CN102747130B - Test evaluation method for sensitization of traditional Chinese medicine injection - Google Patents
Test evaluation method for sensitization of traditional Chinese medicine injection Download PDFInfo
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- CN102747130B CN102747130B CN201210225865.9A CN201210225865A CN102747130B CN 102747130 B CN102747130 B CN 102747130B CN 201210225865 A CN201210225865 A CN 201210225865A CN 102747130 B CN102747130 B CN 102747130B
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Abstract
The invention provides a test evaluation method for sensitization of a traditional Chinese medicine injection. The method comprises the steps of: treating a traditional Chinese medicine injection by ultrafiltration enrichment with 1-3KD to prepare a sensitized large molecular part; then adding the large molecular part into a peripheral blood mononuclear cell (PBMC) solution; carrying out co-culture for 1-3 days; and employing optimized trypan blue or fluorescent dye to dye living cell numbers, in order to calculate and evaluate sensitization strength of the traditional Chinese medicine injection. The method has advantages of convenience, rapidness and sensitivity, can be widely used in test and evaluation on sensitization and irritation of various traditional Chinese medicine injections, and provides scientific basis for safety evaluation and quality control of traditional Chinese medicine injections, and safety of clinical medication.
Description
Technical field
The present invention relates to a kind of detection method, a kind of traditional Chinese medicine injection sensitization, irritating method of determination and evaluation of being widely used in of specific design.
Background technology
Traditional Chinese medicine injection is a kind of modern Chinese herbal medicine preparation developing in modernization of Chinese medicine development, and clinical use is evident in efficacy, is widely used in the fields such as breathing, cardiovascular and cerebrovascular, and has obtained good market economy benefit.But traditional Chinese medicine injection often has untoward reaction to occur when clinical use, has potential safety hazard.The traditional Chinese medicine injection kind of clinical frequent use, as kinds such as SHUANGHUANLIAN, QINGKAILING, Root of Indigowoad, Herba Houttuyniae, potassium dehydroandrographolide succinate, Mailuoning, puerarin, compound Salviae Miltiorrhizaes, all has Reporting of harms.
Untoward reaction type is various, and what the most often occur is that Drug is irritated.For example, the angle constituents that in SHUANGHUANGLIAN ZHUSHEYE, Japanese Honeysuckle contains in chlorogenic acid, QINGKAILING easily causes allergy.Hemolytic reaction is also common in traditional Chinese medicine injection untoward reaction, and for example in Herba Houttuyniae injectio, tween-80 exceeds standard and easily causes haemolysis.In addition also relate to, the untoward reaction of a plurality of systems such as neural system, Digestive tract, cardiovascular systems, sensory organ, urinary system, skin and annex thereof.Drug safety and the new drug development of this veriety have seriously been limited.
For allergenic substance in traditional Chinese medicine injection, effectively check and detection method are most important for controlling its untoward reaction, but the current chemical detection method that still lacks efficient and sensible.Therefore, necessary a kind of reliable and effective traditional Chinese medicine injection sensitization, the irritating method of determination and evaluation researched and developed on the basis of prior art.
Summary of the invention
Goal of the invention: technical problem to be solved by this invention is to overcome the deficiency that prior art can not effectively detect sensitization in traditional Chinese medicine injection, provide a kind of applied widely, efficiently, stable, easy to operate, quick, result is traditional Chinese medicine injection sensitization, irritating detection method accurately.
Technical scheme: in order to realize above object, the technical scheme that the present invention takes is:
(1) use trapped molecular weight for thering is irritating macromole position in 1KD to 3KD ultra-filtration membrane enrichment traditional Chinese medicine injection, standby;
(2) irritating macromole position step (1) being prepared joins in the mononuclearcell (PBMC) in internal organs or peripheral blood source, cultivates altogether 1 to 3 day;
(3) mononuclearcell of getting after step (2) is cultivated adopts the dyeing of trypan blue dye liquor or fluorescent dyeing, and the number of counting cells or functional status carry out the sensitization of detecting and assessing traditional Chinese medicine injection.
As preferred version, the method for determination and evaluation of above-described traditional Chinese medicine injection sensitization, the multiple of step (1) Chinese medicine injection liquid enrichment is 1 to 10000 times, as preferred technical scheme, the multiple of Chinese medicine injection liquid enrichment of the present invention is 20 to 60 times.
As preferred version, the method for determination and evaluation of above-described traditional Chinese medicine injection sensitization, the irritating macromole of step (2) position joins middle the cultivation 2 to 3 days of mononuclearcell (PBMC) in internal organs or peripheral blood source.The present invention screens irritating macromole position incubation time in mononuclearcell (PBMC) by great many of experiments, and experimental result shows, when cultivating 48 to 72 hours hours, can make mononuclearcell under irritating sensitinogen, and cell proliferation reaches the highest.
As preferred version, the method for determination and evaluation of above-described traditional Chinese medicine injection sensitization, wherein said mononuclearcell PBMC cell can be people source or other laboratory animal source, can be from the immunocyte of peripheral blood or internal organs.
As preferred version, above-described ultra-filtration membrane can be used industrial ultra-filtration membrane, also can use the ultra-filtration centrifuge tube of business, and Chinese medicine preparation may produce micro-sensitizing substance in producing and depositing process, and research needs the sensitizing substance of high density.And ultrafiltration is the technology that a kind of effective eliminating small molecules disturbs, and the super filter tube of 3KD can hold back and be enriched to high density sensitizer, has advantages of that loss is little, efficiency is high, simple operation.
The detection method of Chinese medicine injection liquid formulation sensitization is a critical problem, the method of determination and evaluation of traditional Chinese medicine injection sensitization provided by the invention, creationary employing mononuclearcell PBMC activates as model, PBMC cell contains antigen presenting cell, can identify and process sensitinogen and class anaphylactogen (as tannin) and serum protein and be combined into the antigenic information of holoantigen, and submission is to the T in PBMC, bone-marrow-derived lymphocyte.Finally by cell quantity or vitality test, evaluate the pungency of injection liquid sensitizer, there is operational reliability strong, the advantages such as experiment good stability.
The method of determination and evaluation of traditional Chinese medicine injection sensitization provided by the invention, because there is darker color at the macromole position of enrichment, adopting mtt assay to detect cytoactive has obvious interference, and error is larger, and experimental result is inaccurate.The present invention screens by great many of experiments, adopts trypan blue and fluorescent dyeing determination PBMC cell viability; As preferred version, fluorescent probe can be the multiple fluorescent probes such as Hochest, PI, and the detection method of optimization is to detect with fluorescent microscope or fluorescence microplate reader, has detection sensitive, efficiently advantage.
Beneficial effect: compared to the prior art the method for determination and evaluation of traditional Chinese medicine injection sensitization provided by the invention, has the following advantages:
The method of determination and evaluation of traditional Chinese medicine injection sensitization provided by the invention, pass through great many of experiments, optimize macromolecular enrichment times, creationary employing mononuclearcell PBMC is for activating model, and optimize the incubation time of macromole allergenic substance in mononuclearcell PBMC, and adopt preferred trypan blue dye liquor dyeing or fluorescent dyeing method to come number or the functional status of counting cells, have workable, convenient, fast, efficiently, detected result is accurate, can extensively be suitable for and various traditional Chinese medicine injections, the detection evaluation of the sensitization of intermediate etc., many deficiencies of the detection of sensitization in prior art have been overcome, the present invention can be the safety evaluation of traditional Chinese medicine injection, the quality control of traditional Chinese medicine injection, for guaranteeing the security of clinical application, the foundation of science is provided, there is important social effect.
Embodiment:
Below in conjunction with specific embodiment, further illustrate the present invention, should understand these embodiment is only not used in and limits the scope of the invention for the present invention is described, after having read the present invention, those skilled in the art all fall within the application's claims limited range to the modification of the various equivalent form of values of the present invention.
Embodiment 1 Reduning injection sensitization detects
One, method
1, ultrafiltration enrichment traditional Chinese medicine injection sensitization position
100808,111011,101105,101114) and the Reduning injection of testing sample (lot number: 110709,120207,120210,120211) each 100ml get respectively the known Reduning injection that occurs untoward reaction (lot number:, join respectively the super filter tube that molecular weight cut-off is 3KD, 3500 leave heart 40min, through ultrafiltration repeatedly, residue 5ml solution, obtain respectively the macromole sensitization position of 20 times of enrichment methods of above each lot number, sterilizing, 4 degree are preserved.
2, cultivate altogether at spleen mononuclearcell PBMC and pungency position
Get ICR mouse sacrificed by decapitation, be placed in 75% alcohol and soak after 2 minutes and take out, open abdominal cavity and take out spleen, be placed in culture dish, shred rear with nook closing member extruding dispersion, with PBS (5ml) piping and druming, be separated into cell suspension, then cross 300 mesh filter screens, then mix with equivalent PBS solution, 1500 revs/min of the liquid that sieve are centrifugal 10 minutes, after abandoning and adding 2ml erythrocyte cracked liquid to mix after supernatant, centrifugal 8 minutes at 1000 revs/min, after abandoning supernatant, add PBS, mix latter 1000 revs/min centrifugal 8 minutes, and repeat once, abandon supernatant liquor and obtain spleen mononuclearcell PBMC, number cell count are also redissolved in full training (10% foetal calf serum 1640), count standby.
Regulating PBMC concentration is 1 * 10
6cells/ml, is inoculated in the 96 thin plates in hole with 100 μ l/well, adds the sensitization macromole position 20 μ l of the Reduning injection of the different lot numbers that 1 enrichment obtains, and control group adds 20 μ l waters for injection, 10%1640 of 10 μ l foetal calf serums and 70 μ l.Then will under 96 37 ℃ of the thin plates placements in hole, 5%CO2 condition, cultivate 72h.
3, sensitization detects
Get the spleen mononuclearcell PBMC cultivating altogether with pungency position, carry out Trypan Blue and Hochest33342 fluorescent dye.
Trypan Blue: add 0.04% trypan blue dye liquor 20 μ l in every hole, mix 5 minutes, after mixing, draw 20 μ l and add blood counting chamber living cell counting number.
Fluorescent dye: 1.5ml centrifuge tube collect to be cultivated the PBMC after 72h, adds the Hochest33342 of 5 μ g/ml, at 37 ℃, 5%CO2, cultivates after 30min, and 1500 revs/min of centrifugal 5min, PBS washed cell, is applied to slide glass, fluorescence microscope.
With traditional Chinese medicine injection, cell stimulatory multiple is reflected the power of its sensitization.
Pungency multiple=(Chinese medicine preparation group cell count-cellular control unit number)/cellular control unit number
Two, result
The known finished product with clinical adverse appearance is carried out to the ultrafiltration and concentration of 20 times, adopt the PBMC sensitization detection method of above-mentioned foundation to detect.Result shows, with Normal group comparison, known peaceful (the lot number: 100808,111011,101105,101114) all have significant pungency (* * P < 0.01), stimulate multiple to be respectively 2.4,3.4,4.03 and 2.63 of heat poison that occurs untoward reaction.Prove accuracy and the reliability of detection method provided by the invention.
Testing sample heat poison peaceful (lot number: 110709) to mouse PBMC there are no obvious irritation.And the peaceful (lot number: 120207,120210,120211) all detected certain weak pungency, specific experiment result is as shown in table 1 of heat poison.
The clinical heat poison peace hot poison to be tested that occurs untoward reaction of table 1 is rather to PBMC pungency (x ± s, n=5)
*p < 0.01, with Normal group comparison
Embodiment 2 reds sage root, the Radix Astragali, raw arteries and veins, puerarin, XUESAITONG ZHUSHEYE sensitization detect
One, method
1, ultrafiltration enrichment traditional Chinese medicine injection sensitization position
Get the red sage root, the Radix Astragali, raw arteries and veins, puerarin, each 100ml of XUESAITONG ZHUSHEYE, joining molecular weight cut-off is 3KD super filter tube, and 3500 leave heart 40min, through ultrafiltration repeatedly, obtain respectively the macromole sensitization position of 20 and 60 times of enrichment methods, sterilizing, 4 degree are preserved, standby.
2, cultivate altogether at spleen mononuclearcell PBMC and pungency position
Get ICR mouse sacrificed by decapitation, be placed in 75% alcohol and soak after 2 minutes and take out, open abdominal cavity and take out spleen, be placed in culture dish, shred rear with nook closing member extruding dispersion, with PBS (5ml) piping and druming, be separated into cell suspension, then cross 300 mesh filter screens, then mix with equivalent PBS solution, 1500 revs/min of the liquid that sieve are centrifugal 10 minutes, after abandoning and adding 2ml erythrocyte cracked liquid to mix after supernatant, centrifugal 8 minutes at 1000 revs/min, after abandoning supernatant, add PBS, mix latter 1000 revs/min centrifugal 8 minutes, and repeat once, abandon supernatant liquor and obtain spleen mononuclearcell PBMC, number cell count are also redissolved in full training (10% foetal calf serum 1640), count standby.
Regulating PBMC concentration is 1 * 10
6cells/ml, with 100 μ l/well, be inoculated in the 96 thin plates in hole, 20 times and each 20 μ l of enrichment sensitization macromole position of 60 times adding the above-mentioned red sage root preparing, the Radix Astragali, raw arteries and veins, puerarin, XUESAITONG ZHUSHEYE, control group adds 20 μ l waters for injection, 10%1640 of 10 μ l foetal calf serums and 70 μ l.Then the 96 thin plates in hole are put under 37 ℃, 5%CO2 condition and cultivated 72h.
3, sensitization detects
Get the spleen mononuclearcell PBMC that cultivate altogether at above-mentioned each pungency position, carry out respectively Trypan Blue and Hochest33342 fluorescent dye.
Trypan Blue: add 0.04% trypan blue dye liquor 20 μ l in every hole, mix 5 minutes, after mixing, draw 20 μ l and add blood counting chamber living cell counting number.
Fluorescent dye: 1.5ml centrifuge tube collect to be cultivated the PBMC after 72h, adds the Hochest33342 of 5 μ g/ml, at 37 ℃, 5%CO2, cultivates after 30min, and 1500 revs/min of centrifugal 5min, PBS washed cell, is applied to slide glass, fluorescence microscope.
With traditional Chinese medicine injection, cell stimulatory multiple is reflected the power of its sensitization.
Pungency multiple=(Chinese medicine preparation group cell count-cellular control unit number)/cellular control unit number
Two, result
The red sage root, the Radix Astragali, raw arteries and veins, puerarin, XUESAITONG ZHUSHEYE finished product are set up PBMC sensitization detection method and detected.The experimental result of table 2 shows, with Normal group comparison, puerarin injection have significant pungency (
*p < 0.01), stimulating multiple is 3.1.Pungency a little less than Radix Astragali injection has also detected necessarily (
*p < 0.05).The red sage root, raw arteries and veins and XUESAITONG ZHUSHEYE 20 times of enrichment concentration there are no obvious irritation.But all can detect and cause allergy and pungency during by the concentration enrichment to 60 at the red sage root, the Radix Astragali, raw arteries and veins, puerarin, XUESAITONG ZHUSHEYE sensitization position times.The strong and weak order of its moderate stimulation and sensitization is: the raw arteries and veins > of puerarin > Radix Astragali > red sage root > XUESAITONG.Specific experiment result is as shown in table 2:
Table 2 red sage root, the Radix Astragali, raw arteries and veins, puerarin, XUESAITONG ZHUSHEYE are to the irritating test result of PBMC
By above experimental result, show, the method of determination and evaluation of traditional Chinese medicine injection sensitization provided by the invention, have workablely, detected result accurately and reliably, convenient, fast, can extensively be suitable for and various traditional Chinese medicine injections, the detection evaluation of the sensitization of intermediate etc., is the safety evaluation of traditional Chinese medicine injection, the quality control of traditional Chinese medicine injection, for guaranteeing the security of clinical application, provide the foundation of science.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of the principle of the invention and design; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (1)
1. a method of determination and evaluation for traditional Chinese medicine injection sensitization, is characterized in that, comprises the following steps:
(1) use trapped molecular weight for thering is irritating macromole position in 1KD to 3KD ultra-filtration membrane enrichment traditional Chinese medicine injection, standby;
(2) irritating macromole position step (1) being prepared joins in the mononuclearcell in internal organs or peripheral blood source, cultivates altogether 72 hours;
(3) mononuclearcell of getting after step (2) is cultivated adopts the dyeing of trypan blue dye liquor or fluorescent dyeing, and the number of counting cells carrys out the sensitization of detecting and assessing traditional Chinese medicine injection;
The multiple of step (1) Chinese medicine injection liquid enrichment is 20 to 60 times;
The described fluorescence dye of step (3) is Hochest33342;
Described traditional Chinese medicine injection is the red sage root, the Radix Astragali, raw arteries and veins, puerarin, XUESAITONG or Reduning injection.
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| CN104107439A (en) * | 2014-07-28 | 2014-10-22 | 南京中医药大学 | Detection method for evaluating irritation of traditional Chinese medicine |
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| CN103293252B (en) * | 2013-05-09 | 2015-02-04 | 杭州师范大学 | Film enrichment method of traditional Chinese medicines |
| AU2015413017B2 (en) * | 2015-10-30 | 2019-09-19 | Chang Gung Memorial Hospital, Linkou | Method and identification kit for identifying sensitizing drug for drug allergic reaction |
| CN106680410A (en) * | 2017-02-17 | 2017-05-17 | 南京中医药大学 | Screening method for research on compatibility stability of traditional Chinese medicine injection and solvent |
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| CN101955908A (en) * | 2010-07-22 | 2011-01-26 | 中国医学科学院皮肤病研究所 | Artificial epidermis in-vitro model for detecting sensitization of material and screening medicament |
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| CN101955908A (en) * | 2010-07-22 | 2011-01-26 | 中国医学科学院皮肤病研究所 | Artificial epidermis in-vitro model for detecting sensitization of material and screening medicament |
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| CN104107439A (en) * | 2014-07-28 | 2014-10-22 | 南京中医药大学 | Detection method for evaluating irritation of traditional Chinese medicine |
| CN104107439B (en) * | 2014-07-28 | 2016-11-09 | 南京中医药大学 | The detection method that a kind of Chinese medicine excitant is evaluated |
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