CN106176811A - Treatment chronic atrophic gastritis and immunocyte preparation, its preparation method and the application of gastric precancerous lesion - Google Patents
Treatment chronic atrophic gastritis and immunocyte preparation, its preparation method and the application of gastric precancerous lesion Download PDFInfo
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Abstract
The invention belongs to biomedicine technical field, a kind of immunocyte preparation treating chronic atrophic gastritis and gastric precancerous lesion is provided, prepared by following steps: take human peripheral, isolate mononuclearcell, carry out isolated mononuclearcell cultivating with induced amplification, cultivate complete i.e. obtaining and treat chronic atrophic gastritis and the immunocyte preparation of gastric precancerous lesion.The present invention also provides for the application in chronic atrophic gastritis and/or the medicine of gastric precancerous lesion and the medicine treating the digestive tract reaction that chemotherapy causes are treated in preparation of the described immunocyte preparation.The immunocyte preparation that the present invention provides can improve people digest road symptom, evident in efficacy in terms of pathological changes before treatment chronic atrophic gastritis, reversing stomach, treating both the principal and secondary aspects of a disease, good effect, is difficult to recurrence.The preparation method of the present invention can efficiently obtain stable immunocyte preparation, and the leukocyte generally can given up using blood station, as cell derived, is turned waste into wealth, and is suitable to industrial high-volume industrialization and produces.
Description
Technical field
The invention belongs to biomedicine technical field, be specifically related to one and treat chronic atrophic gastritis and gastric precancerous lesion
Immunocyte preparation, its preparation method and application.
Background technology
Gastric cancer is the common cancer of harm human health, and China dies from the patient of gastric cancer every year more than 260,000.Gastric cancer
Front pathological changes is the initial period that gastric cancer occurs, and its year canceration rate progressing to infiltrating cancer a maximum of about of reaches 73%.In recent years, due to
The factor impacts such as environment, diet, operating pressure and helicobacter pylori infection, gastric precancerous lesion sickness rate dramatically increases, therefore, and
The generation and the development that early block gastric precancerous lesion have positive effect to the prevention of gastric cancer.
Chronic atrophic gastritis is important gastric precancerous lesion performance.Digestive system tumor according to WHO announcement in 2000 is sick
Reason and genetic classification, cause the developmental pattern of gastric cancer to be usually the precancerosises such as chronic atrophic gastritis, stomach angle ulcer, polyp of stomach
It is deformed into intestinal epithelial metaplasia and dysplasia, ultimately results in gastric cancer.Chronic atrophic gastritis is important gastric precancerous lesion, and it is sick
Reason change is gastric mucosa parietal cell and chief cell apoptosis causes body of gland atrophy, minimizing.Intestinal epithelial metaplasia is gastric epithelial cell
Substituted by goblet cell and the cylindrical cell of similar intestinal mucosa.Obstinate inflammatory stimulus repeatedly ultimately results in canceration.Therefore have
Effect treatment chronic atrophic gastritis, the pathological change of reverse gastric precancerous lesions are the keys that prevention gastric cancer occurs.
First technical problem, present stage, the treatment for chronic atrophic gastritis includes eliminating pylorus, benefit
Fill antioxidant and trace element, traditional Chinese medical herbal treatment, gastric mucosa protectant, antibiotic etc., but all exist and cure the symptoms, not the disease, no
Can life-time service, weak curative effect, easily repeatedly recur, the problem such as cannot effect a radical cure, and slow for Helicobacter pylori infection
Property patients with atrophic gastritis, even if after killing the infection of helicobacter pylori, the antibody caused due to helicobacter pylori is deposited
, still gastric epithelial is carried out lasting attack, therefore patient's slight illness is not alleviated, and continues to develop to canceration.Stomach glues
Film pathological changes gradually proceeds to intestinal epithelial metaplasia and dysplasia stage, can effectively reverse gastric mucosal lesion for gastric precancerous lesion
The Therapeutic Method of pathological characters and medicine especially almost without, this makes the treatment of gastric precancerous lesion become difficult point.
Another technical problem.Diagnoses and treatment to disease at present comes into molecule and cell epoch.Stem cell, immunity
Cell has the biggest treatment potentiality.A lot of about stem-cell research open source information, such as application number CN201210133427.X
Chinese invention patent application i.e. disclose " a kind of stem cell medicine for wound repair and preparation method thereof ".And about exempting from
Epidemic disease cell still focus mostly in conceptual phase, open source information is less, periodical literature Phenotypic characterization
and identification of cytokine-induced killed cells[J].Exp Hematol,1993；21
(13): 1673-1679, disclose immunocyte application in terms of clinical treatment earlier, specifically disclose use patient certainly
It is used for treating tumor after the outer induced amplification of somatophyte.Present stage other major part is limited to about the research of immunocyte also more
This respect, is all application patients own cells, feeds back patient again after amplification in vitro is induced.Application autologous patient cell therapy,
Cell derived is limited, and treatment is individual limited, and price is high, it is impossible to accomplishes to produce in enormous quantities and industrialized development, also cannot make more
The convenient treatment of many patients.
To sum up, there is not yet the immunocyte preparation of immunocyte preparation especially xenogenic origin both at home and abroad, to chronic
Atrophic gastritis and gastric precancerous lesion carry out treatment report research.
Summary of the invention
In order to solve the problems referred to above that prior art exists, present invention aim at providing one and can reverse precancerosis
Become, make atrophic gastritis become superficial gastritis even to cure and effectively relief of symptoms treatment chronic atrophic gastritis and gastric cancer
The immunocyte preparation of front pathological changes.The present invention provides the preparation method and application of described immunocyte preparation the most simultaneously.This
The preparation method of bright offer can be originated with healthy human peripheral blood leukocyte for immunocyte, expands the source of immunocyte,
It is capable of large batch of production and industrialized development；It is effectively utilized the human peripheral leucocytes portion generally given up blood station simultaneously
Point, turn waste into wealth.
The technical solution adopted in the present invention is:
The treatment chronic atrophic gastritis of present invention offer and the preparation method bag of the immunocyte preparation of gastric precancerous lesion
Include following steps: take people's whole blood, isolate mononuclearcell, carry out cultivating with induced amplification by isolated mononuclearcell,
Cultivate the complete immunocyte preparation i.e. obtaining described treatment chronic atrophic gastritis and gastric precancerous lesion.
The isolated human peripheral leucocytes in blood station is used to replace described people's whole blood；Blood bag is used to take outside the described people of splendid attire
All blood leukocytes, the alcohol wipe blood bag outer wall 2 of concentration 72～75vt%～3 times.The purpose of alcohol wipe is to kill
Bacterium, concentration volume fraction more than 75% ethanol can form protecting film at bacterial cell surface, stops ethanol to enter bacterial cell, difficult
So that antibacterial is thoroughly killed；The alcohol concentration of concentration volume fraction less than 72% is too low, though antibacterial can be entered, but can not be by its body
Interior protein coagulating, thoroughly can not kill antibacterial equally.Therefore, volume-fraction concentration 72～the wipes of alcohol of 75% are used
Wipe blood bag outer wall and be obtained in that optimal bactericidal effect.
The present invention is originated as immunocyte by healthy human peripheral blood leukocyte, expands the source of immunocyte,
And then be beneficial to realize the large batch of production of immunocyte preparation and industrialized development.Meanwhile, the whole blood that blood station is collected generally exists
After isolating the compositions such as red cell suspension, blood plasma, platelet, remaining leucocyte fraction is directly given up.The preparation side of the present invention
Method can well realize recycling to leukocyte, turns waste into wealth.
In order to high-quality, the high efficiency separation completing mononuclearcell and cultivation induced amplification, it is thus achieved that quality
Reliable and stable treatment chronic atrophic gastritis and the immunocyte preparation of gastric precancerous lesion, the present invention is at continuous research and probe
In, it is provided that step and control condition parameter in detail below.
Use Ficoll-hypaque lymphocyte separation medium, isolate described single core with density－gradient centrifuga－tion method thin
Born of the same parents.In described density－gradient centrifuga－tion method, centrifugal rotational speed is 1800～2200rpm/min, and centrifugation time is 18～22min.
After isolating described mononuclearcell with density－gradient centrifuga－tion method, add physiological saline solution centrifuge washing 2～3
Time, the rotating speed of centrifuge washing is 1800～2200rpm/min, and the time of centrifuge washing is 18～22min, trains the most again
Support.The centrifuge washing purpose of multipass is to remove residual platelet and Ficoll-hypaque lymphocyte separation medium.
The concrete steps of described cultivation include: add culture fluid in isolated mononuclearcell and obtain basis cultivation body
System, adds human serum in the cultivating system of basis the most again and IFN-γ obtains a cultivating system, once cultivate body by described
System is placed in 5%CO2Incubator is once cultivated, then in a cultivating system add IL-2, CD3McAb, IL-1,
CD28 antibody and IL-4 configuration obtain second incubation system, and second incubation system is placed in 5%CO2Incubator carries out secondary training
Supporting, within during second incubation every 3 days, add one time of nutrition liquid in second incubation system, described nutritional solution includes culture fluid, people
Serum and IL-2, after second incubation, results isolate cell, then use physiological saline solution washed cell, obtain treatment
The immunocyte preparation of chronic atrophic gastritis and gastric precancerous lesion.
Described culture fluid is GT-T551 culture fluid；Controlled in the cultivating system of described basis by the addition of culture fluid
Cell density is 3.5 × 106～4.5 × 106Individual/ml；In in nutritional solution, the addition of culture fluid controls second incubation system
Cell density be 1.5 × 106～2.5 × 106Individual/ml.
The cultivation temperature of described once cultivation and described second incubation is 37 ± 0.5 DEG C；People in a described cultivating system
The concentration of serum is 8 ± 1wt%, and in a described cultivating system, the concentration of IFN-γ is 1000 ± 100U/ml；Configure described two
During secondary cultivating system: the amount of every milliliter of cultivating system added IL-2, CD3McAb, IL-1, CD28 antibody and IL-4 is respectively
It is followed successively by 500 ± 50U, 100 ± 10ng, 100 ± 10U, 500 ± 50ng, 500 ± 50U；When adding nutritional solution: the most first add every time
The cell density added in culture fluid adjustment second incubation system is 1.5 × 106～2.5 × 106Individual/ml, then every 100ml bis-times
Cultivating system adds 5 ± 1ml human serum, and every milliliter of second incubation system adds the IL-2 of 500 ± 50U；Described once cultivate
Time is 24 ± 2h, and the time of described second incubation is 12～28 days.
Present invention also offers treatment chronic atrophic gastritis and the immunocyte preparation of gastric precancerous lesion, described immunity is thin
The preparation method that born of the same parents' preparation is provided by the invention described above prepares.
After having made further further investigation in terms of the purposes of the immunocyte preparation providing the present invention, the present invention is also
Provide the immunocyte preparation of the aforementioned present invention in the medicine of preparation treatment chronic atrophic gastritis and/or gastric precancerous lesion
Application.Described medicine includes the most acceptable various dosage form, includes but not limited to transfusion etc..Should in order to further facilitate
With, described medicine also includes treat chronic atrophic gastritis and/or the test kit of gastric precancerous lesion.The immunity that the present invention provides
Cell preparation can improve people digest road symptom, and based on this, the immunocyte preparation of the present invention is at other digestive system disease
Aspect also has the digestive tract reaction etc. that significant curative effect, such as chemotherapy cause, and accordingly, present invention also offers described immunocyte system
Agent application in the medicine of the digestive tract reaction that chemotherapy causes is treated in preparation.
Based on discussed above, the invention have the benefit that first, the immunocyte preparation that the present invention provides can improve
People digest road symptom, evident in efficacy in terms of pathological changes before treatment chronic atrophic gastritis, reversing stomach, it is possible to realize specimen and hold concurrently
Control and good effect, be difficult to recurrence.Meanwhile, it is not limited solely to gastric precancerous lesion, chronic atrophic gastritis, owing to changing
Kind people digest road symptom, the immunocyte preparation of the present invention also has significant curative effect, such as in terms of other digestive system disease
The digestive tract reaction etc. that chemotherapy causes.Secondly, the preparation method that the present invention provides can high efficiency acquisition steady quality reliable,
The immunocyte preparation that therapeutic effect is good, and the present invention provide preparation method multiple leukocyte can be had to originate, the most not
It is only limitted to patient itself, also includes relatives and other people donation, be not limited only to peripheral blood, it is also possible to be Cord blood.Further, the present invention
The preparation method provided can be originated using the leukocyte that blood station routine is given up as batch, and while turning waste into wealth, source is more
Extensively, be suitable to industrial production in enormous quantities, reduce price, convenient use, and then make more patient be benefited.
Accompanying drawing explanation
Fig. 1 be the present invention typical clinical case 1 in patient in the gastroscope figure in July, 2013；
Fig. 2 be the present invention typical clinical case 1 in patient in the gastroscope figure of in JIUYUE, 2015.
Detailed description of the invention
Below in conjunction with the accompanying drawings and the present invention is further explained by specific embodiment, if no special instructions, institute in the present invention
All deferring to industry with term it is generally understood that the method equipment used is all industry universal method equipment, used reagent is all
For the common commercial reagent of industry universal.The Whole Blood of Healthy addressed in following example takes from Central Blood Ban, Changchun City；Following reality
Execute the healthy human peripheral blood leukocyte addressed in example take from the whole blood of Central Blood Ban, Changchun City isolate red cell suspension, blood plasma,
The remaining part based on peripheral blood leucocyte after the compositions such as platelet.
Embodiment 1:
The present embodiment provides a kind of immunocyte preparation treating chronic atrophic gastritis and gastric precancerous lesion, by following
Step is prepared: take splendid attire blood station isolated healthy human peripheral blood leukocyte, the wine of concentration 75vt% by blood bag
Essence wiping blood bag outer wall 2 times.Use Ficoll-hypaque lymphocyte separation medium, with density－gradient centrifuga－tion method from the people of blood bag
Peripheral blood leucocyte is isolated mononuclearcell；In density－gradient centrifuga－tion method operation, control centrifugal rotational speed is 2000rpm/
Min, centrifugation time is 20min.After separation, in isolated mononuclearcell, add physiological saline solution centrifuge washing
2 times, the rotating speed of centrifuge washing is 2000rpm/min, and the time of centrifuge washing is 20min.After centrifuge washing, to washed
Mononuclearcell in add GT-T551 culture fluid and obtain basis cultivating system, by the addition of GT-T551 culture fluid, control
Cell density in the cultivating system of system basis is 4 × 106Individual/ml.In the cultivating system of basis, add human serum and IFN-γ obtains
To a cultivating system, in a described cultivating system, the concentration of human serum is 8wt%, IFN-γ in a described cultivating system
Concentration be 1000U/ml.Cultivating system is placed in the 5%CO under the conditions of 37 DEG C2Incubator is once cultivated 24h.
After once cultivating, in a cultivating system, add IL-2, CD3McAb, IL-1, CD28 antibody and IL-4 configuration obtains two
Secondary cultivating system, the amount of every milliliter of cultivating system added IL-2, CD3McAb, IL-1, CD28 antibody and IL-4 depends on respectively
Secondary for 500U, 100ng, 100U, 500ng, 500U.Second incubation system is placed in the 5%CO under the conditions of 37 DEG C2Incubator enters
Row second incubation 20 days, during second incubation, adds one time of nutrition liquid, described nutritional solution in every 3 days in second incubation system
Including culture fluid, human serum and IL-2, when adding nutritional solution: that the most first adds that culture fluid adjusts in second incubation system is thin every time
Born of the same parents' density is 2 × 106Individual/ml, then every 100ml second incubation system adds 5ml human serum, and every milliliter of second incubation system is mended
Add the IL-2 of 500U.After second incubation, gather in the crops and isolate cell, then using physiological saline solution washed cell 3 times, i.e.
The immunocyte preparation of chronic atrophic gastritis and gastric precancerous lesion must be treated.
After the immunocyte preparation that the present embodiment obtains can adjust amount of liquid, directly apply to clinical vein and input to control
Treat chronic atrophic gastritis and/or gastric precancerous lesion；First can also coordinate it with other pharmaceutic adjuvants or other cooperative drugs
After make treatment chronic atrophic gastritis and/or the medicine of gastric precancerous lesion, further for chronic atrophic gastritis and/or
The treatment of gastric precancerous lesion.Same, the immunocyte preparation of the present embodiment is used directly for clinical vein input with treatment
The digestive tract reaction that chemotherapy causes, it is also possible to make, after compounding with other pharmaceutic adjuvants or medicine, the digestive tract that treatment chemotherapy causes
Further use after the medicine of reaction.
Embodiment 2:
The present embodiment provides a kind of immunocyte preparation treating chronic atrophic gastritis and gastric precancerous lesion, by following
Step is prepared: take splendid attire Whole Blood of Healthy by blood bag, the alcohol wipe blood bag outer wall of concentration 72vt% 2 times.Make
Use Ficoll-hypaque lymphocyte separation medium, isolate from the human peripheral leucocytes of blood bag with density－gradient centrifuga－tion method
Mononuclearcell；In density－gradient centrifuga－tion method operation, control centrifugal rotational speed is 1800rpm/min, and centrifugation time is 22min.Point
After from, adding physiological saline solution centrifuge washing 2 times in isolated mononuclearcell, the rotating speed of centrifuge washing is
1800rpm/min, the time of centrifuge washing is 22min.After centrifuge washing, add in washed mononuclearcell
GT-T551 culture fluid obtains basis cultivating system, by the addition of GT-T551 culture fluid, controls in the cultivating system of basis
Cell density is 3.5 × 106Individual/ml.In the cultivating system of basis, add human serum and IFN-γ obtains a cultivating system, institute
Stating the concentration of human serum in a cultivating system is 7wt%, and in a described cultivating system, the concentration of IFN-γ is 900U/ml.
Cultivating system is placed in the 5%CO under the conditions of 36.5 DEG C2Incubator is once cultivated 22h.After once cultivating,
In a cultivating system, add IL-2, CD3McAb, IL-1, CD28 antibody and IL-4 configuration obtains second incubation system, every milli
The amount of cultivating system added IL-2, CD3McAb, IL-1, CD28 antibody and IL-4 of rising be followed successively by respectively 450U, 90ng,
90U、450ng、450U.Second incubation system is placed in the 5%CO under the conditions of 36.5 DEG C2Incubator carries out second incubation 12
My god, during second incubation, within every 3 days, in second incubation system, add one time of nutrition liquid, described nutritional solution includes culture fluid, people
Serum and IL-2, when adding nutritional solution every time: the most first add culture fluid adjust the cell density in second incubation system be 1.5 ×
106Individual/ml, then every 100ml second incubation system adds 4ml human serum, and every milliliter of second incubation system adds the IL-of 450U
2.After second incubation, gather in the crops and isolate cell, then using physiological saline solution washed cell 3 times, must treat chronic
The immunocyte preparation of atrophic gastritis and gastric precancerous lesion.
Embodiment 3:
The present embodiment provides a kind of immunocyte preparation treating chronic atrophic gastritis and gastric precancerous lesion, by following
Step is prepared: take after splendid attire blood station Whole Blood of Healthy isolates the compositions such as red cell suspension, blood plasma, platelet by blood bag
Remaining peripheral blood leucocyte, the alcohol wipe blood bag outer wall of concentration 74vt% 3 times.Use Ficoll-hypaque lymph
Cell separation liquid, isolates mononuclearcell with density－gradient centrifuga－tion method from the human peripheral leucocytes of blood bag；Density gradient
In centrifuging operation, control centrifugal rotational speed is 2200rpm/min, and centrifugation time is 18min.After separation, to isolated
Adding physiological saline solution centrifuge washing 3 times in mononuclearcell, the rotating speed of centrifuge washing is 2200rpm/min, centrifuge washing
Time be 18min.After centrifuge washing, in washed mononuclearcell, add GT-T551 culture fluid obtain basis
Cultivating system, by the addition of GT-T551 culture fluid, the cell density controlled in the cultivating system of basis is 4.5 × 106Individual/
ml.In the cultivating system of basis, add human serum and IFN-γ obtains a cultivating system, human blood in a described cultivating system
Clear concentration is 9wt%, and in a described cultivating system, the concentration of IFN-γ is 1100U/ml.Cultivating system is placed in
5%CO under the conditions of 37.5 DEG C2Incubator is once cultivated 26h.After once cultivating, add in a cultivating system
Entering IL-2, CD3McAb, IL-1, CD28 antibody and IL-4 configuration obtains second incubation system, every milliliter of cultivating system is added
The amount adding IL-2, CD3McAb, IL-1, CD28 antibody and IL-4 is followed successively by 550U, 110ng, 110U, 550ng, 550U respectively.Will
Second incubation system is placed in the 5%CO under the conditions of 37.5 DEG C2Incubator carries out second incubation 28 days, during second incubation,
Within every 3 days, adding one time of nutrition liquid in second incubation system, described nutritional solution includes culture fluid, human serum and IL-2, mends every time
During Ensure Liquid liquid: the cell density the most first added in culture fluid adjustment second incubation system is 2.5 × 106Individual/ml, the most often
100ml second incubation system adds 6ml human serum, and every milliliter of second incubation system adds the IL-2 of 550U.Second incubation is complete
After, gather in the crops and isolate cell, then using physiological saline solution washed cell 2 times, chronic atrophic gastritis and stomach must be treated
The immunocyte preparation of precancerous lesion.
Clinical trial example:
The present invention also further study the immunocyte preparation of the present invention about treatment chronic atrophic gastritis and gastric cancer
The curative effect situation of front pathological changes.Clinical trial process is as follows:
1, physical data: choosing in December, 2014, I accepts for medical treatment at institute (No. 208 Hospital, PLA) in June ,-2016
15 example cases carry out clinical observation, through gastroscope and pathologic finding, all meet that disease for digest branch of Chinese Medical Association formulates is chronic
Precancerous lesion of atrophic gastritis diagnostic criteria, gets rid of the organic disease patients such as liver and gall pancreas.The maleest 10 examples, female 5 example, the age
Minimum 42 years old, maximum 75 years old；15 example patients all have nauseating, abdominal distention, belch, stomachache, early satiety sense, inappetence, dyspepsia, on
In abdominal discomfort and the symptom such as weak at least 3 kinds, wherein with rotten to the corn 4 examples, intestinal epithelial metaplasia 7 example, dysplasia 4 example.Patient
All there are more than 2 years medical histories, and through other treatment method without improving, belong to refractory type case.
2, therapeutic scheme: the immunocyte preparation obtained by the venoclysis embodiment of the present invention 1 method, every day 1 time, every time
Intravenous drip 100ml (cell concentration 2 × 108Individual/100ml), continuous 8 days is a course for the treatment of, carries out every month 1 course for the treatment of, treats 6
The individual course for the treatment of.Treatment terminates 2 months post-evaluation curative effects.Other drug treatment is stopped during treatment.
3, efficacy assessment standard:
Curing: clinical symptoms and sign disappear, appetite recovers normal.Gastroscopy shows that gastric mucosa form is recovered substantially；Sick
Reason inspection shows that gastric mucosa atrophy sexually revises basic disappearance, or only shallow inflammation changes, and dysplasia and intestinal epithelial metaplasia disappear
Lose.
Effective: clinical symptoms and sign disappear substantially, appetite is improved；Gastroscopy shows that gastric mucosa ash white area disappears substantially,
In red and white based on red as, have no blue blood vessel；Pathologic finding show gastric mucosa atrophy, dysplasia and intestinal epithelial metaplasia from
Severe transfers moderate to or transfers to slightly from moderate.
Take a turn for the better (effectively): clinical symptoms and sign substantially alleviate, but still legacy part symptom；Gastric mucosa ash is shown in gastroscopy
White area range shorter, blue blood vessel sees through image；Pathologic finding shows that gastric mucosa atrophy, dysplasia and intestinal epithelial metaplasia take a favorable turn
But it is the most notable.
Invalid: clinical symptoms and sign are without improving or deteriorating；Gastroscopy shows that gastric mucosa atrophy does not alleviates or increases the weight of；Pathology
Inspection shows that intrinsic body of gland atrophy degree and scope are the most unchanged or increase the weight of.
4, therapeutic outcome: total number of cases 15 example, cures 11 example accountings about 73%, and effective 3 example accountings about 20%, effective 1 example accounts for
Ratio about 7%, total effective rate 100%.Patient generally reflects that after treatment remission is obvious, and muscle power is obviously enhanced.According to " chronic
Atrophic gastritis internal medicine Canonical management clinical analysis, theory of medicine and practice, 2016,29 (2), 192-193. " statistics report, when
Front existing methodical clinical cure rate is the highest is about 40-50%, contrasts accordingly, and the clinical cure rate of the present invention is about 73%,
It is significantly better than prior art, achieves the most progressive.
Typical clinical case is as follows:
1, patient Zhang, male, 70 years old, goes to a doctor in July, 2013 cure in Jilin University first because of gastric acid, abdominal distention, stomachache
Institute, gastroscopy shows: mucous membrane of esophagus multiple white hyperplastic nodule；At the bottom of cardia-stomach-gastric body mucosa congestion and edema, gastric is shown on a small quantity
Yellow green muddiness mucus；Stomach angle-antrum congestion and edema, part mucosa is thinning, and submucosal blood vessel net is high-visible, and color and luster is red
Bai Bujun part based on white, multiple smooth erosion；Helicobacter pylori is negative.Diagnosis: chronic atrophic gastritis companion's erosion,
It is specifically shown in Fig. 1.The multi-medicament such as oral WEILEXIN, Ginseng Bolus for tonifying spleen, fragrant sand stomach function regulating ball, thymus protein is treated more than a year without good
Turn.At the beginning of 2015, come my institute (No. 208 Hospital, PLA), give immunocyte preparation 100ml vein of the present invention
Instiling, every day 1 time, continuous 8 days is 1 course for the treatment of, repeats 1 course of therapy every month.After treating for 2 courses for the treatment of, patient's gastric acid, flatulence pain
Substantially alleviate.Continual cure 4 course for the treatment of, in JIUYUE, 2015 check gastroscope shows: esophagus no abnormality seen；At the bottom of cardia-stomach-gastric body mucosa light
Sliding；Stomach angle, antrum sporadically appear lamellar hyperemia speckle, local edema, it is seen that mucus is adhered to, and pale asphyxia mucosa disappears；Pylorus is smooth
Unobstructed.Conclusion: atrophic gastritis and rotten to the corn disappearance.Slight chronic superficial gastritis.It is specifically shown in Fig. 2.Patient's stomach malaise symptoms
Being wholly absent, follow up a case by regular visits to stomach discomfort sense so far and do not recur, living and diet all recovers normal, reaches clinical cure.
2, patient Lee so-and-so, male, 69 years old, went to a doctor in 2009 cure in Jilin University first because of gastric acid, abdominal distention, stomachache
Institute, Gastroscope Diagnosis " chronic atrophic gastritis companion's intestinal epithelial metaplasia, severe dysplasia ", detection HP is positive, applies tetrad medicine
Treating half a year, check HP is negative, but stomach discomfort symptom is not alleviated.In JIUYUE, 2013 gastroscope: pylorus type gastric mucosa is chronic shallow
Table-atrophic gastritis accompanied acute inflammation, body of gland intestinal metaplasia, severe dysplasia.SABC: HP (-), Ki67 (+), P53 is dispersed in
(+), meet precancerous lesion performance.In March, 2014 chemically examines tumor markers carcinoembryonic antigen in the first Affiliated Hospital of Jilin University
CEA6.55 (raises), and in June, 2014 chemically examines CEA6.64 (rising) again in the first Affiliated Hospital of Jilin University, raises earlier above.
Patient's multi-treatment, without taking a turn for the better, uses immunocyte preparation of the present invention to enter to my institute (hospital of PLA the 208th) at the beginning of 2015
Row treatment.In October, 2015 disappears in hospital of Jilin Province check gastroscope display body of gland metaplasia, and dysplasia disappears, and Ki67, P53 are cloudy
Property, chemical examination CEA is down to normally.In January, 2016 is normal (being specifically shown in table 1 below) at I institute (hospital of PLA the 208th) check CEA.
Stomach discomfort symptom is significantly alleviated until disappearing.So far symptom recurs the most again, reaches clinical cure.
The CEA detected value of table 1 clinical case 2 patient's different time
3, patient Liu so-and-so, male, 61 years old, occur that feed is choked with sobs companion's abdominal distention after meal, entirely in January, 2008 without obvious inducement
Body is weak.In Jilin Affiliated Hospital of Beihua University, gastroscopy is shown: atrophic gastritis, and stomach biopsy pathology is shown: chronic active withers
Contracting gastritis, glandular epithelium moderate intestinal metaplasia and dysplasia.In June, 2008, pathology showed in BJ Union Hospital's row gastroscopy:
Gastric mucosa is acute and chronic inflammatory disease accompanies moderate intestinal epithelial metaplasia.Through antiinflammatory, kill helicobacter pylori and protection gastric mucosa etc. is right
Without being clearly better after disease treatment.In April, 2015 my institute (hospital of PLA the 208th), gastroscope shows: chronic erosion-atrophic stomach
Inflammation, accompanies polypoid proliferation, and gastric mucosa interstitial hyperemia is hemorrhage, sees inflammatory cell, and locally thin vessels hypertrophy is like granulation tissue, sees enteric epithelium
Metaplasia is polypoid.Canceration may greatly, and cannot operative treatment, minimal invasive treatment is among fear is worried.Give the present invention to exempt from
Epidemic disease cell preparation is treated, and in December, 2015 shows at my institute (hospital of PLA the 208th) check gastroscope, and gastric mucosa has no that hyperemia goes out
Blood, intestinal epithelial metaplasia reverses and disappears, and polypoid proliferation and granulation tissue disappear, and patients symptomatic is substantially alleviated, in February, 2016,
Patient is choked with sobs, abdominal distention symptom is wholly absent, and muscle power is obviously enhanced, and can do farm work down, follow up a case by regular visits to the most all right, reaches clinical
Cure.
4, patient Zhang, women, 51 years old, more than ten years of epigastric discomfort.In in May, 2014 exacerbation of symptoms, upper abdomen distending pain,
Diffuse and ache to left arcus costarum, shoulder back, accompany acid regurgitation belch.In hospital of Jilin University the 3rd, row gastroscopy is shown: esophagitis, chronic
Atrophic gastritis is the most rotten to the corn, Polypus, and pathology is returned: gastric mucosa chronic inflammatory disease, intestinal epithelial metaplasia, severe dysplasia.Warp
The state of an illness such as oral anti-inflammatory drug, multiple stomach medicine, Chinese medicine are without being clearly better, and in April, 2015, my institute (hospital of PLA the 208th) gives
Giving immunocyte preparation for treating of the present invention, after 2 courses for the treatment of, colic symptoms is significantly alleviated, and acid regurgitation belch alleviates, after continual cure 4 course for the treatment of
In hospital of Jilin University the 3rd, check gastroscope shows: atrophic gastritis rotten to the corn disappearance, slight superficial gastritis, Polypus and weight
Degree dysplasia all disappears.Patient's malaise symptoms complete incidence graph, living and diet recovers normal, reaches clinical cure.
The present invention is not limited to above-mentioned preferred forms, and anyone can show that under the enlightenment of the present invention other are various
The product of form, no matter but in its details, make any change, every have same as the present application or akin technical scheme,
Within all falling within protection scope of the present invention.
Claims (10)
1. the preparation method of the immunocyte preparation for the treatment of chronic atrophic gastritis and gastric precancerous lesion, it is characterised in that include
Following steps: take people's whole blood, isolate mononuclearcell, carry out isolated mononuclearcell cultivating with induced amplification, training
Support the complete immunocyte preparation i.e. obtaining described treatment chronic atrophic gastritis and gastric precancerous lesion.
Preparation method the most according to claim 1, it is characterised in that: use blood station isolated human peripheral leucocytes generation
For described people's whole blood；Blood bag is used to take the described human peripheral leucocytes of splendid attire, the alcohol wipe of concentration 72～75vt%
Blood bag outer wall 2～3 times.
Preparation method the most according to claim 1, it is characterised in that: use Ficoll-hypaque separation of lymphocytes
Liquid, isolates described mononuclearcell with density－gradient centrifuga－tion method；In described density－gradient centrifuga－tion method, centrifugal rotational speed be 1800～
2200rpm/min, centrifugation time is 18～22min.
Preparation method the most according to claim 3, it is characterised in that: isolate described single core with density－gradient centrifuga－tion method
After cell, addition physiological saline solution centrifuge washing 2～3 times, the rotating speed of centrifuge washing is 1800～2200rpm/min, centrifugal
The time of washing is 18～22min, cultivates the most again.
5. according to the arbitrary described preparation method of Claims 1 to 4, it is characterised in that: the concrete steps of described cultivation include: to
Isolated mononuclearcell adds culture fluid and obtains basis cultivating system, in the cultivating system of basis, add human blood the most again
Cleer and peaceful IFN-γ obtains a cultivating system, and a described cultivating system is placed in 5%CO2Incubator is once cultivated,
Then in a cultivating system, add IL-2, CD3McAb, IL-1, CD28 antibody and IL-4 configuration obtain second incubation system,
Second incubation system is placed in 5%CO2Incubator carries out second incubation, during second incubation every 3 days to second incubation body
Adding one time of nutrition liquid in system, described nutritional solution includes culture fluid, human serum and IL-2, and after second incubation, results separate
Going out cell, then use physiological saline solution washed cell, the immunity that must treat chronic atrophic gastritis and gastric precancerous lesion is thin
Born of the same parents' preparation.
Preparation method the most according to claim 5, it is characterised in that: described culture fluid is GT-T551 culture fluid；By training
The cell density that the addition of nutrient solution controls in the cultivating system of described basis is 3.5 × 106～4.5 × 106Individual/ml；Pass through nutrition
Cell density during the addition of culture fluid controls second incubation system in liquid is 1.5 × 106～2.5 × 106Individual/ml.
Preparation method the most according to claim 5, it is characterised in that: described once cultivation and the cultivation of described second incubation
Temperature is 37 ± 0.5 DEG C；In a described cultivating system, the concentration of human serum is 8 ± 1wt%, in a described cultivating system
The concentration of IFN-γ is 1000 ± 100U/ml；When configuring described second incubation system: every milliliter of cultivating system is added
The amount of IL-2, CD3McAb, IL-1, CD28 antibody and IL-4 be followed successively by respectively 500 ± 50U, 100 ± 10ng, 100 ± 10U, 500
±50ng、500±50U；When adding nutritional solution: the cell density the most first added in culture fluid adjustment second incubation system is every time
1.5×106～2.5 × 106Individual/ml, then every 100ml second incubation system adds 5 ± 1ml human serum, every milliliter of second incubation
System adds the IL-2 of 500 ± 50U；The described time once cultivated is 24 ± 2h, and the time of described second incubation is 12～28
My god.
8. treatment chronic atrophic gastritis and the immunocyte preparation of gastric precancerous lesion, it is characterised in that: by claim 1～
8 arbitrary described methods are prepared.
9. immunocyte preparation described in claim 8 is at preparation treatment chronic atrophic gastritis and/or the medicine of gastric precancerous lesion
In application.
10. the answering in the medicine of the digestive tract reaction caused in preparation treatment chemotherapy of immunocyte preparation described in claim 8
With.
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| CN106730015A (en) * | 2017-01-06 | 2017-05-31 | 朱凯林 | A kind of preparation for beauty and preparation method thereof |
| CN108853143A (en) * | 2018-07-04 | 2018-11-23 | 盛春华 | Immunocyte preparation, preparation method and the application for treating psoriasis |
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| CN102641298A (en) * | 2012-05-15 | 2012-08-22 | 祁岩超 | Effector cell combination for preventing and treating tumors and preparation method thereof |
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| CN102641298A (en) * | 2012-05-15 | 2012-08-22 | 祁岩超 | Effector cell combination for preventing and treating tumors and preparation method thereof |
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| CN106730015A (en) * | 2017-01-06 | 2017-05-31 | 朱凯林 | A kind of preparation for beauty and preparation method thereof |
| CN108853143A (en) * | 2018-07-04 | 2018-11-23 | 盛春华 | Immunocyte preparation, preparation method and the application for treating psoriasis |
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Application publication date: 20161207 |