CN1098690C - Microencapsulated bovine adrenal medulla chromaffincell medicine used for treating Parkinson's disease - Google Patents
Microencapsulated bovine adrenal medulla chromaffincell medicine used for treating Parkinson's disease Download PDFInfo
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Abstract
The present invention relates to a microencapsulated bovine adrenal medulla chromaffin cell (BCC for short) medicine for curing parkinson disease. The preparation method of the present invention has the following steps: 1, suspending BCCs in sodium alginate solution; 2, dispersing the suspended liquid in calcium chloride solution to form calcium alginate ball deposits; 3, mixing the deposits with polylysine solution to cover a polylysine film and deposit; 4, mixing the deposits with sodium alginate solution to cover a sodium alginate film; 5, replacing calcium ions in the deposits by sodium citrate to microencapsulate the BCCs; 6, transferring the microencapsulated BCCs into culture solution for use. The cell medicine can secrete dopamine for a long time in the human brain, and can maintain the curative effects for more than 12 months after being implanted into human bodies once.
Description
The present invention relates to the zooblast medicine, more particularly, relate to the purposes that a kind of microencapsulated bovine adrenal medullary substance pheochromocyte suspension is used to prepare treatment parkinson disease medicine.
Parkinson disease a kind of common in, infirmities of age, its main diseases because of being because the hypofunction of dopaminergic neuron in the brain, thereby cause a series of clinical symptoms.Existing drug treatment easily develops immunity to drugs and side effect.Another kind of Therapeutic Method is to the intracerebral transplantation dopaminergic cell, and to replenish the deficiency of Dopaminergics content of material in the parkinson disease human body, this Therapeutic Method is obtained positive effect in animal and a few patients.But the dopaminergic cell source is rare and cause immunological rejection easily, and this two hang-up has limited this The Application of Technology.Dopaminergic cell commonly used now has several, and they respectively have its pluses and minuses: (1) adopts embryo's adrenal medulla tissue or substantia nigra tissue to have source rareness and ethnics Problem though its immunological rejection of alloplast is little; Adopt the parkinson patient that grows up to exist adrenal gland's operation wound patient to be hit problems such as big and cell function difference from body adrenal medulla tissue.(2) the people's pheochromocytoma strain PC12 that adopts microcapsule to wrap up, though can secrete the Dopaminergics material, there is worrying danger in implanted tumor cells.(3) problem that adopts abundant xenogenesis adrenal medulla tissue in source or substantia nigra tissue to have immunological rejection.(4) transgenic cell still is difficult to stably obtain at present, and also has the problem of immunological rejection.At present, have multiple for overcoming the immune isolation technology that immunological rejection adopts in the histiocyte transplanting, for example macrocyst, microcapsule, hollow fiber conduit and perfusion cell etc., microcapsule wherein is beneficial to plantation in the survival of cell and the body because of volume little (less than 1000 μ m).The material of making microcapsule has many kinds, sodium alginate-polylysine for example--sodium alginate (being called for short APA), agarose, chitosan, chitan, polyacrylamide and sodium carboxymethyl cellulose etc., wherein the biocompatibility height of sodium alginate, formed cyst membrane surface is more smooth and help long-time in animal body intact existence.Experiment proves; the APA microcapsule has immanoprotection action preferably; but its cyst membrane cut is greater than the molecule (immunoglobulin and immunologically competent cell can not pass film) of 110,000 Kd, and has better biocompatibility, but in little, large animal body the long period exist in good condition.Proved that in the research of islets of langerhans, hepatocyte, parathyroid gland and recombinant somatropin's secretory cell transplantation treatment disease model animal the APA microcapsule membrane has the protection xenograft and avoids the destructive effect of host's vivo immuning system.But, up to now, do not find to adopt APA microencapsulated bovine adrenal medullary substance pheochromocyte (being called for short BCC) suspension at home and abroad as yet as the Parkinsonian report of Drug therapy.
The purpose of this invention is to provide a kind of cell wide material sources, not by immunologic rejection, action time persistent APA microencapsulation zooblast suspension be used to prepare the purposes for the treatment of Parkinsonian medicine.
The inventor furthers investigate at the situation of above-mentioned prior art, found that, adopts following technical proposals to achieve the above object, thereby has finished the present invention.
Technical scheme of the present invention is as follows:
1, microencapsulated bovine adrenal medullary substance pheochromocyte suspension is used to prepare the purposes of treatment parkinson disease medicine, and wherein, said cell suspending liquid 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 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 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 and cultivates in the cell culture fluid and preserve as microencapsulated bovine adrenal medullary substance pheochromocyte suspension.
2, be used to prepare the purposes of treatment parkinson disease medicine as the 1st described microencapsulated bovine adrenal medullary substance pheochromocyte suspension, wherein, said bovine adrenal medullary substance pheochromocyte preferably has the purity more than 80%.
3, be used to prepare the purposes of treatment parkinson disease medicine as the 1st described microencapsulated bovine adrenal medullary substance pheochromocyte suspension, wherein, the suspension that preferably step (1) is obtained in the preparation process (2) of said cell suspending liquid 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 (2-8 * 10
6Individual cell) inject once in patient's the brain striatum with conventional brain stereotaxic technique and can in 1 week, receive the effect of correcting its Deviant Behavior rapidly, and the therapeutical effect time can keep more than 12 months.
Below technical scheme of the present invention is carried out more detailed explanation.
In several above-mentioned technical schemes, the 1st is necessary to explain feature, and the 2nd, 3 belong to preferred technical characterictic.
Described in the present invention bovine adrenal medullary substance pheochromocyte (BCC) is meant can be by the painted cell of chrome complex dye in the bovine adrenal medullary substance, it can secrete the monoamines material, therefore can be used for replenishing the deficiency of Dopaminergics content of material in the parkinson disease human brain, thereby play the Parkinsonian effect of treatment.
The acquisition of BCC and purification process do not belong to scope of the present invention but belong to routine techniques.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, because BCC accounts for 50-60% in bovine adrenal medullary epithelium sum, can not be applied among the present invention even therefore do not carry out purification yet.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 bluestain) of Placenta Hominis, see " tissue culture's solvent and reagent " (Tissue Culture Mediaand Reagents), the 1566th page.
In belonging to 6 operating procedures of essential features of the present invention, 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 technical scheme 1 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 of pancreatic islet cell:a bio-artificial endocrine pancreas.In:Methods inEnzymology, 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 and experimental example at an easy rate having read above to reach hereinafter.
Confirm by a large amount of zooperies, with the APA microencapsulation BCC of suitable dosage with in the disposable injection Parkinson disease model of the conventional brain stereotaxic technique animal brain striatum, can correct the parkinson disease animal Deviant Behavior, make that the Dopaminergics content of material raises in the cerebral tissue.The APA microcapsule can protect graft to avoid the immune destruction of host, makes that microencapsulation BCC can longer survival in the heterogenous animal body.Microencapsulation BCC intracerebral transplantation is brought into play therapeutical effect well to the parkinson condition of disease, and effect is clear and definite and lasting.
Compare with the similar prior art of this area, the present invention has following beneficial effect:
1, compares with adopting human adrenal gland medullary substance pheochromocyte, can obtain with high-purity ground in large quantity as the bovine adrenal medullary substance pheochromocyte in dopamine secreting cell source.
2, a large amount of experiments confirm that the APA microcapsule that makes by the present invention has good immanoprotection action, thereby can make BCC bring into play therapeutical effect for long periods in the xenogenesis host.
3, the APA microcapsule volume that makes by the present invention little (diameter 180-500 μ m), thereby have 3 advantages at least: (1) is beneficial to the diffusion of nutrient and metabolite, makes the capsule inner cell be easy to longer survival (more than 1 year); (2) make the intracerebral transplantation operation technique easy, little to tissue injury, only need microcapsule to be injected in the cerebral tissue with simple brain stereotaxic technique; (3) stimulation and the contention effect that in brain nervous tissue is produced is less.
4, compare with the immune isolating membrane of other material, the APA microcapsule has good biocompatibility.Promptly have can be mass-produced, easy and simple to handle, curative effect is reliable, steady quality, advantage such as safe in utilization.Can in clinical practice, open up wide use prospect.
5, BCC sustainable secretion Dopaminergics material in host, the therapeutical effect that produce to continue has been avoided long-term prescription and the Drug resistance that produces.
6, can adopt cryogenic technique to preserve microencapsulation BCC, be convenient to long-distance transportation and supply in batches.
Put it briefly, the present invention have the Parkinsonian effect of treatment obviously, long action time, safe in utilization, easy and simple to handle, steady quality, can be mass-produced, characteristics such as with short production cycle.Can in clinical practice, have wide use prospect.
Enumerate embodiment and experimental example below and explain the present invention further, 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, collagenase I (collagenaseI) solution that is 1g/L from adrenal vein implantation concentration, 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 ' s Modified Eagle Medium) culture fluid (wherein also contain 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 dropletgenerator, 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: the application of microencapsulated bovine adrenal medullary substance pheochromocyte intracerebral transplantation treatment parkinson disease (PD) sample rat
1, use 20 Wister strain rats as experimental subject, the weight of every rat is 300 ± 30g.
2, adopting conventional 6-OHDA to bring out method makes 20 rats all become PD original mold type animal.
3, to injecting empty microcapsule (microcapsule that does not contain BCC) in the brain of 5 rats, be the sodium chloride solution 1ml of 9g/L to implantation concentration in the brain of other 5 rats, the result shows that these 10 rats all can not be corrected the Deviant Behavior of PD sample rat.
4,5 rats are implanted the not BCC 3 * 10 of encapsulation
4Individual cell is implanted the BCC 3 * 10 that microcapsule wraps up to other 5 rats
4An individual BCC cell.The result shows that these 10 rats are all corrected its Deviant Behavior rapidly and observe Dopaminergics content of material rising (rising 20-120%) in its cerebral tissue in 1 week.The therapeutic effect of APA microcapsule parcel group (100% PD sample rat is effective) and obviously be better than not encapsulation group (50% PD sample rat effectively, 1-6 month action time) action time (above 10 months), the microcapsule group can be observed microencapsulation BCC intact existence in rat brain in treatment in the time of back 10 months, cerebral tissue does not have tangible inflammatory reaction, does not find obvious toxic and side effects.
Experimental example 2: the application of microencapsulation BCC intracerebral transplantation treatment parkinson disease (PD) sample monkey
1, use 9 Henghe monkeys as subjects.
2, adopting conventional MPTP to bring out method makes these 9 monkeys all become PD original mold type animal.
3, the brain to wherein 1 monkey is implanted into the sky microcapsule, and the result shows that its Deviant Behavior can not obtain correcting.
4, being implanted into not to the brains of wherein 2 monkeys, the BCC of encapsulation (is respectively 2 * 10
6With 8 * 10
6Individual BCC); All the other 6 monkey brain are implanted into the BCC of microcapsule parcel (in 2-8 * 10
6In the individual BCC scope), these 8 monkeys are all corrected the Deviant Behavior of its PD sample monkey about 1 week, and observe in its cerebral tissue Dopaminergics content of material obviously raise (having improved 80-200%).Encapsulation group therapeutical effect time remaining does not disappear after about 1 month, and the therapeutical effect time of APA microcapsule parcel group is above 12 months.Do not find obvious toxic and side effects.
Claims (3)
1, a kind of microencapsulated bovine adrenal medullary substance pheochromocyte suspension is used to prepare the purposes of treatment parkinson disease medicine, and wherein, said cell suspending liquid 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 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 every liter of liquid house 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 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 and cultivates in the cell culture fluid and preserve as microencapsulated bovine adrenal medullary substance pheochromocyte suspension.
2, microencapsulated bovine adrenal medullary substance pheochromocyte suspension as claimed in claim 1 is used to prepare the purposes of treatment parkinson disease medicine, and wherein, said bovine adrenal medullary substance pheochromocyte has the purity more than 80%.
3, microencapsulated bovine adrenal medullary substance pheochromocyte suspension as claimed in claim 1 is used to prepare the purposes of treatment parkinson disease medicine, wherein, the suspension that in the preparation process (2) of said cell suspending liquid step (1) is obtained disperses with the micro-droplet status of 180-500 μ m diameter.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5585183A (en) * | 1994-04-13 | 1996-12-17 | National Science Council | Preparation of liquid-core microcapsules for cell cultures |
| US5730974A (en) * | 1994-11-03 | 1998-03-24 | Jacqueline Sagen | Reversing opiate tolerance by cellular implantation |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5585183A (en) * | 1994-04-13 | 1996-12-17 | National Science Council | Preparation of liquid-core microcapsules for cell cultures |
| US5730974A (en) * | 1994-11-03 | 1998-03-24 | Jacqueline Sagen | Reversing opiate tolerance by cellular implantation |
Non-Patent Citations (1)
| Title |
|---|
| 第二军医大学学报1998,19(3) 1998-03-31 宋玲等 糖皮质激素对大鼠肾上腺髓质嗜铬 细胞内游离钙浓度的快速影响 * |
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