CN1127948C - Microencapsulated bovine adrenal medulla chromaffin cell-drug for alleviating pain - Google Patents
Microencapsulated bovine adrenal medulla chromaffin cell-drug for alleviating pain Download PDFInfo
- Publication number
- CN1127948C CN1127948C CN00801292A CN00801292A CN1127948C CN 1127948 C CN1127948 C CN 1127948C CN 00801292 A CN00801292 A CN 00801292A CN 00801292 A CN00801292 A CN 00801292A CN 1127948 C CN1127948 C CN 1127948C
- Authority
- CN
- China
- Prior art keywords
- pheochromocyte
- precipitate
- solution
- medicine
- concentration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicinal Preparation (AREA)
Abstract
The present invention relates to a microencapsulated bovine adrenal medulla chromaffin cell (BCC) medicine for alleviating pain. 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 pain alleviating materials for a long time in human bodies, and can alleviate pain for more than a plurality of months after being implanted into human bodies once.
Description
Technical field
The present invention relates to the zooblast medicine, more particularly, relate to a kind of micro-capsuled pheochromocyte of bull adrenal medulla as medicine that is used for the treatment of pain.
Background technology
Pain is a kind ofly common can bring the symptom of very big misery to patient by what Different types of etiopathogenises caused.Existing analgesic as the morphine class, though can produce analgesic effect at short notice, easily develops immunity to drugs and addiction after the medication repeatedly.Because adrenal medullary chromaffin cell can secrete some material relevant with analgesic activity, as first deltorphin delta, bright deltorphin delta and monoamines material etc., if this pheochromocyte is implanted under human or animal's the spinal arachnoid mater, just can be as natural " a miniature organism pump ", secrete analgesic matter for a long time consistently, and can not develop immunity to drugs and addiction.So at early eighties in this 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, the three-decker film microcapsule (hereinafter to be referred as the APA microcapsule) that adopts sodium alginate-polylysine-sodium alginate to make, its volume little (diameter is 200-1000um), 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 the report of pain as medicine.
Summary of the invention
The objective of the invention is to provide a kind of biocompatibility height, long action time, the toxic and side effects APA micro-capsuled pheochromocyte of bull adrenal medulla as medicine that is applicable to treatment pain little, easy and simple to handle.
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, be used for the treatment of the micro-capsuled pheochromocyte of bull adrenal medulla as medicine of pain, it is characterized in that, this cell drug 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 in the sodium chloride solution that a kind of concentration is 9g/L and cleans, and at last precipitate is transferred to cultivate in the cell culture fluid and preserve as the micro-capsuled pheochromocyte of bull adrenal medulla as medicine of treatment pain.
2, as the 1st described micro-capsuled pheochromocyte of bull adrenal medulla as medicine, wherein said bovine adrenal medullary substance pheochromocyte preferably has the purity more than 80%.
3, as the 1st described micro-capsuled pheochromocyte of bull adrenal medulla as medicine, 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) injects under the patient's (for example cancer pain patient) who suffers from pain the spinal arachnoid mater, can in 4-24 hour, produce analgesic activity, and a shot can be kept analgesic activity more than 9 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 materials such as monoamines, enkephalin class (comprising first deltorphin delta, bright deltorphin delta) and neurotrophic factor, and these materials have analgesic activity.
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,, 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 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 get final product.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-artificialendocrine pancrea s.In:Methods in 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, use APA microencapsulation zooblast medicine of the present invention (microcapsule diameter 150-1000 μ m) will contain 0.2-1 * 10
5The microcapsule of individual BCC cell injects under the normal rat spinal arachnoid mater, the burning pain threshold value (foot is tested and tail-flick test detects the reaction of rat to acute injury stimulation hot in nature with traditional lifting) of rat is obviously raise (than improving 80%-110% before the injection), and action time was above 9 months.Confirm by treatment, with microencapsulation BCC (2-9 * 10 the cancer pain patient
6Individual cell) inject under the cancer pain patient spinal arachnoid mater, can produce analgesic activity in 4-24 hour, wherein most humans no longer needs to use other analgesic drug products.Patient's pain scores value (being marked by 2 doctors by international VAS point system) obviously descends, and spirit and appetite take a turn for the better, and therapeutic effect has continued to surpass 120 days, and does not have significant side effects.This fact shows; the implantation of APA microencapsulation BCC of the present invention can produce significant analgesia role; and the APA microcapsule can protect graft (being BCC) to avoid the immune destruction of host, make microencapsulation BCC can be in the heterogenous animal body longer survival and bring into play analgesic activity enduringly.
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 (more than 3 months) secretion analgesic matter sustainably in host, the analgesic activity that produce to continue has overcome the undulatory property analgesic effect that clinical application produces;
5, microencapsulation BCC can bring into play analgesic activity for long periods in host, has avoided using analgesic repeatedly and side effect such as the Drug resistance that produces, addiction;
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.
Put it briefly, the effect that the present invention has treatment pain 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.In clinical practice, has wide use prospect.
Preferred forms of the present invention
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 60mi 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 EagleMedium) 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 rat threshold of pain
1, use 10 Wister strain rats as experimental subject, the weight of every rat is 300 ± 30g.
2, making among the embodiment 2 the microencapsulation BCC suspension precipitation that obtains, is the sodium chloride solution washing 3 times of 9g/L with concentration, and acquisition contains the sodium chloride suspension of microencapsulation BCC.This suspension is injected under the spinal arachnoid mater of these 10 rats, injection volume is every rat 1 * 10
5BCC/20 μ l sodium chloride solution, the result shows, all burning pain threshold values (foot is tested and tail-flick test detects the reaction of rat to acute injury stimulation hot in nature with international lifting) of being injected the rat of microencapsulation BCC obviously raise (than improving 80%-110% before the injection), and action time was above 9 months.
Experimental example 2: microencapsulation BCC is applied to treat the cancer pain patient
With APA microencapsulation BCC (about 7 * 10
6Cell) is suspended in the sodium chloride solution that 5 ml concns are 9g/L, injects with the lumbar puncture method of routine under cancer pain patient's that must the life-time service analgesic drug product the spinal arachnoid mater.Choose the pain scores (VAS method) that 10 cancer pain patients inject front and back altogether, the result shows that wherein the pain scores (VAS method) of 9 patients after transplanting (promptly injecting microencapsulation BCC) drops to 0～2 grade by 6～10 grades; Promptly began to stop using analgesic drug product the same day of 4 medications in these 9 patients, other 5 patients began to stop using analgesic drug product next day or the 3rd day.Do not using under the condition of any immunosuppressant, wherein 1 patient's the analgesic activity time surpasses 120 days, and 6 patients' analgesic activity is also above 70 days in addition.9 patients are in high spirits during this period, and appetite increases.Having only 1 example after implantation, still must use analgesic drug product among the 10 routine patients altogether, but consumption has reduced 50%.All 10 routine patients are not all observed obvious toxic and side effects.
Concrete application examples:
(1) Zhang Guigui, man, 42 years old, non_hodgkin lymphoma.
With needing before this medicine, at injection microencapsulation BCC 7 * 10 with oral analgesic Morphine 60mg/ day
6Individual cell is inactive analgesic after 4 hours, and the VAS pain scores drops to 0 grade by 8 grades, and effect now continues above 120 days.
(2) Liu Fengying, woman, 45 years old, He Jiejin lymphomas.
With needing before this medicine, at injection microencapsulation BCC 7.5 * 10 with oral analgesic ibuprofen 300mg/ day
6Individual cell is inactive analgesic after 18 hours, and the VAS pain scores drops to 0 grade by 6 grades, and effect now continues above 90 days.
(3) Zhao Jianhua, the woman, 47 years old, the breast carcinoma bone shifted.
With needing before this medicine, at injection microencapsulation BCC 7 * 10 with oral analgesic Morphine 60mg/ day and ibuprofen 300mg/ day
6Individual cell was kept to after 24 hours only uses ibuprofen 150mg/ day, the analgesic of stopping using fully after 3 days, and the VAS pain scores drops to 1 grade by 10 grades, and effect now continues above 80 days.
(4) Ding Shunxiang, woman, 56 years old, bone metastases of lung cancer.
With needing oral analgesic Morphine 60mg/ day before this medicine, at injection microencapsulation BCC 7 * 10
6Individual cell is kept to Morphine 30mg/ day after 24 hours, the analgesic of stopping using fully after 6 days, and the VAS pain scores drops to 2 grades by 10 grades, and effect now continues above 80 days.
(5) Li Xiangzhi, woman, 60 years old, multiple bone tumor.
With needing before this medicine, in injection microencapsulation BCC7 * 10 with intramuscular injection analgesic morphine 20mg/ day
6Individual cell is kept to morphine 10mg/ day after 24 hours, the analgesic of stopping using fully after 3 days, and the VAS pain scores drops to 1 grade by 10 grades, and effect now continues above 60 days.Statistical effect to treatment pain is as follows:
Up to now 22 routine cancer pain patients are treated, have 21 people to obtain good analgesic effect, total effective rate reaches 95%.Analgesic time by a shot calculates, and has the analgesic time of 3 examples to reach more than 300 days, reaches 570 days most, and majority can reach more than 100 days.
In addition, the treatment to 2 routine intractable neuralgia patients also obtains good analgesic effect.
Industrial applicability
Little (the Zhi footpath of the micro-capsule volume of micro-capsuled pheochromocyte of bull adrenal medulla as medicine of the present invention 180～500um), available straightforward procedure Zhu enters in the cavitas subarachnoidealis spinalis, and is long-term in the capsule inner cell energy Zai human body Survive and long-term performance analgesic activity, do not produce the side effect that stimulates and oppress. In addition, this microencapsulated cell Medicine can obtain and easily long-distance transportation in large quantity.
Claims (3)
1, be used for the treatment of the micro-capsuled pheochromocyte of bull adrenal medulla as medicine of pain, it is characterized in that, this cell drug 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 in the sodium chloride solution that a kind of concentration is 9g/L and cleans, and at last precipitate is transferred to cultivate in the cell culture fluid and preserve as the micro-capsuled pheochromocyte of bull adrenal medulla as medicine of treatment pain.
2, micro-capsuled pheochromocyte of bull adrenal medulla as medicine as claimed in claim 1 is characterized in that, wherein said bovine adrenal medullary substance pheochromocyte has the purity more than 80%.
3, micro-capsuled pheochromocyte of bull adrenal medulla as medicine as claimed in claim 1 is characterized in that, the suspension that in its step (2) step (1) is obtained disperses with the micro-droplet status of 180-500 μ m diameter.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN99109057.8 | 1999-06-16 | ||
| CN99109057A CN1235027A (en) | 1999-06-16 | 1999-06-16 | Micro-capsuled pheochromocyte of bull adrenal medulla as medicine for curing pains |
| PCT/CN2000/000155 WO2000078295A1 (en) | 1999-06-16 | 2000-06-14 | Microencapsulated bovine adrenal medulla chromaffin cell-drug for alleviating pain |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1345234A CN1345234A (en) | 2002-04-17 |
| CN1127948C true CN1127948C (en) | 2003-11-19 |
Family
ID=29550815
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN00801292A Expired - Fee Related CN1127948C (en) | 1999-06-16 | 2000-06-14 | Microencapsulated bovine adrenal medulla chromaffin cell-drug for alleviating pain |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1127948C (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006034601A1 (en) * | 2004-09-27 | 2006-04-06 | Yilong Xue | A new use of medulliadrenal chromaffin cells or peptide functional cells |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100338212C (en) * | 2003-01-29 | 2007-09-19 | 中国科学院大连化学物理研究所 | Microencapsulated cell of human tissue kallikrein, microencapsulating method and application thereof |
| CN102921038B (en) * | 2012-08-06 | 2014-07-09 | 西南交通大学 | Method for preparing porous scaffold with shape memory function |
-
2000
- 2000-06-14 CN CN00801292A patent/CN1127948C/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006034601A1 (en) * | 2004-09-27 | 2006-04-06 | Yilong Xue | A new use of medulliadrenal chromaffin cells or peptide functional cells |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1345234A (en) | 2002-04-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0741550B1 (en) | Multiple layer alginate coatings of biological tissue for transplantation | |
| AU780366B2 (en) | Preparation and xenotransplantation of porcine islets | |
| US10251915B2 (en) | Co-encapsulation of live cells with oxygen-generating particles | |
| US20140017304A1 (en) | Method for encapsulated therapeutic products and uses thereof | |
| CN105832682B (en) | A kind of preparation method of cellular silk fibroin porous microsphere drug slow-released carrier | |
| CN101466360A (en) | Multi-membrane immunoisolation system for cellular transplant | |
| CN1235027A (en) | Micro-capsuled pheochromocyte of bull adrenal medulla as medicine for curing pains | |
| JP2001514880A (en) | Encapsulation of living tissue cells into microspheres | |
| CN1119080C (en) | Implantable agarose-collagen beads containing cells which produce a diffusible biological product, and uses thereof | |
| CN1127948C (en) | Microencapsulated bovine adrenal medulla chromaffin cell-drug for alleviating pain | |
| Kobayashi | Bioartificial pancreas for the treatment of diabetes | |
| WO1995024168A1 (en) | In situ dissolution of alginate coatings of biological tissue transplants | |
| CN100591360C (en) | New application of chromaffin cell of adrenal medulla or opium peptide energy cell | |
| CN1098690C (en) | Microencapsulated bovine adrenal medulla chromaffincell medicine used for treating Parkinson's disease | |
| CN1730099A (en) | Immune isolated cell medicine, its preparing process and its application in killing tumours | |
| CN1837239A (en) | Polypeptide growth factor copolymer and its preparation method and use | |
| US8313741B2 (en) | Use of medulliadrenal chromaffin cells or peptide functional cells | |
| DE60123574T2 (en) | PREPARATION AND XENOTRANSPLANTATION OF PIG ISLANDS | |
| CN114832142A (en) | Chitosan composite dressing and preparation method thereof | |
| Chen et al. | Microencapsulation for cell implants into the central nervous system: the importance of alginate viscosity and related factors | |
| Hwang | A bio-inspired hybrid nanosack for pancreatic islet transplantation in the omentum | |
| CN1408437A (en) | Microcapsule adrenomedullary cell for curing malignant, tumor pain and its preparing method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| ASS | Succession or assignment of patent right |
Owner name: SHANGHAI XI LONG BIOMEDICAL ENGINEERING CO., LTD. Free format text: FORMER OWNER: XUE YILONG Free format text: FORMER OWNER: HE LIMIN Effective date: 20021128 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20021128 Address after: 200122 Quest International Hotel, 778 Dongfang Road, Shanghai, room 2616, Pudong New Area Applicant after: Xilong Biomedical Engineering Co., Ltd., Shanghai Address before: 100853 basic medical research institute, 28 Fuxing Road, Beijing, China Applicant before: Xue Yilong Co-applicant before: He Limin |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C57 | Notification of unclear or unknown address | ||
| DD01 | Delivery of document by public notice |
Addressee: Wang Hong Document name: Notice of conformity |
|
| PP01 | Preservation of patent right |
Effective date of registration: 20050629 Pledge (preservation): Preservation |
|
| C57 | Notification of unclear or unknown address | ||
| DD01 | Delivery of document by public notice |
Addressee: Wang Hong Document name: payment instructions |
|
| C57 | Notification of unclear or unknown address | ||
| DD01 | Delivery of document by public notice |
Addressee: Wang Hong Document name: payment instructions |
|
| PD01 | Discharge of preservation of patent |
Date of registration: 20061230 Pledge (preservation): Preservation |
|
| C57 | Notification of unclear or unknown address | ||
| DD01 | Delivery of document by public notice |
Addressee: Wang Hong Document name: Notification of termination of security procedure Addressee: Wang Hong Document name: payment instructions |
|
| C57 | Notification of unclear or unknown address | ||
| DD01 | Delivery of document by public notice |
Addressee: Wang Hong Document name: Notification of Termination of Patent Right |
|
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |