CN102327462A - Traditional Chinese medicine for treating qi and blood insufficiency asthenia diseases of leukemia patients after chemotherapy and method for preparing traditional Chinese medicine preparations - Google Patents
Traditional Chinese medicine for treating qi and blood insufficiency asthenia diseases of leukemia patients after chemotherapy and method for preparing traditional Chinese medicine preparations Download PDFInfo
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Abstract
The invention discloses traditional Chinese medicine for treating qi and blood insufficiency asthenia diseases of leukemia patients after chemotherapy and a method for preparing traditional Chinese medicine preparations. The traditional Chinese medicine comprises the following raw materials in part by weight: 1 to 98 parts of astragalus mongholicus, 1 to 50 parts of red ginseng, 1 to 98 parts of bighead atractylodes rhizome, 1 to 98 parts of Tuckahoe, 1 to 100 parts of glossy privet fruit, 1 to 100 parts of ophiopogon root, 1 to 100 parts of dried rhizome of rehmannia, 1 to 100 parts of fructus psoraleae, 1 to 98 parts of fleece-flower root, 1 to 98 parts of epimedium and 1 to 98 parts of radix asparagi. The preparation method comprises the following steps of: (1) weighing and taking raw materials; (2) taking the red ginseng, adding ethanol for refluxing extraction twice, filtering the liquid and merging filter liquid, wherein the time of each extraction is 2 hours; and taking red ginseng medicine dregs obtained during filtering to be mixed with rest raw materials, adding water for soaking the medicine materials to be decocted twice, filtering the liquid and merging the decocting liquid, wherein the first decoction time is 2 hours, and the second decoction time is 1.5 hours; and (3) mixing the merged filter liquid and the merged decoction liquid. The traditional Chinese medicine has the advantages that the immunity of patients can be improved, the leukemia recrudescence is prevented or delayed, the life period is prolonged, and the life quality of the patients is improved.
Description
Technical field
The present invention relates to a kind of Chinese medicine medicine and Chinese medicine preparation method for preparing that is used for the leukemia people, particularly the Chinese medicine medicine and the Chinese medicine preparation method for preparing of the deficient disease of insufficiency of vital energy and blood after the treatment leukemia people chemotherapy.
Background technology
The acute leukemia people through chemotherapy obtain alleviate after many places in the minimal residual leukemia state.(minimal residual leukemia is a kind of leukemia state of residual minim leukaemia in the body after treating MRL) to minimal residual leukemia, is the root of leukemia relapse.Up to the present still there is not the diagnostic criteria of MRL both at home and abroad.It is generally acknowledged that the MRL cell accounts for below the 1/1000-1/10000 of medullary cell sum, can not detect that detection method commonly used comprises polymerase chain reaction (PCR) with conventional morphocytology.This moment, mostly patient was the deficient disease of insufficiency of vital energy and blood after the leukemia people chemotherapy.
Chinese medicine thinks that disease is the process of human body struggle between vital QI and pathogen, generation, the development of the power of healthy energy decision disease, changes and lapses to." healthy energy " is appreciated that and is the immune normal function of human body.Diseases such as the existing pale complexion of the deficient disease multilist of insufficiency of vital energy and blood after leukemia people's chemotherapy, lip onyx color be light, dizzy, breathe hard, weak, lassitude loin and legs, with weakened body resistance be main, mostly card genus deficient vital QI leading to lingering of pathogen is deficiency of both QI and YIN, insufficiency of vital energy and blood, hyperactivity of fire caused by deficiency of YIN.There is following technological deficiency in the Chinese medicine medicine that is used to treat the deficient disease of insufficiency of vital energy and blood after the leukemia people chemotherapy in the prior art: 1, leukaemic's survival rate is lower: 5 years survival rates of Chinese child AML are about 20%～30%, and the overall survival rate of Chinese white disorders of blood child is about 50%; Adult AML survival rate is about 10%～35%, becomes the overall survival rate of human leukemia to be about 10%～30%; In addition, 5 years survival rates can reach 40%～50% after relevant research showed present acute leukemia bone marrow transplantation; Though each family's report data has a certain distance in the document, leukemia people living rate is low to be a undisputable fact.2, still do not have effective Chinese medicine medicine for minimal residual leukemia and treat, make the leukemia people can not fundamentally break away from leukemic torment; Particularly for the minimal residual leukemia after the treatment of acute myeloid lineage leukemia process; If the patient can access thorough alleviation; Just can make acute myeloid lineage leukemia on the basis of present complete remission rate 60%～90%, realize preventing or delaying leukemia relapse, thereby prolong patient's life cycle.
Summary of the invention
To the deficiency that exists in the prior art; The objective of the invention is to the acute myeloid lineage leukemia minimal residual leukemia after through treatment, a kind of Chinese medicine medicine and Chinese medicine preparation its preparation method that is used to treat the deficient disease of insufficiency of vital energy and blood after the leukemia people chemotherapy is provided; To improve patient's immunity, prevent or delay leukemia relapse, prolong CCR time and life cycle, improve patient's life quality.
Technical scheme of the present invention is achieved in that a kind of Chinese medicine medicine of treating the deficient disease of insufficiency of vital energy and blood after the leukemia people chemotherapy, is made up of following materials based on weight: 1 part～98 parts of the Radixs Astragali, 1 part～50 parts of Radix Ginseng Rubra; 1 part～98 parts of the Rhizoma Atractylodis Macrocephalaes, 1 part～98 parts in Poria, 1 part～100 parts of Fructus Ligustri Lucidi; 1 part～100 parts of Radix Ophiopogonis, 1 part～100 parts in the Radix Rehmanniae, 1 part～100 parts of Fructus Psoraleaes; 1 part～98 parts of the Radixs Polygoni Multiflori, 1 part～98 parts of 1 part～98 parts of Herba Epimedii and Radix Asparagi.
The Chinese medicine medicine of the deficient disease of insufficiency of vital energy and blood after the above-mentioned treatment leukemia people chemotherapy is made up of following materials based on weight: 25 parts～35 parts of the Radixs Astragali, 1.5 parts～10 parts of Radix Ginseng Rubra; 5 parts～15 parts of the Rhizoma Atractylodis Macrocephalaes, 5 parts～15 parts in Poria, 15 parts～25 parts of Fructus Ligustri Lucidi; 10 parts～20 parts of Radix Ophiopogonis, 15 parts～25 parts in the Radix Rehmanniae, 10 parts～20 parts of Fructus Psoraleaes; 15 parts～25 parts of the Radixs Polygoni Multiflori, 10 parts～20 parts of 5 parts～15 parts of Herba Epimedii and Radix Asparagi.
The Chinese medicine medicine of the deficient disease of insufficiency of vital energy and blood after the above-mentioned treatment leukemia people chemotherapy is made up of following materials based on weight: 30 parts of the Radixs Astragali, 5 parts of Radix Ginseng Rubra; 10 parts of the Rhizoma Atractylodis Macrocephalaes, 10 parts in Poria, 20 parts of Fructus Ligustri Lucidi; 15 parts of Radix Ophiopogonis, 20 parts in the Radix Rehmanniae, 15 parts of Fructus Psoraleaes; 20 parts of the Radixs Polygoni Multiflori, 15 parts of 10 parts of Herba Epimedii and Radix Asparagi.
Utilize the Chinese medicine preparation method for preparing of above-mentioned Chinese medicine medicine, comprise the steps: that (1) takes by weighing raw material respectively by the composition of said Chinese medicine medicine; (2) get said Radix Ginseng Rubra and add alcohol reflux 2 times, each 2 hours, filter merging filtrate; The Radix Ginseng Rubra medicinal residues that obtain when getting filtration and all the other raw materials mix of said Chinese medicine medicine add water logging and cross medical material decoction 2 times, 2 hours for the first time, 1.5 hours for the second time, filter collecting decoction; (3) filtrating after will merging and merge after decocting liquid mix.
The method for preparing of above-mentioned Chinese medicine preparation; Relative density is the clear paste of 1.38-1.40 when being concentrated at 60 ℃ after the decocting liquid after step (3) also comprises the steps: the filtrating after (4) will merge afterwards and merges mixes, 1 part of qinghuo reagent and 2 parts of dextrine powders by weight, and add ethanol; Mix homogeneously; Process granule, drying promptly gets Chinese medicine preparation.
Utilize Chinese medicine medicine of the present invention can be prepared into the dosage form on sugared granule, sugar free granule type, powder, unguentum, oral liquid, syrup, electuary, mixture and other pharmaceutics.
The invention has the beneficial effects as follows: the Radix Ginseng Rubra function is a strongly invigorating primordial QI, and nourishing qi to stop, the Radix Astragali are the panacea of QI invigorating, and the ability tonifying five ZANG-organs is all empty, and two medicines share, and play the merit of QI invigorating primordial QI reinforcing tonify deficiency altogether, are monarch drug in the side; Radix Polygoni Multiflori system usefulness, merit is in invigorating the liver and kidney, benefiting essence-blood, black beard and hair, bone and muscle strengthening, and Radix Asparagi Great Cold, the power of moisturizing of relieving inflammation or internal heat is big than Radix Ophiopogonis; And nourishing kidney-yin, be slightly cold Radix Ophiopogonis, and for poor, it is less that so it grows greasy property than Radix Asparagi for nourshing Yin and drynsessmoistening prescription power; The double relieving restlessness reinforcing stomach reg fluid that clears away heart-fire, Fructus Ligustri Lucidi and Radix Rehmanniae nourishing YIN and benefiting blood, kidney tonifying, essence replenishing, Fructus Psoraleae warming the spleen reinforcing the kidney and supporting YANG is taken in the effect of " treating YANG within YIN " concurrently; The Herba Epimedii kidney-replenishing, bone and muscle strengthening, above medicine is played the effect of tonifying kidney and nourishing yin QI invigorating altogether, all is ministerial drug; The Rhizoma Atractylodis Macrocephalae, Poria are assistant, invigorating spleen to remove dampness, and the Fang Zhongzi that disappears is greasy, and all medicines share, and amount to YIN nourishing and QI supplementing, set upright anti-white function.The present invention is directed to the characteristics of patient's MRL " heresy goes just to decline ", " deficiency of both QI and YIN ",, be particularly useful for the clinical treatment of minimal residual leukemia with supplementing QI and nourishing YIN legislation, merit master's strengthening vital QI to eliminate pathogenic factors.Clinical use and pharmacological research, zoopery and preparatory clinical research through for many years all show: can help body healthy energy; Improve patient's immunity; Prevention or delay leukemia relapse; Prolong CCR time and life cycle, and can improve patient's life quality, thereby reach therapeutic purposes.
The specific embodiment
Effect for further objective evaluation Chinese medicine preparation of the present invention; Carried out clinical trial, accomplished 78 routine clinical trials by 7 three grades of first-class medical institutions such as two garden hospitals of Chinese department of Chinese medicine institute, Shijiazhuang safety hospital, Hospital Attached to Shandong Chinese Medical Univ., Hospital attached to North China Coal Medical College, China People's Liberation Army 210 Hospital (Dalian), Zhengzhou University's medical college first Affiliated Hospital, Langfang City institute of traditional Chinese medicine are common.
Clinical trial one: utilize Chinese medicine Drug therapy medullary system minimal residual leukemia of the present invention clinical research
1. research approach
1.1 diagnostic criteria
By opening south " hematopathy diagnosis and criterion of therapeutical effect "; Acute leukemia reaches fully through treatment to be alleviated the back and accomplishes 3 and consolidate and strengthen course of therapy, or with flow cytometer or part is arranged specific gene changer existing with PCR mensuration minimal residual leukemia cell.
1.2 include the case standard in
Meet the AML diagnostic criteria, and satisfy the MRD definition; Tried voluntarily, and signature Informed Consent Form person all includes the clinical observation case in.
1.3 get rid of the case standard
Age is less than 14 years old or greater than 70 years old; Merge diseases such as severe cardiac, brain, liver, kidney, the psychotic, gestation, women breast-feeding their children, known to this medicine allergy sufferers, and participate in other clinical research person and all should get rid of.
1.4 reject the case standard
, can't not judge clinical efficacy by the regulation medication; Clinical data is incomplete, affects the treatment and safety evaluation; Include in the back find not meet include standard person in all should be as rejecting case.
1.5 end and remove the case standard
Can not adhere to treatment; Serious adverse reaction or severe complication appear in the clinical research process; The state of an illness increases the weight of, and the therapist of need adopting vigorous measures all should be ended clinical research.
The case standard 1.6 come off
The case that comes off is meant that all filled in Informed Consent Form, and no matter when what is because of withdrawing to screen the experimenter of qualified entering clinical research, and the treatment cycle of short of completion scheme defined is the case that comes off.Comprise: (a) experimenter's compliance poor (compliance＜80% or＞120%), or merge the Chinese medicine and western medicine that uses this clinical protocol to ban use of, or change dressings voluntarily midway or add and use other medicines; (b) come off, lose visit naturally in observing and because of not congruent cause influence curative effect of clinical data and safety judgement; (c) serious adverse events and complication taking place, should not continue to accept clinical research and suspended case.
1.7 curative effect determinate standard
By opening south " hematopathy diagnosis and criterion of therapeutical effect ", with continued complete remission time (CCR) and life span (OS) evaluation curative effect; CCR calculates from alleviating fully, and OS calculates from making a definite diagnosis.
2. research method
2.1 case is selected
All patients from year June in February, 2004 to 2010 during to all 7 hematology of tame unit wards and out-patient's totally 78 examples.Male's 37 examples wherein, women's 41 examples; 16～70 years old age; M1 type 1 example wherein, M2 type 21 examples, M3 type 37 examples, M4 type 5 examples, M5 type 12 examples, M6 type 1 example, M7 type 1 example (table 1).
Table 1. goes into to organize minimal residual leukemia patient hypotype distribution situation
2.2 medicine and usage
Provided by the China TCM Academy of Sciences Xiyuan Hospital Drug Manufacturing Room for Chinese medicine medicine instant of the present invention by the reagent thing, Chinese medicine preparation method for preparing of the present invention comprises the steps:
(1) takes by weighing the raw material of following parts by weight respectively by the composition of Chinese medicine medicine: 30 parts of the Radixs Astragali, 5 parts of Radix Ginseng Rubra, 10 parts of the Rhizoma Atractylodis Macrocephalaes, 10 parts in Poria, 20 parts of Fructus Ligustri Lucidi, 15 parts of Radix Ophiopogonis, 20 parts in the Radix Rehmanniae, 15 parts of Fructus Psoraleaes, 20 parts of the Radixs Polygoni Multiflori, 10 parts of Herba Epimedii and 15 parts of Radix Asparagi;
(2) the said Radix Ginseng Rubra that takes by weighing in the step (1) is added alcohol reflux 2 times, each 2 hours, filter merging filtrate; The Radix Ginseng Rubra medicinal residues that obtain when getting filtration and all the other raw materials mix of said Chinese medicine medicine add water logging and cross medical material decoction 2 times, 2 hours for the first time, 1.5 hours for the second time, filter collecting decoction;
(3) filtrating after will merging and merge after decocting liquid mix;
(4) be concentrated into that relative density is the clear paste of 1.38-1.40 in the time of 60 ℃, 1 part of qinghuo reagent and 2 parts of dextrine powders by weight, and add ethanol, and mix homogeneously is processed granule, and drying promptly gets Chinese medicine preparation.Said Chinese medicine preparation is packaged into bag, every bag be equivalent to said Chinese medicine medicine 13 restrain said Chinese medicine preparation; Every day 2 times, each 1 bag, warm water delivery service was taken 3 months continuously.
2.3 observation index: (1) curative effect observation: take behind the Chinese medicine preparation of the present invention 3,5 years complete remission rates, survival rate (more long term survival rate subsequent analysis); (2) safety observation; (3) lab testing: blood, urine, stool routine examination; Liver function (glutamate pyruvate transaminase, glutamic oxaloacetic transaminase, GOT), renal function (blood urea nitrogen, blood flesh liquor-saturated); Electrocardiogram; (4) untoward reaction.
2.4 detection method
2.4.1 routine blood test is measured
Conventional venipuncture venous blood samples 2mL (EDTA anticoagulant) in vacuum test tube, abundant mixing detects BIAO and BEN automatically with cellanalyzer (MEK-6318 type, Japanese Nihon-Kohden company), obtains PBC classification and counting.
2.4.2 lymphocyte subpopulation detects
Conventional venipuncture venous blood samples 2mL (EDTA anticoagulant) in vacuum test tube, fully mixing.With adjustable pipette monoclonal antibody CD3-PC5/CD4-FITC/CD8-PE, CD3-FITC/CD19-PE/CD56-PC5 and homotype contrast IgG1-FITC/IgG1-PE/IgG1-PC5 (U.S. BECMAN-COULTER company) thereof are added test tube (pressing the reagent requirement amount).Get the 100uL anticoagulation, add above-mentioned test tube respectively, abundant mixing, lucifuge incubated at room 30 minutes.Add hemolysin O ptiLyseC (U.S. BECMAN-COULTER company) 0.5mL then, fully mixing was hatched lysed erythrocyte 10 minutes.With centrifugal 10 minutes of high speed low temperature centrifugal machine (RS-20III type, NSK company), 1500rpm removed supernatant.Every pipe adds 500ulPBS (PH7.2), mixing.Open flow cytometer (FPICS-ELITE type, U.S. BECMAN-COULTER company) power supply and hydrogen LASER Light Source (488nm) preheating 30 minutes, simultaneously the instrument auto-initiation; Proofread and correct light path with the standard fluorescence microsphere, the compensation of adjustment fluorescence.Use flow cytometer trichromatic analysis method and detect BIAO and BEN, every part of BIAO and BEN is all regulated the voltage of photomultiplier tube according to the homotype negative control of labelling, and the restriction background fluorescence is below 2%.Take FSvSS to establish a method, confirm lymphocyte populations, detect the percentage ratio of the three fluorescence of labelling in the lymphocyte populations respectively, use EXPO32 software analysis lymphocyte antigen and express.Every part of BIAO and BEN detects 20000 above cells, BIAO and BEN in 24 hours with cells were tested by flow cytometry.
2.5 follow up a case by regular visits to
Go into group from the patient and take Chinese medicine preparation of the present invention and follow up a case by regular visits to from full 3 months, dead patient followed up a case by regular visits to dead day, and the survival patient follows up a case by regular visits to December in 2010 31 days, and is the shortest 3 months, the longest 79 months.
2.6 statistical analysis
Use Kaplan-Meier survival analysis statistical method calculating treatment group patient 3 years and 5 years remission rates and survival rates and draw survival curve; Use the comparison test of log-rank method; Adopt SPSS 13.0 statistical analysis softwares that relevant data is carried out statistical analysis, the P value is less than or equal to 0.05 and will be considered to the difference of checking statistical significance is arranged.
3. result
3 years, 5 years survival rates of treatment group are respectively 85.2%, 79.7% (seeing table 2).
Table 2. was gone into group 78 routine patients 3 years and 5 years continued complete remissions and existence situation
4. conclusion: 3 years, 5 years survival rates of treatment group patient are respectively 85.2%, 79.7% in this research, obviously are superior to Drug therapy of the prior art.
The decoction that obtains after decocting liquid after filtrating after will merging in the step (3) of the invention described above Chinese medicine preparation method for preparing and the merging mixes can directly be taken to the patient as Chinese medicine preparation, can realize goal of the invention of the present invention equally.In addition, Chinese medicine medicine of the present invention can be made up of following materials based on weight: 1 part～98 parts of the Radixs Astragali, 1 part～50 parts of Radix Ginseng Rubra; 1 part～98 parts of the Rhizoma Atractylodis Macrocephalaes, 1 part～98 parts in Poria, 1 part～100 parts of Fructus Ligustri Lucidi; 1 part～100 parts of Radix Ophiopogonis, 1 part～100 parts in the Radix Rehmanniae, 1 part～100 parts of Fructus Psoraleaes; 1 part～98 parts of the Radixs Polygoni Multiflori, 1 part～98 parts of 1 part～98 parts of Herba Epimedii and Radix Asparagi; Preferred said Chinese medicine medicine is made up of following materials based on weight: 25 parts～35 parts of the Radixs Astragali, 1.5 parts～10 parts of Radix Ginseng Rubra, 5 parts～15 parts of the Rhizoma Atractylodis Macrocephalaes; 5 parts～15 parts in Poria, 15 parts～25 parts of Fructus Ligustri Lucidi, 10 parts～20 parts of Radix Ophiopogonis; 15 parts～25 parts in the Radix Rehmanniae; 10 parts～20 parts of Fructus Psoraleaes, 15 parts～25 parts of the Radixs Polygoni Multiflori, 10 parts～20 parts of 5 parts～15 parts of Herba Epimedii and Radix Asparagi; More preferred said Chinese medicine medicine is made up of following materials based on weight: 30 parts of the Radixs Astragali, 5 parts of Radix Ginseng Rubra, 10 parts of the Rhizoma Atractylodis Macrocephalaes, 10 parts in Poria, 20 parts of Fructus Ligustri Lucidi, 15 parts of Radix Ophiopogonis, 20 parts in the Radix Rehmanniae, 15 parts of Fructus Psoraleaes, 20 parts of the Radixs Polygoni Multiflori, 15 parts of 10 parts of Herba Epimedii and gate winters.
Clinical trial two: utilize the influence of Chinese medicine medicine of the present invention to the minimal residual leukemia patient immune function
1. data and method
1.1 clinical data in February, 2004-2010 year December is in China TCM Academy of Sciences Xiyuan Hospital hematology ward and the patient that goes to a doctor of outpatient service; Diagnostic criteria is according to " hematopathy diagnosis and criterion of therapeutical effect "; Acute leukemia reaches through treatment alleviates (complete remission fully; CR) back and accomplish 3 and consolidate and strengthen course of therapy; Or (polymerase chain reaction PCR) measures the existence of MRL cell with polymerase chain reaction with flow cytometer or part the specific gene changer to be arranged.Go into to organize patient's totally 36 examples (MRL group), male 16 examples, women 20 examples; 22～75 years old age, The median age 49 years old; By hypotype classification, M2 17 examples, M3 13 examples, M4 2 examples, M5 3 examples, M7 1 example.Normal healthy controls group 34 examples all are the health examination person, wherein male 19 examples, women 15 examples, 17～60 years old age, The median age 51 years old.
1.2 Therapeutic Method MRL group gives Chinese medicine preparation of the present invention (Drug Manufacturing Room provide by China TCM Academy of Sciences Xiyuan Hospital, and used Chinese medicine medicine composition, Chinese medicine preparation method for preparing and the method for taking are with above-mentioned clinical trial one).The original chemotherapy regimen of patient is constant, and continues to strengthen chemotherapy.In the clinical research process; To complication as infect, heating, agranulocytosis, hemoglobin or thrombocytopenia; Can carry out infection or bring down a fever; Or the use granulocyte colony-stimulating factor, or infusion Red Blood Cells Suspension or platelet and use erythropoietin, anti symptom treatment such as thrombopoietin.In addition, any Chinese and western drugs relevant with this programme all must not use.With 3 months be 1 course of treatment, general treatment is at 1 more than the course of treatment.Go into group from the patient and take Chinese medicine preparation of the present invention and follow up a case by regular visits to from full 3 months, dead patient followed up a case by regular visits to dead day, and the survival patient follows up a case by regular visits to December in 2010 30 days, and the shortest 3 months, the longest 61 months, meta is followed up a case by regular visits to 37 months time.
1.3 experimental technique
1.3.1 reagent source
Used antibody is mouse anti human monoclonal antibody (Beckman-Coulter), OptiLyse C hemolysin (Beckman-Coulter); Cell permeable membrane agent (Beckman-Coulter); PBS:pH 7.2, contain 0.1%NaN
3
Lymphocyte cultivating system (autogamy): (PHORBOL 12-MYRISTATE 13-ACETATE, PMA), ionomycin (IONOMYCIN), monensin (MONENSIN), calf serum and RPMI 1640 liquid are all available from SIGMA company for Buddhist ripple ester.
1.3.2 sample preparations process
Get all patients respectively and go into the group back and 3 months posterior vein blood 2mL that take medicine, get the normal control group and go into to organize posterior vein blood 2mL,, carry out the PBLC counting with cellanalyzer (MEK-6318) with the 2%EDTA-Na2 anticoagulant.
Cell surface antibody labeling: get and add three/four color fluorescent labeling monoclonal antibodies, CD4-fitc/CD8-pe/CD3-pc5, CD3-fitc/CD19-pe/CD56-pc5 in the EDTA anticoagulation; CD3-fitc/CD28-pe/CD8-ecd/CD4-pc5, CD45RA-fitc/CD45RO-pe/CD8-ecd/CD4-pc5, V α 24-fitc/CD161-pe/CD8-ecd/CD4-pc5; Gamma delta T-fitc/CD161-pe/CD8-ecd/CD4-pc5; Lin-fitc/11c-pe/HLA-DR-pc5, Lin-fitc/123-pe/ HLA-DR-pc5, room temperature lucifuge incubation is 30 minutes behind the mixing; Centrifugal after haemolysis, the washing, last machine testing.
Antibody labeling in the cell membrane: add the fluorescent labeling monoclonal antibody in the EDTA anticoagulation; CD8-ecd/CD4-pc5, the room temperature lucifuge was hatched 20 minutes, carried out cell fixation, permeable membrane with the agent of cell permeable membrane; Add Perforin-fitc/Granzyme-pe; Room temperature lucifuge incubation is 30 minutes behind the mixing, and the washing back is centrifugal, last machine testing.
The Th/Tc sample preparations: add the CD4-pc5 monoclonal antibody in the heparin anti-coagulating, the room temperature lucifuge was hatched 30 minutes, and RPMI 1640 washings once add cell in the cultivating system 5%CO
2Incubator is hatched after 18 hours for 37 ℃ and is washed with PBS, adds CD8-ecd, and the room temperature lucifuge was hatched 30 minutes, carried out cell fixation, permeable membrane with the agent of cell permeable membrane; Cell is divided into two pipes, and one of which adds anti-people IL-4-pe and IFN-γ-fitc for measuring pipe, and another pipe adds the homotype contrast, room temperature lucifuge 20 minutes, and PBS washs once, last machine testing.
1.3.3 BIAO and BEN detection method
Adopt FPICS-ELITE flow cytometer (Beckman-Coulter).Proofread and correct light path and liquid road with the standard fluorescence microsphere, use three/four color fluorimetrys, analyze 20000-100000 cell/sample.
1.3.4 data analysis
Take FS vs SS to establish door, use EXPO32 software analysis data result.
1.3.5 cell quantity computational methods: directly read by flow cytometer; Absolute value is drawn by the percentage ratio that PBLC counting absolute value multiply by the corresponding cell of percentage ratio.
1.4 efficacy assessment standard is with reference to acute leukemia continued complete remission (CCR) and leukemia relapse standard, judges MRL patient 3 years and 5 years CCR and recurrence situation.
1.5 statistical method:
Experimental data is represented with average ± standard deviation
.Treatment group and normal control group relatively to accord with normal distribution and satisfy the variable of homogeneity of variance, adopt two sample t check, otherwise adopt rank test.Relatively, difference accord with normal distribution person adopts paired t-test before and after the treatment, otherwise adopts rank test.Adopt SPSS 13.0 statistical softwares to add up.
2. result
2.1 the total cell of lymphocyte subpopulation T, Th cell, Tc cellular change (seeing table 3)
Compare with the total cell of healthy group T, Th cell, Tc cell before and after table 3 MRL group is treated (χ ± s)
Annotate: compare with the health group,
*P＜0.05,
*P＜0.01; Relatively preceding with the treatment of this group, △ P＜0.05, △ △ P＜0.01
MRL patient compares with the health group before using Chinese medicine preparation of the present invention; T cell absolute value obviously reduces (P＜0.01); Th cell percentage ratio and absolute value all reduce (P＜0.01, P＜0.01), and Tc cell percentage ratio increases (P＜0.01); Th/Tc reduces (P＜0.01), explains that the MRL patient immune function obviously lowers; After using Chinese medicine preparation treatment of the present invention; Patient T cell absolute value showed increased (P＜0.01), Th cell percentage ratio and absolute value all increase (P＜0.01, P＜0.05); Tc cell percentage ratio reduces (P＜0.05); Th/Tc increases (P＜0.05), explains that Chinese medicine preparation of the present invention has the MRL of adjusting patient immune function, the effect that improves patient's autoimmunity.
2.2 lymphocyte subpopulation DPT cell, DNT cell, NKT cellular change (seeing table 4)
Compare with healthy group DPT cell, DNT cell, NKT cell before and after table 4 MRL group is treated (χ ± s)
Annotate: compare with the health group,
*P＜0.05,
*P＜0.01; Relatively preceding with the treatment of this group, △ P＜0.05, △ △ P＜0.01
The DNT cell mainly is gamma delta T CR cell (an abbreviation gamma delta T), and gamma delta T cells accounts for 3%～10% of human peripheral lymphocytes sum, express the CD3 molecule, but the overwhelming majority does not express CD4, CD8 molecule.Gamma delta T cells can be with MHC non-limiting way identification tumor antigen, through approach killing tumor cells such as perforin and granzyme, Fas-FasL and TNF-α, and cytokine such as ability secretion of gamma-IFN, promote the activation of other immunocyte.The existing gamma delta T cells of discovering can kill and wound multiple malignant hematologic disease cell external, thereby confirms the immune response of gamma delta T cells participation to the multiple malignant hematologic disease of the mankind.
Peripheral blood DPT cell is a kind of T cell of uniqueness; Be sophisticated memory cell, have typical CD4+T and CD8+T cell function, it is suitable to contain in perforin and granzyme A and the CD8+T cell content in addition in the DPT cell; In some viral infection; The DPT cell proportion increases, and points out this group cell to participate in specific immune response, can cause the quick degraded of virus infected cell and mutant cell.Explain that Chinese medicine preparation of the present invention has the MRL of adjusting patient immune function, the effect that improves patient's autoimmunity.
The NKT cell is a subgroup unique in the T lymphocyte, and the constant expression of people's NKT cell TCRv α 24 expresses the surface marker CD161 of NK cell simultaneously again, and the NKT cell was identified as the 4th quasi-lymphocyte in 2000.Former studies confirms: the NKT cell has the two-ways regulation immunity function, therefore in antitumor, infection, inhibition autoimmune disease etc., plays a significant role.
Table 4 shows: MRL patient compares with the health group before using Chinese medicine preparation of the present invention; Double negative t cells (DNT) absolute value obviously reduces (P＜0.01), and two positive T cells (DPT) and natural killer T cell (NKT) percentage ratio increase (P＜0.01) but both absolute values do not have obvious change (P＞0.05).After using Chinese medicine preparation treatment of the present invention, patient DNT cell absolute value showed increased (P＜0.01), DPT and NKT cell percentage ratio and absolute value all increase (P＜0.01, P＜0.05).Explain that the anti-residual leukemic cell of Chinese medicine preparation of the present invention maybe be through improving cell quantities realizations such as DPT, DNT, NKT.
3. conclusion:
The Chinese medicine preparation that utilizes the Chinese medicine preparation method for preparing of Chinese medicine medicine of the present invention to make can treat MRL, delay MRL patient's recurrence and prolong DFS phase of MRL, thereby realizes prolonging MRL patient's life cycle; Its mechanism possibly be through regulating MRL patient's body autoimmune function, the absolute quantity of raising Th, NKT, DPT and DNT cell, thereby remaining leukaemia in the inhibition MRL patient body.
Claims (5)
1. a Chinese medicine medicine of treating the deficient disease of insufficiency of vital energy and blood after the leukemia people chemotherapy is characterized in that, is made up of following materials based on weight: 1 part～98 parts of the Radixs Astragali; 1 part～50 parts of Radix Ginseng Rubra, 1 part～98 parts of the Rhizoma Atractylodis Macrocephalaes, 1 part～98 parts in Poria; 1 part～100 parts of Fructus Ligustri Lucidi, 1 part～100 parts of Radix Ophiopogonis, 1 part～100 parts in the Radix Rehmanniae; 1 part～100 parts of Fructus Psoraleaes, 1 part～98 parts of the Radixs Polygoni Multiflori, 1 part～98 parts of 1 part～98 parts of Herba Epimedii and Radix Asparagi.
2. the Chinese medicine medicine of the deficient disease of insufficiency of vital energy and blood is characterized in that after the treatment leukemia people chemotherapy according to claim 1, is made up of following materials based on weight: 25 parts～35 parts of the Radixs Astragali; 1.5 parts～10 parts of Radix Ginseng Rubra, 5 parts～15 parts of the Rhizoma Atractylodis Macrocephalaes, 5 parts～15 parts in Poria; 15 parts～25 parts of Fructus Ligustri Lucidi, 10 parts～20 parts of Radix Ophiopogonis, 15 parts～25 parts in the Radix Rehmanniae; 10 parts～20 parts of Fructus Psoraleaes, 15 parts～25 parts of the Radixs Polygoni Multiflori, 10 parts～20 parts of 5 parts～15 parts of Herba Epimedii and Radix Asparagi.
3. the Chinese medicine medicine of the deficient disease of insufficiency of vital energy and blood is characterized in that after the treatment leukemia people chemotherapy according to claim 2, is made up of following materials based on weight: 30 parts of the Radixs Astragali; 5 parts of Radix Ginseng Rubra, 10 parts of the Rhizoma Atractylodis Macrocephalaes, 10 parts in Poria; 20 parts of Fructus Ligustri Lucidi, 15 parts of Radix Ophiopogonis, 20 parts in the Radix Rehmanniae; 15 parts of Fructus Psoraleaes, 20 parts of the Radixs Polygoni Multiflori, 15 parts of 10 parts of Herba Epimedii and Radix Asparagi.
4. utilize the Chinese medicine preparation method for preparing of the arbitrary said Chinese medicine medicine of claim 1-3, it is characterized in that, comprise the steps: that (1) takes by weighing raw material respectively by the composition of said Chinese medicine medicine; (2) get said Radix Ginseng Rubra and add alcohol reflux 2 times, each 2 hours, filter merging filtrate; The Radix Ginseng Rubra medicinal residues that obtain when getting filtration and all the other raw materials mix of said Chinese medicine medicine add water logging and cross medical material decoction 2 times, 2 hours for the first time, 1.5 hours for the second time, filter collecting decoction; (3) filtrating after will merging and merge after decocting liquid mix.
5. the method for preparing of Chinese medicine preparation according to claim 4 is characterized in that, relative density is the clear paste of 1.38-1.40 when being concentrated at 60 ℃ after the decocting liquid after step (3) also comprises the steps: the filtrating after (4) will merge afterwards and merges mixes; 1 part of qinghuo reagent and dextrine powder are 2 parts by weight; And adding ethanol, mix homogeneously is processed granule; Drying promptly gets Chinese medicine preparation.
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| CN107753602A (en) * | 2017-11-30 | 2018-03-06 | 毛小达 | A kind of Chinese medicine composition for treating leukaemia and preparation method thereof |
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Non-Patent Citations (3)
| Title |
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| 刘永华等: "中医药治疗急性白血病研究进展", 《江西中医药》 * |
| 许勇钢等: "扶正抗白冲剂对微小残留白血病模型小鼠免疫功能及生存期的影响", 《中国中西医结合杂志》 * |
| 麻柔等: "扶正抗白冲剂对急性白血病患者长期存活影响的临床研究", 《中国中西医结合杂志》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107753602A (en) * | 2017-11-30 | 2018-03-06 | 毛小达 | A kind of Chinese medicine composition for treating leukaemia and preparation method thereof |
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