CN101745128A - Method utilizing Co60-Gamma ray to carry out cold sterilization on Zilongjin medicine solution for cell culture - Google Patents
Method utilizing Co60-Gamma ray to carry out cold sterilization on Zilongjin medicine solution for cell culture Download PDFInfo
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- CN101745128A CN101745128A CN201010001094A CN201010001094A CN101745128A CN 101745128 A CN101745128 A CN 101745128A CN 201010001094 A CN201010001094 A CN 201010001094A CN 201010001094 A CN201010001094 A CN 201010001094A CN 101745128 A CN101745128 A CN 101745128A
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Abstract
The invention relates to a method utilizing Co60-Gamma ray to carry out cold sterilization on Zilongjin medicine solution for cell culture. The Co60-Gamma ray is irradiated for 1 to 12 hours, and the radiation dosage is within 2 to 8kGy. The method is suitable for quickly and effectively carry out sterilization on different cell culture solution with different medicine concentration under the premise of not affecting the medicine effect. The method has the characteristics of simple as well as convenient operation, high universality, no toxin and low cost. The irradiated Zilongjin medicine solution can be used for later-period cell culture after being completely sterilized.
Description
Technical field
The present invention relates to a kind of method of the Co60-of utilization radiation gamma sterilization, relate in particular to the sterilizing methods that utilizes the purple imperial golden drug solution of Co60-radiation gamma.
Background technology
Purple imperial gold plaque is that subordinate enterprise of Tianjin Zhongxin Pharmaceutical Group Co., Ltd. is grand along banyan pharmaceutical factory and Beijing Inst of Tumor Prevention and Treatment cooperative development development, three class PTS of National Drug Administration's approval.The basic research of the main pharmacodynamics material and the mechanism of action is sturdy, and is quite thorough to the research of main Chinese medicinal materials such as the Radix Astragali, Radix Angelicae Sinensis, Radix Salviae Miltiorrhizae etc., has benefiting QI and nourishing blood, heat-clearing and toxic substances removing, the effect of regulating the flow of QI to dissipate blood stasis.The adjuvant drug that this product is held concurrently stagnant heat card patient chemotherapy for the pulmonary carcinoma QI and blood deficiency, the tool clinical symptoms that has some improvement, the effect of muscle power situation scoring, immune indexes NK cell, cd4 cell etc. there is the improvement effect, can reduce clinical responses such as peripheral hemogram reduction, hepatic and renal function injure and nausea and vomiting due to the chemotherapy, alopecia, its multinomial research has international most advanced level through expert testimony.
Chinese medicine effect characteristics are the pleiotropy that acts on, the complexity of dose-effect relationship, the relative instability of effect, the dual regulation of some Chinese medicine etc.; The characteristic that has many compositions, too many levels, many target spots simultaneously on the Chinese medicine model of action again.The characteristics that these are complicated, drug research method in the past be difficult to from integral body to the cellular level, or even molecular level is studied all sidedly.The clear and definite drug effect of the research of modern pharmacology molecular level all has its " target gene ", and target gene also is the most essential index of Chinese medicine effect.
Appear as the biochip technology of one of molecular biology method that we understand the action target spot of Chinese medicine and mode at gene level, metabolic pathway provides strong tool.With thousands of kinds of nucleic acid probe molecules orderly be arranged on the little carrier material of area and with the sample nucleic acid hybridization of sign, show by autoradiography or fluorescent scanning, can once obtain a large amount of useful bio informations.It has been for to have erected a bridge block from molecular level to the recurrence of integral level research biosis, it be one can be with the biological high-tech of the molecular biology of the round pcr that matches in excellence or beauty developing " milestone " level.Its practicality is extremely strong, has been used for the discovery and the every field such as expression study, sequencing by hybridization and polymorphism detection of gene at present, is cited as one of the most rising new and high technology of 21 century.
Expression profiles of gene chip is to be most widely used in the gene chip, also is the high flux, microminiaturization and the automatization's feature that best embody gene chip.It is just bringing into play important role in the research of functional genomics.Chip of expression spectrum also is in the starting stage in the Chinese medicine Application for Field, and many technical methods also are in the stage of fumbling, and theoretical, method still owes ripe, mainly concentrates on aspects such as herbal pharmacology research at present.Can predict, biochip technology will be exhibited one's skill to the full in modernization of cmm and exploitation.Utilize the chip of expression spectrum analysis platform to experimentize, at first will solve the purple imperial golden drug solution sterilization problem that is used for cell culture.
The normal sterilizing methods that adopts has in the medicament production process: autoclaving, and free flowing steam sterilization, boiling sterilization filters sterilization and gas sterilization etc.Co60-gamma-rays (cobalt 60-gamma-rays) irradiation sterilization is a kind of comparatively rapidly sterilizing methods of development in recent years.It is strong that it has penetration power, easy and simple to handle, and speed is fast, can sterilize at normal temperatures, and radiation dose is suitable, can not destroy the effective ingredient of medicine, also can not produce injury to the people, and the advantages such as propagation again of sterilization back long period control antibacterial are arranged.
Summary of the invention
The objective of the invention is to overcome the deficiency that prior art exists, provide a kind of Co60-gamma-rays cold sterilization that utilizes easy and simple to handle, easy row to be used for the method for the purple imperial golden drug solution of cell culture, wherein shone the Co60-gamma-rays 1-12 hour, radiation dose is 2-8kGy, and the sharp purple imperial golden drug solution after the sterilization can be used for the later stage cell culture.
In the preferred embodiment of the present invention, irradiation Co60-gamma-rays 2-8 hour, more preferably, irradiation Co60-gamma-rays 4 hours.
In the preferred embodiment of the present invention, radiation dose is 4kGy.
Contain at least a in antibacterial, fungus or the mycete in the imperial golden drug solution of purple of the present invention; The imperial golden medicine of described purple is dissolved at least a among cell culture fluid modified form α-MEM, modified form RPMI-1640 or the HAM ' S/F-12; Drug level is 4.125mg/ml-133mg/ml, is preferably 66mg/ml, 33mg/ml, 22mg/ml, 16.5mg/ml, 8.25mg/ml.
In the preferred embodiment of the present invention, the method for utilizing the purple imperial golden drug solution sterilization of Co60-radiation gamma and being used for the later stage cell culture mainly comprises step:
1) utilizes the 2YT culture of Gram-positive and Gram-negative (G+, G-) bacterium to do simulation experiment, grope the condition of Co60-radiation gamma;
2) gains with step 1) install in the 50ml centrifuge tube with the 40ml branch;
3) with step 2) gains irradiations Co60-gamma-rays 4 hours, radiation dose is 4kGy;
4) gains of step 3) are got 100ul and be coated with flat board, put 37 degree constant temperature culture 24h;
5) gains with step 4) all change in the sterilization triangular flask, put 37 degree constant temperature culture 24h, 48h, 72h, 96h;
6) with step 4) and 5) gains observe not have the growth sign;
7) with Co60-radiation gamma 4 hours, radiation dose is that 4kGy selects parameter irradiation irradiation to be dissolved in the purple imperial golden drug solution of different cell culture fluids and variable concentrations gradient at last.
Experimental result of the present invention shows, method of the present invention is applicable to that different pharmaceutical concentration, different cell culture fluid are under the prerequisite that does not influence drug effect, sterilization fast and effectively, easy and simple to handle, highly versatile, nontoxic and expense is low, the imperial golden drug solution of postradiation purple is sterilized fully and be can be used for the later stage cell culture.
The following example is intended to further illustrate, rather than restriction the present invention.Under the spirit and principles in the present invention prerequisite, all will fall in the claim scope of the present invention inventing any change that indivedual technical steps carry out and changing.
Description of drawings
Figure 1A-Figure 1B represents to mix purple imperial gold solution with escherichia coli (DH5 α) simulations, and to carry out before and after the Co60-radiation gamma 4 hours plate count result: Figure 1A be that escherichia coli (DH5 α) are simulated and mix purple imperial gold solution but do not carry out the Co60-radiation gamma, is coated with dull and stereotyped incubated overnight result; Figure 1B is that escherichia coli (DH5 α) simulations is mixed purple imperial gold solution but do not carried out the Co60-radiation gamma after 4 hours, is coated with dull and stereotyped incubated overnight result.
Fig. 2 A-Fig. 2 B represents to mix purple imperial gold solution with staphylococcus aureus simulation, and to carry out before and after the Co60-radiation gamma 4 hours plate count result: Figure 1A be that staphylococcus aureus is simulated and mixes purple imperial gold solution but do not carry out the Co60-radiation gamma, is coated with dull and stereotyped incubated overnight result; Figure 1B mixes purple imperial gold solution for the staphylococcus aureus simulation but does not carry out the Co60-radiation gamma after 4 hours, is coated with dull and stereotyped incubated overnight result.
Fig. 3 A-Fig. 3 B represents that purple imperial gold solution carries out the cultivation results of Co60-radiation gamma front and back cell (human embryonic lung fibroblast MRC-5) in modified form α-MEM: Figure 1A does not carry out Co60-radiation gamma cell culture result for purple imperial gold solution, because of bacterial growth media has become muddy yellow; Fig. 2 A is that purple imperial gold solution carries out cell culture result behind the Co60-radiation gamma, and culture medium is as clear as crystal aubergine.
The specific embodiment
The present invention is described in further detail below by most preferred embodiment.Following examples are only used for explanation rather than restriction the present invention.
Embodiment 1
Carry out Co60-radiation gamma experiment plate count with escherichia coli (DH5 α) simulation
Complete step comprises:
1) escherichia coli (DH5 α) glycerol preservation strain is got 20ul and connect in the 20ml 2YT culture medium, incubated overnight, 37 degree, 150rpm;
2) antibacterial of incubated overnight was diluted by 1: 10, dilute each 50ml, dilute 8 gradients 10 with 2YT
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, 10
-8, get 10 respectively
-6, 10
-7, 10
-8Each 100ul carries out coated plate, put 37 degree constant temperature culture 24h after, counting;
3) will dilute the good dense gradient of each bacterium, and parallelly respectively get 3 parts, and send Tianjin physics Institute to carry out the experiment of Co60-radiation gamma, irradiation time is 1h, 2h, 4h, 8h;
4) get the 100ul coated plate, put 37 degree constant temperature culture 24h after, counting.
Embodiment
2 carry out the Co60-radiation gamma with the staphylococcus aureus simulation tests plate count
Complete step comprises:
1) staphylococcus aureus glycerol preservation strain is got 20ul and connect in the 20ml 2YT culture medium, incubated overnight, 37 degree, 150rpm;
2) antibacterial of incubated overnight was diluted by 1: 10, dilute each 50ml, dilute 8 gradients 10 with 2YT
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, 10
-8, get 10 respectively
-6, 10
-7, 10
-8Each 100ul carries out coated plate, put 37 degree constant temperature culture 24h after, counting;
3) will dilute the good dense gradient of each bacterium, and parallelly respectively get 3 parts, and send Tianjin physics Institute to carry out the experiment of Co60-7 roentgenization, irradiation time is 1h, 2h, 4h, 8h;
4) get the 100ul coated plate, put 37 degree constant temperature culture 24h after, counting.
Embodiment 3
Mix purple imperial gold solution with escherichia coli (DH5 α) simulation and carry out Co60-gamma-rays photograph
Penetrate experiment and shake the bottle counting
Complete step comprises:
1) escherichia coli (DH5 α) glycerol preservation strain is got 20ul and connect in the 20ml 2YT culture medium, incubated overnight, 37 degree, 150rpm;
2) antibacterial of incubated overnight was diluted by 1: 10, dilute each 50ml, dilute 8 gradients 10 with 2YT
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, 10
-8, get 10 respectively
-6, 10
-7, 10
-8Each 100ul carries out coated plate, put 37 degree constant temperature culture 24h after, counting;
3) will dilute the good dense gradient of each bacterium, and parallelly respectively get 3 parts, and send Tianjin physics Institute to carry out the experiment of Co60-radiation gamma, irradiation time is 1h, 2h, 4h, 8h;
4) get the 100ul coated plate, all the other change in the aseptic triangular flask, 37 degree, and 150rpm cultivates, and continues observed result in 24h, 48h, 72h, 96h.
Embodiment 4
The staphylococcus aureus simulation is carried out the experiment of Co60-radiation gamma and is shaken the bottle counting
Complete step comprises:
1) staphylococcus aureus glycerol preservation strain is got 20ul and connect in the 20ml 2YT culture medium, incubated overnight, 37 degree, 150rpm;
2) antibacterial of incubated overnight was diluted by 1: 10, dilute each 50ml, dilute 8 gradients 10 with 2YT
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, 10
-8, get 10 respectively
-6, 10
-7, 10
-8Each 100ul carries out coated plate, put 37 degree constant temperature culture 24h after, counting;
3) will dilute the good dense gradient of each bacterium, and parallelly respectively get 3 parts, and send Tianjin physics Institute to carry out the experiment of Co60-radiation gamma, irradiation time is 1h, 2h, 4h, 8h;
4) get the 100ul coated plate, all the other change in the aseptic triangular flask, 37 degree, and 150rpm cultivates, and continues observed result in 24h, 48h, 72h, 96h.
Embodiment 5
Carry out the experiment of Co60-radiation gamma with the imperial golden drug solution of purple
Complete step comprises:
1) purple imperial gold plaque main component is the Radix Astragali, Radix Angelicae Sinensis, Herba Solani Lyrati, Herba Solani Nigri etc.;
2) obtain solution: take by weighing purple imperial golden tablet 13.3g and be dissolved in the corresponding cell culture medium, wait dissolving fully after, change over to and carry out standardize solution 100ml in the volumetric flask, packing 1.2ml carries out the experiment of Co60-radiation gamma in the 1.5ml centrifuge tube then; Other drug concentration 66mg/ml, 33mg/ml, 22mg/ml, 16.5mg/ml, 8.25mg/ml, 4.125mg/ml dilute with the mother solution of 133mg/ml and form, and carry out the every pipe packing of 1.2ml then;
3) will dilute each good dense gradient, and parallelly respectively get 3 parts, and send Tianjin physics Institute to carry out the experiment of Co60-radiation gamma, irradiation time is 4h;
4) handle cell with the purple imperial golden drug solution behind the Co60-radiation gamma of variable concentrations, each culture bottle adds 9ml culture medium (2% serum, 1% pair anti-), 1ml medicine, 37 degree 5%CO
2Leave standstill cultivation;
5) continue observed result in 24h, 48h, 72h, 96h.
Claims (7)
1. a method of utilizing the cold sterilization of Co60-gamma-rays to be used for the purple imperial golden drug solution of cell culture is characterized in that, irradiation Co60-gamma-rays 1-12 hour, and radiation dose is 2-8kGy.
2. method according to claim 1 is characterized in that, irradiation Co60-gamma-rays 2-8 hour.
3. method according to claim 2 is characterized in that, irradiation Co60-gamma-rays 4 hours.
4. method according to claim 1 is characterized in that, radiation dose is 4kGy.
5. according to each described method of claim 1-4, it is characterized in that, contain at least a in antibacterial, fungus or the mycete in the imperial golden drug solution of described purple.
6. method according to claim 5 is characterized in that, the imperial golden medicine of described purple is dissolved at least a among cell culture fluid modified form α-MEM, modified form RPMI-1640 or the HAM ' S/F-12.
7. method according to claim 5 is characterized in that, the concentration of the imperial golden drug solution of described purple is 4.125mg/ml-133mg/ml.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102172251A (en) * | 2011-03-24 | 2011-09-07 | 西南大学 | Method for prolonging preservation period of fresh konjak |
| CN110607273A (en) * | 2019-09-19 | 2019-12-24 | 上海长征医院 | Cell strain capable of being passaged for long time after gamma ray irradiation of mouse embryo fibroblast and construction method thereof |
-
2010
- 2010-01-21 CN CN201010001094A patent/CN101745128A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102172251A (en) * | 2011-03-24 | 2011-09-07 | 西南大学 | Method for prolonging preservation period of fresh konjak |
| CN110607273A (en) * | 2019-09-19 | 2019-12-24 | 上海长征医院 | Cell strain capable of being passaged for long time after gamma ray irradiation of mouse embryo fibroblast and construction method thereof |
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Application publication date: 20100623 |