove supernatant after precipitation is fully, obtained precipitate;
(4) precipitate that step (3) is obtained joins in the solution of sodium alginate that a kind of concentration is 1.0-2.0g/L, and the ratio of the two is for guaranteeing that every liter of liquid contains 0.2-2 * 10
8Individual cell, mix homogeneously was placed 3-15 minute, treated to remove supernatant after precipitation is fully, obtained precipitate;
(5) precipitate that step (4) is obtained joins in the sodium citrate solution that a kind of concentration is 40-70mmol/L, and the ratio of the two is for guaranteeing that every liter of liquid contains 0.2-2 * 10
8Individual cell, mix homogeneously was placed 5-20 minute, treated to remove supernatant after precipitation is fully, obtained microencapsulated bovine adrenal medullary substance pheochromocyte or opium peptide cell precipitate;
(6) precipitate that step (5) is obtained joins the sodium chloride solution cleaning that a kind of concentration is 9g/L, at last precipitate is transferred to cultivate in the cell culture fluid and preserve as the microencapsulated bovine adrenal medullary substance pheochromocyte or the opium peptide cell medicine of treatment pain.
According to the present invention, adrenal medullary chromaffin cell can be from adrenal medullary chromaffin cell or the genetically engineered cell of mammal such as people, cattle, sheep, pig or Mus among the present invention, preferred bovine adrenal medullary substance pheochromocyte.
According to the present invention, adrenal medullary chromaffin cell of the present invention or opium peptide cell use with the microencapsulation form, the bovine adrenal medullary substance pheochromocyte of preferred APA microencapsulation.
Further, the microencapsulated bovine adrenal medullary substance pheochromocyte among the present invention preferably has the purity more than 80%.
According to the present invention, opium peptide cell of the present invention says it is to modify the cell with opioid peptide ability that obtains by mammal such as people or bovine adrenal medullary epithelium are carried out genetic engineering for example.
According to micro-capsuled pheochromocyte of bull adrenal medulla as medicine of the present invention, the suspension that preferably step (1) is obtained in its step (2) disperses with the micro-droplet status of 180-500 μ m diameter.
In use, as long as with APA microencapsulation BCC of the present invention (2-9 * 10
6Individual cell) injection has under withdrawal syndrome patient's the spinal arachnoid mater, can produce therapeutical effect in 4-24 hour, and a shot can be kept therapeutical effect more than 9 months.
Further say, described in the present invention bovine adrenal medullary substance pheochromocyte (BCC) is meant in the bovine adrenal medullary substance can modify the opium peptide cell that obtains by the painted cell of chrome complex dye or by genetic engineering, and it can secrete materials such as monoamines, enkephalin class (comprising first deltorphin delta, bright deltorphin delta) and neurotrophic factor.
The acquisition of BCC and purification can carry out with means known in the art.For example, can use collagenase (Collagenase) and the adrenal gland of cattle to act on, make wherein collagen tissue decomposition, the cooperative mechanical method becomes individual cells with the bovine adrenal separate tissue then, then use the screen filtration of 170 orders (88 μ m) mesh, the bovine adrenal medullary epithelium (contains pheochromocyte, endotheliocyte, fibroblast and hemocyte) pass filter screen, liquid off the net is centrifugal, abandoning supernatant, promptly obtained the precipitate of above-mentioned various bovine adrenal medullary epitheliums, wherein, pheochromocyte accounts for the 50-60% of total cellular score, the two accounts for the 40-50% of total cellular score altogether endotheliocyte and fibroblast, because the form of hemocyte is very little, therefore it is not calculated in total cell usually.Should be noted that,, also can not be applied among the present invention even therefore do not carry out purification because BCC accounts for 50-60% in bovine adrenal medullary epithelium sum.But, preferably be purified to BCC and account for more than 80% of total cellular score in order to improve therapeutic effect.As the purification process of BCC, for example can adopt conventional adherent method of purification, its principle is to separate according to the adherent tendency that different types of cell has in various degree.For example for the mixed liquor that contains pheochromocyte, endotheliocyte, fibroblast and hemocyte, in culture bottle in a few hours, most of fibroblast is adherent, therefore and most of pheochromocyte, endotheliocyte and hemocyte are not adherent, can remove most of fibroblast when changing bottle.Again because of hemocyte adherent growth not, thus can be by cultivating the long period (for example 24-48 hour), treat that pheochromocyte and endotheliocyte change bottle once more after adherent and can remove most of hemocyte.So changing bottle through twice can make the purity of BCC reach more than 80% of total cellular score (except the hemocyte).
About the method for counting of cell, can adopt the method that microscopically is observed counting after the conventional dyeing.For example can adopt the blue staining (Trypan blue stain) of Placenta Hominis, see " tissue culture's solvent and reagent " (Tissue Culture Media and Reagents), the 1566th page.
In the preparation of microsaccular adrenal gland medullary substance pheochromocyte of the present invention or the opium peptide cell medicine that obtains by methods such as genetic engineerings, each step solutions employed amount can be grasped according to the cell number that limits.For example, in the suspension that the step (1) of the invention described above medication preparation is obtained every liter contain 0.1-1 * 10
10Individual cell, and in the mixed liquor of step (2) every liter contain 0.1-1 * 10
8Individual cell, just the cell concentration in the suspension of step (1) is equivalent to about 100 times of cell concentration in the mixed liquor of step (2), therefore, if from the suspension of step (1), take out 1ml, then in step (2), preferably it is distributed in the calcium chloride solution of 100ml.The rest may be inferred by analogy.
The effect that forms the calcium alginate pearl in step (2) is to create conditions for microcapsule that acquisition in step (5) contains bovine adrenal medullary substance pheochromocyte.In step (5), the sodium ion of sodium citrate will just form the microcapsule with many fine pores after the displacement of the calcium ion in the calcium alginate pearl.In these microcapsules, contain many BCC, and around BCC, be wrapped in sodium alginate.
Formation method to the calcium alginate pearl does not have particular determination, so long as can will contain the sodium alginate suspension of BCC be scattered in the calcium chloride solution with enough small drop and both can.The diameter of this sodium alginate suspension microdroplet usually should be in the scope of 150-1000 μ m, preferably in the scope of 180-500 μ m.If diameter of droplets is more than 1000 μ m, the then last microcapsule that obtains is excessive, causes easily when being injected into human or animal's body and breaks, and is therefore bad.Normally used drop process for dispersing has needle injection method, nebulization etc., nebulization preferably, the static microcapsule generator that most preferably utilizes University of Toronto to make (is seen " Enzymology method " " microencapsulation islet cells: a kind of biological endocrine pancreas " SUN, A.M.Micro-encapsulation ofpancreatic islet cell:a bio-artificial endocrine pancreas.In:Methodsin Enzymology, the 137th volume, the 575-580 page or leaf, 1988) method of the spraying carried out.
Above technical scheme of the present invention has been carried out more detailed explanation, those skilled in the art can understand the present invention after the appended examples at an easy rate having read above to reach hereinafter.
Compare with the existing similar technology of this area, the present invention has following good effect:
Confirm by zoopery, the opium peptide cell that uses APA microsaccular adrenal gland medullary substance pheochromocyte of the present invention or obtain by methods such as genetic engineerings can excretory for a long time slightly endogenous opiatepeptide class material, acts on patient's opiate receptor.Lasting, a small amount of of the cell performance " miniature organism pump " of implanting are secreted effect, in a period of time, when clinical withdrawal symptom is alleviated, can reduce negative feedback inhibition effect to the emiocytosis of patient self endogenous opiate energy, make that the ability of its secretion EOP is recovered gradually, thereby reach the purpose of drug rehabilitation.
Compared with prior art, the present invention has following characteristics:
1, the APA microcapsule volume of making by the present invention little (diameter 180-500um), thereby have 3 advantages at least, promptly (1) is beneficial to the diffusion of nutrient and metabolite, makes the capsule inner cell be easy to longer survival; (2) do not need hollow fiber conduit to be implanted, only need microcapsule to be injected in the cavitas subarachnoidealis spinalis with the simple waist method of wearing with the method for operation, little to tissue injury; (3) in cavitas subarachnoidealis spinalis, be difficult for spinal nerves is produced the side effect that stimulates and oppress;
2, compare with the immune isolating membrane of other material, the APA microcapsule has good biocompatibility;
3, a large amount of experiments confirm that APA microcapsule of the present invention has good immanoprotection action;
4, BCC material of (more than 3 months) secretion antagonism withdrawal syndrome sustainably in host produces treatment that continues and/or the effect of alleviating withdrawal syndrome;
5, microencapsulation BCC can bring into play the effect of treatment and/or alleviation withdrawal syndrome for long periods in host;
6, compare with adopting human adrenal gland medullary substance pheochromocyte, bovine adrenal medullary substance pheochromocyte can obtain in enormous quantities;
7, can adopt cryogenic technique to preserve microencapsulation BCC, be convenient to long-distance transportation and supply in batches.
The specific embodiment
Enumerate embodiment, experimental example and application examples below and further explain the present invention, but the present invention is not subjected to the qualification of these concrete examples.
The isolation and purification of embodiment 1:BCC
1, get 12 fresh bovine adrenals (Ischemia Time at room temperature is no more than 1 hour) from the slaughterhouse, it is standby to transport laboratory under refrigerated condition rapidly back.
2, the collagenase I that is 1g/L from adrenal vein implantation concentration (collagenase I) solution, each adrenal gland injects collagenase I solution 5ml, places 30 minutes down at 37 ℃ then, so that allow collagenase and the pericellular colloid of bovine adrenal fully react.
3, cut cortex along adrenal gland's longitudinal axis, isolate medullary substance and it is shredded.
4, adding 60ml concentration in the medullary substance tissue that has all shredded is the collagenase I solution of 1g/L, places 30 minutes again.
5, with a mesh be the steel mesh filtration of 170 orders (88 μ m), liquid under the collecting net.
6, with liquid centrifugalize off the net, abandoning supernatant obtains to contain the bovine adrenal medullary epithelium precipitate of (comprising BCC, endotheliocyte, fibroblast and hemocyte).
7, count with the blue staining of platform dish, the total cellular score of acquisition (except the hemocyte) is 5.8 * 10
7, cell survival rate is 90%.
8, cell transfer is gone in 2 culture bottles, every bottle adds DMEM (Dulbeco ' sModified Eagle Medium) culture fluid (wherein also containing penicillin 100 units/ml, streptomycin 100 μ g/ml, calf serum 10 volume %) 20ml, placing a temperature is 37 ℃, contains 5 volume %CO
2Cultivate in the incubator, change bottle after 5 hours, continue to cultivate, the BCC overwhelming majority is affixed on bottle wall, stand-by.
Usually need at least to cultivate more than 24 hours, when being used to prepare microencapsulation zooblast medicine, only need the solution removal in the culture bottle is collected BCC and can be further processed with conventional trypsin digestion from culture bottle.
Should illustrate that the method for embodiment 1 belongs to routine techniques, it does not play any qualification effect to the present invention.
Embodiment 2: the preparation of microencapsulation zooblast medicine
1, the BCC that uses the method press embodiment 1 to obtain, centrifugalize obtains the precipitate of BCC, washs and is diluted to 1ml with normal saline, and it is moved in the centrifuge tube.
2, with the blue staining counting of platform dish, the total cellular score that obtains is 3 * 10
6Individual cell, wherein the purity of BCC is 82%.
3, to wherein adding the solution of sodium alginate that 1ml concentration is 15g/L, be made into suspension with paddling process.
4, with static microcapsule generator (electrostatic droplet generator, University of Toronto makes) suspension is injected in the calcium chloride solution that 100ml concentration is 100mmol/L, having obtained the celliferous calcium alginate pearl precipitation of a kind of diameter in the 180-500 mu m range after 10 minutes, treat to remove supernatant after precipitation fully.
5, the calcium alginate pearl is joined in the polylysine solution that 50ml concentration is 0.5g/L, mix homogeneously was at room temperature placed 10 minutes, treated to remove supernatant after precipitation fully.
6, the precipitate that step 5 is obtained joins in the solution of sodium alginate that 60ml concentration is 1.5g/L, and mix homogeneously was at room temperature placed 10 minutes, treats to remove supernatant after precipitation fully.
7, the precipitate that step 6 is obtained joins in the sodium citrate solution that 60ml concentration is 55mmol/L, at room temperature places 10 minutes, treats to remove supernatant after precipitation fully, obtains to contain the microencapsulation zooblast precipitate of BCC.
8, with concentration be the sodium chloride solution washing precipitate of 9g/L, then this precipitate be transferred in the described DMEM culture fluid of step 8 of embodiment 1 and cultivate, stand-by as injection microencapsulation BCC medicine.
Experimental example 1: microencapsulation BCC is to the influence of the withdrawal syndrome of rat generation
Experiment material
1 experimental drug: morphine hydrochloride, methadone, naloxone hydrochloride, normal saline, NO test kit, SOD test kit, prostaglandin E
2Radioimmunity medicine box, heparin sodium injection, indometacin injection, chloral hydrate injection.
2 microencapsulation pheochromocytes.
3 laboratory animals: the Wistar rat, about 250g.
Experimental technique
1 rat morphine relies on the foundation (with reference to the method for Ling GSF etc.) of model: get body weight and be totally 150 of Wistar rats about 250g, male and female half and half are divided into 5 groups at random, every group each 30.1-4 group relies on model group for morphine, 2 subcutaneous injection morphines every day (morning 8:00 and afternoon 6:00), and dosage increases the 20mg/kg body weight every day since 20mg/kg body weight every day, administration 5 days, final dose reaches 100mg/kg body weight every day.4h after last injection morphine, lumbar injection naloxone hydrochloride 4mg/kg body weight is observed 1h, and every withdrawal symptom and scoring respectively organized in record, and integration is above person's modelling success at 5 minutes.
The transaction module of 3 each group is set up successfully, and back 16h (6 days) begins each group is carried out different disposal.
The microcapsule group: every rat subarachnoid space is implanted 2*10
5APA-BCC stops to inject morphine;
Self withdrawal group: every rat is injected cavitas subarachnoidealis spinalis with the 20ul normal saline, stops to inject morphine;
4 laboratory observations and data acquisition
Urge the addiction experiment for the 1st time: transplant next day (7 days) certainly, in the end 3h-4h carries out following experiment behind 1 injection of morphia: lumbar injection naloxone hydrochloride 4mg/kg body weight, observe lh, write down every withdrawal symptom and scoring, measure NO, SOD, prostaglandin E in body weight, the blood
2Content.
Urge addiction experiment for the 2nd time: above experiment is after 4 days, and lumbar injection naloxone 4mg/kg body weight is urged addiction, observed and respectively organize withdrawal symptom for the 2nd time.
Attached 1: withdrawal symptom standards of grading (with reference to the method for Ling GSF etc.) are formulated
(l) abnormal posture is 2 minutes, height excitation, touching excitation 1 minute, close excitation 2 minutes.
(2) grit one's teeth: discontinuity 0.5 minute, seriality 1 minute.
Deviant Behavior 1h keeps the score once.
(3) shed tears 4 fens.
(4) diarrhoea: soft stool 4 minutes, shapeless 8 minutes.
(5) sialorrhea: slight 1 minute, obvious 2 minutes.
The every 15min of autonomic nervous system symptom keeps the score 1 time.
4 data collections:
Gather respectively and organize respectively that rat is urged addiction for the first time, the scoring of withdrawal symptom when urging addiction for the second time; Urge and respectively organize rat body weight in the different time after the addiction and alleviate comparison; Urge after the addiction and respectively organize NO in the rat blood, SOD, PGE in the different time
2Changes of contents.
5 row statistical procedures.
Experimental result
Microencapsulated bovine pheochromocyte subarachnoid space is transplanted the influence that morphine is relied on rat
Get 15 body weight and be totally 10 of 240g left and right sides Wistar rats, all male, successfully made morphine with 5 days and rely on model, withdrawal symptom scoring average out to 24 minutes.Be divided into 2 groups at random, 5 every group.The 1st group is the microcapsule group, and the 2nd group is self withdrawal group.
Experimental result is as follows:
Table 1 is respectively organized the scoring of rat withdrawal symptom relatively
Annotate with the microcapsule group and compare
*P＜0.01
*P＜0.05
Table 2 is respectively organized the comparison that loses weight of rat different time
Annotate with the microcapsule group and compare
*P＜0.01
*P＜0.05
Table 3 is urged after the addiction and is respectively organized NO SOD assay result in the rat blood in the different time
Annotate with the microcapsule group and compare
*P＜0.01
*P＜0.05
There is marked difference (have statistical significance) between microcapsule group and self withdrawal group at withdrawal symptom score value, body weight and NO aspect horizontal as can be seen from the above table.The result shows: microencapsulated bovine pheochromocyte subarachnoid space is implanted and can obviously be alleviated the withdrawal symptom that morphine relies on rat.
Embodiment 3: the foundation of immortal human adrenal medullary chromaffin cell system
It is foster that human adrenal gland medullary substance pheochromocyte former is commissioned to train: get the fresh adult or the fetal adrenal of no adrenal gland diseases, adopt machinery and chemical method to obtain human adrenal gland medullary substance pheochromocyte (HCC) and cultivation; Detect the biological property of HCC, prove the ability that possesses catecholamine secretion and enkephalin.
The foundation of human adrenal gland medullary substance pheochromocyte system: adopt the liposome transfection method, the eukaryon expression plasmid PCIneo-hTERT of coding hTERT and neo gene is imported the former foster HCC that is commissioned to train, screen positive cell clone with G418, adopt the RT-PCR method to detect the positive colony telomerase activation, checking external source hTERT activates the ability of purpose cell telomerase activation; Be chosen in long-term long-living good clone under the G418 environment, detect mRNA and the proteic expression of positive cell clone hTERT respectively by RT-PCR and WesternBlot.
Should illustrate that the method for embodiment 3 belongs to routine techniques, it does not play any qualification effect to the present invention.
Embodiment 4: the preparation of microencapsulation genetically engineered cell medicine
1. use the HCC of the method acquisition of pressing embodiment 3, pancreatin separates, and obtains the precipitate of HCC, washs with normal saline.
2. to wherein adding the sodium alginate soln that 1ml concentration is 15g/L, it makes suspension.
3. with static microcapsule generator (electrostatic droplet generator) suspension is injected in the calcium chloride solution that 100ml concentration is 100mmol/L, obtain the celliferous calcium alginate pearl precipitation of a kind of diameter in the 180-500 mu m range, treat to remove supernatant after precipitation fully.
4. the calcium alginate pearl is joined in the polylysine solution that 50ml concentration is 0.5g/L, mix homogeneously was at room temperature placed 5-10 minute, treated to remove supernatant after precipitation fully.
5. the precipitate that step 4 is obtained joins in the solution of sodium alginate that 60ml concentration is 1.5g/L, and mix homogeneously was at room temperature placed 5-10 minute, treats to remove supernatant after precipitation fully.
6. the precipitate that step 5 is obtained joins in the sodium citrate solution that 60ml concentration is 55mmol/L, at room temperature places 5-10 minute, treats to remove supernatant after precipitation fully, obtains microencapsulation HCC precipitate.
7. be the sodium chloride solution washing precipitate of 9g/L with concentration, this precipitate be transferred in the DMEM culture fluid cultivate then, stand-by as injection microencapsulation HCC medicine.
Embodiment 5: microencapsulation HCC subarachnoid space is transplanted the influence that morphine is relied on the rat withdrawal syndrome
Experiment material and method: identical with experimental example microencapsulation BCC to the influence of the withdrawal syndrome of rat generation, but experimental drug is microencapsulation HCC (APA-HCC).
Get body weight and be totally 10 of 240g left and right sides Wistar rats, all male, successfully made morphine with 5 days and rely on model, withdrawal symptom scoring average out to 24 minutes.Be divided into 2 groups at random, 5 every group.The 1st group is the microcapsule group, and the 2nd group is self withdrawal group.
Experimental result shows urges for the second time after the addiction scoring of microcapsule group rat withdrawal symptom obviously to reduce (P＜0.01) than self withdrawal group scoring; Transplant beginning in back 1 day, microcapsule group rat body weight alleviates degree and is starkly lower than self withdrawal group (P＜0.01).Prompting microencapsulation HCC subarachnoid space is implanted and can obviously be alleviated the withdrawal symptom that morphine relies on rat.
Claims (9)
1. the opium peptide cell that obtains of method such as adrenal medullary chromaffin cell or genetic engineering is used for the treatment of and/or alleviates purposes in the product of the withdrawal syndrome that produces because of drug dependence or abuse in preparation.
2. treatment and/or alleviation are because of the method for drug dependence or abuse generation withdrawal syndrome, and it comprises to medicine addiction or misuser injects the opium peptide cell that methods such as adrenal medullary chromaffin cell or genetic engineering obtain.
3. be used for the treatment of or the product of the disconnected syndrome of alleviation formula, it comprises the especially opium peptide cell that obtains of method such as APA microsaccular adrenal gland medullary substance pheochromocyte or genetic engineering of opium peptide cell that methods such as adrenal medullary chromaffin cell or genetic engineering obtain.
4. be used for the treatment of or alleviate the opium peptide cell medicine that methods such as the microencapsulated bovine adrenal medullary substance pheochromocyte of withdrawal syndrome or genetic engineering obtain, it is characterized in that this medicine follows these steps to make:
(1) opium peptide cell that methods such as bovine adrenal medullary substance pheochromocyte or genetic engineering are obtained and a kind of concentration are that the solution of sodium alginate of 10-20g/L is mixed, and make suspension, make to contain 0.1-1 * 10 in every liter of suspension
10Individual cell;
(2) utilize suspension that sprayer unit obtains step (1) to be scattered in the calcium chloride or calcium lactate solution that a kind of concentration is 80-120mmol/L with the micro-droplet status of 150-1000 μ m diameter, the ratio of two kinds of liquid is for guaranteeing that every liter of mixed liquor contains 0.1-1 * 10
8Individual cell was placed 5-20 minute, treated to remove supernatant after precipitation fully, obtained to contain the calcium alginate pearl precipitation of the opium peptide cell that methods such as bovine adrenal medullary substance pheochromocyte or genetic engineering obtain;
(3) precipitate that step (2) is obtained joins in the polylysine solution that a kind of concentration is 0.3-0.7g/L, and the ratio of the two is for guaranteeing that every liter of liquid contains 0.2-2 * 10
8Individual cell, mix homogeneously was placed 5-20 minute, treated to remove supernatant after precipitation is fully, obtained precipitate;
(4) precipitate that step (3) is obtained joins in the solution of sodium alginate that a kind of concentration is 1.0-2.0g/L, and the ratio of the two is for guaranteeing that every liter of liquid contains 0.2-2 * 10
8Individual cell, mix homogeneously was placed 3-15 minute, treated to remove supernatant after precipitation is fully, obtained precipitate;
(5) precipitate that step (4) is obtained joins in the sodium citrate solution that a kind of concentration is 40-70mmol/L, and the ratio of the two is for guaranteeing that every liter of liquid contains 0.2-2 * 10
8Individual cell, mix homogeneously was placed 5-20 minute, treated to remove supernatant after precipitation is fully, obtained the opium peptide cell precipitate that methods such as microencapsulated bovine adrenal medullary substance pheochromocyte or genetic engineering obtain;
(6) precipitate that step (5) is obtained joins the sodium chloride solution cleaning that a kind of concentration is 9g/L, at last precipitate is transferred to and cultivates in the cell culture fluid and preserve as the opium peptide cell medicine that methods such as microencapsulated bovine adrenal medullary substance pheochromocyte for the treatment of pain or genetic engineering obtain.
5. according to the purposes of claim 1, wherein the opium peptide cell that obtains of method such as adrenal medullary chromaffin cell or genetic engineering can be from adrenal medullary chromaffin cell or the genetically engineered cell of mammal such as people, cattle, sheep, pig or Mus.
6. according to the purposes of claim 5, wherein adrenal medullary chromaffin cell is a bovine adrenal medullary substance pheochromocyte.
7. according to the purposes of claim 1, wherein adrenal medullary chromaffin cell uses with the microencapsulation form.
8. according to the purposes of claim 7, wherein adrenal medullary chromaffin cell is the bovine adrenal medullary substance pheochromocyte with the APA microencapsulation.
9. according to the purposes of claim 1, wherein microencapsulated bovine adrenal medullary substance pheochromocyte has the purity more than 80%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200410068367A CN100591360C (en) | 2003-09-16 | 2004-08-31 | New application of chromaffin cell of adrenal medulla or opium peptide energy cell |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN03157131 | 2003-09-16 | ||
| CN03157131.X | 2003-09-16 | ||
| CN200410068367A CN100591360C (en) | 2003-09-16 | 2004-08-31 | New application of chromaffin cell of adrenal medulla or opium peptide energy cell |
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| Publication Number | Publication Date |
|---|---|
| CN1626245A true CN1626245A (en) | 2005-06-15 |
| CN100591360C CN100591360C (en) | 2010-02-24 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN200410068367A Expired - Fee Related CN100591360C (en) | 2003-09-16 | 2004-08-31 | New application of chromaffin cell of adrenal medulla or opium peptide energy cell |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101805396B (en) * | 2007-06-28 | 2011-09-14 | 北京大学 | Anti-opioid peptide antagonist peptide and use thereof |
| CN101817877B (en) * | 2007-06-28 | 2011-09-14 | 北京大学 | Active fragment of anti-opioid peptide |
| CN102127151B (en) * | 2007-06-11 | 2012-07-04 | 北京大学 | Antagonistic peptide of anti-opioid peptide and application thereof |
| CN102127152B (en) * | 2007-06-11 | 2012-07-04 | 北京大学 | Antagonistic peptide of anti-opioid peptide and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1235027A (en) * | 1999-06-16 | 1999-11-17 | 薛毅珑 | Micro-capsuled pheochromocyte of bull adrenal medulla as medicine for curing pains |
-
2004
- 2004-08-31 CN CN200410068367A patent/CN100591360C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102127151B (en) * | 2007-06-11 | 2012-07-04 | 北京大学 | Antagonistic peptide of anti-opioid peptide and application thereof |
| CN102127152B (en) * | 2007-06-11 | 2012-07-04 | 北京大学 | Antagonistic peptide of anti-opioid peptide and application thereof |
| CN101805396B (en) * | 2007-06-28 | 2011-09-14 | 北京大学 | Anti-opioid peptide antagonist peptide and use thereof |
| CN101817877B (en) * | 2007-06-28 | 2011-09-14 | 北京大学 | Active fragment of anti-opioid peptide |
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| Publication number | Publication date |
|---|---|
| CN100591360C (en) | 2010-02-24 |
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