CN105255828A - Preparation method of extract for improving proliferation rate of CIK (Cytokine Induced Killer) cells - Google Patents
Preparation method of extract for improving proliferation rate of CIK (Cytokine Induced Killer) cells Download PDFInfo
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Abstract
The invention discloses a preparation method of an extract for improving the proliferation rate of CIK (Cytokine Induced Killer) cells. The preparation method is characterized by comprising the following steps: firstly, preparing the following raw materials in parts by weight: 8 parts of radix astragali, 10 parts of liquorice, 12 parts of red ginseng, 15 parts of flos carthami, 20 parts of dried ginger, 30 parts of divaricate saposhnikovia root, 3 parts of radix aconiti preparata, 13 parts of white chinaure herb and 6 parts of Tetrapanax papyriferus; secondly, preparing according to the following specific steps: firstly, adding water into the raw materials for decocting, wherein the addition amount of water is 5 to 10 times of the weight of the raw materials, and decocting time is 3 to 4 hours; secondly, filtering, concentrating and collecting an extracting solution I; secondly, precipitating the extracting solution with ethanol, then filtering, and recovering ethanol to obtain an extracting solution II; thirdly, enabling the extracting solution II to pass through macroporous adsorption resin, then eluting with distilled water to be colorless, eluting with ethanol, and collecting eluent. According to the extract prepared by the preparation method disclosed by the invention, the proliferation rate of the CIK cells can be greatly improved.
Description
Technical field
The present invention relates to a kind of method for preparing extractive for improving CIK cell multiplication rate.
Background technology
CIK is a group foreign cell obtained after human peripheral blood single nucleus cell being used in vitro cytokine profiles (as CD 3-resisting monoclonal antibody, IL-2 and IFN-γ etc.) co-cultivation for some time.Because this kind of cell expresses CD3+ and CD56+ two kinds of membrane protein molecules simultaneously, therefore the NK cell sample T lymphocyte that is otherwise known as, the non-MHC of the anti-tumor activity powerful with T lymphocyte and NK cell is restricted kills knurl advantage.
CIK cell advantage is as follows: 1.CIK cell proliferation rate is fast, and anti-tumor activity cell can be bred in a large number, and cytoactive also strengthens greatly.2.CIK cell has the mechanism identifying tumour, to normal cytotoxic effect.3. kill knurl spectrum wide, can be used for the treatment of the kinds of tumors such as leukemia, lymphoma, lung cancer, cancer of the stomach, intestinal cancer, responsive equally to multidrug resistant tumour cell.4. are typical personalized biological Therapeutic mode.After this kind of cell is fed back, body immunity can also be made to improve, produce special antivirus action, thus dual effect is imposed to oncotherapy.5., due to the autogenous cell that CIK cell is activation, use very safe.
Tumor biotherapy is a large focus in current tumor research field, tumor biotherapy comprises the methods such as cytokine, gene therapy, monoclonal antibody, tumor vaccine and adoptive immunotherapy, wherein tumour cell adoptive immunotherapy in tumor biotherapy in occupation of critical role.Adoptive cellular immunotherapy is that at present research is more and entered the Biotherapy method of clinical trial.It is by the autologous of Activation In Vitro or alloimmune effector cell infusion to patient, kill the tumour cell in patient body; Be applicable to the low patient of cellular immune function, as after high-dose chemotherapy, radiotherapy, after bone marrow transplantation, the patient of virus infection damage immunocyte quantity and function.The appearance of cytokine induced kill cell CIK, for tumour adoptive immunotherapy brings new hope.CIK cell is equaled within 1991, to take the lead in finding by the SchmidtWolf of Stanford Univ USA, human peripheral blood mononuclear cell is obtained a group foreign cell with after cytokine profiles co-cultivation for some time by vitro, this cell co expression CD3 and CD56 two kinds of membrane protein molecules, be a kind of tumor biotherapy effector cell that at present known killing activity is the strongest, this cell has that the non-MHC of the powerful anti-tumor activity of T lymphocyte and NK cell is restricted kills the advantages such as knurl concurrently.In conjunction with treatments such as excision, intervention, radio frequency, argon helium knifes, can remove and with the oncocyte deposited loose in the atomic tubercle stove of excision or body, can not delay or stoping in the transfer of tumour or recurrence to play an important role; Temporarily be not suitable for performing an operation for part, get involved or the tumour patient of other treatment, also first can carry out CIK cell treatment, improve physical function situation, quality of making the life better, strives for other treatment machine meeting.
Traditional CIK cell in vitro long-term cultivation method adopts the static mode with intermittently changing liquid, tradition training method is in order to obtain the cell of sufficient amount, treat a patient and often need process up to a hundred culturing bottles or culture bag, easily cause pollution, affect cell quality and stability, therefore from industrialization and clinical requirement very away from.Therefore, how external a large amount of propagation of CIK cell, namely improve its in-vitro multiplication speed further, is the key point using CIK cell immunotherapy on a large scale.
Summary of the invention
The object of the present invention is to provide a kind of method for preparing extractive that greatly can improve CIK cell multiplication rate.
In order to realize above object, technical scheme of the present invention is as follows: a kind of method for preparing extractive for improving CIK cell multiplication rate, it is characterized in that: the raw material first preparing following weight part: the Radix Astragali 8 parts, 10 parts, Radix Glycyrrhizae, red ginseng 12 parts, 15 parts, safflower, rhizoma zingiberis 20 parts, windproof 30 parts, Radix Aconiti Preparata 3 parts, Herba Boenninghauseniae Albiflorae 13 parts, the stem pith of the rice-paper plant 6 parts; Then prepare according to following steps;
The first step, by above raw material boiling; Wherein, amount of water is 5-10 times of raw material weight, and decocting time is 3-4 hour; Then filter, concentrate and collect extracting solution I;
Second step, by extracting solution I alcohol settling, then filters, and reclaims ethanol, obtains extracting solution II;
3rd step, extracting solution II will be refined through macroporous adsorbent resin, then be eluted to colourless with distilled water, use ethanol elution again, collect elutriant, then filter through 0.45 μm of millipore filtration, filtrate is concentrated into 5g crude drug/ml, adjust pH, then through 0.22 μm of millipore filtration Entkeimung, namely last packing obtains Chinese medical extract.
Further, second step, the consumption of the ethanol adopted in the 3rd step and extracting solution same volume.
Further, in the first step, amount of water is 8 times of raw material weight.
Compared with prior art, beneficial effect of the present invention: Chinese medical extract of the present invention is compared with other chemical induction multiplicaiton factor, and to human non-toxic's side effect, therefore the CIK cell after proliferative induction directly may be used for clinical trial.(2) Chinese medical extract of the present invention, has the effect significantly improving CIK cell multiplication rate, the cell killing curative effect after activation higher than former without Chinese medical extract CIK cell result for the treatment of.
Embodiment
The invention will be further described below.
Embodiment: the raw material first preparing following weight part: Radix Astragali 8g, Radix Glycyrrhizae 10g, red ginseng 12g, safflower 15g, rhizoma zingiberis 20g, windproof 30g, Radix Aconiti Preparata 3g, Herba Boenninghauseniae Albiflorae 13g, stem pith of the rice-paper plant 6g; Then prepare according to following steps; The first step, by above raw material boiling; Wherein, amount of water is 1000g, and decocting time is 3.5 hours; Then filter, concentrate and collect extracting solution I; Second step, by extracting solution I alcohol settling, then filters, and reclaims ethanol, obtains extracting solution II; 3rd step, extracting solution II will be refined through macroporous adsorbent resin, then be eluted to colourless with distilled water, use ethanol elution again, collect elutriant, then filter through 0.45 μm of millipore filtration, filtrate is concentrated into 5g crude drug/ml, adjust pH, then through 0.22 μm of millipore filtration Entkeimung, namely last packing obtains Chinese medical extract.Wherein, the consumption of the ethanol of employing and extracting solution same volume.
Experiment divides two groups, and first group is that conventional CIK cell cultural method is cultivated, and second group is the extract adding above preparation toward conventional RPMI1640 complete culture solution, add-on is respectively 3%, and 5%, 7% (3%, 5%, 7% is the per-cent accounting for nutrient solution weight).Often organize the identical culture vessel of employing 3 to cultivate.
Conventional CIK cell cultural method belongs to existing known technology, is just not described in detail the method for cultivation here, just mentions once.
The amplification times detected result display of the 14th day, containing cell absolute number ratio in the substratum of 3%, 5%, 7% Chinese medical extract without traditional Chinese medicine extraction object height 2.1,3,3.3 times.Respectively to the cell after 3%, 5%, 7% Chinese medical extract proliferative induction carry out the kill rate of tumour cell detection find all more than 98% is reached to the kill rate of tumour cell.
Claims (3)
1. for improving a method for preparing extractive for CIK cell multiplication rate, it is characterized in that: the raw material first preparing following weight part: the Radix Astragali 8 parts, 10 parts, Radix Glycyrrhizae, red ginseng 12 parts, 15 parts, safflower, rhizoma zingiberis 20 parts, windproof 30 parts, Radix Aconiti Preparata 3 parts, Herba Boenninghauseniae Albiflorae 13 parts, the stem pith of the rice-paper plant 6 parts; Then prepare according to following steps;
The first step, by above raw material boiling; Wherein, amount of water is 5-10 times of raw material weight, and decocting time is 3-4 hour; Then filter, concentrate and collect extracting solution I;
Second step, by extracting solution I alcohol settling, then filters, and reclaims ethanol, obtains extracting solution II;
3rd step, extracting solution II will be refined through macroporous adsorbent resin, then be eluted to colourless with distilled water, use ethanol elution again, collect elutriant, then filter through 0.45 μm of millipore filtration, filtrate is concentrated into 5g crude drug/ml, adjust pH, then through 0.22 μm of millipore filtration Entkeimung, namely last packing obtains Chinese medical extract.
2. the method for preparing extractive for improving CIK cell multiplication rate according to claim 1, is characterized in that: second step, the consumption of the ethanol adopted in the 3rd step and extracting solution same volume.
3. the method for preparing extractive for improving CIK cell multiplication rate according to claim 1, is characterized in that: in the first step, amount of water is 8 times of raw material weight.
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| CN102755512A (en) * | 2011-04-28 | 2012-10-31 | 陈春 | Traditional Chinese medicine extract capable of improving CIK cell proliferation rate as well as preparation method and application of same |
| CN103705726A (en) * | 2013-12-31 | 2014-04-09 | 车艳艳 | Cell-mediated immunity therapy traditional Chinese medicine tea for preventing and treating tumors |
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| CN104651310A (en) * | 2015-02-28 | 2015-05-27 | 杭州中德贝尔生物科技有限公司 | NK cell in-vitro induced amplification method |
| CN104873777A (en) * | 2015-05-28 | 2015-09-02 | 湖南中医药大学 | Traditional Chinese medicine composition for treating hepatitis and preparing method of traditional Chinese medicine composition |
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2015
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| CN1362059A (en) * | 2001-01-05 | 2002-08-07 | 唐山王清任制药有限公司 | Nano Chinese medicine piece and its prepn |
| CN102755512A (en) * | 2011-04-28 | 2012-10-31 | 陈春 | Traditional Chinese medicine extract capable of improving CIK cell proliferation rate as well as preparation method and application of same |
| CN103705726A (en) * | 2013-12-31 | 2014-04-09 | 车艳艳 | Cell-mediated immunity therapy traditional Chinese medicine tea for preventing and treating tumors |
| CN104593326A (en) * | 2014-12-30 | 2015-05-06 | 杭州特马赛生物技术有限公司 | Method for preparing enhanced DC-CIK cell induced by traditional Chinese medicines and application of enhanced DC-CIK cells induced by traditional Chinese medicines |
| CN104651310A (en) * | 2015-02-28 | 2015-05-27 | 杭州中德贝尔生物科技有限公司 | NK cell in-vitro induced amplification method |
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| 赵俊峰 等: "CIK细胞结合中药扶正固本治疗中晚期肾脏肿瘤", 《中国现代医学杂志》 * |
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